Human recombinant mannan-binding lectin

FIELD: pharmaceuticals.

SUBSTANCE: claimed method includes composition containing mannan-binding lectin and treatment of conditions associated with deficit of mannan-binding lectin, causing susceptibility to infective disorders. Also disclosed is method for production of new expression structure encoding human mannan-binding lectin.

EFFECT: method for production of new human mannan-binding lectin.

47 cl, 8 ex, 5 dwg

 

The present invention relates to a method for preparing a composition/mixture/composition, including mannan-binding lectin, and treatment of diseases and disorders of the immune system, including the treatment of conditions associated with a decrease in immunity. In particular, the present invention relates to the treatment of conditions or latent conditions associated with deficiency of mannan-binding lectin, which is associated with increased susceptibility to infection. The present invention also relates to the preparation/creation of new constructs for expression of the gene encoding mannan-binding lectin person, a new recombinant method for preparing the mannan-binding lectin of human rights and, in addition, to the use of this compound in the treatment of conditions related to immunosuppressive chemotherapy, and/or conditions associated with deficiency of mannan-binding lectin, including latent States, i.e. those that do not occur in the present time. This treatment applies to the people and animals whose immune system functions in the same way as the human immune system.

BACKGROUND of INVENTION

The immune system consists of many cellular and molecular mechanisms that recognize and attack a pathogen or infected them with cells. Recently manna is-binding lectin began to show great interest as an important part of the innate immune system, which is the immune system, already present at birth, in contrast to the adaptive immune protection, which is formed/started to operate only during early childhood (Janeway and others, 1999). After binding of hydrocarbons on the surface of microbes mannan-binding pectin stimulates the activation of complement that triggers an enzymatic cascade, which causes the label to target for destruction by phagocytosis or lysis of the microorganism (Law and Reid. 1995). The complement system is activated by at least three separate ways, representing the classical path, an alternative path and the path of the mannan-binding lectin (Janeway and others, 1999). The classical path is enabled when the factor of complement (C1) detects surface-bound immunoglobulin. Complex C1 consists of two proteolytic enzymes SG and 1s and nonenzymatic part of C1q, which includes recognizing immunoglobulin domains. In the electron microscope, it is seen that the structures of both molecules, as C1q and mannan-binding lectin, reminiscent of a bouquet. As C1q, mannan-binding lectin is in complex with two proteolytic enzymes - proteases. associated (linked) with mannan-binding lectin. All three paths reproduce the Mac factor of complement is 3 (C3 convertase), associated with activating the surface of the target pathogenic microbe. The transformation of C3 in surface-associated 3b is essential in the process of destroying pathogenic microbes by phagocytosis or lysis (Janeway and others, 1999).

Mannan-binding lectin, also known as mannan-binding protein or mannose-binding protein, was first identified in rabbits (Kawasaki and others, 1978). Mannan-binding lectin belongs to the group of soluble CA2+-dependent (type C) pectin containing the C-terminal domain recognition hydrocarbon and collagenopathy region, characterized by the triple repetition of parts of glycine (Gly), followed by two neglicence amino acids. Thus, the mannan-binding lectin belongs to the group of collectino. i.e. the type lectins With collagenopathy areas, which additionally contain a surfactant proteins lung a and D, as well as conglutinin and CL-43, which, however, were identified only in cattle (Holmskov and others, 1994). Protein mannan-binding lectin person consists of identical polypeptide chains of molecular weight 32 kDa each, up to 18 (Lu and others, 1990). each of which includes a short N-terminal segment of 21 amino acids, including three cysteine residue, followed by 7 collagen repeats the Vienna Gly-X-Y, interrupted Gln residues, followed by other repetitions of the 12 links of Gly-X-Y. a Small area of 34 residues connects the C-terminal domain of CA2+-dependent lectin from 93 amino acids of collagen molecules (Sastry and others, 1989). These chains of protein mannan-binding lectin are combined into a subunit of mannan-binding lectin. Mannan-binding lectin consists of branches, including subunit mannan-binding lectin, United at the base of the bouquet, and the length of the branches reaches 15 nm, and their number reaches six. Later, the mannan-binding lectin was identified in rodents (Mizono and others, 1981, Oka and others, 1985), cattle (Holmskov and others, 1993, Kawai and others, 1997) and in chickens (Oka and others, 1985, Laursen and others, 1995, Lamsen and others, 1998). In rodents (Drickamer and others, 1986) and in rhesus monkeys (Mgues and others, 1996) identified two types of genes mannan-binding lectin, usually denoted as a and C. In rats was detected pseudogene "In" mannan-binding lectin (Drickamer and others, 1986), and recently pseudogene mannan-binding lectin was also cloned from the human genome, the sequence analysis, it was concluded that it is the remains of a gene And the mannan-binding lectin primates (Guo and others, 1998 g). Mannan-binding lectin person was identified Kawasaki and others in 1983, was identified only Odie the gene mannan-binding lectin person, in composed of four exons with three intermediate introns of the gene mannan-binding lectin, covering approximately 6 kb, which is located in the area 10q11.2-q21 (Sastry and others, 1989, Taylor and others, 1989).

The collagen region of three polypeptide chains form a subunit. which covalently stabilized disulfide bridge links. Individual subunits are joined together by a disulfide bridge links, as well as through non-covalent interactions (Lu and others, 1990).

The position of these disulfide bridge links is not quite defined. The analysis SDS-PAGE in the environment of mannan-binding lectin, not having reducing ability, revealed ligament with a molecular mass of more than 200 kDa. presumably represented by the blocks 3, 4, 5 and even 6 United subunits (Lu and others, 1990).

The actual number of subunits in the natural protein mannan-binding lectin of human remains controversial. The authors Lipscombe and others in 1995 using ultracentrifugation received evidence from which it can be assumed that 25% of the mannan-binding lectin serum human form bundles, consisting of 2-3 subunits and only a small fraction of them reaches 6 subunits. Relative quantitative analysis was performed by densitometry of Western blots. received chemiluminescent way the m Lower efficiency arising on the transfer of protein of high molecular weight membrane, in comparison with proteins of smaller molecular weight complicate the analysis using this methodology. The authors Lu and others in 1990 by analysis of the fractions by ion-exchange chromatography method SDS-PAGE was found that the prevailing chains covalently linked soyadiniz mannan-binding lectin consisted of tetramers, while the complement activated only pentamers or review of the complexes. In contrast, the analysis by means of gel chromatography showed that the size of the mannan-binding lectin is comparable with the size of the complex C1. Analysis by the method of gel chromatography can be performed under conditions allowing the study of the importance of weak interactions of the protein with the protein in the formation of molecules mannan-binding lectin and in combination with the standard analysis of mannan-binding lectin, also allows you to objectively determine the content of mannan-binding lectin in fractions of gel chromatography.

Obviously, in vivo mannan-binding lectin in relation to the innate immune system plays the role of a humoral factor stimulating some antimicrobial activity that does not require the formation of contributing itself/mesmorising as the adaptive immune system protection, based on the recognition of T - and b-cells (Janeway and others, 1999, Vorup-Jensen and others, 1998). Identify targets for binding of mannan-binding lectin occurs through the lectin domain of type C. the Calcium-dependent domain type have been found in proteins that are common both in phylogeny and during further operation. In the case of mannan-binding lectin calcium-dependent domain recognizes mainly hexose Equatorial groups 3 and 4 HE, for example, minasny glucosamine and N-acetylglucosamine, whereas hydrocarbons, which do not fulfill this requirement, for example, galactose and D-fucose, remain unbound (Weis and others, 1992).

Terminal calcium-dependent domains are distributed in such a way as to allow binding of a target surfaces of all three domains representing binding sites (sites) with spacer elements area of about 53 Å (Sheriff and others, 1994, Weis and Drickamer, 1994). This property is "pattern recognition" may further contribute to the selective binding of the surfaces of microbes. Selectivity/hydrocarbon selectivity is an important aspect of self/desmoralizaci via the mannan-binding lectin and possibly mediated by differences in the prevalence of N-acetylglucosamine on the surfaces of microbes, one example of which is the high content of mannose in the cell wall of the yeast type Saccharomyces ceravisiae and Candida albicans. Hydrocarbon structures in the glycosylation of mammalian proteins usually end sialic acid, which prevents the binding of mannan-binding lectin with these oligomeric hydrocarbons and, thus, prevent the recognition of "their" surfaces mannan-binding lectin. Also the three-dimensional structure of each subunit of the mannan-binding lectin can be very important for target recognition.

Structure and biosynthesis of mannan-binding lectin was investigated using in vitro synthesis systems. Structure of calcium-dependent domain mannan-binding lectin And rats was determined using a crystallization of recombinant protein produced by the bacteria E. coli. (Weis and others, 1992), and the structure of the mannan-binding lectin With rats was determined using recombinant material (Ng and others, 1996). In more recent studies, the crystal structure of the trimer of calcium-dependent domains were collected from the expression as "the neck", and the calcium-dependent domain, which confirmed the results of earlier studies, according to which the area of the neck" is the result of information together, the three chains through hydrogen bonds between twisted α-spirals (Sheriff and others, 1994, Weis and Drickamer, 1994). Calcium-dependent domains and fragments of calcium-z is independent of the domain area "cervical" other collectino also were expressionand in systems based on the bacteria E. coli. without a requirement to enhance the functional activity. Thus, in-vitro synthesis of domains mannan-binding lectin can be referred to as functional properties recognition of hydrocarbon and structural properties of trimerization, effectively carried out in prokaryotic or simple eukaryotic expression systems.

In-vitro synthesis of full collectino using recombinant techniques was undertaken mainly using cell lines of mammalian, e.g., ovary cells Chinese hamster or COS cells as host cells. Attempts to Express the mannan-binding pectin in insect cells resulted in the receipt of mannan-binding lectin low molecular weight (MA and others, 1996), while recombinant proteins was almost entirely composed of subunits of the dimers and trimers chain subunits mannan-binding lectin.

There have been several studies of recombinant synthesis of mannan-binding lectin human cell lines mammals. In studies of opsonic function, i.e. the ability to improve the absorption rate of using macrophages mannan-binding lectin, the authors Kuhlman and others in 1989, used recombinant mannan-binding lectin obtained in the ovary cells Chinese hamster, which showed the same opson the economic activity, as natural mannan-binding lectin. Identification of the structure and posttranslational modifications of recombinant mannan-binding lectin was not conducted. Similarly, the authors Schweinie and others in 1993 by stable transfection ovary cells Chinese hamster got recombinant mannan-binding lectin and its shortened form without the collagen tail. The activity of the recombinant proteins was measured by the deposition of C3 on Salmonella montevideo, pre survive with mannan-binding lectin and diluted human serum depleted antibodies against Salmonella montevideo as a source of complement. Unexpectedly, the activity of recombinant full-mannan-binding lectin was as high as the activity of natural mannan-binding lectin, although the analysis iodirovannoi recombinant mannan-binding lectin using ultracentrifugation showed that the majority of protein molecular mass was small. Not a comparison was made of the amount of recombinant mannan-binding lectin derived from ovary cells Chinese hamster size natural mannan-binding lectin or method SDS-PAGE, either by ultracentrifugation or gel chromatography. When the study authors and other Super in 1992 cells hybridoma mouse Sp2/0Ag14 were obtained the s profiles getpropecia chromatography of recombinant mannan-binding lectin, with the help of which it was shown that the size distribution of the molecules of recombinant mannan-binding lectin purified from culture supernatant using hydrocarbon affinity chromatography, was significantly different from the distribution of the sizes of the molecules of recombinant mannan-binding lectin, isolated using affinity chromatography based on antibodies against the mannan-binding lectin.

