CN102559615A - Preparation method of EV71 vaccine and vaccine prepared by preparation method - Google Patents
Preparation method of EV71 vaccine and vaccine prepared by preparation method Download PDFInfo
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Abstract
The invention discloses a preparation method of an EV71 vaccine and the vaccine prepared by the preparation method, relating to the preparation method of taking a Pichia yeast as an expression system for co-expression of P1 and 3CD proteins of the EV71 after codon optimization by utilizing different promoters, and obtaining the viral like particle vaccine with immunogenicity, and also relating to the vaccine prepared by the preparation method.
Description
Technical field
The present invention relates to the pichia spp is expression system, P1 and the 3CD albumen of the EV71 after utilizing the different promoters coexpression codon optimized, and obtain having the preparation method of immunogenic virus sample particle vaccines.Also relate to vaccine through this method preparation.
Background technology
Enterovirns type 71 (EV71) belongs to the Picornaviridae enterovirus genus; Main hand foot mouth disease and the central nervous system diseases such as aseptic meningitis, BBE and poliomyelitis appearance paralysis of causing; Disable and case fatality rate higher, be hazard rating be only second to poliovirus have a liking for the nervosa enterovirus.EV71 is unique host with the mankind, and main route of transmission is a fecal-oral transmission.The crowd is to the general susceptible of EV71, but its mainly to infect object be infant below 5 years old.Since reported first in 1974; EV71 has caused more than ten scale property outburst in the whole world; Only in China; Just once caused tens of thousands of people's subinfection on Taiwan and Shandong and other places respectively with 2007-2008, and in some RR in 1998, EV71 at the geographic sickness rate of Chinese part up to 10%.
The EV71 virion is made up of 60 identical subunits for no coating icosahedron symmetry spheroid.Its viral genome is the sub-thread positive chain RNA; Be about 7400bp, have only one to read frame and be positioned at 5 ' and 3 ' non-coding region, the polyprotein of encoding; Comprise 3 precursor protein P1-P3; Four capsid proteins of P1 coding VP1-VP4, P2 and 7 Nonstructural Proteins of P3 coding comprise 2A, 2B, 2C, 3A, 3B, 3C, 3D.Wherein 3CD is the specific proteins lytic enzyme, identification Gln-Gly site, and decomposed P 1 precursor protein makes it discharge the VP1-VP4 that forms viral capsid subunit, finally accomplishes the assembling of virion.
According to gene order homology difference, can EV71 be divided into A, B, three kinds of genotype of C, wherein Type B and C type can further be subdivided into B1-B5, C1-C4 hypotype again.The fashion trend of different genotype presents certain areal variation, and areal different time popular EV71 genotype is different.But since 1997, EV71 was more stable in the fashion trend of China, was main with the C4 type mainly.
Because the pathogenesis of EV71 and immunologic mechanism remain further to be disclosed, so up to the present, still do not have the vaccine of effective antiviral and prophylaxis of viral infections.In recent years, also there are many researchs to be illustrated in and obtained some progress on the EV71 vaccine research, comprise inactivated vaccine, subunit vaccine, dna vaccination, polypeptide vaccine and attenuated live vaccine.Wherein efficient and the safest vaccine research direction is recombinant virus appearance particle (VLPs) vaccine.
Virus-like particle is the grain pattern that virus-free genomic capsid protein with complete three-dimensional arrangement is formed; Both there be not infectivity; Kept the epitope on the capsid protein again, and Stability Analysis of Structures, therefore have security more reliably compared to traditional attenuation or inactivated vaccine; And compared to other recombiant vaccines, its immunogenic advantage is also very obvious.Found since 1985 hepatitis B virus (HBV) surface antigen (Surface Antigen, HBsAg) particle comes to light and can be used as since the Hepatitis B virus vaccine, the VLPs technology develops rapidly in the research of new virus vaccine.More typical successful use virus-like particle is human papillomavirus (HPV) virus-like particle as the example of vaccine; Many results of study show; But the many expression systems that comprise intestinal bacteria, yeast and baculovirus are express recombinant HPV late protein L1 and L2 all, the L1/L2-VLPs or the L1-VLPs that obtain having virus particle structure, and these virus-like particles are behind the immune host of aluminium adjuvant; Can induce body to produce the neutralizing antibody of high titre, reach good protective action.
