RU2126045C1 - Expression method - Google Patents

Abstract

FIELD: biotechnology and genetic engineering. SUBSTANCE: host mammalian cells are transfected with vector adapted for expression of aminopeptidase antigen H 110D or its fragment. Prior to transfection, host cells are freed of endogenous enzymes possessing the same function and the same integration with cell membrane or its absence as aminopeptidase antigen H 110D or its fragment. Host cell is COS-1 or CH-0 cell. Aminopeptidase antigen H 110D or its fragment is expressed integrated with host cell membrane and/or soluble cytoplasmic enzyme. EFFECT: enabled expression of antigens integrated with membrane of helminth aminopeptidases. 5 cl, 2 dwg, 4 tbl, 2 ex

Description

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

 Parasites cause a wide range of diseases in both humans and pets. To date, chemotherapeutic methods have been used to treat these diseases, however, more recently, immunological methods have begun to be used. Despite the fact that parasites at most stages of their life cycle are relatively large biological objects that are affected by the cellular immune defense of the host animal system, they can be sensitive to antibodies that act in such a way that they inactivate the vital functions of the parasite. In particular, it has been shown that the host animal can be immunized with antigens bound to the surface of the gut of the parasite, so that the antibodies generated in this way bind to antigens on such an inner membrane when body fluids containing antibodies are swallowed by parasites. Such antigens may be called "latent antigens" because they do not increase natural immunity against parasites.

 We have found that the “hidden antigen”, which is the helminthicidal antigen H110D isolated from Halmonchus contortus, which was described in WO 088/00835 and 90/11086, is particularly effective. In WO 93/23542, we described our further results related to the fact that H110D is a membrane-bound aminopeptidase from the helminth gut. It is believed that this enzyme is necessary for the conversion of protein nutrients into amino acids for absorption in the intestines, while its inactivation by the action of antibodies to H110D causes the death of the parasite from impaired absorption of nutrients. Further, we found that a large number of parasites demonstrates sensitivity to the same strategy based on the “hidden antigen” applied to them, in the sense that enzymes bound to the intestinal membrane are necessary for the absorption and further metabolism of protein components. Such enzymes include aspartyl proteases (as described in PCT / GB 93 01521) and thiol proteases, in particular cathepsin. These enzymes are located on the gut of a large number of parasites, such as helminths, including various species of the families Halmonchus, Ostertagia, Trichostroogylus, Nematodirus, Dictyocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris, Strongylus and Fasciola; and arthropod species, especially representatives of the arachnids and insects class, and in particular ectoparasites, such as blood-sucking insects (including representatives of two groups of insects, both with incomplete and complete conversion cycles), and dipterans, such as beetles (Lucilia ), miyazy flies (myiais jlies), horseflies, lice, ticks, fleas, sheep runes and bedbugs.

 The above WO 93/23542 also describes the production of H110D antigen fragments using recombinant DNA technology. In this work, H110D material was obtained by expression in E. coli using a pGEX vector injected into sheep, which led to an increase in the level of antibodies to H110D. In further work, we used baculoviruses in insect cells (Sf 9 cells), and it was possible to express a 3.5 rb H110D gene bug. Its nucleotide sequence is shown in FIG. 1 (seg. 1D NO: 1). The corresponding translation product is shown in FIG. 2 (seg. 1D NO: 2).

 However, for the production of vaccines for use in animals, it is preferable to express the antigen in mammalian cells. Such a system is characterized by better reproduction of the native form and protective epitopes of the antigen, since the eukaryotic expression system allows the glycosylation process, the formation of disulfide bonds and other post-translational modifications to be much closer to what is needed than in E. coli, in which as a result of expression, an insoluble protein requiring folded restructuring, while this E. coli system is characterized by a low level of reproduction of the native form .

