CN102838668A - Plasmodium falciparum pfs25 protein dipolymer derivative and application thereof - Google Patents

Abstract

The invention belongs to the field of communicable disease prevention and cure, and particularly relates to a candidated autoantigen of vaccine for blocking malignant malaria transmission. According to the invention, a gene recombination method is used for constructing a plasmodium falciparum pfs25 protein dipolymer derivative, and the dipolymer derivative comprises two pfs25 protein units which are connected through a connection arm. The obtained dipolymer derivative strengthens immunogenicity of pfs25 proteins, and can arouse high antibody response in an animal body. Dipolymers can be produced by recombination yeast cells in a high-expression mode and secreted to a culture medium. An expression product with even compositions is obtained after purification, and the expression product can be applicable to mass production. The plasmodium falciparum pfs25 protein dipolymer derivative is expected to be candidated antigens of blocked vaccines of the malignant malaria transmission.

Description

A kind of plasmodium falciparum pfs25 albumen dimer derivate and uses thereof

Technical field

The invention belongs to the transmissible disease prevention and control field, be specifically related to the control of pernicious malaria, relate in particular to candidate's target antigen of the vaccine that is used to block malaria transmission.

Background technology

Malaria is the parasitosis of propagating through by mosquito bite, and its pathogenic agent is a plasmodium.The World Health Organization's 2009 years report shows that 2.43 hundred million malaria clinical cases, dead 86.3 ten thousand people (WHO.World Malaria Report 2009 [EB/OL] .) are arranged global every year.Given this, use vaccine, become and one of world's " three is big " vaccine project that AIDS, Vaccinum Calmette-Guerini are arranged side by side to the very high pernicious malaria of mortality ratio exploitation prevention.In first developing project is listed malaria vaccine by the World Health Organization, plan agency for development of United Nations.Bill's Gates Foundation also is provided with " malaria vaccine special topic ".

Pfs25 albumen is the gametophyte surface protein of plasmodium falciparum; And continue to be present in zygote and vermicule phase; Pfs25 be the mediation vermicule pass through peritrophic membrane (peritrophic membrane, PM) and the key albumen that mosquito midgut wall basilar membrane is invaded, its antibody can suppress the growth of zygote in the mosquito enteron aisle; Therefore play the effect of propagating blocking-up, so pfs25 albumen is considered to the important candidate antigens of pernicious malaria vaccine.When this vaccine and other malaria vaccines or antimalarial drug combined utilization, capable of blocking those are escaped the vaccine protection or medicine are produced resistance and the protozoacide that survives propagation.

Reported first such as Kaslow DC in 1988 Pfs25 protein, the result shows that this protein is made up of 217 amino acid, is rich in halfcystine, its characterization of molecules is to have four " structural domain of EGF appearance " (Nature.1988 May 5; 333 (6168): 74-6.); Barr PJ in 1991 etc. have reported that reorganization Pfs25 albumen is as propagating effect (the The Journal of experimental medicine.1991 Nov 1 of blocking-up vaccine on experimental animal; 174 (5): 1203-8.); More than research is laid a good foundation for further developing propagation blocking-up type pernicious malaria vaccine.After this; A lot of researchists express pfs25 in multiple systems and estimate; People such as Takafumi Tsuboi have expressed Pfs25 albumen in Fructus Hordei Germinatus cell free system (Wheat Germ Cell-Free System); This recombinant protein can suppress plasmodial growth (Infection and Immunity, 2008,76 (4): 1702-1708.); Godfree Mlambo etc. has expressed Pfs25 albumen (Vaccine 2010,28:7025 – 7029) in the baculovirus expression system; Christine E. etc. has reported that pfs25 expresses in plant expression system, and verified blocking activity that pernicious malaria is propagated (Human Vaccines, 2011, Volume 7 Supplement, DOI:10.4161/hv.7.0.14588.).

In various expression systems, yeast expression system is widely used, and the expression product of acquisition also is applied to human clinical's research.1993, Kaslow DC etc. disclosed and has adopted Yeast system to express the method for Pfs25 protein mutant, and this two mutants replaces to L-Ala (U.S. Pat 5217898) with the 112nd, 165,187 l-asparagine of pfs25 albumen native sequences; Zou L etc. is reported in the another kind of pfs25 protein mutant of expressing in the pichia spp, and this two mutants replaces to Stimulina (Vaccine.2003 Apr2 with the 112nd, 165,187 l-asparagine of Pfs25 albumen native sequences; 21 (15): 1650-7.).On the basis of above work, and the Pfs25 protein mutant that people such as Yimin Wu recombinate yeast respectively (Zou L etc., Vaccine.2003Apr 2; 21 (15): 1650-7.) and with Montanide ISA 51 adjuvant mixing manufacture vaccines carried out the I clinical trial phase; The result of this project test shows; Pfs25 protein can excite immunne response in human body, still, serum antibody titer is lower; It is of short duration to hold time, referring to PloS one.2008; 3 (7): e2636. discloses.

As candidate vaccine, the proteinic less immunogenic of pfs25 awaits further enhancing.Feng Qian etc. have obviously improved the proteic immunogenicity of pfs25, referring to Vaccine.2007 through pfs25 albumen and Pseudomonas aeruginosa exotoxin A (ExoProtein A) coupling; 25 (20): 3923 – 3933 disclose.Joanna Kubler-Kielb etc. with ovalbumin (OVA), circumsporozoite protein (CSP) and pfs25 albumen self as carrier proteins; Obtain serial pfs25 albumen and carrier protein couplet thing; Can promote the proteic immunogenicity of pfs25 and keep the immunne response of long period, specifically referring to PNAS.2007; 104 (1): 293-298 discloses.

Above-mentioned research work; All adopt chemical reagent to carry out coupling at protein level; Though the immunogenicity of pfs25 has obtained enhancing; But its defective is: pfs25 albumen-the carrier conjugates structure is uncertain, coupling efficiency is low, complex manufacturing, thereby has increased the difficulty of Quality Control in the production process.Therefore, the pfs25 that this area the presses for development of novel classes antigen of deriving is used to prevent and treat malaria.

Summary of the invention

The technical problem that the present invention will solve provides a kind of new plasmodium falciparum Pfs25 protein derivatives, as the candidate antigens of propagating the blocking-up vaccine, with the problem a little less than the solution Pfs25 protein immunization originality.

First aspect of the present invention provides a kind of plasmodium falciparum Pfs25 protein derivatives, and particularly, this verivate is the Pfs25 albumen dimer of recombination, has following structure:

pfs25-L-pfs25

Wherein pfs25 represents the pfs25 protein mutant, and L represents connecting arm (linker).