The beginning of the nineties; People such as David C.Ansardi successfully express the VLPs that obtains very similarly belonging to together with the EV71 virus structure poliovirus of Picornaviridae in the Hela cell; Its main policies is the recombinant vectors that cotransfection has coding poliovirus P1 precursor protein and P3 precursor protein gene; The result proves the 3CD proteolytic enzyme complexes exert specific proteins hydrolytic enzyme functional in the P3 precursor protein; The P1 precursor protein of coexpression is decomposed into VP1, VP0 and VP3 albumen, and the final virus-like particle that in electron microscopic observation, obtains the form homogeneous.In 2003, after people's cotransfections in insect cell such as Yuchen Hu have the recombinant baculovirus of EV71P1 and 3CD gene, obtain the EV71 virus-like particle equally.But the main drawback of insect cell expression system is a transient expression; Virus infection finally can cause the host expresses necrocytosis; Therefore must restart infection, each takes turns proteinic production all must infect new cultured cells again, and cost is expensive; Production cycle is long, and the about 10mg/10 of EV71 virus-like particle expression amount of the use insect cell of bibliographical information
9Cell, expression amount is low, and certain problem is arranged in suitability for industrialized production.
Summary of the invention
The present invention with P1 precursor protein encoding sox and the gene clone of 3CD protease-encoding to yeast expression vector; Successfully obtain the form stable homogeneous behind the DE and have immunogenic virus-like particle, this conclusion has proved that also the assembling of EV71 virion only needs P1 precursor protein and 3CD proteolytic enzyme.
In order to satisfy the suitability for industrialized production of EV71 vaccine research and development, the present invention selects pichia yeast expression system for use, also is to utilize this system expression to obtain the research of EV71 virus-like particle first.That pichia yeast expression system has is easy and simple to handle, expression amount is high, with low cost, advantage such as growth cycle is short, is applicable to the large-scale production recombinant protein.And be more suitable in pichia spp, expressing after the optimization of the proteic DNA sequences encoding codon of P1 and 3CD, ensured the high expression level amount of recombinant virus appearance granule protein effectively.
In the host, translate the situation with expression for simulated virus infects later stage P1 and 3CD albumen, be cloned into carrier respectively through codon optimized P1 and 3CD gene, pair carriers carry out the cotransformation of pichia spp then.In addition; 3CD is handled by weak promoter pYPT1; Expression amount is low relatively, and P1 then uses strong promoter pAOX1, and expression amount is higher relatively; Control different copy numbers after P1 and two carriers of 3CD gene employing make it be integrated into the pichia spp genome, be convenient to regulate and control simultaneously two kinds of expression of recombinant proteins amounts to reach the more purpose of effective expression recombinant virus appearance granule protein.
First aspect of the present invention discloses a kind of preparation EV71 recombinant virus appearance particulate method; This method to yeast expression vector, obtains P1 precursor protein encoding sox and the gene clone of 3CD protease-encoding the form stable homogeneous and has immunogenic virus-like particle behind the DE.
In a preferred embodiment, the sequence of said P1 precursor protein encoding sox and 3CD protease-encoding gene is with the codon optimized sequence of pichia spp.In a preferred embodiment, the sequence of said P1 precursor protein encoding sox is SEQ ID NO:5, and the sequence of 3CD protease-encoding gene is SEQ ID NO:6.
In a further preferred embodiment, said P1 precursor protein encoding sox and 3CD protease-encoding gene are cloned in the yeast expression vector through different carriers.
In another preferred embodiment, said different expression vectors comprise the promotor of varying strength.In a preferred embodiment; The expression vector of said P1 precursor protein encoding sox is for inserting the recombinant vectors that SEQ ID NO:5 is constituted in the BstBI/KpnI site of pPICZ α B carrier; Said 3CD protease-encoding expression carrier is for inserting SEQ ID NO:6 and the pAOX1 promotor being replaced with the recombinant vectors that the promotor (this paper called after pYPT1 promotor, SEQ IDNO:13) of YPT1 gene is constituted in the BstBI/KpnI site of pPICZ α B carrier.
