CN1187851A - Modified polypeptides for enhanced immunogenicity - Google Patents

Abstract

The invention relates to a recombinant polypeptide showing enhanced immunogenicity, comprising a glycosyl-phosphatidylinositol structure. The polypeptide is for example an antigen, such as a parasite polypeptide, in particular a Plasmodium falciparum polypeptide, such as the circumsporozoite protein (CSP). The invention further relates to a recombinant DNA vector for expressing the polypeptide, methods of inducing an immune response directed to one or more epitopes of the polypeptide, and methods for the production of the modified polypeptides vaccines, comprising the polypeptide.

Description

Immunogenic modified polypeptide with raising

The present invention relates to demonstrate the immunogenic recombinant chou polypeptide of raising, particularly relate to polypeptide by the fat structural modification.The present invention relates to parasite polypeptide specifically, more specifically to the circumsporozoite protein matter of the Plasmodium falciparum by such fat structural modification by the fat structural modification.The vaccine that the invention still further relates to the immunoreactive method of inducing, the method that is used to produce said polypeptide, carrier and host and comprise said polypeptide at described modified polypeptides.

The system of immunity is a mixture, and is also insufficient to its understanding.Foreign immunologic is former only to be had part to illustrate by the host immune system identification mode.The mammiferous immune response of contact exogenous protein can change to a great extent.The preparation have increase immunogenic polypeptide any method all highly significant, it can overcome other faint defective that causes the immune epitope site.

A kind of method of improving the immunization that adopts recombinant protein is to produce a plurality of epi-positions site from more than one protein that derive from one or more infectious agents.This method makes can obtain not expensive multivalence and more effective vaccine, thereby can cause simpler and safer immune medication system.Yet, but such method means all capacity production of every peptide species, and every kind of specific purification scheme of polypeptide needs that is used for immunity is to guarantee optimal quantity.Find that following method is a kind of challenge: wherein can induce the immune response of the anti-specificity epitope of intensive, but immunogen quantity is low or be present in other polypeptide mixture, under some interior state, observes as it.

Plasmodium falciparum is modal malaria disease substance, finds with different forms in insect and human host.In Mammals, use the parasite form of deactivation to demonstrate up-and-coming result as vaccine.Yet main restriction is such fact: its sporozoite can not be cultivated, and must separate from mosquito sialisterium, and this has hindered the parasitic use that obtains the deactivation of protection with this.

In order to address this problem, the gene of expressing the specific protein of the different plasmodium form of coding in the allos recombination system also successfully is used as the potential host antigen to a certain extent.In addition, make the zone corresponding with limiting peptide antigen be used for protection research, this has demonstrated the limitation of such immunity.Circumsporozoite protein matter (CSP) is one of antigen that is present on the Plasmodium falciparum sporozoite surface of finding in tissue after insect bite infects.This protein is synthesized as polypeptide precursor, and it is formed by amino terminal signal sequence (add and remove man-hour), being called area I and area I I (they are included in the conserved sequence between the different plasmodium species) lateral big central repeating structure territory and hydrophobic terminal carboxyl(group) structural domain.It is effective B-cell epitope of Plasmodium falciparumCSP that the repeating structure territory NANP that is made up of the linking repeat region of amino acid family l-asparagine-L-Ala-l-asparagine-proline(Pro) (ASN-ALA-ASN-PRO) demonstrates.The synthetic peptide that comprises such repeat region is to a certain extent successfully as the subunit vaccine in the protection research.The proteinic T-cell response of CSP element is positioned at repeats pulsating outside.In all experiments up to now, carried out with the antigenic immunization experiment of large-scale purification.

An object of the present invention is to provide the protein of modification, as Plasmodium falciparum protein, even parasite antigen does not have purifying, this protein also can cause the intensive immune response.This method is for further exploitation is not too expensive but the whole-cell vaccines of more effective immune medication system are favourable.

According to the present invention, now found Portugal base phosphatidylinositols (GPI) anchor is added into and to have caused the immune response higher in the antigen than the corresponding antigens of no anchor.

Thereby the invention provides the immunogenic recombinant polypeptide that demonstrates raising, this polypeptide comprises increase is compared in initiation with the corresponding polypeptide of no anchor immunoreactive Portugal base-phosphatidylinositols structure.Described polypeptide can be an antigen, and parasite antigen preferably is as the Plasmodium falciparum antigen of Plasmodium falciparum circumsporozoite protein matter (CSP) or its modifying protein and so on.

