BG61574B1 - Expression of protective antigens - Google Patents

Abstract

The invention relates to method for the expression ofhelminthic integral membrane aminopeptidase antigens. The enzymeantigen in nature is parasite gut membrane bound enzyme or afragment thereof having similar enzyme and/or antigenic activity,wherein mammalian host cells are transfected with a vector adaptedto express said enzyme antigen or fragment thereof, characterisedin that prior to transformation the host cells are substantiallyfree from endogenous enzymes having the same function and the samecell membrane integration or lack of integration, as said parasiteenzyme antigen or fragment thereof.

Description

The invention relates to the production of protective antigens of proven quality by recombinant deoxyribonucleic acid (DNA) technology.

Parasites are responsible for causing many diseases in humans and pets. The treatment so far has been mostly through chemotherapy, but lately immunological methods have been applied. Although parasites, in most stages of their life cycles, are relatively large in size and not readily distributed to the host cell's immune system, they may be susceptible to antibodies that act otherwise. 20 identify the most important functions of the parasite. In particular, it has recently been found possible to immunize host animals with antigens that are membrane-bound on the intestinal surface of the parasite, such that 25 antibodies induce binding to the antigens on such internal membranes where the body fluids containing the antibodies serve as food for the parasite. Such antigens can be defined as "hidden antigens" since they do not elicit natural immunity to the parasite.

Characteristics of the Prior Art 35

We have found that a particularly effective "hidden antigen" is the anthelminthic antigen H110D derived from Haemonchus contortus, as described in WO88 / 00835 and 90/11086. WO93 / 23542 discloses that Hl 10D is a membrane-bound aminopeptidase from the worm intestine. This enzyme acts as the major enzyme for the conversion of nutrients of a protein nature into amino acids 45 for digestion through the intestine and, when inactivated by the anti-HHOD antibody, causes the death of the parasite due to impaired food absorption. We have further found that a large number of parasites are acceptable with respect to the same "hidden antigen" strategy, in which intestinal membrane-bound enzymes are particularly important for the processing of protein nutrients. Such enzymes include aspartyl proteases (as described in PCT / GB93 01521) and thiol proteases, such as cathepsin. They are present in the gut of a large number of parasites, such as different species of worms from the families Haemonchus, Ostertaqia, Trichostrongylus, Nematodirus, Dictvocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris, Strogylus and Fasciola; and arthropod species, especially members of the spider and insect classes and in individual ectoparasites, such as blood sucking insects (eg members of the exopterigot and endopterigot classes), flies (Lucilia), flies causing myosis, and parasites, lice, worms, worms bed bugs.

SUMMARY OF THE INVENTION

WO93 / 23542, already mentioned above, also describes the production of antigenic fragments from H110D by recombinant DNA technology. In this invention, the Hl 10D material was expressed by E. coli using the pGEX vector and by injecting sheep and triggering the production of antiH110D antibodies. In further work, using baculovirus in insect host cells (Sf9 cells), we successfully expressed a 3.5 Kb clone of the Hl 10D gene. Its nucleotide sequence is shown in Figure 1 (seq. ID No: l). The corresponding translation is shown in Figure 2 (seq. ID No: 2).

However, it is preferable for the preparation of the vaccines to be administered to animals to express the antigen in mammalian host cells. This provides good reproductive properties of the natural form and the protective epitopes of the antigen, while the eukaryotic expression system will give an increase in similar glycosylation structures, disulfide bridges and other post-translational modifications, such as E.coli, which produces insoluble protein requiring refolding and refolding the natural form.

In addition, mammalian glycosylation is unlikely to induce an immune response resulting from the protective antiprotein response that can be encountered in insect cell line material due to its very different ticolysis structure. For the protection of humans and animals, it is preferable to use human or animal fibroblasts or a myeloma cell line, e.g. such as Hela-a human cell line; Baby hamster BNK cells; VERO u COS, monkey kidney cell line; FR3T3, rat fibroblasts; N1H3T3 mouse fibroblast cell line; C127I, mouse mammary tumor cell line; CV-1, African Green Monkey Kidney Fibroblasts; 3T6, mouse embryo fibroblasts; L cells, mouse cell line, CHO, Chinese hamster ovary cell line; NSO HSI, SP2 and other murine myeloma cell lines and rat myeloma cell lines, such as YB2 / O and Y3.

Because the hidden antigens to be produced are paramount for the processing of nutrients from the parasite, enzymes or other functional proteins with the same activity are usually widespread in the available eukaryotic cell lines and not only impede the selection of antigen-producing clones , but also make it difficult to purify the desired antigen from cellular products. In addition, the cell lines selected for the host will inevitably be genetically similar, at the ends of the protein sequence, to the animals to be protected, so that contamination of the desired foreign external antigen with such an endogenous antigen is likely to produce an undesired autoimmune reaction. Furthermore, qualitative control analysis of the expressed hidden antigen will be more difficult.

Detailed description of the invention

The present invention is based on the understanding of the expression of recombinant DNA of the desired external enzyme "hidden antigen" in a transformed mammalian host cell line, which when both enzymes are associated with the cell membrane or both are cytoplasmic and thus excluded for physico-chemical separation, is essentially free of endogenous antigens having the same enzymatic function as the external "hidden antigen".

According to the invention we provide me with a method for the expression of an enzyme antigen, which is inherently a parasitic intestinal membrane-bound enzyme or instead a fragment having similar enzymatic and / or antigenic activity in which the mammalian host cells are transfected with a vector adapted to express said enzyme antigen or, instead, a fragment characterized before transformation, the host cells are substantially free with respect to endogenous enzymes having (a) the same function and (b) the same cell membrane their integration or lack of integration, such as said parasitic enzyme antigen or fragment thereof.