Described pfs25 protein mutant comprises 23-193 amino acids fragment and the non-glycosylated two mutants thereof that is equivalent to pfs25 albumen native sequences; Promptly removed 24 amino acid whose film calmodulin binding domain CaMs of 22 amino acid whose signal peptide sequences of pfs25 native sequences N end and C end, and/or the l-asparagine that is equivalent to the 112nd, 165,187 of native sequences has been carried out point mutation.

In a preferred scheme, described pfs25 protein mutant comprises the 23-193 amino acids sequence that is equivalent to pfs25 albumen native sequences, but the l-asparagine that wherein is equivalent to the 112nd, 165,187 of native sequences replaces with L-Ala.

In a preferred scheme, described pfs25 protein mutant comprises the 23-193 amino acids sequence that is equivalent to pfs25 albumen native sequences, but the l-asparagine that wherein is equivalent to the 112nd, 165,187 of native sequences replaces with Stimulina.

In a preferred scheme, described pfs25 protein mutant comprises the 23-193 amino acids sequence that is equivalent to pfs25 albumen native sequences, but the l-asparagine that wherein is equivalent to the 112nd, 165,187 of native sequences replaces with L-glutamic acid.

Described connecting arm is made up of glycocoll, Serine, has following structure: S _a-(G _bS) _c, wherein S represents Serine, and G represents glycocoll, a=0 or 1, b=3 or 4, the integer of c=1-4.

In a preferred scheme, connecting arm has the structure of SGGGGS.

In a preferred scheme, connecting arm has (GGGGS) ₃Structure.

In a preferred scheme, connecting arm has (GGGS) ₄Structure

In preferred scheme, plasmodium falciparum Pfs25 protein derivatives has the aminoacid sequence shown in the sequence table Seq.No.5.

In preferred scheme, plasmodium falciparum Pfs25 protein derivatives has the aminoacid sequence shown in the sequence table Seq.No.7.

Second aspect of the present invention provides the purposes of plasmodium falciparum Pfs25 albumen dimer derivate, is used to prepare the propagation blocking-up vaccine that prevents subtertian malaria.

(pfs25) of the present invention ₂Protein derivatives is to form dimer by two pfs25 protein protomers that the polypeptide connecting arm connects on the molecular structure, and the polypeptide connecting arm has kept the character of pfs25 albumen self to the not influence of biological activity of two pfs25 protein protomers.The data presentation of this research, (pfs25) ₂Albumen can with the standard monoclonal antibody 4B7 specific reaction of anti-pfs25, this has reflected (pfs25) ₂Molecule has kept the proteic immunoreactivity of pfs25; (pfs25) ₂Behind the molecule inoculation mouse, can the higher immunne response of rate of induced polarization pfs25 protein monomer, proved (pfs25) ₂Molecule possesses stronger immunogenicity.In preferred embodiment of the present invention, the present invention has expressed (pfs25-3E) ₂Dimer, this dimer can with anti-pfs25 standard monoclonal antibody 4B7 specific reaction, explain that this verivate has kept the proteinic immunogenicity of pfs25.In the mouse in vivo tests, the antibody horizontal that this dimer excites becomes positive correlation with dimeric immunizing dose.With the monomeric simultaneous test of pfs25-3E in, under same dose, the antibody horizontal that dimer excites has improved more than 10 times than monomer.

(pfs25) of the present invention ₂Protein derivatives utilizes the recombination means, in host, directly expresses the dimer protein that becomes pfs25, can obtain purity very high (pfs25) through purification process ₂Protein dimer, its production technique is simple, controllability is high, the vaccine composition homogeneous.In preferred embodiment of the present invention, (pfs25-3E) ₂Dimer is high expression level in yeast expression system; Its expression product accounts for more than 65% of total protein content in the yeast fermentation supernatant; Through ion exchange chromatography, gel permeation chromatography two-step purifying, can obtain purity and reach the target protein more than 95%, be fit to scale operation.

Comparatively speaking, the external chemical coupling method that adopts in the prior art though increased immunogenic molecular weight, has improved immunogenicity, because the randomness and the uncontrollability of linked reaction, makes the product heterogeneity.With pfs25 albumen self coupling is example, finally can obtain pfs25 dimer, pfs25 tripolymer, the pfs25 tetramer even to ten aggressiveness.Only with regard to the dimer that coupling obtains,, in fact comprised the isomer of various not isomorphic maps because link coupled position difference takes place.The complicacy of this coupled product causes the uncertainty of product Quality Control.In addition,, cause that linked reaction is wayward, production cost is high, be unfavorable for the industrialization amplification because the complicacy of coupling process and linked reaction is insufficient.

In general, the present invention (pfs25) ₂The protein derivatives immunogenicity is strong, the product structure homogeneous, and the production technique simple controllable is expected to become the candidate antigens to the propagation blocking-up type vaccine of pernicious malaria.

Description of drawings

Fig. 1: recombinant expression plasmid pfs25-3E/pGAPZ α A enzyme is cut evaluation

Swimming lane 1:DL2000 DNA marker, 2000,1000,750,500,250,100

The two pfs25-3E/pGAPZ α A recombinant plasmids of cutting of swimming lane 2:Xho I and Xba I

Fig. 2: pfs25-3E/pGAPZ α expressed supernatant SDS-PAGE in A/GS11572 hour and analyzes

Swimming lane 1:protein marker (kd): 96,67,43,31,21,14

Swimming lane 2:pfs25 standard protein

Swimming lane 3-8:pfs25/pGAPZ α A/GS115 positive colony expression product

Swimming lane 9:pGAPZ α A/GS115 yeast (negative control)

Fig. 3: pfs25-3E/pGAPZ α A/GS115 reorganization bacterium expression product Western-blot identifies

Swimming lane 1:pGAPZ α A/GS115 Pcihia pastoris (negative control)

Bacterium was expressed in 24 hours in swimming lane 2:pfs25/pGAPZ α A/GS115 reorganization

Bacterium was expressed in 48 hours in swimming lane 3:pfs25/pGAPZ α A/GS115 reorganization

Bacterium was expressed in 72 hours in swimming lane 4:pfs25/pGAPZ α A/GS115 reorganization

Swimming lane 5:pfs25 standard protein

Fig. 4: pfs25-3Q/pGAPZ α A/GS115 reorganization bacterium expression analysis

Swimming lane 1:pfs25 standard substance;