In this specification sheets and claims, use phrase " or its modifying protein " to be meant and mix any derivative of the circumsporozoite protein matter of the enough immunogenicities that demonstrate the induction of immunity reaction.Therefore, the present invention not only comprises complete protein, and comprises its fragment or mutein.

In this article by the present invention being described with reference to Plasmodium falciparumCSP protein.Yet, the invention is not restricted to this specific antigen.For those of skill in the art, can not carry out too much experiment with the alternative CSP of other desirable antigen usually and obtain advantage of the present invention.The present invention is based on and find that interpolation GPI-anchor to any polypeptide can improve its immunogenicity.

In allos recombination system (intestinal bacteria, yeast, vaccinia virus, baculovirus, Salmonellas, dictyostelium discoideum), obtained the expression of CSP.The species that have now found that mucus mould dictyostelium (Dictyostelium) can be used as the effective eukaryotic expression system that produces recombinant protein.And, to compare with other expression system, completely stable CSP polypeptide can produce (Fasel, N., Begdadi-Rais, C. in dictyostelium, Bernard, M., Bron, c., cORRADIN, g. and Reymoud, C.D., (1992), gene, 111,157-163).Like this, this system can be used to obtain strong and persistent immunoprotection, because full CSP carries B-and T-cell epitope.

And dictyostelium has the biotechnology potentiality.It is the organism of free living, is easy to growth and maintenance.Bacterial strain can be grown on bacteria culture medium, and the doubling time is approximately 3 hours, can grow to high-density and (reach every liter 10 in bacterial suspension ¹⁰Individual cell) or in the semisynthetic medium that comprises glucose, peptone and yeast extract grow, the doubling time is about 12 hours.

Be made up of the g and D phase life history of dictyostelium.Growth period starts by hungry, it is characterized in that previous individual cells polymerization to form multicellular organism, and this multicellular organism differentiation produces spore then.On bacterium or germinate in the presence of the ripe spore abundant half a lifetime and cause upgrading growth.In the growth cycle process, produce propagable factor, be attached on its acceptor one of them induces the genetic transcription (referring to Loomis, the growth of dictyostelium discoideum, Acad. press, 1982) of a group-specific at least.Growth properties of dictyostelium discoideum (Dictyostelium discoideum) and conversion capacity provide the possibility of expressing exogenous protein, because cell can be grown with low cost concerning bacterium, the expression of specific protein can be by the control of the hunger in simple culture media.At last, the free living organism of safe, nontoxic, the no pathogenicity of dictyostelium discoideum, it is the candidate (if can not be the mankind, at least can for the animal doctor's) of whole-cell vaccines exploitation.

Like this, dictyostelium discoideum can by with the suitable carriers transformant, allowing under the environment of expression of polypeptides culturing cell and not essential ground isolated polypeptide to be used to produce modified polypeptide of the present invention.Suitable carriers comprises the dna sequence dna of coded polypeptide, and this dna sequence dna adds sequence with the glycolipid anchor that is positioned its downstream and is operably connected, and suitable transcription initiation also operationally is connected with it with terminator sequence.The dna sequence dna parasite antigen of preferably encoding is as the Plasmodium falciparum antigen of Plasmodium falciparum circumsporozoite protein matter (CSP) or its modifying protein and so on.

Many cell surface proteinss in various organisms are anchored in the film bimolecular lipid membrane by Portugal's base phosphatidylinositols (GPI) structure.This composite structure is synthetic and transfer on the glycoprotein in endoplasmic reticulum as the precursor glycolipid.The GPI anchor of implementing only transfer on the specific polypeptide in appropriate signals sequence (this paper as " the glycolipid anchor adds sequence) is included in the C-stub area of target protein, be only possible.This signal sequence demonstrates by one group of 10-12 residue in the upstream of hydrophobic C-end sequence to be formed.In dictyostelium, specific protein demonstrates the grappling by GPI.C-end sequence antigenic determinant is defined as one of these protein, promptly contact site A (Noegel, A., Gerisch, G., Stadler, J. and Westphal, M., (1986), the EMBO magazine, 5,1473-1476).The GPI anchor that shifts polypeptide can pass through specificity Phospholipid hydrolase (GPI-Phospholipase C or D) cracking, causes as separate the modification of the protein hydrophobic matter that detects by the difference of specific washing agent TX-114.