Thus, if the host cell contains an endogenous enzyme, such as an aminopeptidase in the cytoplasm, while the external enzyme is expressed by a transmembrane sequence that becomes localized in the membrane of the host cell, there is no difficulty in separating endogenous and endogenous processing cells to separate membrane fragments, for example by centrifugation. On the other hand, the outer antigen can be modified to produce a fragment with a missing membrane binding region and if the host cell membrane carries an endogenous enzyme having the same activity as the outer enzyme, this will allow it to affect the separation by separating the cell membrane material. ; thus, the coding sequence for the transmembrane region of the parasite enzyme can be shifted to the gene to be expressed by a secretion signaling sequence.

Of partial interest are helminth aminopeptidase antigens such as H110D mentioned above and fragments thereof. They can be expressed in a wide variety of mammalian host cells using appropriate vectors. For the expression of H.contortus antigen H110D, which exhibits predominant aminopeptidase A-like or M-like activity and thus cleaves predominantly methionine and leucine peptide bonds, we have found COS-1 cells for use in accordance with the invention, because they lack significant Ap-like or M-like aminopeptidase activity. They appear to have an aminospididase enzyme that breaks down alanine peptide bonds, and this is poorly associated with the cell membrane, so there is no difficulty in separating the endogenous enzyme from the expressed H110D, which is localized to the cell membrane.

Proteolytic enzymes, such as trypsin, have been shown to cleave the parasite antigen H110D from the membrane, yielding H1 lOF-soluble (H11S). Such enzymes can thus be used to differentially degrade an external latent antigen, separate from an endogenous enzyme with similar activity.

Suitable cell lines for use in accordance with the invention may be selected from pre-existing strains by screening for their profile of appropriate enzyme activity and / or localization by an appropriate appropriate enzyme in association with the cell membrane or cytoplasm. In a later case, association with the cell membrane can be established by extracting lysed cells first with detergent such as TWEEN, which does not integrally extract membrane enzymes, and then with detergent such as TRITON, which can release such enzymes.

It is also possible to create a mammalian cell line with reduced enzyme content. One way in which this can be done is as follows: antibodies are derived from a mammalian enzyme for deletion. Cell lines were modified by radiation treatment or by chemicals to induce point mutations. The cells were cultured in a suitable nutrient medium to compensate for lost enzyme activity. Cells were then exposed to fluorescently labeled antibodies and passed through a fluorescently activated cell sorter (FACS). Fluorescently negative cells were cloned and reselected and stability of enzyme loss was established.

Cells can also be modified by gene deletion or rational (direct) mutation technologies to remove or mutate the gene for the relevant endogenous enzyme.

Suitable vectors for different classes of mammalian cell lines are well known in the art. Generally speaking, they include a promoter and / or snanssr operatively linked to a gene expressing the enzyme antigen or fragment thereof. So, partly

A 3.5 kb fragment of the Hl 10D gene can be linked in a structure with a suitable promoter. Suitable promoters include the SV40 early or late promoter, for example, the PSVL vector, the cytomegalovirus (CMV) promoter, the mouse metallothionic I promoter, and the mouse mammary tumor virus with a long recurrent terminal. The vector preferably includes a suitable marker such as a dihydrofolate reductase gene or glutamine synthetase. Vectors of this type are described in WO86 / 05807, WO87 / 04462, WO89 / 01036 and WO89 / 10404.

Host cell transfection can be accomplished by the use of standard technologies, for example, using calcium phosphate, DEAE dextran, polybren, protoplast mixture, liposomes, direct microinjection, gene canon, or electrical purification. Newer technologies are preferably, methods of transfection of mammalian cell lines using electrical purification are described by Andreason G.L. in Evans G.A., Introduction and expression of DNA molecules in eukaryotic cells by electroporation, Biotechniques 6, 650, 1980. In general, linear DNA is more readily introduced than circular DNA. The H110D antigen exhibits leucine aminopeptidase (M-like) and methionine (A-like) aminopeptidase activity and is generally preferred because host cells are free from at least these types of aminopeptidase activity.

The following examples are for illustrative purposes only. In these examples, the figures represent:

Figure 1 shows the DNA sequence of

3.5 Kb PCR clone 2 (sequence ID No: l); and

Figure 2 shows the amino acid translation of 3.5 Kb PCR clone 2 (sequence ID No: 2).

Example 1. Enzyme analysis of Cos-1 cells.

Cos-Ι cells were obtained from University of Surrey in 5 ml DMEM culture media, the cells were separated into two 200 ml flasks, each containing 11 ml from the medium in which the cells gave growth to confluence. Cos cells were adhered and then separated from the flask by aspiration using a glass pipette in 10 ml PBS buffer. When all cells are in suspension, they are transferred to a universal tube and frozen at - 20'C. The cells were repeatedly frozen and mixed in liquid nitrogen until disrupted, then rotated to release the PBS supernatant (PLS). For the resulting extract, 500 ml of 0.1% Tween and PBS 2% Triton were used to give TwLS and TrLS supernatants. All supernatants were concentrated to 200 ml using Millipore micro concentrators. PBS extracts enzymes that are free in the cytoplasm, Tween extracts enzymes that may be weakly bound to the cell membrane, while Triton extracts whole membrane proteins.

The supernatants were analyzed at 25 mM against: phenylalanine, gamaglutamic acid, leucine, lysine. methionine, alanine, alphaglutamic acid, glycine-proline and aspartic acid para-nitroanilidene (pH) substrates at pH 7.0 in HEPES bicarbonate buffer. No activity was detected after 30 min incubation at 37 ° C, so analysis was followed by incubation for 18 h to determine the presence of any enzymatic activity. The specific activities were calculated and the results are shown in Table 1.

