BG99887A - Expression of protective antigens - Google Patents

Abstract

A method of expressing an enzyme antigen which is substantially a parasitic intestinal membrane-bound enzyme or a fragment thereof with similar enzyme and / or antigenic 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 transformation, the host cells are substantially free of endogenous enzymes and have the same function and the same cell membrane integration or lack of integration as the parasitic enzyme or the fragment with similar enzyme and / or antigenic activity.

Description

FIELD OF THE INVENTION

The present invention relates to the production of protective antigens of proven quality by recombinant deoxyribonucleic acid (D H K) 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, they are of relatively large size and are not distributed · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-ί ..

readily with respect to the cellular host defense system of the host animal, they may be susceptible to antibodies that act by inactivating 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 to the intestinal surface of the parasite, such that antibodies induce binding to the antigens on such internal membranes where the body fluids containing the antibodies serve to

food of the parasite. Such antigens can be defined as hidden antigens as they do not elicit natural immunity to the parasite.

Characteristics of the Prior Art hOqLty-iQM—

We have found that a particularly effective latent antigen is the anthelminthic antigen H11OD derived from Haemonchus contortus, as described in WO88 / 00835 and 90/11086. In W093 / 23542 we have described the following

finding that H110D is a membrane-bound aminopeptidase □ T worm intestine. This enzyme acts as a major enzyme for the conversion of nutrients of a protein nature into amino acids 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 susceptible to the same strategy of latent antigen 6 that 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 intestinal worms ·· · *.

• · ·.

• proteases such as cathepsin. • ··.

• ··.

• · 1 · · · · · · · · For example, a large number of parasites from families

T r: i. hos t ron cj y 1 us,

Nematodirus,

Ascaris, Dirofilaria, Trichuris, · · · · · · · · · · · · ·:

·· * ··

They are present 6 as e.g. different species

Haemonchus,

Ostertaqia,

Dictyocaulus.

Cooperia,

Strong ⁴ / lus and Fasciola;

arthropod species, especially members of the spider and insect classes and in individual ectoparasites, such as mating axis insects (eg members of the exopterigote and endopterigot classes), flies (Lucilia), flies causing myosis, and parasites, lice , worms, fleas and bed bugs

SUMMARY OF THE INVENTION

WO93 / 23542, already mentioned above, also describes the preparation of antigenic fragments from H11OD by recombinant DNA technology-. In this invention, the H11OD material was expressed by E. coli using the pGEX vector and by injecting sheep and triggering the production of anti-HllOD antibodies. In further work, using baculovirus in insect host cells (Sf9 cells), we successfully expressed the 3.5 Kb clone of the H11OD gene. Its nucleotide sequence is shown in Figure 1 (seq. ID Nosl). The corresponding translation is shown in Figure 2 (seq. ID Nos2).

However, it is preferable for the preparation of 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 bonds and other post-translational modifications, such as E.

coli, which produces an insoluble protein that requires refolding and produces a scarce reproduction of the native form.

In addition, mammalian glycosylation is unlikely to induce an immune response resulting from the protective anti-protein response that can be encountered in insect cell line material due to its very different glycosylation structure.

For

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 single human cell line; BNK - Hamster Baby Kidney Cells; VERO and COS, monkey kidney cell line; FR3T3,

Rat fibroblasts; NIH3T3. mouse Fibroblast cell line;

C127I murine breast tumor cell line;

CV-1, African Green Monkey Kidney Fibroblasts;

3T6, mouse embryo fibroblasts; L cells, mouse cell

CHO line, Chinese hamster ovary cell line;

NSO NSI, SP2 and other murine myeloma cell lines and rat myeloma cell lines, such as YB2 / 0 and Y3.

Because the hidden antigens that will be produced are paramount for the processing of nutrients by parasites, enzymes or other Functional proteins having the same activity, the ~ 6 eukaryotic cell lines available are widespread and not only impede the selection of clones, producing the desired antigen, 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 close, at the ends of the protein sequence, to the animals to be protected, so that contamination of the desired foreign external antigen with such endogenous antigen is likely to produce an adverse autoimmune reaction.

Moreover, 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 expression by 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, so is excluded -chemical separation is essentially

free from endogenous antigens having the same enzymatic function as the external hidden antigen.

According to the present invention, we therefore provide 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 integration or lack of integration as said parasitic enzyme antigen or Fragment instead.

Thus, if the host cell: contains an endogenous enzyme, such as an aminopeptidase in the cytoplasm, while the outer enzyme is expressed by a trans-membrane sequence that becomes localized in the host cell membrane, there is no difficulty in extrinsic endogenous separation. cell processing enzymes to separate membrane fragments,

for example, through CentersFunction. Alternatively, the external antigen may be modified to produce a Missing Fragment Membrane Binding Region and if the host cell membrane carries an endogenous enzyme having the same activity as the external enzyme, this will allow it to effect cell division separation. material;

thus, the coding sequence for the transmembrane region of the parasite enzyme can be shifted into the gene to be expressed by a secretion signaling sequence.

Of particular interest are helminth aminopeptidase antigens such as H11OD mentioned above and Fragments thereof. They can be expressed in a wide variety of mammalian host cells using appropriate vectors. For the expression of the H11OD antigen by H. contortus, 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 administration, 6 in accordance with the invention, therefore. that they lack significant

A-like or M-like aminopeptidase activity.

They appear to possess an aminopeptidase enzyme that breaks down alanine peptide bonds, which is poorly associated with the cell membrane, so there is no difficulty in separating the endogenous enzyme from the expressed H11OD that is localized to the cell membrane.

