CN104610456A - Preparation method and application of subunit vaccine for H7N9 subtype avian influenza - Google Patents
Preparation method and application of subunit vaccine for H7N9 subtype avian influenza Download PDFInfo
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Abstract
The invention discloses a preparation method and an application of a subunit vaccine for H7N9 subtype avian influenza. The subunit vaccine for the H7N9 subtype avian influenza is fusion protein, and the fusion protein comprises H7N9 subtype avian influenza virus antigen HA1-2 protein and salmonella typhi type-I flagellin (fliC). The fusion protein has the following amino acid sequences: (1) protein comprising an amino acid sequence as shown in SEQ ID NO.2; (2) an amino acid sequence which has the homology of 80-100% together with the amino acid sequence defined in SEQ ID No.2 and is used for coding proteins with same functions; or (3) protein which has the same activity after the amino acid sequence as shown in the SEQ ID No.2 is subjected to addition, deletion or substitution of one or more amino acids and is derived from the protein (1). The submit vaccine is short in expression cycle of the fusion protein and good in immunogenicity.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of preparation method and application of H7N9 subtype avian influenza subunit vaccine.
Background technology
Influenza virus (Influenza Virus) is the pathogenic agent of influenza, classification belongs to orthomyxovirus section (Orthomyxovoridae), and influenza A belongs to.Viral genome is made up of the RNA fragment of 8 sub-thread minus strands, and respective encodes functional protein is respectively PB1, PB2, PA, HA, NP, NA, M and NS gene.Generally spherical in shape, diameter 80nm ~ 120nm, has cyst membrane, cyst membrane surface mainly contains 2 kinds of glycoprotein, i.e. hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA), 16 kinds of HA hypotypes and 9 kinds of NA hypotypes have been found so far.Influenza virus is by multiple gene-determined to the pathogenic of host, wherein the albumen of HA gene and coding thereof is particularly important, at viruses adsorption and wear in membrane process and play keying action, is important surface antigen, it stimulates body to produce neutralizing antibody, can neutralize the infection of virus.
Hemagglutinin is the glycoprotein containing 562-566 amino acid (Amino acid, aa), and its precursor molecule HA0 is HA1 and HA2 two portions by host protein enzymatic lysis.Wherein HA1 contains 319-326 aa, HA2 and contains 221-222 aa.The spherical district of HA monomer is made up of the major part of HA1, and bulbous district forms primarily of HA2, also containing sub-fraction HA1.Although HA1 is poorer than HA2 conservative property; but after natural infection or vaccination, most of protectiveness neutralizing antibody is the spherical district formed for HA1, in addition; X-ray crystalline diffraction result shows, HA (57-264aa and 63-286aa) can form the higher structure close to native state.
In by the end of February, 2013, H7N9 subtype avian influenza virus is found at first Chinese, epidemic situation development is subsequently very fast, has the Laboratory Diagnosed case that 453 routine people infect H7N9 subtype avian influenza virus, comprising dead 175 examples (http://www.who.int/) in October, 2014.Gao etc. take the lead in reporting that this viral all gene fragment all derives from avian influenza virus, not containing the gene fragment of anyone influenza virus, H7N9 subtype avian influenza virus is a kind of novel reassortant influenza vims, there is the influenza virus (LPAI) of low pathogenicity in poultry, but pathogenic higher than H5N1 to people, be difficult to detect and prevention, therefore attract wide attention.
Vaccine is one of most effective means controlling virus infection; but the antigenicity of H7N9 subtype avian influenza virus is different from the past popular avian influenza virus; therefore; original Seasonal Influenza Vaccine and avian influenza vaccine lack immanoprotection action to it; the influenza vaccines that current China has got permission to go on the market all adopt traditional deactivation or cracking technology, there is the production cycle long and shortcoming such as to yield poorly.The subunit vaccine of recombinant subunit vaccine, particularly expression in escherichia coli, has the advantages that protective epitope is many, expression efficiency is high, zymotechnique is ripe, manufacturing cost is low and be easy to scale operation.And subunit vaccine has higher security, be easy to be accepted.But it is weak that the main drawback of subunit vaccine is immunogenicity, often need effective adjuvant to assist and just can cause desirable immunne response.
Flagellin is the principal structural component of bacterial flagellum, is a kind of albumen of high conservative.It is as a kind of special inflammatory molecule, and existing antigenicity has adjuvant effect again, and its adjuvant effect realizes mainly through TLR5 signal transduction pathway.Large quantity research shows that flagellin is the very potential adjuvant of one, is not only the inductor producing innate immunity, and contributes to inducing adaptive immunity response.
At present, a kind of preparation method and the application with good immunogenic H7N9 subtype avian influenza subunit vaccine is lacked.
Summary of the invention
Technical problem to be solved by this invention there is provided a kind of preparation method and the application with good immunogenic H7N9 subtype avian influenza subunit vaccine.