Greater activity is shown in Tween extraction, where the highest activity is found in alanine p-HA, activity in phenylalanine, gamaglutamic acid, leucine, lysine and methionine pH substrates has been demonstrated. PLS and TrLS contain low activity only assuming gamma GTP and residual lysine AP and alanine AP.

Table 1.

Specific activity (optical density / min / mg protein) of Cos cells

district of Substratum PLS TwLS extraction TrLS Phenylalanine 0,0000 0,089x10 ' 0.000 Homaglutamine acid 0,0015x10 ' 0,165x10 ' 0,033x10 ' Levcin 0,0012x10- ' 0,066x10 ' 0.000 Lysine 0,0029x10 ' 0,273x10 ' 0,010x10 ' Methionine 0,0011x10 ' 0,083x10 ' 0.000 Alanine 0,0270x10 ' 1,110x10 ' 0,159x10 ' Alphaglutamine acid 0,0000 0.000 0.000 Glycine proline 0,007x10 ' 0.000 0.000

ml of each Cos-Ι cell supernatant were added to 25 mM p-nitroanilide substrate in 250 ml of HEPES bicarbonate buffer at pH 7.0 and incubated at 37 ° C for 18 h.

Enzyme analysis of Chinese Hamster Ovary (CHO) cells and NSO

Extracts from cultured CHO cells and NSO cells were obtained by the same method as described above for COS-1 cells. These extracts were analyzed against the following paranitroanilide substrates: alanine, arginine, glycine, alpha-glutamic acid, gamma-glutamic acid, leucine, lysine, methionine, phenylalanine, shedding and glycine-proline, all at 25 mM in HEPES bicarbonate buffer pH 7, 0. The assays were incubated for 30 min at 37 ° C, where the final optical density was taken and the specific activity was calculated.

Table 2 showed CHO cell soluble and membrane associated extracts and contained low levels of enzyme activity, with the exception of TwLS activity against the glycine-proline substrate. In contrast, the TrLS ex-5 tract has low activity in all cases except for the arginine substrate. Thus, the latter activity is different from Hl 10D aminopeptidase activity.

Table 2.

Specific activity (optical density / min / mg protein) of CHO cell supernatants

p-NA substrate PLS TwLS extraction TrLS Levcin 0,070x10 ³ 0,147x10 ³ 0,106x10 ³ Phenylalanine 0,059x10 ³ 0,052x10 ³ 0.000 Lysine 0,178x10 ³ 0,123x10 ³ 0,106x10 ³ Methionine 0,130x10 ³ 0,118x10 ³ 0,106x10 ³ Alanine 0,148x10 ³ 0,169x10 ³ 0,137x10 - ³ Glycine 0.000 0,052x10 ³ 0,068x10 ³ Arginine 0,078x10 ³ 0,118x10 ³ 0,889x10 Proline 0,024x10 ³ 0,030x10 ³ 0,046x10 ³ Arginine Proline 0,024x10 ³ 0,052x10 ³ 0,061x10 - ³ Glycine proline 0,074x10 ³ 0,405x10 ³ 0,068x10 ' Alphaglutamine acid 0,011x10 ³ 0,052x10 ³ 0,144x10 ³ G amaglutamine acid 0,030x10 ³ 0.000 0.053x10 - ³

NSO cells and PLS and TwLS extract had the same aminopeptidase activity in part 35 with respect to leucine and methionine (Table 3). In contrast, TrLS have low levels of such activity, suggesting purification, incl. Triton extraction, as such has recently been applied to H110D, will result in very little contaminated activity of the endogenous enzyme.

Table 3.

Specific activity (optical density / min / mg protein) of NSO cell supernatants

p-NA substrate PLS TwLS extraction TrLS Levcin 0,640x10 ³ 1,840x10 ³ 0,137x10 ³ Phenylalanine 0,108x10 ³ 0,205x10 ³ 0.087x10 ³ Lysine 0,136x10 ³ 0,188x10 ³ 0.093x10 - ³ Methionine 0,280x10 ³ 0,764x10 ³ 0,099x10 ³ Alanine 0,047x10 ³ 0,150x10 ³ 0,067x10 ³

G.chici n 0,014x10 ' 0,045x10 ³ 0,019x10 ³ Arginine 0,110x10 ⁵ 0,114x10 ³ 0,081x10 ³ Proline 0,014x10 ³ 0,034x10 ³ 0,019x10 ³ Arginine Proline 0,043x10 ³ 0,085x10 ³ 0,050x10 ³ Glycine i-proline 0,058x10 ³ 0,063x10 ³ 0,050x10 ³ Alphaglutamine acid 0,017x10 ³ 0,270x10 ³ 0,019x10 ³ Homaglutamine acid 0,017x10 ³ 0,040x10 ³ 0,019x10 ³

Comparative example.

BHK cells were obtained by the same method as the Cos-Ι cells described in Example 1, despite the larger quantities (2x1 L cylindrical cylindrical cylinders in 100 ml of medium). Supernatants were analyzed with 25 mM against: phenylalanine, leucine, gamma-glutamic acid, alanine, arginine asparagine, lysine, methionine, glycineproline and alpha-glutamic acid paranitroanilidene (p-HA) substrates at pH 7.0. The assays were incubated for 30 min at 37 ° C, where final optical density was taken and specific activity was calculated.

