CN102559615B - EV71 vaccine preparation method and the vaccine prepared by the method - Google Patents
EV71 vaccine preparation method and the vaccine prepared by the method Download PDFInfo
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Abstract
EV71 vaccine preparation method and the vaccine prepared by the method.The present invention relates to Pichia sp. as expression system, P1 Yu the 3CD albumen of the EV71 after utilizing different promoters coexpression codon optimized, and obtain the preparation method with immunogenic virus sample particle vaccines.Further relate to the vaccine prepared by the method.
Description
Technical field
The present invention relates to Pichia sp. as expression system, the EV71 after utilizing different promoters coexpression codon optimized
P1 Yu 3CD albumen, and obtain the preparation method with immunogenic virus sample particle vaccines.Further relate to by the party's legal system
Standby vaccine.
Background technology
Enterovirns type 71 (EV71) belongs to Picornaviridae enterovirus genus, main initiation hand-foot-mouth disease and aseptic brain
The central nervous system disease such as film inflammation, brain stem encephalitis and poliomyelitis sample paralysis, disable and case fatality rate are higher, be the extent of injury
It is only second to the Neural invasion enterovirus of poliovirus.EV71 with the mankind for unique host, major transmission path be excrement-
Oral instructions are broadcast.Crowd is the most susceptible to EV71, but its main infection object is 5 years old Infants Below.From reported first in 1974 with
Coming, EV71 has caused more than ten scale outburst in the whole world, only in China, the most once in 1998 and 2007-2008 fraction of the year
Do not cause tens of thousands of people's subinfection on Taiwan and Shandong and other places, and in some research report, EV71 sending out in selected areas of China
Sick rate is up to 10%.
EV71 virion is without peplos icosahedral symmetry spheroid, is made up of 60 identical subunits.Its virus base
Because group is for single-stranded positive RNA, be about 7400bp, only one of which reading frame and be positioned at 5 ' and 3 ' noncoding region, encode more than one
Polyprotein, comprises 3 precursor protein P1-P3, P1 and encodes tetra-capsid proteins of VP1-VP4, P2 and P3 encodes 7 non-structural proteins
In vain, including 2A, 2B, 2C, 3A, 3B, 3C, 3D.Wherein 3CD is specific proteolytic enzyme, identifies Gln-Gly site, decomposed P 1
Precursor protein so that it is discharge the VP1-VP4 of composition viral capsid subunit, be finally completed the assembling of virion.
According to gene homology difference, EV71 can be divided into tri-kinds of genotype of A, B, C, wherein Type B and c-type can be entered again
One step is subdivided into B1-B5, C1-C4 hypotype.The fashion trend of different genotype presents certain areal variation, and areal is not
The EV71 genotype popular with the time is the most different.But from the beginning of 1997, EV71 is more stable in the fashion trend of China,
Main based on C4 type.
Owing to the pathogenesis of EV71 needs to be disclosed further with immunologic mechanism, so up to the present, still without effective
Antiviral drugs and the vaccine of prophylaxis of viral infections.In recent years, many research is also had to show to achieve on EV71 vaccine research
Some progress, including inactivated vaccine, subunit vaccine, DNA vaccination, polypeptide vaccine and attenuated live vaccine.The most efficiently and peace
Full vaccine research direction is recombinant virus sample granule (VLPs) vaccine.
Virus-like particle is the grain structure of the capsid protein composition with complete stereochemical structure of virus-free genome, both
Without infectious, remain again the epitope on capsid protein, and Stability Analysis of Structures, therefore compared to tradition attenuation or inactivation epidemic disease
Seedling has more reliable safety, and compared to other recombiant vaccinies, its immunogenic advantage is the most clearly.Since 1985
Year find hepatitis B virus (HBV) surface antigen (Surface Antigen, HBsAg) granule be found can as Hepatitis B virus vaccine with
Coming, VLPs technology develops rapidly in the research of new virus vaccine.Successfully utilize virus-like particle as epidemic disease than more typical
The example of Seedling is human papillomavirus (HPV) virus-like particle, and many results of study show, including escherichia coli, yeast and bar
Shape virus all can express restructuring HPV late protein L1 and L2 at interior many expression systems, obtains having virus particle structure
L1/L2-VLPs or L1-VLPs, these virus-like particles after immunity host, can induce body to produce height together with aluminium adjuvant
The neutralizing antibody of titre, reaches good protective effect.
