The manner of expression


(57) Abstract:

The invention relates to biotechnology and genetic engineering. The method allows for the expression of antigens that are integrated with the membrane aminopeptidase of worms. Spend the transfection of the host mammalian cell vector. The vector is adapted for expression aminopeptidases antigen 110D or its fragment. Before transfection of the host cell release from endogenous enzymes having the same function and the same integration with the cellular membrane or its absence, as aminopeptidases antigen 110D of the parasite or its fragment. The host cell is a OS-1 or Cho cells. Aminopeptidases antigen 110D or its fragment expressed integrated with the cellular membrane of the host cell and/or in the form of a soluble cytoplasmic enzyme. 4 C.p. f-crystals, 2 ill., table 4.

The invention relates to the production of specific protective antigens using recombinant DNA technology.

Parasites cause a wide range of diseases, both in humans and in domestic animals. Up to the present time for the treatment of these diseases was used chemotherapeutic methods, but is the most stages of their life cycle are relatively large biological objects impacted by the cellular immune defense system of an animal host, they can show sensitivity to antibodies, which act in such a way that cause inactivation of the vital functions of the parasite. In particular, it was shown the possibility to immunize the body of an animal host antigens associated on the surface of the gut parasite, which is generated when antibodies bind to antigens on such internal membrane, when body fluids containing antibodies ingested parasites. Such antigens can be called "hidden antigens, because they do not increase the natural immunity against parasites.

We have found that particularly effective has "hidden antigen, which is helminthology antigen H110D isolated from Halmonchus contortus, which was described in WO 088/00835 and 90/11086. In the work WO 93/23542 we have described our further results related to the fact that H110D represents the membrane-bound aminopeptidase from the gut helminth. It is believed that this enzyme necessary for the conversion of protein nutrients into amino acids for absorption in the intestine, while its inactivation under the action of antibodies to H110D cause the death of the parasite is t sensitivity to applied to them the same strategy on the basis of "hidden antigen", in the sense that the enzymes associated to the membrane of the intestine, are necessary for the absorption and subsequent metabolism of protein components. Such enzymes include aspartate (as described in PCT/GB 93 01521) and terproteksi, in particular cathepsin. These enzymes are localized in the intestine of a large number of parasites, such as helminths, including various types of collections Halmonchus, Ostertagia, Trichostroogylus, Nematodirus, Dictyocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris, Strongylus and Fasciola; and species of arthropods, especially representatives of the class Arachnida and insects, and in particular ectoparasites, such as blood-sucking insects (including representatives of the two groups of insects with incomplete and complete metamorphosis), and dipterans, such as gorzalka (Lucilia), mesnie flies (myiais jlies)kanaki, lice, ticks, fleas, sheep runty and bugs.

In the above work WO 93/23542 also describes the production of fragments of antigen H110D using recombinant DNA technology. In this work, the material H110D was obtained by expression in E. coli using the pGEX vector, injectable in sheep, which led to increased levels of antibodies to H110D. In future work, we used baculoviruses in insect cells (Sf-9 cells), while UDA is, 1 (seg. 1D NO:1). The appropriate product broadcast shown in Fig. 2 (seg. 1D NO:2).

However, for the production of vaccines for use in animals, preferably the expression of antigen in mammalian cells. This system has the best performance reproduction native form and protective epitopes of the antigen, since the expression system of eukaryotes allows much closer to the process of glycosylation, formation of disulfide bonds and other post-translational modifications than is the case in E. coli, which is formed as a result of expression of insoluble protein requiring folded restructuring, with this system of E. coli is characterized by a low level of reproduction native form.

In addition, glycosylation in the system of mammals, apparently, does not induce an immune response, distracting response function antiproteinase, and this situation takes place in the material obtained by the use of cell lines insects, which is explained by the different nature of glycosylation. To protect humans and domestic animals is preferable to use a cell line vibrola baby hamsters; VERO and COS - cell line of monkey kidney; FR3T3 - rat fibroblasts Fisher; NIH3T3 cell line of fibroblasts of mice; C1271 - cell line tumors in the mammary gland of mice; CV - 1 fibroblasts kidney African green monkey; 3T6 - embryonic fibroblasts of mice; L - cells of the cell line of mice; CHO - cell line Chinese hamster ovary; NSI NSO, SP2 and other myeloma cell lines, mice and cell lines myeloma rats, such as YB 2/0 and Y3.