BHK cell extracts have a significant enzyme activity that is present in all supernatants (Table 4). There is little activity on substrates of gamma-glutamic acid, asparagine or alpha-glutamic acid. All supernatants show good lysine pH activity (which is highest in PLS), leucine, alanine and glycine-proline. Methionine p-HA activity is negligible in PLS and maximal in TrLS supernatant. BHK cells will not be suitable for the expression of Hl 10D, whereas high levels of A-like and M-like aminopeptidase activity are found in the cytoplasm and integrally in the membrane.

Table 4.

Specific activity (optical density / min / mg protein) of BHK cell supernatants

p-NA substrate PLS TwLS extraction TrLS Phenylalanine 0,102x10 ³ 0,080x10 ³ 0,113x10 ³ Levcin 0,320x10 ³ 0,340x10 ³ 0,433x10 - ³ Homaglutamine acid 0,006x10 ³ 0.000 0,004x10 ³ Alanine 0,260x10 ³ 0,220x10 ³ 0.328x10 - ³ Arginine 0,150x10 ³ 0,200x10 ³ 0,344x10 ³ Asparagine 0,017x10 ³ 0,015x10 ³ 0.008x10 - ³ Lysine 0,330x10 ³ 0,259x10 ³ 0,420x10 - ³ Methionine 0,020x10 ³ 0,185x10 - ³ 0,355x10 ³ Glycine proline 0,312x10 ³ 0,318x10 - ³ 0,225x10 ³ Alphaglutamine acid 0,014x10 ³ 0.008x10 - ³ 0.008x10 - ³

Example 2. Cloning of the H110D sequence into a mammalian expression vector

For cloning DNA with a cloned gene

3.5 Kb PCR clone 2 Η110D, described in WO93 / 23542. The DNA sequence (sequence ID no: 1) of this insert obtained by polymerase chain reaction (PCR) is shown in FIG. 1, the amino acid translation of (sequence ID no: 2) in Fig. 2. This DNA was excised from the vector pT7Blue-TVector (Novagene) by BamHI digestion and cloned into the BamHI region of the multiplicated cloned region of vector pSPT 18 (Boehreringer Mahneim) to give clone pSPT18-3.5-2. Partial BamHI digestion from clone pSPT 18-3.5-2 was performed and the linear DNA was purified twice by gel electrophoresis. The "sticky" ends are blunted with dNTPs using a Klenow enzyme (DNA polymerase, large fragment) and an Ncol linker containing ATG (Boehreringer Mahnheim Cat no: 1171 160) ligated into a plasmid. The clones were screened by restriction analysis for those having this linker at the 5, end of the 3.5 Kb insert, giving them in the ATG framework an initiation site under the control of the T7 promoter. The modified 3.5 Kb insert from one such clone (clone pSPT18-3,5-2N44) was excised and then subcloned into a mammalian pRC / CMV expression vector using the following strategy:

1. The DNA of clone pSPT18 (T7) -3,5-2N44 is digested with the restriction enzyme Smal;

2. The Notl linker is ligated into this modified Smal region to give the growth of the Notl clones at the 5 'end of the insert preceding the Ncol linker containing the ATG start region;

3. Purified DNA from a suitable clone was digested with NotI u Xbal to release 3.5 Kb insert. The Pvul enzyme was added for incubation to cut the pSPT 18 vector DNA in half, which is necessary because of the similar size of the vector and the insert, which hinders purification.

4. A 3.5 Kb insert was purified on 0.60.7% agarose gel.

5. The mammalian expression vector pRC / CMV was digested with NotI u Xbal and the linear plasmid band was purified on an agarose gel.

6. Ligation was performed between 3.5 Kb insert and the linearized vector.

7. Suitable clones are selected to have 3.5 Kb when digested with Noil and Xbal. The clones are called pRC / C.M V-3.5-2.

Mammalian transfection of COS-1 cells

The DNA of the mammalian expression vector with the H110D insert clone pRC / CMV-3.5-2 was purified by centrifugation in a cesium chloride gradient. (Sambrook J., Fritsch E.F. in Maniats J. Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbor Press, 1989).

Transient expression of H110D can be obtained using such purified DNA from clone pCMV-3.5-2 to transfect COS-1 cells (obtained from ECACC, Porton). Transfection was performed by DEAE-dextran (Cullen BR, Use of Eukaryotic expression technology in functional analysis of Cloned genes, Methods in Enzymology: A Guide to Molecular Cloning Techniques, Eds SL Berger and AR Kimmal, Academic Press, 1987, pp 684-704 ). Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM, Gibco BRL) and 10% fetal fetal serum. (FCS, Gibco BRL) and expression analyzed followed by 48-72 h incubation.

Chinese Hamster Ovary (CHO) cell transfection

The DNA vector was transfected into CHO cells (obtained from ECACC, Porton) using a calcium phosphate method (as in the method described by Cullen B.R.). Eukaryotic expression technology is applied in the functional analysis of gene cloning. Methods in Enzymology: A Guide to Molecular Cloning Techniques, Eds S.L. Berger and A.R. Kimmal, Academic Press, 1987, pp. 684-704). The transformed cells were cultured in DMEM and 10% FCS (Gibco BRL). Geneticin (G418) gives good growth on only transformed cell lines and does not yield on untransformed ones, the latter can be up to 800gg / ml. The transformed cells were then cloned by limited dilution into microtiter plates.

Mammalian transfected cells assay

Transformed and untransformed CHO cells can be transferred to growth on growth glass for immunofluorescence analysis. Growth takes the form of small colonies fixed with methanol and probes with sheep anti-Hl 10D antisera, followed by: - fluorescently stained (eg FTTCt conjugated anti-ulcer immunoglobulin antiserum. A fluorescence microscope was applied to look for positive colonies.