At the beginning of the nineties, David C.Ansardi et al. successfully expresses in Hela cell and obtains and EV71 virus structure
The VLPs of the very similar poliovirus belonging to Picornaviridae together, its main policies is that cotransfection is with coding ridge
Tephromyelitis virus P1 precursor protein and the recombinant vector of P3 precursor protein gene, result proves the 3CD egg in P3 precursor protein
White combined enzyme agent plays specific proteolytic enzyme function, and the P1 precursor protein of coexpression is decomposed into VP1, VP0 and VP3 egg
In vain, and finally in electron microscopic observation, obtain the virus-like particle that form is homogeneous.In 2003, Yuchen Hu et al. was thin insecticide
In born of the same parents after the cotransfection recombinant baculovirus with EV71P1 Yu 3CD gene, it is similarly obtained EV71 virus-like particle.But insecticide
The major defect of cell expression system is transient expression, and virus infects and eventually results in host expression cell death, it is therefore necessary to
Restarting infection, each production taking turns protein all must infect the cell of new cultivation, cost intensive again, and the production cycle is long,
And the EV71 virus-like particle expression about 10mg/10 using insect cell of document report9Cell, expression is low,
Industrialized production has some problems.
Summary of the invention
P1 precursor protein encoding gene and 3CD proteinase encoding genes are cloned into yeast expression vector by the present invention
In, successfully obtain form stable homogeneous after double expression(DE) and there is immunogenic virus-like particle, this conclusion also demonstrates EV71
The assembling of virion only needs P1 precursor protein and 3CD protease.
In order to meet the industrialized production of EV71 vaccine research and development, the present invention selects pichia yeast expression system, is also first
This system expression is utilized to obtain the research of EV71 virus-like particle.Pichia yeast expression system have easy and simple to handle, expression is high,
The advantages such as with low cost, growth cycle is short, it is adaptable to large-scale production recombiant protein.And the DNA sequences encoding of P1 Yu 3CD albumen
It is more suitable in Pichia sp. after codon optimization expressing, has effectively ensured the high expressed of recombinant virus sample granule protein
Amount.
In order to simulated virus infects the situation that later stage P1 translates in host with 3CD albumen and expresses, through codon optimized
P1 Yu 3CD gene be cloned into carrier respectively, then complex carries carries out the cotransformation of Pichia sp..Additionally, 3CD is by weak startup
Sub-pYPT1 handles, and expression is relatively low, and P1 then uses strong promoter pAOX1, and expression is of a relatively high, P1 Yu 3CD gene uses
Two carriers make it be integrated into the copy number that the control of Pichia sp. postgenome is different, it is simple to two kinds of recombiant protein tables of regulation and control simultaneously
The amount of reaching is to reach more effectively to express the purpose of recombinant virus sample granule protein.
A first aspect of the present invention discloses a kind of method preparing EV71 recombinant virus sample granule, and the method is by P1 precursor
Protein coding gene and 3CD proteinase encoding genes are cloned in yeast expression vector, obtain form homogeneous after double expression(DE)
Stablize and there is immunogenic virus-like particle.
In a preferred embodiment, described P1 precursor protein encoding gene and the sequence of 3CD proteinase encoding genes
For the sequence codon optimized with Pichia sp..In a further preferred embodiment, described P1 precursor protein encoding gene
Sequence be SEQ ID NO:5, the sequence of 3CD proteinase encoding genes is SEQ ID NO:6.
In a further preferred embodiment, described P1 precursor protein encoding gene passes through with 3CD proteinase encoding genes
Different carriers is cloned in yeast expression vector.
