Method of increasing the productivity of birds, fused heterologous protein and method thereof

 

(57) Abstract:

The invention relates to the field of biotechnology. The proposed method is that the bird is administered an effective amount of a heterologous protein, alpha subunit protein inhibin birds or immunogenic fragment and a carrier protein. Heterologous protein can be either inhibin, conjugated to protein carrier, or inhibin, fused with a protein carrier. A method of obtaining a fused heterologous protein includes the introduction of double-stranded complementary DNA encoding an inhibin, or a fragment, a vector that encodes a carrier protein such as maltose binding protein or bovine serum albumin. After the introduction of the vector into the expression system it is expressed fused heterologous protein. After purification of expressed heterologous protein an effective amount of this protein designate the animal so that the animal there is a response of the immunological reaction. The invention allows to increase the production of eggs in birds, to accelerate the onset of egg production and increase the duration of the period of egg production. 3 S. and 17 C.p. f-crystals, 3 ill., table 4.

The invention is a partial prodoljaetsa in General to a method of accelerating puberty in birds, in particular, basilevych and Psittaciformes, by assigning xenogenic protein containing protein inhibin or its fragment and a carrier protein. The present invention relates also to a method of increasing the production of eggs in animals, in particular birds, especially in basilevych, by assigning xenogenic protein containing protein inhibin or its fragment and a carrier protein. The present invention relates further to xenogenic protein containing protein inhibin or its fragment and a carrier protein, and a method for producing xenogeneic protein, which inhibin or merged, or anywhereman with protein carrier.

Becciliya are unable to fly are usually large cursorial birds, consisting of several units, including species such as ostrich, EMU, Rhea, cassowary and kiwi. EMU (Dromiceius novaehollandiae) is an Australian baselevel bird, which is distinguished by the presence rudimentry wings and the fact that her head and neck are covered with feathers. The increase in the average adult EMUs is approximately 6 feet (180 cm) and weighs approximately 150 pounds (68 kg). Ostrich (Struthio camelius) is a large, cursorial bird with short wings and a thick powerful legs. The normal growth of the ostrich is bring the new order of birds Rheiformes.

Rheiformes are a troop of South American cursorial birds, called American ostriches, which differ from the true ostriches amongst its smaller size, by the presence of feathers on the head and neck and three-toed feet. Ostrich and EMUs have long appreciated in their natural environment, respectively, in South America and in Australia. The products obtained from ostrich, demand more than a hundred years, and around the world find sale of their skins, meat and feathers. For example, the skin of an ostrich are used in the manufacture of shoes, handbags, jackets, attaché cases, wallets and many other products. Ostrich feathers are used to trim clothing, dresses and for the manufacture of filters. For example, the ostrich feathers are sold in Europe from about 60 to 1200 dollars per kilogram. In addition, it is believed that the ostrich feathers are "magnetic dust", and therefore it is widely used in the U.S. and abroad in computer engineering and in the automotive industry.

On the contrary, EMU has recently appeared on the market. Valued the same their products, as well as essential oil that is used in the cosmetic industry. The EMU oil, which is heated with a thick layer of subcutaneous fat, has the ability to glue the e compounds to prevent the formation of wrinkles. We are currently exploring the possibility of using EMU oil in medicine, for example in the treatment of arthritis. A typical adult EMUs can reach heights of from 1.6 to 1.9 m or more and a weight of 30 to 45 kg or more. Mature EMU at the age of approximately one year and prepubescent and postpubescent EMUs do not show sex-linked phenotype differences. Similarly, the ostriches in the last few years have seen a rapid growth in the EMU population in the United States. In 1994 in the United States was only about 150000 EMU, including a 15,000 breeding pairs. Assume that the number of EMUs in 1995 will increase to a value of from 500,000 to 750,000 people are birds of the 45000 will be breeding pair.

In several countries, including Australia, Belgium, Israel, Canada, Holland, Namibia, South Africa and Zimbabwe, there is a growing demand for products derived from basilevych. So, for several years in the domestic market experiencing rapid growth in the demand for ostriches and EMUs, and to a lesser extent on Rhea. Over the past five years, the number of breeding pairs of ostriches and the total number of birds in the United States increased, respectively, 7.5 times and 20 times. Estimates in 1995 is Connected to the x due to the high cost adults as well as young animals and is particularly established breeding pairs of ostriches, which price is 75,000 dollars, and the price of a pair of EMUs is $ 30,000 or more. The price of young ostriches aged three to four months is about 7500, and the cost of young EMUs estimated to be approximately $ 5,000. Most animals are buying when their age ranging from three to six months.

Next, a huge interest in beschreven as an alternative to more traditional forms of livestock. Several inherent beschreven factors provide them with an advantage over more traditional forms of livestock (i.e. cattle, pigs and sheep). These factors are: a higher degree of digestion, better suitability for intensive cultivation, large animals, high performance and exceptional nutritional value of their meat.

For example, ostrich meat, which is meat a red color, resembling beef, contains considerably less fat, calories and cholesterol than meat chickens and turkeys. In particular, the portion of ostrich meat weighing 85 grams contains the RA, 59 mg of cholesterol and 135 calories. The portion of chicken meat weighing 86 grams contains 3 grams of fat, 73 mg cholesterol and 140 calories. The portion of beef (for frying) weight 85 grams contains 15 grams of fat, 77 mg cholesterol and 240 calories. Finally, the portion of pork weighing 85 grams contains 19 grams of fat, 84 mg cholesterol, 275 calories. (Data for ostrich meat obtained from a Report 0800100 laboratory quality AMSI. Data for other types of meat taken from the Handbook U. S. D. A. Handbook No. 8, "Nutritive Value of Foods".) Like ostriches, EMU meat is a red meat that is low in fat. In particular, the portion of meat EMU weighing 100 grams contains 1.7 grams of fat, of 57.5 mg of cholesterol and 109 calories. (Data for EMU meat obtained from the company "Siliker Laboratories of Texas, Inc.").

In addition, becciliya, such as ostrich, give approximately 100 pounds (45,3 kg) of meat at the age of 12 months, and therefore produce a significant amount of meat in a relatively short period of time.

Illustration of how great is the advantage basilevych over more traditional forms of husbandry, is the following comparison between the ostrich and cow. First, ripening period and incubation for the ostrich is 42 days, while EN is d, while the cow produces only one calf. Thirdly, the degree of digestion of an ostrich is less than 2:1, while the degree of assimilation of food for cows is 5:1. Fourth, the number of days from fertilization of the egg to slaughter approximately 407 days for ostrich compared to 645 days for cows. Finally, ostriches, in addition to meat and skin, give the feathers, while cows do not give any products other than meat and skin.

Given these circumstances and the increasing demand of the world's population in the meat, which has nutritional value, but has nevertheless low in fat and cholesterol, which can be efficiently produced with minimal negative impact on the environment, industry basilevych has a high potential for growth in the future.

Currently, the demand for basilevych far exceeds the supply. However, breeders basilevych is limited by the fact that the production of many eggs breeding females is less than optimal. Depending on the form of the most basilevych in captivity lay on average 10-20 eggs per year, while their genetic potential for production of eggs, believed to exceed 60 eggs per year. Nab is approximately every 48 hours and high-performance EMUs in the wild is able to lay in the spawning period egg every 72 hours. On the contrary, in captivity, the ostrich is often required from 5 to 10 days to lay an egg, and EMU to lay an egg, it takes from 4 to 8 days.

Market basilevych intended for slaughter, will not be able to grow until every year there will be a sufficient number of offspring. According to some estimates, to maintain the level of market basilevych intended for slaughter each year should appear on the light, at least 250000 animals. For this reason, the method of increasing the production of eggs will significantly increase the growth of the market. Thus, a need exists for compositions and method of increasing egg production in birds, in particular basilevych, such as ostriches and EMUs.

There is also a need for a composition and method of increasing the production of eggs of exotic birds, such as Psittaciformes . Psittaciformes include parrots and are composed of the same family, the order of birds that are zygodactyly and have a strong curved beak. Parrot is defined as a member of a family of birds Psittacidae (the only family Psittaciformes), which is characterized by short, strong, strength is increasing ovulation in mammals. Inhibin is a peptide hormone that is produced mainly sexual glands, in particular, by the growing follicles. In mammals, it performs the function of inhibitory regulator of the secretion of pituitary follicle-stimulating hormone, which performs the function of feedback. Although the existence of inhibin postulated more than 60 years ago, its allocation chemical methods implemented only recently.

The mammalian inhibin is a dimeric protein hormone, which consists of a-subunit (molecular weight 18000) and subunit (molecular weight 14000). -Subunit is unique to inhibin as dimers-subunit form activin, a hormone that releases follicle-stimulating hormone from the pituitary gland-Subunit exists in two forms (AandIN), which are different, but very similar. For this reason, depending on the cash-subunit inhibin may exist as inhibin-a or inhibin-B. Both subunits when they are connected by disulfide bonds necessary for the manifestation of biological activity in suppressing the secretion of follicle-stimulating hormone from the pituitary. The sequence of amino acid OST the ESA, mice and domestic chickens. Excellent reviews on identification, acquisition, analysis and biological effects of inhibin is Risbridger et al., "Current Perspectives of Inhibin Biology", Acta Endocrinologica (Copenh), 122: 673-682 (1990); and C. Rivier et al., "Studies of the Inhibin Family of Hormones: A Review", Hormone Research, 28: 104-118 (1987), which are given here for reference. Animals and birds-stimulating hormone plays a role in the growth and development of the follicle, while the luteinising hormone is believed, induces ovulation. Several factors of the brain and gonads (peptide and steroid hormones) concolorous among themselves for control of the release of the hormone gonadotropin. Of these factors gonadotropin-releasing hormone and inhibin exert opposite regulatory effects on the pituitary secretion of follicle-stimulating hormone in mammals. Gonadotropin releasing hormone is Decapeptide brain, which stimulates the secretion of follicle-stimulating hormone and luteinising hormone, while inhibin is a hormone gonads, which is probably aimed at the selective inhibition of the secretion of follicle-stimulating hormone in mammals.

Basic knowledge ovulation in birds. Growing follicles functionally Mature ovaries of hens exist in a particular hierarchy. A typical ovary contains from four to six large (two to four inches in diameter) filled with yolk follicle (F1 through F4, F6), which is accompanied by a larger number of smaller (from two to ten millimeters) yellow follicle and a lot of very small white follicles. Most pre-follicle (F1) is used for ovulation to the next tribute, the second largest (F2) - another day (approximately 26 hours later) and so on. Monitoring the recovery of the follicle and their development within a specified hierarchy are not yet clarified. Part of gonadotropin from the pituitary has been proven, however, the role of inhibin for controlling the secretion of gonadotropin birds and control of ovulation remains unclear.

A recent strategy to induce hyperovulation in mammals was to develop methods that are in the neutralization activity of endogenous inhibin. For example, consider the active immunization of mammals against inhibin-containing compounds. Immunoneutralization inhibin accompanied povyshennogo vaccine antigenic preparations inhibin, acceleration ovulation is believed to be the result of increased levels of plasma follicle-stimulating hormone, which leads to increased development of follicles in the ovary. These studies have used different vaccine antigens that have proven increase in the rate of ovulation in mammals. Some of the investigated antigens include fragments of the-subunit of inhibin derived from recombinant DNA (Wrathall et al., "Effects of active immunization against a synthetic peptide sequence of inhibin-subunit of plasma gonadotropin concentrations, ovulation rate and lambing rate in ewes", J. Reprod. Fert., 95: 175-182, 1992; Meyer et al. , "Antiserum to an Inhibin Alpha-Chain Peptide Neutralizers Inhibin Bioactity and Increases Ovulation Rate in Sheep", Scientific Journal Series of the Minnesota Agric. Exp. Sta. paper N. 17103, 1991), synthetic replica of the N-terminal sequence - subunit bovine inhibin associated albumin (Glencross et al., "Effects of active immunization of heifers against inhibin of plasma FSH concentrations, ovarian follicular development and ovulation rate", Journal of Endocrinology, 134, 11-18, 1992): synthetic peptide sequence - subunit bovine inhibin associated albumin human serum (Morris et al., "Effect of immunization against synthetic peptide sequences of ovine inhibin-subunit of ovulation rate and twin-calving rate in heifers", Journal of Reproduction and Fertility, 97: 255-261, 1993), and partially purified inhibin from follicular fluid of the cow (Morris on conflicting information about how to change the content of follicle-stimulating hormone during the ovulation cycle, all of the cycles studied mammal immunoneutralization endogenous inhibin leads to a significant increase in the development of follicles in the ovaries and increase in the rate of ovulation, regardless of antigen or mammalian species, which have introduced a sample.