Therefore, according to the present invention, the glycolipid anchor that described recombinant DNA carrier comprises derived from dictyostelium discoideum contact site A adds sequence.

The present invention also provides the method that produces at the antibody of one or more epi-positions of polypeptide, and this method comprises separates consequent polypeptide with polypeptide immune appropriate host of the present invention (for example Mammals) and subsequently not essentially.Thing can use whole serum as an alternative.Preferably, Mian Yi polypeptide takes the full cell of the host cell of express polypeptide to split born of the same parents' product form.

An example in the present invention is, by the immune response in the full cellular lysate initiation mouse of injecting the CSP dictyostelium of expressing Portugal's base phosphatidylinositols grappling.And specific immune response has only acquisition when the CSP epi-position is connected with GPI, demonstrates immunoreactive reinforcement.

The invention still further relates to the vaccine of immune mammalian hosts.This vaccine comprises the modified polypeptide of the present invention that exists with immune protective number and suitable vehicle.The immunoprotection amount comprises, for example, and about 1-5 * 10 ⁷, particularly 2 * 10 ⁷Individual said polypeptide of coding and the glycolipid anchor used adds the content of peptides of the carrier transformed host cells of sequence.

The following example is used for illustrating the present invention, but should not be considered to the restriction to its scope.

Embodiment

1. foreword

The following example has been instructed by the glycolipid anchor being added dictyostelium discoideum into and being used for the production method of Plasmodium falciparumCSP of the modification of immune medication system.In detail, add signal sequence expression CSP polypeptide by in mucus mould dictyostelium discoideum, merging leading peptide with the Portugal's base-phosphatidylinositols (GPI) that contacts site A derived from dictyostelium.

With the full cellular lysate immune mouse of dictyostelium of expressing this GPI modified polypeptide.Two different districts of the antibody recognition polypeptide that produces.Like this, the GPI modified polypeptide can be expressed in the dictyostelium cell.The polypeptide of unpack format and the cell that comprises polypeptide can be used for having immunity, the immunization protocol of diagnostic test or fundamental research potentiality.

2. material and method

The method of using in the following experiment is made up of many technology that know and easy handling concerning the technician of molecular biology, protein chemistry and field of immunology.It is not the such method of always detailed description.

Enzyme uses from the commercial source acquisition and according to supplier's scheme.

Bacteria culture medium and current clone technology have description in (molecular clonings: laboratory manual, CSH press, 1989) such as Sambrook.

Monoclonal antibody and NANP are from F, and Sinaglia (Beffman La Roche company limited, Basel) obtains.

3. the structure that comprises the CS of plasmid

3.1.pEDII-CS?49

Expression vector pEDI-CS is by comprising breeding in prokaryotic hosts and keeping pVEII carrier (Maniak and the Nellen of important element (replication orgin and ampicillin resistance gene), (1990), nucleic acids research, 18,5375) and the coding neomycin resistance gene (under the control of dictyostelium Actin muscle 15 transcription units) of giving the Tn903 coding of eukaryotic cell Geneticin (G418) resistance form.The existence of Discoidin1 promotor makes the growth that can carry out downstream sequence and the expression of Actin muscle 8 sequences control, and guarantees the correct termination of RNA.

For the structure of pEDI-CS expression vector, at first 1161bp restricted fragment (Caspers, the P. of the HaeIII+RsaI of CS NF54 gene, Gentz, R., Matile, H., Pink, J.R. and Sinigaglia, F., (1989), Mol.Biochem.Parisitol., 35,185-189) after filling up, be inserted in the Asp718+BamHI site of pVEII by the Klenow archaeal dna polymerase.At Applied Biosystem Model, composite coding contact site A (CsA) leading peptide adds the DNA chain of the sequence of 3 seed amino acids on the 380B dna synthesizer subsequently.The nucleotide sequence of synthetic leading peptide is as follows:

5-ATGTCTAGATTTTTAGTATTGATAATATTATATAATTTT

AAATAGTGCACATTCAGCTCCAACCCAGGATCCATG-3 and by importing flat terminal fragment in the M13mp18 replication form in the SmaI site, carrying out dna sequencing then and confirm.