To achieve these goals, the present invention is achieved through the following technical solutions: the subunit vaccine that the invention provides a kind of H7N9 subtype avian influenza, it is fusion rotein, and described fusion rotein comprises H7N9 subtype avian influenza virus antigen HA1-2 albumen and has the Salmonella typhimurtum I type flagellin fliC of adjuvant effect.
Further, described fusion rotein has following aminoacid sequence:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.2; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.2 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.2 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more amino acid.
A kind of nucleic acid molecule of the present invention, its coding described in fusion rotein.
Further, described nucleic acid molecule, its nucleotide sequence is as shown in SEQ ID No.1.
The present invention prepares the method for the subunit vaccine of described H7N9 subtype avian influenza, comprises the following steps:
(1) gene of albumen described in overlap pcr amplification claim 1,
(2) be connected to pCold carrier, obtain recombinant plasmid pCold-HA1-2-fliC,
(3) transformed Host Strains E.coli BL21 (DE3), obtained positive recombinant bacterium DE3 (pCold-HA1-2-fliC),
(4) H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and the amalgamation and expression of Salmonella typhimurtum I type flagellin fliC in escherichia expression system; By IPTG abduction delivering target protein HA1-2-fliC, adopt Ni-NAT affinity chromatography purifying with the object fusion rotein of His label.
Further, in step (1), the acquisition of H7N9 subtype avian influenza virus HA1-2 gene: the extraction of a.H7N9 subtype avian influenza virus geneome RNA;
B.RNA reverse transcription synthesis cDNA;
The clone of c.H7N9 subtype avian influenza virus HA1-2 gene; The pcr amplification of fliC gene; The overlap pcr amplification of HA1-2-fliC gene; The retrieve and purification of PCR primer.
Further, in step (2), by the PCR primer HA1-2-fliC reclaimed through 1% agarose gel electrophoresis, be connected with prokaryotic expression carrier pCold, obtain recombinant plasmid pCold-HA1-2-fliC;
In step (3), connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms Host Strains E.coli DH5a, obtains positive recombinant bacterium DH5a (pCold-HA1-2-fliC); The double digestion of recombinant plasmid and order-checking qualification;
Connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms expresses Host Strains E.coli BL21 (DE3), obtains positive recombinant bacterium DE3 (pCold-HA1-2-fliC);
In step (4), the expression of fusion rotein HA1-2-FliC; The purifying of fusion rotein HA1-2-FliC.
Further, in step (1), the clone of H7N9 subtype avian influenza virus HA1-2 gene:
According to the nucleotide sequence of H7N9 subtype avian influenza virus hemagglutinin HA in GenBank, design two pairs of primers, HA1-2-F '/HA1-2-R ' and HA1-2-F/HA1-2-R;
Wherein 5 ' the end of HA1-2-F ' and the 3 ' end of HA1-2-R ' introduce restriction enzyme site Sac I, Hind III respectively, are the primers for the independent HA1-2 gene that increases; And the 5 ' end of primer HA1-2-F and HA1-2-R introduces restriction enzyme site EcoR I and flexible peptide (Gly respectively
4ser)
3encoding gene; For the primer merging HA1-2 gene in fragment that increases;
Primer HA1-2-F ' has the nucleotide sequence of SEQID NO.3;
Primer HA1-2-R ' has the nucleotide sequence of SEQID NO.4;
Primer HA1-2-F has the nucleotide sequence of SEQID NO.5;
Primer HA1-2-R has the nucleotide sequence of SEQID NO.6.
Further, in step (1), the pcr amplification of fliC gene: according to Salmonella typhimurtum I type flagellin fliC gene order design primer fliC-F and fliC-R, wherein 5 ' the end of primer fliC-F and fliC-R introduces flexible peptide (Gly respectively
4ser)
3encoding gene and restriction enzyme site Xba I;
Primer fliC-F has the nucleotide sequence of SEQID NO.7;
Primer fliC-R has the nucleotide sequence of SEQID NO.8.
The application of subunit vaccine of the present invention in the immunoprophylaxis of H7N9 subtype avian influenza.
Beneficial effect: the protective antigen of the subunit vaccine of the high immunogenicity that the present invention obtains is HA1-2-fliC fusion rotein, and the cycle of its expressed fusion protein is short, and expression product has the biological characteristics similar to natural product and good immunogenicity.H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene is connected to the N end of salmonella typhimurium flagellin gene by the present invention by flexible peptide, by escherichia coli prokaryotic expression system, flagellin and HA1-2 albumen are carried out amalgamation and expression, overcome traditional deactivation or cracking technology, production cycle is long, yield poorly, subunit vaccine immunogenicity is shortcoming not fully, therefore has great importance to the exploitation of H7N9 subtype avian influenza new generation vaccine.