As indicated earlier, part of inhibin in the regulation of reproductive function species of birds remains unclear. Currently published messages are limited reproductive function inhibin in poultry. Much of this literature supports theory that inhibin probably plays in poultry the same physiological role, as established for mammals: chicken inhibin may serve as a regulator recovery and/or development of the follicle. However, the birds part inhibin in monitoring the speed of ovulation may be or may not be due to the suppression of the secretion of pituitary follicle stimulating hormone. For example, although females with low egg production in plasma was found higher content of inhibin and layers of granular cells in pre follicles, h is on, speed-related eggs, not found. Wang et al., "Increase in Ovarian-Inhibin Gene Expression and Plasma Immunoreactive Inhibin Level is Correlated with a Decrease in Ovulation Rate in the Domestic Hen", General and Comparative Endocrinology, 91, 52-58 (1993). In this publication, therefore, it is suggested that related to the speed of ovulation in chickens changes in gene expression-subunit of inhibin and levels of immunoactive inhibin in plasma does not have a direct impact on the rate of ovulation by modulation content in plasma follicle-stimulating hormone. Further, in the publication by R. A. Jonson, "Inhibin in the Hen", Poultry Sciences. 72: 955-958 (1993), in which for a successful definition of immunoactive inhibin in plasma of chickens used bullish system radioimmunoassay, however, not found a significant peak of immunoactive inhibin during the ovulation cycle, despite the pre-surge of luteinising hormone. Thus, the role of inhibin in the development of the follicle in birds remains unclear. Recently - subunit of inhibin chick was successfully subjected to cloning and sequencing amino acid residues. Wang and Jonson, "Complementary Deoxyribonucleic Acid Cloning and Sequence Analysis of the-Subunit of Inhibin from Chicken Ovarian Granulosa Cells", Biology and Reproduction, 49, 1-6 (1993), which palestini-subunits inhibin mammals shows 86-89% gomologichnosti. The analysis method Naser-blotting using two selected probes (cINA6 and cINA12) revealed that the-subunit of inhibin is expressed in the granular cells of the ovary chickens, but is not expressed in the brain, kidneys, liver or spleen of chickens.

Thus, biology inhibin in birds is still poorly understood, and no attempt was made to trace the response of birds to the introduction of provocative tests antigenic inhibin. Thus, as the market basilevych largely limited to less than optimal, the rate of egg laying, many ostrich, EMU and Rhea, the required composition or a method of increasing the production of eggs from these birds. For this reason, the required composition and method of accelerating the start of egg laying in birds. Also required composition and method for increasing the period of poultry egg production in the course of her life. In addition, for a long time remains a need for rapid, simple, reliable and inexpensive way to determine whether the bird to give immunological response to the introduction of provocative tests in the form of a composition antigenic inhibin. In addition, you need fast, simple, reliable and inexpensive method to define the The need for a composition and method of increasing the production of eggs is not limited to birds. Remains a need for effective compositions and method of increasing the production of oocytes of many animals. For example, for a long period of time, there is still a need to increase the production of eggs in most animals bred in agriculture, such as pigs, cows, sheep and chickens. For a long time, there remains a need to increase the reproduction of oocytes from animals with fur, such as mink, Fox, otter, skunk and raccoon, as well as high demand for other animals, the skins of which are used for decorative purposes. Also required composition and method of increasing the production of eggs to increase the population of many animals, such as exotic species or endangered species, to prevent their extinction. Next, for a long time, there remains a need to increase the reproduction of oocytes from animals that are used in the races for entertainment or show (competition), such as horses, dogs, cats, animals, contained in zoos and circus animals. As evidenced by the many examples of the treatment of infertility in humans, there is a need to increase Woe ovulation in many animals.

The present invention relates to a composition and method for producing xenogeneic protein containing inhibin or its fragment and a carrier protein. Protein inhibin or its fragment can be inhibin birds, mammalian inhibin or inhibin reptiles. The carrier protein includes, among other things, but they are not limited to, binding protein maltose, or bovine serum albumin. The preferred protein carrier is a protein that binds maltose.

Xenogenic protein can be either inhibin, conjugated to protein carrier, or inhibin, fused with a protein carrier. The method of obtaining merged xenogenic protein includes the introduction of a complementary DNA that encodes the expression of inhibin or its fragment, a vector containing the coding information for protein carrier. After the introduction of the vector into the expression system in this system is expressed fused xenogenic protein. Mainly xenogenic protein includes inhibin basilevych, such as inhibin ostrich, inhibin EMUs and inhibin Rhea.

The present invention also relates to the increase in the production of oocytes from animals by assigning xenogenic protein on the present izobreteny the number of animal protein, so the reproduction of the eggs of the animal increases. Mainly the animal occurs immunological response directed against the protein. More preferably the immunological response directed against the protein inhibin produced animals (endogenous inhibin). The method leads to an increase in the production of eggs in females animals that produce inhibin, such as mammals, reptiles and birds, in particular, becciliya birds. In particular, this method increases the production of eggs and females basilevych, such as ostrich, EMU and Rhea.

The present invention relates to a method for accelerating the start of egg production in female birds, which includes a step of assigning the female bird an effective amount of xenogenic proteins containing alpha-subunit protein inhibin birds or its fragment and a carrier protein, so that the start of egg production in birds is accelerated. The present invention relates also to a method of increasing the number of eggs with low cholesterol content, which are produced by female birds, and includes a step of assigning the female bird an effective amount of xenogenic proteins containing and the number of eggs with low cholesterol. The present invention relates also to a method of increasing the overall duration of the period of egg production in female birds, which includes a step of assigning the female bird an effective amount of xenogenic proteins containing alpha-subunit protein inhibin birds or its fragment and a carrier protein, so that the total duration of the period of egg production in birds increases.

The present invention relates also to a method for producing antibodies directed against xenogenic protein of the present invention. In General, the method of obtaining antibodies directed against xenogenic protein, is the appointment to the animal an effective amount of xenogenic protein containing protein inhibin or its fragment and a carrier protein, so that the animal occurs immunological response directed against xenogenic protein. Finally, the animal take a blood sample from serum secrete antibodies directed against inhibin. Antibodies are mainly separated from the whey, passing the serum through a column containing an effective amount of carrier protein, with the aim of separating antibodies from the serum.

Further, the present invention relates to bistroomrade high or low egg production. The method consists in determining the number of inhibin produced by animals, which correlated with the ability of the animal to produce eggs. Briefly, the method for determining the number of inhibin in the blood of the animal is the selection of a sample of blood from the animal and contacting the blood sample with antiinsulinovykh antibodies that are directed against specific endogenous inhibin animal. The blood sample is in contact with antiinsulinovykh antibodies under conditions that allow the antibody to selectively interact with inhibin, which is present in the sample. Then not entered into the interaction of the antibody is separated from the entering into co-antibodies and determine the number of antibodies that have entered into cooperation.

The present invention relates also to a rapid, simple, reliable and inexpensive method of determining whether an animal's immunological response to the introduction of provocative tests in the form of compositions containing inhibin. In brief, the method involves associating inhibin or xenogenic protein of the present invention with the solid phase and contacting the immobilized inhibin with the sample of blood of the animal, which pH inhibin selectively interacts with antiinsulinovykh antibodies in the sample. After removal has not entered into the interaction of antibodies from the sample add a quantity containing the label antibodies of a second animal that is directed against a class of antibodies of the first animal. Containing the label antibodies, which are antibodies directed against the animal then selectively interact with antibodies that bound to the immobilized inhibin. After removal of unreacted antibodies containing the label, determine the presence or amount entered in the interaction of the antibody containing the label, by visualizing the label. So, the way allows you to set the presence and quantity of antibodies directed against inhibin animal, and therefore allows to determine whether the animal is to give immunological response to the introduction of a composition containing inhibin.

Thus, the aim of the present invention is containing inhibin composition that induces an immunological response in an animal when it is the introduction of the animal.

Another objective of the present invention is xenogenic protein containing inhibin or its fragment and a carrier protein.

Another objective of the present invention is a composition in the plants is also a method of obtaining a xenogeneic protein, in which one of the proteins contains inhibin or its fragment.

Another aim of the invention is a method of obtaining a merged xenogenic protein, in which one of the proteins contains inhibin or its fragment.

Another objective of the present invention is a method for xenogenic protein containing inhibin or its fragment and a carrier protein.

The aim of the present invention is a method of accelerating the start of egg production (puberty) in birds.

The aim of the present invention is a method of accelerating the start of egg production (puberty) basilevych, such as ostriches or EMUs.

Another objective of the present invention is a method of accelerating the start of egg production (puberty) in Psittaciformes.

Another objective of the present invention is a method of increasing the overall duration of poultry egg production in the course of her life.

Another objective of the present invention is a method of reducing or eliminating the need for shedding altenessen birds.

Another objective of the present invention is a way to force the birds to lay eggs, which contain less kolichestvennie is a way to increase the level of reproduction of oocytes from animals.

Another objective of the present invention is a method of increasing the production of oocytes in mammals.

Another objective of the present invention is a method of increasing the level of reproduction of eggs at people.

Another objective of the present invention is a method of increasing the level of reproduction of oocytes from cows.

Another objective of the present invention is a method of increasing the production of eggs of reptiles.

Another objective of the present invention is a method of increasing the production of eggs in birds.

Another objective of the present invention is a method of increasing the production of eggs basilevych, such as the ostrich and EMU.

Another objective of the present invention is a method of increasing the production of eggs and chickens.

Another objective of the present invention is fast, simple, reliable and inexpensive method of determining whether an animal's immunological response to the introduction of provocative tests in the form of a composition containing inhibin or its fragment.

Another objective of the present invention is to mysterylady high or low egg production.

Another objective of the present invention is fast, simple, reliable and inexpensive method of determining whether a bird hormonal predisposition to having high or low egg production.

Another objective of the present invention is a method of obtaining antibodies directed against inhibin or its fragment.

Another objective of the present invention is a method of obtaining antibodies directed against xenogenic protein containing inhibin or its fragment.

Another objective of the present invention is a method for the accurate determination of quantity of inhibin contained in the blood of the animal.

Another objective of the present invention is a composition comprising antibodies, antibodies directed against another animal.

Another objective of the present invention is a method of producing animal antibodies, antibodies directed against another animal.

These and other objectives, features and advantages of the present invention will become apparent after considering the following detailed description of embodiments of the present invention and the attached claims.

On figet antibody anti (inhibin chicken maltose binding protein), In corresponds to the plasmid pMAL-C, used as a standard vector, corresponds to a protein molecule that is used as the standard of mass, D is a real vector pMAL-C, used to get the merged xenogenic protein, E corresponds to the purified fused protein inhibin chicken - maltose binding protein (xenogenic protein) of the present invention, a F corresponds eluent after cleaning, which was not loaded with xenogenic protein.

Fig. 2 illustrates the effect of immunization with a subunit of inhibin on the number of eggs, put the chickens per day ("HDEP) for Japanese quail using maltose binding protein fused with protein, codenamed INA515. In particular, Fig. 2 is a graphical representation of the data in Table 2 of Example 8.

Fig. 3 illustrates the effect of immunization with a subunit of inhibin on the number of eggs, put the chickens per day ("HDEP) for Japanese quail using maltose binding protein fused with protein, codenamed cINA515, when there are only two injections; control and immunization by inhibin. In particular, Fig. 3 is a graphical performance, the I in the present description to refer to a member of the class Aves animals they differ in that they are warm-blooded animals an egg-laying animals, mainly adapted for flying. The term "becciliya" is used in this description to refer to the group is not able to fly, mainly large cursorial birds, consisting of several units and including EMUs, kiwis and ostriches, cassowaries. The term "Psittaciformes" in the context of the present invention refers to parrots, which are containing a single family order of birds, are zygodactyly and have a strong curved beak. Under the "parrot" you see any member of the family of birds Psittacidae (the only family Psittaciformes), which differ in the short, robust, strongly curved beak.

The term "egg" is used in this description to denote a large female egg enclosed in a porous limestone or leather shell, which is produced by a bird or reptile. The expression "is produced by a bird or reptile" is used in this description to refer to the act of laying eggs, poultry, or egg laying". The term "ovum" is used to denote the female reproductive cell, also known as the egg. Thus, the reproduction of eggs at all W Eloy egg from the ovary, or "ovulation". Thus, it should be understood that the term "egg" is used to denote a large female reproductive cells, enclosed in a porous limestone or leather shell, which is produced by a bird or a reptile, or a Mature egg, which is produced by all other animals.

The terms "laying eggs" or "sex was ripened" in relation to the birds used in the present description as equivalent and refer to the moment when the bird will lay her first egg. Thus, the expression "speed start" eggs or sexual maturity birds in the context of the present invention refers to the induction of an earlier date of first clutch of eggs compared to the date when the bird has delayed the egg under normal conditions.

It should be understood that the method of reducing the cholesterol in the egg in the context of the present invention refers to a way to get the bird to postpone one or more eggs with less cholesterol than the average cholesterol content in the eggs, put the birds of the same species.

The expression "the total duration of egg production during the life of birds specifies the total number of eggs, put the bird is the present invention denotes the number of eggs, postpone a specific group of chickens a day.

Unlike term bird or poultry, the term "mammal" is used in this description to refer to a member of the class Mammalia which represents a large class of warm-blooded vertebrate animals, including animals characterized by the presence of mammary glands, hair covering the body, three bones in the middle ear, a muscular diaphragm separating the lung and abdomen, the red blood cells with no nuclei, and embryonic development in allantoine and amniotic membranes.

The term "reptiles" is used in this description to refer to any member of the class Reptilia, which is a class of terrestrial vertebrate animals characterized by the absence of fur, feathers and breast, while their skin is covered with cushwa, they have a three-chambered heart, and lung and abdominal cavity are not separated from each other.