As produced hidden antigens vital for the process of absorption and assimilation of parasite nutrients, enzymes and other functional proteins that have the same activity, are common to a large number available for the study of eukaryotic cell lines and they not only interfere with breeding bugs, producing the desired antigen, but also create additional difficulties in the selection of the desired antigen from the cell products and further purified. In addition, the selected cell line owner must be genetically similar from the point of view of protein sequence to that animal, which serves as the object of protection, so the contamination of the desired alien hidden antigen is therefore endogenous to the Antiga is Auda control studies quality expressed "hidden antigen".

The invention is based on the concept that recombinant DNA carries the expression of the desired alien enzymatically active "hidden antigen in transformed cell lines of the host mammal, which in those cases when both enzyme or associated with the cellular membrane, or both localized in the cytoplasm, which is equally hinder their physico-chemical separation, essentially free from endogenous antigens having the same enzymatic functions as alien "hidden antigen".

In accordance with the present invention, we provide a way of expression of the enzyme-antigen, which in its natural state is an enzyme associated with the membrane of the gut of the parasite or its fragment having similar enzymatic and/or antigenic activity in the host cell is introduced vector adapted for expression of the above-mentioned enzyme-antigen or its fragment, which is characteristic for this case is the fact that before the transformation of the host cell does not inherently contain endogenous enzymes that perform (s) the same function and is characterized by (b) the presence of the same integration with the cellular membrane or the absence of such online is Yaya cell contains cytoplasm endogenous enzyme, such as aminopeptidase, and because the foreign enzyme is expressed from the TRANS-membrane sequence and is localized on the membrane of the host cell, it is not difficult to conduct separation of endogenous and foreign enzymes through a process leading to the separation membrane fragments, i.e., by centrifugation. On the other hand, a foreign antigen can be modified with the aim of producing a fragment lacking the site necessary for binding to the membrane, if the membrane of the host cell carries endogenous enzyme having the same activity as alien fragment, it is also possible to influence the separation by removing material of the cell membrane; therefore, the coding sequence for the transmembrane region of the fragment of the parasite can be substituted in expressed gene signal sequence, which affects the secretion.

Of particular interest present antigens amino peptidases helminths, such as the above H110D and its fragments. Such antigens and fragments thereof can be expressed in a wide range of mammalian cells the BL is but aminopeptidase activity of a-type or M-type and capable for this reason, split methioninol and lacinova peptide bond, we have demonstrated the possibility of using COS-I cells in accordance with the present invention because they lack a significant aminopeptidase activity of a-type and M-type. It seems that they have aminopeptidase activity that breaks down alanine peptide bond, which in a weak degree associated with the cell membrane, so that in this case there are no problems with the separation of endogenous enzyme and downregulation of the enzyme H110D, which is localized on the cell membrane.

It has been shown that proteolytic enzymes such as trypsin, split on the membrane of parasite antigen H110D with the production of soluble H110D (H11S). Such enzymes can be used for selective cleavage alien "hidden antigen" and separate it thus from endogenous enzyme with similar activity. Suitable cell lines for use in accordance with the present invention can either be selected from existing strains by screening on the basis of the outer enzymatic activity and/or on the basis of the localization of any relevant enzyme or on the cell membrane or in the cytoplasm. In the latter case, communication with the cellular membrane , the which is not to extract integral membrane enzymes, and then, for example, Triton, which releases these enzymes.

It is also possible to create a cell line mammals with low content of interest to the enzyme. One such method may include the following procedures: increase the level of antibodies to the mammalian enzyme, which must be removed. Then cell line is subjected to modification by irradiation or chemicals for induction of point mutations. Cells are cultivated in an environment that appropriately selected so as to compensate for the loss of enzymatic activity. After that, cells treated with fluorescent labeled antibodies and passed through the laser analyzer (sorter) cells according to the intensity of fluorescence (FACS). Cells with no fluorescence, clone, of them are re-selected cells, devoid of the above-mentioned enzyme, while continuously monitoring the stability of such loss.