Accompanying drawing explanation
The electrophorogram of gene PCR amplified production for the purpose of Fig. 1;
Fig. 2 is that recombinant plasmid pCold-HA1-2 enzyme cuts qualification figure;
Fig. 3 is that recombinant plasmid pCold-HA1-2-fliC enzyme cuts qualification figure;
Fig. 4 is the SDS-PAGE result figure of recombinant bacterium DE3 (pCold-HA1-2) expression product;
Fig. 5 is the SDS-PAGE result figure of recombinant bacterium DE3 (pCold-HA1-2-fliC) expression product;
Fig. 6 is the SDS-PAGE result figure of albumen after purifying;
Fig. 7 is the Western blot detection figure of albumen HA1-2, HA1-2-fliC;
Fig. 8 is the TLR5 biological activity assay figure of fusion rotein HA1-2-fliC;
Fig. 9 two to exempt from, three exempts from the IgG antibody detection figure that after 12 days, in mice serum, HA1-2 is special;
Figure 10 is H7N9 subtype avian influenza virus hemagglutination test detection figure;
Figure 11 three to exempt from after 12 days hemagglutination inhibition antibody titre detection figure in mice serum;
Figure 12 three exempts from antibody horizontal dynamic change figure in rear mice serum.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
According to technical scheme of the present invention, contriver provides following concrete Application Example, it should be noted that, following examples are only illustrative, and the present invention is not limited to these embodiments.
The electrophorogram of gene PCR amplified production for the purpose of Fig. 1; Wherein, M1:DL2000DNA marker, M2: λ-EcoT14digest DNA marker, 1:HA1-2 amplified production, 2:fliC amplified production, 3:HA1-2-fliC amplified production.
Fig. 2 is that recombinant plasmid pCold-HA1-2 enzyme cuts qualification figure; Wherein, M1: λ-EcoT14digest DNA marker, M2:DL 2000DNA marker, 1:pCold-HA1-2 digestion products.
Fig. 3 is that recombinant plasmid pCold-HA1-2-fliC enzyme cuts qualification figure; Wherein, M: λ-EcoT14digest DNA marker, 1:pCold-HA1-2-fliC digestion products.
Fig. 4 is the SDS-PAGE result figure of recombinant bacterium DE3 (pCold-HA1-2) expression product; Wherein, M:protein marker, 1: empty carrier, 2: do not induce, 3: induced precipitation, 4: induction supernatant.
Fig. 5 is the SDS-PAGE result figure of recombinant bacterium DE3 (pCold-HA1-2-fliC) expression product; Wherein, M:proteinmarker, 1: empty carrier, 2: do not induce, 3: induced precipitation, 4: induction supernatant.
Fig. 6 is the SDS-PAGE result figure of albumen after purifying; Wherein, M:protein marker, 1:HA1-2 albumen, 2:HA1-2-fliC fusion rotein.
Fig. 7 is the Western blot detection figure of albumen HA1-2, HA1-2-fliC;
Fig. 8 is the TLR5 biological activity assay figure of fusion rotein HA1-2-fliC;
Fig. 9 two to exempt from, three exempts from the IgG antibody detection figure that after 12 days, in mice serum, HA1-2 is special;
Figure 10 is H7N9 subtype avian influenza virus hemagglutination test detection figure;
Figure 11 three to exempt from after 12 days hemagglutination inhibition antibody titre detection figure in mice serum;
Figure 12 three exempts from antibody horizontal dynamic change figure in rear mice serum.
The invention provides a kind of subunit vaccine of H7N9 subtype avian influenza, it is fusion rotein, and described fusion rotein comprises H7N9 subtype avian influenza virus antigen HA1-2 albumen and has the Salmonella typhimurtum I type flagellin fliC of adjuvant effect.HA1-2 albumen in this fusion rotein is made up of the 62-284 amino acids of the main protection antigen hemagglutinin of H7N9 subtype avian influenza virus, and flagellin enhances the immunne response of HA1-2 albumen as adjuvant, has stronger immunogenicity.
Described fusion rotein has following aminoacid sequence:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.2; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.2 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.2 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more amino acid.
A kind of nucleic acid molecule of the present invention, the fusion rotein of its coding described in claim 1 or 2.Described nucleic acid molecule, its nucleotide sequence is as shown in SEQ ID No.1.
The present invention prepares the method for the subunit vaccine of described H7N9 subtype avian influenza, comprises the following steps:
One, build the recombinant plasmid containing H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene and Salmonella typhimurtum I type flagellin fliC gene, concrete steps are as follows:
The acquisition of 1.H7N9 subtype avian influenza virus HA1-2 gene: the extraction of (1) H7N9 subtype avian influenza virus geneome RNA, (2) clone of RNA reverse transcription synthesis cDNA, (3) H7N9 subtype avian influenza virus HA1-2 gene;
The pcr amplification of 2.fliC gene;
The overlap pcr amplification of 3.HA1-2-fliC gene;
The retrieve and purification of 4.PCR product;
5. ligation: by through 1% agarose gel electrophoresis reclaim PCR primer HA1-2 and HA1-2-fliC, be connected with prokaryotic expression carrier pCold respectively, obtain recombinant plasmid pCold-HA1-2 and pCold-HA1-2-fliC;
6. connect the conversion of product: by recombinant plasmid pCold-HA1-2 and pCold-HA1-2-fliC, transform Host Strains E.coliDH5a respectively, obtain positive recombinant bacterium DH5a (pCold-HA1-2) and DH5a (pCold-HA1-2-fliC).