Xenogenic protein in the context of the present description refers to a protein containing inhibin or its fragment and a carrier protein. It should be understood that the term "inhibin" and "fragment inhibine" are interchangeable in the composition xenogenic protein, the method

You should also understand that "cINA515" is used to denote a sequence with 515 base pairs (Sequence ID number 1), or to denote a sequence with 516 base pairs (Sequence ID number 3), or to denote the corresponding sequence cINA6 instructions on page 3 (from base pair number 790 to about a couple of grounds with a number 1310) article Wang and Johnson, "Complementary Deoxyribonucleic Acid Cloning and Sequence Analysis of the-Subunit of Inhibin from Chicken Ovarian Granulosa Cells", Biology of Reproduction, 49, 1-6 (1993),

which set forth fully herein for reference, and obtained from the genomic DNA of a chicken. cINA515 encodes part of the alpha-subunit of inhibin ostrich, such as a Sequence number identifying 2 or Sequence ID number 4. In the context of the present description "MBP-cINA515" denotes the conjugate of a protein, which is expressed from a recombinant host cell after cloning cINA515 in the recombinant cell host and expression merged xenogenic protein containing maltose binding protein and a fragment of the alpha-subunit protein inhibin encoded cINA515. Thus, "cINA515" refers to a sequence nucleot the description is defined as a protein, containing inhibin, or a fragment, fused with a protein carrier. Merged xenogenic protein is expressed from expression systems, including fused gene product, which contains the gene encoding the protein expression of inhibin or its fragment, fused with the gene coding for the expression of a protein carrier. The expression "fused gene product" in the context of the present invention means a product resulting from the fusion of the gene encoding the protein expression of inhibin or its fragment and the gene encoding the expression of a protein carrier.

Conjugated xenogenic protein in the context of the present description refers to a protein containing inhibin, or a fragment, conjugated to a protein carrier. Conjugated xenogenic protein get, carrying out a chemical reaction in which a protein inhibin connected with protein carrier through covalent bonds.

Immunological response of the animal to a substance that he introduced, in the context of the present description denotes the adjustable cells and/or humoral response of the animal, which are specific directed against the introduced substances.

The expression "selectively interacts" in the context of the communication, non-covalent connection, electrostatically, the mechanism of interaction of the receptor-ligand, on the mechanism of interaction of the enzyme-substrate or through another type of link or connection. The binding is selective in the sense that two objects interact-specific, specific place or only with each other.

The present invention relates in General to compositions used in the method of accelerating the start of egg laying in birds. The composition includes xenogenic protein containing inhibin or its fragment and a carrier protein. Inhibin may be obtained from any animal species, which produces inhibin. Inhibin includes among others, but they are not limited to, inhibin birds, mammalian inhibin, inhibin reptiles, inhibin amphibians or inhibin fish. More specifically mammalian inhibin can be selected, but not limited to, from inhibin cows, inhibin person, inhibin horses, inhibin cats, inhibin dogs, inhibin sheep, inhibin mink, inhibin foxes, inhibin otters, inhibin ferret, inhibin raccoon and inhibin pigs. Inhibin birds can be selected, but not limited to, from inhibin ostrich, inhibin EMU, taciformes. The preferred inhibin is inhibin birds. The preferred inhibin is inhibin basilevych. Even more preferred inhibin is inhibin ostrich. Other preferred inhibin is inhibin EMU. Another preferred inhibin is inhibin Rhea. Another preferred inhibin is inhibin chicken. Most preferably xenogenic protein of the present invention contains alpha-subunit protein inhibin or its fragment and a carrier protein.

Xenogenic protein of the present invention containing protein inhibin or its fragment and a carrier protein used in the method of increasing the duration of the total period of poultry egg production in the course of her life. This composition is also used in the method of increasing the level of egg production in animals. This composition is then used in the method of reducing cholesterol in eggs of birds. This composition is also used in the method of reducing or eliminating the need molting birds. These methods and the way to accelerate the start of egg laying in birds will be more discussed below.

Inhibin or its fragment can be isolated from the liquid is synthetically through a series of chemical reactions. In particular, the fragment inhibin can be selected, but not limited to, of the following compositions: -subunit of inhibin; -subunit of inhibin; fragments; -subunit of inhibin or-subunit of inhibin obtained using recombinant DNA; synthetic replica fragments-subunit of inhibin or-subunit of inhibin; synthetic replica of the N-terminal sequences-subunit of inhibin or-subunit of inhibin; fragments of partially purified inhibin from follicular fluid; fragments of endogenous subunit of inhibin or - subunit of inhibin; and fragments of exogenous - subunit of inhibin or - subunit of inhibin. As indicated previously, the fragment inhibin most preferably represents - subunit of inhibin or its fragment.

Inhibin in xenogenic protein or merged, or anywhereman with protein carrier, as described next. If inhibin fused with protein carrier, xenogenic protein is a "merged xenogenic protein". If inhibin anywhereman with protein carrier, xenogenic protein is a "conjugated xenogenic protein". Preferred xenogenic protein is fused xenogene. In xenogenic protein can be used any known from the technical field of the carrier protein. The carrier protein, which can be used according to the present invention, can be selected, but not limited to, the following groups: protein, maltose binding; bovine serum albumin; hemocyanin lymph snails; ovalbumin; flagellin; serum albumin of any animal species; gamma-globulin of any animal species; isogenic cells; isogenic cells containing Ia antigens; and polymers of D - and/or L-amino acids. The preferred protein carrier is a protein that binds maltose. Another preferred protein carrier, if xenogenic protein will not be assigned a cow or a horse, is bovine serum albumin. Finally, another preferred protein carrier, if xenogenic protein will not be assigned the bird is ovalbumin. The most preferred protein carrier is a protein that binds maltose. The carrier protein should preferably be immunogenic with respect to the animal to which it is assigned.

The present invention relates also to a method for producing conjugated xenogenic protein of the present invention. How podrobno described in the publication "Antibodies, A Laboratory Manual" (Editors Ed Harlow &David Lane) Cild Spring Harbor Lab (1988), which is reproduced here for reference.

Although conjugated proteins can be used in the present invention, the slit proteins are preferred. In particular, the slit xenogenic proteins give a homogeneous product in which the various segments of proteins are always merged in the same position and merged the same number of segments of proteins. In addition, the slit xenogenic proteins homogeneous, inexpensively and in large quantities. In contrast, conjugated xenogenic proteins are not as homogeneous as fused proteins. For example, depending on, any conjugation of proteins is carried out, the conjugation reaction can lead to a mixture of proteins having one or more ties or proteins have contacts in different places, either unconjugated proteins remain. Further, some mates can cause xenogenic protein cannot be used due to steric interaction (e.g., due to steric interaction becomes unavailable immunogenic portion of the protein). In addition, when carrying out reactions of conjugation of its conditions and reagents can lead to degradation of the resulting proteins. For example, in reactions conjugal is when you obtain them in large quantities is much more expensive than fused proteins.

The present invention relates also to a method for producing fused xenogenic proteins. Merged xenogenic proteins expressed fused gene product containing the gene encoding the protein expression of inhibin or its fragment and the gene encoding the expression of a protein carrier. Fused gene product and method for producing fused gene product are described in more detail hereinafter. Briefly, the method of obtaining merged xenogenic protein of the present invention includes the introduction phase fused gene product in the coding region of the plasmid, transfection of a host cell with the plasmid and expression of the slit xenogenic protein from the host cell using methods well known from the field of technology.

From the field of technology there are many ways to get merged xenogenic proteins. Therefore, to obtain merged xenogenic protein of the present invention can use any method known from the technical field. To obtain a merged xenogenic protein of the present invention can be used in many commercially available sets of vectors and expression systems. An example of such a set of vectors and expression systems is the pMAL-c company New England Biolabs" (Bever get merged xenogenic protein from a set of pMAL-c described later in Examples 1 and 2. Other sources of vectors and expression systems that can be used to get the merged xenogenic protein of the present invention, include, but not limited to: Pharmacia Biotech" (Piscataway, NJ) and "Clontech" (Palo Alto, California).

The present invention relates further to a fused gene product containing the gene encoding the protein expression of inhibin or its fragment and the gene encoding the expression of a protein carrier. Gene inhibin can be obtained from any species of animal that produces inhibin. Gene inhibin may be amongst the genome inhibin poultry genome inhibin mammal, the genome of inhibin reptile, genome inhibin an amphibian or genome inhibin fish. More specifically gene inhibin mammal may be selected from the group including, but not limited to, gene inhibin cows, gene inhibin human gene inhibin horses, gene inhibin cat gene inhibin dogs, gene inhibin sheep, gene inhibin mink, gene inhibin Fox, gene inhibin otters, gene inhibin ferret, gene inhibin raccoon and gene inhibin pigs. Gene inhibin birds can be selected, but not limited to, gene inhibin ostrich, gene inhibin EMU, gene inhibin homemade chicken and gene inhibin any member of the squad Psittaciformes, gene inhibin any of the Falconiformes, gene inhibin any of the Piciformes, gene inhibin any of Strigiformes, gene inhibin any of Coraciformes gene inhibin any of Ralliformes, gene inhibin any of the Passeriformes, gene inhibin any of the cuculiformes, gene inhibin any of Serves, gene inhibin any of Galliformes (poultry), gene Inguna any of the Anseriformes (geese, ducks and other domestic waterfowl) gene inhibin any of Herodiones and gene inhibin any of the following birds: Falcon, eagle, hawk, dove, long-tailed parrots, cockatoos, parrot and bird sitting on the perch (such as songbirds, Jay, Blackbird, Finch, Warbler and Sparrow). Preferred genome inhibin is gene inhibin birds. More preferred genome inhibin is gene inhibin basilevych. Especially preferred genome inhibin is gene inhibin ostrich. Other preferred genome inhibin is gene inhibin EMU. Another preferred genome inhibin is gene inhibin Rhea. Another preferred genome inhibin is gene inhibin chicken.

A complementary DNA clone-subunit of inhibin chicken (cINC6; Wang and Johnson, "Complementary deoxyribonucleic acid cloning and sequence analysis of the-subunit from chicken ovarian granulosa cells", Biology of Reproducti(Cornell University). Clone cINA6, specially heriditary for the genomic DNA of the ostrich, shows the analysis by the method of southern blotting significant gomologichnosti DNA between these two samples (Chouljenko et al. , "Expression and purification of chicken-inhibin as a fusion protein with E. coli maltose Binding protein", Poultry Science, 73 (Suppl. 1): 84, 1994). The DNA fragment ("cINA515") cut out from a clone cINA6 using fermentation with Pst 1. Fragment cINA515 accompanies most-subunits inhibin Mature hens.

Sequence - subunit of inhibin ostrich receive by the method of polymerase reaction synthesis circuit, well known from the technical field. In particular, design the following primers based on the sequences provided in article Wang, and used in the polymerase reaction synthesis circuit with the genomic DNA of the ostrich: 5'-TCTTTCGAGGAGCTGGGCTGG-3' and 3' GGGCCGTGGTACGCGAGTGACGCA-5'. Approximately 75% of the DNA sequence cINA515 compared with the genomic DNA of the ostrich. Thus, the comparison between the DNA of the chicken and the ostrich shows 100% gomologichnosti.

As indicated above, it should be understood that the carrier protein is not the critical aspect of the present invention. For this reason, in the present invention can be any gene that encodes the expression of belciu the following proteins: protein binding maltose; bovine serum albumin; hemocyanin lymph snails; ovalbumin; flagellin; serum albumin of any animal species; gamma-globulin of any animal species; isogenic cells; isogenic cells containing Ia antigens; and polymers of D - and/or L-amino acids. Preferred gene of carrier protein is the gene encoding the expression of the binding protein maltose. Other preferred gene of the protein carrier, if the resulting xenogenic protein will not be assigned a cow or a horse, is a gene of bovine serum albumin. Finally, another preferred gene of the protein carrier, if the resulting xenogenic protein will not be assigned the bird is the ovalbumin gene. The most preferred genome of carrier protein is the gene encoding the expression of the binding protein maltose. Preferred gene of carrier protein encodes proteins that increase both the magnitude and duration of immune responses of the host to the action of the protein inhibin.

The present invention relates also to a method for producing a fused gene product, comprising the fusion gene encoding the protein expression of inhibin or its fragment with the gene, codelocal stage scroll to the desired complementary DNA inhibin, obtain double-stranded DNA inhibine, the merging of double-stranded DNA inhibin double-stranded DNA of carrier protein so that the fused DNA was allowed to spend the expression of the slit xenogenic protein containing inhibin or its fragment and a carrier protein.

From the field of technology there are many ways to select genes and gene products merge. Cm. , for example, Sambrook, Fritsch &Maniatis, "Molecular Cloning, A Laboratory Manual", 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, Vol. I, II, III. Thus, there may be used any known from the technical field of a method of obtaining a fused gene product of the present invention. To obtain the fused gene product of the present invention can be used in many commercially available sets of vectors. An example of such a commercially available set of vectors is the pMAL-c company New England Biolabs (Beverly, Massachusetts). A method of obtaining a fused gene product from a set of pMAL-c are described in detail hereinafter in Example 1. Other sources of sets of vectors that can be used to obtain a fused gene product of the present invention include, but not limited to: Pharmacia Biotech" (Piscataway, shtam new Jersey,) and Clontech) (Palo Alto, California).

Unexpectedly, it was found that the composition according to the present invention accelerates the start of egg laying in birds. The present invention relates also to a method for accelerating start laying eggs or puberty females birds by assigning an effective amount of xenogenic protein of the present invention (containing inhibin or its fragment and a carrier protein) so that the start of egg laying in birds is accelerated. The term "accelerated" means that the day of the first clutch of eggs from treated birds occurs at least 3% before the eggs would come th least about 5%, more preferably occurs at least about 7% previously. Even more preferably the beginning of egg laying occurs at least 10% and preferably occurs at least approximately 13% earlier than this laying Netopia would in normal circumstances have not processed poultry. It should be understood that "processed" bird means the bird, which designate xenogenic protein of the present invention. Preferably the immunological response occurs in birds against inhibin. More preferably the immunological response directed against endogenous inhibin produced by the bird.