Separate the XbaI/BamHI restricted fragment that comprises the GsA leading peptide then and also insert on the XbaI/BamHI site that is present in the said carrier, to produce expression vector pEDII-CS.In the pEDII-CS expression vector, substitute the natural UAG terminator codon of CSP with the UAA terminator codon.Finish this step by using following oligonucleotide to substitute most of CSP coding region by the dna fragmentation amplification:

5 amplimers (place CsA leader peptide sequences downstream and comprise the BamHI site):

5-ACCCAGGATACCCTTATTCCAG-3

3-amplimer (corresponding but comprise UAA terminator codon and SacI site) with the last codon of CSP gene:

These oligonucleotide of 5-AAAGCCGAGCTCTTAATTAAGGAACAAGAAGGATAAT-3 carry specificity restriction site BamHI and SacI, and they also are present among the pEDII-CS and are used for substituting the CSP gene segment of pEDII-CS.Use this strategy, we have obtained expression vector pEDII-CS49, and its generation has the CSP protein of its initial C-terminal polypeptide.

3.2.pERIV-CS

In order to express the GPI modified forms of CSP, make up plasmid pERIV-CS.This plasmid-derived is from self pERIV derived from pERII, and pERII is dictyostelium ras ras promoter fragment, CsA signal peptide, Actin muscle 6 terminator sequences and the combination of neoR box in the pGEM3 carrier.Said neoR box comprises dictyostelium Actin muscle 15 promotors, bacterium Tn903 resistant gene, dictyostelium Actin muscle 15 terminator sequences, adopt EcoRV with it from pDneo2 (Witke, W. and Nellen, A. (1987), the EMBO magazine 4143-4148) separates and is inserted among the PGEM3 (Promega company).Then the dictyostelium ras promotor that derives from pERI-CS-CsA signal peptide fragment (Fasel etc., the same) is inserted between EcoRI and the BamHI site.At first clone in the HindIII site of pGEM4 (Promega company) into so that second BamHI site to be provided, and then separate and be inserted in the BamHI site that is positioned behind the CsA signal peptide extracting from Actin muscle 6 terminator sequences of pDneo2.The construct that comprises Actin muscle 6 terminator sequences of correct direction is called PERII.Digest this plasmid by EcoRV+XhoI, the dna fragmentation that the amplification of using suitable site to be inserted through the zone of coding Portugal base phosphatidylinositols anchor interpolation sequence obtains.Two kinds of oligonucleotide that are used to increase have following sequence:

5 amplimers (comprising the EcoRV site):

5-CCCGGTACCAGGCCTGATATCTCCAACTCCAACTGAAAC-3

3 amplimers (comprising the XhoI site):

5 '-CGGCTCGAGTTAAATTAATAAAACAAAAGAAATG-3 ' is with Asp718+EcoRV digestion pERIV plasmid and insert CSP NF54 allelotrope.By using the amplification of following amplimer to obtain CSP DNA (comprise the CSP gene but do not comprise the terminal signal peptide of N-and the terminal hydrophobic CSP coded slices of C-):

5 amplimers (comprising the Asp718 site):

5′-CCCGGTACCATTATTCCAGGAATACCAGTGC-3′

3 ' amplimer (comprising the HaeIII site that can merge with the EcoRV site):

5 '-ATAGGCCACATTTTTCCATTTTACAAATTTTTTTTTC-3 after with Asp718 and HaeIII digestion, between the Asp718 of pERIV and EcoRV site, insert the DNA of amplification.The plasmid that obtains is named and is pERIV-CS.

4. the cultivation of dictyostelium cell, conversion and expression

Dictyostelium is being cultured to 5 * 10 on the HL-5 substratum in vibration suspension ⁶Cell/ml and in PDF (liner diluted liquid) hungry tool (Sussman, M., (1987), cell biology method (Spudich, J.A., ed.), pp9-29, academic publishing company, Orlando, FL).Import various carriers by electroporation, as in (Nellen, W. and Firtel, R.A. (1985), gene, 39,155; Howard, P.K., Ahern, K.G. and Firtel, A. (1988), nucleic acids research, 16, select express cell described in 2613-1623).