7. the double digestion of recombinant plasmid is identified with order-checking
8. connect the conversion of product: by recombinant plasmid pCold-HA1-2 and pCold-HA1-2-fliC, transform respectively and express Host Strains E.coli BL21 (DE3), obtain positive recombinant bacterium DE3 (pCold-HA1-2) and DE3 (pCold-HA1-2-fliC).
Two, H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and the amalgamation and expression of Salmonella typhimurtum I type flagellin fliC in escherichia expression system:
1. the expression of albumen HA1-2 and fusion rotein HA1-2-FliC;
2. the purifying of albumen HA1-2 and fusion rotein HA1-2-FliC;
3.Western blotting identifies;
4.TLR5 biological activity assay.
Three, the immunogenicity of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurtum I type flagellin fliC fusion expressed product:
6-8 C3H/HeJ female mice in age in week 18, mouse is divided into 3 groups at random, often organize 6, immunization route is abdominal injection, immunity point is carried out for three times, to detect after immunity the dynamic change in time in vivo of antibody horizontal and antibody in each immune group mice serum, also suppress to tire to detect to the blood clotting of Serum Antibody.
Embodiment 1
The present invention prepares the method for the subunit vaccine of described H7N9 subtype avian influenza, comprises the following steps:
One, build the recombinant plasmid containing H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene and Salmonella typhimurtum I type flagellin fliC gene, concrete steps are as follows:
The acquisition of 1.H7N9 subtype avian influenza virus HA1-2 gene
(1) extraction of H7N9 subtype avian influenza virus geneome RNA:
Livestock and poultry loimology emphasis open laboratory of the Ministry of Agriculture of Yangzhou University gives the chick embryo allantoic liquid containing deactivation H7N9 subtype avian influenza virus, gets 500 μ L and extracts RNA.
1. to having added
the allantoic fluid of Reagent add 200 μ L chloroforms (
1/5 volume of Reagent), fully vibrate, solution to be emulsified is that after milky white shape, room temperature leaves standstill 5min;
2. centrifugal (12000g, 15min, 4 DEG C);
3. carefully centrifuge tube is taken out, now homogenate is divided into three layers (colourless supernatant liquor, middle white egg white, coloured lower floor organic phases), and Aspirate supernatant is transferred in another new centrifuge tube and (must guard against sucking-off white middle layer);
4. in supernatant, add isopyknic Virahol, after the centrifuge tube that turns upside down fully mixes, at 15 ~ 30 DEG C, leave standstill 10min; Centrifugal (12000g, 10min, 4 DEG C);
5. the cleaning of RNA precipitation: careful supernatant discarded, slowly add the ethanol of 1mL 75% along centrifugal tube wall, wash tube wall gently, centrifugal (12000g, 5min, 4 DEG C), discard ethanol;
6. the dissolving of RNA: drying at room temperature is precipitated to transparent, adds appropriate RNase-free water dissolution precipitation, dissolves completely ,-70 DEG C of preservations;
7. take a morsel RNA solution detectable level.
(2) RNA reverse transcription synthesis cDNA
By PrimeScript
tMrequirement preparation RT reaction system (10 μ L) of RT reagent kit is as follows:
Note: 10 μ L system RNA can not more than 1 μ g.
37 DEG C of water-bath 15min, 85 DEG C of water-bath 5s deactivation ThermoScript II.
(3) clone of H7N9 subtype avian influenza virus HA1-2 gene:
According to influenza virus in GenBank (A/Hangzhou/1/2013 (H7N9)) hemagglutinin HA nucleotide sequence, design two pairs of primers, HA1-2-F '/HA1-2-R ' and HA1-2-F/HA1-2-R.Wherein 5 ' the end of HA1-2-F ' and the 3 ' end of HA1-2-R ' introduce restriction enzyme site Sac I, Hind III (underscore place) respectively, are for the independent HA1-2 gene that increases.
HA1-2-F′：5′-CCC
GAGCTCAAAGGGAAAAGGACAGTTGACC-3′ SEQID NO.3
HA1-2-R′：5′-CCC
AAGCTTGGCATCAACCTGTACT-3′ SEQID NO.4
And the 5 ' end of primer HA1-2-F, HA1-2-R introduces restriction enzyme site EcoR I and flexible peptide (Gly respectively
4ser)
3encoding gene (underscore place) merges the HA1-2 gene in fragment for increasing.
HA1-2-F：5′-CCG
GAATTCAAAGGGAAAAGGACAGTTGACC-3′ SEQID NO.5
HA1-2-R：5′-
CACCTCCGCTTCCACCTCCACCGGCATCAACCTGTACT-3′ SEQID NO.6
Amplification HA1-2 gene PCR reaction system (20 μ L) is as follows:
Reaction system is mixed brief centrifugation, is placed in PCR instrument and increases.HA1-2 Gene response condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 50s, 59 DEG C of annealing 50s, 72 DEG C of extension 50s, 30 circulations, 72 DEG C of ends extend 10min.