Cells can also be modified by deletions in the gene or rational (directed) technology making mutations for deletion or mutation engine of the gene responsible for the synthesis releva the ranks classes of cell lines mammals. In General, they should include a promoter and/or an amplifier, operatively associated with the gene responsible for the expression of an enzyme-antigen or its fragment. Thus, in particular, the fragment of the gene for H110D 3.5 kb can be linked in reading frame with an appropriate promoter. Suitable promoters include the early or late promoter, SV-40, i.e., PSVL vector, the promoter of cytomegalovirus (CMV) promoter metallothionein I mice and terminal sequence of the virus tumors in the mammary gland of mice. Preferably, the vector included suitable marker such as a gene encoding dihydrotetrazolo or the gene encoding glutamylcysteine. The vectors of these types are described in WO 86/05807, WO 87/04462, WO 89/01036 and WO 89/10404.

The level of transfection host cell can be enhanced through standard technologies using, for example, calcium phosphate, DEAE-dextran, polybrene, the method of fusion of protoplasts, liposomes, targeted microinjection, mail bombing of a gene or electroporation. The latter method is preferable, and the ways transfection of cell lines, ispolzuemykh electroporation in the literature (Andreason, G. L., and Evans, G. A., Introduction and Expression of DNA molecules in encaryotic cells by electroporation, Biotechnigues, 6, 65, 1980). In common with the s (M-type) and methioninamide activity (a-type) and, in General, preferably, to the host cell does not contain at least these two types aminopeptidase activity. The following examples are given only for illustrative purpose. In these examples, presented the following:

in Fig. 1 and Fig.1.1 shows the DNA sequence of size 3.5 kb for polymerase chain reaction (CPD), clone 2 (sequence ID NO:1);

in Fig. 2 shows the amino acid composition of the product broadcast clone 2 3.5 kb for polymerase chain reaction (sequence ID NO:2).

Example 1. Determination of enzyme activity in COS-I cells.

COS-I cells were obtained from the University Suree (Surrey University) in 5 ml of growth medium CUMD (DMEM). Cells were divided into two 200-ml flasks for cultivation, each contained 11 ml of medium in which the cells grew up to clumping. COS cells stick together, so that the surface of the bulb can be removed by suction using a glass Pasteur pipette with 10 ml PBS buffer. When the suspension will contain all cells, it is transferred into a conventional tube and frozen at -20oC. the Frozen cells are thawed and frozen several times in liquid nitrogen for cell disruption, then out to separate PBS supeene TwLS and TrLS of supernatant. All supernatant concentrated to a volume of 200 μl using Millerovskij of microconcentrators. Extracts only PBS contain enzymes that were free in the cytoplasm extracts on the basis of Twin contain enzymes that were weakly associated with the cellular membrane, and extracts, based on the Triton contain proteins that are integrated with the membrane.

Supernatant investigated against 25 mm: the Palestinian national authority (pNA) (p-nitroaniline) substrates, represented by phenylalanine, gamma-glutamic acid, leucine, lysine, methionine, alanine, alpha-glutamic acid, Gli-Pro and aspartic acid, bicarbonate HEPES buffer at pH 7.0. After 30 minutes incubation at 37oC found no apparent enzymatic activity, to evaluate which the incubation period was increased to 18 hours. Were counted value of the specific activity, the results are presented in table 1.

The highest activity was obtained in the extract based on the Twin, the highest activity was shown to alanine pHA substrate, some activity was also observed relative to phenylalanine, based on gamma-glutamine acid, latinoware, lysine, matio is-GTP and residual activity of the lysine AP and alanine AP.

10 µl of each supernatant of COS-I cells added to 25 mm p-nitroanilide substrate in 250 μl of bicarbonate HEPES buffer at pH 7.0 and incubated at 37oC for 18 hours.

Determination of enzyme activity in cells Chinese hamster ovary (CHO) and NSO cells.