In yet another preferred embodiment, described different expression vector comprises the promoter of varying strength.At one more
In preferred embodiment, the expression vector of described P1 precursor protein encoding gene is the BstBI/KpnI at pPICZ α B carrier
The recombinant vector that SEQ ID NO:5 is constituted is inserted in site, and the expression vector of described 3CD proteinase encoding genes is at pPICZ α
The BstBI/KpnI site of B carrier is inserted SEQ ID NO:6 and pAOX1 promoter is replaced with the promoter of YPT1 gene (herein
Named pYPT1 promoter, SEQ IDNO:13) recombinant vector that constituted.
A second aspect of the present invention discloses EV71 recombinant virus sample granule prepared by the method by the present invention.
A third aspect of the present invention discloses the vaccine of the EV71 recombinant virus sample granule comprising the present invention.At one preferably
In embodiment, described vaccine also comprises aluminum hydroxide adjuvant.
A fourth aspect of the present invention discloses the EV71 recombinant virus sample granule purposes as vaccine of the present invention.
A fifth aspect of the present invention discloses a kind of DNA molecular, and its nucleotides sequence is classified as with Pichia sp. codon optimized
Coding EV71 virus P1 precursor protein or the nucleotide sequence of 3CD albumen.In a preferred embodiment, described DNA divides
The nucleotides sequence of son is classified as SEQID NO:5 or SEQ ID NO:6.
A sixth aspect of the present invention discloses a kind of recombinant vector, wherein comprises the nucleotides sequence of fifth aspect present invention
Row.
In one embodiment, described recombinant vector is pPICZ α B.
In a preferred embodiment, the pAOX1 promoter of pPICZ α B is replaced with weak promoter.More excellent at one
In the embodiment of choosing, described weak promoter is pYPT1 promoter.
A seventh aspect of the present invention discloses the host cell of the recombinant vector comprising sixth aspect present invention.Excellent at one
Selecting in embodiment, described host cell is Pichia pastoris.
Accompanying drawing explanation
Fig. 1 shows the sepharose electrophoresis detection that the double digestion (HindIII+KpnI) of restructuring P1-pYPT1 is identified.1:
250bp ladder DNA marker；2: restructuring P1-pPICZ α B plasmid (non-enzyme action)；3: restructuring P1-pPICZ α B plasmid
(HindIII+KpnI)。
Fig. 2 shows the sepharose electrophoresis detection that the double digestion (HindIII+KpnI) of restructuring 3CD-pPICZ α B is identified.1:
250bp ladder DNA marker；2: restructuring 3CD-pPICZ α B plasmid (non-enzyme action)；3: restructuring 3CD-pPICZ α B plasmid
(HindIII+KpnI)。
Fig. 3 shows the sepharose electrophoresis detection that the PCR of restructuring 3CD-pYPT1 identifies.1:DL2000Marker；2: restructuring
3CD-pYPT1 plasmid is the pYPT1PCR product of template；3:ddH2O is the pYPT1PCR product of template.
Fig. 4 shows that the Western-blot of P13CD-pYPT1-X33 abduction delivering identifies.1-2.P13CD-pYPT1-
X33 induces bacteria break supernatant albumen after 72 hours；3-4.P1-pPICZ-X33 induces bacteria break supernatant albumen after 72 hours；5-6.X-33
Bacteria break supernatant albumen after inducing 72 hours；7.EV71 virus；8.PageRuler Prestained ProteinLadder.
Fig. 5 shows that (a.10000 again P13CD-pYPT1 expresses the electron microscopic observation of gained restructuring EV71 virus-like particle；
B.40000 again).