 In addition, glycosylation in the mammalian system does not seem to induce an immune response that distracts the response function of antiproteins, namely this situation occurs in the material obtained using the insect cell line, which is explained by the different nature of glycosylation. To protect humans and domestic animals, it is preferable in this regard to use human or animal fibroblast or myeloma cell lines, such as Hela, the human cell line; VNK - renal cells of young hamsters; VERO and COS - monkey kidney cell line; FR3T3 - rat Fisher fibroblasts; NIH3T3 - mouse fibroblast cell line; C1271 - mouse mammary tumor cell line; CV - 1 African green monkey kidney fibroblast; 3T6 — embryonic mouse fibroblasts; L - cells - cell line of mice; CHO - Chinese hamster ovary cell line; NSO NSI, SP2, and other mouse myeloma cell lines and rat myeloma cell lines, such as YB 2/0 and Y3.

 Since the produced hidden antigens are vital for the process of absorption and assimilation of nutrients by the parasite, enzymes and other functional proteins that have the same activity are common to a large number of eukaryotic cell lines available for study and they not only interfere with the selection of bugs producing the desired antigen, but also also create additional difficulties in the selection of the desired antigen from cellular products and further purification. In addition, the selected host cell line must be genetically close from the point of view of the protein sequence to the animal that is being protected, so that the contamination of the desired foreign latent antigen by such an endogenous antigen is likely to lead to an undesirable autoimmune reaction of the host. In addition to this, control studies of the quality of the expressed “latent antigen” are greatly complicated.

 The invention is based on the concept that recombinant DNA expresses the desired foreign enzymatically active “hidden antigen” in a transformed mammalian host cell line, which when both enzymes are either connected to the cell membrane or both are located in the cytoplasm, which interferes equally their physico-chemical separation is essentially free of endogenous antigens having the same enzymatic functions as the alien “hidden antigen”.

 In accordance with the present invention, we propose a method for expressing an antigen enzyme, which in its natural state is an enzyme associated with the intestinal membrane of the parasite, or a fragment thereof having similar enzymatic and / or antigenic activity, and a vector adapted to the expression of the aforementioned antigen enzyme or its fragment, characteristic of this case is the fact that, prior to transformation, the host cells essentially do not contain endogenous enzymes, boiling (a) the same function and characterized by (b) the presence of the same cell membrane integration or lack of integration such as that of said parasite enzyme antigen or fragment thereof.

 Thus, if the host cell contains an endogenous enzyme, such as aminopeptidase, in the cytoplasm, and since the foreign enzyme is expressed with a trans-membrane sequence and is localized on the host cell membrane, it is not difficult to separate endogenous and foreign enzymes through a process leading to the separation of membrane fragments , i.e. by centrifugation. On the other hand, a foreign antigen can be modified to produce a fragment in which there is no site necessary for binding to the membrane, and if the host cell membrane carries an endogenous enzyme having the same activity as the foreign fragment, it is also possible to act on separation by removal of cell membrane material; thus, the coding sequence for the transmembrane region of the parasite fragment can be replaced in the expressed gene with a signal sequence that affects secretion.

 Of particular interest are helminth aminopeptidase antigens, such as the above H110D and its fragments. Such antigens and fragments thereof can be expressed in a wide range of mammalian cells using appropriate vectors. For the expression of the H. contortus H110D antigen, which predominantly exhibits A-type or M-type aminopeptidase activity and is therefore capable of cleaving methionine and leucine peptide bonds, we have shown the possibility of using COS-I cells in accordance with the present invention, since they lack significant aminopeptidase activity of A-type and M-type. It seems that they have aminopeptidase activity that cleaves alanine peptide bonds, which is weakly associated with the cell membrane, so that in this case there is no problem in the separation of the endogenous enzyme and the expressed H110D enzyme, which is localized on the cell membrane.