Swimming lane 2-10:Q2-Q10 clone reorganization bacterium expression product

Fig. 5: pfs25-3E/pGAPZ α A/GS115 reorganization bacterium is expressed the supernatant ion exchange chromatography

Swimming lane 1:1.0M NaCl elution peak;

Swimming lane 2:0.3M NaCl elution peak

Swimming lane 3: stream is worn the peak; Swimming lane 4: appearance before the post

Fig. 6: pfs25-3E/pGAPZ α A/GS115 reorganization bacterium is expressed the supernatant gel permeation chromatography

Swimming lane 1-6: the 1st to 6 pipe pipe sample of fraction collection

Fig. 7: overlapping PCR obtains (pfs25-3E) 2 genes

Swimming lane 1:DNA Marker DL2000;

Swimming lane 2: the PCR result of primer a and primer b

Swimming lane 3: the PCR result of primer c and primer d;

Swimming lane 4: the PCR result of primer a and primer d

Fig. 8: recombinant expression plasmid (pfs25-3E) 2/pPICZ α A enzyme is cut evaluation

Swimming lane 1:DNA Marker DL2000;

Swimming lane 2: the PCR result of primer a and primer d

Swimming lane 3:Xba I and Xho I double digestion (pfs25-3E) 2/pPICZ α A

Swimming lane 4:Xba I single endonuclease digestion (pfs25-3E) 2/pPICZ α A

Swimming lane 5:DNA Marker D507A:11849,10085,8023,6133,5026,3997,3049,2087

Fig. 9: SDS-PAGE analyzes (pfs25-3E) 2/pPICZ α A/GS115 recombination microzyme and expressed supernatant in 72 hours

Swimming lane 1,2: two different (pfs25-3E) 2/pPICZ α A/GS115 reorganization bacterium clones;

Swimming lane 3:pPICZ α A/GS115;

Swimming lane 4: standard protein molecular weight (Kd)

Figure 10: Western-blot identifies (pfs25-3E) 2/pPICZ α A/GS115 recombination microzyme expression supernatant

The yeast expressed supernatant of swimming lane 1:pPICZ α A/GS115

Swimming lane 2: (pfs25-3E) 2/pPICZ α A/GS115 recombination microzyme was expressed supernatant in 72 hours

Figure 11: ion exchange chromatography purifying (pfs25-3E) 2/pPICZ α A/GS115 reorganization bacterium is expressed supernatant

The yeast expressed supernatant of swimming lane 1:pPICZ α A/GS115;

Swimming lane 2: standard protein molecular weight (Kd): 96,67,45,30,20,14

Swimming lane 3: (pfs25-3E) 2/pPICZ α A/GS115 recombination microzyme is expressed supernatant

Swimming lane 4:0.1mol/L NaCl elution peak;

Swimming lane 5:0.3mol/L NaCl elution peak

Swimming lane 6:1.0mol/L NaCl elution peak

Figure 12: gel permeation chromatography purifying (pfs25-3E) 2 albumen

Swimming lane 1: standard protein molecular weight (Kd);

Swimming lane 2: (pfs25-3E) 2 protein

Embodiment

Propagate blocking-up type pernicious malaria vaccine

Gamophase and the cell early stage in imperfect stage and the not synantigen of erythrocytic phase to the plasmodium perfect stage; Malaria vaccine divides three major types: and phase vaccine, erythrocytic stage vaccine and propagation blocking-up type vaccine in the liver (transmission blocking vaccines, TBVs).Propagate the blocking-up type vaccine and be polypide surface protein with mosquito stage plasmodium specifically expressing as the antigen immune human body, make it to produce the specific antibody of anti-mosquito stage plasmodium surface protein.After mosquito sucked the human blood of quilt immunity, plasmodium that grows in the mosquito midgut and people's antibody produced antigen antibody reaction, thereby caused interior plasmodium syngenesis of mosquito body and sporogony to be blocked.The more propagation blocking-up vaccine candidate antigen of research at present has plasmodium falciparum GAP-associated protein GAP Pfs25, Pfs28, Pfs48/45 and Pfs230, vivax malaria GAP-associated protein GAP Pvs25, Pvs28, Pvs48 etc.

Can not be infected with malaria by the epidemic prevention individuality though propagate the blocking-up type vaccine, also can not alleviate the malaria symptom, can block anopheles plasmodium is transmitted to another host from a host.When this vaccine and malaria protectiveness vaccine or antimalarial drug combined utilization, the TBVs plasmodial propagation that those survive to medicine generation resistance capable of blocking.Another purposes of TBVs is that the traveller is carried out immunity, brings plasmodium into non-popular district to prevent them, causes popular.

Because phase vaccine, erythrocytic stage vaccine candidate antigen molecule are exposed to the human immune system in the liver, receive the influence of factors such as human immunity pressure, there is gene pleiomorphism widely in candidate antigens.The TBVs candidate antigens mainly is expressed in mosquito stage plasmodium surface; This type of antigen is not under the vertebrate immune system selective pressure; So these albumen demonstrate low-down polymorphic level, antigenic variation can not occur, help immune effect of vaccine; Being considered to block effectively malaria from the propagation of mosquito matchmaker to the people, is one of focus of current malaria vaccine research.

Natural Pfs25 antigen and two mutants thereof

Pfs25 (Plasmodium falciparum s25) is plasmodium falciparum zygote or the main surface protein of vermicule, is the key albumen that the mediation vermicule is passed through peritrophic membrane and mosquito midgut wall basilar membrane is invaded.Natural pfs25 protein is present in plasmodium falciparum sporozoite cyst membrane surface, is made up of 217 amino acid, and molecular weight 25Kd is rich in halfcystine.Structurally, 22 amino acid of the N of Pfs25 native protein end are signal peptide sequences, and 24 amino acid of C end are the film calmodulin binding domain CaMs, and there be four " structural domain of EGF appearance " in this albumen, and 23 halfcystines have formed nine pairs of disulfide linkage.Pfs25 is a gp, and one has four glycosylation sites, wherein; 112,165,187 and 202 l-asparagine (Asn; N) formed the N type sugar chain on, (Ser has formed the sugar chain (GPI-anchor) of glycosylation PI grappling on S) to 196 Serine.Specifically referring to Kaslow DC etc. at Nature.1988 May 5; 333 (6168): 74-6. discloses.