In 1999, the authors Ohtani et al. published data on recombinant mannan-binding lectin obtained in the ovary cells Chinese hamster, the output of which amounted to 120 μg per 1 ml of culture medium. The results were obtained using the expression vector providing for selection of transfectants by G418 resistance and further amplification of the gene by breeding sulfoximine methionine. Functional activity on hydrocarbon selectivity was similar to the activity of the natural protein, however, measurements by lysis of erythrocytes showed some differences in the ability to activate complement. Both methods, as helpanimals chromatography, and a method of SDS-PAGE showed the presence of forms of mannan-binding lectin with a higher molecular weight, although, as noted by the authors, was observed too big (huge, total) the difference in size in comparison with mannan-connect the non-lectin, purified from plasma. Interestingly, the mechanism of hydroxylation essentially was similar to the profile hydroxylation natural mannan-binding lectin even in the absence of additives ascorbic acid. With regard to post-translational modifications, it should be noted that research hydroxylation characteristic of recombinant mannan-binding lectin does not confirm the similarity (similarity) of its structure with that of the natural mannan-binding lectin.

The locus of mannan-binding lectin human polymorphic and has three known mutations, located in the region encoding the protein (Sumia and others, 1991, Lipscombe and others, a, 1992b, Madsen and others, 1994), and others affecting the regulation of gene elements (Madsen and others, 1994). It is obvious that all mutations lead to significantly lower levels of mannan-binding lectin in body fluids from diseased mammals. Thus, the results of measurements of the variation of the content of mannan-binding lectin is about three orders of magnitude from 2-5 µg mannan-binding lectin 1 ml less than 10 ng mannan-binding lectin 1 ml in homozygous mammals susceptible to mutations that affect the coding of the protein. Experimental data on the deficiency of mannan-binding lectin associated with recurrent infections in children is th, which was diagnosed as suffering from opsonic defect (Super and others, 1989, Sumia and others, 1991), have demonstrated a correlation between levels of deficiency of mannan-binding lectin and reduced protection from microorganisms. Additionally, it was confirmed that when interacting with microorganisms collective stimulate anti-virus protection (Hartshorn and others, 1993, Malhotra and others, 1994). In-vitro studies on the interaction with the human immunodeficiency virus showed that the mannan-binding lectin inhibits infection of T-positive CD4 cells and U937 cells. Clinical studies have also confirmed that the mannan-binding lectin plays a role of the first line of protection against human immunodeficiency virus. The period of time from the onset of symptoms of AIDS until the final stage in patients with deficiency of mannan-binding lectin is different from the period of time that is characteristic for patients with normal levels of mannan-binding lectin. It is also clear that susceptibility to infection infection is significantly higher among individuals with deficiency of mannan-binding lectin as a deficiency of the mannan-binding lectin is present more frequently in patients infected with human immunodeficiency virus than in healthy control patients (Neilsen and others, 1995, Garred and others, 1997). To whom it was shown for the cases of deficiency of C1, C2 and C4 (analysis authors Tan and Amett. 1998) in the development of systemic lupus erythematosus contributes inherited deficiency of complement. In some studies it was confirmed that, with the development of systemic lupus erythematosus deficiency of mannan-binding lectin is also a risk factor (Davies and others, 1995, Lau and others 1996, Ip and others, 1998), although the role of mannan-binding lectin is not quite defined. In accordance with one assumption, following the explanations of the other deficiencies of complement as a cause for systemic lupus erythematosus, defect of complement fixation caused by deficiency of mannan-binding lectin, leads to leads to inefficient working of the immune complex (Ip and others, 1998), which indicates that the mannan-binding lectin as a participant in maintaining homeostasis. In accordance with other data from clinical studies of mannan-binding lectin plays an important role in reproductive biology. As shown by recent reports, there is a correlation between recurrent miscarriages and levels of mannan-binding lectin (Kilpatric and others, 1995, Christiansen and others, 1999).

Strategy for the treatment of mammals with symptoms of deficiency of mannan-binding lectin, aimed at the restoration path mannan-binding lectin, is described in only two studies. Opsonizes the second defect, observed in some children only during the clinical symptoms frequent infections, were treated by the introduction of plasma (Soothill and Harvey, 1976), while in more recent studies, two-year-old girl was introduced mannan-binding lectin derived from plasma (Valdimarsson and others, 1998). In both studies noted improvement in health, although a small number of patients involved in these studies, it is not possible to consider the mannan-binding lectin as a therapeutic agent. No clinical studies with recombinant mannan-binding lectin. The study authors MA et al. in 1999 showed antitumor activity of recombinant mannan-binding lectin in mice, stimulated expression system of the virus.

From the foregoing it is obvious that there have been attempts to get in in-vitro systems recombinant mannan-binding lectin or in the form identical to the natural protein, either in the form of a high degree of similarity (affinity) with him several attempts were made. However, obtaining such recombinant mannan-binding lectin in currently known in-vitro systems synthesis faced significant restrictions.

SUMMARY of the INVENTION

In one aspect the present invention provides a new form of m is non-binding lectin, which is prepared by recombinant DNA technology in-vitro or in-vivo. More specifically the present invention relates to recombinant mannan-binding lectin in a new form, open the inventors during the synthesis in accordance with the following method.

A method of obtaining a composition of recombinant mannan-binding lectin person with such a profile size distribution of molecules in which at least 50% identical to the profile size distribution of molecules of natural mannan-binding lectin person, including:

- creating constructs for expression of the gene encoding the peptide mannan-binding lectin person or its functional equivalent;

- the transformation of the culture of the host cell with the help of this design;

- cultivating a culture of the host cell, receiving with the release of the polypeptide into the culture medium,

- affinity chromatography specified culture medium using a matrix derived from a hydrocarbon, and the affinity of this matrix with tetramine, pentamerone and/or hexamine subunit mannan-binding lectin, at least two times greater than the dimer subunit of mannan-binding lectin;

- obtaining of the eluate containing the composition of recombinant mannan-binding lectin person.

Under the functionality is determined as being equivalent to the mannan-binding lectin person refers to a molecule, perform the function of mannan-binding lectin person, for example, the binding of hydrocarbons to microbial stimulation opsonizing activity and/or activation of complement, which can be demonstrated by the deposition of C4 on the surface coated with mannan.

Functional activity of mannan-binding lectin can be evaluated for its ability to form complexes (mannan-binding lectin)/MAR, leading to activation of the system of compliments. The cleavage of complement C4 complex (mannan-binding lectin)/MASP allocated ether thiol and complement C4 forms a covalent bond with the nearest nucleophilic groups. A significant part of the complement 4b forms of communication with the hole, covered with plastic, and can be detected by antibody against complement C4.

Quantitative analysis TRIFMA used to determine the functional activity of mannan-binding lectin, includes the following steps: 1) applying a layer of mannan on microtiter wells at a rate of 1 mg of mannan in 100 ml of buffer solution; 2) blocking with Tween-20: 3) application of samples, for example, preparations of a dilute solution of mannan-binding lectin; 4) application of serum with deficiency of mannan-binding lectin (this leads to the formation of the complex (mannan-binding lectin)/MASP); in another case before Nan is the group on the surface of microtiter wells mannan-linking pectin and whey with deficiency of mannan-binding lectin can be mixed; 5) deposition of complement factor C4 concentration of 5 mg/ml; 6) exposure for one hour at 37°S: 7) the application of antibodies against complement C4, labeled Eu: 8) applying the amplifying solution; 9) reading Eu using temporary fluorescence analyzer. Between every two phases of the plate is kept at room temperature and washed with the exception of stages 8 and 9.

The evaluation by the method of ELISA can be performed in the same way, for example, by applying labeled with Biotin anticomplement C4 in step 7; 8) applying avidin labeled with alkaline phosphatase; 9) application substrate; 10) read the intensity of the colors. The calibration curve can be constructed using breeding, taken from a normal plasma. In the present invention the sample is serum plasma pool plasma pool) LJ 6,57 28/04/97. Functionality can be expressed as the specific activity of mannan-binding lectin, such as units of activity of mannan-binding lectin 1 ng mannan-binding lectin.

Another criterion is the functional equivalent of mannan-binding lectin is the ability to form on the cells due to the receptor/receptors.

The interaction of mannan-binding lectin-receptor (receptors) on the cell can be analyzed is by using cytofluorometry. 1) Mannan-binding lectin at a concentration of 50 μm/ml is maintained with 2×105 cells. Linking is carried out in a phosphate buffered salt solution containing 1% FCS and 0.1% sodium azide. 2) To detect the associated cell mannan-binding lectin is used biotinylated antibody against mannan-binding lectin. 3) then add streptavidin-PTS and 4) the resulting mixture was investigated using fluorescence analysis.

A functional equivalent of the present invention corresponds to at least one of the criteria discussed above, for example, or the activation of C4, or interaction with receptor/receptors on cells, respectively. In a preferred embodiment of the invention meet both criteria.

By "recombinant mannan-binding lectin person" means mannan-binding lectin person who Express created from genetic engineering, nucleic acids, and under other designs for gene expression mannan-binding lectin" refers to the expression vector suitable for expression in host cells.

The term "composition with the same profile size distribution of molecules in which at least 50% identical to the profile size distribution of molecules of natural mannan-binding lectin person" means that according the analysis of SDS-PAGE, the size distribution of molecules of different oligomers mannan-binding lectin 50% identical to the size distribution of molecules of natural mannan-binding lectin person, purified from plasma using hydrocarbon affinity chromatography. Under the identity of 50% means that the average molecular weight of at least 50% of oligomers, obtained by the method of SDS-PAGE and/or Western blotting, greater than 200 kDa. When determining the number of mannan-binding lectin average molecular weight of greater than 200 kDa, can be used densitometry. in which the gel SDS-PAGE stained protein bands, for example, silver or Coomassie Blue (Coomassie brilliant blue), or specific staining of Western blots using antibodies specific for the mannan-binding lectin.