Detailed description of the invention
The present invention utilizes pichia yeast expression system expression obtain form stable homogeneous and have immunogenicity and exempt from first
The EV71 virus-like particle of epidemic disease protectiveness.And have the advantage that (1) pichia yeast expression system is easy and simple to handle, it is with low cost,
Yield is high, product property stable homogeneous, beneficially large-scale production；(2) express 3CD protease and ensure that the P1 albumen of coexpression is divided
Solve the VP1-VP4 albumen being to can be assembled into viral capsid；(3) P1 Yu 3CD gene inserts different carriers, controls two genes respectively
Copy number, it is simple to simultaneously regulation and control two kinds of expression of recombinant proteins amounts；(4) P1 with 3CD uses strong promoter pAOX1 respectively and weak opens
Mover pYPT1, makes P1 expression in host of a relatively high, and simulated virus infects the later stage and mainly expresses the feelings of capsid protein
Condition；(5) adapt to pichia yeast expression system after P1 Yu 3CD gene codon optimizes, reach the purpose of high efficient expression.
The present invention is illustrated by following example by way of example, but is not limiting as the scope of the present invention.
Embodiment
1. gene Selection and codon optimized design
Synthesized by technology well known in the art with reference to C4 type strain in China BJ08-Z020-1 (GenBank:FJ606449.1)
The DNA sequence of coding EV71P1 Yu 3CD.BJ08-Z020-1 wild type P1 and 3CD DNA sequence are SEQ ID NO:1 and SEQ
1D NO:2, both aminoacid sequences of coding are SEQ ID NO:3 and SEQ ID NO:4.DNA sequence to wild type P1 Yu 3CD
Row are transformed, and codon all uses and uses the codon that frequency is higher in Pichia sp., obtains optimization SEQ ID NO:5
With SEQ ID NO:6.
2.P13CD-pPEXZ recombinant expression carrier builds
First by the way of gene chemical synthesis well known in the art, P1 Yu 3CD gene is obtained according to above-mentioned synthetic method, so
Rear structure P1-pPICZaB Yu 3CD-pPICZaB recombinant vector.Again the AOX1 promoter of 3CD-pPICZaB is replaced with pYPT1
Promoter, obtains 3CD-pYPT1.It is embodied as step as follows:
2.1P1-pPICZ α B builds
The P1 sequence of synthesis gained SEQ ID NO:5 is cloned into pPICZ α B carrier (Invitrogen) by following method.
With the P1 sequence of SEQ ID NO:5 as template, forward primer: 5 ' CCAAGCTCTTCGAAACGATGGGTTCTCAAG
TCT 3 ' (SEQ IDNO:7)；Reverse primer: 5 ' AGCGGTACCCTATTATAAAGTAGTA 3 ' (SEQ ID NO:8).Pass through
PCR mode expands to be respectively provided with the P1DNA fragment of BstBI and KpnI to two ends.All primers mentioned by the present invention are
Designed, designed, entrusts Shanghai Ying Jun Bioisystech Co., Ltd to synthesize.PCR program: 94 DEG C 5 minutes, 94 DEG C 30 seconds, 57 DEG C
30 seconds, 72 DEG C 2 points 15 seconds circulate 30 times, 72 DEG C 10 minutes, 10 DEG C 10 minutes, end of run.PCR primer is with agarose gel electricity
Swimming is identified and reclaims band at about 2600bp (Qiagen glue reclaims test kit).Reclaim fragment with pPICZ α B with BstBI and KpnI
(restricted enzyme involved in the present invention is purchased from NEB company) associating enzyme action, agarose gel electrophoresis is identified and distinguishes
Reclaim about 2600bp and about 3300bp fragment.After recovery, P1 fragment and pPICZ α B are with ratio T4 ligase that mol ratio is 5: 1
(Takara) 16 DEG C overnight connect, and within second day, connecting product, to be transformed into E.coli DH5 α (limited purchased from Beijing ancient cooking vessel state biotechnology
Company), coat less salt LB (1% tryptone, 0.5% yeast powder, 0.5%NaCl) flat board (containing 25 μ g/ml Zeocin),
37 DEG C of incubated overnight.Picking Partial Conversion rear clone extracting plasmid, double digestion (HindIII+KpnI) qualification, sepharose electrophoresis is examined
Survey (Fig. 1).Identify that gained Positive recombinant clones preserves after DNA sequencing checking is correct, this recombinant vector named P1-pPICZ α
B。
2.23CD-pPICZ α B builds
The 3CD sequence of synthesis gained SEQ ID NO:6 is cloned into pPICZ α B carrier by following method.