The method according to the present invention can be used to accelerate the start of egg laying females birds of any species that produce inhibin. Female birds can choose, but not limited to, basilevych, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Serves, Galliformes (poultry), Anseriformes (geese, ducks and other waterfowl poultry) and Herodiones. More specifically, the female bird you can choose, but not limited to, ostrich, EMU, Rhea, kiwi, cassowary, turkeys, confused is such as songbirds, Jay, Blackbird, Finch, Warbler and Sparrow) and any representative of the order Psittaciformes. The preferred bird is beskriva bird. The preferred bird is the ostrich. Another preferred baselevel bird is the EMU. Another preferred baselevel bird is Rhea. Another preferred bird is any member of the squad Psittaciformes. Another preferred bird is a chicken. Another preferred bird is quail. The method according to the present invention can also be used to accelerate the start of egg laying bird species at risk. These endangered birds include, but not limited to, eagles, hawks, condors and owls.

Inhibin and protein carrier in the composition xenogenic protein of the present invention vary depending on what species of bird it is. In compositions that are assigned to the birds, preferably using inhibin birds and binding protein maltose. If the composition is assigned beschreven, the preferred inhibin is chicken inhibin or inhibin baselevel birds. The preferred inhibin, if the composition is assigned strapnom protein does not have to be obtained from the same animal species, which xenogenic protein will be appointed. For example, xenogenic protein, which designate the ostrich, can contain inhibin chicken and a carrier protein. It should also be understood that the composition may further include an auxiliary connection, preservatives, diluents, emulsifiers, stabilizers, and other known components that are used in the art for the preparation of vaccines. In the composition of the present invention may use any auxiliary system known from the technical field. Preferred auxiliary system is the incomplete beta-blockers. Another preferred auxiliary system is full of beta-blockers. Finally, another preferred auxiliary system is polydispersions acetylated - (1,4) - linked mannan ("Acemannan").

Composition xenogenic protein of the present invention may be administered to the female birds are all known from the technical field methods. For example, the composition may be administered subcutaneously, intraperitoneally or intramuscularly. Preferably the composition is introduced in the form of subcutaneous injections. The composition can be administered to poultry in the form of one or more doses. Gets re-immunization.

The composition of the present invention should be assigned to the female bird before the bird comes the ability to lay eggs or sexual maturity. Preferred age, when the composition of the present invention for the first time is assigned to the animal depends on the type of animal mating animal (if any), size of the bird, identity components (inhibin and protein carrier) and composition.

The number assigned to the female birds xenogenic protein of the present invention varies depending on the species, its age and weight of the birds, whether appointed protein during the breeding season (if the birds are breeding period) and how much protein will be appointed. In addition, the implementation schedule appointments or schedule processing varies depending on the species, the average age at the onset of which begins oviposition in this species, the history of the family of birds (prehistory beginning of egg laying in this family), time of year, when the bird was removed, the level of feeding birds (birds that are well fed, reach sexual maturity earlier than those who do not finish feeding), General health of the birds at the beginning of ritalinic adverse weather conditions, such as rain, heat, or windy weather, to which the bird is not used), conditions (overcrowding) and lack of ability to move.

Specialist in the art in accordance with the description of the present invention can be determined by conventional tests, the number of xenogenic protein, which is necessary in order to cause the bird response immunological reaction to a protein.

For example, the following briefly describes the method according to the present invention for accelerating the onset of sexual maturity in Japanese quail, which is more detailed in Example 8. The average age of puberty for nepodvizhnykh processing quail is approximately six to eight weeks. The following is the schedule of treatments of Japanese quail with an average weight in the range from 0.1 to 0.25 lbs (45 g-113 g): primary (first) injection of 0.75 mg xenogenic protein of the present invention on the 25th day of the birth of the birds and revaccination in the amount of 0.375 mg on 32nd, 39th, 46th, 53rd, 60th and 90th day from the birth of birds.

In particular, when they reach the age of 25 days, 100 quail randomly and evenly divided into the following four groups of the(1,4)-linked mannan ("Acemannan"), immunostimuliruyushhim media ("MBP-cINA515/ACE"),

(2) MBP - cINA515 in Freund's adjuvant ("MBP-cINA515/FRN"),

(3) Acemannan (control immunostimulant; "ACE") or

(4) beta-blockers (control adjuvant; "FRN"). Each bird, which conduct immunization against inhibin (Groups 1 and 2), designate approximately 0.75 mg of MBP-cINA515 in the appropriate test media. Equivalent volumes (0.2 ml) injection carrier ACE or FRS are used, respectively, in Groups 1 and 3 and Groups 2 and 4. All injections carry subcutaneously with a syringe for injection of tuberculin with needles number 25. Over the next five weeks each bird subcutaneously re-vaccination to 0.375 mg MBP-cINA515 or the corresponding control provocative tests.

When the bird age, day 41 (designated as Day 1 of the cycle of egg laying) for 12 consecutive weeks are registering daily egg laying chickens ("HDEP) and mortality ("MORT"). Next, for each of the four groups to determine the average age when the first clutch of eggs ("FIRST") and the age when 50% of the birds start to lay eggs ("FIFTY "). Data HDEP, as will be more described in Example 8, parsed in two different ways.

At present lie is generalnyh intervals of egg-laying (day 0, 7, 14, 21 and 35) during the first 35 days of the study is shown in Fig. 2 (four groups with different injections are considered exposed to various types of processing) and Fig. 3 (compare reimmunization (control) and inhibin-immunogenic groups).

Average daily egg laying chickens for Week 1, Week 2, Week 3, Week 4. Week or the combined data for weeks from the first to the fifth to the four groups who had an injection and considered independently from each other, are shown in Table 2. In particular, the values in Table 3 are presented as percentage levels of oviposition. For example, in Week 1 of the study, only 5.8% of the birds in the group of ACE laid an egg per day. During Week 2 the research 39,56% of birds in the group of ACE laid an egg per day. (39,56% Week 2 includes and 5.8% Week 1).

Table 2 also shows that during the Week, group 4, treated with injection of MBP-cINA515/FRN, reached a peak level of eggs, amounting to 96,57%. Moreover, in accordance with the data of Table 2, the other three groups, which were administered injections, not peaked in Week 4, as the percentage of eggs continued to grow in the period between Week 4 and injections, during the period from Week 1 to Week 5.

Table 3 shows the average daily egg laying chickens in percent during the same time as in Table 2, and the data are summarized for comparison of control groups and immunogenic groups. In the column under data for Weeks 1-5 shows the average percentage level of egg laying for the combined control groups in relation to the United immunogenum groups over the period from Week 1 to Week 5.

Table 4 contains data for the first to lay eggs for both of the considered aggregation processing results (i.e., the comparison for the four groups who had an injection, and comparing the combined data). The age of first egg laying in Table 4 is the average of first clutch of eggs for the birds in each group. It should be noted that the data of Table 5 indicate more than five days to accelerate the start of egg laying between birds group FRN and bird groups MBP-cINA515/FRN. Table 5 also shows more than three acceleration start laying eggs between birds of the control group and the birds impunitivnoj group. (See Tables 2, 3 and 4 in Example 8).

There is the primary positive effect of immunization angioi egg laying hens (HDEP) (table 3) and data for the occurrence of the first clutch (FIRST) (table 4), where the increase in the number of observations increases the importance of applied statistical methods.

Most referred to the time of biological responses to treatments were studied relative to the beginning, the magnitude and duration of response. The data given in the present description, are preliminary (i.e., information about the whole cycle of laying eggs (approximately 12 weeks from the start of egg laying) has not yet been received. For this reason yet to reveal the hidden mechanisms of how changes in productive capacity quail, immunogenic against inhibin. However, in the present description, the data do demonstrate that the influence of immunoneutralization inhibin on the first part of the cycle of egg laying (i.e., puberty).

The data show that the onset of puberty in groups subjected to processing by inhibin, is accelerated. This is evidenced by how the data HDEP and data FIRST. The value of statistical significance associated with a high average level value HDEP limit for treated inhibin quail during Weeks 1 study, significant (P<0, utime treatment groups, observed in Week 2, may mean that the clutch of eggs, though delayed, begins to intensify in quails control group (see Fig. 2). Therefore it can be expected that the magnitude of statistical differences found between the two test groups (groups with inhibin in relation to the group without inhibin) in Week 1, will be somewhat blurred with the advent of the day of egg laying. In fact, when the data obtained during Weeks 1-5, unite, chickens, processed by inhibin, observed significantly higher levels HDEP when the average value of statistical significance (P<0,0763).

Additional evidence supports the conclusion that immunoneutralization inhibin accelerates puberty in quail. So, all (100%) chickens, who were prescribed inhibin already (i.e., 76 day from birth or on Day 35 from the beginning of the experiment) began to lay eggs, while two hens of the control groups have not yet started to lay eggs. This forces us to assume that if the two remaining chicken will never begin to lay eggs, immunoneutralization inhibin could induce the laying of eggs in laying hens, which otherwise wouldn eggs were not laid. Moreover, if the same two birds will never lay eggs or if the remaining two hens will begin to lay eggs earlier the end of the studies (by the age of 124 days), the difference is 3+ in the data for the first day of egg laying (FIRST), in the present description, will inevitably increase.

Further, although the statistical processing of data not yet FIFTY privo these birds, characterized the later laying eggs (approximately 57%) are members of the control (not exposed immunization) groups. This observation is further confirmation that the data HDEP and FIRST, indicating the onset of sexual maturity was accelerated in female quail, which were immunoneutralization inhibin.

Thus, the above data indicate that containing inhibin composition of the present invention accelerates the onset of puberty in Japanese quail. Since the Japanese quail is a satisfactory animal model for hens in relation to their reproductive systems, the above data indicate that the method according to the present invention accelerates the start of egg laying hens. Thus, the method according to the present invention enables suppliers of eggs to produce more eggs and less food.

The following is a brief description of the method of the present invention to accelerate puberty, ostriches, which is discussed in Example 9. The average age of puberty in nepodvizhnykh processing ostrich is in the interval from 150 to 300 pounds (68 to 136 kg): primary (first) injection of 5.0 mg xenogenic protein of the present invention to 26 months from birth; and re-immunization in the amount of 2.5 mg on the 27th, 28th, 30th, 32nd, 34th and 36th month from birth.

The following is a brief description of the method of the present invention to accelerate puberty EMU, which is discussed in Example 10. The average age of puberty in nepodvizhnykh processing EMU is approximately 20 months. The following is a schedule of treatments for EMU with a weight approximately in the range of from 50 to 90 pounds (23 to 41 kg): primary (first) injection of 3.0 mg xenogenic protein of the present invention to 18 months from birth, and re-immunization in the amount of 1.5 mg on the 19th, 20th, 22nd, 24th, 26th and 30th month from birth.

The following is a brief description of the method of the present invention to accelerate puberty in domestic chickens, which is discussed in Example 11. The average age of puberty in nepodvizhnykh processing chickens is approximately 20 weeks. The following is a schedule of treatments for chickens weighing approximately in the range of from 2.0 to 3.5 pounds (0.9 to 1.6 kg): primary (first injection 1.5 mg xenogenic protein of the present invention on a 15 week from birth, and re-immunization in the amount of 0.75 mg on the 17th,the invention to accelerate puberty, turkeys, which is discussed in Example 12. The average age of puberty in nepodvizhnykh processing turkeys is approximately 30 weeks. The following is a schedule of treatments for turkeys weighing approximately in the range of from 9.0 to 12 pounds (4.1 to 5.4 kg): primary (first injection of 2.0 mg xenogenic protein of the present invention to 28 week from birth, and re-immunization in the amount of 1.0 mg on the 29th, 30th, 34th, 38th, 46 and 54 weeks from birth.

The following is a brief description of the method of the present invention to accelerate puberty, parrots, which is discussed in Example 13. The average age of puberty in nepodvizhnykh processing parrots is approximately 20 months. The following is a schedule of treatments for parrots weighing approximately in the range from 0.5 to 1.25 pounds (225 to 570 g): primary (first) injection of 0.375 mg xenogenic protein of the present invention to 28 months from birth, and re-immunization in the amount of 0.375 mg on the 29th, 30th, 32nd, 34th, 36th and 38th month from birth.

Another variant implementation of the present invention relates to a method for increasing egg production in animals by assigning effective is Sitel), so the production of eggs a bird increases. The term "increased" means that the production of eggs processed birds increases at least approximately 3% compared to the number of eggs, put not processed birds. Production of eggs is preferably increased at least about 7%, and more preferably increased to at least approximately 12%. It should be understood that "processed" is a bird, which designate xenogenic protein of the present invention. The animal is preferably occurs the response, the immunological reaction against inhibin. Immunological response is mainly directed against endogenous inhibin produced animals.