The term "purified recombinant mannan-binding pectin" refers to recombinant mannan-binding lectin purified from the supernatant or body fluids or tissues of transgenic animals using hydrocarbon affinity chromatography.

In the present invention proposed form of mannan-binding lectin, which is similar to natural mannan-binding lectin person profile size distribution of molecules in its purified form, which is closer to the profile size distribution of molecules of natural mannan-binding lectin person than it was observed in other forms known today. Thus, other aspects the present invention relates to a composition of recombinant mannan-binding lectin person, which contains oligomers subunits mannan-binding lectin, and the profile size distribution of the molecules, oligomers, at least 50% identical to the size distribution of molecules mannan-binding lectin human plasma.

In this context, the term "oligomer" refers to different "...measures" mannan-binding lectin, such as monomer, dimer, trimer, tetramer, pentamer and hexamer. The monomer consists of three identical polypeptide chains and in this context is considered as a subunit. Other oligomers are formed as a combination of 2-6 subunits.

As for the expression of recombinant mannan-binding lectin, the present invention relates to constructions for the expression of the gene encoding the recombinant polypeptide mannan-binding lectin person, including

- at least one intron sequence of a gene mannan-binding lectin person or its functional equivalent;

- at least one exon of the gene sequence mannan-binding lectin person or its functional equivalent;

the promoter other than the promoter mannan-binding lectin person;

the expression vector.

Preferably, the expression was carried out, for example, in cells melchorita, the drug according to the present invention obtained by using the expression vector, including intron sequence (sequence) of the gene mannan-binding lectin person and at least one exon sequence. Regarding transgenic animals as expression systems, this term in this context refers to genetically modified animals, whose cells contain and Express a gene of mannan-binding lectin person or his likeness.

The term "recombinant mannan-binding lectin with structural properties under conditions that do not result in denaturation, similar to natural mannan-binding lectin person" refers to recombinant mannan-binding lectin extracted in a similar way by using gel chromatography in the form of mannan-binding lectin person present in the serum. The term "recombinant mannan-binding lectin with structural properties under conditions of denaturation, similar to natural mannan-binding lectin person" means purified mannan-binding lectin with the size distribution of the molecules obtained by the method of SDS-PAGE, 50% identical to the size distribution of molecules mannan-binding lectin person, purified from plasma by hydrocarbon affinity chromatography (see figure 1). Thus, the term "recombinant mannan-binding Le the tin" refers to the mannan-binding lectin, substantially free of any impurities that are usually present in the mannan-binding lectin purified from plasma.

In this regard, a protease associated with mannan-binding lectin (MASP), are not considered as impurities.

Therefore, another aspect of the present invention relates to a method for production of recombinant mannan-binding lectin person, comprising the following steps:

- creating constructs for expression of the gene encoding the peptide mannan-binding lectin person or its functional equivalent;

- transformation of the culture of the host cell with the help of this design;

- cultivating a culture of the host cell, with the release of the polypeptide into the culture medium, with

- obtaining a culture medium containing recombinant mannan-binding lectin.

Another aspect of the present invention is a method of separation or purification of mannan-binding lectin person in order to obtain the composition of the polypeptides with the size distribution of the molecules, which, at least 50% identical to the size distribution of molecules mannan-binding lectin human plasma. Thus, the invention relates to a method of selection of the composition of the oligomers mannan-binding lectin person, not less than 50% of which have a size distribution ID is but natural (natural) contained in the plasma of man, including:

- a drug oligomers mannan-binding lectin person;

- affinity chromatography specified drug using matrix obtained from a hydrocarbon, and the affinity of this matrix with tetramine, pentamerone and/or hexamine subunit mannan-binding lectin, at least two times greater than the dimer subunit of mannan-binding lectin;

- obtaining of the eluate containing composition selected recombinant mannan-binding lectin person.

Another aspect of the present invention relates to mannan-binding lectin, including recombinant mannan-binding lectin, its fragments or its likeness or imitation, for use in therapy of cancer and diseases and disorders, for example, immune system and reproductive system, with this therapy involves the creation, reconstruction, strengthening and/or promote opsonic effect and bactericidal activity of the complement system, i.e. strengthening the ability of the immune system to recognize and kill pathogens. The use of recombinant mannan-binding lectin similar to natural mannan-binding lectin, when this therapy is great progress compared with the well-known mA the NAS-binding lectin, in particular, the mannan-binding lectin of human plasma, as this therapy with the use of recombinant mannan-binding lectin much reduces the risk of viral infections.

This therapy may include treatment and/or prevention of diseases, disorders and/or conditions, if necessary, therapy of cancer and diseases and disorders, for example, immune system and reproductive system in humans or animals, these functional units activated in this regard, as well as in humans. Under the condition that requires therapy, refers to any condition associated with existing and/or anticipated need for treatment or the need to improve the normal state. In particular, this therapy is therapy of a condition associated with a deficiency of mannan-binding lectin.

The present invention also relates to pharmaceutical compositions containing mannan-binding lectin, prepared in accordance with the present invention.

Another aspect of the present invention relates to the use of mannan-binding lectin, prepared in accordance with the present invention, including its fragments or its likeness or imitation in the manufacture of a drug or pharmaceutical composition for the treatment, including treatment who/or prevention of diseases and disorders, for example, immune system and reproductive system in humans or animals, these functional units, acting in this respect as a person.

In these diseases, disorders and/or conditions applied therapy with the use of the compositions according to the present invention, for example, therapy of conditions associated with deficiency of mannan-binding lectin, cancer therapy and infections associated with immunosuppressive chemotherapy, including, in particular, infection-related conditions that occur during cancer therapy or implantation and/or organ transplantation. The present invention also includes treatment of conditions associated with recurrent miscarriages.

DETAILED description of the INVENTION

The immune system has the ability to prevent infections, which is very important for homeostasis and subsequent survival and the preservation of life. Therefore, it is interesting to identify compounds that are active in such processes. Intensive research in recent decades has led to the mannan-binding lectin, which is a very versatile macromolecule, showing, apparently, their activity, for example, in natural (innate) immune system.

In accordance with the present invention can receive the composition/blend/composition of recombinant mannan-binding lectin person with the distribution of the sizes of molecules, which, as described above, at least 50% identical to the size distribution of molecules of natural mannan-binding lectin person.

First, as described above receive a composition/mixture/composition mannan-binding lectin, and then for the Department of higher oligomers mannan-binding lectin from other oligomers perform affinity chromatography.

Cultural environment, including a variety of oligomers mannan-binding lectin, is subjected to affinity chromatography on a matrix obtained from a hydrocarbon.

Affinity chromatography is a well-known method of purification of proteins from the protein matrix. The affinity matrix derived from a hydrocarbon, with tetramine, pentamerone and/or hexamine subunit mannan-binding lectin, at least two times greater than the dimer subunits mannan-binding lectin. In a preferred embodiment of the present invention the affinity matrix derived from a hydrocarbon, with tetramine, pentamerone and/or hexamine subunits mannan-binding lectin, at least three times larger than dimers mannan-binding lectin. The affinity matrix derived from a hydrocarbon, with tetramine, pentamerone and/or hexamine subunits mannan-binding lectin may be, at least twice, che is with subunits mannan-binding lectin.

Thus, a composition/mixture/composition mannan-binding lectin has a size distribution of molecules, at least 50, 60, 70, 80, 90, 95% coinciding with the distribution of the sizes of the molecules of natural mannan-binding lectin. Preferably, the size distribution of molecules as much as possible coincided with the distribution of the sizes of the molecules of natural mannan-binding lectin, which means match at least 99%.

The matrix obtained from a hydrocarbon essentially has no affinity with the subunits and/or dimers of subunits mannan-binding lectin. Preferably, the matrix obtained from a hydrocarbon, was largely identical only tetramer, pentamers and/or review of the subunits of recombinant mannan-binding lectin.

Preferably, the composition/mixture/composition was dominated by higher oligomers subunit type tetramers, pentameron and/or hexamers. Thus, it is desirable that in such a composition ratio of the number of tetramers, pentameron and/or hexamers to the number of subunits and/or dimers was at least 2:1, preferably at least 5:1.

In a preferred embodiment of the invention, it is desirable that the ratio of the sum tetramers, pentameron and/or hexamers to the amount subunits and dimers was at least 2:1, but better at least 5:1.

The matrix can be obtained from any hydrocarbon or mixture of hydrocarbons, associated with mannan-binding pectin and for which a binding higher oligomers mannan-binding lectin is preferred. Preferably, the hydrocarbon matrix, matrix was obtained from hexose, such as mannose or N-acetyl-glucosamine matrix, but it is preferable mandonna matrix.

The selectivity of the matrix obtained from a hydrocarbon, provided that the matrix as such, i.e. the matrix obtained not from a hydrocarbon, has no affinity with subunits mannan-binding lectin, in particular, has no affinity with trimers or smaller oligomers mannan-binding lectin. This is achieved when the matrix is free from hydrocarbons. In particular, the matrix should not contain separate. Preferably, the matrix consisted of polymeric materials that do not contain hydrocarbons, the type of pellet Fractogel®TSK.

The matrix can be of any shape suitable for chromatography, in most cases in the form of a pellet type plastic balls.

After applying the source of mannan-binding lectin column is washed, preferably with the use of buffer solutions, not leading to denaturation, composition, pH value and ionic strength are provided which indicate the absence of proteins without elution of higher oligomers mannan-binding lectin. As the buffer solution can be used Tris-buffered saline (TBS). You elution mannan-binding lectin using selective Stripping agent capable of efficient elution of higher oligomers mannan-binding lectin, such as Tris-buffered saline containing deformirujuschij agent type add (for example, add concentration 5 mm) or mannose (for example, a solution of mannose concentration 50 mm) and then the oligomers mannan-binding lectin going.

In accordance with the present invention the sequence of the gene mannan-binding lectin can be obtained from gene mannan-binding lectin of the person or of the genes mannan-binding lectin other species of animals whose immune system in this respect acts as the human immune system. A preferred variant of the preparation of recombinant mannan-binding lectin in accordance with the present invention described in example 1 below, in accordance with which the indicated recombinant mannan-binding lectin get with the use of the expression vector, comprising a gene mannan-binding lectin person. The expression vector of the specified preferred variant of the method of obtaining considered in example 1 and Phi is ur 2.

The present invention also relates to the use of expression vectors, which are composed of sequences that are functional derivatives of the sequences of the gene mannan-binding lectin person. Under these functional derivatives are understood as sequences that alternate key pair, which causes functional or primarily non-functional characteristic deviation of the expression vector, and the functionality of the thus obtained mannan-binding lectin, is comparable to the functionality of the mannan-binding lectin, obtained using the expression vector, which consists of an unmodified gene sequence mannan-binding lectin person.

Preferably, in addition to the method of purification of higher oligomers, would be provided and also ionic design expression and host cell.

