CN101487016B - Avian influenza vaccine and preparation thereof - Google Patents
Avian influenza vaccine and preparation thereof Download PDFInfo
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- CN101487016B CN101487016B CN2008101613322A CN200810161332A CN101487016B CN 101487016 B CN101487016 B CN 101487016B CN 2008101613322 A CN2008101613322 A CN 2008101613322A CN 200810161332 A CN200810161332 A CN 200810161332A CN 101487016 B CN101487016 B CN 101487016B
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Abstract
The invention relates to an avian influenza vaccine and a preparation method thereof, in particular to the preparation of a recombinant plasmid and a recombinant virus in the avian influenza vaccine preparation. The invention provides a recombinant gene and a preparation method thereof, and the recombinant gene is formed by fusing a baculovirus envelope protein gp64 signal peptide gene sequence, a major antigen gene HA sequence of an avian influenza virus and a baculovirus envelope protein gp64 transmembrane gene sequence. The invention further provides a recombinant plasmid that is constructed by recombining the recombinant gene and a pBacPAK8 plasmid, the recombinant bombyx mori baculovirus BmHA that contains the recombinant gene and a virus preparation method. The recombinant bombyx mori baculovirus BmHA that contains the recombinant gene is preserved in China General Microbiological Culture Center of Microbial Culture Collection Management Committee with the preservation number of CGMCC No.2316. The invention further provides an expression protein of the recombinant gene. The recombinant bombyx mori baculovirus BmHA and the protein that are provided by the invention can be used for preparing the avian influenza vaccine. The methods of the invention are applicable to large-scale production, and have lowered cost, high yield and large application value of the produced avian influenza vaccine.
Description
Technical field
The present invention relates to a kind of avian influenza vaccine and preparation thereof, relate in particular to recombinant plasmid and the preparation of recombinant virus in the avian influenza vaccine preparation.
Background technology
Bird flu is the acute respiratory transmissible disease that is caused by some strains in some hypotype of influenza virus A avian.Animal bird flu large-scale outbreak not only brings heavy losses to the raiser, also will bring heavy losses to national economy, simultaneously, also will jeopardize human existence safety.
The animal avian influenza vaccine is prevention and a kind of efficient ways of treatment animal bird flu.
Currently available vaccines, existing vaccines is with the production of chicken embryo, and the manufacturer need shift to an earlier date 12 months and order the chicken embryo.Virus could be grown in the chicken embryo after needing to adapt to the chicken embryo, and the production cycle of vaccine is longer.Virus virus in the process that goes down to posterity of chicken embryo can morph, and the difference between vaccine strain and the epidemic strain can reduce the immune protective effect of vaccine.Bird flu has easy pathogenic to the chicken embryo, be easy to cause the death of chicken embryo.
The human influenza vaccines of the medical council in Europe approval, the vaccine that MDCK (MDCK) produces, but industrial scale is limited, only reaches 1,000,000 people's using dosages at present.
Treanor (2007) with the inactivation of viruses split vaccine with 90,45,15,7.5ugHA antigen intramuscular injection immunity twice, 18-64 year 451 people participate in experiment.Experimental result shows that the antibody response effect of immune induction is not good enough.
Summary of the invention
The purpose of this invention is to provide a kind of animal avian influenza vaccine that overcomes above-mentioned defective.Recombinant vaccine of the present invention utilizes silkworm baculovirus surface display technology and the characteristics of sprouting, and produces the pseudo-virus of band cyst membrane; Because the silkworm baculovirus host is narrow and small, no matter be that it all has the traditional vaccine of ratio on producing and using; Security is good, and utilizes silkworm biological reactor great expression target protein, and production cost is low; Output is high, and is easy to operate, is applicable to large-scale production.
Technical scheme of the present invention is following:
The present invention provides a kind of recombination, is merged by baculovirus envelope protein gp64 signal peptide gene sequence (shown in the SEQ ID NO:7), bird flu virus major antigen gene HA sequence (shown in the SEQ ID NO:8) and baculovirus envelope protein gp64 membrane-spanning domain gene order (shown in the SEQ ID NO:9) to form.