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

Appropriate cell lines for application 6 according to the invention can be selected from pre-existing strains by screening for their profile of appropriate enzyme activity and / or localization by a corresponding appropriate enzyme in relation to the cell membrane or cytoplasm. In a later tear, 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 with 6 suitable nutrient media to compensate for lost enzyme activity. Cells were then exposed to fluorescently labeled antibodies and passed through a fluorescently activated cell sorter '(FACS).

Fluorescently resected detected by mutation for the relevant ·· ··.

«· · • ·!

• ··

-8negative and stability

Cells also gene divisions ·· ··. • · ·. * • ···

J · ·· • · ·.

· · · · Cells of can or · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · загу загу клетки загу загу загу са са са са са са загу са endogenous enzyme

Under? ··; Plowing vectors for different classes of mammalian cell lines are well known in the art.

Generally speaking, they will include a promoter and / or enhancer operatively linked to the gene expressing the enzyme antigen or fragment thereof. Thus, in part, the 3.5 kb fragment of the H11OD 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 WD86 / 05807, W087 / 04462, W089 / 01036 and W089 / 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 applying electrospinning are described by Andreason G.L. and Evans Introduction and expression of DNA molecules in eukaryotic cells by electroporation, Biotechniques ό, 650, ♦ · ··.

• · ·· • ·· • ·· • ·· ···· ·· ·· ·· • ·· -. ♦ • · ·.

• · · · · · · · · · · · · · · · · ·:.

·· ···

-91980.

In general, linear dna is more readily introduced than circular DNA

The antigen

H11OD shows leucine amino peptidase (M-like) methionine (A-like) aminopeptidase activity and is generally preferred because cells are host *: free from at least these types of aminopeptidase activity

The following examples are given for illustration purposes only. In these examples, the figures represent

Figure 1 shows a DNA sequence of 3.5 Kb PCR clone 2 (sequence ID Nosl); and

Figure 1 shows the amino acid translation of

3.5 Kb PCR clone 2 (sequence ID No: 2)

EXAMPLE 1

Enzyme analysis of Cos-Ι cells

Cos-Ι cells were obtained from University of

Surrey in 5 ml DMEM culture media, the slides were divided into 6 two 200 ml flasks, each containing 11 ml from the medium where the cells gave growth to confluence. Cos cells were pooled and then removed from the flask by aspiration using a glass pasteur pipette 6 10 ml PBS buffer. When all cells were 6 suspensions, they were transferred to a universal tube and frozen at - 20 degrees Celsius. The cells were repeatedly frozen and thawed with 6 liquid nitrogen until the cells were destroyed, then rotated to release PBS supernatant (PLS). For the resulting extract, 500 ml of 0.1% Tween and PBS 27. 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, whereas Triton extracts whole membrane proteins.

Spernatants were analyzed at 25 mM against: phenylalanine, gamma-glutamic acid, leucine, lysine, methionine, alanine, alpha-glitamin k, glycine-proline and aspartic acid para-nitroanilidene (p-HA) substrates at pH 7.0 6 HEPES bicarbonate bead. No activity was detected after 30 minutes of incubation at 37 degrees Celsius, so the assay was followed by incubation for 18 hours to determine the presence of any enzyme activity. The specific activities were calculated and the results are shown in Table Να 1.

Higher activity was shown in Tween extraction, where the highest activity was found at

alanine p-HA, activity was demonstrated on Phenylalanine, gamma-glutamic acid, leucine, lysine and methionine p-HA substrates. PLS and TrLS contained little activity only with the assumption of gammaST3P and residual lysine AP and alanine AP.

Table 1

Specific activity (Optical Density / minute / mg protein)

of Cos cells p-ON SUBSTRATE Yield PLS Twl ... S TrLS Phenylalanine 0.0000 0.089 x ΙΟ' — 3 0.000 gamma-glutamic acid 0.0015 x .10 --- 3 0.165 x 10--3 0.033 x 10--3

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-11 (continued) p-NA SUBSTRATE

Yield

PLS TwLS TrLS LEVTSIN 0.0012 X 10--3 0.066 x 10--3 0.000 LYSINE 0.0029 X 10--3 0.273 x 10-3 0.010 x 10— Methionine 0.0011 X 10--3 0.083 x 10-3 0.000 ALANIN 0.0270 X 10--3 1,110 x 10 · -—3 0.159 x 10-- alpha-glutes- 0.0000 0.000 0.000 NEW ACID GLYCIN-PROLINE 0.0007 10 ^l -3 0.000 0.000

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

Enzyme analysis of ovarian cells from

Chinese Hamster (CHO) and NSO to leaflet and

Extracts of cultured CHOs were obtained by the same method described above for C0S-1 cells

These k leaflet and method as extracts

NSOs have already been analyzed against the following paranitroanilide substrates:

alanine, arginine, glycine, alpha-glutamic acid, gamma-glutaminobic acid, leucine, lysine, methionine,

Phenylalanine, proline and glycine-proline, all at mm in

HEAK bicarbonate buffer pH 7.0. The assays were incubated for 30 minutes at 37 degrees Celsius, where the final optical density was taken and the specific activity was calculated

Table 2 shows that CHO cell soluble and membrane associated extracts contained low levels of enzyme activity, except for TwLS activity against glycine-proline substrate

In contrast,

The TrLS extract had low activity in all cases with the exception of activity. • · · · · ······· · ······· · · · · · · · · · · · · · · · The arginine substrate.

Thus, the latter was different from H110D aminopeptidase activity.