Therefore, it is preferable that the composition constructs for expression of a gene comprised of at least one intron sequence of a gene mannan-binding lectin person or its functional equivalent.

In addition, in the design for gene expression may include at least two exon gene sequence mannan-binding lectin person or fu is tsionaljnogo equivalent. Most preferably, if the design for gene expression includes at least three exons of the gene sequence mannan-binding lectin person or its functional equivalent. In the case of more than one exon preferably exon sequences were lined up in the same way as in the gene mannan-binding lectin person.

Although it is preferable that the composition of this sequence was part of intron sequences, in some cases it may be convenient to have part of the structure of the expression was part of the DNA sequence, complementary to the coding subunit of mannan-binding lectin or its functional equivalent.

The present invention relates to the use of rather constructions gene expression mannan-binding lectin than the structures of DNA, complementary to the coding mannan-binding lectin for the expression of recombinant mannan-binding lectin in mammalian cell lines or transgenic animals, with the aim of obtaining recombinant mannan-binding lectin with structural properties under conditions not leading and leading to denaturation, which is largely similar to the natural mannan-binding lectin person. By "recombinant mannan-binding lectin h the rights" means the mannan-binding lectin person, which expresses created from genetic engineering, nucleic acids, and under "design for gene expression mannan-binding lectin" refers to the expression vector suitable for expression in the cell lines of the host cell, comprising exon sequence and at least one intron sequence of a gene mannan-binding lectin of human genes or mannan-binding lectin other animal species, which include (but are not limited to chimpanzees and rhesus macaques.

Preferably, the DNA sequence encodes the polypeptide sequence (e.g., sequence No. 1) or its functional equivalent, and the notion of a functional equivalent is defined above. Sequence No. 1 corresponds to the sequence of mannan-binding lectin with access to a database No. R. The equivalent can be obtained by modification of the peptide sequence shown in sequence No. 1, for example, change the relevant properties of the sequences of the present invention, which is substituted by one or more amino acids.

Preferably, the functional equivalent included conservative substitution, i.e. the substitution of one or more amino acids amino acid region is non similar properties, for example, specialists in the chemistry of proteins can obtain secondary and tertiary structure of the protein, which does not change. Amino acids suitable for conservative substitutions include those chains that have a functional similarity of these circuits. For example, hydrophobic residues, glycine, alanine, valine, leucine, isoleucine and methionine can replace other similar residues. Similarly, conservative substitutions can include interchangeable hydrophilic residues (e.g., arginine and lysine, glutamine and asparagine, threonine and serine), basic residues (e.g. lysine, arginine and histidine), and/or acidic residues (e.g., aspartic acid and glutamic acid). Functional equivalents may also be modified, for example, by removing or adding amino acids or chemical modification of amino acids, while maintaining the function of the polypeptide.

The selected peptide mannan-binding lectin, including any of its functional equivalents, may in one variation of the invention to include at least 80 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, at least 200 amino acid residues, at least 220 amino acid residues, at least 250 amino acid residues.

In predpochtite the flax embodiment of the present invention the expression vector is suitable for expression in cell lines mammal or a transgenic animal, cells which contain exon sequence and at least one intron sequence of a gene mannan-binding lectin of human genes or mannan-binding lectin other animal species, which include (but are not limited to chimpanzees and rhesus macaques. In one of the examples of the present invention, the culture of the host cell is grown in cells cranganore animal. Under transgenic animals in this context, see genetically modified animal cells which contain and Express a gene of mannan-binding lectin person or its fragments or its likeness or imitation.

In a preferred embodiment of the present invention in the composition constructs for expression of the gene of the present invention includes a viral vector type of viral vector DNA, a viral vector RNA or chimeric viral vector. Examples of DNA viruses include cytomegalovirus, herpes simplex virus, Epstein-Barr, simian virus SV-40, the human papilloma virus bovine adeno-associated virus, adenovirus, vaccinia virus, Kunjin (Kunjin virus, Sendai (Sendai) virus and baculovirus.

In the cells of the host mammal can be used a number of systems-based viruses. In cases when, as the expression vector is adenovirus, NAA molecule is einevoll acid according to the present invention can be crosslinked with the control complex of transcription/translation of adenovirus, for example, the late promoter and tripartite leader sequence. Then this chimeric gene may be inserted in the adenovirus genome by in vitro or in vivo recombination. This insert in nonprincipal/essentialyou region of the viral genome (e.g., region E1 or E3) leads to a recombinant virus in infected host cells (e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659, 1984). For efficient translation of inserted nucleic acid molecules may also require specific signals beginning. These signals include the codon that initiates antithymocyte globulin (ATG), and neighboring sequences. In cases where a gene or complementary DNA, including its own initiator codon and adjacent sequences, is inserted into the appropriate expression vector, no need for additional translational control signals. However, in cases when inserted only part of the coding sequence required exogenous translational control signals, including, perhaps, the codon (ATG). In addition, to ensure translation of the entire insert the initiating codon must be in the same phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiating codons can have the RA is ing the origin, including both natural and synthetic. The efficiency of expression may be enhanced by enabling the respective amplifying the transmission elements, transcription terminators, etc. (see Bitter et al., Methods in Enzymol. 153:516-544, 1987).

Examples of the RNA virus is a virus Simlike Forest, Sindbis, Poko, rabies virus, influenza virus, SV5, respiratory intitially virus, the virus encephalitis Venezuelan horses, vesicular stomatitis virus, Sendai virus, Kunjin (Kunjin virus, and retroviruses.

Examples of chimeric viruses include adenovirus, Sindbis virus and adenovirus - adeno-associated virus.

With regard to specific vectors, then it is written about them in the work of the author Markides S.C. "Components of vectors for gene transfer and expression in mammalian cells (Components of the vectors for transfer and expression of a gene in mammalian cells).

In particular, use of the origin of replication of Epstein-Barr or its functional derivatives, or its likeness/imitation, including vector pREP9.

In one aspect the present invention provides a construction for the expression of the gene encoding mannan-binding lectin person, the hallmark of which is the content of one or more intron of posledovatelnostei gene mannan-binding lectin of human rights, including its functional derivatives. In addition, it includes the area ol the motor, taken from the genes of a virus or eukaryotes, including mammals and insects.

Preferably, the promoter region was different from the promoter mannan-binding lectin person. The question of optimizing the output of mannan-binding lectin and the size distribution of oligomers mannan-binding lectin preferably the promoter region be chosen in such a way as to ensure the optimal functioning with the vector and the host cell.

In a preferred embodiment of the invention field of the promoter is selected from the group comprising the promoter of the long terminal repeat of Rous sarcoma virus, and immediate early promoter of cytomegalovirus, and alpha promoter, elongation factor 1.

In another example of the invention field of the promoter is selected from the genes of microorganisms, such as viruses, yeast and bacteria.

To increase the yield of recombinant mannan-binding lectin promoter region may include reinforcing elements, such as element QBI SP163, consisting of a 5'-terminal untranslated region vascular endothelial gene growth factor mouse. This structure is used to transform host cells to obtain a cell culture of the host, is capable of expression of mannan-binding lectin.

Synthesis of recombinant mannan-binding lectin can b the th cultures used in-vivo or in-vitro. Preferably, the culture of the host cell was eukaryotic. In this context, the transformation of eukaryotic cell culture refers to the introduction of recombinant DNA into cells. Design for gene expression, used in this way, characterized by the presence of the field, the coding mannan-binding pectin, extracted from mammalian genes, including genes human genes, strongly reminding them, for example, the genes of chimpanzees. Applied design for gene expression is also characterized by a region of the promoter taken from the genes of viruses or eukaryotic, including mammalian cells and insect cells.

In the present invention considers such recombinant mannan-binding lectin, which is a form of mannan-binding lectin similar to natural mannan-binding lectin person the fact that the size distribution of molecules in its purified form is the most close to the natural profile of the size distribution of molecules among all known forms.

Method of production of recombinant mannan-binding lectin of the present invention is characterized by the fact that the culture of the host cell is eukaryotic, for example, culture of mammalian cells. Preferably, the culture of the host cell was a culture of human kidney cells, but it is even more preferred I have is, to this culture of host cell consisted of embryonic human kidney cells (SOME cells). In the invention discusses the use of cell lines NEC for the preparation of recombinant mannan-binding lectin person. The term "cell line NECK" refers to any cell line derived from embryonic stem cells human kidney (but not limited to) type cell lines stored in the American Type Culture Collection number CRL-1573 and CRL-10852.

Other cells can be embryonic fibroblast cells of the ovary hamster cells of the kidneys of a young hamster cell carcinoma of the uterus human cells, human melanoma cells, human liver, endothelial cells of the umbilical vessels, the endothelial cells of the brain, the tumor cells of the oral cavity of the person, the kidney cells of monkeys, mouse fibroblast, kidney cells mouse cells of the connective tissues of the mouse, oligodendrocyte cells of a mouse, the mouse macrophage, fibroblast mouse neuroblastoma cells mouse pre-b cells of the mouse, the mouse lymphoma cell, cells plasmacytoma mouse cells teratocarcinoma mouse cell astrocytomas rats, epithelial cells of the mammary gland of the rat, COS cells, CHO, KSS, 293, VERO, HeLa, MDCK, WI38, and IH3T3.

In addition, can be selected such strain of host cells that modulates the expression of the inserted sequences or special the way modifies and processes the gene product. Such modifications (e.g. glycosylation) gene product and processing (e.g., cleavage) of protein products may be important for function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of the expressed foreign protein can be selected appropriate cell line or system owner. For appropriate treatment type primary transcription, glycosylation and phosphorylation of the gene product can be used in eukaryotic host cell with the appropriate cellular mechanisms. The above types of mammalian cells are types of cells that can be used as host cells.

In another aspect of the present invention considered the mannan-binding lectin, which is produced by recombinant DNA technology, for example, in mammalian cells or transgenic animals. In particular the present invention relates to recombinant mannan-svyazyvaysya the lectin secreted by:

- the described method constructs for expression of the gene encoding the polypeptide mannan-binding lectin person or functionallyequivalent;

- broadcast culture of the host cell with the help of this design;

- cultivation of the culture of the host cell, receiving, from the selection of the polypeptide into the culture medium, with

- obtaining a culture medium containing recombinant mannan-binding lectin.

In addition, the present invention relates to the synthesis of mannan-binding lectin, which is implemented through:

(a) creating designs for gene expression. encoding mannan-binding lectin person;

(b) transforming the culture of eukaryotic cells using the above-mentioned structure, expression and reception, thus, culture eukaryotic recombinant host cells;

(c) cultivation of this culture eukaryotic recombinant host cells either in vitro or in cells of transgenic animals and gene expression, thus, specified the mannan-binding lectin person;

(d) collecting the specified mannan-binding lectin of human rights and its treatment using the above-described affinity chromatography.