The method according to the present invention can be used to increase the production of eggs by females of all species of animals that produce inhibin. The animal can be, among others, birds, mammals, reptiles, amphibians or fish. More specifically, the mammal may be selected from the group including, but not limited to, cow, human, horse, cat, dog, sheep, mink, Fox, is not limited to, from basilevych, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Serves, Galliformes (poultry), Anseriformes (geese, ducks and other waterfowl poultry) and Herodiones. More specifically, the female bird you can choose, but not limited to, ostrich, EMU, Rhea, kiwi, cassowary, turkeys, quail, chicken, Falcon, eagle, hawk, dove, long-tailed parrots, cockatoos, parrots and birds sitting on the roost (such as songbirds, Jay, Blackbird, Finch, Warbler and Sparrow) and any representative of the order Psittaciformes. The preferred bird is beskriva bird. The preferred bird is the ostrich. Another preferred baselevel bird is the EMU. Another preferred baselevel bird is Rhea. Another preferred bird is any member of the squad Psittaciformes. Another preferred bird is the domestic chicken. Another preferred bird is quail. The method according to the present invention can also be used to increase egg laying bird species at risk. These endangered birds include, but not limited to, eagles, hawks, condors and owls.

Inhibin and the carrier protein to which the shaft is intended. Xenogenic protein of the present invention are discussed in detail previously. In compositions that are assigned to the birds, preferably using inhibin birds and binding protein maltose. If the composition is assigned beschreven, the preferred inhibin is homemade chicken inhibin or inhibin baselevel birds. The preferred inhibin, if the composition is assigned to the ostrich is the homemade chicken inhibin or inhibin ostrich. It should be understood that inhibin in xenogenic protein does not have to be obtained from the same animal species, which xenogenic protein will be appointed. For example, xenogenic protein, which designate the ostrich, can contain inhibin homemade chicken and a carrier protein. It should also be understood that the composition may further include an auxiliary connection, preservatives, diluents, emulsifiers, stabilizers, and other known components that are used in the art for the preparation of vaccines. In the composition of the present invention may use any auxiliary system known from the technical field. Preferred auxiliary system is the incomplete beta-blockers. Another preferred auxil which the invention may be administered to any animal known from the technical field methods. For example, the composition may be administered subcutaneously, intraperitoneally or intramuscularly. Preferably the composition is introduced in the form of subcutaneous injections. The composition can be administered to the animal in the form of one or more doses. Preferably the composition is assigned to the animal in several doses, for primary immunization followed by repeated immunization.

The composition can be administered to the female animal in any period of time before she will stop ovulation due to illness or age. The preferred age at which you want to assign to the animal a composition of the present invention depends on the type of animal mating animal (if any) and destination track.

For example, if the composition is administrated to increase the production of oocytes cultured in agriculture animals that have periods of reproduction, then the preferred destination time composition precedes the breeding period. On the contrary, if the composition is assigned a Mature animal with a low level of reproduction of the oocyte, the composition is administered in a time when this low level will cause problems.

edocfile is immunization birds during the six-month period, prior to the first breeding season birds. Specialists in the field of technology it is known that on average female birds begin to lay eggs during the first breeding period. Therefore, it is preferable to immunization birds approximately six months before the onset of the first period of breeding birds, and then repeated immunization with intervals of one month before the onset of the first period of breeding birds. Even more preferred is immunization birds approximately six months before the onset of the first period of breeding birds, and then repeated immunization with intervals of one month within six months. The number assigned to the animal xenogenic protein of the present invention varies depending on the type of animal, its age and weight of the animal, whether appointed protein during the breeding season (if the animal has the breeding period) and how much protein will be appointed. Specialist in the art, using this description, it is easy to determine using conventional tests, the number of xenogenic protein, which is necessary to suppress the immunological response in the animal compared to the USA, primary immunization ostrich spend approximately 6 months before the first breeding season, and then for six months with intervals of one month is repeated immunization. Primary immunization consists of assigning approximately from 0.5 to 4.5 mg xenogenic protein of the present invention. Re-immunization include the appointment of from about 0.30 to 3.0 mg xenogenic protein of the present invention. Primary immunization mainly includes the appointment of approximately 1.5 to 3.0 mg xenogenic protein of the present invention. Re-immunization include the appointment of from about 0.75 to 1.5 mg xenogenic protein of the present invention. Xenogenic protein preferably emuleret in complete Freund's adjuvant (Sigma Chemical Co." St. Louis, Missouri) for primary immunization and re-immunization with xenogeneic protein preferably emuleret in incomplete Freund's adjuvant (Sigma). Even more preferably the composition xenogenic protein is administered as subcutaneous injections. Even more preferably the composition xenogenic protein is administered as subcutaneous injections in three places at the top of the thigh ostrich.

The way to increase aizens is s not subjected to genetic selection from the point of view of fertile eggs. For example, domestic chickens genetically selected from the point of view of maximum egg production from the late 1920's. See, for example, M. A. Jull, 1932, "Poultry Breeding, John Wiley & Sons. Due to the short life time of hens, from about 1928 until the present time is doing a great job on breeding birds from the point of view of their egg production. On the contrary, becciliya and Psittaciformes and most exotic birds were not selected genetically from the point of view of fertile eggs. Similarly, from the point of view of the fertile eggs are not genetically selected birds at risk. Thus, as domestic chickens already genetically possess excellent egg production, the improvement that can occur when using the method according to the present invention, is limited in comparison with birds that have genetically bad or average egg production. For this reason, a significantly larger improvement in the level of egg production when using the method according to the present invention there is for birds that are not genetically selected with respect to their fertility, egg laying, such as becciliya, Psittaciformes, and other exotic birds and birds at risk.

Another example FPIC is consistent xenogenic protein administered to the mammal so what mammal response occurs immunological reaction against xenogenic protein. Xenogenic protein mainly consists of inhibin mammal conjugated with protein, maltose binding. Another preferred xenogenic protein consists of inhibin birds or reptiles and binding protein maltose.

Immunization with xenogeneic animal protein of the present invention causes the animal to produce antibodies that selectively directed against xenogenic protein. Immunization preferably also causes the animal to produce antibodies that selectively directed against endogenous inhibin. The production of such antibodies bird leads to acceleration of puberty or early production of oocytes. The production of these antibodies in animals also increases the ability of the animal to produce the egg, because the antibodies neutralize the biological activity of inhibin present in the blood of the animal.

Without assuming the following assumption, we believe that the - subunit of inhibin binds the receptor, follicle-stimulating hormone and, thus, completely inhibits the binding of follicular who can contact the sites of the receptor, increases in animal biological effects of follicle-stimulating hormone, as competition for the sites of the receptor, follicle-stimulating hormone decreases. We believe that antibodies neutralize inhibin due to the interaction with circulating in the body by inhibin, creating, thus, steric obstacles joined with them in the interaction of the inhibin contact sites of the receptor, follicle-stimulating hormone.

It was unexpectedly found that the composition according to the present invention can also be used to increase the total duration of the egg production of birds in their lifetime. The term "increased" means that the total period of egg production processed poultry increases at least by 3% compared with the total period of egg production have not processed poultry. Preferably the total period of egg production is increased at least about 7%, more preferably increases at least approximately 12%. Even more preferably the total period of egg production is increased at least 15%. It should be understood that "processed" bird means the bird, which designate xenogenic beffective number of xenogenic protein of the present invention, to raise an immunological response against xenogenic protein, and then, if

the bird prescribe the effective amount xenogenic protein (hold reimmunization) in order to maintain the maximum or higher than the conventional egg production. In particular, if xenogenic protein of the present invention for a long period of time is assigned to the female birds in the dosages described in the previously described ways to accelerate the start of egg laying or puberty, the total period of poultry egg production increases compared to a bird that has not been subjected to the processing composition of the present invention.

In the General case, the method according to the present invention leads to a noticeable increase in the total period of egg production in birds that have not been subjected to genetic selection with respect to their fertility while laying eggs. The total period of poultry egg production is limited by the number of female gametes that are formed in birds. However, some birds were subjected to genetic selection on the subject of maximum of ovulation or egg laying. As indicated previously, for example, domestic chickens from the late 1920-ies subjected to genetic selection from the point of view m is asego time is doing a great job on breeding birds from the point of view of their egg production. On the contrary, becciliya and Psittaciformes and most exotic birds were not selected genetically from the point of view of fertile eggs. Similarly, from the point of view of the fertile eggs are not genetically selected birds at risk. Thus, as domestic chickens already genetically possess excellent egg production, the improvement that can occur when using the method according to the present invention, is limited in comparison with birds that have genetically bad or average egg production. For this reason, a significantly larger increase in the total period of egg production when using the method according to the present invention there is for birds that are not genetically selected with respect to their fertility while laying eggs, such as becciliya, Psittaciformes, and other exotic birds and birds at risk. Thus, the increase in the total period of egg production results in a greater number of eggs from the same number of birds that allows breeders of chickens to get more profit.

Unexpectedly, it was found that the composition according to the present invention can also be used to reduce or mouth of oznachaet the female bird, as described in the above method, the level of poultry egg production in comparison with the bird, which does not prescribe the composition of the present invention, remains quite high, so there is a need for shedding birds to increase her egg production. Practice molting females birds, such as domestic chickens (onegreeneye white Leghorn, which are producers of table eggs), is normal, when egg production is reduced, so that the cost of the poultry exceed the economic benefits derived from eggs. To "molt" domestic chickens, birds do not give food for about four to fourteen days before until she starts shedding, i.e., to lose their feathers. During the moulting bird stops to lay eggs. After the birds continue to give a normal amount of food clutches of eggs in the meantime. The whole molting takes approximately two months since the cessation of feeding and prior to the beginning of a new cycle of egg laying. As a result, the level of poultry egg production rejuvenated. However, after a molting birds level of egg production in the next cycle will not reach the level of egg production in the first the EP, domestic chickens begin to lay eggs after about 20 weeks and reach economically viable egg production in approximately 40-50 weeks. When reaching a peak of egg production, domestic chickens lay from eight to nine eggs every ten days. However, approximately 50 weeks of egg laying, the level of egg production is reduced approximately to the value component of 60% from peak values. At this point, the cost of food for chickens exceeds the value they produce eggs. It is common practice moulting birds at this time so that, when the chicken will continue laying eggs, the level of egg production has increased.

Thus, the composition of the present invention, since it retains a higher level of egg production compared to the case when the bird is not exposed to the specified composition reduces or eliminates the need for molting birds. Reducing or eliminating the need molting birds leads to significant savings. Moreover, during the time when the bird is subjected to shedding, it is not productive towards the cost of its power in the period before the molt, and then she is not productive for iget need these unproductive phases birds, thus reducing the costs of breeder hens and increasing its revenues, maintaining egg production at high levels leads to a further increase profits breeder hens, as the level of egg production of hens after molting is not equal to the level of egg production during the first cycle of egg laying, as noted earlier.

In short, the level of egg production birds increase, eliminating the need for molting birds, by assigning an effective amount of xenogenic protein of the present invention, to cause the attitude response of the immunological reaction, and then assign an effective amount of xenogenic protein (re-vaccination) to maintain a larger than normal level of egg production.

Another surprising aspect of the present invention is that the composition of the present invention results in a greater number of eggs with low cholesterol, compared to egg laying birds not exposed to processing. In particular, if the above-mentioned composition designate the female birds, as described previously, the cholesterol in eggs, put this is the position under the present invention. Thus, the composition according to the present invention increases or leads to the production of more eggs with less cholesterol.

The terms "increase" or "more" means that the number of eggs with low cholesterol derived from processed poultry increases, at least about 5% compared with the number of eggs with low cholesterol content, which lays not processed bird. The number of produced eggs with low cholesterol content is preferably increased at least about 10%, and more preferably increases by at least approximately 15%. It should be understood that "processed" bird means the bird, which designate xenogenic protein of the present invention. The term "low cholesterol" or "low cholesterol" means that the cholesterol in eggs is lower than the average cholesterol content in the eggs, put the birds in the course of her life, at least 10%. Preferably the cholesterol in eggs with low cholesterol content is lower than the average content of about 20%. E. the possession, approximately 30%.

It is known that hens for the first five or six eggs, set aside by the female bird (chicken) after it reaches sexual maturity, contain less cholesterol than eggs that she lays later. The composition according to the present invention causes of female birds to lay eggs with low cholesterol content over a longer period of time. As a result of consequences affecting health that are associated with high cholesterol in the blood, there is a need to obtain eggs with low cholesterol. Thus, the composition according to the present invention allows to obtain eggs, the greater number of which are required types of eggs.

Thus, the methods of the present invention to increase the production of eggs and accelerate the start of egg laying will lead to a significant increase in growth of population, and therefore market basilevych, such as the ostrich and EMU, because of their insufficient at the present time, the level of egg production will be increased thanks to the method according to the present invention. The method according to the present invention will satisfy the constant demand in domestic birds such as chickens, current not limited to the laying of eggs by birds. The method according to the present invention to increase the production of eggs can be used to improve the production of oocytes of many animals. For example, the method according to the present invention can be applied to increase the production of eggs in most animals, cultivated in agriculture, such as pigs, cows, sheep and chickens. In addition, the method according to the present invention can be applied to increase the reproduction of oocytes from animals with fur, such as mink, Fox, otter, skunk, and raccoon, and other animals, the skins of which are used for decorative purposes. Next, the method of increasing the production of eggs according to the present invention can also be used to increase the population of many animals, such as exotic species or endangered species, to prevent their extinction. The method according to the present invention can also be used to increase the production of eggs from animals that are used for racing, entertainment or show (competition), such as horses, dogs, cats, animals, contained in zoos and circus animals. Further, the method according to the present invention can be used in cobretti is a method of obtaining antibodies against xenogenic protein of the present invention. In General, the method of obtaining antibodies directed against xenogenic protein comprises the following stages: assigning the animal an effective amount of xenogenic protein containing protein inhibin or its fragment and a carrier protein, so that the animal occurs immunological response directed against xenogenic protein: selection of the blood sample from the animal; a selection from the serum of the blood sample of any of antibodies directed against inhibin. Antibodies predominantly isolated from serum, passing the serum through a column containing an effective amount of carrier protein, with the aim of separating antibodies from serum. Methods used for obtaining and purification of antibodies directed against xenogenic protein of the present invention, well-known specialists in this field of technology.