 It has been shown that proteolytic enzymes such as trypsin are cleaved into the H110D membrane parasitic antigen to produce soluble H110D (H11S). Such enzymes can be used to selectively cleave a foreign “latent antigen” and thus separate it from an 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 for external enzymatic activity and / or for the localization of any relevant enzyme either on the cell membrane or in the cytoplasm. In the latter case, communication with the cell membrane can be established by extracting the lysed cells first with a detergent such as tween, which does not extract integral membrane enzymes, and then, for example, with a triton, which releases such enzymes.

 It is also possible to create a mammalian cell line with a low content of the enzyme of interest. One of these methods may include the following procedures: increase the level of antibodies to that mammalian enzyme that needs to be removed. Cell lines are then modified — by irradiation or chemical reagents — to induce point mutations. Cells are cultured in an appropriate medium so as to compensate for the loss of enzymatic activity. After that, the cells are treated with labeled fluorescent antibodies and passed through a laser analyzer (sorter) of cells by fluorescence intensity (FACS). Cells that do not have fluorescence are cloned, cells that lack the enzyme are re-selected from them, and the stability of such a loss is constantly monitored.

 Cells can also be modified by deletion in the gene or by a rational (directed) mutation technique to remove or mutate the gene responsible for the synthesis of the relevant endogenous enzyme.

 Vectors suitable for introducing them into various classes of mammalian cell lines are well known in the art. In general, they should include a promoter and / or amplifier operably linked to the gene responsible for the expression of the antigen enzyme or fragment thereof. Thus, in particular, a gene fragment for H110D with a size of 3.5 kb can be bound in the reading frame to the corresponding promoter. Suitable promoters include the SV-40 early or late promoter, i.e. PSVL vector, cytomegalovirus promoter (CMV), mouse metallothionein I promoter, and terminal mammary tumor virus sequence. Preferably, the vector includes a suitable marker, such as a gene encoding a dihydrofolate reductase or a gene encoding a glutamine synthetase. Vectors of these types are described in WO 86/05807, WO 87/04462, WO 89/01036 and WO 89/10404.

The level of transfection of host cells can be enhanced by standard technologies using, for example, calcium phosphate, DEAE-dextran, polybrene, protoplast fusion, liposomes, targeted microinjection, gene bombardment or electroporation. The latter method is preferred, with methods for transfecting cell lines using electroporation in the literature (Andreason GL and Evans GA, Introduction and Expression of DNA molecules in encaryotic cells by electroporation, Biotechnigues, 6, 65, 1980). In general, linear DNA is introduced into cells more easily than circular DNA. The H110D antigen exhibits leucine aminopeptidase (M-type) and methionine aminopeptidase (A-type) activity and it is generally preferred that host cells do not contain at least these two types of aminopeptidase activity. The following examples are given for illustrative purposes only. The following are presented in these examples:
in FIG. 1 and FIG. 1.1 shows a 3.5 kb DNA sequence for polymerase chain reaction (PPR), clone 2 (sequence ID NO: 1);
in FIG. 2 shows the amino acid composition of the translation product of clone 2 of size 3.5 kb for the polymerase chain reaction (sequence ID NO: 2).

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

COS-I cells were obtained from the University of Surrey (Surrey University) in 5 ml of growth medium CUMD (DMEM). The cells were divided into two 200 ml flasks for growing the culture, each containing 11 ml of medium in which the cells grew until they clumped. COS cells stick together, so that they can be removed from the surface of the flask by aspiration using a glass Pasteur pipette with 10 ml of PBS buffer. When the suspension contains all the cells, it is transferred to a standard tube and frozen at -20 ^° C. Frozen cells are thawed and frozen several times in liquid nitrogen to destroy the cells, after which they are unscrewed to separate the PBS supernatant (PLS). For subsequent extraction, 500 μl of PBS with 0.1% Tween and PBS with 2% Triton are used to obtain TwLS and TrLS supernatants, respectively. All supernatants were concentrated to a volume of 200 μl using Milliporov microconcentrators. PBS-only extracts contain enzymes that were free in the cytoplasm, Tween-based extracts contain enzymes that were weakly bound to the cell membrane, and Triton-based extracts contain proteins integrated with the membrane.