The culture of the host cell can be cultured in an appropriate culture medium. An example of a culture medium can serve as RPMI-1640 or DMEM supplemented, for example, insulin, transferrin. selenium and fetal calf serum.

The present invention also includes the step of clearing or releasing sananvapaudessa lectin person, for example, mannan-binding lectin man of culture supernatants or body fluids or tissues of the transgenic animals by use of hydrocarbon affinity chromatography, as described above. In a preferred embodiment of the invention affinity chromatography is performed using matrices mannose, hexose or matrix obtained from N-Camilluccia that are suitable for affinity chromatography, as described above. In particular, this applies affinity chromatography, matrix, which are obtained by mannose. Before the matrix, obtained with the help of mannan was used for collection of recombinant mannan-binding lectin from the culture supernatant. While the matrix obtained from the mannan bind both forms of mannan-binding lectin with large and small molecular weight, the use of matrices obtained by the method described above, for example, using mannose, provides significant advantages in purification of recombinant mannan-binding lectin, since these matrices are selectively bind forms of recombinant mannan-binding lectin, similar to the natural mannan-binding lectin. The degree of affinity with the natural mannan-binding lectin obtained when measuring the size distribution of molecules using the method of SDS-PAGE, should be the m ore than 50%, followed by protein staining of the gel, as described, for example, the authors of MA and others in 1997

Purified recombinant mannan-binding lectin in this context should be understood as recombinant mannan-binding lectin purified from cell culture supernatants, or body fluids, or tissues of the transgenic animals, the cleansing is done with the use of hydrocarbon affinity chromatography.

Preferably, the process of separation or purification of the composition of the oligomers mannan-binding lectin person with the distribution of the sizes of molecules, at least 50% identical to the size distribution of molecules of natural mannan-binding lectin plasma, included:

- a drug oligomers mannan-binding lectin person;

- affinity chromatography specified drug using matrix obtained from the derivative of a hydrocarbon, and the affinity of this matrix with tetramine, pentamerone and/or hexamine subunit mannan-binding lectin, at least two times greater than the dimer subunit of mannan-binding lectin;

- obtaining of the eluate containing composition selected recombinant mannan-binding lectin person.

In the present invention proposes a new form composition of recombinant mannan-binding Lek is in, containing oligomers subunits mannan-binding lectin, and the profile size distribution of the molecules of these oligomers, at least 50% identical to the profile size distribution of molecules mannan-binding lectin human plasma. In a preferred embodiment of the invention, the size distribution of the molecules of these oligomers, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, coincides with the distribution of the sizes of the molecules of natural mannan-binding lectin person.

Preferably, the present composition was dominated by higher oligomers subunits, such as tetramer, pentamers and/or hexamers. Thus, it is preferable that in such a composition ratio of the number of tetramers, pentameron and/or hexamers to the number of subunits and/or dimers was at least 2:1, although preferably not less than 5:1.

In a preferred embodiment of the invention, it is desirable that the ratio of the sum tetramers, pentameron and/or hexamers to the amount subunits and dimers was at least 2:1, but rather that this ratio was not less than 5:1.

When assessing the size distribution of the molecules, oligomers mannan-binding lectin was used Western blotting by SDS-PAGE. Thus it is obvious that under the identity of 50% understands what I the average molecular weight of at least 50% of oligomers, obtained by the method of SDS-PAGE and/or Western blotting, greater than 200 kDa.

The composition of recombinant mannan-binding lectin can be purified by any medium suitable for the culture medium, such as any physico-chemical method of allocation, including, but not limited to, filtration methods, chromatography, such as ionoobmennaya chromatography based on size, penetrating gel affinity chromatography. The composition of recombinant mannan-binding lectin preferably clear from the culture medium by affinity chromatography, as described above.

Preferably, the composition of recombinant mannan-binding lectin would provide functionality similar to the functionality of the mannan-binding lectin serum or plasma. In this context, the functionality of the mannan-binding lectin is the ability to activate the complement system, which is discussed above in relation to functional equivalents. Functionality can be expressed as a specific (specific) activity of mannan-binding lectin in the form of a unit activity of mannan-binding lectin 1 ng mannan-binding lectin. It is desirable that the functionality of the composition of recombinant mannan-binding is found lectin, expressed in terms of specific activity, was not less than 25% of the specific activity of mannan-binding lectin purified from serum, at least 50% of the specific activity of mannan-binding lectin purified from serum, at least 75% of the specific activity of mannan-binding lectin purified from the serum.

The composition of the mannan-binding lectin of the present invention substantially free of any impurities naturally associated with mannan-binding lectin derived native host organism, as well as any impurities naturally associated with mannan-binding lectins purified from serum. The term native host organism is understood that the mannan-binding lectin secreted by the cell, the normal way of expressing the mannan-binding lectin.

The eluate obtained using affinity chromatography, can be used to prepare pharmaceutical compositions, or the eluate before use may be subjected to additional treatment in several stages.

The composition of the mannan-binding lectin obtained according to the present invention, can be used to prepare pharmaceutical compositions for the prevention and/or treatment of various viral diseases or conditions.

In addition to oligomers man is EN-binding lectin, these pharmaceutical compositions may contain pharmaceutically acceptable substances-media and/or means of delivery.

In particular for the stabilization of proteins mannan-binding lectin can optionally enter a stabilizing agent. As a stabilizing agent, you can use the alcohol derived from sugar, saccharide, protein and/or amino acids. An example of a stabilizing agent may be albumin or maltose.

Depending on, for example, the form of the drug, as additives you can use other traditional additives to pharmaceutical compositions.

In one embodiment of the present invention the pharmaceutical composition is injected in a form suitable for injection. Can also be used such traditional substance-media as an isotonic saline solution.

In another embodiment of the present invention the pharmaceutical composition is applied in a form suitable for introduction through the lungs, or for administration in the form of powder for inhalation, or cream, or liquid for topical application.

The present invention is based on the synthesis of a new form of recombinant mannan-binding lectin, which is more close to the natural mannan-binding lectin person than known by now in shape, and which can be synthesized using in-vivo or in-vitro technology. This proposed form of recombinant mannan-binding lectin provide great opportunities for the La therapy of such diseases, as cancer and infections associated with immunoparalysis chemotherapy. Structural differences previously known preparations of recombinant mannan-binding lectin from drugs from natural forms of mannan-binding lectin make such a therapy maloobeshchajushchim. In addition, the use of recombinant mannan-binding lectin similar to natural mannan-binding lectin, in these therapies is of great progress compared with the well-known mannan-binding lectin, in particular, the mannan-binding lectin of human plasma, as this therapy with the use of recombinant mannan-binding lectin much reduces the risk of viral infections.

This therapy should not be therapy diagnosed disease, disorder or condition in the case of immediate or obvious therapy, and can be used for prevention of the disease or condition.

Under therapy in this context refers to the treatment and/or prevention of, for example, immune system and reproductive system in humans or animals, the mechanism of action of these functional units is identical to similar mechanisms in humans. Under the condition that requires treatment, not necessarily means a condition that should be treated in the present lie is, rather, the state associated with the existing and/or perceived need or need to improve the normal state. In particular, this therapy is therapy of a condition associated with a deficiency of mannan-binding lectin.

Another aspect of the present invention relates to the manufacture of a medicinal product consisting of the above-mentioned pharmaceutical compositions of the mannan-binding lectin, including the composition of the fragments and the similarity of recombinant mannan-binding lectin, intended for the treatment of conditions including the treatment and/or prevention of diseases and disorders, for example, immune system and reproductive system in humans or animals, these functional units operate in this regard the animal as well as humans.

These diseases, disorders and/or conditions applied therapy with the use of the compositions according to the present invention, for example, therapy of conditions associated with deficiency of mannan-binding lectin, cancer therapy and infections associated with immunosuppressive chemotherapy, including, in particular, infection-related conditions that occur during cancer therapy or implantation and/or organ transplantation. The present invention also includes treatment of conditions associated with recurrent what akinyemi.

Thus, the pharmaceutical composition can be used for therapy and/or prevention of clinical disease type infections, deficiency of mannan-binding lectin, cancer, diseases associated with chemotherapy, types of infections, conditions associated with human immunodeficiency virus (HIV), conditions associated with congenital or acquired immunodeficiency. In particular, such diseases as chronic inflammatory demyelinizing polyneuropathy, multi focal motor neuropathy, multiple sclerosis, severe myasthenia syndrome Itena-Lambert, inflammation of the optic nerve, epilepsy, syndrome of primary antiphospholipid. rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis, Wegener's Werner, Sjogren syndrome, chronic rheumatoid arthritis in children, autoimmune neutropenia, autoimmune hemolytic anemia, neutropenia, Crohn's disease, ulcerative colitis, intestinal disorders, asthma, syndrome, septic shock, chronic fatigue syndrome, psoriasis, syndrome toskicheskih shock, diabetes, sinusitis, advanced cardiomyopathy, endocarditis, atherosclerosis, primary Hypo/agammaglobulinemia, including common variable immunodeficiency syndrome Viskota-Aldrich and combined immunodeficiency, secondary Hypo/agammaglobulinemia in patients with khron the ical lymphatic leukemia and multiple myeloma, acute and chronic idiopathic thrombocytopenic purpura, allogeneic bone marrow transplantation, Kawasaki disease, Guillain-Barre syndrome.

The drug may be administered by any suitable means, such as, intravenous, intramuscular, subcutaneous or intradermal. Can also be used a drug through the lungs or local administration of the drug.

In particular the composition of the mannan-binding lectin can be entered for the prevention and/or treatment of infections in patients with clinical syndromes associated with congenital or acquired deficiency of mannan-binding lectin or at risk of developing the syndrome. When various conditions occur, for example, as a result of chemotherapy or other types of cytotoxic therapy, cancer, AIDS, genetic predisposition, chronic infections and neutropenia, in individuals with deficiency of mannan-binding lectin may increase susceptibility to infections.

It is obvious that patients with cancer undergoing chemotherapy, are often prednisoloni to infection caused by side effects of drugs on cells of the immune system, which is the basis for therapy with the use of mannan-binding lectin for the treatment of such diseases. In accordance with the observations of low concentration (the lower the 500 ng/ml) mannan-binding lectin plasma indicate increased susceptibility to clinical infections and immune protection of these patients can be enhanced by the introduction of recombinant or natural (natural) mannan-binding lectin obtained from plasma.

Thus, the pharmaceutical composition may be injected during the period preceding the beginning of chemotherapy, or during at least some part of chemotherapy.

In particular the composition of the mannan-binding lectin can be entered as the main booster injection" before chemotherapy or only the patients who are at risk of developing deficiency of mannan-binding lectin. Before therapy, patients of the risk group is determined by measuring levels of mannan-binding lectin and therapy with those who have levels of mannan-binding lectin below. For most groups the lower limit of the content of mannan-binding lectin was estimated to be less than 500 ng/ml Levels of mannan-binding lectin may be determined by the method TRIFMA (time resolved immunofluorescent assay - immunofluorescence analysis with a time resolution), as described in example 9, the method of ELISA, RIA or nephelometry.