The preparation method of above-mentioned recombination: adopt with SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 are the method that primer carries out pcr amplification.
The present invention also provides the recombinant plasmid of above-mentioned recombination and pBacPAK8 plasmid recombination to construct.
The preparation method of above-mentioned recombinant plasmid, BamH I and Not I double digestion pBacPAK8 plasmid and BamH I and the described recombination of NotI double digestion claim 1 are recombinated.
The present invention also provides the silkworm with recombinant baculovirus that contains above-mentioned recombination BmHA; This virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Classification called after Bombyx mori nuclear polyhydrosis virus (Bombyx morinucleopolyhedrovirus); Preservation date is on December 28th, 2007, and deposit number is CGMCC No.2316.
The preparation method of above-mentioned silkworm with recombinant baculovirus BmHA:
(1) the weighting profit requires 3 described recombinant plasmids and the DNA of the viral BmBacPAK6 that cuts through the Bsu36I enzyme, and the substratum mixing that adds serum-free is processed the pBacHA mixed solution;
(2) get Dosper, add the substratum of serum-free, mixing is processed the Dosper mixed solution;
(3) will cultivate the BmN cell with the washing of the substratum of serum-free after, dropwise add the pBacHA mixed solution and the Dosper mixed solution of above-mentioned preparation, 27 ℃ of cultivations;
(4) collect supernatant in the petridish, infect the BmN cell, agarose is cultivated;
(5) picking plaque divides into groups to infect the BmN cell, preserves supernatant;
(6) infected B mN cell DNA is used for Southern blot dot hybridization;
(7) go bail for that the supernatant of positive colony cell carries out plaque screening in the dot hybridization of depositing;
(8) get the supernatant infected silkworm cell of the positive colony cell of plaque screening.
The present invention also provides the albumen of above-mentioned recombinant gene expression, and its aminoacid sequence is shown in SEQ ID NO:10.
Above-mentioned proteic preparation method:
(1) the described silkworm with recombinant baculovirus BmHA of claim 5 is infected the BmN cell and carries out virus amplification,
(2) stab inoculation inserts silkworm and plays silkworm and pupa five ages,
(3) collect the said albumen of claim 7 of expressing.
The present invention further provides above-mentioned silkworm with recombinant baculovirus BmHA application in preparation animal avian influenza vaccine.
The present invention also provides the application of above-mentioned albumen in preparation animal avian influenza vaccine.
The technique effect that the present invention realizes is following:
Utilize silkworm larva, pupa as bio-reactor, baculovirus expression vector system efficiently expresses the method with high clinical value animal avian influenza vaccine.Present method is applicable to scale operation, has reduced cost, and output is high, and the animal avian influenza vaccine using value of being produced is big.
Description of drawings
Fig. 1: the recombinant plasmid pGEM-HA that contains recombination SP-HA-TM;
Fig. 2: contain recombination SP-HA-TM baculovirus transferring plasmid pBacHA;
P:polyhedrin, polyhedrin gene promoter; A:Polyhedin Poly A
+Signal, the polyhedron gene polyadenylation signal; M13ori:M13 phage replication starting point; Amp: penbritin gene; The ori:pUC replication origin.
Embodiment
The method of utilizing silkworm biological reactor animal avian influenza vaccine of the present invention is the recombination that obtains bird flu virus major antigen HA gene through the method for PCR, and introduces BamH I and Not I restriction enzyme site respectively at its 5 ' and 3 ' end; PBacPAK8 is connected with the silkworm baculovirus carrier, connects product and wild-type silkworm baculovirus BmBacPAK6 homologous recombination takes place in bombyx mori cell, through plaque select; Obtain the silkworm with recombinant baculovirus of band recombination SP-HA-TM; Artificial inoculation silkworm larva, pupa were collected larva body fluid and pupal cell, homogenate after 5-7 days; Carry out centrifugal; Separation and purification, lyophilize processes under the aseptic condition that animal avian influenza vaccine lyophilized powder realizes.
Set forth particular content of the present invention in detail below in conjunction with embodiment:
Experimental example 1: the structure of recombination SP-HA-TM.