The pcr amplification of 2.fliC gene:
According to Salmonella typhimurtum I type flagellin fliC gene order design primer fliC-F, fliC-R, wherein 5 ' the end of primer fliC-F, fliC-R introduces flexible peptide (Gly respectively
4ser)
3encoding gene and restriction enzyme site Xba I (underscore place).
fliC-F：5′-
AGGTGGAAGCGGAGGTGGTGGAAGCATGGCACAAGTCATTAATA-3′
SEQID NO.7
fliC-R：5′-CCG
TCTAGATTAACGCAGTAAAGAGAGGACG-3′SEQID NO.8
Amplification fliC gene PCR reaction system (20 μ L) is as follows:
Reaction system is mixed brief centrifugation, is placed in PCR instrument and increases.FliC Gene response condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 50s, 56 DEG C of annealing 50s, 72 DEG C of extension 2min, 30 circulations, 72 DEG C of ends extend 10min.
The overlap pcr amplification of 3.HA1-2-fliC gene
With above-mentioned PCR primer (HA1-2 gene and fliC gene) for template, HA1-2-F and fliC-R is primer, amplifies goal gene HA1-2-fliC by overlapPCR method.
Amplification HA1-2-fliC gene PCR reaction system (20 μ L) is as follows:
Reaction system is mixed brief centrifugation, is placed in PCR instrument and increases.HA1-2-fliC Gene response condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 50s, 56 DEG C of annealing 50s, 72 DEG C extend 2min 30s, totally 30 circulations, 72 DEG C of ends extend 10min.
In above step, PCR primer is respectively got 2 μ L and is added the agarose gel electrophoresis that appropriate Loading Buffer carries out 1%.Result shows, and occur specific band at about 669bp, 1488bp and 2187bp place respectively after HA1-2, fliC and HA1-2-fliC gene product electrophoresis of pcr amplification, conform to expected results, result as shown in Figure 1.
The retrieve and purification of 4.PCR product
After nucleic acid electrophoresis terminates, under ultraviolet lamp, cut out the gel containing object sheet segment DNA with clean blade.Reclaim test kit with DNA gel and reclaim goal gene, operation reference dna reclaims test kit.
5. ligation
By through 1% agarose gel electrophoresis reclaim PCR primer HA1-2 and HA1-2-fliC, carry out double digestion respectively with prokaryotic expression carrier pCold; 16 DEG C, the object fragment of recovery and carrier is spent the night and is connected.
Ligation system (10 μ L) is as follows:
6. connect the conversion of product
1. a 1.5mL dactylethrae containing 100 μ L competent cell E.coli DH5a is got from-70 DEG C, ice bath melted.
2. add 10 μ L connecting fluids under aseptic condition, ice bath 30min, period must guard against vibration.
3. 42 DEG C of heat shock 90s, do not vibrate.
4. ice bath 10min.
5. add nonreactive LB substratum 890 μ L, 37 DEG C, 200rpm cultivates 1-2h.
6. centrifugal (4000rpm, 5min).
7. stay 200 μ L supernatants, abandon redundance, mixing, is applied to containing ammonia benzyl microbiotic LB agar plate.
8. 37 DEG C of incubators are just being put without obvious liquid-flow to flat board, are inverted overnight incubation, about 16-20h,
7. the double digestion of recombinant plasmid is identified with order-checking
(1) recombinant bacterium is through extracting plasmid, and double digestion is identified
Reaction system (20 μ L) is as follows:
After 37 DEG C of water-bath 3h, carry out agarose electrophoresis observation.
Result shows, and the object fragment of about 669bp and the carrier segments of 4407bp appear in recombinant plasmid pCold-HA1-2 after enzyme is cut, and conform to, the results are shown in Figure 2 with expected results.Equally, after enzyme is cut, there is the object fragment of about 2187bp and the carrier segments of 4407bp, the results are shown in Figure 3 in recombinant plasmid pCold-HA1-2-fliC.
(2) recombinant plasmid order-checking and sequential analysis
Double digestion is accredited as positive clone DH5a (pCold-HA1-2) and DH5a (pCold-HA1-2-fliC), incubated overnight, extracts recombinant plasmid and send Nanjing Genscript Biotechnology Co., Ltd. to check order.
8. the recombinant plasmid checking order correct by double digestion imports E.coli BL21 (DE3) competent cell, by qualification correct recombinant bacterium called after DE3 (pCold-HA1-2) and DE3 (pCold-HA1-2-fliC).