Transformed cell lines were broken into RIPA buffer (150 mM sodium chloride 1% Nonidet P40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 50mM TR1S-HC1 pH 8.0) by removing the culture medium from the cells and after this gentle shaking of cells for 5 min in RIPA buffer. The cells were then transferred to a tube and rotated in a microfuge tube at full speed for 15 min to obtain a clear lysate, which was transferred to a new tube. Aliquots of this lysate equivalent to 2 x 10 ⁵ cells were electrophoresed on SDS polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins in the gel were transferred to a nitrocellulose membrane by Western blot. The membrane is broken down, it is possible to include a period with the treatment of periodate and analysis with antiserum with increase to different forms of Hl 10D antigen. Periodontal treatment destroys carbohydrate epitopes; Mammalian carbohydrate epitopes may be significantly different from natural helminthic carbohydrate. A Western blot of transformed cells shows the presence of a protein recognized by an antiserum specific for H110D.

Extracts of transformed and untransformed cells obtained by the method described in Example 1 and lysates obtained using RIPA were subjected to enzyme activity assay, just as described in Example 1. Cells transformed with H110D showed higher levels of aminopeptidase activity compared to untransformed cells.

The presence of the transfected DNA vector containing 3.5 Kb PCR clone 2 in Geneticin resistant CHO cell lines was determined by Southern analyzes of DNA preparations from these cell lines. DNA was extracted from cells using the Sambrook method. J .. Fritsch, E.F. in Manitias, T. Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Press, 19S9. 10-20 g of DNA were digested with restriction enzymes and then electrophoresed on agarose gel and DNA transferred to the membrane via Southern Blot [Southern, E. Detection of specific sequences among DNA fragments separated by gel electrophoreses. J.Mol.Biol. 98 p. 503, 19751. This membrane was hybridized with a probe encoding 3.5 Kb PCR clone 2, and after intensive washing, the membrane underwent autoradiography. Specific bands for 3.5 Kb PCR clone 2 are represented in transformed cells.

The expression of 3.5 Kb PCR clone 2 at RNA level in transformed mammalian cells was determined by Northern analysis of RNA isolated from these cells. RNA was extracted from cells using RNAzol Cinna / Biotech, Texas) following the instructions given, and 20 μg were transferred to agarose gel and transferred to a membrane via Northern blot [Sambrook, J., Fritsch, E.F. in Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Ed. Cold Spring Harbor Press, 1989]. This membrane was then hybridized with a probe encoding 3.5 Kb PCR clone 2, and after intensive washing, the membrane underwent autoradiography. A specific hybridization band for 3.5 Kb PCR clone 2 was observed in transformed cells expressing 3.5 Kb PCR clone 2.

List of sequences

Information about SEQ ID # 1:

(i) Sequence characteristics

A. Length: 3303 Base Pairs B. Type: Nucleic Acid

B. Branch: Single chain D. Topology: Linear (I) Type of molecule: kDoxyribonucleic acid (iii) Sequence description: SEQ ID # 1:

C1NLL1 CIA AcICCAAICC 81 (., IT AT I GT CGCAT1ATIT CI AG IAGC I G C1GCAGfCGG CCTCICTAT Γ 120 AGGGAAGGAT 180 GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC GATACTGGTG GCAAGGACAA AGACATTCCT CCCTCTGCGG CGGAAC1ACT CCTTCCAAGT 240 AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT ATCTACCTGC TTAT'GTGGAC 300 TTCCCACCGG AGAAAAACCT CACATTCGAT GGGCGTGTGG AAATATCAAf GGTTGTAATT 360 GAGCCAACAA AGGTTATCGT ACTCAATTCA AAGAAGATCT CTGTAATACC CCAAGAATGT 420 C3AACTGGTAT CGGGCGATAA AAAACTCGAA ATTGAAAGTG TAAAGGAGCA CCCAAGACTG 480 GAAAAGGTTG AGTTTCTTAT CAAAAGCCAA CTGGAAAAAG ATCAACAAAT CTTGCTCAAG 540 GTCGGCTACA TCGGTCTCAT CAGCAACAGC TTTGGTGGAA TCTACCAGAC CACTTATACC 600 ACCCCGGATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT AGATGCTCGT 660 CGAATGGTAC CATGCATGGA TGAACCGAAA TACAAAGCAA ACTGGACCGT TACTGTCATT 720 CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA TGGAGAGATC 780 AGTGGTGATT GGATCACATC GAAGTTCTTG ACTACTCCAC GGATGTCATC CTACTTGTTG 840 GCAGTTATGG TTTCAGAATT TGAATACATC GAAGGTGAAA CAAAGACGGG TGTTCGGTTC 900 CGTATATGGT CACGCCCAGA GGCAAAGAAG ATGACACAAT A TGCTCTGC A ATCTGGTATC 960 AAGTGCATAG AATTCTACGA AGATTTCTTT GATATCAGAT TCCCTCTGAA GAAACAAGAT ATGATTGCCC TTCCTGATTT CTCTGCCGGT GCCATGGAGA ATTGGGGCCT