Bird flu virus major antigen gene is the HA gene, holds the membrane-spanning domain gene order of the signal peptide gene sequence that is connected the baculovirus envelope protein gp64 that encodes respectively and the baculovirus envelope protein gp64 that encodes at 5 ' and 3 ' of HA gene, makes up recombination.Through design of primers, introduce BamH I and Not I endonuclease digestion site respectively at 5 ' and 3 ' end of this recombination.Be 3 pairs of primers of stencil design with the signal peptide gene sequence of baculovirus AcNPV envelope protein gp64, the HA gene order of avian influenza virus H 5 N 1 and the membrane-spanning domain gene order of baculovirus AcNPV envelope protein gp64 respectively; Pcr amplification purpose fragment at first: gp64 signal peptide gene sequence (sp), HA gene and gp64 membrane-spanning domain gene order (tm), primer and template make up recombination SP-HA-TM each other to utilize each purpose fragment then.
Design of primers is following:
Pspl(SEQ?ID?NO：1)：
5’TCGGATCCATGCTACTAGTAAATCAGTC3’
Psp2(SEQ?ID?NO：2)：
5’GTGTAACAGTAACGTTCTTTTCCGCAAAGGCAGAATGCGCCGC3’
Phal(SEQ?ID?NO：3)：
5’TAAAAGAACGTTACTGTTACTGTTACACATG3’
Pha2(SEQ?ID?NO：4)：
5’AATGCAAATTCTGCATTGTAACGAC3’
Ptml(SEQ?ID?NO：5)：
5’TCGTTACAATGCAGAATTTGCATTGGTGATACTGGGCTATCCAAAAAT3’
Ptm2(SEQ?ID?NO：6)：
5’AGGCGGCCGCTTACTTTCCAAGTCGGTTCAT3’。
(1) amplification of gp64 signal peptide gene sequence (sp)
Dna sequence dna with gp64 is a template, and the PCR reaction parameter is designed to, 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 30s, 68 ℃ of renaturation are extended 30s, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following component:
10×PCR?Buffer 10μl
25mmol?MgCl
2 8μl
2.5mmol?dNTPs 8μl
Pspl 1μl
Psp2 1μl
Template 1 μ l
KOD-Plus archaeal dna polymerase 1 μ l
Add aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed amplified fragments.
(2) amplification of gp64 membrane-spanning domain gene order (tm)
Dna sequence dna with gp64 is a template, and the PCR reaction parameter is designed to, 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 30s, 68 ℃ of renaturation are extended 30s, 30 circulations, 68 ℃ are extended 5min.
The reaction system of 100ul is the same, and the primer is Ptml and Ptm2, behind each component mixing, puts into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the order amplified fragments.
(3) amplification of HA gene
With avian influenza virus H 5 N 1 HA gene is template, and the PCR reaction parameter is 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
The reaction system of 100ul is the same, and the primer is Phal and Pha2, behind each component mixing, puts into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.
(4) gp64 signal peptide sp gene and HA gene Fusion
The PCR reaction parameter is 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following component:
10×PCR?Buffer 10μl
25mmol?MgCl
2 8μl
2.5mmol?dNTPs 8μl
Pspl 1μl
Pha2 1μl
Template sp 1 μ l
Template HA 1ul
KOD-Plus archaeal dna polymerase 1 μ l
Add aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed amplified fragments.
Because the downstream primer Psp2 of sp the time has added the partial sequence of HA gene 5 ' end in design,, utilize PCR method just can amplify recombination sp-HA sequence once more so that sp fragment 3 ' terminal sequence that pcr amplification goes out and HA gene 5 ' terminal sequence exist is overlapping.
(5) recombination sp-HA-tm is constructed in the fusion of recombination sp-HA and tm
The PCR reaction parameter is 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1.5min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl
2 8μl
2.5mmol?dNTPs 8μl
Pspl 1μl
Ptm2 1μl
Template sp-HA 1 μ l
Template tm 1ul
KOD-Plus archaeal dna polymerase 1 μ l
Add aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR appearance, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.