Extracts of cultured CHO and NSO cells is prepared in a manner analogous to what was described above in the section on COS-I cells. These extracts were analyzed against the following paranitroaniline substrates: alanine, arginine, glycine, alpha-glutamic acid, gamma-glutamic acid, leucine, lysine, methionine, phenylalanine, Proline and Gli-Pro - all the substrates are taken in quantities of 25 mm bicarbonate HEPES buffer. Incubation is conducted at 37oC for 30 minutes, after which recorded the final values of SNPS on the basis of which the calculated specific activity of the enzyme.

From table. 2 shows that extracts of soluble and membrane-bound components CHO cells have a low level of enzymatic activity, except in the case of determination of activity in TwLS against Gli-Pro substrate. And TrLS extract shows low activity in all cases, except Argi is Asano, as PLS and TwLS extracts NSO cells have aminopeptidase activity, it is particularly high against leucine and methionine (table. 3). In contrast, TrLS extract has low activity, suggesting that the cleanup process, including extraction of Triton, which was used in the case of H110D, leads to the achievement of a very low level of contamination of this extract endogenous enzymatic activity

Comparative examples.

EXAMINE the cells using the same method as COS-I cells as described in example 1, however, in larger quantities (2 x 1 l round bottles with their cells in 100 ml medium). Supernatant researched against: phenylalanine, latinoware, on the basis of gamma-glutamic acid, alanine, arginine, based on aspartic acid, lysine, methioninol, Gli-Pro and on the basis of alpha-glutamic acid PNA substances with pH 7.0. Testing was conducted under conditions of incubation at 37oC for 30 minutes with the registration of the final values of OD.

Extracts KSS cells contain significant enzymatic activity, which is found in all supernatant (PL. 4). Little activity is observed only did supernatant was marked by high activity for lysine PNA substrate (which was the largest in PLS supernatant), Latino, alanine and Gli-Pro. Activity relative to methioninamide PNA substrate, which was insignificant in PLS the supernatant was minimal in TrLS the supernatant. It is seen that such KSS cells are not suitable for expresii H110D, because a high level aminopeptidase activity of a-type and M-type was found both in the cytoplasm and associated with the membrane-integrated form.

To 10 µl of each supernatant KSS cells add 25 mm p-nitroanilide substrate in 250 μl of bicarbonate HEPES buffer at pH 7.0 and incubated the mixture at pH 7.0 for 30 minutes at 37oC.

Example 2. Cloning HIIOD sequence of the expression vector of mammals.

DNA, which is subjected to cloning, which is a clone of the 2 PCR (polymerase chain reaction) gene H11OD size of 3.5 kb was described in WO 93/23542. The DNA sequence (Sequence ID NO: 1) of such insert) received during the polymerase chain reaction (PCR), shown in Fig. 1, and the amino acid sequence of the product of translation (Sequence ID NO: 2) of Fig. 2. This DNA was cut from the vector pT7Blue-T vector (Novagene) by splitting BamH1 and cloned at the BamH1 site multiple Cloner using BamH1 clone pSPT18-3,5-2, and the resulting linear DNA was twice subjected to purification using gel electrophoresis. "Sticky" ends were blunted" by introducing dNTP using enzyme maple (large fragment of DNA polymerase), and using Ncol linker containing the ATG (Boehringer Mannheim Cat N 1171160), blunt ends were connected with obtaining the plasmid. The clones were subjected to screening using restriction analysis on the basis of the presence of the linker on the 5' end of the insert size of 2.5 kb, which gave vnutriramochnym site initiation ATG under the control of T7 promoter. Modified insert size of 3.5 kb from one such clone (clone pST18-3,5-2 N 44) was cut out and then subcloned in the expression vector mammalian pRC/CMV using the following strategies:

1. DNA clone pSPT18 (T7-3,5-2 N 44) were cleaved with restriction enzyme Smal.

2. Notl linker connect with this modified Smal site that allows the emergence of clones with Notl site at the 5' end of the insert, which is preceded by the linker Ncol containing the website start reading the APG.

3. Purified DNA from the appropriate clone split using as Notl and Xbal to release the insert size of 3.5 kb.

To the incubation medium add the sector and paste complicate the cleaning process.