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCTCAT CAGTGGTTCG GCGACTTGGT 1140 TATTGGAGCT GGTCAGATAA CTCAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA 1260 TGTACTTGAA 1320 AATCGACAAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG GCTGCAGAAG TTGAAGAAGC CTTTGAT6AT ATCACATACG CCAAAGGAGC 1380 TTCTGTTCTT ACTATGCTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC 1440 GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1560 TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG IGGAGAAAGA 1680 GAAGTACCGT CACCCGAAAT ACGGATTTAA ATGGGATATT CCACTGTGGT ATCAGGAAGG 1740 CGATAAGAAG GAGATAAAGC GAACATGGTT GAGAAGAGAT GAACCGCTTT ACTTGCATGT 1800 TAGTGATGCT GGCGCTCCCT TTGTGGTGAA CGCAGACCGC TATGGATTTT ATCGACAAAA I860 TCATGACGCT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGATAATC ATGAGGTTTA 1920 CAGTCCCCGG ACAAGAAATG CCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC 1980 CAGTCCCCGG ACAAGAAATG CCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC 1980 AATTGAGTAT GAGACTGTAT TTGAACTTCT GAATTATGCC GAAAAAGAAA CGGAAT AT CT 2040 ACCATTAGAA ATCGCAATGT CCGGGATCTC TTCGATTTTG AAATACTTCG GTACCGAGCC 2100 AGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAACCGATGT ATGAAAAAAG 2160 CAGTATCGAC TTCATTGCCA ATAACTACAG AAATGACAAG CTGTTTTTCC AAATCAACCT 2220 CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTCGGATCG CAAGACTGCA GGAAGAAATA

TOOAAASTT TTCGATGACG AAGTCATGAA CAAAIGCABG GATGG1CAAG CAGCAACCGA

23-40

ATGCGTAAGA ATSVSSBSTS CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 2400

CGGTGATTAT GCTTCCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC 1CGCCC! AGA 2460

AAAAGACTTC CTACGCCTAG CATTGGGATG TCATAAAGAT GTTACTGCTT TGAAAGGACT 2520

TCTCTTGCGG GCTCTGGACA GGAA! TCGTG GTTCGTACGT ATGCAGGATA TCCCAAGTGC 2580

TTTCAATGAT GTAGCAGCAA ATCCTATCGG CGGAGAATTC ATTTTCAATT TCCTTATTGA 2640

AAGATGGCCA GATATCATTG AAAGTATAGG AACGAAGCAC ACATACGTTG AGAAAGTGAT 2700

ACCAGCCTGC ACTTCAGGAA TCCGCTCACA ACAGCAGATT GACCAGCTGA AGAATCTGCA 2760

GAAAAATGGC ATGAACGCTC GTCAATTCGG TGCATTCGAT AAAGCAATCG AACGAGCACA 2820

AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT TCAAGAAAGC 2880

CACCTTGTAA TTCGAATTAC ATTGCCAGTA ATCCAGATCT TAAAGTTCAT (3AAGGAATAT 2940

GACAGGGAAC TGACTGTCTG TTGGTCACTG TTCCACTGAA TGGAAGTTTT TACCTACAAA 3000

AATPTGATS GTTATATTTG CCTTCCGTGA GGGGTCATTG TTGTCACTTG AATAGTAAAC

3060

AAAGCTCAGT ATTGCAACCA GTGAACAATA TTACTTTCGC TTCATCAAAT TGTTATCTTC 3120

CCTATACTCT CTTCCTAACT GAATTCGGAA ATTTGTTCAT ATTCGTTTGT AGTCTGTTGC 3180

TCAGAACACT TTCTCCTCAA TAGCTTCTTG TTTGTTTTTT TTTGATTGTA TTGATCGTTT 3240

TACAATTGTA TAGATTAGTT ATCTTATAAA TATTGATGGT TAAAAAAAAA AAAAAAAAAA

3330

AAA

2. Information about SEQ ID # 2:

(i) Sequence characteristics

A. Length: 962 amino acids

B. Type: Amino acid

C. Branching: Single chain

D. Topology: Linear (s) Molecule Type: Peptide (xi) Sequence Description: SEQ 1D No. 2:

ieC'juH Asparagin Oevtsin Tresna Lrolin Isolevia Arlyain Levia Isolevtsin Valia Alanine Levia venila.lanin Ulcers »Valia Alanine 15 1015

Alanine Alanine Wales G-Levin Left Series Islevcin Glycine Vevcin Threosin Tyrosine Veilylalanine Threonine lrginine Visions

25J0

Alanine Vanimlanche Spamgiyva-a-threonine Series Glaminova a-a Yaizsh Golin Gliyin Yaizin Asparagin-a Asparaginova a-a 25 «

Treonia Gliyin Gliyin Vision Asp.a.-Vizin Aspl.-Aspazigin Proline Proline Series Alanine Adashi Gl.a.-Levi Levi Levi

L5 50 55 «0

Grolin Asparagin Isoleucin Series Vision (Eolia Vevtin Tyrosine Series Asp.a. Yevin Treonia Isolation Vision Threonine Tyrosine Vezcin 45 7075

Glycine Strait Tirsin Valia Asp-a-Oshalaniya Drolin Drolich St-Via Asparagin Vevin Threonine Lenilalanin Asp-A Gliyin 80 8500

And

Argcn * · g <'nn Glut.k-izlebtsin Seven Methonias Velin Wali Isolebiin Glut.k-na% omi Truan Vl> Sv itew & u · Va »i> - 1ln? 5 155 155110

Asparagia Series, 1 Issues Visions Isolation Session Valka Izolevtsn Proline Glutaiin Glzt-l Cysteine Glut, l-ie 1ebiin Vel-Seoin Gl-tsn

115 125125

Yaiz Asp.k.-x Obesity of Obebia Glut.k.-Izolevtsa Glut.k-Series Valim Viain Glut.k.-Histioin Tsyul Arginin Vebim Glut.k-na

135 13519 »

Visions Valym Glut.k-of Venilalanin Vevin Izolevtsin Vyakn Series Glutanya Yaevtsin Glut.k-of Vizin Asp.-glutania Glutinin Izolebtsya

195 15 »155165

1e5tschn Yaevtsin Yaizin Valya Glitsin Tyrosine Isolevitsn Glyn Yaebtsm Izolevtsin Asparagia Series Venilalanin Glshin Glycin