With the 3CD sequence of SEQ ID NO:6 as template, with forward primer: 5 ' TTTAGTTCTTCGAAGCTAGCATGGGTC
CATCTCTGG 3 ' (SEQID NO:9) and reverse primer: 5 ' GGCGGTACCCTATTATTGTTCTGAA3 ' (SEQ ID NO:
10) the 3CD DNA fragmentation of BstBI and KpnI restriction enzyme site is expanded to be respectively provided with to two ends by PCR mode.PCR program: 94
DEG C 5 minutes, 94 DEG C circulated 30 times for 30 seconds, 55 DEG C for 30 seconds, 72 DEG C for 50 seconds, 72 DEG C 10 minutes, 10 DEG C 10 minutes, end of run.PCR
Product is identified with agarose gel electrophoresis and reclaims band at about 600bp (Qiagen gel extraction kit).Reclaim sheet
Section combines enzyme action with pPICZ α B with BstBI and KpnI, and about 600bp peace treaty is identified and be separately recovered to agarose gel electrophoresis
3300bp fragment.After recovery, 3CD fragment and pPICZ α B are with 16 DEG C of mistakes of ratio T4 ligase (Takara) that mol ratio is 5: 1
Night connects, and within second day, connects product and is transformed into E.coli DH5 α, coats less salt LB flat board (containing 25 μ g/ml Zeocin), and 37
DEG C incubated overnight.Picking Partial Conversion rear clone extracting plasmid, double digestion (HindIII+KpnI) qualification, sepharose electrophoresis detects
(Fig. 2).Identify that gained Positive recombinant clones preserves after DNA sequencing checking is correct, this recombinant vector named 3CD-pPICZ α
B。
2.33CD-pYPT1 build
With X-33 Pichia sp. genomic DNA as template, with forward primer: 5 ' TTTAGATCTCCATATGATGAGTCACA
AT 3 ' (SEQ ID NO:11)；Reverse primer: 5 ' AAAGCTAGCCTATTATCTCTGTGTGTAT 3 ' (SEQ IDNO:12) lead to
Cross PCR mode to expand to be respectively provided with to two ends the pYPT1 promoter of BglII and NheI (GenBank numbers: AF027960)
DNA fragmentation (SEQ ID NO:13), named pYPT1 promoter.PCR program: 94 DEG C 5 minutes, 94 DEG C 30 seconds, 55 DEG C 30 seconds,
72 DEG C 50 seconds circulate 30 times, 72 DEG C 10 minutes, 10 DEG C 10 minutes, end of run.
PYPT1 with 3CD-pPICZ α B combines enzyme action with BglII and NheI, and agarose gel electrophoresis is identified and is separately recovered
The about 3900bp fragment of about 900bp and the 3CD-pPICZ α B of pYPT1.After recovery pYPT1 fragment and 3CD-pPICZ α B with mole
Overnight connect than the ratio T4 ligase (Takara) 16 DEG C being 5: 1, within second day, connect product and be transformed into E.coli DH5 α,
Coat less salt LB flat board (containing 25 μ g/ml Zeocin), 37 DEG C of incubated overnight.Picking Partial Conversion rear clone extracting plasmid,
PCR identifies the pYPT1 fragment in recombiant plasmid, sepharose electrophoresis detection (Fig. 3).Identify that gained Positive recombinant clones is surveyed through DNA
Preserve after sequence checking is correct, the named 3CD-pYPT1 of this recombinant vector.
