Expression Purification And Immunogenicity Of The Recombinant H5 Avian Influenza Virus Protein Epitope Vaccine

Avain influenza is a general infection disease caused by A subtype avian influenza virus in bird population. This disease has prevailed more one hundred years since it burst out in 1878 in Italy . Every epidemic has made not only great economic loses but also consume of human and matter resources. There are several reports that people were infected by H9N2,H7N7 and H5N1 subtype virus, and the condition become more serious increasingly. There are several reports that human have AI in provinces in our country since 2009. Highly pathogenic avian influenza A (HPAI) viruses, specifically H5N1 strains, cause widespread morbidity and mortality in domestic and wild bird populations, and recent outbreaks have resulted in severe economic losses. Avian influenza virus(AIV) belongs to the influenza A genus of the family Orthomyxoviridae and is the only type can infect birds. AIV can be classified some subtypes by different HA (hemagglutinin) and NA (neuramidinase) molecular constitutions. There are 16 kinds of HA (hemagglutinin) and 10 kinds of NA (neuramidinase). These molecules combine different subtypes. H5 subtype and H7 subtype are highly pathogenic strains, and H9 subtype is lowly pathogenic stain. There are very weak cross protection among different antigen subtypes. H5N1 is the major subtype that cause HAI infects human.The specific humoral immunity induced by HA can prevent the virus get into the cells. 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. An important principle to prevent the infection of AI by using vaccine is to induce HA specific immune responses.There are some kinds of avian influenza vaccines: inactivated whole virus vaccine; DNA vaccine; recombinant active vector vaccine; reverse genetic engineering vaccine. The inactivated whole virus vaccine is the most often used one. This kind of vaccine's forte is better effects, and the demerit is there are many dangers during making vaccine. DNA vaccine and recombinant active vector vaccine use a lowly pathogenic virus or another virus to insert some gene which can express some viral protein in eukaryocyte which can induce b eneficial specific responses to the virus. DNA vaccine and recombinant active vector vaccine can do their work for a long time, but they are also not very safe as they might cause tumor or induce immune tolerance. Genetically engineering vaccine which uses a virus as a vector do not very good when host have infected vector virus because of interference phenomenon. Genetically engineering vaccine is used reverse genetic technique to make a new kind of vaccine. This is a good way to reform a new strain which can be a safe and effective vaccine. Genetic engineering subunit vaccine is an antigen which comes from pathogenic microorganism mixing with adjuvant. This kind of vaccine is a purified antigen. And it can also induce the specific immune response analogous to natural infection. This kind of vaccine's forte is safety and utility. This vaccine can not induce an immune response to internal antigens. So this vaccine's effects are different to natural infection, and it can not confuse monitoring of AI.Our object is to build a prokaryotic expression vector which can express a recombinant protein having antigenicity of H5, and we can use it as a gene recombination subunit vaccine.1. Construction of expression vectorWe have analyzed the spatial structure of H5(influenzaAvirusA/Goose/Guangdong/1/96(H5)-NCBI:AAD51927) and pretest epitopes which can be binded with T-cell and B-cell first. Then we choose 4 sites to compound in series again and again. These works have been finish by XuLiu in 2008.2. Identification of recombinant plasmidThere were two steps in identification. First, we used the EcoRⅠand HINDⅢto cut the recombinant plasmid, and measure the segments by agarose gel electrophoresis. If the sizes of segments were quiet accord to our expecting, we amplified this plasmid and did sequencing. Finally, we got the right recombinant plasmid.3. Expression, assessment and purification of recombinant vaccinePut the right recombinant plasmids into BL21, and choose the clones which plasmids shift in the cells successfully to amplify them. The expression bacteria containing pET28a-(B1C1B2C2)3 were induced by IPTG. The insert in high-level protein expression bacteria strain was sequenced and confirmed. A great quantity of recombinant H5 AIV protein vaccine was expressed with the high-level protein expression bacteria strain. The best conditions of induction contain that : 37℃, IPTG 0.4mmol/L. Do SDS-PAGE after bacteria done with ultrasound to prove that the recombinant protein expressed as inclusion body. The recombinant protein has two His-tag, the recombinant H5 AIV protein vaccine was purified by Ni2+ affinity chromatography. We prove the time and flow rate conditions in purification. The protein fractions collected were detected by SDS-PAGE and purity of the purified protein monomer was up to 95%.4. Immune animals and analysis the immunogenicity of recombinant vaccineTo verify the immunogenicity of the recombinant H5 AIV protein vaccine, Balb/c mice were immunized. The H5 AIV specific IgG antibodies induced by this recombinant vaccine were detected using indirect ELISA.We have built a stabile system to detect the specific IgG antibodies of virus. Immuned by recombinant vaccine mice's sera have rising antibodies of virus at the 14th day and 21st day. After the 28th day, the antibodies were not rising. But the quantity was not big enough to get a positive results. This phenomena told us that the recombinant vaccine's immunogenicity is more thinness than natural H5 protein.Next, we can do these jobs: redesigning the vaccine to improve its immunogenicity; testing better immune programme; analysising another type of virus antibodies.