Second aspect of the present invention discloses the EV71 recombinant virus appearance particle through method preparation of the present invention.
The third aspect of the invention discloses and has comprised EV71 recombinant virus appearance particulate vaccine of the present invention.In a preferred embodiment, said vaccine also comprises aluminum hydroxide adjuvant.
Fourth aspect of the present invention discloses the purposes of EV71 recombinant virus appearance particle of the present invention as vaccine.
The 5th aspect of the present invention discloses a kind of dna molecular, and its nucleotides sequence is classified as with pichia spp codon optimized coding EV71 virus P1 precursor protein or the proteic nucleotide sequence of 3CD.In a preferred embodiment, the nucleotides sequence of said dna molecular is classified SEQID NO:5 or SEQ ID NO:6 as.
The 6th aspect of the present invention discloses a kind of recombinant vectors, wherein comprises the nucleotide sequence of fifth aspect present invention.
In one embodiment, said recombinant vectors is pPICZ α B.
In a preferred embodiment, the pAOX1 promotor that replaces pPICZ α B with weak promoter.In a preferred embodiment, said weak promoter is the pYPT1 promotor.
The 7th aspect of the present invention discloses the host cell of the recombinant vectors that comprises sixth aspect present invention.In a preferred embodiment, said host cell is the pichia spp cell.
Description of drawings
The agarose electrophoresis that Fig. 1 shows double digestion (HindIII+KpnI) evaluation of reorganization P1-pYPT1 detects.1:250bp ladder DNA marker; 2: reorganization P1-pPICZ α B plasmid (enzyme is not cut); 3: reorganization P1-pPICZ α B plasmid (HindIII+KpnI).
The agarose electrophoresis that Fig. 2 shows double digestion (HindIII+KpnI) evaluation of reorganization 3CD-pPICZ α B detects.1:250bp ladder DNA marker; 2: reorganization 3CD-pPICZ α B plasmid (enzyme is not cut); 3: reorganization 3CD-pPICZ α B plasmid (HindIII+KpnI).
The agarose electrophoresis that Fig. 3 shows the PCR evaluation of reorganization 3CD-pYPT1 detects.1:DL2000Marker; 2: reorganization 3CD-pYPT1 plasmid is the pYPT1PCR product of template; 3:ddH
2O is the pYPT1PCR product of template.
The Western-blot that Fig. 4 shows the P13CD-pYPT1-X33 abduction delivering identifies.1-2.P13CD-pYPT1-X33 induce and break white protein on the bacterium after 72 hours; 3-4.P1-pPICZ-X33 induce and break white protein on the bacterium after 72 hours; 5-6.X-33 induce and break white protein on the bacterium after 72 hours; 7.EV71 virus; 8.PageRuler Prestained ProteinLadder.
Fig. 5 shows electron microscopic observation that P13CD-pYPT1 expresses gained reorganization EV71 virus-like particle (a.10000 doubly; B.40000 doubly).
Embodiment
The present invention utilizes pichia yeast expression system to express first and obtains the form stable homogeneous and have immunogenicity and the EV71 virus-like particle of immune protective.And have following advantage: (1) pichia yeast expression system is easy and simple to handle, with low cost, and output is high, and the product property stable homogeneous is beneficial to large-scale production; (2) express 3CD proteolytic enzyme and guarantee that the P1 albumen of coexpression is broken down into the VP1-VP4 albumen that can be assembled into viral capsid; (3) P1 and 3CD gene insert different carriers, control the copy number of two genes respectively, are convenient to regulate and control simultaneously two kinds of expression of recombinant proteins amounts; (4) P1 and 3CD use strong promoter pAOX1 and weak promoter pYPT1 respectively, make P1 expression amount in host higher relatively, and simulated virus infects the later stage and mainly expresses the situation of capsid protein; (5) adapt to pichia yeast expression system after P1 and the optimization of 3CD gene codon, reach the purpose that efficiently expresses.
Following examples are illustrated the present invention by way of example, but are not intended to limit scope of the present invention.