Pfs25 albumen adopts yeast expression, the inhomogenous problem of the glycosylation modified and expression product of over-drastic usually occurs.In existing research; Kaslow DC has designed a kind of non-glycosylated two mutants pfs25-B, and this two mutants is equivalent to the 22-188 amino acids of pfs25 native sequences, and is L-Ala (Ala with the asparagine mutation in 112,165,187 3 sites; A), make its forfeiture glycosylation function.Pfs25-B obtains the expression product than homogeneous at yeast expression, referring to Nature.1988 May 5; 333 (6168): 74-6. and US5217898.With the monoclonal antibody 4B7 of this two mutants preparation, become the proteic stdn monoclonal antibody of pfs25 at present and supplied global researchist to use, referring to The Journal of experimental medicine.1991 Nov1; 174 (5): 1203-8.2003, Zou L designed another kind of non-glycosylated two mutants, and this two mutants is equivalent to the 23-193 amino acids of pfs25 native sequences, and with the asparagine mutation in 112,165,187 3 sites be Stimulina (Gln, Q).This two mutants is successful expression in yeast saccharomyces cerevisiae and pichia spp, and America NI H has carried out I phase clinical study based on this two mutants, the result referring to Zou L etc. at Vaccine.2003 Apr2; 21 (15): the open and Wu Y of 1650-7. etc. are at PloS one.2008; 3 (7): e2636. discloses.

The inventor is in previous work; Designed another kind of non-glycosylated two mutants, be equivalent to the 23-193 amino acids of pfs25 native sequences, and be L-glutamic acid (Glu the asparagine mutation in 112,165,187 3 sites; E); Successful expression in pichia spp, the pfs25 protein mutant that obtains can with the 4B7 specific reaction, and can excite immunne response behind the mouse in inoculation.

It is used that the said mutation body is the present invention, abbreviates pfs25-3A two mutants, pfs25-3Q two mutants and pfs25-3E two mutants respectively as.

The Pfs25 protein derivatives that Chemical Crosslinking Methods obtains

Pfs25 protein can excite immunne response in human body, still, serum antibody titer is lower, and it is of short duration to hold time.As candidate vaccine, the proteinic less immunogenic of pfs25 awaits further enhancing.Feng Qian etc. carry out chemically crosslinked with pfs25 albumen and reorganization Pseudomonas aeruginosa exotoxin A (rEPA) external, and the immunogenicity of the cross-linking agent pfs25-rEPA that obtains is apparently higher than pfs25 albumen itself, specifically referring to Vaccine.2007; 25 (20): 3923 – 3933.Joanna Kubler-Kielb etc. has studied a series of pfs25 protein conjugate; Comprise that pfs25 and self-crosslinking, pfs25 and ovalbumin (OVA) are crosslinked, pfs25 and reorganization Pseudomonas aeruginosa exotoxin A (rEPA) be crosslinked; Can adopt different connected cliques; Make to be connected with different modes such as amido linkage, hydrazone key, thioether bonds between pfs25 and carrier proteins or self, obtain multiple connection product.Cross-linking agent carries out injecting immune 2-3 time to the NIH mouse, and the antibody horizontal in March, July is higher than the antibody horizontal in a week after the last immunity after the last immunity, specifically referring to PNAS.2007; 104 (1): 293 – 298.

The Pfs25 protein derivatives that gene recombination method obtains

Utilization DNA recombinant technology; Can the protein structure domain fragment reorganization with difference in functionality be become fusion rotein; For example; To have not the antibody variable region sequence of synantigen binding characteristic and recombinate and obtain bi-specific antibody, and outer soluble fragments of the born of the same parents of some acceptor and antibody Fc fragment fusion will be obtained new fusion rotein or the like.Have when recombinating operation between the protein structure domain fragment of difference in functionality; Usually need between two function fragments, increase a connecting arm (linker); This connecting arm only plays the effect of physical connection for two connected protein function fragments generally speaking; And do not give fusion rotein new function, do not influence two functions that connected protein fragments yet.

The design of connecting arm, the more options molecular weight is little, the inactive glycocoll of character (Gly, G), L-Ala (Ala; A) and Serine (Ser; S) constitute linker, for example, Ni Jianfeng etc. are when design " anti--GD2/ resists-CD16 " bispecific single-chain antibody; Adopt " SGGGGS " as linker (" biomedical engineering journal, 2007; 24 (3): 659-663.); Trinh R etc. have adopted (GGGGS) 3 as linker (Mol Immunol.2004 Jan between VH and VL when making up anti-HER2/neu single-chain antibody; 40 (10): 717-22.); Xi Y etc. select for use (GGGGS) 4 as linker (J Immunother.2006Nov-Dec when making up HLA-B's 7-2 extracellular region and ETA two mutants (PE40KDEL) fusion rotein; 29 (6): 586-95.); Under the few cases, also have the researchist use l-asparagine (Asn, N), proline(Pro) (Pro, P), Methionin (Lys, K) and Xie Ansuan (Val V) waits other amino acid to constitute connecting arms (linker).For example, Lu P etc. have adopted linker (the Appl Microbiol Biotechnol.2008 Jun of two types of (GGGGS) n (n≤3), (EAAAK) n (n≤3) when design beta-glucanase and the zytase fusion rotein; 79 (4): 579-87.); Xu J etc. make up the fusion rotein of AnsB-C and GnRH3-hinge-MVP hybrid polypeptide, insert the unsettled dipeptides linker of Asp-Pro acid between the two.

In above research, the linker that glycocoll and Serine constitute forms the polypeptied chain of one section flexibility, does not have the fixed space structure; The linker that comprises L-glutamic acid, L-Ala, Methionin then forms the α spiral secondary structure of relative rigidity like (EAAAK) n (n≤3); The linker that comprises proline(Pro) has the effect that intensive interrupts the polypeptide secondary structure, is used to separate closely adjacent polypeptide.The two kinds of linker in back possibly give the new function of fusion rotein or influence the function of two pfs25 subunits.

The present invention is intended to two pfs25 series connection becoming dimers, in the hope of increasing the immunogenicity of pfs25.Connecting arm of the present invention only plays the physical connection effect, does not give fusion rotein new function, does not also influence the function of two pfs25 subunits.Therefore, glycocoll, Serine constitute, and the linker that can form flexible structure is adapted to the present invention.The various combination of glycocoll and Serine and linker length are not fixed, as long as satisfy the object of the invention.

Expression plasmid of yeast pPICZ α A, pGAPZ α A, microbiotic Zeocin and bacterial strain GS115 are all available from Invitrogen company; Restriction enzyme Xho I, Xba I, BstX I and T4DNA ligase enzyme are all available from TaKaRa company; The Taq archaeal dna polymerase is available from Roche company; Plasmid extraction kit is available from the vast Imtech in Beijing; PCR product purification test kit and glue reclaim test kit available from QIAGEN company; The standard protein molecular weight is available from GE company.(Malaria Research and Reference Reagent Resource Center NIH) provides by U.S. MR4 for anti-pfs25 monoclonal antibody 4B7 and pfs25 standard protein; HRP mark rabbit anti-mouse igg is available from Sigma company; Other reagent are import or homemade analytical pure.