You should also understand, xenogenic protein of the present invention may be administered to any animal, depending on the type of the required antibodies. You should also understand that inhibin may be exogenous or endogenous. Thus, the type inhibin and xenogenic protein of the present invention and a view of hydrated protein containing inhibin homemade chicken and maltose binding protein can be administered to an ostrich, with the aim of obtaining the ostrich antibody inhibin antiquita. Similarly, xenogenic protein containing inhibin ostrich and maltose binding protein can be administered to an ostrich, with the aim of obtaining the ostrich antibody inhibin ancistrus.

The present invention relates to a method of determining the number of inhibin produced animals that allows you to define ascensus the ability of the animal. Briefly, the method for determining the number of inhibin in the blood of an animal includes the following stages: collection of blood sample from the animal; contacting the blood sample with antiinsulinovykh antibodies directed against specific endogenous inhibin animal, under conditions that allow the antibody to selectively interact with inhibin, if present in the sample; separating any not entered into the interaction of antibodies from entering into co-antibody; and determining the amount of antibodies, which came into interaction.

To a person skilled in the art it is obvious that the methods of immunoassay that can be used in the above method, the e visualization, known from the field of technology can be applied in the above way. A preferred immunoassay is ELISA (enzyme immunosorbent assay), and the preferred label is peroxidase from horseradish. Another preferred label is a colored latex bead. Arseny latex ball can have any color required for the implementation of the visualization. Latex ball preferably has a yellow, red, blue or green color. Latex ball may be hollow or solid, but preferably it is hollow to minimize weight. The size of the latex bead varies depending on the purpose of its use in the immunoassay. Experts in the art using standard tests will select the largest size of a ball, which is still visible, but does not provide steric barriers for the reactions immunoassay. Preferably the diameter of the latex bead is not more than 0.5 μm, and most preferably it is less than 0.2 μm.

For example, the concentration of circulating in the blood of birds inhibin can be installed using standard methods sandwich enzyme immunosorbent assay. the tablet for micrometrology. After washing and blocking tablet add some amount of blood taken from the tested birds. Give inhibin, which may be contained in the sample, selectively react with immobilized antiribosomal antibody, and then the sample is washed from the cells of the tablet. Later in the cell type labeled antiinvasive antibodies, which are directed to another part of inhibin or its fragment, compared to the antibody immobilized in the cell. The antibody may be labeled with any label, known from the technical field, such as peroxidase from horseradish. Give labeled antiribosomal the antibody to interact with the immobilized possible by inhibin, and then unreacted labeled antiinvasive antibodies are removed by washing. The number present in the plasma sample inhibin determine, using a suitable imaging technique label, which is used in enzyme immunosorbent analysis to quantify immobilized antiribosomal antibodies in the cell. At the same time in adjacent cells analyzed with negative and positive control samples.

It should be understood that the above method allows you to define the structure can be used to determine the number of inhibine, produced by female animal of any kind that produces inhibin. The animal can be, among others, birds, mammals, reptiles, amphibians or fish. More specifically, the mammal may be selected from the group including, but not limited to, cow, human, horse, cat, dog, sheep, mink, Fox, otter, ferret, raccoon and a pig. The bird you can choose, but not limited to, ostrich, EMU and chicken. Preferred animal is a bird. Preferred animal is beskriva bird. The most preferred animal is the ostrich. Another preferred animal is EMU. Another preferred animal is a domestic chicken.

Animals with a high level of inhibin have low reproductive potential and, depending on species and whether they are grown for agricultural purposes, such animals that produce few eggs, can be sent to slaughter, and not to keep for breeding. On the contrary, bred for agricultural purposes animals with low levels of inhibin are good producers of eggs and they are usually used for breeding and not sent to slaughter.

esmerine (if any). Thus, identification of the potential of an animal for the production of eggs can be determined relative to the aforementioned factors, and measurement of levels of inhibin the animal has the greatest value when compared with the average levels of inhibin those of the same species of animal, roughly the same age, at the same time of year, if the animal is breeding period.

Because the number of inhibin produced by bird, varies depending on these factors, the relative amount of inhibin below for different age groups of birds. Table 1 illustrates the differences in basilevych, such as EMU and ostrich, with respect to the reproduction of their inhibin depending on whether they are bad or good egg production, and depending on the age baselevel birds.

In table 1 the level of inhibin provided on the generalized value of 1 for females ostriches, postponing average for the season from 50 to 60 eggs, which is a good indicator, and a value of 7 for functionally unproductive females basilevych, postponing for the season less than 5 eggs, that is a bad indicator.

Another aspect of the present invention assetsall briefly, the method of obtaining the animal antibodies directed against immunoglobulins of an animal, includes the following stages: the use of effective amounts of a class of antibodies of the first animal, such that you receive the above-described method, the second animal, so that the second beast arises immunological response against the antibody of the first animal; the sampling of blood in the second animal; and the selection of antibodies of a second animal from the serum of the blood sample, as described previously. The second beast is preferably an animal of another species in relation to the first animal.

The present invention relates also to a method of determining whether an animal's immunological response to the introduction of provocative tests in the form of a composition containing inhibin. This method uses antibodies of a second animal, directed against a class of antibodies of the first animal, as described previously. In brief, the method involves associating inhibin or xenogenic protein of the present invention with the solid phase and contacting the immobilized inhibin with a blood sample from the test animal. The sample is in contact with the immobilized inhibin in terms topivshih in the interaction of the antibody from the sample and rinse add a quantity containing the label antibodies of a second animal, which are directed against a class of antibodies of the first animal. Containing the label antibodies directed against antibodies of the animal, and then selectively interact with antibodies that bound to the immobilized inhibin. After removal of unreacted antibodies containing the label, determine the presence or amount entered in the interaction of the antibody containing the label, by visualizing the label. Thus, the method allows us to establish the presence and quantity of antibodies directed against inhibin animal, and therefore allows to determine whether the animal is to give immunological response to the introduction of a composition containing inhibin.

It should be understood that the above method of determining whether an animal's immunological response to the introduction of provocative tests in the form of a composition containing inhibin can be made for the female animal of any kind. The animal can be, among others, bird, mammal or reptile. More specifically, the mammal may be selected from the group including, but not limited to, cow, human, horse, cat, dog, sheep, mink, Fox, otter, ferret, raccoon and a pig. The bird using the preferred animal is beskriva bird. The most preferred animal is the ostrich. Another preferred animal is EMU. And another preferred animal is Rhea.

To a person skilled in the art it is obvious that the methods of immunoassay that can be used in the above method, well known from the technical field. Therefore, any immunoassay method, any method of forming the label and its visualization, known from the field of technology can be applied in the above way. A preferred immunoassay is an enzyme immunosorbent analysis, and the preferred label is peroxidase from horseradish. Another preferred label is a colored latex bead. Colored latex bead can have any color required for the implementation of the visualization. Latex ball preferably has a yellow, red, blue or green color. Latex ball may be hollow or solid, but preferably it is hollow to minimize weight. The size of the latex bead varies depending on the purpose of its use in the immunoassay. Experts in the art using standard tests will select the largest size is edocfile diameter latex ball is not more than 0.5 μm, and most preferably it is less than 0.2 μm.

Another variant of implementation of the present invention associated with the above-mentioned method of determining whether an animal's immunological response to the introduction of provocative tests in the form of a composition containing inhibin, the method immunoassay modify as follows. In brief, the method involves the selection of the animal's blood sample and contacting it with labeled inhibin animal or its fragment. The sample is in contact with the labeled inhibin animal under conditions in which the inhibin animal selectively interacts with any antiinsulinovykh antibodies in the sample. After removal of the sample entering into co-labeled inhibin the presence or amount of entering into co-labeled inhibin determined by visualization of the label. Inhibin used in the specified way is chosen, but not limited to, fused xenogenic protein inhibin of the present invention; endogenous inhibin or its fragment; and exogenous inhibin or its fragment. The preferred inhibin, containing the label, is an endogenous inhibin.

The present invention daltoniana. On the contrary, it should be apparent that the present description is the source of other possible ways of implementation, modifications or equivalents of the present invention that after reading this description can be developed by experts in the field of technology, but they do not contradict the essence of the invention and/or the volume of claims by the claims.

Example 1. A method of obtaining a fused gene product containing the gene encoding the expression of inhibin chicken, and the gene encoding the expression of the binding protein maltose

Below is a method of obtaining a fused gene product containing the gene (INA515) that encodes the expression of a fragment of the alpha-subunit of inhibin chicken (Sequence ID number 2 or Sequence ID number 4) and the gene encoding the expression of the binding protein maltose. Fused gene product of the present invention is obtained from the set of vectors, pMAL-C, which is manufactured by New England Biolabs(Beverly, Massachusetts).

The pMAL vectors allow expression of the protein subjected to cloning a gene or open reading frame of the genetic information. Subject who then leads to the expression of the fused proteins, binding maltose ("MBP-cINA515"). This method allows high level expression of cloned sequences and one-step purification of fusion protein, MBP-cINA515 using the affinity of MBP with respect to maltose.

The following is a method of ligating a gene of inhibine, cINA515, the vector pMAL-c.

1. Fermented 0.5 mg DNA plasmids pMAL 20 ál with genome inhibin chicken, cINA515.

2. Fermented 20 µg DNA from clone gt 11 200 ál with genome inhibin chicken, cINA515.

3. Add to 0.05 units intestinal alkalinity calf (NEB # 290) to the product of fermentation of DNA. Maintained at a temperature of 37oC for one hour.

4. Check the completeness of the course of fermentation by studying 4 μl of the reaction mixture pMAL and 15 μl of the reaction mixture by 0.8% agarose gel. Add to the products of the fermentation and pMAL gtll respectively 1 μl and 8 μl of 0.5 M solution etc.

5. The restriction products of fermentation, add an equal volume mixture of phenol/chloroform (1:1), mix, remove the aqueous (top) phase and placed in a fresh tube. Repeat with one chloroform.

6. Add to vector fermentation of 10 ág of glycogen or tRNA as a carrier. To both products fermentans. Leave at room temperature for 10 minutes.

7. Placed in microcentrifuge for 15 minutes. The liquid above the precipitate is drained, washed tablet 70% ethanol and allowed to dry.

8. Once again suspended each sample in 25 μl of 10 mm solution of Tris-C1, 1 mm solution edtc with pH 8.0.

8. Mix: 1 μl of the product of fermentation vector; 6 μl of the product of fermentation insertions; add 8 ál of water, and then heat the mixture of DNA to a temperature of 65oC for 5 minutes. Cooled on ice, and then add 4 ml of buffer 5x ligase; 0,5 ál ligase NEB T4 (# 202; 200 units); and incubated at a temperature of 16oC for at least 2 hours or leave overnight.

10. Heated for 5 minutes to a temperature of 65oC, cooled on ice.

11. Mixed with 25 µl of competitive TB1 (or any lacZ complementary chain) and incubated on ice for 5 minutes. Heated for 2 minutes to a temperature of 42oC.

12. Add 0.1 ml of LB and incubated at temperature 37oC for 20 minutes. Placed on LB tablet containing 100 mg/ml ampicillin. Leave overnight in the incubator at a temperature of 37oC. Selected colonies using a sterile wooden stick is the C thermostat at a temperature of 37oC for 8 to 16 hours. Compute the phenotype tablet Lac and emit white clones from the reference tablet.

13. Analyze for the presence of indels in one or both of the following ways:

A. Get minireport DNA. Fermented with a suitable restriction enzyme, to determine the presence and orientation of the insertion.

C. i) Grow a 5 ml culture in LB broth amp to a value of approximately 2108Jr.

ii) Take the sample in the amount of 1 ml was Placed in microcentrifuge for 2 minutes, the liquid above the precipitate is drained and the cells are again suspended in 50 μl of buffer for electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate.

iii) To the remaining culture add isopropyl-thiogalactoside to a concentration of 0.3 mm, for example 15 ál of 0.1 M stock solution. Incubated at temperature 37oC with good aeration for 2 hours.

iv) Take samples of 0.5 ml Microcentrifuge within 2 minutes, the liquid above the precipitate is drained and the cells are again suspended in 100 μl of buffer for electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate.

v) Boil the samples for 5 minute sodium. Stain the gel with diamond blue Kumasi. Put the strip is easily visualized in the position corresponding to the molecular weight of the protein fusion. The molecular weight of the binding protein maltose is 42000 Dalton.

Example 2. The method of obtaining merged xenogenic protein, "MBP-cINA515" containing inhibin chicken and protein, maltose binding

Below is the method of obtaining merged xenogenic protein containing inhibin chicken and binding protein maltose, "MBP - cINA515". Fused gene product of Example 1 expresses merged xenogenic product maltose binding protein - inhibin, "MBP-cINA515", as follows:

1. Bring in 80 ml of enriched nutrient medium containing galactose and ampicillin (see below section Environment and solutions) 0.8 ml seasoned overnight culture of cells containing the plasmid merge in Example 1.