4. Box 3.5 cleaned 0.6-0.7 per cent agarose gel.

5. The expression vector mammalian pRC/CMV digested with Notl and Xbal, and the band corresponding to the linear plasmids, clean agarose gel.

6. Spend ligation of insert size 3.5 kb and translated in a linear form of the vector.

7. Select the appropriate clones, which have a size of 3.5 kb splitting using Notl and Xbal. These clones designated as pRC/CMV-3,5-2.

Transfection of COS-1 cells of mammals.

DNA expression vector mammalian inserted in it a clone of antigen HIIOD designated as pRC/CMV-3,5-2, is subjected to a high degree of purification by centrifugation in a gradient of cesium chloride. (Sambrook, J., Fritch, E. F. and Maniatis T. Molecular Cloning, A Laboratory Manual, Second edition, Cold Spring Harbar Press, 1989).

Temporal expression HIIOD can be achieved using this purified DNA clone pRC/CMV-3,5-2 for transfection of COS-1 cells (obtained from ECACC, Porton). Transfection is performed using DEAE-dextran (Cullen B. R., Use of Eucaryotic expression technology in the functional analysis of cloned genes, Metods in Enzymology: Guide to molecular cloning techniques, Eds S. L. Berger and A. K. Kimmal, Academic Press, 1987, pp 684-704). Cells cultured in the medium Needle in the modification of Dulbecco (DMEM) [Flexibly BRL (Gibco BRL)] containing 10% embryonictissue.

Transfection of CHO mammalian cells (ovary cells Chinese hamster).

Vector DNA is introduced into CHO cells (obtained from ECACC, Porton) using the calcium phosphate method (this, in particular, as the method described in the work of Cullen B. R Use of eucaryotic compression technology in the functional analysis of cloned genes. Methods in Enzymology: Guide to Molecular Cloning Techniques, Eds S. L. Berger and A. R. Kimmel, Academic Press, 187, pp. 684-704). Transformed cells are cultivated in DMEM medium containing 10% fetal calf serum (Flexible BRL). Geneticin-resistant line (G418) is a single transformed cell line, in contrast to normal cells, growing the cells are grown in culture to a concentration of 800 μg/ml Then transformed cell clone by the method of limited dilution in microtiter plateau.

Analysis of mammalian cells subjected to transfection.

Transformed and untransformed CHO cells can be transferred to growth on top of the glass with the purpose of immunofluorescence analysis. The cells allow to rise to the formation of small colonies, fix them with methanol and treated sheep anticorodal to HIIOD and then fluorescent paints is using a fluorescent microscope.

Cells in transformed cell lines destroy in RIPA buffer (150 mm sodium chloride, 1% Nonidet P [Nonidet P40], 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mm Tris-HCl, pH 8.0) when removing from the mass of cells in growth medium and then by careful grinding of the cells for 5 minutes in RIPA buffer. The cells are then transferred into a test tube for microcentrifuge and dismantle them in microcentrifuge at full speed for 15 minutes with fresh lysate, which is transferred into a clean tube. Aliquots of the lysate, containing 2105cells subjected to electrophoresis in SDS-polyacrylamide gel (SDS-PAGE) and proteins from the gel transferred to nitrocellulose membrane by Western blotting. The membrane process, possibly with the inclusion of controllable periodic destruction stages of processing, and analyze it against antisera obtained to various forms HIIOD antigen. Controllable periodic destruction processing destroys the carbohydrate epitope; carbohydrate epitopes mammals may differ significantly from native carbohydrates helminths. The fragments obtained in Vester-blotting transformed cells show the presence of protein, recognized by antisera specific to H11OD.

Extracts transformationalist for the presence of enzymatic activity in exactly the as described in example 1. Transformed cells containing HIIOD, demonstrate a higher level aminopeptidase activity than untransformed cells.