Table 2

Specific activity (Optical to CHO cell

p-ON SUBSTRATE PLS / LEVTSIN 0.070 x 10-3 Phenylalanine 0.059 m 10'-3 lysine 0.178 x 10--3 Methionine 0.130 x 10-3 ALANIN 0.148 x 10-3 GLYCIN 0.000 ARGININE 0.078 X 10-3 PROLIN 0.024 x 10-3 ARGININE-PROLINE 0.024 x 10-3 GLYCIN-PROLINE 0.074 x 10-3 alpha-glutes- 0.011 x 10-3 NEW ACID gamma-glutes- 0.030 x 10-3 NEW ACID

Density / minute / mg protein) with nutrient

Yield

TwLS TrLS

0.147 X ιο- «Y 0.106 X 10-3 0.052 X ί ο- -3 0.000 0.123 X ι о- · -3 0.106 X 10-3 0.118 X ιο- ~ r 0.106 X 10-3 0.169 X ί Ο- r3 0.137 X 10-3 0.052 X ι о- -3 0.068 X 10-3 0.118 X 1 Ο- -3 0.889 X 10-3 0.030 X ι 0-- -3 0.046 X 10-3 0.052 X 1 ο- -3 0.061 X 10-3 0.405 X ι 0-- —3 0.068 X 10-3 0.052: X 10-- 3 0.144 X 10-3 0.000 0.053 X 10-3

NSO cells and PLS and TwLS extracts had the same aminopeptidase activity partly against leucine and methionine

(Table 3), . In contrast, TrLS had low levels of such activity, prompting for purification, incl. Triton extraction, as such has recently been applied to the H110D, will happened very low endogenous contaminated activity

enzyme

Table 3

Specific Activity (Optical Density / minute / mg protein) of NSO cell supernatants

p-ON SUBSTRATE PLS LEVTSIN 0.640 x 10 · -3 Phenylalanine 0.108 x 1O'-3 LYSINE 0.136 x 10'3 Methionine 0.280 x 10--3 ALANIN 0.047 x ΙΟ · -3 GLYCIN 0.014 x 10 · -3 ARGININE 0.110 x ίο · -—3 PROLIN 0.014 x IO · -3 ARGININE-PROLINE 0.043 x 10 · -—3 GLYCIN-PROLINE 0.058 x 10 --- 3 alpha-glutamic acid 0.017 x 10--3 gamma-glutamic acid 0.017 x 10--3

Yield

TwLS TrLS 1.840 X ιο- •• jr 0.137 X 10- -3 0.205 X ί Ο- -3 0.087 X ιο- * y 0.188 X ι о- 0.093 X ί ο- • γ 0.764 X 10- · “3 0.099 X ιο- -3 0.150 X 1 O'- 3 0.067 X ί ο- -3 0.045 X ίο ·· ' —.T. 0.019 X ι о- · - Τ% - · 0.114 X 10- "3 0.081 X 1 Ο- -3 0.034 X 1 Ο- —3 0.019 Ι Ο- -3 .0.085 X Ι Ο- “3 0.050 X Ι 0- -3 0.063 In m Ι 0- 0.050 X 10- -3 0.270 X 10 · -— “T. 0.019 X 10- -3 0.040 X 10 · - · _Ί 0.019 X 10 · - · „-Η ·

COMPARATIVE EXAMPLE

BHK cells were obtained by the same method as the Cos-Ι cells described in Example 6, despite the larger quantities (2x1 L cylindrical cylindrical cylinders in 100 ml of medium). The supernatants were analyzed with 25 mM against: Phenylalanine, leucine, gamma-glutamic acid, alanine, arginine asparigine, lysine, methionine, glycine-proline and alpha-glutamic acid para-nitroanilidene (p-HA) substrates at pH 7.0. Analyzes were incubated for 30 minutes at 37 degrees C. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-: 1.4Celsius, where the ultimate optical density was taken and the specific activity was calculated.

BHK cell extracts had significant enzyme activity that was present in all supernatants (Table 4). There was little activity on substrates of gamma-glutamic acid, asparagine or alpha-glutamic

acid. All spernatants showed good activity against lysine pH (which is highest in PLS), leucine, alanine and glycine-proline. Methionine p-HA activity was negligible in F'LS and maximal in TrLS supernatant. ' It will be seen that BHK cells will not be suitable for H11OD expression, 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 / minute / mg protein) of BHK cell supernatants

p-ON SUBSTRATE PLS Phenylalanine 0.102 x 10-3 LEVTSIN 0.320 x 10 ^L -3 gamma-glutamic acid 0.006 x ίο- · —3 ALANIN 0.260 x 10 ^L -3 ARGININE 0.150 x ΙΟ -— 3 ASPARAGIN 0.017 x 10 ^L -3 LYSINE 0.330 x ίο · -— 3 Methionine 0.020 x 10 · -—3 GLYCIN-PROLINE 0.312 x 10--3

Yield

TwLS TrLS 0.080 V 10 ^l —S 0.113 x 10--3 0.340 X 1 o'- -3 0.433 x 10--3 0.000 0.004 x 10--3 0.220 X ίο- ·· —3 0.328 X 10--3 0.200 X ιο- -3 0.344 x 10-- 3 0.015 X ί ο- —3 0.008 x 10--3 0.259 X ι Ο- -3 0.420 x ίο-— 3 0.185 >: ι Ο- 0.355 x 10 · -— 0.318 X Ι ο- 0.225 x 10--3

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-15alpha-GLUTAMY- 0.014 x 10 ^l -3 0.008 x 10-3 0.008 x K> -3 NEW ACID ml of each BHK cell supernatant was added to 25 mM p-nitroancidide substrate in 250 ml of HEPES bicarbonate buffer at pH 7.0 and incubated at 37 degrees Celsius for 30 minutes.