The same, because the upstream primer Ptml of tm design the time has added the partial sequence of HA gene 3 ' end,, utilize PCR method just can amplify recombination sp-HA-tm so that tm fragment 5 ' terminal sequence that pcr amplification goes out and HA gene 3 ' terminal sequence exist is overlapping.
Experimental example 2: the structure of cloned plasmids pGEM-HA
The recombination sp-HA-tm that pcr amplification is gone out is connected on the pGEM-T vector:
The said components mixing, 25 ℃ of reaction 1h.
Reaction mixture is converted among the competent cell E.coli DH5 α, is coated with flat board, in 37 ℃ of cultivations, bacterium colony can occur after 12-16 hour.Picking list bacterium colony extracts plasmid, carries out enzyme and cuts and check order, and sequence is entirely true, obtains plasmid pGEM-HA.
Experimental example 3: contain the acquisition of the baculovirus transferring plasmid pBacHA of recombination SP-HA-TM
Plasmid pGEM-HA is through BamH I and Not I double digestion; Downcut recombination SP-HA-TM fragment; Be cloned in the plasmid pBacPAK8 of BamH I and Not I double digestion, (polyhedrin is ph) under the gene promoter control to make recombination SP-HA-TM place polyhedrin; Be built into recombinant transfer plasmid pBacHA, correct through restriction analysis identified gene sequence.
Experimental example 4: contain the acquisition of the silkworm with recombinant baculovirus BmHA of recombination SP-HA-TM
Get recombinant transfer plasmid pBacHA and through the viral BmBacPAK6DNA of Bsu36I linearization for enzyme restriction, TC-100 substratum (GIBCOBRL company) mixing that adds the 100ul serum-free is processed the pBacHA mixed solution.Get the Dosper (Bao Ling Man) of 6ul, add the TC-100 substratum of 100ul serum-free, mixing is processed the Dosper mixed solution.With cultivating BmN cell in the 35mm plate in advance with the TC-100 substratum washed twice of serum-free, dropwise add the pBacHA mixed solution and the Dosper mixed solution of above-mentioned preparation, cultivated 4-5 days for 27 ℃.Collect supernatant and carry out first round plaque screening.Get the 5ul supernatant and infect the BmN cell in the 35mm plate, supernatant discarded adds the TC-100 substratum and the low melting-point agarose of balanced mix after 1 hour.Picking plaque after 4-5 days; Divide into groups to infect the BmN cell 3-4 days; Preserve supernatant, cell is used for Southern blot dot hybridization with the NaOH cracking, makes template with random primer probe mark test kit (Bao Ling Man) label probe with recombination SP-HA-TM; Hybridizing method is according to " molecular cloning " (Science Press, 1995).The supernatant of positive colony cell of going bail in the dot hybridization of depositing carries out second and takes turns plaque screening.Get the supernatant infected silkworm cell of positive colony cell, obtain a large amount of recombinant baculovirus BmHA that contains recombination SP-HA-TM with amplification.This virus ability infected silkworm cell, the bombyx mori cell that infects at microscopically is the morbidity shape.
The expression of experimental example 5:HA fusion rotein in silkworm 5 instar larvaes and pupa
Recombinant virus BmHA is infected the BmN cell with 10MOI carry out virus amplification, by 1 * 10
6PFU/ bar stab inoculation inserts silkworm and plays silkworm or pupa five ages, after infecting 5-7 days, takes silkworm body or pupal cell homogenate; (12000rpm 30min), gets supernatant through high speed centrifugation; The HA that surveys in the hemolymph is active, and at the 6th day that infects, the HA activity in the hemolymph reached 6 * 10
4U/ml.Supernatant behind the high speed centrifugation adds isopyknic 2 * protein sample-loading buffer (100MmTris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), and 100 ℃ of heating 10min get 20ul and carry out the SDS-PAGE analysis.The result shows that recombinant virus has been expressed the HA fusion rotein.The protein sequencing result shows that its aminoacid sequence is as shown in SEQ ID NO:10.