Two, H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and the amalgamation and expression of Salmonella typhimurtum I type flagellin fliC in escherichia expression system
1. the expression of albumen HA1-2 and fusion rotein HA1-2-FliC
Picking recombinant bacterium DE3 (pCold-HA1-2) and DE3 (pCold-HA1-2-fliC) plants in the LB liquid nutrient medium of amicillin resistance, and 37 DEG C of shaking culture are to OD
600be that to add IPTG to final concentration after 0.6 ~ 0.8,15 DEG C of standing 30min be 0.5mM, 15 DEG C of shaking culture 24h inducible proteins are expressed.Centrifugal after induced product ultrasonic treatment, respectively induced product cracking supernatant and precipitation are carried out SDS-PAGE analysis, judge the solubility of albumen.Result display is located to occur obvious target protein band at about about 26kDa and 82kDa respectively, and in the same size with expection albumen, show that successful expression goes out HA1-2 albumen and HA1-2-fliC fusion rotein, result as shown in Figure 4 and Figure 5.
2. the purifying of albumen HA1-2 and fusion rotein HA1-2-FliC
Continue the expression of inducing a large amount of recombinant bacterium (800mL) according to method in 1, according to Novagen company
the explanation of Purification Kit carries out purifying to albumen, and after getting partial purification, albumen carries out SDS-PAGE qualification.Result display is located to occur single target protein band at about about 26kDa and 82kDa respectively, and with expection albumen is in the same size and purity is higher, result as shown in Figure 6.
3.Western blotting identifies
By the fusion rotein HA1-2-fliC of purifying and albumen HA1-2 after SDS-PAGE electrophoresis, be transferred to nitrocellulose filter, 2h is closed with containing 5% skim-milk room temperature, respectively using little mouse-anti H7N9 subtype avian influenza virus polyvalent antibody and anti-flagellin polyvalent antibody as primary antibodie, with 1:500 dilution, 4 DEG C of overnight incubation; Next day with the sheep anti-mouse igg that HRP marks be two resist, 1:5000 dilute, after incubated at room 2h ECL/DAB colour developing.Result shows, albumen HA1-2 and HA1-2-fliC all can react with little mouse-anti H7N9 subtype avian influenza virus polyvalent antibody, fusion rotein HA1-2-fliC also can with little mouse-anti flagellin polyvalent antibody generation specific reaction, illustrate that albumen all has good immunoreactivity, as shown in Figure 7, Fig. 7 is the Western blot detection figure of albumen HA1-2, HA1-2-fliC to result.
4.TLR5 biological activity assay
Every hole 5 × 10
4individual HEK293-mTLR5 cell (100 μ L) is placed in 96 orifice plate overnight incubation.Next day, commercial flagellin, HA1-2 albumen, HA1-2-fliC fusion rotein are stimulated HEK293-mTLR5 cell with the concentration of 100ng/mL, collect supernatant after 5h, detect IL-8 secretion level with Human IL-8ELISA kit, the TLR5 evaluating target protein is active.Result shows, the fusion rotein HA1-2-fliC energy high-caliber IL-8 of induced cellular secretion (2200pg/mL), and HA1-2 albumen stimulating group only secretes extremely low-level IL-8 (100pg/mL), utilize GraphPad Prism 5.0 software to analyze result, the level pole of fusion rotein HA1-2-fliC induced cellular secretion IL-8 is significantly higher than HA1-2 (P<0.001).As shown in Figure 8, Fig. 8 is fusion rotein HA1-2-fliC TLR5 biological activity assay figure to result.
Three, the immunogenicity of H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and Salmonella typhimurtum I type flagellin fliC fusion expressed product
1. material
Laboratory animal: 6-8 C3H/HeJ female mice in age in week 18, purchased from Nanjing biological medicine research institute of Nanjing University.
Immune protein: restructuring target protein HA1-2 and HA1-2-fliC utilizing His tag purification in previous experiments.
2. mouse immunization protocol
18 mouse are divided into 3 groups at random, and often organize 6, immunization route is abdominal injection, and 200 μ L/ only immunity point carry out for three times, and head exempts from not carry out antibody test, and two exempt from rear according to antibody test result, and carry out three and exempt from, immunization method and dosage are exempted from head, as shown in table 1:
Table 1
3. experiment mice blood sampling and serum are separated
Second and third immunity carries out orbital vein blood sampling to each immune group in latter 12 days, and blood is put 4 DEG C of placements and spent the night, 4 DEG C, the centrifugal 15min of 4000rpm/min, the transparent serum in careful sucking-off upper strata again, the serum of sucking-off is put-20 DEG C frozen, separation of serum is used for antibody test and hemagglutination-inhibition test.