EXAMPLE 2

Cloning of the H110D sequence 6 mammalian expression vector

For DNA cloning, it was

Kb PCR clone 2 H110D described in W093 / 23542

DNA (Sequence

ID No

1) that of the cloned gene 3.5

The sequence of the polymerase chain reaction (PCR) insert is shown in

Figure 1, the amino acid translation of (Sequence ID Nos

2) in Figure 2. This DNA will be excised from the vector

pT7B1ie-T Vector (Novagene) by BamHI digestion and cloned into the BamHI region of the multiple cloned vector region pSPT18 (Boehreringer Mahnheim) to give clone pSPT18-3.5-2. Partially BamHI, digestion from clone pSPT18-3.5-2 was carried out and the linear DNA was purified twice by gel electrophoresis. The adhesive ends were bluntly terminated with dNTF's 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 who had this linker at 5 'end at 3.5 Kb insert, giving them in the ATG framework an initiation site under the control of a T7 promoter.

The modified 3.5 KB b insert from one such clone (clone pSPTlS3.5-2N44) was: · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · mammalian cell using the following strategy:

1. DNA of clone pSPT18 (T7) -3.5-2N44 was digested with the restriction enzyme Smal.

2. The Notl linker was ligated '6 this modified Smal region to give the growth of the Notl branches at the 5' end of the insert preceding the Ncol linker,

containing ATG start region.

3. Purified DNA from a suitable clone was digested with Not1 and Xbal to release 3.5 Kb insert. The F′vul enzyme was added to the incubation to cut the pSF'TIS vector DNA in half, which was necessary because of the similar size of the vector and the insert that made purification difficult.

4. The 3.5 Kb insert was purified on 0.6

0.77. agarose gel.

5. The mammalian expression vector pRC / CMV

was digested with Not1 and Xbal, and the linear plasmid band was purified on an agarose gel.

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

7. Suitable clones were selected to have 3.5 Kb when digested with Notl and Xbal. The clones were named pRC / CMV-3.5-2.

Transfection of mammalian C0S-1 cells

The DNA from the mammalian expression vector with the H110D insert clone pRC / CMV-3.5-2 was precisely purified by centrifugation in a cesium chloride gradient. (Sambrook J., ·· ·· • · · · · · · · · · · · · · · · · ·

--17 ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Fritsch E.F. in Maniats T. Molecular

Cloning, A Laboratory

Cold Spring Harbor Press, 1989

Transient expression of H11OD can be obtained using such purified DNA from clone pCMV-3.5-2 to transfect C0S-1 cells ((obtained from

ECACC, Porton)

Transaction Faction performed by (Cullen B.R., Use of Eukaryotic expression technology in the functional analysis of cloned genes.

Methods: i.n

Guide to mo 1 is si 1 ar c 1 on i n g techniques., Eds S.L

Berger and A.R l <i mma 1., A cad em i c:

Press, 1987, pp. 684-704)

The cells were cultured in

Dulbecco's Modified Eagles

Medium (DMEM,

Gibco

BRL) and 107.

Veal Fetus Serum (FCS, Gibco BRL) and expression analyzed followed by

48-72 hours incubation.

Transfection of Chinese Hamster Ovary (CHO) cells

The DNA vector was transfected into CHO cells (obtained from ECACC, F'orton) using a calcium phosphate method (using the method described by Cullen BR. Eukaryotic expression technology is applied in the functional analysis of gene cloning. Methods in Enzymology: Guide to molecular cloning techniques, Eds SL Berger and A .. R. Kimmal, Academic Press, 1987, pp 684-704). The transformed cells were cultured in DMEM and 107.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 800 pg / ml. The transformed cells were then cloned by limiting dilution 6 microtiter plates.

····

-18-Analysis of transfected mammalian cells

Transformed and untransformed CHO cells can be transferred to growth on growth glass for immunofluorescence analysis. Growth took the form of small colonies fixed with methanol and probed with general anti-HHOD antisera, followed by fluorescently stained (eg FTTC) conjugated anti-ovarian immunoglobulin antiserum. A fluorescence search microscope was applied

of positive colonies.

Transformed cell lines were broken into RIPA buffer (150 mM sodium chloride 17. Nonidet P40, 0.57. Deoxycholate, 0.17. Sodium dodecyl sulfate, 50 mM TRIS-HC1 pH 8.0) by removing the culture medium from the cells and then gently shaking the cells. for 5 minutes in RIPA buffer. The cells were then transferred to a tube and rotated in the microfuge at full speed for 15 minutes to give a clear lysate, which was

walls in a new tube equivalent to 2 x 10 ^l 5

Aliquots of this lysate cell were electrophoresed on SDS polyacrylamide gel electrophoresis (SDS-PAGE) and the 6 gel proteins were transferred to a nitrocellulose membrane by

Western blot. The membrane is broken down, it is possible to include a period treatment period and analysis with antiserum with growth to different forms of H110D 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.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ······· 4 · · · · · · · · · · · · · ·

-19Extracts of transformed and untransformed cells prepared according to Example 1 and lysates obtained using RIPA were subjected to enzyme activity assay, just as described in Example 1. Cells transformed with H11OD showed higher levels of aminopeptidase activity compared to the untransformed leaflet and.