Embodiment
1. gene Selection and codon optimized design
With reference to the dna sequence dna of C4 type strain in China BJ08-Z020-1 (GenBank:FJ606449.1) through technological composite coding EV71P1 well known in the art and 3CD.BJ08-Z020-1 wild-type P1 and 3CD dna sequence dna are SEQ ID NO:1 and SEQ 1D NO:2, and both amino acid sequence coded are SEQ ID NO:3 and SEQ ID NO:4.Dna sequence dna to wild-type P1 and 3CD is transformed, and codon all adopts the higher codon of frequency of utilization in the pichia spp, the sequence that is optimized SEQ ID NO:5 and SEQ ID NO:6.
2.P13CD-pPEXZ recombinant expression vector makes up
At first obtain P1 and 3CD gene through gene synthetic mode well known in the art, make up P1-pPICZaB and 3CD-pPICZaB recombinant vectors then according to above-mentioned compound method.AOX1 promotor with 3CD-pPICZaB replaces with the pYPT1 promotor again, obtains 3CD-pYPT1.The practical implementation step is following:
2.1P1-pPICZ α B makes up
The P1 sequence of synthetic gained SEQ ID NO:5 is cloned into pPICZ α B carrier (Invitrogen) through following method.
P1 sequence with SEQ ID NO:5 is a template, forward primer: 5 ' CCAAGCTC
TTCGAAACGATGGGTTCTCAAGTCT 3 ' (SEQ IDNO:7); Reverse primer: 5 ' AGC
GGTACCCTATTATAAAGTAGTA 3 ' (SEQ ID NO:8).Increase through the PCR mode and to obtain the P1DNA fragment that two ends have BstBI and KpnI respectively.All mentioned primers of the present invention are design voluntarily, entrust Shanghai Ying Jun Bioisystech Co., Ltd to synthesize.The PCR program: 94 ℃ 5 minutes, 94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 2 minutes 15 seconds the circulation 30 times, 72 ℃ 10 minutes, 10 ℃ 10 minutes, end of run.About 2600bp place band (Qiagen glue reclaims test kit) is identified and reclaimed to the PCR product with agarose gel electrophoresis.Reclaim fragment and pPICZ α B and cut with BstBI and KpnI (among the present invention related restriction enzyme all available from NEB company) associating enzyme, agarose gel electrophoresis is identified also the about 2600bp of recovery and about 3300bp fragment respectively.The ratio that reclaimed back P1 fragment and pPICZ α B and with mol ratio be 5: 1 is spent the night for 16 ℃ with T4 ligase enzyme (Takara) and is connected; Connect product in second day and be transformed into E.coli DH5 α (available from Beijing ancient cooking vessel state Bioisystech Co., Ltd); Coat less salt LB (1% Tryptones; 0.5% yeast powder, 0.5%NaCl) dull and stereotyped (containing 25 μ g/ml Zeocin), 37 ℃ of incubated overnight.Picking partly transforms rear clone extracting plasmid, and double digestion (HindIII+KpnI) identifies that agarose electrophoresis detects (Fig. 1).Identify the preservation after the dna sequencing checking is correct of the positive recombinant clone of gained, this recombinant vectors called after P1-pPICZ α B.
2.23CD-pPICZ α B makes up
The 3CD sequence of synthetic gained SEQ ID NO:6 is cloned into pPICZ α B carrier through following method.
3CD sequence with SEQ ID NO:6 is a template, uses forward primer: 5 ' TTTAGTTC
TTCGAAGCTAGCATGGGTCCATCTCTGG 3 ' (SEQID NO:9) and reverse primer: 5 ' GGC
GGTACCCTATTATTGTTCTGAA3 ' (SEQ ID NO:10) increases through the PCR mode and obtains the 3CD dna fragmentation that two ends have BstBI and KpnI restriction enzyme site respectively.The PCR program: 94 ℃ 5 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 50 seconds the circulation 30 times, 72 ℃ 10 minutes, 10 ℃ 10 minutes, end of run.The PCR product identifies and reclaims about 600bp place's band (Qiagen gel extraction kit) with agarose gel electrophoresis.Reclaim fragment and pPICZ α B and cut with BstBI and KpnI associating enzyme, about 600bp and about 3300bp fragment are identified and reclaimed respectively to agarose gel electrophoresis.The ratio that reclaimed back 3CD fragment and pPICZ α B and with mol ratio be 5: 1 is spent the night for 16 ℃ with T4 ligase enzyme (Takara) and is connected, and the connection product was transformed into E.coli DH5 α in second day, coated less salt LB flat board (containing 25 μ g/ml Zeocin), 37 ℃ of incubated overnight.Picking partly transforms rear clone extracting plasmid, and double digestion (HindIII+KpnI) identifies that agarose electrophoresis detects (Fig. 2).Identify the preservation after the dna sequencing checking is correct of the positive recombinant clone of gained, this recombinant vectors called after 3CD-pPICZ α B.