Experimental example 6: the acquisition of animal avian influenza vaccine
(1) separation and purification HA fusion rotein (animal avian influenza vaccine) from silkworm chrysalis
A. get the silkworm chrysalis sample that infects through BmHA in right amount, after thawing under 4 ℃, mixed in right amount with 0.85% saline water, homogenate to slurries are careful evenly to get final product;
B. homogenate is added in the 500ml centrifuge tube, trim, the centrifugal 20-60min of 3000rpm gets supernatant, carefully discards grease;
C. the 3000rpm centrifuged supernatant is poured in the new 500ml centrifuge tube, trim, the centrifugal 20-60min of 6000rpm gets supernatant, abandons grease;
D.6000rpm the centrifugal supernatant is poured the 50ml centrifuge tube into, trim, and the centrifugal 30-120min of 12000rpm gets supernatant, abandons grease;
E.12000rpm centrifuged supernatant changes new 50ml centrifuge tube over to, trim, and the centrifugal 30-120min of 18000rpm gets supernatant, abandons grease;
F.18000rpm the supernatant after centrifugal is sub-packed in the ultra in pipe of sterilization, balance, the centrifugal 30-90min of 35000rpm on super clean bench;
G.35000rpm after centrifugal, get supernatant, carry out ultrafiltration.
(2) ultrafiltration
Use hollow-fibre membrane to carry out ultrafiltration.Select suitable hollow-fibre membrane specification (molecular weight cut-off is 750kD), clean the back application of sample, with an amount of aseptic ultrapure water ultrafiltration.
(3) animal avian influenza vaccine biological activity is identified
Sample after the ultrafiltration is surveyed work, and measuring method for activity adopts hemagglutination test (HA test).Get 24 porocyte culture plates, after each row's 1 to 6 hole (A1-A6) adds 250 μ l saline water (0.85%), add 250 μ l samples in first hole; Do doubling dilution; Negative control hole is set simultaneously, adds the liquid that does not contain sample and do doubling dilution, add 1% chicken erythrocyte suspension, 250 μ l again to each hole; The light shaking culture plate, in 37 ℃ hatch 4 hours after observations.
With Bradford method (Xylene Brilliant Cyanine G method) test sample protein content.
It is following that the animal avian influenza vaccine is calculated (mg/mL) formula than work: the animal avian influenza vaccine is than living=2
n* 4/ sample concentration, the hole count of RCA for taking place in n.The animal bird flu virus that this method obtained can reach more than 20 than work.
(4) preserve and detect
Than the higher sample of living, aseptic subpackaged, animal avian influenza vaccine lyophilized powder is processed in-50 ℃ of lyophilizes.Simultaneously, identify lyophilized powder again and reach 20 above persons than living, getting.The 12%SDS-PAGE electrophoresis detection has the expression of target protein at the 45kD place.
(5) vaccine effect
The animal avian influenza vaccine carries out testing in the animal body, subcutaneous injection, and ID is 0.1-50mg/kg, 2-4 detects antibody titer after week.
Produce antibody in 4 ages in week in the chicken body, its protection antibody titer is greater than 1:150, and the challenge test result shows that this vaccine can produce neutralizing antibody and have the viral effect of obvious opposing.Safe pharmacology result shows that this vaccine does not have obviously influence to cardiovascular, the respiratory system of chicken, neural system, body temperature etc.Long malicious result shows that this vaccine is to the chicken nontoxicity.Anxious malicious result shows that this vaccine carries out subcutaneous injection to chicken, and maximum tolerated dose is greater than 153mg/kg.Mutagenicity test result shows that this vaccine does not have mutagenicity.
It is thus clear that this vaccine can produce antibody in animal body, have obvious provide protection, have no side effect, peace comments the result to show that this vaccine safety is effective.
SEQUENCE?LISTING
< 110>Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang
< 120>avian influenza vaccine and preparation method thereof
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<151>2007-09-30
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< 213>gp64 membrane-spanning domain gene
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<400>10
Claims (6)
1. silkworm with recombinant baculovirus BmHA, this virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2316.