U5 17 »175

Isolebiin Tyrosine Gluten Threonine Threonine Tyrosine TriptoVan TriptoVan Glyulin Asp.k-of Glycine Triptavan Preley Yaizin Slaughterhouse

185 185195

Alanine Wali Seeli Gluttony Asparagia Gl.G.Celin Isolebiin Asp.klan Alanine Arginine Arginine Iethionine Velin Prola Cysteia

1'5 25 »255

Yethionin Asp.k-on Glut.k-Poolin Yaisin Tyrosine Yaisin Alani Asparapn TriptoVan Threonine Wale Threonia Wale yuolebci Brushes

210 21522 »tyoun Vizin Glitsya Toknya Yaizin Alanin Velin Series Asparapn Glytsin Jazolevtsi Glut.k-Valia Asparagia Glitsn Asp.k-na

225 23 »235248

Gliish Glut.k.-Izolevtsin Serai Glycin Asp.-Trstovan Neolevtsi Tresn Series Visions Venilalanin Yaebia Threonine Threonine Tsulay

295 25 »255

Arpnin Legions Serai Tyrosine Series YaEvi Fruits Alenia Valia Netionia Velin Glut.k.-Venilalanin Gl.T. Tyrosin

260 26527 »

Aeolebcn Glutin-Glitsin Glutin-Gl Treya Visions Threonin Glycin Velin Argynya Venilalanin Argynya Izlebytsia Tryggavan Seoya Argynyya 275 28 »285

Proline Glat.-Deer Yaiaya Yaizin Netionia Treonia Glutanin Tiroaya Alanine Yaevtsin Glugan Serin Clay Ieolebsh Visions 2? »2953» »

Cysteine Izlebtsin Glat.k-na Venilalanin Tyrosine Glut.kma Asp.k-na Venilalanin Venilalanin Asp.k-Iulebshn Arpma Venilalanya Poolin 305 31 »315

Yaebiyi Visions Visions Vulgaria Asp.k-na Netisnin Izolebiin Alanin Aebtsin Proshch Asp.k-vshchalanan Sarsh Alenya Glytsin Alenya Netionya 32 »325 33» 335

Asparagi Gl.k.TryptoVan Glycin Webb Izolebets Treonin Tiroaya Arpnn Asparapn Gl.k. Yaevtsin Series Egg Tyrosin Asp.kn 391 39555 »

Asp.k-na Arginin Venilalanya Tyrosan Alenya Prolya Netionin Asparagus Visions Mug Arpmya Izolevtsin Alanin Arpmya Izmebshi Valan 355 36 »3 * 5

Reindeer Brush Glacier Webcn Deer IucruguH Glutam TragtoVai Venilalanya Glitzm Asp.Kar Webb Walka Treonia Netionia Visions

37 »375ZM

TriptoVan TriptoVan Asp.k-of Asparipa Yaebets TraptoVan YMtsin Asparagi Glut-k Glitsa Venilalanin Alanine Arpnin Venilalanya 385 39 »595

TrajatoVan Glut.k-na Venilalanin Izlebvdn Glitsin Alenya Glitsn Glutanin Isolebiin Threonin Glutakya Asp.k Asp.k-alanin Arpnin 905 905910

Nethionine Arpin Asparagi Tyrosine Venilalanin Singers Izolebtsi Asp.k.-Valia Aebtsin Glut.k-Arginin Alanin Yaevtsin Vision Alanin 915 92 »925

Asp.k.-Seren Valia Alanin Series Serine lucmqiH tyoAUH Webcin Serin Venilalanya Arginia Isolebiin Acn.r-of Visions Alanya Alanine

430 955 '990995

Glutl-on Glutl-on Alanine Veyulania fcn.x-on Aspl-on IsleLin 'genius Tigzzin Alanine tajn Glycine Alann Series Valin (eZia

450 «55 <60

Threonine Methionine Levshm Arginine Alanine Yaecin Isolevine Glynn Glut.x-glugl-lysine Histioin Lysine lucruow Alann Va »in '. Eoin W; 4? 0 <'5 <30

Glutinine Tyrosine Levcin Lysine Lysine (Enimnine Stan Thioosin Series Aspl-alanin Glutl-alanin Tresn nnpl-Levits (35 <30 <95

TriptoOan Alanine Valin (Enilanal Aspl-on Glugl-Valin Val <and Tresnin Aspl-Valin Glutl-on Gladia (Ulin Aspl-on Glycine

500 505514

Lysine Dyul1M Metisnin Lysine Threonine Threonine Glugl-w xlzlanin Alann Series Glutinine Tryptophanes Threonine Toonin Glutinine Metisnya 515 520525

Glynne Booklinan Proline Valia Isolevtsin Seoin Valin Alanine Glutl-on Bookallan Asparapn Seny Threonine Threonine Levin Liznn 554 535544

Levad Threonine Glutinine Seia Arginine Thioosin Glugl-on Alanine Asparagine Lysine Aspl-on Alanine Valgn Glutl-on Lysine Glugl-on

545 550 55556 »

Lysine Tyrosine Arginine (Aegidia Proline League Tiroan Gliad Bookland Yaisn Tripto4an Asple-na Ikmevn Proline Yaevad T (hgto4an

565 57 »575

Tyrosine Glutans Glutanin Gliad Aspl-on Lysine Lysine Glutl-on Isalevad Lizig Arpn Threonine TriptoOan Levcin Arpn Arginine

584 58553 »

Aspl-na Glugl-na Prolin Lentils Tyrosine Levin Kisgidin Valin Serin Aspl-na Alann Glitsya Alann Zulgn (enilanania Valin Valin