Supernatants were tested against 25 mM: pNA (pNA) (p-nitroanilide) substrates represented by phenylalanine, gamma-glutamic acid, leucine, lysine, methionine, alanine, alpha-glutamic acid, Gly-Pro and aspartic acid, in HEPES bicarbonate bicarbonate pH 7.0. After 30 minutes of incubation at 37 ^° C, no apparent enzymatic activity was detected, to evaluate which the incubation period was increased to 18 hours. The specific activity values were calculated, the results are presented in table 1.

 The highest activity was obtained in the extract based on Tween, while the highest activity was shown for alanine pHA substrate, some activity was also noted relative to phenylalanine, based on gamma-glutamic acid, leucine, lysine and methionine pHA substrates. PLS and TrLS were not very active, apparently with some level of gamma-GTP activity and residual activity of lysine AP and alanine AP.

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

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

Extracts of cultured CHO and NSO cells are prepared in a similar manner to that described above in the section on COS-I cells. These extracts were analyzed against the following paranitroanalide substrates: alanine, arginine, glycine, alpha-glutamic acid, gamma-glutamic acid, leucine, lysine, methianine, phenylalanine, proline and Gly-pro - all substrates were taken in an amount of 25 mM in HES bicarbonate. Incubation is carried out at 37 ^o C for 30 minutes, after which the final OD values are recorded, based on which the specific activity of the enzyme is calculated.

 From the table. Figure 2 shows that extracts of soluble and membrane-bound components of CHO cells have a low level of enzymatic activity, with the exception of the case of determining activity in TwLS against Gly-Pro substrate. And TrLS extract shows low activity in all cases, except for the use of arginine substrate. The latter activity differs from the aminopeptidase activity of H110D.

It was shown that both PLS and TwLS extracts of NSO cells have aminopeptidase activity; it is especially high against leucine and methionine (Table 3). In contrast, TrLS extract has a low activity, which suggests that the purification process, including triton extraction, which was used in the case of H110D, leads to a very low level of contamination of this extract with endogenous enzymatic activity
Comparative examples.

VNIK cells are obtained using the same method as the COS-I cells described in Example 1, however, in a larger quantity (2 x 1 L round bottles with adherent cells in 100 ml of medium). Supernatants were tested against: phenylalanine, leucine, based on gamma-glutamic acid, alanine, arginine, based on aspartic acid, lysine, methionine, Gli-Pro and based on alpha-glutamic acid, PNA substrates at pH 7.0. Testing was carried out under incubation conditions at 37 ^o C for 30 minutes with registration of the final OD value.

 Extracts of BHK cells contain significant enzymatic activity, which is found in all supernatants (table. 4). Insignificant activity is noted only for such substrates as gamma-glutamic acid, aspartic acid or alpha-glutamic acid. For all supernatants, high activity was observed for the lysine PNA substrate (which was the highest in the PLS supernatant), leucine, alanine, and Gly-Pro. Activity relative to the methionine PNA of the substrate, which was negligible in the PLS supernatant, was minimal in the TrLS supernatant. It is seen that such BHK cells are not suitable for H110D expression, since a high level of A-type and M-type aminopeptidase activity was found both in the cytoplasm and in the integrated form associated with the membrane.

To 10 μl of each supernatant of BHK cells add 25 mm p-nitroanilide substrate in 250 μl of bicarbonate HEPES buffer at pH 7.0 and incubate the resulting mixture at pH 7.0 for 30 minutes at 37 ^o C.

 Example 2. Cloning of the HIIOD sequence of a mammalian expression vector.