3.P13CD-pYPT1 recombinant strains construction and expression
Host Strains used by the present invention is pichia pastoris X-33 bacterial strain (purchased from Invitrogen company).P13CD-pYPT1 finishes
The red concrete construction step of yeast expression bacterial strain is as follows:
With this area conventional laboratory conditions with SacI linearisation P1-pPICZ α B and 3CD-pYPT1 carrier, common-battery turns complete red
Yeast X-33, coats YPDS (1% yeast powder, 2% peptone, 2% glucose, 1M sorbic alcohol, 2% agar powder) flat board and (contains
200 μ g/mlZeocin), to cultivate 3 days for 30 DEG C, total hectogram is grand.Therefrom the tens of clone of picking be inoculated in YPD (1% yeast powder,
2% peptone, 2% glucose, 2% agar powder) flat board (containing 1500 μ g/mlZeocin), screening plasmid height copy bacterial strain, 30 DEG C
Cultivate 2 days.The several clone of picking is inoculated in 4ml YPD fluid medium, changes BMMY (1% yeast powder, 2% egg after 24 hours
White peptone, 100mM phosphate buffer pH6.0,1.34%YNB, 0.5% methanol, 0.00004% biotin) culture medium, 0.5%
Methanol induction collects thalline after 72 hours.Thalline is after bead is broken, and centrifugal gained supernatant is identified with Western-blot
(Fig. 3, merely illustrates the result of two picked clones (1-2), and the result of other picked clones is not shown).Used one resists for passing through
The rabbit multi-resistance of the homemade anti-VP1 albumen of technology well known in the art, two anti-be that goat anti-rabbit igg-HRP is (public purchased from Calbiochem
Department).With P1-pPICZ α B-X33 and X-33, under similarity condition, expression of results is as negative control (Fig. 4 _ 3-4 and 5-6), with EV71
Virion is positive control (Fig. 4 _ 7).P1-pPICZ α B-X33 due to lack 3CD protease, its induction after whole cell albumen
Amixia bar (Fig. 4 _ 3-4) near the 34kD of VP1, and hybridising band is had in the position that molecular weight of albumen is bigger, illustrate finishing
Red yeast is only expressed the VP1-VP4 albumen that P1 cannot obtain having virion assembling function；Relative, P13CD-pYPT1-
X33 has obvious hybridising band (Fig. 4 _ 1-2) near 34kD, hybridizes gained band with the natural EV71 virion of positive control
Identical (Fig. 4 _ 7), it was demonstrated that the 3CD protease of P1-pPICZ α B and 3CD-pYPT1 coexpression has played proteolytic enzyme function, will
VP1 releases out from P1.Take a P13CD-pYPT1-X33 bacterial strain (but if it find that VP1 expression and other bacterial strain phases
Lower than abnormal, give up), the such as bacterial strain shown in Fig. 4 _ 1 or 2, frozen in-80 DEG C, as fermentor cultivation work seed.
4.EV71 the fermentor cultivation of recombiant protein
Take 1 strain glycerol cryopreservation tube from work seed bank, draw 100 μ L after thawing and access 5ml YPD culture medium,
280rpm, cultivates 20 hours for 30 DEG C.OD6001～2.Activating solution 1ml qualified for microscopy is accessed 500ml YPD culture medium,
280rpm, cultivates 20 hours for 30 DEG C.OD6002～6.Microscopy is without living contaminants.Use BIOENGINEERINGNLF22 fermentation
Tank, basal salt media BSM is used in fermentation1(K2SO4273g, MgSO4109g, CaSO4.2H2O 17.6g, H3PO4400.5ml, KOH
62g, glycerol 600g, PTM160ml, bubble enemy 1ml, deionized water adds to 15L).By 1: 15 inoculation.Fermentation temperature is 30.0 DEG C, just
Beginning pH5.00, rotating speed 300rpm cultivate, ventilation 0.5vvm, DO100%, add PTM1(CuSO4.5H2O 6.0g, NaI
0.008g, MnSO43.0g, NaMoO40.2g, H3BO30.02g, ZnSO420.0g, CoCl20.5g, FeSO4.7H2O 65.0g,
Biotin 0.2g, H2SO45.0ml, deionized water adds to 1L) trace salt.Initial about the about 20 hours multiplicative stage, maintain
Oxygen dissolving value is higher than 30%, and when carbon source is exhausted, thalline weight in wet base reaches about 100g/L.Add the glycerite (every liter of 50%
Add 12ml PTM1).By regulation speed of agitator, air mass flow, tank pressure (＜ 0.8bar) make dissolved oxygen level maintain 20% with
On.Add about 8 hours, during thalline weight in wet base about 350g/L.PH value is controlled to be adjusted to 6.00 simultaneously, add methanol (every liter of interpolation
12mlPTM1) induction.Maintaining oxygen dissolving value higher than 20%, temperature 30 DEG C, it is 6.00 that pH value controls.During 40 hours fermentation ends of induction
Release fermentation liquid.Fermentation liquor 4800rpm, 20 minutes, 4 DEG C of collected after centrifugation bacterium mud, frozen in-20 DEG C.