Another indication for the introduction of mannan-binding lectin is levels of mannan-binding lectin below 50% of normal levels or below 300 ng/ml, or below 200 ng/ml.

The composition of the mannan-binding lectin enter the appropriate doses per hour the particular usually at intervals of time, for example, once or twice a week during the course of chemotherapy.

Usually one dose contains from 1 to 100 mg of the drug, or from 2 to 10 mg, or more often from 5 to 10 mg is Injected usually 0.1 mg of the drug per 1 kg of body weight of the patient.

Thus, another aspect of the present invention relates to mannan-binding lectin, including recombinant mannan-binding lectin, its fragments or the like for use in therapy of cancer and diseases and disorders, for example, immune system and reproductive system, these therapy involves the creation, reconstruction, strengthening and/or promote opsonic effect and bactericidal activity of the complement system, i.e. strengthening the ability of the immune system to recognize and kill pathogens.

In addition, one aspect of the present invention relates to the use of recombinant composition of the present invention in the composition of other medications. Such medicines may be antimicrobial drug type of antibiotic.

As for abortion, it was noted that patients with recurrent miscarriages often observed low levels of mannan-binding lectin plasma that is the background against which in the case of restoring the levels of mannan-binding is found lectin by introduction of recombinant mannan-binding lectin prone to miscarriage decreases.

With regard to the nature of the compositions according to the present invention, it is obvious that in a broad aspect the present invention relates to compounds acting as opsonins, can increase the uptake by macrophages or by direct interaction between composition and macrophage, or through an intermediate deposition of complement on the target surface. A concrete example of such composition is mannan-binding lectin, its fragment or the like. The present invention is based on the discovery of the synthesis of recombinant mannan-binding lectin man, whose structure is, obviously, closer to the natural structure of the mannan-binding lectin than patterns previously known forms of mannan-binding lectin.

The present invention provides an explanation and evaluation of the various aspects and details, and in addition to the figures 1 and 2 are illustrations of the present invention; consider also the examples of the preferred variants of the invention.

BRIEF DESCRIPTION of FIGURES

The figure 1 shows a comparative analysis of the purified recombinant mannan-binding lectin and natural mannan-binding lectin in unabridged form, obtained by the method of SDS-PAGE and silver staining (including concentrating gel). On the track "S" is visible natural mannan-binding Lek is in the amount of 24 ng (MO-04, (State serum Institute, Copenhagen, Denmark). On the track "SOME" visible recombinant mannan-binding lectin in the amount of 60 ng received in the cells of SOME 293EBNA, and on the track "Hyb" visible recombinant mannan-binding lectin in the amount of 12 ng obtained in cells Sp2/0Ag14 (presented by Dr. R.A.B.Ezekowitz). The arrow indicates the position of the dominant forms of recombinant mannan-binding lectin obtained from cells hybridoma. Although the number of recombinant mannan-binding lectin on the track "SOME" five times more than the number of recombinant mannan-binding lectin on the track "Hyb", on the track "SOME" do not see a strip the size of 55 kDa, which suggests that there are significant differences in the formation of disulfide bridges these two subunits of recombinant mannan-binding lectin (see example 6).

Figure 2 schematically shows the construction of expression in accordance with the present invention, namely design (mannan-binding lectin)/pREP9 discussed in example 1.

The figure 3 presents the concentration of mannan-binding lectin in the fractions (elution volume)obtained by gel chromatography (example 7).

The figure 4 shows the results of the analysis of recombinant mannan-binding lectin method Western blotting, when et is m recombinant mannan-binding lectin was purified using beads mannose-TSK, granules mannan-TSK, granules mannose-sepharose, granules mannan-sepharose and non-derivative granules sepharose 4 C. For comparison we have included the supernatant nefrackzionirovannam culture of cells transfected design (mannan-binding lectin)/pREP9 (example 3).

The figure 5 shows the results of analysis by the method of Western blotting of two preparations of recombinant mannan-binding lectin in unrestored 50 ng of recombinant mannan-binding lectin received in accordance with these invention (cells 293 EBNA) and 50 ng of recombinant mannan-binding lectin obtained in accordance with the method described by the authors of Super and others, 1992 (cells Sp2/0Ag14). Natural mannan-binding lectin purified from human serum as described by the authors Lu and others, 1990 ("Natural MBL") (example 6).

EXAMPLES

EXAMPLE 1

Design for gene expression mannan-binding lectin

Human genomic DNA was extracted from 20 ml of blood, entered in ethylenediaminetetraacetic acid (adtc). Etc and the blood was mixed with 80 ml of cold (4° (C) Rasbora Tris-HCl (concentration 10 mm), MgCl2concentration of 5 mm, 1% (vol.) Triton X-100, sucrose concentration of 0.32 mm, and all of this was stored at 4°C for 30 minutes with occasional stirring. The samples were tsentrifugirovanie acceleration of 1000 g for 30 minutes is ri 4° S, after which the supernatant was separated and nucleated residue was washed in 10 ml of NaCl concentration of 0.9% (mass fraction to volume). The collected cell nuclei were resuspendable in 6 ml of NaCl solution concentration of 75 mm, EDD concentration of 24 mm (pH 8.0) and 460 μl of 10% solution (the ratio of the mass fraction to volume) didierslach sodium and added 1 mg pronase (cat. No. 165921, Boehringer-Mannheim, Mannheim, Germany). After holding at room temperature under mild stirring was added 2 ml of saturated solution (˜6 M) NaCl. After intensive mixing residues were removed by centrifugation at acceleration of 1000 g. To the supernatant at a ratio of 1:1 was added to isopropanol and the contents of the vessel were slightly usbutils up until did not precipitate the DNA. Filiform precipitate was collected by Pasteur pipette, washed in 70% (vol.) ethanol, dried in air and was resuspendable in 1 ml of buffer solution. Performed PCR amplification of the gene mannan-binding lectin involving sense-primer modified to make it consisted of the XhoI site: 5'-AGATTAACCTTCCtcGAGTTTTCTCACACC-3'. Anti-sense primer was modified to make it consisted site BamHI: 5'-TTACggaTCCACTCCATTATACATACAC-3' (restriction sites (underlined)introduced in the sequence using genetic engineering modified in pairs than the AI with the natural sequence of genes in accordance with the publication authors Sastry and others, 1989 and are shown as lowercase letters in both sequences of the primers. Primers obtained through DNA technology, Aarhus, Denmark). Sense primers and anti-sense primers are located in the 5' and 3' UTR. Reaction conditions for PCR consisted of 200 μm dATP. 200 μm dCTP, 200 μm dGTP, 200 μm dTTP (cat. No. 1969064, Boehringer-Mannheim, Mannheim, Germany), reaction buffer with the value of the ionic strength is equal to 1, of 0.75 μl of enzyme (cat. No. 1732641, Expand High Fidelity™, Boehringer-Mannheim, buffer response, supplied with enzyme), genomic DNA (100 ng), and water, brought to a final volume of 50 µl. Heat the PCR program was performed 1 cycle at 96°C for 3 min, 10 cycles at 94°C for 15 s, 60°C for 30 s, at 72°C for 5 min, then 20 cycles at 94°C for 15 s, 60°C for 30 s, at 68°C for 5 min in increments of time, equal to 20 s per cycle, followed by a final elongation at 72°C for 5 min (sensor thermal cycles Omn-E™, Hydaib, Ashford, UK). In accordance with the manufacturer's recommendations were carried out cloning of the product of 6.2 kb PCR vector pCR2 using kit TA cloning (cat. No. K-01, Invitrogen, Leek, The Netherlands). By digestion of the vector with the enzymes NotI and BumHI (cat. No. 15441-025 and 15201-023, GibcoBRL, Paisley, UK) for 6 h at 37°in the corresponding buffer of the vector pCR2 the separation is the very gene mannan-binding lectin. The insert DNA (i.e. a fragment of the 5' - NotI - mannan-binding lectin - BamHI - 3') were extracted from 1% agarose gel (ratio of mass fraction to volume) using a set of Pharmacia's Bandprep™ (cat no.27-9285-01, Pharmacia, Uppsala, Sweden). Vector pREP9 (cat. No. V009-50, Invitrogen) was digested as described for the vector (mannan-binding lectin)/RSR and restriction of the product, i.e. the linearized vector was isolated from the agarose to minimize contamination undigested vector. In a final volume of 10 μl, 1 μl (˜100 ng) of digested vector pREP9, 4 μl (˜400 ng) fragment 5' - NotI - mannan-binding lectin - BamHI - 3', buffer for ligation with the value of ionic strength equal to 1, and 20 units of T4 ligase (cat. No. 202S, New England Biolabs, Beverly, buffer response submitted together with the enzyme) was aged under 14°C. E. coli Cells. TOPF10 prepared for transformation by penetration CaCl2and to be transformed by the ligation product using a heat stroke, as described by Hanahan (1983). Bacteria were inoculated on plates with the medium LB-agar with the addition of 50 μg of ampicillin per 1 liter of medium and after exposure at 37°appeared colonies.

The figure 2 shows schematically a preferred variant of the present invention, namely patterns (mannan-binding lectin)/pREP9. Amplificatory by PCR part of the gene mannan-binding Le is Tina (the location of the sense primers and anti-sense primers are shown as black arrows) contained four exons, including the known exon 3 netransliruemye exon 4, as described by the authors Sastry and others, 1989 Fragment was combined with the expression vector pREP9 on restriction sites (stations) endonuclease NotI and BamHI (showing restriction endonuclease sites EcoRV, XbaI, Bg/II, XmnI, ClaI, StuI and SacI vector sequence). Transcription of a fragment of mannan-binding lectin, cloned in the expression vector pREP9, was controlled by the promoter of the long terminal repeat of Rous sarcoma virus (PRVSlocated above cloning site (arrows indicate the direction of transcription of the vector elements) and the polyadenylation site of the monkey virus (SV40). Bacteria, which are the product of the vector DNA in the bacterium E. coli., was obtained using marker of resistance to ampicillin (Amp) and the prokaryotic "originalcolor" CoIE1. The conservation of the vector in cells HEK293EBNA was provided by originalaction virus Epstein-Barr (OriP) and the expression of nuclear antigen 1 of Epstein-Barr (EBNA-1), which attaches the vector to the chromosomes of the host cell.