Express for Discoidin I promotor dependency, unless otherwise indicated, with the dictyostelium discoideum cell with 5 * 10 ⁶The density of cell/ml in PDF in vibration suspension (160rpm) 4 hours (Fasel etc., the same) of hunger.

For the ras promoter construct, unless otherwise indicated, with cell in PDF with about 5 * 10 ⁶Hungry 6 hours of/ml is by adding DIF (differentiation inducing factor) (Morris, H.R., Taylor, the G.W. of 200 μ M and 10nM, Massento, M.S, Jermyn, K.A., Kay, R.R. (1987), nature, 328,811) one hour inducible transcription (Louvion, J.F., Scholder, J.C., Pinaud, S., and Reymond, C.D., (1991), nucleic acids research, 19,6133-6138).

5. protein analysis

By 10%SDS-PAGE separate (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989), molecular cloning: laboratory manual, second edition, cold spring harbor laboratory, the cold spring port, NY) in 1 * Laemmli damping fluid, boil 5 minutes derive from 2 * 10 ⁶The protein of individual cell (groove that every 4mm is wide).With protein electrotransfer (Immunoblots) to nitrocellulose membrane.The anti-NANP monoclonal antibody (Sp3E9) of 50 μ g/ml (Boulanger, N., Matile, H. and Betschart, B. (1988), Acta.Tropica, 45,55-65) add on the filter membrane and be incubated overnight at room temperature.Protein and the chemiluminescence reaction (Amersham) of using alkaline phosphatase to put together disclose anti-NANP combination.

6.GPI-lecithinase D is measured

By at 20mM Tris/HCl pH 7.5,0.1M CaCl ₂, freeze to conciliate the GPI-PLD susceptibility that the CSP that test cell line modifies with GPI is separated in bursting by freezing by four cycles among the 0.008%TX-100.Add the GPI-PLD enzyme (Boehringer Mannheim) of 1 or 5 unit and 37 ℃ of following incubation extracts 1 hour.Then the TX-114 in the 1 * TBS that comprises 1mM EDTA is added into 1% ultimate density, water phase separated and washing agent are mutually.In 10% sds page, also use sample dissolution the Sp3E9 monoclonal antibody to analyze as mentioned above by immunoblotting.

7.ELISA

Measure serum and the monoclonal antibody that produces anti-N-end (amino acid 22-125), NANP repetition peptide or C-end (amino acid 289-390) segment peptide by ELISA.In brief, the ethene flat board applies, washs and sealing with the 1%BSA in PBS with different peptides.With the 1%BSA/PBS serial dilution mono-clonal or the serum antibody that comprise 0.05% Tween 20.Add to dilute serum in the hole that antigen applies and incubation 1 hour at room temperature.Dull and stereotyped with the PBS washing that comprises 0.05%Tween 20, add the proper diluent and the incubation 1 hour at room temperature of anti-IgG of the species specificity of peroxidase-put together.The peroxidase substrate solution of 100ml is added in each hole, and measure A410.The ELISA titre of design mice serum is so that serum dilution produces the absorbance of comparison according to the big 2D of mean value of mouse.

8. immunity of animal and antiserum(antisera) analysis

Twice 25 μ l or 1: 1 incomplete Freund adjuvant and 2 * 10 of one times of 50 μ l ⁷The subcutaneous respectively or peritoneal injection of the mixture of the sonication of individual cell advances in the Balb/C mouse.After 4 weeks, carry out booster immunization, collect serum again after 10 days and pass through elisa assay with the material that is equal to.

9. result

For the exploitation of living vaccine or diagnostic test, express the CSP that modifies through GPI.Like this, use terminal hydrophobic fragment (last 23 amino acid) (Noegel etc., the same) (Figure 1A and B) of last 49 amino acid replacement CSP-of the contact site A (CsA) that comprises GPI anchoring structure territory.Insertion CSP/GsA fusion gene under the control of ras promotor (Louvion etc., the same, Fasel etc., the same).Construct is imported dictyostelium discoideum cell (pERIV-CS).Use the immunofluorescence of anti-CSP antibody to be used for determining exist (data not shown) of CSP at the pERIV-CS cell surface.Because the use of ras promotor only 20% of inducing cell to 40% demonstrates expression, this be those cells that are divided into pre-stalk cell (prestalk) (Reymond, C.D., Gomer, R.H., Mehdy, C. and Firtel, R.A, (1984) 39,141-148).