5.EV71 virus-like particle is gathered in the crops
By P13CD-pYPT1-X33 thalline after fermentation inducement by 1: 3 addition cleaning buffer solution (100mM PB pH7.0,
0.15M NaCl) mixing, fully mix, in 8000rpm, centrifugal 5 minutes, collect cell, repeat above operation two times.To clean
After cell by 1: 5 addition broken bacterium buffer mixing, fully after mixing, high pressure crushes above cell suspension, and repetitive operation, makes
The cell breakage of 90%.The broken bacterium solution broken by high pressure, in 9000rpm, 30 minutes, 10 DEG C of centrifugations, collect on after being centrifuged
Clear liquid.Taking 1ml and break supernatant 40000rpm after bacterium, 3 hours, 4 DEG C of ultracentrifugations, supernatant discarded solution, precipitation was with 100 μ l PBS
(10mM, pH7.4) is resuspended.Transmission electron microscope observing result shows, presents form homogeneous in the bacteria break supernatant liquid after ultracentrifugation
Stable virus-like particle (Fig. 5), particle diameter is between 20-30nm.
Every liter of fermentation liquid can gather in the crops 300 grams of thalline, after purification step, it is thus achieved that pure VLP, according to the product of final VLP
The yield of amount and purification step calculates the VLP expression of the inventive method and is about 150mg/ liter fermentation liquid.People in the art
Member can understand, the recombinant virus sample granule protein expression of this order of magnitude can meet the requirement of industrialized production, with existing skill
Art is compared and is achieved unforeseeable technological progress.
Prepared by 6.EV71 virus sample particle vaccines
" recombinant hepatitis B vaccine (wine brewing ferment with reference to the Pharmacopoeia of the People's Republic of China the 3rd (version in 2010)
Female) " chapter, the method for page 126, the EV71 virus-like particle protein absorption aluminum hydroxide adjuvant that purification is obtained, it is prepared as pre-
The candidate vaccine of anti-EV71 virus.
7.EV71 virus-like particle immunogenicity and the mensuration of immune protective
Choose the SPF level BALB/c mouse (Shanghai western pul-Bi Kai laboratory animal company limited) of 6～8 week old, be divided into 2
Group, often 8 mices of group.1st group of mice carries out immunity (as negative control group) with the buffer that 0.5ml contains aluminium adjuvant, and the 2nd
Group 0.5ml concentration is the VLP (as detection group) of the Al adsorption adjuvant of 20 μ g/ml, exempts from respectively at the 0th, 14 days subcutaneous injections
Epidemic disease, altogether immunity twice, second time immunity blood sampling two weeks after.After the blood collected is placed 2h in 37 DEG C, 4000g is centrifuged
10min, draws supernatant, i.e. obtains Mus polyvalent antibody, deposit in-20 DEG C, and detects the antibody titer of Mus serum and neutralize EV71
Antiviral antibody titre, concrete grammar is as follows:
Antibody titer assay method is as follows: be coated liquid dilution purification EV71 virus vlps to 1 μ g/ml, every to ELISA Plate
Respectively adding 0.1ml in individual shrinkage pool, 4 DEG C overnight.Remove and be coated liquid, wash with 0.3ml PBST (10mM PBS+0.05%Tween-20)
Wash shrinkage pool.It is incubated 2 hours in 37 DEG C with 0.3ml confining liquid (5% defatted milk powder+PBST).Every shrinkage pool addition dilution buffer
(2% defatted milk powder+PBST) is each with the tested serum (diluting gradient from 1: 100 to 1: 3278800) of different gradient dilutions
0.1ml, removes serum solution after being incubated 1 hour in 37 DEG C, washs shrinkage pool with 0.3ml cleaning mixture.Then add with dilute to every shrinkage pool
Releasing the buffer each 0.1ml of goat anti-mouse igg of HRP labelling with 1: 5000 dilution, 37 DEG C of insulations removed enzyme mark after 0.5 hour
Liquid, washs shrinkage pool with 0.3ml cleaning mixture；Then after adding 0.1ml DAB nitrite ion, room temperature lucifuge effect 20 minutes in shrinkage pool
Add 2M H2SO40.05ml stop buffer terminates reaction, and measures OD with enzyme mark colour comparatour450Value, according to OD450Reading value calculates serum
Antibody titer value.