2.33CD-pYPT1 make up
With X-33 pichia spp genomic dna is template, uses forward primer: 5 ' TTTAGATCTCCATATGATGAGTCACAAT 3 ' (SEQ ID NO:11); Reverse primer: 5 ' AAAGCTAGCCTATTATCTCTGTGTGTAT 3 ' (SEQ IDNO:12) increases through the PCR mode and obtains pYPT1 promotor (the GenBank numbering: dna fragmentation AF027960) (SEQ ID NO:13), called after pYPT1 promotor that two ends have BglII and NheI respectively.The PCR program: 94 ℃ 5 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 50 seconds the circulation 30 times, 72 ℃ 10 minutes, 10 ℃ 10 minutes, end of run.
PYPT1 and 3CD-pPICZ α B cut with BglII and NheI associating enzyme, and about 3900bp fragment of about 900bp and the 3CD-pPICZ α B of pYPT1 is identified and reclaimed respectively to agarose gel electrophoresis.The ratio that reclaimed back pYPT1 fragment and 3CD-pPICZ α B and with mol ratio be 5: 1 is spent the night for 16 ℃ with T4 ligase enzyme (Takara) and is connected; Connect product in second day and be transformed into E.coli DH5 α; Coat less salt LB dull and stereotyped (containing 25 μ g/ml Zeocin), 37 ℃ of incubated overnight.Picking partly transforms rear clone extracting plasmid, and PCR identifies the pYPT1 fragment in the recombinant plasmid, and agarose electrophoresis detects (Fig. 3).Identify the preservation after the dna sequencing checking is correct of the positive recombinant clone of gained, this recombinant vectors called after 3CD-pYPT1.
3.P13CD-pYPT1 recombinant strains construction and expression
The used host bacterium of the present invention is pichia spp X-33 bacterial strain (available from an Invitrogen company).The concrete construction step of P13CD-pYPT1 Pichiapastoris expression strain is following:
With SacI linearizing P1-pPICZ α B and 3CD-pYPT1 carrier, common-battery changes pichia spp X-33, coats YPDS (1% yeast powder with this area normal experiment condition; 2% peptone; 2% glucose, 1M sorbic alcohol, 2% agar powder) dull and stereotyped (containing 200 μ g/mlZeocin); Cultivated 3 days for 30 ℃, the total hectogram is grand.Therefrom the tens of clones of picking are inoculated in YPD (1% yeast powder, 2% peptone, 2% glucose, 2% agar powder) dull and stereotyped (containing 1500 μ g/mlZeocin), and the high copy of screening plasmid bacterial strain was cultivated 2 days for 30 ℃.Several clones of picking are inoculated in 4ml YPD liquid nutrient medium, change BMMY (1% yeast powder, 2% peptone after 24 hours; 100mM phosphate buffered saline buffer pH6.0,1.34%YNB, 0.5% methyl alcohol; 0.00004% vitamin H) substratum, 0.5% methanol induction was collected thalline after 72 hours.Thalline is after the granulated glass sphere fragmentation, and centrifugal gained supernatant is identified (Fig. 3 only shows the result that two pickings are cloned (1-2), and other pickings clone's result is not shown) with Western-blot.Used one is anti-for how anti-through the proteic rabbit of the homemade anti-VP1 of technology well known in the art, and two to resist be goat anti-rabbit igg-HRP (available from Calbiochem company).With P1-pPICZ α B-X33 and X-33 negative contrast of expression of results (Fig. 4 _ 3-4 and 5-6) under similarity condition, with the positive contrast of EV71 virion (Fig. 4 _ 7).P1-pPICZ α B-X33 is owing to lack 3CD proteolytic enzyme; It induces near the back whole cell albumen amixia bar (Fig. 4 _ 3-4) 34kD of VP1; And the hybridization band is arranged in the bigger position of molecular weight of albumen, explain and in pichia spp, only express the VP1-VP4 albumen that P1 can't obtain having the virion assembling function; Relative; P13CD-pYPT1-X33 has obvious hybridization band (Fig. 4 _ 1-2) near 34kD; Natural EV71 virion hybridization gained band identical (Fig. 4 _ 7) with positive control; The 3CD proteolytic enzyme of proof P1-pPICZ α B and 3CD-pYPT1 coexpression has been brought into play the proteolytic ferment function, and VP1 has been discharged from P1.Get a P13CD-pYPT1-X33 bacterial strain (if but find VP1 expression amount compare unusual low then give up with other bacterial strains), for example the bacterial strain shown in Fig. 4 _ 1 or 2 is frozen in-80 ℃, as fermentor cultivation work seed.