The CSP/CsA fusion rotein that produces in dictyostelium discoideum has amphiphilic feature (Bordier, C. (1981) journal of biological chemistry, 256 that are contemplated to the GPI modifying protein, 1604-1607, Conzelmann, A., Spiazzi, A., Hyman, R. and Bron, C. (1986), EMBO magazine, 5,3291-3296), because it distributes (Fig. 2) at the TX-114 washing agent in mutually.In order to determine the existence of GPI anchor on the CSP/CsA fused protein, by 3 cycles dissolvings in 0.008%TX-100, freezing and thaw, with the GPI-PLD processing cellular lysate of 1 or 5 units 1 hour.The fat of CSP/CsA is partly removed the hydrophobic character that has changed it with the GPI-PLD of 5 units and is made it distribute water inlet phase (Fig. 2), but limited with the effect of the GPI-PLD incubation of 1 unit.These results have shown there is the GPI structure on the CSP that expresses that in the dictyostelium cell this shows: dictyostelium discoideum can produce, process and modify allogenic parasite protein matter by adding the GPI anchor.

For the CSP that estimates the GPI modification causes immunoreactive ability, use and incomplete Freund adjuvant blended 2 * 10 ⁷Following or immune 8 the Balb/C mouse of peritonaeum of individual full cell skin.After injection for the second time ten days, the elisa assay humoral immune reaction (table 1) of the synthetic peptide by resisting the different zones that derives from CSP.Detect the NANP iteron of the anti-advantage immunity of antibody, the terminal non-iteron (amino acid 289-390) of anti-C-but the synthetic peptide of the terminal 22-125 of not anti-N-.What is interesting is that the existence of specific antibody and antibody titers are not injected the influence (table 1) of route.The control experiment mouse is with expressing the dictyostelium injection cell that passes through pEDII-CS49 synthetic CSP.In this case, CSP has its initial peptide C-terminal fragment, and not modified by the GPI anchor.In ELISA, detected not at the different pulsating antibody (table 2) of anti-CSP.

10. legend

Fig. 1 circumsporozoite protein matter (CSP) expression vector

A. in text, provided the detailed description that these two kinds of carriers produce.In brief, be fused in the CsA leading peptide lacking its initial 18 amino acid whose CSP with meeting frame, form pEDII-CS, initial Plasmodium falciparumUAG substitutes the formation that UAA causes construct pEDII-CS 49.PERIV-CS derives from pERII (referring to text), comprises Tn5 neoR gene in this pERII between Actin muscle 15 promotors and terminator sequence.The proteinic GPI anchoring structure of CsA territory is at the C-of CSP end replaced in frame (referring to figure B).Symbol: " Discoidin I " and " ras " are the sequences that promotion is transcribed in dictyostelium.Arrow shows transcription initiation site.I, II and III have shown the CSP zone of high conservative.

B. in pERIV-CS, with proteinic last 49 the amino acid replacement CSP hydrophobic domains of CsA.During the clone between CSP (line) and the CsA sequence, add other proline(Pro) (P).

Fig. 2. the CSP's that expresses in the dictyostelium discoideum cell is phospholipid modified

GPI-PLD with 1 unit (road 1) or 5 units (road 2) handles from about 2 * 10 ⁶The protein that extracts in the individual cell one hour carries out TX-114 and is separated (Bordier, the same), and passes through immunoblotting assay.In brief, the hydrolysis-stable cell transformed is also passed through SDS PAGE and is separated, and protein transduction is moved on on the nitrocellulose membrane.By using (NANP) ₅₀The immunodetection of monoclonal antibody Sp3E9 shows CSP.Use molecular weight standard to estimate the apparent molecular weight (62kDa) of CSP.Molecular weight standard is as follows: phosphorylase b (97kDa), bovine serum albumin (66kDa), ovalbumin (42.7kDa).Aq: water, Tx:TX-114 washing agent phase.0: without the sample of GPI-PLD processing.

11. table

Table 1: with (NANP) ₅₀, M1 (189-390) peptide mice immunized pERIV-CS serum ^aELISA.