NAT assay method is as follows: in 96 porocyte culture plates, and serum to be checked carries out gradient dilution, dilute
Releasing multiple from 1: 8 to 1: 512, each dilution factor takes EV71 virus liquid (titre 100CCID of 0.05ml and 0.05ml50/
0.05ml) mixing, mixed liquor put into 37 DEG C hatch 2 hours after, add concentration be 2 × 105The RD cell suspension of individual/ml, then
Put in 37 DEG C of C02 incubators and hatch cultivation.Use inverted microscope to observe CPE (cytopathic effect) every day, and record disease
Poison titration results, was in final serum and anti-with the inverse of suppression 50% cytopathic serum highest dilution after 6-7 days
Body titre value.
The result of serum antibody titer and neutralization antiviral antibody titre is as shown in table 1, uses from the results shown in Table 1
The EV71 vaccine immune mouse of virus-like particle has the strongest immunogenicity and the immune protective of 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, refers to that those of ordinary skill in the art can be according to experiment purpose from ability
The technology that can find or draw in the documents such as dictionary, textbook, patent, paper disclosed in territory or association area, such as at " molecule
Clone " described in technology.
Claims (6)
1. the method preparing EV71 recombinant virus sample granule, the method includes EV71 virus P1 precursor protein encoding gene
It is cloned in Pichia sp. by different expression vectors from 3CD proteinase encoding genes, after double expression(DE), obtains form stable homogeneous
And there is immunogenic virus-like particle,
The expression vector of wherein said P1 precursor protein encoding gene is to insert in the BstBI/KpnI site of pPICZ α B carrier
The recombinant vector that SEQ ID NO:5 is constituted, the expression vector of described 3CD proteinase encoding genes is at pPICZ α B carrier
BstBI/KpnI site is inserted SEQ ID NO:6 and pAOX1 promoter replaces with the restructuring load that pYPT1 promoter is constituted
Body.
2. the EV71 recombinant virus sample granule prepared by the method for claim 1.
3. a vaccine, it comprises the EV71 recombinant virus sample granule of claim 2.
4. the vaccine of claim 3, also comprises aluminum hydroxide adjuvant.
5. the EV71 recombinant virus sample granule of claim 2 is for preparing the purposes of vaccine.
6. comprising the Pichia pastoris of two kinds of different recombinant vectors, one of which is the BstBI/KpnI at pPICZ α B carrier
The recombinant vector that SEQ ID NO:5 is constituted is inserted in site, and another kind is to insert in the BstBI/KpnI site of pPICZ α B carrier
PAOX1 promoter is also replaced with the recombinant vector that pYPT1 promoter is constituted by SEQ ID NO:6.
Priority Applications (1)
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| SG11201609062PA (en) * | 2014-04-29 | 2016-11-29 | Nat Health Research Institutes | Adenoviral vector-based vaccine against enterovirus infection |
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