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 method of Sambrook, J., Fritsch, E.F. and Manitias, T. Molecular Cloning, ALaboratory Manual, Second Ed Cold Spring Harbor Press, 1989. 10-20 pg 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 DMA fragments separated by gel electrophoreses. J. Mol. Biol. 98 p. 503, 1975). 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 anal is of RNA isolated from these cells. RNA was extracted from cells using RNAsol Cinna / Biotecx, 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. and 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

1. GENERAL INFORMATION (i) APPLICANT OF THE INVENTION:

A. NAME: PITMAN-MOORE, INC.,

B. STREET: 421 EAST HAWLEW STREET

B. CITY: MUNDELEIN

D. STATE: ILLINOIS

E. COUNTRY: USA

F. POST CODE: 60062

APPLICANT OF THE INVENTION:

A. NAME: THE AGRICULTURAL AND FOOD RESEARCH COUNCIL

B. STREET: BABRAHAM HALL, BABRAHAM

B. CITY: CAMBRIDGE

D. COUNTRY: UNITED KINGDOM

E. POST CODE: GB2 4AT

Applicant HA INVENTION:

A. NAME: EDWARD ALBERT MUNN

B. STREET: 72 STATION ROAD, FULBOURN /

B. CITY: CAMBRIDGE

D. COUNTRY: UNITED KINGDOM

E. POST CODE: GB1 5ES

Applicant HA INVENTION:

A. NAME: MARGARET GRAHAM

B. STREET: 17 BAWTREE CRESCENT, LINTON

B. CITY: CAMBRIDGE

D. COUNTRY: UNITED KINGDOM

E. POSTAL KDA: GB1 6X0

Applicant HA INVENTION:

A. NAME: TREVOR STANLEY SMITH

B. STREET: 14 THE GROVE, LINTON

B. CITY: CAMBRIDGE

D. COUNTRY: INITED KINGDOM

E. POST CODE: 6B16UQ

APPLICANT HA OF THE INVENTION;

A. NAME: TIMOTHY PETER ROLPH

B. STREET: 42 LITTLEWORTH

B. TOWN: WHEATLEY

D. STATE: OXON

E. COUNTRY: UNITED KINGDOM

F. POST CODE: 0X33 ITR

Applicant HA INVENTION:

A. NAME: SUSAN ELIZABETH NEWTON

B. STREET: 3 LEBANON STREET

B. CITY: STRATHMORE

G. STATE: VICTORIA

E. COUNTRY: AUSTRALIA

F. POST CODE: 3401 (ii) TITLE OF THE INVENTION: EXPRESSION OF PROTECTIVE

ANTIGENS (iii) SEQUENCE NUMBERS: 2 (iv) COMPUTER CONFIGURATION FORM:

A. ENVIRONMENTAL TYPE: DISC FLOP

B. COMPUTER: IBM PC COMPATIBLE

B. OPERATION SYSTEM: PS-DOS / MS-DOS

G. SOFTWARE PRODUCT: Patentin Release # 1.0,

Version «1.25 (EPO) (v) APPLICATION DETAILS:

A. APPLICATION NUMBER: GB 9302302.6

B. DATE OF THE APPLICATION: FEBRUARY 05, 1993

2. INFORMATION ON SEQ ID NO:

(i) HA SEQUENCY CHARACTERISTICS

A. LENGTH: 3303 BASIC Pairs

E. TYPE: NUCLEIC ACID

B. CONNECTION: SINGLE CHAIN

D. TOPOLOGY: LINEAR (ii) MOLECULLA TYPE: kDE30XYROBYNUCLEIC ACID (iii) SEQUENCE DESCRIPTION: SEQ ID No 1:

GCTGAATCTA ACTCCAATCC GTCTTATTGT CGCATTATTT CTAGTAGCTG CTGCAGTCGG 60 SSTSTATT 120 AGGGAAGGAT GGTCTCACCT ATTACTTTAC TCGCAAAGCG 1TCGATACCT CAGAAAAGCC GATACTGGTG GCAAGGACAA AGACATTCCT CCCTCTGCGG CGGAACTACT ISO CCTTCCAAGT AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT ATCTACCTGC 240 TTATGTGGAC TTCCCACCGG AGAAAAACCT CACATTCGAT GGGCGTGTGG AAATATSAAG 300 GGTTGTAATT GAGCCAACAA AGGTTATCGT ACTCAATTCA AAGAAGATCT CTGTAATACC 360 CCAAGAATGT GAACTGGTAT CGGGCGATAA AAAACTCGAA ATTGAAAGTG TAAAGGAGCA 420 CCCAAGACTG GAAAAGGTTG AGTTTCTTAT CAAAAGCCAA CTGGAAAAAG ATCAACAAAT 480 CTTGCTCAAG GTCGGCTACA TCGGTCTCAT CAGCAACAGC TTTGGTGGAA TCTACCAGAC 540 CACTTATACC ACCCCGGATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT 600 AGATGCTCGT CGAATGGTAC CATGCATGGA TGAACCGAAA TACAAAGCAA ACTGGACCGT 660 TACTGTCATT 720 TGGAGAGATC CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA AGTGGTGATT GGATCACA7C GAAGTTCTTG ACTACTCCAC GGATGTCATC 780 CTACTTGTTG GCAGTTATGG TTTCAGAATT TGAATACATC GAAGGTGAAA CAAAGACGGG 840 TGTTCGGTTC CGTATATGGT CACGCCCAGA GGCAAAGAAG ATGACACAAT ATGCTCTGCA 900 ATCTGGTATC AAGTGCATAG AATTCTACGA AGATTTCTTT GATATCAGAT TCCCTCTGAA 960 GAAACAAGAT ATGATTGCCC TTCCTGATTT CTCTGCCGGT GCCATGGAGA ATTGGGGCCT 1080