Synthetic and the Yeast expression carrier of embodiment 1, Pfs25 protein mutant gene makes up

(1) the mutant gene design is with synthetic

According to the pfs25 gene information of announcing among the GenBank (GenBank locus X07802), the 23rd in the natural pfs25 albumen of its coding is following to the 193rd amino acids sequence:

KVTVDTVCKRGFLIQMSGHLECKCENDLVLVNEETCEEKVLKCDEKTVNKPCGDFSKCIK

IDGNPVSYACKCNLGYDMVNNVCIPNECK

VTCGNGKCILDTSNPVKTGVCSCNIGKVP

NVQDQNKCSKDGETKCSLKCLKE ETCKAVDGIYKCDCKDGFIID

ESSICT

Wherein being equivalent to the 112nd, 165,187 in natural pfs25 albumen is l-asparagine (N), is the proteic glycosylation site of pfs25.

The non-glycosylated two mutants of pfs25 albumen of the present invention design abbreviates pfs25-3E as, and this two mutants is equivalent to the aminoacid sequence of natural Pfs25 protein 23-193; The and the 112nd; 165,187 asparagine mutation is a L-glutamic acid, specifically sees sequence table SEQ .NO.1.According to the nucleotide sequence of pichia spp hobby codon design code book invention two mutants (SEQ.NO.1), see sequence table SEQ .NO.2.

According to Zou L etc. at Vaccine.2003Apr 2; 21 (15): the disclosed pfs25 protein mutant of 1650-7. sequence, abbreviate pfs25-3Q as, specifically see sequence table SEQ .NO.3.The present invention has designed the nucleotide sequence that meets pichia spp hobby codon, sees sequence table SEQ .NO.4.

Above-mentioned SEQ.NO.2, SEQ.NO.4 nucleotide sequence entrust Shanghai living worker's biotechnology ltd synthetic; Worker's biotechnology synthetic SEQ.NO.2 of ltd is given birth in Shanghai, the SEQ.NO.4 nucleotide sequence is cloned into the pUC57 carrier respectively; Be called pfs25-3E/pUC57, pfs25-3Q/pUC57, order-checking is correct; Synthetic primer provides with lyophilized form.

(2) Yeast expression carrier makes up

Comprise the cloning vector pfs25-3E/pUC57 and the Yeast expression carrier pGAPZ α A that comprises glyceraldehyde 3-phosphate dehydrogenase (GAP) promotor of goal gene, carry out the double digestion operation with restriction enzyme Xho I and Xba I respectively.Utilize the T4DNA ligase enzyme that the goal gene subclone is obtained recombinant plasmid pfs25-3E/pGAPZ α A to pGAPZ α A; Transformed E .coli Top10F '; Extract plasmid, the enzyme evaluation positive colony of cutting and check order, the Xho I of pfs25-3E/pGAPZ α A plasmid and Xba I double digestion electrophoresis are seen Fig. 1.

Adopt same procedure, the pfs25-3Q gene clone to the Yeast expression carrier pGAPZ α A that comprises glyceraldehyde 3-phosphate dehydrogenase (GAP) promotor, is obtained recombinant plasmid pfs25-3Q/pGAPZ α A.

Above-mentioned two kinds of expression vector Pfs25-3E/pGAPZ α A, pfs25-3Q/pGAPZ α A identify that through order-checking the result is correct.Pfs25-3E two mutants dna sequence dna is seen SEQ.NO.2, and pfs25-3Q two mutants dna sequence dna is seen SEQ.NO.4.

The expression of embodiment 2, Pfs25-3E two mutants and the evaluation of expression product

Positive recombinant expression plasmid Pfs25-3E/pGAPZ α A after BstX I enzyme tangent lineization, electrotransfection method transformed competence colibacillus pichia spp GS115; With blank plasmid pGAPZ α A transfection yeast, preparation negative control bacterial strain.Electricity commentaries on classics condition: 1.5kV, 250 μ F, 200 Ω.Electric shock adds 800 μ l 1M sorbyl alcohols after finishing immediately, and mixing was placed 2 hours for 30 ℃, got an amount of conversion product and coated on the YPD solid screening culture medium that contains 100 μ g/ml Zeocin, and 30 ℃ of wet boxes leave standstill, and visible mono-clonal bacterium colony occurs after 3-5 days.The picking positive colony is yeast reorganization bacterium Pfs25-3E/pGAPZ α A/GS 115.

Picking Pfs25-3E/pGAPZ α A/GS115 positive colony is transferred in the 20ml triangular flask of 3mlYPD liquid nutrient medium, and 30 ℃, 250r/min shaking table are cultivated, expressed, and every at a distance from the 24h sampling, the centrifugal 3min of 8000r/min gets supernatant-20 ℃ preservation.Negative control bacterial strain pGAPZ α A/GS115 synchronized culture also keeps sample.

The expression supernatant of all reorganization bacterium and negative bacterium different times kept sample carry out the electrophoresis operation, the concentration of separation gel is 10%, and the concentration that concentrates glue is 5%, adopts Xylene Brilliant Cyanine G as the protein staining agent.As positive control, negative bacterium is expressed supernatant as negative control with the pfs25 standard substance, and the expression supernatant electrophoresis result that pfs25-3E/pGAPZ α A/GS 115 recombination microzymes were cultivated 72 hours is seen Fig. 2.All reorganization bacterium have the target protein band in the position of 21kd, this explanation pfs25 protein by the recombination microzyme secreting, expressing in substratum.

The expression supernatant that pfs25-3E/pGAPZ α A/GS115 was cultivated 24,48,72 hours carries out SDS-PAGE, expresses supernatant and compares with pfs25 standard protein, pGAPZ α A/GS115 negative bacterium.The SDS-PAGE electrotransfer reacts with 4B7 monoclonal antibody and HRP mark rabbit anti-mouse igg to pvdf membrane one by one, chemoluminescence method colour developing observations.The result sees Fig. 3, and the expression product of visible different incubation times has identical specific reaction with the pfs25 standard protein, and along with incubation time prolongs, expression amount increases.

The expression of embodiment 3, Pfs25-3Q two mutants

Adopt the conversion processes pfs25-3Q/pGAPZ α A/GS115 reorganization bacterium of embodiment 2, select 9 clones altogether, be expressed as clone Q2-10 respectively.