2. the preparation method of the said silkworm with recombinant baculovirus BmHA of claim 1:
(1) method through pcr amplification obtains to make up the recombination that forms by baculovirus envelope protein gp64 signal peptide gene sequence, bird flu virus major antigen gene HA sequence and baculovirus envelope protein gp64 membrane-spanning domain gene order;
(2) after 5 ' and 3 ' end of step (1) gained recombination is introduced BamH I and Not I restriction enzyme site respectively, be connected to silkworm baculovirus carrier pBacPAK8, recombination is placed under the polyhedrin gene promoter control, be built into recombinant plasmid;
(3) get the recombinant plasmid of step (2) gained and the DNA of the viral BmBacPAK6 that cuts through the Bsu36I enzyme, the substratum mixing that adds serum-free is processed the pBacHA mixed solution;
(4) get Dosper, add the substratum of serum-free, mixing is processed the Dosper mixed solution;
(5) will cultivate the BmN cell with the washing of the substratum of serum-free after, dropwise add the pBacHA mixed solution and the Dosper mixed solution of above-mentioned preparation, 27 ℃ of cultivations;
(6) collect supernatant in the petridish, infect the BmN cell, agarose is cultivated;
(7) picking plaque divides into groups to infect the BmN cell, preserves supernatant;
(8) infected B mN cell DNA is used for Southern blot dot hybridization;
(9) go bail for that the supernatant of positive colony cell carries out plaque screening in the dot hybridization of depositing;
(10) get the supernatant infected silkworm cell of the positive colony cell of plaque screening.
3. the described silkworm with recombinant baculovirus BmHA of claim 1 expressed proteins, its aminoacid sequence is shown in SEQ ID NO:10.
4. said proteic preparation method of claim 3:
(1) the described silkworm with recombinant baculovirus BmHA of claim 1 is infected the BmN cell and carries out virus amplification,
(2) stab inoculation inserts silkworm and plays silkworm and pupa five ages,
(3) collect the said albumen of claim 3 of expressing.
5. the application of the said silkworm with recombinant baculovirus BmHA of claim 1 in preparation animal avian influenza vaccine.
6. the application of the described albumen of claim 3 in preparation animal avian influenza vaccine.
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| CN2008101613322A CN101487016B (en) | 2007-09-30 | 2008-09-19 | Avian influenza vaccine and preparation thereof |
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| CN200710164215 | 2007-09-30 | ||
| CN200710164215.7 | 2007-09-30 | ||
| CN2008101613322A CN101487016B (en) | 2007-09-30 | 2008-09-19 | Avian influenza vaccine and preparation thereof |
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| CN101487016B true CN101487016B (en) | 2012-04-18 |
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| AT510585B1 (en) * | 2010-11-18 | 2012-05-15 | Apeptico Forschung & Entwicklung Gmbh | COMPOSITION COMPRISING A PEPTIDE AND AN INHIBITOR OF VIRAL NEURAMINIDASE |
| CN102533677B (en) * | 2012-01-10 | 2013-07-24 | 特菲(天津)生物医药科技有限公司 | Malaria vaccine and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1918188A (en) * | 2004-02-05 | 2007-02-21 | Lg化学株式会社 | Method of emulsion polymerization using liquid miniemulsion as seed particle |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1918188A (en) * | 2004-02-05 | 2007-02-21 | Lg化学株式会社 | Method of emulsion polymerization using liquid miniemulsion as seed particle |
Non-Patent Citations (3)
| Title |
|---|
| Ding-Gang Yang等.Avian Influenza Virus Hemagglutinin Display on Baculovirus Envelope: Cytoplasmic Domain Affects Virus Properties and Vaccine Potential.《Molecular Therapy》.2007,第15卷(第5期),989－996. * |
| Liqun Lu等.Baculovirus surface-displayed hemagglutinin of H5N1 influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity.《Biochemical and Biophysical Research Communications》.2007,第358卷404-409. * |
| Md. Masmudur Rahman等.Bombyx mori nucleopolyhedrovirus-based surface display system for recombinant proteins.《Journal of General Virology》.2003,第84卷2023－2031. * |
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