· · · · · · · · · · · ·

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCTCAT CAGTGGTTCG GCGACTTGGT 1140 TATTGGAGCT GGTCAGATAA CTCAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA 1200 TGTACTTGAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG 1320 AATCGACAAA GCTGCAGAAG TTGAAGAAGC CTTTGATGAT ATCACATACG CCAAAGGAGC 1380 TTCTGTTCTT ACTATGCTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC 1440 GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1'560 TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG TGGAGAAAGA 1680 GAAGTACGGT CACCCGAAAT ACGGATTTAA ATGGGATATT CCACTGTG6T ATCAGGAAGG 1740 CGATAAGAAG GAGATAAAGO 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 CGGAATATCT 2040 ACCATTAGAA ATCGCAATGT CCGG6ATCTC TTCGATTTTG AAATACTTCG GTACCGAGCC 2100 AGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAACCGATGT ATGAAAAAAG 2160 CAGTATCGAC TTCATTGCCA ATAACTACAG AAATGACAAG CTGTTTTTCC AAATCAACCT 2220 CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTCGGATCG CAAGACTGCA GGAAGAAATA 2280 TAAAAAACTT TTCGATGACG AAGTCATGAA CAAATGCAGG GATGGTCAAG CAGCAACCGA 2340 ATGCGTAAGA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 24O0 CGGTGATTAT GCTTCCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TCGCCCTAGA 2460 AAAAGACTTC CTACGCCTAG CATTGGGATG TCATAAAGAT GTTACTGCTT TGAAAGGACT 2520 TCTCTTGCGG GCTCTGGACA GGAATTCGTC 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 GAAGGAATAT 2940 GACAGGGAAC TGACTGTCTG TTGGTCACTG ITCCACTGAA TGGAAGTTTT TACCTACAAA 3000

AATTTTATTS GTTATATTTG 3060 AAAGCTCAGT ATTGCAACCA 3120 STATISTICS STSTSTAST 3180 TCAGAACACT TTSTSTSAA 3240 TACAATTGTA TAGATTAGTT 3330

GTGAACAATA TTACTTTCGC

GAATTCGGAA ATTTGTTCAT TAGCTTCTTG TTTGTTTTTT ATCTTATAAA TATTGATGGT ·· ····

• · · • ··· · tvTSASTTb AATAGTAAAC TTSATSAAAT TGTTATCTTC ATTCGTTTGT AGTCTGTTGC TTTGATTGTA TTGATCGTTT TOOAAAAAAA AAAAAAAAAA

AAA

3303

2. INIRKY 3A SEO 10 Ho 2:

(i) CHARACTERISTICS OF THE HA PRIEST

A. IMWift: W AMIGISELM

B. VIL: AMNHISELMNA

C. BRANCH: ELECTRICAL CHAIN

D. Topology: Linear (ii) Mouse Type: PEPTI1 (j1 SEQUENCE DESCRIPTION: SEC ID Ito 2:

Levcin Asparagine Levcin Threonine Proline Ivolevitchn Arginine Levcin Izolevtsin Valin Alanin Jaevitzn Venilalanin Levitzin Valian Alanine

5 $ 101

Alam »Alanine To You» Glitsnn Lionesses Isolevtsin Series Glycine Singers Threonine Thyroid Thyroid Venylalanine Threonine Arginine Lysine

2530

Alanine Venilalanin Asparaginova Threonin Serin Glitaninova Yaivin Folin Gmtz »Lizin Asparaginova Asparaginova

3500

Threonine Glycine Glycine Lysine Asp.k.a. Yaevin Asp.k.-Asparigin Serin Proshn Serin Alanin Alanine Glnt.k.-Levitsi Levtsm Kevtsin 45 50 55M

Promm Serin Asparagin Ivshevtsin Kivin Proline Kevtsin Serin Thyroin Asp.k.-Levits Threonine Isoleucine Lysine Threonine Thyroin Levits 05 7075

Proline Glycine Ttsuvin Valin Isp.k-na venilalanin Proline Tsulin Tynlin Glnt.k-Aiain Asparagin Levcin Threonine venilalanin Asp.k-na Glycin 80 8570

Arginine Valin Glnt.k-on Isolevtsin Series Mionin Valin Valin Iolevad Glnt.k-folin Threonine Lysine Serine Ivolevitn Valin Levcin

100 105110

Asparagine Serine Lysine Lysine Isoleucine Serine Valine Isoleucine Proline Glianin Glt.k-cysteine Glt.k.-levi * »Valin Serin Glitznn

115 120125

Lysine Glsin Lysine Kevtsin Glt.k Izolevtsin Gln.k Serine You »Lysine Gltz Histidine Proline Arginine Levcin Glnk

130 135100

Lysine Valin Glnt.k-of-Venylalanine Levzn Isoleucine Lysine Serine Glntamin Levcin Glnt-k-of Lysine ftcn.K-of Glntanii Glntanin Isolevts 105 150 155H

Levcin Levin Jawin Valin Glycine Tiroain Isolevtz in Glitsn Levin Isolevtsin Serine Asparagine Serine Venylalanine Glycine Glycine

105 170175

Tyrosine Isolation Glanthanine Threonine Threonine Thyroid Triptovan Tryptophan Prom »Glycine Asp. Tryptophan Prolin Yaivin Isolevcin Alanine

185

170 ·· ·· ····

Alanine Valley Serine Glantanin Asparagine Glint Proline Izolebtsin Asp Alanine Arginine Arginine Methionine Valine Proline Cysteine

195 200205

Metiont Asp.k-on Glug.k-on Proline Yaisin Tyropn Yaisin Alanine Asparagine Tryptophan Threonine Valine Threonine Valine Isolebcin Histidine