The DNA that is cloned and which is clone 2 PCR (polymerase chain reaction) of the 3.5 kb H11OD gene was described in WO 93/23542. The DNA sequence (Sequence ID NO: 1) of such an insert) obtained during the polymerase chain reaction (PCR) is shown in FIG. 1, and the amino acid sequence of the translation product (Sequence ID NO: 2) in FIG. 2. This DNA was cut from the pT7Blue-T vector of the vector (Novagene) by digesting BamH1 and cloning at the BamH1 site of the multiple cloned site of the pSPT18 vector (Beringer Mannheim) to obtain clone pSPT18-3.5-2. Partial digestion with BamH1 of clone pSPT18-3.5-2 was performed, and the resulting linear DNA was purified twice by gel electrophoresis. The “sticky” ends were “blunted” by introducing dNTP using the Klenov enzyme (large fragment of DNA polymerase), and using the Ncol linker containing ATG (Beringer Mannheim Cat N 1171160), the blunt ends were connected to obtain a plasmid. Clones were screened using restriction analysis based on the presence of the indicated linker at the 5 'end of the 2.5 kb insert, which gave an intra-frame ATG initiation site under the control of the T7 promoter. A modified 3.5 kb insert from one such clone (clone pST18-3.5-2 N 44) was excised and then subcloned into the mammalian expression vector pRC / CMV using the following strategy:
1. The DNA of clone pSPT18 (T7-3.5-2 N 44) was digested with restriction endonuclease Smal.

 2. The Notl linker is connected to this modified Smal site, which allows clones to appear with the Notl site at the 5 'end of the insert, which precedes the Ncol linker containing the ATG reading start site.

 3. Purified DNA from a suitable clone was digested with both Notl and Xbal to release a 3.5 kb insert.

 The Pvul enzyme is added to the incubation medium in order to cut the DNA of the pSPT18 vector in half, which is necessary since the close sizes of the vector and the insert complicate the cleaning process.

 4. Insert size 3.5 clean in 0.6-0.7% agarose gel.

 5. The mammalian expression vector pRC / CMV was digested with Notl and Xbal, and the strip corresponding to the linear plasmid was purified on an agarose gel.

 6. Ligation of the insert of size 3.5 kb and the linearized vector is carried out.

 7. Select appropriate clones that are 3.5 kb in size when digested with Notl and Xbal. These clones are designated as pRC / CMV-3,5-2.

 Transfection of COS-1 mammalian cells.

 The mammalian expression vector DNA with the HIIOD antigen clone designated pRC / CMV-3,5-2 inserted therein 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).

 Transient expression of HIIOD can be achieved using this purified DNA of clone pRC / CMV-3,5-2 for transfection of COS-1 cells (obtained from ECACC, Porton). Transfection is performed using DEAE-dextran (Cullen BR, Use of Eucaryotic expression technology in the functional analysis of cloned genes, Methods in Enzymology: Guide to molecular cloning techniques, Eds SL Berger and AK Kimmal, Academic Press, 1987, pp 684-704 ) Cells were cultured on Eagle's medium in a Dulbecco modification (DMEM) [Flexo BRL (Gibco BRL)] containing 10% fetal calf serum (FCS) [Flexo BRL (Gibco BRL)] and the expression product was analyzed 48-72 hours after the start of incubation .

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

 Vector DNA is introduced into CHO cells (obtained from ECACC, Porton) using a calcium phosphate method (such as, for example, the method described in Cullen BR Use of eucaryotic compression technology in the functional analysis of cloned genes. Methods in Enzymology: Guide to Molecular Cloning Techniques, Eds SL Berger and AR Kimmel, Academic Press, 187, pp. 684-704). Transformed cells are cultured in DMEM medium containing 10% fetal calf serum (Flexo BRL). The geneticin-resistant line (G418) is the only transformed cell line, unlike non-transformed cells, which grows well; cells are grown in culture to a concentration of 800 μg / ml. Then the transformed cells are cloned using the method of limited dilutions on microtiter plateaus.