EXAMPLE 2

Expression of recombinant mannan-binding lectin in mammalian cells

Cells HEK293EBNA grown to confluence in the flask with an area of 150 cm2(cat. No. 9075, TPPAG)containing DMEM (Biological Industries), with the addition of 10% FBS, glutamine, 0418 200 ál/ml (Gneticin™, cat. No. G9516, Sgma) and antibiotics (10,000 units of penicillin and 1% streptomycin (ratio of mass fraction to volume)), was going through treatment with trypsin and 24 hours before transfection were inoculated at a density four times lower than previous (˜25% of the state merge) into the flask with an area of 150 cm2(cat. No. 90150, RTR). Three hours before transfection, the whole environment was updated. When full 1680 µl was mixed with 60 µg design (mannan-binding lectin)/pREP plasmid DNA (see example 1) with a NaCl concentration of 150 mm, EDD concentration of 1 mm. The Tris-HCl concentration of 10 mm (pH 7,12), CaCl2the concentration of 268 mm. The DNA solution with calcium was added to the solution, volume 1680 µl containing HEPES concentration of 50 mm, NaCl concentration of 250 mm, Na2HPO4+NaH2PO4concentration of 1.5 mm with a pH brought to 7.12. The cells were maintained for 14-16 h of deposition of DNA/Sa·PO4, were washed twice in phosphate buffer and again was aged in medium RPMI-1640 (50 ml capacity) supplemented with glutamine, antibiotics (penicillin and streptomycin) and a solution of insulin-transferrin-selenium (ITS) (cat. No. 51300-44, GibcoBRL) for 6-8 days.

EXAMPLE 3

Purification of recombinant mannan-binding lectin with matrices derived from hydrocarbons

Granules Fractogel®TSK HW-75 (cat. No. 14985, Merck KgaA, Darmstadt, Germany) was activated by mixing 50 ml of the pellet with 50 ml of a solution of Na2CO3concentration of 0.5 mm (pH 11) and 10 ml of vinylsulfonic (cat. No. V370-, Alrich, Milwaukee, steveascension, USA) followed by exposure with stirring for 1.5 h at room temperature. Pellets were collected on a glass filter and were washed once with water and once in a solution of Na2CO3concentration of 0.5 mm (pH 11). Then the granules were resuspendable in a solution of Na2CO3concentration of 0.5 mm (pH 11) and mannose was added to obtain a final concentration of 10% (mass fraction to volume). The mixture was maintained with stirring at room temperature. After that, the mixture was washed in water, remains chemically active groups were blocked with the shutter pellets in a solution of ethanolamine concentration of 0.1 M (pH 9) for 2 hours After washing in water and a solution of TBS (Tris-buffered saline solution) granules TSK-75 was applied to the chromatographic column and was washed in 15 ml of glycine concentration of 0.1 M (pH 3.0) and cited in equilibrium in a solution of TBS-Tween 20™ (Tris-buffered saline containing 0.05% Tween 20™ (the ratio of the mass fraction to volume)and the solution of CaCl2until then, until stabilized absorption of the ultraviolet spectrum. Culture medium from cells transfected with the construct (mannan-binding lectin)/pREP9, purified by centrifugation at acceleration 5000 g, passed through a chromatographic columns is at a flow rate of 0.4 ml/min The column was washed using 75 ml TBS and a solution of CaCl2concentration of 2 mm, and related recombinant mannan-binding lectin was elyuirovaniya in a solution of TBS and add concentration of 5 mm and was collected in fractions of 500 µl. To obtain pellets of Fractogel®TSK, mannan (purified from yeast as described by Nakajima and Ballou, 1974) pellets were activated as described above, and was aged in a solution of Na2CO3concentration of 0.5 M (pH 11)containing 25 mg of mannan on 1 ml, followed by the same processing as that for the preparation of pellets, derived from mannose. Pellet sepharose 4 (cat. No. 17-0120-01, Pharmacia, Uppsala, Sweden) were obtained with mannose and mannanam as described for granules Fractogel®TSK. The eluate from the three matrices were analyzed using Western blotting as described in example a, by downloading 7 ng of recombinant mannan-binding lectin purified granules mannose-TSK, granules mannan-TSK, granules mannose-sepharose and granules mannan-sepharose, on separate tracks of the gel SDS-PAGE. For comparison on one of the tracks was loaded ultrareactionary culture supernatant, corresponding to 7 ng of recombinant mannan-binding lectin. The results of the Western blot shown in figure 4.

EXAMPLE 4

Analysis of mannan-binding lectin under conditions that do not result in denaturation, with what omashu gel chromatography

On the chromatographic column Superose-6 (Superose-6™ HR 10/30, cat. No. 17-0537-01, Pharmacia) was performed penetrating chromatography purified recombinant mannan-binding lectin or culture medium containing recombinant mannan-binding lectin, are in equilibrium in solution edtc concentration of 5 mm, and the solution of TBS-Tween 20™ (Tris-buffered salt solution containing 0,01% (vol.) Tween 20™) (pH 7.4). Samples of 100 µl were passed through the column with a flow rate of 0.5 ml/min fractions of 5 ml were collected in a volume of 250 ml.

EXAMPLE 5

Analysis of mannan-binding lectin in the conditions that lead to denaturation, using the method of SDS-PAGE with silver staining

SDS-PAGE-gels with a gradient from 4 to 20% were prepared and used in the electrophoresis, as described by the authors Jensen and others, 1997 Procedure for silver staining was borrowed from authors Nesterenko and others, (1994). The PAGE gels were fixed in 100 ml of 50% (vol.) the acetone solution, and 6.25 ml of trichloroacetic acid, 0,85% (vol.) formaldehyde for 15 min, followed by a triple rinse in water and another rinsing in water for 15 minutes the Gel was aged in 100 ml of 50% (vol.) solution of acetone for 15 min, then was aged for 1 h in a solution of Na2S2About3the concentration of 0.67 mm and were washed three times in water. Soaking the gel nitrate sulfur is RA was performed in a solution of AgNO 3concentration of 15 mm, containing 0.9% (vol.) formaldehyde, followed by a triple rinse in water. The gel was obtained by soaking in a solution of Na2CO3the concentration of 0.19 mm, including a 0.9% (vol.) formaldehyde and Na2S2CO3the concentration of 0.67 mm, within about 3 minutes the deposition Process was stopped by aging the gel for 3 min in 1% (vol.) acetic acid solution, suspension in 25% (vol.) ethanol, 3% (vol.) glycerol.

EXAMPLE 6

Analysis of recombinant mannan-binding lectin using the method of SDS-PAGE with subsequent Western blotting

In accordance with the description given by the authors Sambrook and others, were prepared with Tris-buffered saline (TBS) and phosphate buffered saline solution (PBS) with pH brought to 7.4. To block nonspecific protein interactions in buffered solutions were administered a solution containing 0.05% (vol.) Tween 20™. SDS-PAGE-gels with a gradient from 4 to 20% were prepared and used in the electrophoresis, as described by the authors Jensen and others, 1997 Recombinant mannan-binding pectin mass of 50 ng received in accordance with real invention in the cells of SOME 293EBNA, recombinant mannan-binding lectin mass of 50 ng received in accordance with the method described by the authors of Super and others, 1992, and mannan-binding lectin m is Soi 50 ng, derived from human plasma (MO, (State serum Institute, Copenhagen. Denmark), were loaded in the gel in the environment, not having reducing ability. Using semi-dry blotting for 60·h in solution-based preparation of Tris concentration of 50 mm, containing 40 mm glycine, and 3.7% (mass fraction to volume) sodium dodecyl sulfate, 20% (vol.) ethanol was performed transfection of proteins on the membrane Amersham's Hybond™ P. Nonspecific protein interactions were blocked by exposure of the membrane for 1.5 h in 0.1% solution of Tween 20™. For the detection of mannan-binding lectin membrane was maintained at room temperature in a solution containing a monoclonal antibody, raised against the mannan-binding lectin person (hybrid 131-1, (State serum Institute, Copenhagen. Denmark), diluted to a concentration of 1 μg/ml in Tris-buffered salt solution containing 0.05% Tween 20™, 1 mm add, 1 mg HSA/mg (cat. No. 440511, (State serum Institute, Copenhagen, Denmark), 1 mg of human immunoglobulin in 1 ml (cat. No. 007740, Centeon Pharma GmbH, Marburg, Germany). After three washes in Tris-buffered saline solution containing Tween 20™, the membrane was kept for 2 h at room temperature with horseradish peroxidase conjugated rabbit artemisinin immunoglob is Lin G (cat. No. R, Dako A/S, Copenhagen, Denmark), diluted in the ratio 1:2500 in Tris-buffered saline solution containing Tween 20™, 1 mm etc and 100 mg of human immunoglobulin in 1 ml solution. Finally, the membrane was washed 5 times in Tris-buffered saline solution containing Tween 20™ and developed using chemiluminescent substrate firm Pierce's (SuperSignal™, cat. No. 34080, Pierce, Rockford, stillings, USA) on the photographic film Kodak.

EXAMPLE 7

Analysis of recombinant mannan-binding lectin using gel chromatography

On the chromatographic column Superose-6 (Superose-6™ HR 10/30, cat. No. 17-0537-01, Pharmacia) was performed helpanimals chromatography purified recombinant mannan-binding lectin or culture medium containing recombinant mannan-binding lectin, are in equilibrium in solution edtc concentration of 5 mm, and the solution of TBS-Tween 20™ (Tris-buffered salt solution containing 0,01% (vol.) Tween 20™) (pH 7.4). Samples with a volume of 100 μl (corresponding to 100 ng of recombinant mannan-binding lectin or natural mannan-binding lectin) was passed through the column with a flow rate of 0.5 ml/min fractions of 5 ml were collected in a volume of 250 ml. Fractions were diluted in the ratio 1:2 in Tris-buffered saline solution containing Tween 20™. 10 mm etc, analisia is ALIS method TRIFMA (time resolved immunofluorescent assay - immunofluorescent analysis with a time resolution), as described in example 8.

EXAMPLE 8

Analysis of recombinant mannan-binding lectin using the method TRIFMA (time resolved immunofluorescent assay - immunofluorescence analysis with a time resolution)

Microturbulence plate Fluorosorp™ (cat. No. 437958, Nunc, Kamstrup, Denmark) was coated with 100 μl (per well) monoclonal antibodies 131-1 against mannan-binding lectin (State serum Institute. Copenhagen, Denmark) at a concentration of 5 μg per 1 ml of phosphate buffered saline (PBS) and were washed three times in Tris-buffered saline solution containing Tween 20™. Standard serum with a known mannan-binding lectin concentration of 3.6 μg per 1 ml of solution was diluted in the ratio 1:20 in 20 mm etc, Tris-buffered saline solution containing Tween 20™ and when further diluted in the ratio 1:5 was used in duplicate wells containing 100 μl of a solution. There were three control sera diluted in a ratio of 1:20 in Tris-buffered saline solution containing Tween 20™, add: one with a high concentration of mannan-binding lectin (1.2 µg per 1 ml of solution), one with an average concentration of mannan-svyazivalsa on lectin (250 ng per 1 ml of solution) and one with low concentrations of mannan-svyazivayuschego lectin (50 ng h is 1 ml). Serum deficiency of mannan-binding lectin was diluted in the ratio of 1:20 and added to the plate as a control sample with a negative result.