215 215225

Prolin Yaisin Glycine Threonine Drink Alanine Walcht Serine Asparagin Glycine Isilebcin Glt.k-valin Asparagin Glycine Asp.k-na

225 '235 235240

Glycine Glnt.k-of Isolebtsin Serine Glycine Asp.-k of Tryptophan Isolebtsin Threonine Serine Yaisin Benylanthele Yaebtsin Threonine Threonine Proline

255 255255

Arginine Methiontin Serine Serine Tyrosine Yaebtsin Yaebtsin Alanine Valin Mepinin Valin Serin Glzt.k-of Benilalanin Glnt.k-Tyrosin

245 245275

Isolebcin Glnt.k-Glycine Glnt.k-th Threonine Yaisin Threonine Glycine Valin Arginine Benylalanine Argine Isolebcin Tryptophan Serine Arginine

275 285285

Proline Glnt.k-na Alanine Yaisin Yaisin Methionine Threonine Glntanine TRrosin Alanine Yaebtsin Glntanin Serine Glycine Isolebcin Yaisin 295 295355

Cysteine Isolebcin Glnt.k-of benylalan Tyrosine Glnt.k-of Asp.k-of benylalanine benylalan Asp.-k Isolebcin Arginine benylalan Proline 305 315315

Yaebtsin Yaizin Yaizt Glntanin Asp.k-na Methionine Isolebtsin Alamzh Yaebtsin Proline Asp.k-na benilalanin Serine Alanine Glycine Alanine Methionine 325 325 335335

Asparagine Glt. Tryptophan Glycine Yaebtsin Isolebtsin Threonine Tyrosine Arginine Asparagine Gls. Serin Yaebtsin Yaebtsin Tyrosine Asp.kn 340 345355

Asp.k-na Argint Benilalanin Tyrosine Alanine Proline Methionine Asparagine Yaizin Glntanine Arginine Isolebcin Alanine Arginine Isolebcin Valine 355 345345

Alanine Histidine Glnt.k-na Yaebtsht Alanin Histidin Glntanin TriptoFan benilalanin Glycine Asp.k-na Yaebtsin Walsh Threonine Methionine Jaisin

375 375385

TriptoFan TriptoFan Asp.k Asparip Yaebtsin Tryptophan Yaebtsin Asparagin Glnt Glycine Benilalanin Alanine Arginine Benilalanin

385 390395

TriptoFan Glnt.k-of benilalanin Isolebtsin Glycine Alanine Glycine Glntanin Ioolebstt Threonine Glntanin Asp.k.sp.sp.-Alanine Arginine 400 405415

Methionine Arginine Asparagine Tyrosine Benylalanine Yaebtsin Isolebtsin Asp.k.-Valin Yaebtsin Glt.k.-Arginin Alanin Yaebtsin Yaizin Alanin 415 425425

Serin Valin Alanin Aspen Serin Serin Histidine Proline Yaebtsn Ser Benilalanin Arginin Izolebtsin Aspen Jaenin Alanin Alanin 435 435 440445

Glit.k-na Glnt.k-na Alanine Benilalanin Asp.k-Asp Asp.k-Izolebtsin Threonine Tyrosine Alanine Yaizht Glycine Alanine Serine Valin Yaebtsin

455 455445

Threonine Methionine Yaebstn Arginine Alanine Yaebtsin Isolebcin Glycine Gln.k.-Gln.k.-Yaizin Histidine Jaisin Histidine Alanine Valin Serine 445 475 475485

Gluganin Tyrosin Yaebtsin Yaizin Yaisin benilalanin Serin Tyrosine Serin Asp.k.-alanin Glit.k.-alanin Treonin Asp.k.-yaebtsin

485 495495

Tryptophan Alanine Valia Benilalanin Asp.k.-Glnt.k.-Valin Valin Threonin Asp.k.-Valin Glnt.-Glycin Prolin Asp.k.-Glycin

555 555515 ·· ····

Jaiain Proline Methionine Jaiain Threonine Threonine Glnt.k-of Venylalanine Alanine Serine Glntanin Tryptophan Threonine Threonine Glntanine Methionine

515 525525

Glycine Vekmalanin Proline Valin Iaolevtsin Serin Valin Alanine Glnt.k-na Venilalanin Asparagin Serin Threonine Threonine Yaevtsin Yia <m 535 535550 feflUPH Threonine Glntam® Serine Arpnm Thyroniman Glakn-Alanine Alanine on Yaiain Glnt.k-on

545 555 555560

Yiaia Thiroin Arginine Cystodine Proline Yaiia Thiroain Gad Venilalanin Yiaiain TriptoFan Asp.k-na Iklevtsin Proline Yaevtsin Tryptophan

565 575575

Thyroin Glntamsh Glntanin Glycine Asp.k-na Yaizin Yaiein Glnt-kk Iaolevtsin Yaiein Arginine Threonine Tryptophan Yaevtsin Arginine Argins

5S5 58559 «

Glnt. Asp. Of Prol. Jaevitzn Tiroain Yaevtsin Histidin Valin Serin Asp. Of Alanine Glycine Alanine Proline venilalanin Valin Valin 555 655655

Asparagine Alanine Asp.k.-arginine Tiroein Glycine Venilalanin Tiroain Arginine Glantanin Asparagine Histidine Asp.k-alanin Asparagine 615 615625

Glycine Tryptophan Yaiain Yaiein Yaolevtsin Yaolevtsin Yaiein Glntanin Yaevtsin Yaiein Asp.K-Asparagine Histidine Glnt.k-Valin Tiroain