4.EV71 the fermentor cultivation of recombinant protein
Get 1 frozen pipe of bacterial classification glycerine from the work seed bank, melt the back and draw 100 μ L access 5ml YPD substratum, 280rpm cultivated 20 hours for 30 ℃.OD
6001～2.The activation solution 1ml that microscopy is qualified inserts 500ml YPD substratum, and 280rpm cultivated 20 hours for 30 ℃.OD
6002～6.Microscopy does not have living contaminants.Use the BIOENGINEERINGNLF22 fermentor tank, fermentation is with basic salt culture medium BSM
1(K
2SO
4273g, MgSO
4109g, CaSO
4.2H
2O 17.6g, H
3PO
4400.5ml, KOH 62g, glycerine 600g, PTM160ml, bubble enemy 1ml, deionized water adds to 15L).By inoculation in 1: 15.Leavening temperature is 30.0 ℃, initial pH5.00, and rotating speed 300rpm cultivates, air flow 0.5vvm, DO100% adds PTM
1(CuSO
4.5H
2O 6.0g, NaI 0.008g, MnSO
43.0g, NaMoO
40.2g, H
3BO
30.02g, ZnSO
420.0g, CoCl
20.5g, FeSO
4.7H
2O 65.0g, biotin 0.2g, H
2SO
45.0ml deionized water adds to 1L) the trace salt.Initial about about 20 hours of the multiplicative stage, keep oxygen dissolving value and be higher than 30%, when carbon source was exhausted, the thalline weight in wet base reached about 100g/L.Add 50% glycerine solution (every liter is added 12ml PTM1).Through regulate mixing speed, air flow quantity, tank pressure (＜0.8bar) dissolved oxygen level is maintained more than 20%.Add about 8 hours, during the about 350g/L of thalline weight in wet base.Simultaneously the control of pH value is adjusted to 6.00, adds methyl alcohol (every liter is added 12mlPTM1) and induce.Keep oxygen dissolving value and be higher than 20%, 30 ℃ of temperature, the pH value is controlled to be 6.00.Emit fermented liquid when inducing 40 hours fermentation ends.Fermented liquid is through 4800rpm, and 20 minutes, bacterium mud is collected in 4 ℃ of centrifugal backs, and was frozen in-20 ℃.
5.EV71 virus-like particle results
P13CD-pYPT1-X33 thalline behind the fermentation inducement was added cleaning buffer solution (100mM PB pH7.0,0.15M NaCl) by 1: 3 mix, abundant mixing, in 8000rpm, centrifugal 5 minutes, collecting cell repeated above operation two times.Cell after cleaning was added broken bacterium damping fluid by 1: 5 mix, fully behind the mixing, the above cell suspension of high pressure fragmentation, and repetitive operation make 90% cytoclasis.The broken bacterium liquid that high pressure is broken, in 9000rpm, 30 minutes, centrifugal back supernatant was collected in 10 ℃ of spinnings.Get supernatant 40000rpm behind the broken bacterium of 1ml, 3 hours, 4 ℃ of ultracentrifugations, supernatant discarded solution, deposition with 100 μ l PBS (10mM, pH7.4) resuspended.The transmission electron microscope observing result shows, presents the virus-like particle (Fig. 5) of form stable homogeneous in the broken bacterium supernatant behind ultracentrifugation, and particle diameter is between 20-30nm.