The positive yeast reorganization of picking Q2-10 bacterium is transferred in the 20ml triangular flask of 3mlYPD liquid nutrient medium, and 30 ℃, 250r/min shaking table are cultivated, expressed, sampling in 72 hours, and the centrifugal 3min of 8000r/min gets supernatant and carries out SDS-PAGE and detect.All reorganization bacterium are equipped with the target protein band in the corresponding positions of standard protein, but expression amount is lower, and concrete outcome is seen Fig. 4.

The reorganization pfs25-3E protein purification that embodiment 4, fermentor tank are expressed

Adopt 10 liters of fermentor tanks that Pfs25-3E/pGAPZ α A/GS115 reorganization bacterium is carried out fermentation culture.

Select the mono-clonal bacterium colony and in 100mLYPD, cultivate, 29.0 ℃, pH6.0 ~ 4.0, the rotating speed of 250rpm was cultivated after 18 hours, and the seed that microscopy is qualified is inoculated in the fermentor tank, with the ratio inoculation of 1:15.

Fermentation parameter is: 29.0 ℃, and pH6.0 ~ 4.0, dissolved oxygen>20%, the about 350rpm of rotating speed, ventilation speed 350L/h, Antifoam 204 is as skimmer (SIGMA).During fermentation expression, fermentor tank is added the glucose of 50% (V/V) automatically, makes the glucose final concentration in the fermented liquid maintain 0.5-1g/L, expresses after 24 hours, and is centrifugal with 4000rpm, 15min, collects supernatant as expression product.

Collect and express 6.5 liters of supernatants, concentrated after tentatively filtering with the hollow fiber ultrafiltration membrane of 0.45um with the ultra-filtration membrane of 5kD, obtain 200 milliliters of filtered solutions.Appearance ion exchange chromatography on the filtered solution, chromatography media is SP Sepharose Big Beads (GE healthcare), with NaCl solution gradient wash-out, carries out wash-out with 0.3M NaCl earlier, collects elutriant; Carry out wash-out with 1.0M NaCl again, collect elutriant; Get the electrophoretic examinations of part elutriant, visible 1.0M NaCl elution peak is a target protein, and purity reaches more than 95%; 0.3M the NaCl elution peak then contains more foreign protein, specifically sees Fig. 5.

0.3M NaCl elutriant is carried out the second step gel permeation chromatography, and medium is superdex 200, and elutriant is the NaCl of 0.1mol/L, the fraction collection elutriant; Get the electrophoretic examinations of gel-filtration elutriant, visible is target protein at the 4-6 pipe, and purity reaches more than 95%; The 1-3 pipe is specifically seen Fig. 6 for foreign protein.

The elutriant that 1.0M NaCl is carried out ion exchange chromatography elutriant and gel permeation chromatography 4-6 pipe merges, and ordinary method is dialysed, freeze-drying, obtains the pure article albumen of pfs25 of 280mg.

The structure of the synthetic and expression vector of the pfs25 dimer gene of embodiment 5, employing (GGGGS) 3 pentadecapeptide connecting arms

(1), (pfs25-3E) ₂The structure of dimer gene

With the pfs25-3E/pUC57 vector plasmid is that template is carried out overlapping PCR (overlapping PCR) operation; The pfs25 gene of two copies is through pentadecapeptide " connecting arm (linker) series connection the becoming dimer gene; the G of connecting arm represents glycocoll, and S represents Serine of (GGGGS) 3.

Utilize four primers of primer-design software Primer premier5.0 design, entrust Shanghai to give birth to worker's biotechnology ltd and synthesize.

Primer a:

GGA CTCGAGAAAAGAGAGGCTGAAGCTATGAAAGTAACAGTCGATACT (underscore is an Xho I recognition site);

Primer b:

AGATCCACCGCCTCCGGAACCACCACCCCCAGAACCGCCACCTCCAGTACATATTGATGATTCCTCGTCGATGATAAATC；

Primer c:

GGAGGTGGCGGTTCTGGGGGTGGTGGTTCCGGAGGCGGTGGATCTAAAGTAACAGTCGATACTGTGTGTAAGAGAGGCTT；

Primer d:GG TCTGATTAAGTACATATTGATGATTCCTCGTCGATG (underscore is an Xha I recognition site)

The overlapping PCR of goal gene divided for four steps carried out:

PCR for the first time: with " primer a " and " primer b ", template is the pfs25-3E/pUC57 vector plasmid;

PCR for the second time: with " primer c " and " primer d ", template is the pfs25-3E/pUC57 vector plasmid;

PCR for the third time: do not add primer, PCR mixes 10 circulations with the product of PCR for the second time with the first time;

The 4th PCR: with " primer a " and " primer d ", template is a PCR product for the third time.

PCR reaction conditions: 95 ℃, 4min; 95 ℃, 60 seconds, 55 ℃, 60 seconds, 72 ℃, 70 seconds, 30 circulations; 72 ℃, 7min.

Two copy pfs25-3E genes through four PCR operations obtain abbreviate pfs (25-3E) as ₂, electrophoresis result is seen Fig. 7.

Behind overlapping PCR product purification, carry out ligation according to test kit specification sheets and pMD18-T Simple Vector.Connect product and transform TOP10F ' competence bacterium, the LB solid medium of coating Amp resistance carries out PCR, Xho I and Xba I double digestion to the clone who is obtained and identifies and sequencing analysis, and it is exactly (pfs25-3E) that the result is the male clone ₂/ pMD18-T recombinant plasmid.

With restriction enzyme Xho I and Xba I respectively to pfs (25-3E) ₂/ pMD18-T plasmid and pPICZ α A vector plasmid carry out the double digestion operation, utilize the T4DNA ligase enzyme with (pfs25-3E) ₂Gene is connected with pPICZ α A carrier segments, spends the night (pfs25-3E) that obtains ₂/ pPICZ α A recombinant expression plasmid transforms TOP10F ' competence bacterium, and the LB solid medium that coating zeocin resistance is selected extracts plasmid, PCR, the enzyme evaluation positive colony of cutting and check order.

Recombinant expression plasmid (pfs25-3E) ₂/ pPICZ α A carries out PCR, Xho I and Xba I double digestion through primer a and primer d, behind the electrophoresis, in the position of 1000bp the goal gene band is arranged, and the result sees Fig. 8.(pfs25-3E) ₂The dna sequencing result sees SEQ.NO.6, and the protein sequence of supposition is seen SEQ.NO.5.