Animal (NANP) 50M1 (289-390) ELISA titre ELISA titre B (ip) bI/iooo 1/900 G (sc) 1/5,000 1/900 R (ip) 1/5,000 1/900 Y (sc) I/iooo 1/300 Y (ip) 1/5,000 1/300 W (ip) I/iooo 1/900 contrastc ＜1/10 ＜1/10

^aSerum is with 2 * 10 ⁷After strengthening, the Balb/C mouse single of individual cellular immunization obtained in 7 days ^bMouse subcutaneous (sc) or peritonaeum (ip) immunity ^cControl animal is only with incomplete Freund adjuvant injection

Table 2: with (NANP) ₅₀, M1 (289-390) and M2 (22-125) peptide mice immunized pEDII-CS 49 serum ^aELISA.

Animal (NANP) 50M1 (289-390) M2 (22-125) ELISA titre ELISA titre ELISA titre Nr.1 b＜1/10＜1/10＜1/10 Nr.2＜1/10＜1/10＜1/10 Nr.3＜1/10＜1/10＜1/10 contrast c ＜1/10 ＜1/10 ＜1/10

^aSerum is with 2 * 10 ⁷After strengthening, the Balb/C mouse single of individual cellular immunization obtained in 7 days ^bMouse subcutaneous (sc) or peritonaeum (ip) immunity ^cControl animal is only with incomplete Freund adjuvant injection

Claims (18)

1. demonstrate the immunogenic recombinant polypeptide of raising, this recombinant polypeptide comprises Portugal's base-phosphatidylinositols structure.

2. as the desired polypeptide of claim 1, wherein said polypeptide is a kind of antigen.

3. as the desired polypeptide of claim 2, wherein said antigen is a kind of parasite polypeptide.

4. as the desired polypeptide of claim 3, wherein said parasite antigen is the Plasmodiumfalciparum polypeptide.

5. as the desired polypeptide of claim 4, wherein said Plasmodium falciparum antigen is circumsporozoite protein matter (CSP) or its modifying protein.

6. express recombinant DNA carrier as arbitrary desired polypeptide of claim 1-5, this carrier comprises the dna sequence dna of coding said polypeptide, this dna sequence dna operationally adds sequence with the glycolipid anchor that is positioned at its downstream and is connected, and suitable transcription initiation all operationally is connected with it with terminator sequence and dispensable leader peptide sequences.

7. as the desired recombinant DNA carrier of claim 6, wherein said dna sequence encoding parasite antigen.

8. as the desired recombinant DNA carrier of claim 7, wherein said parasite antigen is a Plasmodium falciparum antigen.

9. as the desired recombinant DNA carrier of claim 8, wherein said Plasmodiumfalciparum antigen is circumsporozoite protein matter (CSP) or its modifying protein.

10. as the arbitrary desired recombinant DNA carrier of claim 6-9, wherein said glycolipid anchor adds sequence source from dictyostelium discoideum (Dictyostelium discoideum) contact site A.

11. comprise recombinant host cell as arbitrary desired recombinant DNA carrier of claim 6-10.

12. as the desired recombinant host cell of claim 11, wherein said host is net post bacterium subclass host.

13. as the desired recombinant host cell of claim 12, wherein said net post bacterium subclass host is dictyostelium (Dictyostelium) species, is dictyostelium discoideum specifically.

14. induce the immunoreactive method at one or more epi-positions of polypeptide, this method comprises with the arbitrary desired polypeptide immune mammalian hosts as claim 1-5, separates consequent antibody thereafter.

15. as the desired method of claim 14, the wherein said polypeptide that is used for immunity adopts the full cell lysate form of the host cell of expressing described polypeptide.

16. be used to produce method as the desired modified polypeptide of claim 1-5, this method by with as the desired suitable carrier of claim 6-10 transform proper host cell, making that culturing cell also separates said polypeptide not essentially under the environment that said polypeptide can be expressed.

17. a vaccine, this vaccine comprise the immunoprotection amount as the arbitrary desired polypeptide of claim 1-5 and suitable vehicle.

18. as the desired vaccine of claim 17, wherein said immunoprotection amount comprises about 1-5 * 10 ⁷, particularly 2 * 10 ⁷Individual content of peptides as desired host cell among the claim 11-13.

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