Design, Construction, Expression And Antigenecity Of The Recombinant H5 Avian Influenza Virus Protein Vaccine

Avian influenza (AI) is an acute infectious disease that affects various avian species and also several mammals including human, and is caused by type A influenza viruse. HPAI is continuously occurring more and more frequently and the number of animal species infected with AIV is increasing.Avian influenza viruse (AIV) belongs to the influenza A genus of the family Orthomyxoviridae and is negative-sense, single-stranded RNA viruse. The genome of influenza A viruse consists of eight negative-sense, single-stranded RNA segments that encode nine structural proteins and two non-structural proteins. The structural proteins HA and NA are the major targets of the protective immune response and the genes of them are variable. Especially, HA can induce organism to produce neutralization antibodies and is the major protective antigen of influenza A virus. HA is cleaved into HA1 and HA2 subunits when influenza A virus infects host cell and this cleavage is essential for the infectivity of influenza A viruse. HA1, locating on the head of HA, is capable of inducing to produce neutralization antibodies. The receptor binding sites are located on the tip of HA1 and comprised of the 190-helix, the 130-loop and the 220-loop.Influenza virus infection of the host cell can be prevented by HA-specific antibodies. During infection, influenza viruses attach to the cell surface through binding of the HA to cell surface sialic acid receptors and are internalized through endocytosis and fusion between host and viral membranes. Therefor, antibodies specific for HA block virus attachment, thereby preventing infection of cells. But antibodies to NA and M2 proteins prevent release of the viruses and the cell-mediated immune response lyses the infected cells only after the cells become infected.Inoculation with the type-specific avian influenza vaccine is still the main measure to prevent and control HPAI. So far, most avian influenza vaccines applied are whole inactivated virus influenza vaccine. The inactivated influenza viruses are very easy to spread during the production of inactivated vaccines; moreover, discrimination between vaccinated and virus-infected animals is confusing due to application of the inactivated vaccines during disease monitoring. In order to overcome these disadvantages, we constructed a new type of recombinant H5 AIV protein vaccine. B1, B2, C1 and C2 epitopes of the virus A/Goose/ Guangdong/1/96(H5) - NCBI:AAD51927 were determined through bioinformatics and analysis of the H5 HA structure. The four epitopes were ligated by the means of cloning and subcloning to construct the gene encoding recombinant H5 AIV protein vaccine that was expressed successfully in E.coli. Balb/c mice were inoculated with the recombinant H5 AIV protein vaccine and H5 AIV specific antibodies induced by this recombinant vaccine were detected using indirect ELISA.1. Determination of B cell epitopes and T cell epitopes of H5 AIVTo determine B epitopes of H5 subtype AIV, the structure of H5 HA was analyzed with the software Cn3D at first. Then the second structure, flexible regions, hydrophilicity, antigenic index and surface accessibility of HA1 amino acid sequence of the A/Goose/Guangdong/1/ 96(H5)-NCBI: AAD51927 (H5N1 AIV) were analyzed with the software DNAStar. The amino acid sequences of HA1 in thirty-six H5N1 AIVs were contrasted with the software BioEdit. Finally the epitopes were predicted with the website Bepipred. Four B cell epitopes– B1, C1, B2 and C2 were determined by synthesizing all results.T cell epitopes were predicted with the website Propred. The result indicates that C1 and C2 epitopes also contain the genes encoding T cell epitopes.2. Design of the recombinant H5 AIV protein vaccineThe genes of B cell epitopes and T cell epitopes were ligated repeatedly to construct the gene encoding recombinant H5 AIV protein vaccine. There are several patterns to construct recombinant vaccine because of different ligations and linkers. Their secondary structures were analyzed with DNAStar and spatial structures were predicted by Geno3D. The optimal construct-(B1C1BB2C2)3 was determined finally.3. Construction of the gene encoding recombinant H5 AIV protein vaccineTo construct the recombinant plasmid pMD18-T-(B1C1B2C2)3, the coding genes of B1, C1, B2 and C2 were synthesized in two round of PCR at first. The PCR products were confirmed by agarose gel electrophorosis and ligated into pMD18-T vector. The ligates were subsequently transformed into the competent cells JM109. After antibiotic screening and plasmids extracting, the recombinant plasmids were digested by EcoR I and the corresponding gene segments were released. The sequence of the insert was confirmed by DNA sequencing. Utilizing the isocaudarner, the genes of B1, C1, B2 and C2 epitopes were repeatedly ligated in tandom to construct the gene encoding recombinant H5 AIV protein vaccine.To construct the expression plasmid pET28a-(B1C1B2C2)3, the recombinant plasmid pMD18-T-(BB1C1B2BC2)3 and pET28a were both digested by EcoR I and Hind III at first. The gene (B1C1BB2C2)3 and pET28a vector obtained were ligated and the product of ligation transformed into the clone bacteria JM109. After antibiotic screening and plasmids extracting, the plasmids were digested by EcoR I and Hind III to identify the recombinant plasmid pET28a-(B1C1B2C2)3.4. Expression, identification and purification of the recombinant H5 AIV protein vaccineTo obtain the recombinant H5 AIV protein vaccine, the recombinant plasmid pET28a-(B1C1B2C2)3 was transformed into the expression bacteria BL21. After antibiotic screening, plasmids were extracted and then digested by EcoR I and Hind III to identify the recombinant plasmid pET28a-(BB1C1B2BC2)3. The expression bacteria containing pET28a-(B1C1B2C2)3 were induced by IPTG. The insert in high-level protein expression bacteria strain was sequenced and conffirmed. A great quantity of recombinant H5 AIV protein vaccine was expressed with the high-level protein expression bacteria strain.To detect whether the recombinant H5 AIV protein vaccine was expressed as inclusion body, the bacteria was lysed by sonication. The expression proteins were located in precipitate of the lysed bacteria by analysis of SDS-PAGE, indicating the recombinant H5 AIV protein vaccine was expressed as inclusion body.The recombinant H5 AIV protein vaccine was purified by Ni2+ affinity chromatography. The protein fractions collected were detected by SDS-PAGE and purity of the purified protein was up to 90%.5. Antigenicity of the recombinant H5 AIV protein vaccineTo verify the antigenicity of the recombinant H5 AIV protein vaccine, Balb/c mice were immunized. The H5 AIV specific antibodies induced by this recombinant vaccine were detected using indirect ELISA.The aqueous phase collected from the divalent oil emulsion inactivated vaccine of AIV (H5N1,strain Re-1 and Re-4) after freeze thawing was used as the coating antigen in ELISA and the sera of the immunized mice were diluted 1:400. The result showed that: On day 14 after first immnunization with the recombinant H5 AIV protein vaccine, the H5 AIV specific antibodies increase slightly in two mice. But on day 7 after booster vaccination, the H5 AIV specific antibodies in three mice increased significantly and dramatically declined on day 14 after booster vaccination.This study provides evidence for the development of efficient recombinant vaccines for H5 and other subtypes.