(2), the structure of Pfs25-3Q dimer gene

With the Pfs25-3Q/pUC57 plasmid is template, and overlapping PCR (overlapping PCR) makes up (pfs25-3Q) ₂Gene.Article four, overlapping PCR primer, wherein primer a is identical with Pfs25-3E with primer c, and primer b is different with Pfs25-3E with primer d, is specially:

Primer b:

AGATCCACCGCCTCCGGAACCACCACCCCCAGAACCGCCACCTCCAGTACATATTGATGATTC CTGGTCGATGATAAATC；

Primer d:GGTCTAGATTAAGTACATATTGATGATTC CTGGTCGATG

The triplet code that underscore indicates among primer b and the primer d, different with the primer of Pfs25-3E, specifically corresponding to the Stimulina of 187 of Pfs25-3Q protein mutants.

With reference to (pfs25-3E) ₂The structure of dimer gene and clone's step obtain (pfs25-3Q) ₂/ pMD18-T plasmid with (pfs25-3Q) ₂/ pPICZ α A recombinant expression plasmid, correct through the order-checking qualification result.(pfs25-3Q) ₂The dna sequencing result sees sequence table SEQ .NO.8, and the protein sequence of supposition is seen SEQ.NO.7.

The structure of the synthetic and expression vector of the pfs25 dimer gene of embodiment 6, employing (GGGS) 40 six peptide connecting arms

With the pfs25-3E/pUC57 vector plasmid is that template is carried out overlapping PCR (overlapping PCR) operation; The pfs25 gene of two copies is through 16 peptides " connecting arm (linker) series connection the becoming dimer gene; the G of connecting arm represents glycocoll, and S represents Serine of (GGGS) 4.

Utilize four primers of primer-design software Primer premier5.0 design, entrust Shanghai to give birth to worker's biotechnology ltd and synthesize.

Primer a:

GGA CTCGAGAAAAGAGAGGCTGAAGCTATGAAAGTAACAGTCGATACT (underscore is an xho I recognition site)

Primer b:

AGAGCCACCTCCAGAGCCACCTCCAGAGCCACCTCCAGAGCCACCTCCAGTACATATTGATGATTCCTCGTCGATGATAAATC

Primer c:

GGAGGTGGCTCTGGAGGTGGCTCTGGAGGTGGCTCTGGAGGTGGCTCTAAAGTAACAGTCGATACTGTGTGTAAGAGAGGCTT

Primer d:

GG TCTAGATTAAGTACATATTGATGATTCCTCGTCGATG (underscore is an xba I recognition site)

With reference to the clone operations step of embodiment 5, made up comprise (GGGS) 4 as the dimeric recombinant expression plasmid of the pfs25-3E of linker (pfs25-3E) ' ₂/ pPICZ α A, correct through the order-checking qualification result, (pfs25-3E) ' ₂The dna sequencing result see SEQ.NO.10, the protein sequence of supposition is seen SEQ.NO.9.

Embodiment 7, recombination microzyme (pfs25-3E) ₂Structure and the evaluation of/pPICZ α A/GS115

Positive recombinant expression plasmid (pfs25-3E) ₂/ pPICZ α A after BstX I enzyme tangent lineization, electrotransfection method transformed competence colibacillus pichia spp GS115.Electric shock adds 800 μ l 1M sorbyl alcohols after finishing immediately, and mixing was placed 2 hours for 30 ℃, got an amount of conversion product and coated on the YPD solid screening culture medium that contains 100 μ g/ml Zeocin, and 30 ℃ of wet boxes leave standstill, and visible mono-clonal bacterium colony occurs after 3-5 days.The some colony inoculations of picking are in the 3mlYPD substratum at random, and 30 ℃ of grow overnight are collected thalline, carry out PCR with primer a, primer d and identify, positive yeast reorganization bacterium is (pfs25-3E) ₂/ pPICZ α A/GS115.

Picking (pfs25-3E) ₂/ pPICZ α A/GS115 recombination microzyme bacterium colony, in 2ml YPD nutrient solution, 30 ℃, the 250r/min overnight cultures, centrifugal collection thalline was transferred and in 3ml YPD nutrient solution, was continued to cultivate next day.It is 2% that every separated 24h adds methyl alcohol to final concentration, and keeps sample.Cultivation and abduction delivering are collected supernatant to 72h.PPICZ α A/GS115 bacterium is as the synchronous abduction delivering of negative control.

The expression supernatant of all reorganization bacterium and negative bacterium different times kept sample carry out the electrophoresis operation, the concentration of separation gel is 10%, and the concentration that concentrates glue is 5%, adopts Xylene Brilliant Cyanine G as the protein staining agent, and the result sees Fig. 9.There is target protein matter the position that the result is presented at 45kd, this explanation (pfs25-3E) ₂Protein by the recombination microzyme secreting, expressing in substratum.

Utilize among the professional result of gel electrophoresis strip analysis software ImageQuant TL (GE Healthcare company) (pfs25-3E) Fig. 9 ₂Proteic content is analyzed, and the result sees table 1.Can see (pfs25-3E) ₂Albumen (band 4) accounts for 68% of expression of recombinant yeast supernatant total protein, and this expression amount can be used for following large-scale industrial production fully.

Table 1:ImageQuant TL software analysis (pfs25-3E) ₂/ pPICZ α A/GS115 recombination microzyme is expressed supernatant

SDS-PAGE is identified that the male sample carries out the SDS-PAGE electrophoresis again; With the pfs25 standard substance as positive control; Negative bacterium is expressed supernatant as negative control; To pvdf membrane, react ECL chemoluminescence method colour developing observations one by one with 4B7 monoclonal antibody and HRP mark rabbit anti-mouse igg through electrotransfer.The result sees Figure 10.

The result shows, in this research (pfs25-3E) ₂Gene is successful expression in pichia spp, and expression product possesses good immunoreactivity.

Employing makes up (pfs25-3Q) 2/pPICZ α A/GS115 recombination microzyme with quadrat method, expresses and cultivates, and this recombination microzyme expression product and 4B7 monoclonal antibody are positive.

Embodiment 8, (pfs25-3E) ₂Dimeric expression and purifying

Picking (pfs25-3E) ₂/ pPICZ α A/GS115 recombination microzyme bacterium colony, in 2ml YPD nutrient solution, 30 ℃, overnight cultures.Ratio with 1:200 is inoculated in the 200ml YPD substratum 30 ℃ of overnight cultures.For the first time add methyl alcohol and begin to induce, afterwards, it is 2% that interval 24h adds methyl alcohol to final concentration, expresses to 72h the supernatant of culture medium that results are expressed.