Every liter of fermented liquid can be gathered in the crops 300 gram thalline, through behind the purification step, obtains pure VLP, and the VLP expression amount that calculates the inventive method according to the yield of the output of final VLP and purification step is about 150mg/ and rises fermented liquid.Those skilled in the art can understand, and the recombinant virus appearance granule protein expression amount of this order of magnitude can satisfy industrial production requirement, has compared with prior art obtained unforeseeable technical progress.
6.EV71 virus sample particle vaccines preparation
" recombinant hepatitis B vaccine (yeast saccharomyces cerevisiae) " chapter with reference to the 3rd one of the Pharmacopoeia of the People's Republic of China (version in 2010); 126 pages method; With the EV71 virus-like particle protein absorption hydrogen aluminum adjuvant that purifying obtains, be prepared into the candidate vaccine of prevention EV71 virus.
7.EV71 the mensuration of virus-like particle immunogenicity and immune protective
Choose the SPF level BALB/c mouse (west, Shanghai pul-Bi Kai laboratory animal ltd) in 6～8 ages in week, be divided into 2 groups, every group of 8 mouse.The 1st group of mouse carries out immunity (as negative control group) with the damping fluid that 0.5ml contains aluminium adjuvant; The 2nd group is used 0.5ml concentration is the VLP (as test set) of the Al adsorption adjuvant of 20 μ g/ml; Respectively at subcutaneous injection immunity in the 0th, 14 day, immunity twice altogether, immunity back two weeks blood sampling for the second time.With the blood that collects in 37 ℃ place 2h after, the centrifugal 10min of 4000g draws supernatant, promptly obtains the mouse polyvalent antibody, deposit in-20 ℃, and detect mouse serum antibody titers and in EV71 antiviral antibody titre, concrete grammar is following:
The antibody titers measuring method is following: EV71 virus vlps to the 1 μ g/ml with coating buffer dilution purifying, in each shrinkage pool of enzyme plate, respectively add 0.1ml, and 4 ℃ are spent the night.Remove coating buffer, with 0.3ml PBST (10mM PBS+0.05%Tween-20) washing shrinkage pool.With 0.3ml confining liquid (5% skim-milk+PBST) be incubated 2 hours in 37 ℃.Every shrinkage pool adds with dilution buffer liquid (2% skim-milk+PBST) with each 0.1ml of seized serum (the dilution gradient was from 1: 100 to 1: 3278800) of different gradient dilutions, remove serum solution in 37 ℃ of insulations after 1 hour, with 0.3ml washings washing shrinkage pool.Add with dilution buffer liquid each 0.1ml of goat anti-mouse igg with the HRP mark of dilution in 1: 5000 to every shrinkage pool then, enzyme mark liquid is removed in 37 ℃ of insulations after 0.5 hour, with 0.3ml washings washing shrinkage pool; In shrinkage pool, add 0.1ml DAB colour developing liquid then, the effect of room temperature lucifuge adds 2M H after 20 minutes
2SO
40.05ml the stop buffer termination reaction, and with enzyme mark photoelectric color comparator mensuration OD
450Value is according to OD
450Reading value calculates the antibody titers value of serum.
The NAT measuring method is following: in 96 porocyte culture plates, serum to be checked is carried out gradient dilution, extension rate was from 1: 8 to 1: 512, and each extent of dilution is got EV71 virus liquid (the titre 100CCID of 0.05ml and 0.05ml
50/ 0.05ml) mix, mixed solution put into 37 ℃ hatch 2 hours after, adding concentration is 2 * 10
5The RD cell suspension of individual/ml is put into 37 ℃ of C02 incubators then and is hatched cultivation.Using inverted microscope to observe CPE (cytopathic effect) every day, and record titration of virus result, was final serum NAT value with the inverse that suppresses the high dilution of 50% cytopathic serum after 6-7 days.
Serum antibody titer with in as shown in table 1 with the result of antiviral antibody titre, can find out that from the result of table 1 the EV71 vaccine immune mouse that adopts virus-like particle has the immune protective of very strong immunogenicity and external virus.