2. Grown at a temperature of 37oC with good aeration to the value of 2 x 108cells/ml (A600of ~ 0.5). Take a sample volume of 1 ml and placed in microcentrifuge for 2 minutes (neindutsirovannom cells). The liquid above the precipitate is drained and the cells are again suspended in 50 μl of buffer for electrophoresis on polyacrylamide geobased isopropylthioxanthone to a final concentration of 0.3 mm, in particular in 0.24 ml of uterine 0.1 M solution in water (see section Environment and solutions). Continue incubation at a temperature of 37oC for 2 hours. Take a sample volume of 0.5 ml and placed in microcentrifuge for 2 minutes (induced cells). The liquid above the precipitate is drained and the cells are again suspended in 100 μl of buffer for electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. Mix and place on ice.

4. Divide the culture into two aliquots. Separating the cells by centrifugation with acceleration 4000 x g for 10 minutes. The liquid above the precipitate is drained. One tablet (sample A) re-suspended in 5 ml of buffer for holding lysis (see Environment and solutions). The second tablet (sample) re-suspended in 10 ml of 30 mm solution Tric-Cl containing 20% sucrose, pH 8(8 ml for each 0.1 g cells in raw form).

5. Freeze the sample in a bath of dry ice - ethanol (or left overnight at a temperature of -28oC). Thaw in cold water (at 20oC more effective than the 70oC, but takes more time).

6. Subjected to ultrasonic processing, controlling the process of destruction of the cells by determining the amount of released balastegui maximum. Add 0.6 ml of 4 M solution of sodium chloride.

7. Centrifuged with acceleration 9000 x g for 20 minutes. Decanted liquid above the precipitate (crude extract 1) and stored on ice. The tablet again suspended in 5 ml of buffer for holding lysis. Receive a suspension of insoluble substances (crude extract 2).

Column purification obtained previously xenogenic product maltose binding protein-inhibin, "MBP-cINA515 which are as follows:

1. Give the amylose resin (1.5 g) to swell for 30 minutes in 50 ml of buffer for carrying out column chromatography (see Environment and solutions) in a flask with a capacity of 250 ml of Conduct degassing using an aspirator. Placed in a column (2.5 x 10 cm column is Washed three volumes with the same buffer + 0.25% of Tween 20.

The required amount of resin depends on the number of obtained protein binding. The resin binds about 3 mg/ml bottom of the column so that the column capacity 15 ml should be enough for up to 45 mg fusion protein per liter of culture. The syringe 50 ml tube of silanizing fibers can be replaced by a column of 2.5 see the Ratio of column height to diameter should be no more than 4.

2. speed [10x(diameter of the column in cm)2] ml/hour. It is approximately 1 ml/min for column 2,5 see

The dilution of the crude extract is performed to reduce the concentration of the protein to a value of approximately 2.5 mg/ml If the crude extract less concentrated, it should not be so dilute. A good rule of thumb of the right hand lies in the fact that 1 g wet cells gives approximately 120 mg of protein.

3. Washed with two column volumes of buffer solution of + 0.25% Tween 20.

4. Washed with three column volumes of the buffer without Tween 20.

5. Elute protein fusions, "MBP-cINA515", column buffer + 10 mm maltose + 0,1% sodium dodecyl sulfate (optional 10 mm beta-mercapto - ethanol, 1 mm EGTA). Select 10-20 fractions in 3 ml.

Analyze fractions for protein, i.e., method

Bradford or A260'in the fractions containing the fusion protein, it is easily detected. Protein merge eluted shortly after undergoing the void volume of the column.

Environment and solutions

Enriched medium + glucose and ampicillin = per litre:

10 g Triplane, 5 g yeast extract, 5 g sodium chloride, 2 g of glucose. Withstand autoclave; add sterile ampicillin to concealed water up to 50 ml Filtered and sterilized.

0.5 M buffer solution of sodium phosphate, pH 7,2 (mother solution) =

(A) of 69.0 g of NaH2PO4H2O to 1 l H2O.

(B) to 70.9 g of Na2HPO4to 1 liter of H2O.

Mix 117 ml (A) and (383 ml (B). the pH of the mother liquor should be approximately a 7.2. In diluted up to 10 mm column buffer pH should be between 7.0.

Buffer for lysis.

Per liter final concentration

20 ml of 0.5 M solution of Na2HPO4- 10 mm phosphate;

of 1.75 g NaCl, 30 mm sodium chloride;

10 ml of 25% aqueous Tween 20 - 0,25% Tween 20;

0.7 ml beta-mercaptoethanol (optional) - 10 mm beta-mercaptoethanol;

20 mm 0.5 M solution edtc (pH 8) - 10 mm etc;

10 ml of 1 M solution of EGTA (pH 7) - 10 mm EGTA.

Adjusted to pH 7.0 using HCl or NaOH.

Column buffer.

Per liter final concentration:

20 ml of 0.5 M solution of sodium phosphate, pH 7,2 - 10 mm phosphate;

the 29.2 g NaCl - 0.5 M sodium chloride;

1 ml of 1 M solution of sodium azide 1 mm azide;

0.7 ml beta-mercaptoethanol (optional) - 10 mm beta-mercaptoethanol;

1 ml of 1 M solution of EGTA (pH 7) (optional) - 1 mm EGTA,

Adjusted to pH 7.0 if necessary.

Column buffer with a low content of the BR>
of 1.75 g NaCl, 30 mm sodium chloride;

1 ml of 1 M solution of sodium azide 1 mm azide;

0.7 ml beta-mercaptoethanol (optional) - 10 mm beta-mercaptoethanol;

1 ml of 1 M solution of EGTA (pH 7) (optional) - 1 mm EGTA.

Adjusted to pH 7.0 if necessary.

Purity fused xenogenic protein inhibin chicken - MBP. "MBP-cINA515", after passing through the column illustrated in Fig. 1 track "E". The track "F" denotes the eluent from the column when it is not loaded xenogenic protein (negative control sample). Tracks marked as "B" correspond to the standards Pismenovo vector pMAL-c. Tracks marked as "C" correspond to the molecular weight standards. Tracks marked as "D" correspond to the real vector pMAL-c, which is used to get the merged xenogenic protein inhibin chicken-MBP, "MBP-cINA515", prior to the insertion of the gene of inhibin as described in Example 2. The above proteins are subjected to electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate using gel electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, and stained with brilliant blue Kumasi.

Example 3. How and what to know immunization ostrich spend approximately 6 months before the first breeding season, and then within six months with intervals of one month is repeated immunization. Thus, it is preferable to set a primary immunization of an ostrich, when his age to be approximately 18 to 24 months. Primary immunization consists of assigning approximately 1.5 to 3.0 mg merged xenogenic protein containing inhibin (a fragment of the alpha-subunit) and binding protein maltose, according to the method described in Examples 1 and 2. Re-immunization include the appointment of from about 0.75 to 1.5 mg merged xenogenic protein. For primary immunization merged xenogenic protein emuleret in complete Freund's adjuvant (Sigma Chemical Co.", St. Louis, Missouri), and the repeated immunizations merged xenogenic protein emuleret in incomplete Freund's adjuvant (Sigma). The composition of the merged xenogenic protein is injected subcutaneously in three places in the upper leg of the ostrich.

Example 4. A method of producing antibodies ostrich selectively directed against inhibin

The following is a method of obtaining antibodies ostrich (antiquita antibody inhibin ostrich"), which selectively directed against xenogenic protein of the present invention, containing inhibin CNY protein is a protein, containing inhibin chicken and binding protein maltose obtained by the methods described in Examples 1 and 2. With the aim of obtaining antibodies, ostrich immunizat xenogenic protein inhibin chicken - MBP obtained by the method described in Example 3.

The amount that is administered to the ostrich, should be enough to provoke an immune response in ostrich against xenogenic protein. Taken from ostrich approximately 5 ml of blood and separated from the obtained blood sample antibodies against inhibin. To select antibodies can use any method known from the technical field. Preferably use standard methods of enzyme immunosorbent assay.

Example 5. A method of producing antibodies goat selectively directed against antibodies ostrich.

The following is a method of obtaining antibodies goat selectively directed against the ostrich antibodies, including antibodies obtained in Example 4, in particular, antibodies of IgG ostrich. In this way the goat immunized immunoglobulin G ostrich in the amount of from 0.5 to 3.0 mg, so that the goat there is a response of the immunological response against IqC-ostrich. IgG-ostrich allocated from the Fund savor the standard methods, well known from the technical field.

The Fund serum purified using standard methods, which include precipitation with 50% ammonium sulfate, and then subjected to fractionation using a column of protein-A on sepharose. Deposition of IgG is preferably carried out as follows. Twelve milliliters of serum containing IgG, diluted 1: 1 with 50 mm Tris solution (pH 8.0). Then slowly with stirring, add 24 ml of a saturated solution of ammonium sulfate (all operations is carried out at a temperature of 4oC) and the resulting mixture was stirred for approximately 2 hours. Centrifuged the mixture with a speed of 10,000 rpm for 10 minutes to precipitate. He was again suspended in 50 mm solution of Tris/saline solution (12 ml) and subjected to dialysis overnight against 2 l of 50 mm Tris at a temperature of 4oC, while the final volume is 20 ml

Subsequent fractionalization on a column of protein-A on sepharose preferably carried out as follows. Column contains protein-A on sepharose CL-4B (Pharmacia Biotech., Inc.", Piscataway, new Jersey). In column placed approximately 5 milliliters of sediment ammonium sulfate/serum. Allow the sample to contact with protein-A on sepharose for the solution of glycine (pH of 2.8). Finally, obtained by elution fraction is neutralized by adding a few drops of 1 M solution of Tris - HCl (pH 9). The quality of purified IgG ostrich, before being appointed to the goat, assessed by visualization after electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. The method of immunization and excipients

are not critical to the present invention and therefore can be used any known from the technical field method immunization and any known from the technical field system auxiliary substances. Preferably the purified IgG ostrich enter the goat in the form of a subcutaneous injection. Repeated injections mainly prescribed intervals in four weeks, using the incomplete beta-blockers. Purified IgG prescribed along with complete adjuvant's adjuvant or hunter's Titermax (Sigma Chemical Co.", St. Louis, Missouri). A satisfactory response is formed from a goat after three or four injections.

Then the goat take 5-10 ml of blood and separated from the sample antibodies goat anti IgG ostrich. For the selection of the sample of blood antibodies goat apply any of the methods well known from the field of technology. The preferred method of untimely goat wash from the column with glycine buffer solution with pH 8.0.

Example 6. Method of controlling immunological responses ostrich after vaccination merged xenogenic protein.

The following is a method of controlling immunological responses ostrich after he injected the vaccine merged xenogenic protein containing inhibin chicken and binding protein maltose obtained by the method described in Example 1 and 2, while the response of the immunological response control using antibodies goat, obtained in Example 5. To determine whether the ostrich immunological response to the introduction of the slit xenogenic protein inhibin-ICBM, first connect xenogenic protein to the solid phase. Then the ostrich, which was introduced vaccine xenogenic protein, take 5 - 10 ml of blood and separate the serum. Immobilized xenogenic protein is in contact with the serum in the conditions under which xenogenic protein selectively interacts with any antiinsulinovykh antibodies serum. After washing, add antibody goat, obtained in Example 5, which contains a label peroxidase from horseradish. Containing the label antibodies goat selectively interact with ostrich antibodies that bound to the immobilized amount entered in the interaction of antibodies, containing the label of peroxidase from horseradish, by visualizing the label by adding nitrotetrazolium blue, which is the substrate for peroxidase from horseradish.

To a person skilled in the art it is obvious that the methods of immunoassay used in the above method, well known from the technical field. Therefore, any immunoassay method, methods of forming labels and visualization, known from the field of technology can be applied in the above way. A preferred immunoassay is an enzyme immunosorbent analysis, and the preferred label is peroxidase from horseradish. Another preferred label is a colored latex bead.

Example 7. The method of determining the reproductive potential of ostrich

Below is the method for determining the reproductive potential of ostrich by conducting a qualitative assessment of the number of inhibin in the blood of the ostrich. The concentration of circulating in the blood ostrich inhibin can be installed using standard methods sandwich enzyme immunosorbent assay. First link antiinvasive antibodies obtained in Example 4, in the cells of the tablet for microti rocky, taken from the ostrich. Give inhibin, which may be contained in the sample, selectively react with immobilized antiinsulinovykh antibodies, and then the sample is washed from the cells of the tablet. Next, in a cell type other antiinvasive antibodies other than antiinsulinovykh antibodies obtained in Example 4, which conjugated with horseradish peroxidase. Conjugated with peroxidase from horseradish antiinvasive antibodies differ from immobilized antiinsulinovykh antibodies to the fact that their action is selectively directed to other parts of inhibin. Give labeled antiribosomal the antibody selectively interact with the immobilized possible by inhibin, and then unreacted labeled antiinvasive antibodies are removed by washing. The number present in the plasma sample inhibin determined by adding to the cell, nitrotetrazolium blue and visualize the number of labeled antiribosomal antibodies to the cell. At the same time in adjacent cells analyzed with negative and positive control samples. Analysis of negative and positive control samples in adjacent cells should be carried out in parallel. From techniques well known to many methods is t be used in any immunoassay techniques, methods of forming the label and its visualization.