625. 635 635645

Serine Proline Arpnm Threonine Arpmin Asparagine Alanine Iaolevtsin Iaolevtsin Serin Asp.k-alanin Venilalam Alanin Alanine Alanine Alanine

645 650655

Alanine Threonin Asp.k-alanin Iaolevstmg Glnt.k.-Tyroein Glnt.k.-threonin Valin Venilalanin Glnt.k-yaevtsin Yaevtsin Asparagin Tyros

660 665670

Alanine Glnt.k.a. No. Aain Gl.nt.k.-Threonine Glnt.k.-Thyroein Yaevtsin Tsyulin Yaevtsin Glnt.k.a. Iaolevtsin Alanine Methionine Serine Glycin

675 680685

Yaolevtsin Serin Serin Yaolevtsin Yaevtsin Yaiain Thyroin Venylalanine Glycine Threonine Glt.k-on G | rollin Glnt.k-alanin Yaiain Proline 650 655700

Alanine Glountnn Threonine Thyroin Nethionine Methionine Asparagine Yaolevtsin Yaevtsin Yaiain Proline Methionine Thyroaine Glnt.k-Yaiain Serin

705 710 715720

Serine Iaolevtsin Asp.k-na Venilalanin Iaolevtsin Alanin Asparagin Asparagin Tiroain Arpmin Asparagin Asp.k-Yaiain Yaevtsin Venilalanin

725 730735

Venilalanin Glantanin Iaolevtsin Asparagin Yaevtsin Glntanin Yaiain Asp.k.-Valin Iaolevtsin Asp.k.-Metshnin Venilalanin Cysteine Alanine 740 745750

Yaevtsin Glycine Serine Glntanin Asp.k.-cysteine Arginine Yaiain Yaiain Tyroain Yaiain Yaiain Yaevtsin Venilalanin Asp.k-Asp Asp.k Glnt.k-na 755 760765

Glt.k-na Valin Methionm Asparagin Yaiain Cysteine Arpmin Asparapm Glycin Glntanin Alanine Alanine Threonine Glt.k-cysteine Valin Argon 770780

Yaolevtsin Alanine Alanine Proline Yaevcin Arpmin Serine Serine Valine Thyroin Cysteine Thyroin Glycine Valin Yaiain Glnt.k-on Glycine 785 750 755800

Glycine Asp.k.-Tiroain Alanine Serin Asp.k.-Jaiain Valin Methionine Glnt.k.-Yaevtsin Tiroin Threonine Alanine Glnt.k.-Threonine

805 810815

Yaevtsin Alanin Yaevtsin Glnt.k-na Yaiin Asp.k-na Venilalanin Yaevtsin Arpmin Yaevtsin Alanin Yaevtsin Glycine Cysteine Histidine Yaiain 820 825830

ф ф ф •

FF • f f •

·· ···· f fff f

• • Ф ф · ф ф · ф

Ф • ф ф · ·· ······

-26Asp.k-na Wali Threonine Alanin Yaevtsin Yaizin Glycine Yaevtsin № Ytsin Yaevtsin Arginine Alanin Yaevtsn Asp.k -s Arginin Asparagin 835 840845

Serin Series Oenilalanin Valim Arginine Netionin Glzamin Asp.k.-Izolevtsin Proline Serin Alanin Oenilalanin Asparagin Asp.k.-Valim 850 8 »860

Alanine Alanine Asparagine Proline Isoleucine Glycine Glycine Glzt.k-of Oenylalanine Isoleucine Oenylalanine Asparagine Oenalalanm Yaevtsin 865 870875

Isolevtsin Glzt.k-na Arginin TriptoFan Proline Asp.k.-Izolevtsh Izolevtsin Glzt-kk Serin Izolevtsin Glycine Threonine Jiain Histdin 880 885800

Threonine Tyrosine Valim Glzt.k-of Vision Valim Isolevtsin Proline Alamn Tsistesh Threonine Serine Glycine Isoleucine Arginine Series Glutamine 895 900 905910

Glutamine Glutamine Izolevtsin Asp.k-na Glztamin Yaevtsn Yaizin Asparagin Yaevtsin Glztamin Yaizin Asparagin Glycine Netionin Asparagine Alanine 915 920925

Arginine Glutamine Oenylalanine Glycine Alanine Fenilalanche Asp.k.a. Yaizin Alanine Izolevtsin Glzt.k.-Argimm Alanin Glztashn Asparagin

930 935900

Arginine Valin Asp Tryptophan Izolevtsin Yaizin Yaizin Histidine Oenylalanine Glutamine Yaizin Yaevirn Alanine Alanine Phenylalazine Oenylalanine 945 950955

Yaisin Yaisin Alanine Threonine Yaevtsn

960

Claims (4)

Patent CLAIMS! 1. A method for the expression of an enzyme antigen, which is inherently a parasitic intestinal membrane-bound enzyme or instead of a Fragment, you have, like, enzymatic and / or antigenic activity, 6 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 actually free of endogenous enzymes having (a) the same function and (5) the same cellular membrane integration or lack of integration as said pa a developing enzyme antigen or a Fragment instead. The method of claim 1, wherein said antigen is a helminth enzyme. The method of claim 1 or 2, wherein said antigen is an aminopeptidase enzyme. 4. The method of claims 1, 2 or 3, wherein said antigen is H110D or instead an antigenic fragment. The method of claims 3 or 4 in which the aforementioned host cells do not have significant aminopeptidase A-like or aminopeptidase M-like activity. The method of claim 5, wherein said host cells are COS-1 or CHO to the leaflet and 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