4. two to exempt from, three exempt from HA1-2 specific IgG antibodies in latter 12 days mice serums and detect
Indirect ELISA detecting step is as follows:
1. quilt is wrapped: dilute GST-HA1-2 albumen to 1.5 μ g/mL with carbonate buffer solution the day before yesterday in test, with micro sample adding appliance every hole application of sample 100 μ L, 4 DEG C are spent the night;
2. wash: next day rinses enzyme plate with the PBST containing 0.05%Tween-20, washs 4 times altogether, each 5min;
3. close: add in every hole containing confining liquid (PBST of 1%BSA) 200 μ L with micro sample adding appliance, 2h is closed in 37 DEG C of water-baths;
4. wash: PBST rinses enzyme plate, wash 4 times altogether, each 5min;
5. primary antibodie is hatched: dilute serum to be checked to working concentration (1:100 dilution) with confining liquid, every hole adds 100 μ L serum dilution to be checked, is diluted to suitable concentration successively by 2 times of dilution methods, 37 DEG C of water-bath effect 2h;
6. wash: PBST rinses enzyme plate, wash 5 times altogether, each 5min;
7. two anti-to hatch: with confining liquid, the sheep anti-mouse antibody that HRP marks is diluted to working concentration, IgG (1:10000 dilution), adds 100 μ L with the every hole of micro sample adding appliance, 37 DEG C of water-bath effect 1h;
8. wash: PBST rinses enzyme plate, wash 6 times altogether, each 5min;
9. develop the color: add the colour developing of tmb substrate damping fluid, 100 μ L/ holes, 37 DEG C of lucifuge colour developing 10min; 2M H
2sO
4solution termination reaction, 50 μ L/ holes;
10. reading: enzyme linked immunological reading apparatus reads OD
450value
Result shows, the mean level (ML) of the two HA1-2 specific IgG antibodies of exempting from 12 rear HA1-2-fliC immune group mice serums reaches 12800, three exempt from 12 days after reach 48640, through GraphPad Prism 5.0 software analysis, HA1-2-fliC immune group is all significantly higher than HA1-2 immune group (P<0.05), as shown in Figure 9, Fig. 9 two to exempt from, three exempts from the IgG antibody detection figure that after 12 days, in mice serum, HA1-2 is special result.
5. three hemagglutination-inhibition tests of exempting from latter 12 days mice serums
(1) hemagglutination test
1. in blood-coagulation-board, every hole adds 25 μ L PBS;
2. the first hole adds deactivation H7N9 subtype avian influenza virus, makes 2 times of serial dilutions successively, sets up Positive control wells and negative control hole simultaneously;
3. every hole adds 25 μ L 1% chicken red blood cell suspension, and l ~ 2min that horizontal oscillator tube vibrates mixes, result of determination after 37 DEG C of standing 30min.
Hemagglutination test result: deactivation H7N9 subtype avian influenza virus HA-HI test is 2
8, result as shown in Figure 10.Figure 10 is H7N9 subtype avian influenza virus hemagglutination test detection figure;
(2) hemagglutination-inhibition test
1. prepare 4 unit virus liquids according to result in (1): using the HA-HI test of deactivation H7N9 subtype avian influenza virus divided by 4 as the extent of dilution of 4 unit virus liquids, dilute with PBS;
2. in blood-coagulation-board, every hole adds 25 μ L PBS, and the first hole adds 25 μ L serum to be checked, makes 2 times of serial dilutions successively, sets up Positive control wells and negative control hole simultaneously;
3., except negative control hole, every hole adds 25 μ L, 4 unit virus liquids; After putting l ~ 2min that horizontal oscillator tube vibrates, 37 DEG C of standing 15min;
4. every hole adds 25 μ L 1% chicken red blood cell suspension, and l ~ 2min that horizontal oscillator tube vibrates mixes, result of determination after 37 DEG C of standing 30min.
Result shows, and the three hemagglutination inhibition antibody average titers of exempting from latter 12 days HA1-2-fliC immune group mice serums reach 2
5, through GraphPad Prism 5.0 software analysis, be significantly higher than HA1-2 immune group (P<0.01), as shown in figure 11, Figure 11 three to exempt from after 12 days hemagglutination inhibition antibody titre detection figure in mice serum to result.
6. three exempt from Antibody dynamics change in rear mice serum
Three exempt from 0 day (namely two exempting from latter 12 days) detects antibody titer in mice serum, three exempt from after every 12 days, orbital vein blood sampling is carried out to each immune group, Antibody dynamics change in rear mice serum is exempted from monitoring three, result shows, after exempting from three 12 days, in HA1-2-fliC immune group mice serum, HA1-2 specific antibody level reaches the highest (average out to 48640), antibody horizontal is all on a declining curve subsequently, until three to exempt from latter 84 days HA1-2-fliC immune group mice serums HA1-2 specific antibody level still 10
4left and right, is all significantly higher than HA1-2 immune group (P<0.05), and as shown in figure 12, Figure 12 three exempts from antibody horizontal dynamic change figure in rear mice serum to result.
The protective antigen of the subunit vaccine of the high immunogenicity that the present invention obtains is HA1-2-fliC fusion rotein, and the cycle of its expressed fusion protein is short, and expression product has the biological characteristics similar to natural product and good immunogenicity.H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 gene is connected to the N end of salmonella typhimurium flagellin gene by the present invention by flexible peptide, by escherichia coli prokaryotic expression system, flagellin and HA1-2 albumen are carried out amalgamation and expression, overcome traditional deactivation or cracking technology, production cycle is long, yield poorly, subunit vaccine immunogenicity is shortcoming not fully, therefore has great importance to the exploitation of H7N9 subtype avian influenza new generation vaccine.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and application claims protection domain is defined by appending claims, specification sheets and equivalent thereof.