Example 8. The method of acceleration of egg laying quail

As indicated previously, the complementary DNA clone-subunit of inhibin chicken (cINA6), introduced in position EcoR 1 Bluescript provided by P. A. Johnson (Cornell University). The DNA fragment ("cINA515") cut out from a clone cINA6 fermentation using Pst 1. DNA cINA515 accompanies most-subunits inhibin Mature chickens. This fragment (cINA515) clone in plasmid p-MALTM-c in frame with the protein, maltose binding ("MBP"), and fusion protein of the desired size (path "E", Fig. 1) is determined after induction with isopropyl --D-thiogalactopyranoside and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The obtained protein conjugate ("MBP-cINA515") is used as antigen for immunization prepubescent female Japanese quail (Coturnix coturnix japonica) against circulating inhibin, as described below.

A flock of quails grown in battery brooders firm "Petersime model 2S-D) adapted for quail. The initial experiment on the cultivation is carried out at a temperature of 37.8oC, weekly, reducing the temperature by 2.8oC up until it reaches room temperature. In perioada and use continuous periods of scattered light (22 Lux) with a ratio of 14 hours of light time (from 280 to 300 Lux): 10 hours of dark time. When they reach the age of 25 days, 100 quail randomly and evenly divided into the following four groups for injection (25 birds in each group):

(1) MBP-cINA515 in polydispersion acetylated -(1,4)-linked mannan ("Acemannan"), immunostimuliruyushhim media (Chinnah et al. , "Antigen dependent adjuvant activity of polydispersed -(1,4)-linked acetylated mannan (acemannan)", Vaccine, Vol. 10, Issue 8, 1992, p. 551-557 ("MBP - cINA515/ACE"),

(2) MBP-cINA515 in Freund's adjuvant ("MBP-clNA515/FRN"),

(3) Acemannan (control immunostimulant; "ACE") or

(4) beta-blockers (control adjuvant; "FRN").

Birds who are immunized against inhibin (Groups 1 and 2), designate approximately 0.75 mg of MBP-cINA515 each in the appropriate test media. Equivalent volumes (0.2 ml) injection carrier ACE or FRN used, respectively, in Groups 1 and 3 and Groups 2 and 4. All injections carry subcutaneously with a syringe for injection of tuberculin with needles number 25. After the initial injection, the quail wings tied ribbons to identify them before placing individually in cages for egg laying. Over the next five weeks (i.e., at the age of 32, 39, 46, 53 and 60 days) each bird Pocono re-vaccination puwalu growth diet (21% CP, 2750 kcal ME/kg of food) and water. When the bird age, day 41 (designated as Day 1 of the cycle of egg laying) for 12 consecutive weeks are registering daily egg laying chickens ("HDEP) and mortality ("MORT"). Next, for each of the four groups to determine the average age when the first clutch of eggs ("FIRST") and the age when 50% of the birds start to lay eggs ("FIFTY ").

Data on daily egg-laying hens analyze two different methods of variational analysis, each of which includes a complete randomized disaggregated data and drawing on the graph. In the first method, variational analysis of the primary graph includes four injection (MBP-cINA515/ACE, MBP-cINA515/FRN, ACE or FRN, and the breakdown includes 12 periods of 7 days each. In the second method, variational analysis for a fuller response when immunoneutralization in relation to the response with the introduction of the reference sample, combine the data of two treatments a and inhibin data for the two control treatments. Thus, the main graph includes two types of injections (combined data MBP-cINA515/ACE and MBP-cINA515/FRN against the combined data ACE or FRR) for 12 periods eggs for 7 days.

Currently BP is generalnyh intervals of egg-laying (day 0, 7, 14, 21 and 35) during the first 35 days of the study is shown in Fig. 2 (where four groups with different injections are considered exposed to various types of processing) and Fig. 3 (where a comparison is made reimmunization (control) and inhibin-immunogenic groups). Average daily egg laying chickens for Week 1, Week 2, Week 3, Week 4, Week 5, or the combined data for weeks from the first to the fifth to the four groups who had an injection and that are considered independently from each other, are shown in Table 2.

In Table 3 for the same time intervals presents the average levels of oviposition, and the data are summarized for comparison of control groups and immunogenic groups. Table 4 contains data for the first day of oviposition (FIST) for both the considered aggregation processing results (i.e., the comparison for the four groups who had an injection, and comparing the combined data).

To date, mortality is not a significant factor, since he died only four birds (three control and one treated). Such mortality (4%) is quite fall within the expected bounds for quail, aged 76 days. Further, the cases shown the second probability of occurrence of such cases, however, they are evenly distributed among all four treatment groups (i.e., immunization with inhibin does not change the amount set aside by the birds of defective eggs).

There is a primary positive effect of immunoneutralization inhibin on producing ability. This effect is particularly noticeable for generalized data HDEP (table 3) and FIRST (table 4), for which a greater number of results strengthens the statistical significance of the applied tests.

The data show that the onset of puberty has been handling groups of birds accelerated. This is obvious for data HDEP and data FIRST. The value of statistical significance associated with a high average level value HDEP limit for treated inhibin quail during Weeks 1 study, significant (P<0,0095); this difference is not so clear (P<0,0888) in Week 2 (table 3). This edge is the difference between treated groups observed during Week 2, may mean that the clutch of eggs, though delayed, begins to intensify in quails control group (see Fig.2).

Therefore, it can be expected that the value of statistical dif is Uchenie Weeks 1, will be somewhat blurred with the advent of the day of egg laying. In fact, when the data obtained during Weeks 1 - 5, unite, chickens, processed by inhibin, observed significantly higher levels HDEP when the average value of statistical significance (P<0,0763).

Example 9. The method of acceleration of egg laying ostriches.

Conjugate protein (MBP-cINA515) is used as antigen for immunization paupertate female ostrich against circulating inhibin and, thus, to accelerate the start of egg laying processed ostriches. Use the method described in Example 8, with the following exceptions. The average age of puberty in nepodvizhnykh processing ostriches is approximately 28 to 32 months. The following is primary) injection of 5.0 mg xenogenic protein of the present invention at the age of 26 months; and re-immunization in the amount of 2.5 mg at the age of 27, 28, 30, 32, 34, and 36 months.

Example 10. The method of acceleration of egg laying EMUs.

Conjugate protein (MBP-cINA515) is used as antigen for immunization prepubescent female EMUs against circulating inhibin and, thus, to accelerate the start of egg laying processed EMU. Use the method described in Example 8, with the following exceptions. The average age of puberty in nepodvizhnykh processing EMU is approximately 20 months. The following is a schedule of treatments EMU, the weight of which is approximately from 50 to 90 pounds (23 to 41 kg): the first (primary) injection of 3.0 mg xenogenic protein of the present invention at the age of 18 months; and re-immunization in the amount of 1.5 mg at the age of 19, 20, 22, 24, 26 and 30 months.

Example 11. The method of acceleration of egg laying hens.

Conjugate protein (MBP-cINA515) is used as antigen for immunization prepubescent chickens against circulating inhibin and, thus, to accelerate the start of egg laying processed chickens. Use the method described in Example 8, with the following exceptions. The average age of puberty , the EU which is approximately from 2.0 to 3.5 pounds (0.9 to 1.6 kg): the first (primary) injection of 1.5 mg xenogenic protein of the present invention at the age of 15 weeks; and re-immunization in the amount of 0.75 mg at the age of 17, 20, 24, 30, 40 and 50 weeks.

Example 12. The method of acceleration of egg laying turkeys.

Conjugate protein (MBP-cINA515) is used as antigen for immunization prepubescent turkeys against circulating inhibin and, thus, to accelerate the start of egg laying processed turkeys. Use the method described in Example 8, with the following exceptions. The average age of puberty in nepodvizhnykh processing turkeys is approximately 30 weeks. The following is a schedule of treatments turkeys, weighing approximately from 9.0 to 12 pounds (4 to 5.4 kg): the first (primary) injection of 2.0 mg xenogenic protein of the present invention at the age of 28 weeks; and re-immunization in the amount of 1.0 mg at the age of 29, 30, 34, 38, 46 and 54 of the week.

Example 13. The method of acceleration of egg laying parrots.

Conjugate protein (MBP-SNA) is used as antigen for immunization prepubescent female parrots against circulating inhibin tion in Example 8, the following is the exception. The average age of puberty in nepodvizhnykh processing parrots is approximately 30 months. The following is a schedule of treatments parrots, the weight of which is approximately from 0.5 to 1.25 pounds (225 to 570 g): the first (primary) injection of 0.75 mg xenogenic protein of the present invention at the age of 28 months; and re-immunization in the amount of 0.375 mg at the age of 29, 30, 32 34, 36 and 38 months.

It should be understood that all the foregoing relates only to preferred variants of implementation of the present invention and can be performed numerous modifications and changes do not contradict the essence of the present invention and the scope of the claims described in the claims.

1. Method of increasing the productivity of birds, characterized in that the method comprises the introduction of the bird an effective amount of a heterologous protein, alpha subunit protein inhibin birds or immunogenic fragment and a carrier protein.

2. The method according to p. 1, where an increase in the productivity of birds includes increased egg laying, the acceleration of the start of egg laying, the maximum expediting the start of egg laying, the increase in the total periodic eggs, improving the quality of shell eggs, accelerating the onset of puberty and/or prolonged persistence of ovulation in birds.

3. The method according to p. 1, wherein the bird is selected from the group including basilevych, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, CuCuliformes, Serves, Galliformes, Anseriformes and Herodiones.

4. The method according to p. 3, characterized in that baselevel bird selected from the group including African ostrich, EMU, Rhea, kiwi and cassowary.

5. The method according to p. 3, wherein the bird is selected from the group including chicken, Turkey, parrot, parakeet, Macao, Falcon, eagle, quail, hawk, dove, cockatoos, songbirds, Jay, Blackbird, Finch, Warbler, goose, duck, pigeon, lane, Toucan and a Sparrow.

6. The method according to p. 1, wherein the carrier protein is chosen from the group containing binding protein maltose, bovine serum albumin, ovalbumin, flagellin, hemocyanin lymph mollusk, serum albumin, gamma globulin, thyroglobulin and polymers of amino acids.

7. The method according to p. 1, characterized in that the introduction heterological protein increases the production of eggs.

8. The method according to p. 7, wherein the bird is selected from the group fluorogenic protein administered before puberty.

10. The method according to p. 1, wherein the heterologous protein is administered during puberty.

11. The method according to p. 1, wherein the heterologous protein is fused protein.

12. The method according to p. 1, wherein the heterologous protein is conjugated protein.

13. The method according to p. 1, characterized in that impose additional adjuvants, preservatives, diluents, emulsifiers or stabilizers.

14. The method according to p. 1, wherein the protein is administered after puberty.

15. The method according to p. 1, wherein the alpha-subunit of protein inhibin birds or immunogenic fragment contains the amino acid sequence of SEQ ID N0:2 or SEQ ID N0:4.

16. Fused heterologous protein of General formula A-B, where a is the alpha-subunit of protein inhibin birds or immunogenic fragment, such as a protein comprising the amino acid sequence shown in SEQ ID N0:2 or SEQ ID N0: 4; B is a carrier protein, such as binding protein maltose, bovine serum albumin, ovalbumin, flagellin, hemocyanin lymph mollusk, serum albumin, gamma globulin, thyroglobulin and polymers of amino acids.

17. The method of obtaining protein, including the UNT; (b) obtaining a vector that encodes a carrier protein; (C) ligation of cDNA alpha-subunit of inhibin in the vector; (d) the introduction of a very vector expressing the system, and (d) the expression of the fused heterologous protein, alpha subunit protein inhibin or its fragment and a carrier protein, characterized in that the protein is a fused heterologous protein under item 16.

18. The method according to p. 17, wherein the cDNA alpha-subunit protein inhibin contains the sequence SEQ ID N0:1 or SEQ ID N0:3.

19. The method according to p. 17, wherein the alpha-subunit protein inhibin or immunogenic fragment expressed fused heterologous protein containing SEQ ID N0:2 or SEQ ID N0:4.

20. The method according to p. 17, wherein the carrier protein is chosen from the group comprising binding protein maltose, bovine serum albumin, ovalbumin, flagellin, hemocyanin lymph mollusk, thyroglobulin, serum albumin, gamma globulin or polymers of amino acids.

 

Same patents:

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions

The invention relates to new biologically active soedineniya, namely the fullerene derivative glycopeptide GlcNAc-(1 _4)-MurNAc-Ala-D-iGln[Lys-- CO(CH2)5-NHC60-H] formula I described in the claims, having adjuvant activity

The invention relates to the field of organic chemistry, to antibacterial agents and may find application in medicine and veterinary medicine

The invention relates to the field of synthesis of physiologically active peptides, specifically to an improved method of obtaining muramylpeptides General formula I:

< / BR>
where R is hydrogen orAnd the residue of the amino acid or peptide

The invention relates to organic chemistry, specifically to a method for producing triterpene glycopeptides derived glycyrrhizic acid (GA) using unprotected glycoside (carboxy-component) and free amino acids or dipeptides (aminoquinones AK)

New peptides // 2162855

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the activity IL4

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions

The invention relates to a new radiopharmaceutical drugs, which are radioactivedecay cyclic compounds containing carbocyclic or heterocyclic ring system and acting as antagonists of glycoprotein complex IIb/IIIa

The invention relates to biotechnology and medicine
Up!