535 604 * 05

Asparagine Alanine Asple-na Arpn Tirozsch Glischn canalnand Tschuzin Arginine Glstamn Asparapn Zistidgn Asple-na Alanne Asparagin 61 »61562»

Glyn Trigtoban Lysine Lysine Isolevin Isolevin Lysine Glutann Levia Lysine Aspl-as Asparagine lucrjgw Glutl-Valin Tyrosine

625 630 635440

Seren Dulln Arpnen Threonine Arpvsh Asparapn Allan Isolevin Isolevin Serine Asple-na Allan * e »yelanin alan alan alanin

635 65 »655

Allan Threonine Aspl-on Allan Isolevin Glutl-on Tyrosine Glutl-on Tresn Valin Booklett Glutl-on Left Levi Asparagin Quiet »66» 64567 «

Alan Glutl-on Lizn Glugl-on Threonine Glztl-on Tyrone Levi fyaxw Levcin Glutl-on Isolevin Alan Metisnn Seris Glycine

675 6 (4685

Isalevin Serine Serine Isolevin Levcin Lizzie Tyrosine Bookworm Glyn Tresn Glutl-on fyoAW Glutl-Alanine Lizch Falin

630 6357 »»

Alann Glutanin Treonv Tyrosine Nepknn Methionine Asparapn Isalevin Levi Lysine Pralig Methionn Tiroain Glutl-on Lykne Series

785 71 71572

Serine Isolevin Aspl-on Bookstore Isolevin Alanine Asparapn Asparagine Tyrone Arpn Asparapn Asp-na Lian Levin (violin

725 7I715 bookstore Glutanin Isoleucine Asparagine Levi Glupnan Lian Asple-on Valin Izolevin Asple-on Imagine bookstore Cystan Alan 7 «74575»

Leucine Glynn Serine Swallowing Aspl-na Cystan Arpnn Lian Lian Tiroan Lian Lian Levi (Asplan-on Aspl-na Glutl-na 755 76 »765

Glugl-on Valley Methionine Asparapn Lysine Cysteine Arpn Asparapn Glyn Glugann Alanine Alan Tpeaw Glutl-on Listen Valin Argon 77078 »

Isolevin Alanine Alanine G ^ olin Levin Arpnn Serig Serine Valin Thyroid Cysteine Tiroan Clay Valin Lian Glutl-on Gladi

785 790 795I »»

Tirzgi Tasgi Ironing Alan Seren Asping Lysine Valin Methionine Glztl-Levzin Tyroan Tresn Alnan Glutl-Tresn

905 810815

Levi ** Achi> Lzvztsi!> «.« - «Wires of Asgl-ia · 9 * ιαιμμ» ι Le & wn Argyin Mt »<Amchr Schw Glitz Kuctwh lucwguH Yiao

820 82583 «

Asg-.k-sch vachl-: 'qeoHjH Αλιχιη Yaevtsn Hmschtn Levvdn 1g5tsh Levtsh rhizh Al * their Levsn km-i »ispin kraoir>

255 8 «8« 5

Eshalinia Series 81Lin Ysghim Yaethiohi Glappia Asp.G. Isolation of Lrolia The Alznia Series ени Enilalashn Asgimgin kp. - Cha fexun 850 8 880

Ayash Amhjh Acntpuw G ^ olin Isolevtsi Glitsy Clay Glzt.k-na «ellllnh № olevtsm (eilalamh isparagia (enamnsh Singers 865 87« 875

Isolevtsi Glut.k-sh Arpn Tpunrofin Prom Asp.k-n Izolevtsch Isolvts Glut-nz Series Isolevtsh Glits Tresni Lizin lucrugm 880 8858? «

Tresin Tysyuzin Ylin Gly.k-hj Lysine Vzlsz Isolewc phallin Ainin Shtkwi Treschin Sarsh Clay Isolation of the Arpia Series of the Gluzia 885 8W »0581«

Glutzin Glossy Isolation Mr. Mr. »Glutton Levcin Lysine Istrapi Singers Silly Piai Jotrzgin Clay Ketoni Asgarapi Alshi 815» 28825

Arpmi Glugmn (Enilzlshn Glitsi Alanin 1enilalmn Asp.k-na Nizh Alanin Isolation of Glutl-sh Argia Alami Glutai Asparagh 83 «835 8«

Arginia Wales Asple-on Tritoni Iulebtsi Lysine Liai Listidine Venilayain Glutantine Lysine Singers Alanine Alanine Venchlani Venilayai

885 85 « 855 Lysine Lysine Ainh Treoni Lobsch 860

Claims

Claims (8)

Claims 1. A method for the expression of an enzyme antigen, which is essentially a parasitic intestinal membrane-bound enzyme, or a fragment thereof, with similar enzyme and / or antigen activity, in which the mammalian host cells are transfected with a vector adapted to express the enzyme antigen or fragment , characterized in that, prior to their transformation, the host cells are substantially free of endogenous enzymes and having the same function and the same cellular membrane integration or lack of integration as the parasitic enzyme a an antigen or fragment having similar enzymatic and / or antigenic activity. The method of claim 1, wherein said antigen is a helminth enzyme. The method of claims 1 or 2, wherein said antigen is an aminopeptidase enzyme. The method of claims 1, 2 or 3, wherein said antigen is H1OD or an antigenic fragment. The method of claims 3 or 4, wherein said host cells do not exhibit significant aminopeptidase A-like or aminopeptidase M-like activity. The method of claim 5, wherein said host cells are COS-1 or CHO cells. The method of claims 1 to 6, wherein said antigen is expressed integrally with the cell membrane of the host cells. The method of claims 1 to 6, wherein said antigen is expressed as a soluble cytoplasmic enzyme.