 Analysis of mammalian cells transfected.

 Transformed and non-transformed CHO cells can be transferred to a coverslip for growth for immunofluorescence analysis. Cells are allowed to grow until small colonies are formed, fixed with methanol and treated with sheep’s antiserum to HIIOD and then fluorescent dye (i.e. FTCC) conjugated with sheep’s immunoglobulin antiserum. To search for positive colonies using a fluorescence microscope.

Cells in transformed cell lines are destroyed in RIPA buffer (150 mM sodium chloride, 1% Nonidet P40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, pH 8.0) when removed by weight of the cells of the growth medium and then by carefully grinding the cells for 5 minutes in RIPA buffer. Then the cells are transferred to microcentrifuge tubes and unscrew them in a microcentrifuge at full speed for 15 minutes to obtain a fresh lysate, which is transferred to a clean tube. Aliquots of this lysate containing 2 × 10 ⁵ cells are subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins from the gel are transferred to the nitrocellulose membrane by Western blotting. The membrane is treated, possibly with the inclusion of a periodic processing stage, and analyzed against antisera obtained to various forms of HIIOD antigen. Periodic treatment destroys the carbohydrates of the epitope; mammalian epitope carbohydrates can be significantly different from native helminth carbohydrates. Fragments obtained by Western blotting of transformed cells show the presence of a protein recognized by H11OD-specific antisera.

 Extracts of transformed and non-transformed cells were prepared as described in Example 1, and the lysates obtained using RIPA were analyzed for the presence of enzymatic activity exactly as described in Example 1. Transformed cells containing HIIOD showed a higher level of aminopeptidase activity than untransformed cells.

 The presence of the introduced vector DNA, which contains clone 2 PCR of 3.5 kb in size, resistant to the genetin of the CHO cell line, is determined by southern blotting in DNA preparations obtained from these cell lines. DNA is extracted from cells using a known method (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 is digested with restriction endonucleases and then agarose gel electrophoretically separated by 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 is hybridized with a probe encoding clone 2 PCR of 3.5 kb, which, after thorough washing, is subjected to autoradiography. In transformed cells, bands specific for clone 2 PCR of 3.5 kb were detected.

 The expression of clone 2 PCR of 3.5 kb in size at the RNA level in transformed mammalian cells is determined using Northern analysis of RNA isolated from these cells. RNA was extracted from cells using RNAzol (Cinna Bioteks, Texas (Cinna (Biotecx, Texas)), provided with manufacturer's instructions, the mentioned RNA in an amount of up to 20 μg was dispersed in agarose gel, and then transferred to the membrane using Northern blotting (Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989.) This membrane is then hybridized with a probe encoding a 3.5 kb PCR clone 2, which After thorough washing, they are examined by autoradiography. Specific hybrid is observed in transformed cells. -ionized band corresponding to the PCR clone 2 size 3,5 kb.

Claims (5)

1. A method of expressing an aminopeptidase antigen H11OD or a fragment thereof having a similar antimatic and / or antigenic activity, comprising transfection of host mammalian cells with a vector adapted for expression of said aminopeptidase antigen H11OD or a fragment thereof, characterized in that the host cells are essentially transformed before being transformed exempt from endogenous enzymes having
(a) the same function and
(b) the same integration with the cell membrane or its absence as the aminopeptidase antigen of the H11OD parasite or its fragment.  2. The method according to claim 1, characterized in that the host cells lack significant activities like aminopeptidase A and aminopeptidase M.  3. The method according to claim 2, characterized in that said host cell is a COS-1 or CHO cell.  4. The method according to any one of claims 1 to 3, characterized in that the said aminopeptidase H11OD antigen or a fragment thereof is expressed integrated with the host cell’s cell membrane.  5. The method according to any one of claims 1 to 3, characterized in that the said aminopeptidase antigen H11OD or a fragment thereof is expressed as a soluble cytoplasmic enzyme.

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