The expression supernatant of collecting with the centrifugal 30min of 4000rpm, is collected supernatant.After carrying out further clarification filtration with the hollow fiber ultrafiltration membrane of 0.45um; Ultra-filtration membrane with 5kD concentrates and replaces in the 15mM citric acid-sodium citrate damping fluid (PH4.5); Carry out ion exchange chromatography; Chromatography media is: SP Sepharose Big Beads (GE healthcare) is a basal liquid with the 15mM citric acid-sodium citrate, carries out 0.1mol/LNaCl, 0.3mol/L NaCl and 1.0mol/L NaCl gradient elution.Each wash-out part SDS-PAGE analyzes and sees Figure 11, and near the target protein the 45KD mainly is included in 0.3mol/LNaCl and the 1.0mol/LNaCl gradient elution peak, and a small amount of foreign protein is only arranged.

Collect 0.3mol/L NaCl and 1.0mol/L NaCl elution peak and carry out gel permeation chromatography, medium is: superdex 200, and elutriant is the NaCl of 0.1mol/L, and SDS-PAGE analyzes and sees Figure 12.Collection comprises the elution peak of purpose product, ordinary method dialysis, freeze-drying, and the pure article 68mg of accumulative total preparation (pfs25-3E) 2 albumen analyzes through HPLC, and purity is higher than 95%.

Embodiment 9, the proteic immunogenicity of pfs25 detect

Select the 12-14g female NIH mice as animal subject.2.5ug pfs25-3E and 7.5ug, 2.5ug, 0.83ug (pfs25-3E) are set ₂Four experimental group, every group of 10 mouse.In addition, Al (OH) 3 adjuvants, two control groups of saline water are set, immune programme for children is 0,0.5, pin inoculation in January three.Accomplish inoculation and gathered the mouse blood sample in back one month, the Pfs25 standard protein that provides with U.S. MR4 carries out the indirect ELISA detection.

Pfs25-3E monomeric protein group detected result is seen table 2, (pfs25-3E) ₂Three various dose group detected result data are seen table 3.In all four experimental group, except (pfs25-3E) ₂0.8ug mouse antibodies is outside sun changes in the dose groups, the antibody of all experimental mice all sun changes.Meanwhile, in the mouse of two contrasts of saline water and adjuvant group, the pfs25 antibody test is all negative.

The Pfs25-3E monomeric protein is compared with dimer protein, 0.8ug (pfs25-3E) ₂The antibody horizontal of the antibody horizontal of experimental mice and 2.5ug pfs25-3E experimental mice is suitable, all 10 ²The order of magnitude; When inoculating the antigen of 2.5ug dosage equally, (pfs25-3E) 2 albumen are than the high one magnitude of the proteic antibody horizontal of pfs25-3E.And in inoculation (pfs25-3E) ₂Between antigenic three various dose groups, pfs25 antibody horizontal and antigenic dosage of inoculation are linear positive correlation.Above digital proof, (pfs25-3E) ₂Albumen has stronger immunogenicity than pfs25-3E albumen.

Table 2:ELISA detects the anti-pfs25 protein antibodies in the pfs25 albumen inoculation mice serum

Table 3:ELISA detects (pfs25) ₂Anti-(pfs25) in the albumen inoculation mice serum ₂Protein antibodies

Embodiments of the invention are used for explanation preferred embodiment, and unrestricted protection scope of the present invention.The objective of the invention is to utilize the recombination means to make up the pfs25 gene that comprises two copies; Connect two pfs25 unit-segment through connecting arm in the expression product and form dimer, each pfs25 unit-segment comprises the part of removing natural pfs25 albumen n end signal peptide and C end transmembrane domains.Preferred form is to remove 22 amino acid of N end and 24 amino acid of C end, obtains being equivalent to the part of natural pfs25 protein 23-193 amino acids.But the present invention is not limited to remove accurately the embodiment of N end signal peptide and C end transmembrane domains; For example; Adopt Kaslow DC to design non-glycosylated two mutants pfs25-B; This two mutants is equivalent to the 22-188 amino acids of pfs25 native sequences, and is L-Ala (Ala, A) (Nature.1988 May 5 with the asparagine mutation in 112,165,187 3 sites; 333 (6168): 74-6.; US5217898), be built into the dimer that comprises two molecule pfs25-B, can realize the object of the invention equally, this is that those skilled in the art understand easily.Similarly, connect the connecting arm of two pfs25 unit-segment, be not limited to the cited mode of the embodiment of the invention.As long as this connecting arm can form the flexible polypeptide fragment that does not have fixed sturcture, when playing the physical connection effect, do not influence the immunogenicity of pfs25 unit-segment, do not increase new function yet, can realize the object of the invention.Any identical in fact distortion for pfs25 unit-segment of the present invention, connecting arm falls in protection scope of the present invention equally.

Claims (10)

1. proteic dimer derivate of plasmodium falciparum Pfs25 has following structure:

pfs25-L-pfs25

Wherein pfs25 represents the pfs25 protein mutant, and L represents connecting arm (linker);

Described pfs25 protein mutant comprises 23-193 amino acids fragment and the non-glycosylated two mutants thereof that is equivalent to pfs25 albumen native sequences;

Described connecting arm has following structure: S _a-(G _bS) _c, wherein S represents Serine, and G represents glycocoll, a=0 or 1, b=3 or 4, the integer of c=1-4.

2. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that the 112nd, 165,187 the l-asparagine that described pfs25 protein mutant is equivalent to natural Pfs25 sequence replaces with L-Ala.

3. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that the 112nd, 165,187 the l-asparagine that described pfs25 protein mutant is equivalent to natural Pfs25 sequence replaces with Stimulina.

4. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that the 112nd, 165,187 the l-asparagine that described pfs25 protein mutant is equivalent to natural Pfs25 sequence replaces with L-glutamic acid.

5. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that described connecting arm has the structure of SGGGGS.

6. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that described connecting arm has (GGGGS) ₃Structure.

7. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that described connecting arm has (GGGS) ₄Structure.

8. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that the proteic dimer derivate of Pfs25 has the aminoacid sequence shown in the sequence table Seq.No.5.

9. according to the proteic dimer derivate of the Pfs25 of claim 1, it is characterized in that the proteic dimer derivate of Pfs25 has the aminoacid sequence shown in the sequence table Seq.No.7.

10. according to the purposes of the proteic dimer derivate of each described Pfs25 of claim 1-9, be used to prepare the vaccine that prevents pernicious malaria.

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