Table 1EV71 virus-like particle immune mouse gained serum antibody titer value and NAT value
" technology well known in the art " described herein; Be meant the technology that those of ordinary skills can find or draw according to experiment purpose from documents such as this area or the disclosed dictionary of association area, textbook, patent, paper, for example the technology described in " molecular cloning ".
Claims (13)
1. one kind prepares EV71 recombinant virus appearance particulate method; This method comprises EV71 virus P1 precursor protein encoding sox and the gene clone of 3CD protease-encoding to yeast expression vector, obtains the form stable homogeneous behind the DE and have immunogenic virus-like particle.
2. the process of claim 1 wherein that the sequence of said P1 precursor protein encoding sox is SEQ ID NO:5, the sequence of 3CD protease-encoding gene is SEQ ID NO:6.
3. claim 1 or 2 method, wherein said P1 precursor protein encoding sox and 3CD protease-encoding gene through different expression carrier clonings to yeast expression vector.
4. the method for claim 3, wherein said different expression vectors comprise the promotor of varying strength.
5. the method for claim 4; The expression vector of wherein said P1 precursor protein encoding sox is for inserting the recombinant vectors that SEQ ID NO:5 is constituted in the BstBI/KpnI site of pPICZ α B carrier, and said 3CD protease-encoding expression carrier is for inserting SEQ ID NO:6 and the pAOX1 promotor is replaced with the recombinant vectors that the pYPT1 promotor is constituted in the BstBI/KpnI site of pPICZ α B carrier.
6. through each the EV71 recombinant virus appearance particle of method preparation of aforementioned claim.
7. vaccine, it comprises the EV71 recombinant virus appearance particle of claim 6.
8. the vaccine of claim 7 also comprises aluminum hydroxide adjuvant.
9. the EV71 recombinant virus appearance particle of claim 6 is as the purposes of vaccine.
10. dna molecular, its nucleotides sequence is classified SEQ ID NO:5 or SEQ IDNO:6 as.
11. a recombinant vectors is the pPICZ α B carrier of the nucleotide sequence that comprises claim 10.
12. the recombinant vectors of claim 11, wherein the pAOX1 promotor is replaced by the pYPT1 promotor.
13. comprise the pichia spp cell of the recombinant vectors of claim 11 or 12.
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| CN102925476A (en) * | 2012-11-15 | 2013-02-13 | 中国科学院上海巴斯德研究所 | Preparation method and application of self-combined immunogenic enterovirus-like particles in yeast |
| CN103255163A (en) * | 2013-05-15 | 2013-08-21 | 北京民海生物科技有限公司 | EV71virus-like particles as well as preparation method and application thereof |
| CN104945472A (en) * | 2014-12-31 | 2015-09-30 | 苏州偲聚生物材料有限公司 | Polypeptide, detection device comprising polypeptide, and detection kit comprising device |
| CN105087505A (en) * | 2015-07-23 | 2015-11-25 | 山东大学 | Human enter ovirus 71 3C chimeric virus as well as rescue method and application thereof |
| CN105087503A (en) * | 2015-07-23 | 2015-11-25 | 山东大学 | Human enter ovirus 71 3CD chimeric virus as well as rescue method and application thereof |
| CN105802923A (en) * | 2016-04-14 | 2016-07-27 | 中国科学院生物物理研究所 | Recombinant poliomyelitis virus-like particles and preparation method and application thereof |
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| JP2018191645A (en) * | 2012-09-05 | 2018-12-06 | メディカゴ インコーポレイテッド | Production of picornavirus-like particles in plants |
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| CN105087505A (en) * | 2015-07-23 | 2015-11-25 | 山东大学 | Human enter ovirus 71 3C chimeric virus as well as rescue method and application thereof |
| CN105087503A (en) * | 2015-07-23 | 2015-11-25 | 山东大学 | Human enter ovirus 71 3CD chimeric virus as well as rescue method and application thereof |
| CN105802923A (en) * | 2016-04-14 | 2016-07-27 | 中国科学院生物物理研究所 | Recombinant poliomyelitis virus-like particles and preparation method and application thereof |
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