Supernatant culture, or containing FBS. or not containing serum, as usual, it was diluted in the ratio of 1:20 and 1:100. After exposure, the plates were washed three times in Tris-buffered saline solution containing Tween 20™and each hole was covered with 100 μl of monoclonal antibodies 131-1 against mannan-binding lectin labeled with isotope Eu3+at a concentration of 125 μg per 1 ml of Tris-sabourenkov saline solution containing Tween 20™, 25 µg etc. Labeled antibody was obtained in accordance with the Protocol of the company Wallac (Wallac, Turku, Finland). Development and measurement of the bound europium was carried out in accordance with the standard procedure for the method TRIFMA (Hemmila and others, 1993).

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Sheriff, S., Cnang, C.Y. & R.A.B. Ezekowitz (1994) Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple a-helical coiled-coil. Structural biol. 1:789-794.

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Captions to figures

Fig 1, Fig. 2, 3

1 - concentration of mannan-connect the surrounding lectin, mg/ml

2 - elution volume, ml

3 - cells Sp2/0Ag14

4 - SOME 293EBNA

5 - natural mannan-binding lectin

Figure 4.

1 - granules mannose-TSK

2 - granules mannan-TSK

3 - sepharose 4B

4 - granules mannose-sepharose

5 - granules mannan-sepharose

6 - supernatant nefrackzionirovannam culture of cells transfected structure (mannan-binding lectin)/pREP9

Figure 5.

1 - cells Sp2/0Ag14

2 - SOME 293EBNA

3 - natural mannan-binding lectin

1. A method of obtaining a composition of recombinant mannan-binding lectin person with such a profile of the size distribution of molecules in which the average molecular weight of at least 50% of oligomers larger than 200 kDa, which includes the following steps:

creating constructs for expression of the gene encoding the peptide mannan-binding lectin person or its functional equivalent;

the transformation of the culture of the host cell with the help of this design;

the cultivation of the culture of the host cell so that is the expression and secretion of the polypeptide into the culture medium;

affinity chromatography specified culture medium using a matrix, the derivative of a hydrocarbon, having essentially affinity with what jedinica mannan-binding lectin person and/or dimers of subunits, where the specified matrix obtained from a hydrocarbon, has essentially affinity only with tetramine, pentamerone and/or hexamine subunits of recombinant mannan-binding lectin person, or matrix, free from hydrocarbon;

receipt of the eluate containing the composition of recombinant mannan-binding lectin person, where the average molecular weight of at least 50% of oligomers larger than 200 kDa.

2. The method according to claim 1, characterized in that the design for gene expression includes at least one intron sequence from the gene in the mannan-binding lectin person or its functional equivalent.

3. The method according to claim 1, characterized in that the design for gene expression includes at least two exon sequences of the gene mannan-binding lectin person or its functional equivalent.

4. The method according to claim 1, characterized in that the construction for the expression of a gene contains the sequence complementary to the sequence of DNA that encodes a subunit of the mannan-binding lectin or its functional equivalent.

5. The method according to claim 1, characterized in that the culture of the host cell grown in vitro.

6. The method according to claim 1, characterized in that the culture of the host cell is eukaryotic.

7. The method according to claim 1, characterized in that the culture of the host cell is a cell of a mammal.

8. The method of selection of the composition of the oligomers mannan-binding lectin person with such a profile of the size distribution of molecules in which the average molecular weight of at least 50% of oligomers larger than 200 kDa, including:

obtaining the composition of the oligomers mannan-binding lectin person;

affinity chromatography of the above-mentioned composition using the matrix obtained from a hydrocarbon, having essentially affinity with subunits mannan-binding lectin person and/or dimers of subunits where the specified matrix obtained from a hydrocarbon, has essentially affinity only with tetramine, pentamerone and/or hexamine subunits of recombinant mannan-binding lectin person, or matrix, free from hydrocarbon;

receipt of the eluate containing composition selected recombinant mannan-binding lectin person.

9. The method according to any of the preceding paragraphs, characterized in that the matrix as such is free from hydrocarbons.

10. The method according to claim 9, characterized in that the matrix consists of non-hydrocarbon polymer material.

11. The method according to claim 10, characterized in that the matrix consists of plastic granules.

12. The method according to any of the preceding paragraphs, characterized in that the separation of the polypeptide composition is performed on the matrix, the scientists from hexose.

13. The method according to any of the preceding paragraphs, characterized in that the separation of the polypeptide composition is performed on a matrix derived from mannose.

14. The method according to any of the preceding paragraphs, characterized in that the matrix obtained from a hydrocarbon, is not essentially affinity with the dimer subunits mannan-binding lectin.

15. The method according to any of the preceding paragraphs, characterized in that the matrix obtained from a hydrocarbon, has essentially affinity only with tetramine, pentamerone and/or hexamine subunits of recombinant mannan-binding lectin.

16. The method according to any of the preceding paragraphs, characterized in that the matrix obtained from a hydrocarbon, has essentially affinity only with tetramine subunits of recombinant mannan-binding lectin.

17. The method according to any of the preceding paragraphs, characterized in that the matrix obtained from a hydrocarbon, has essentially affinity only with pentamerone subunits of recombinant mannan-binding lectin.

18. The method according to any of the preceding paragraphs, characterized in that the matrix obtained from a hydrocarbon, has essentially affinity only with hexamine subunits of recombinant mannan-binding lectin.

19. The method according to any of the preceding paragraphs, characterized in that the average molecular weight, the least 70% of oligomers larger than 200 kDa.

20. The method according to any of the preceding paragraphs, characterized in that the average molecular weight of at least 80% of oligomers larger than 200 kDa.

21. The method according to any of the preceding paragraphs, characterized in that the average molecular weight of at least 90% of oligomers larger than 200 kDa.

22. The method according to any of the preceding paragraphs, characterized in that the average molecular weight at least 99% of oligomers larger than 200 kDa.

23. A method of obtaining a composition of recombinant mannan-binding lectin person with such a profile of the size distribution of molecules in which the average molecular weight of at least 50% of oligomers larger than 200 kDa, which includes the following steps:

creating constructs for expression of the gene encoding the polypeptide mannan-binding lectin person, which includes:

at least one intron sequence of a gene mannan-binding lectin person or its functional equivalent;

at least one exon of the gene sequence mannan-binding lectin person or its functional equivalent;

the area of a promoter other than the promoter mannan-binding lectin person; and

the expression vector;

the transformation of the culture of the host cell using this is the second design;

the cultivation of the culture of the host cell, so that is the expression and secretion of the polypeptide into the culture medium, followed by;

obtaining a culture medium containing recombinant mannan-binding lectin person;

purification of recombinant mannan-binding lectin man from the specified culture medium.

24. Construction for the expression of the gene encoding the polypeptide of recombinant mannan-binding lectin person, which is a component of the mannan-binding lectin that has this profile size distribution of molecules in which the average molecular weight of at least 50% of oligomers larger than 200 kDa, which includes:

at least one intron sequence of a gene mannan-binding lectin person or its functional equivalent.

at least one exon of the gene sequence mannan-binding lectin person or its functional equivalent;

the area of a promoter other than the promoter mannan-binding lectin person;

the expression vector.

25. Design for gene expression in paragraph 24, wherein the promoter region selected from the genes of eukaryotes, such as mammals and insects.

26. The design for expressiion in paragraph 24, characterized in that region of the promoter chosen from viral genes.

27. Design for gene expression in p, characterized in that region of the promoter chosen from the group comprising the promoter of the long terminal terminal repeat of Rous sarcoma virus, pretani the cytomegalovirus promoter and alpha promoter, elongation factor 1.

28. Design for gene expression in paragraph 24, wherein the promoter region selected from the genes of microorganisms, such as viruses, yeast or bacteria.

29. Design for gene expression in paragraph 24, characterized in that the promoter region contains enhancer elements.

30. Design for gene expression in paragraph 24, characterized in that as a vector using the vector pREP9.

31. Method of production of recombinant mannan-binding lectin person, which is a component of the mannan-binding lectin that has this profile size distribution of molecules in which the average molecular weight of at least 50% of oligomers larger than 200 kDa, which includes the following steps:

creating designs for gene expression in one of p-30 that encodes a polypeptide mannan-binding lectin person or its functional equivalent;

the transformation of the culture of the host cell with the help of this design;

cultivation to the cultural host cell so what happens expression and secretion of the polypeptide into the culture medium, followed by:

obtaining a culture medium containing recombinant mannan-binding lectin person.

32. Pharmaceutical composition for treating conditions associated with deficiency of mannan-binding of pectin containing pharmaceutically acceptable substance carrier and the composition of recombinant mannan-binding lectin person, at least 50% of oligomers which have a molecular weight of more than 200 kDa and where this composition is essentially free of impurities naturally associated with mannan-binding pectin person received in their host organism.

33. The pharmaceutical composition according p, characterized in that at least 70% of the oligomers have a molecular weight of more than 200 kDa.

34. The pharmaceutical composition according p, characterized in that at least 80% of the oligomers have a molecular weight of more than 200 kDa.

35. The pharmaceutical composition according p, characterized in that the ratio of tetramers, pentameron and/or hexamers to the dimer subunits mannan-binding lectin person is at least 2:1, mostly at least 5:1.

36. The pharmaceutical composition according to any one of p-35, characterized in that the distribution of RA is a measure of the molecules is estimated using the method of SDS-PAGE and/or Western blotting.

37. The pharmaceutical composition according to any one of p-36, located in a non-denatured state.

38. The pharmaceutical composition according to any one of p-36, located in the denatured state.

39. The pharmaceutical composition according p, wherein the subunit of the mannan-binding lectin consists of three peptide sequences, which include the sequence of SEQ ID NO:1 or its functional equivalent.

40. The composition according to p in a form suitable for injection.

41. The composition according to p in the form, characterized in that the carrier substance is a salt solution, albumin human serum or mannose.

42. The composition according to p in a form suitable for insertion through the lungs.

43. The composition according to p in the form of a powder for inhalation.

44. The composition according to p in the form of a cream or lotion for topical application.

45. Composition according to any one of p-40 for intravenous, intramuscular, subcutaneous or intradermal injection.

46. Design for gene expression, characterized in PP-30 to obtain a composition of recombinant mannan-binding lectin person.

47. The composition of recombinant mannan-binding lectin of the person obtained by the method according to any one of claims 1 to 7, 23 or 31 to prevent or treat conditions associated with a deficiency of mannan-binding lectin.

Priority items:

14.05.1999 according to claims 1, 7-8, 23-25, 27, 31-32, 42, 45-46;

10.05.2000 according to claim 2 to 6, 9-22, 26, 28-30, 33-41, 43-44;

20.10.1999 on p.



 

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