Claims (10)
1. a subunit vaccine for H7N9 subtype avian influenza, is characterized in that: it is fusion rotein, and described fusion rotein comprises H7N9 subtype avian influenza virus antigen HA1-2 albumen and has the Salmonella typhimurtum I type flagellin fliC of adjuvant effect.
2. the subunit vaccine of H7N9 subtype avian influenza according to claim 1, is characterized in that: described fusion rotein has following aminoacid sequence:
(1) protein be made up of the aminoacid sequence shown in SEQ ID No.2; Or
(2) amino acid sequence homology limited with sequence SEQ ID No.2 is encoded 80% to 100% the aminoacid sequence of identical function protein; Or
(3) aminoacid sequence shown in SEQ ID No.2 has the albumen derivative by (1) of same isoreactivity through increasing, lacking or replace one or more amino acid.
3. a nucleic acid molecule, the fusion rotein of its coding described in claim 1 or 2.
4. nucleic acid molecule according to claim 3, its nucleotide sequence is as shown in SEQ ID No.1.
5. prepare the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 1, it is characterized in that comprising the following steps:
(1) gene of albumen described in overlap pcr amplification claim 1,
(2) be connected to pCold carrier, obtain recombinant plasmid pCold-HA1-2-fliC,
(3) transformed Host Strains E.coli BL21 (DE3), obtained positive recombinant bacterium DE3 (pCold-HA1-2-fliC),
(4) H7N9 subtype avian influenza virus hemagglutinin fragment HA1-2 and the amalgamation and expression of Salmonella typhimurtum I type flagellin fliC in escherichia expression system; By IPTG abduction delivering target protein HA1-2-fliC, adopt Ni-NAT affinity chromatography purifying with the object fusion rotein of His label.
6. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 5, is characterized in that: in step (1), the acquisition of H7N9 subtype avian influenza virus HA1-2 gene: the extraction of a.H7N9 subtype avian influenza virus geneome RNA;
B.RNA reverse transcription synthesis cDNA;
The clone of c.H7N9 subtype avian influenza virus HA1-2 gene; The pcr amplification of fliC gene; The overlap pcr amplification of HA1-2-fliC gene; The retrieve and purification of PCR primer.
7. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 6, it is characterized in that: in step (2), by the PCR primer HA1-2-fliC reclaimed through 1% agarose gel electrophoresis, be connected with prokaryotic expression carrier pCold, obtain recombinant plasmid pCold-HA1-2-fliC;
In step (3), connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms Host Strains E.coli DH5a, obtains positive recombinant bacterium DH5a (pCold-HA1-2-fliC); The double digestion of recombinant plasmid and order-checking qualification;
Connect the conversion of product: by recombinant plasmid pCold-HA1-2-fliC, it transforms expresses Host Strains E.coli BL21 (DE3), obtains positive recombinant bacterium DE3 (pCold-HA1-2-fliC);
In step (4), the expression of fusion rotein HA1-2-FliC; The purifying of fusion rotein HA1-2-FliC.
8. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 7, is characterized in that: in step (1), the clone of H7N9 subtype avian influenza virus HA1-2 gene:
According to the nucleotide sequence of H7N9 subtype avian influenza virus hemagglutinin HA in GenBank, design two pairs of primers, HA1-2-F '/HA1-2-R ' and HA1-2-F/HA1-2-R;
Wherein 5 ' the end of HA1-2-F ' and the 3 ' end of HA1-2-R ' introduce restriction enzyme site Sac I, Hind III respectively, are the primers for the independent HA1-2 gene that increases; And the 5 ' end of primer HA1-2-F and HA1-2-R introduces restriction enzyme site EcoR I and flexible peptide (Gly respectively
4ser)
3encoding gene; For the primer merging HA1-2 gene in fragment that increases;
Primer HA1-2-F ' has the nucleotide sequence of SEQID NO.3;
Primer HA1-2-R ' has the nucleotide sequence of SEQID NO.4;
Primer HA1-2-F has the nucleotide sequence of SEQID NO.5;
Primer HA1-2-R has the nucleotide sequence of SEQID NO.6.
9. the method for the subunit vaccine of H7N9 subtype avian influenza according to claim 8, it is characterized in that: in step (1), the pcr amplification of fliC gene: according to Salmonella typhimurtum I type flagellin fliC gene order design primer fliC-F and fliC-R, wherein 5 ' the end of primer fliC-F and fliC-R introduces flexible peptide (Gly respectively
4ser)
3encoding gene and restriction enzyme site Xba I;
Primer fliC-F has the nucleotide sequence of SEQID NO.7;
Primer fliC-R has the nucleotide sequence of SEQID NO.8.
10. the application of subunit vaccine in the immunoprophylaxis of H7N9 subtype avian influenza described in any one of claim 1 or 2.
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| CN117069860B (en) * | 2023-07-06 | 2024-03-12 | 华南农业大学 | Molecular adjuvant, chimeric avian influenza virus-like particle, vaccine, and preparation and application thereof |
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