The presence of introduced vector DNA that contains a clone of the 2 PCR 3.5 kb, resistant to geneticin cell lines CHO, determined by southern blotting in the DNA preparations obtained from these cell lines. DNA extracted from cells using known methods (Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989). 10-20 μg of DNA digested with restriction endonucleases and then carry out electrophoretic separation in agarose gel, transferring the DNA to the membrane using southern blotting (Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoreses, J. Mol. Biol., 1975, 98, p. 503). This membrane hybridized with sample coding clone 2 PCR 3.5 kb, which after thorough washing is subjected to autoradiography. In transformed cells find bands that are specific for clone 2 PCR size of 3.5 kb.

Expression of clone 2 PCR 3.5 kb RNA level in transformed mammalian cells is determined using Northern blot analysis of RNA isolated from these cells. RNA is extracted and is of Italia, mentioned RNA in an amount up to 20 µg dispersed in agarose gel, then transferred onto the membrane using Northern blotting (Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989). This membrane then hybridizing with sample coding clone 2 PCR 3.5 kb, which after washing explore method autoradiography. Transformed cells are marked specific hybridization bands corresponding to clone 2 PCR size of 3.5 kb.

1. The manner of expression aminopeptidases antigen NOD or its fragment having similar ensimaticheskoi and/or antigenic activity, including transfection of the host mammalian cell vector adapted for expression mentioned aminopeptidases antigen NOD or its fragment, characterized in that prior to transformation of the host cell, essentially free from endogenous enzymes with

(a) the same function and

(b) the same integration with the cellular membrane or its absence, as aminopeptidases antigen NOD of the parasite or its fragment.

2. The method according to p. 1, characterized in that in the above-mentioned host cells no significant activity the host cell is a CS-1 or Cho cells.

4. The method according to any one of paragraphs.1 to 3, characterized in that the said aminopeptidase antigen NOD or its fragment expressed integrated with the cellular membrane of the host cell.

5. The method according to any one of paragraphs.1 to 3, characterized in that the said aminopeptidase antigen NOD or fragment is expressed in the form of a soluble cytoplasmic enzyme.


Same patents:

The invention relates to biotechnology preparative and can be used to obtain a purified preparation of collagenase (collaz) of animal origin for use in cellular biotechnology, cosmetics and medicine
The invention relates to biotechnology and is designed for production of protein hydrolysates

The invention relates to biotechnology, and in particular to methods of obtaining protein hydrolysates from meat, meat and bone, bone raw slaughter of animals, and can be used in food and medical industries

The invention relates to biotechnology, namely, preparative biochemistry, and can be used in cell biology, medical practice for the treatment of burns, osteomyelitis, bedsores and scar removal collagen for cosmetic purposes, cosmetic and perfume industry in the manufacture of creams and ointments for rejuvenation of the skin, in the food industry for processing of meat to improve its quality and nutritional properties, leather and fur production to obtain an elastic, thin and durable materials, as well as in scientific research to study the microstructure of collagen, when working with cellular structures, as well as various tissues and organs for individual intact cells
The invention relates to biotechnology, food, medical and pharmaceutical industries, relates to a method of receiving a drug that has collagenolytic activity from hepatopancreas crab Paralihodes camtschatica (complex trypsin and chymotrypsin-like proteases) and can be used in the fur and leather industry, veterinary science and medicine

The invention relates to biotechnology, and in particular to methods of obtaining cultures of cells from organs and tissues of human and animal
The invention relates to the meat industry and is intended for the manufacture of concetrated about the purpose of their further use in sausage, dumplings and meat-packing production
The invention relates to the meat industry and is intended for the manufacture of concentrates to further their use in sausage, dumplings and meat-packing production
Fibrin-isopeptides // 2049819
The invention relates to a new native enzyme isolated from the medicinal leech Hirudomedicinalis, fibrin-isopeptidases

The invention relates to methods of producing rennet from Sychugov calves and lambs dairy and can be used in the production of milk-clotting drugs for cheese making in the dairy industry, and in medicine

The invention relates to biotechnology and is designed to produce human erythropoietin

The invention relates to biotechnology and molecular biology, namely, genetic and cell engineering, of interest to obtain recombinant erythropoietin (EP) of a person, which can be used in medical and research purposes

The invention relates to genetic engineering in particular the production of mutant proteases in Bacillus cells

The invention relates to genetic engineering and medicine