The Design, Preparation And Animal Experiment Research Of H5N1Avian Influenza Virus Genetic Engineering Vaccine

Avian flu is a deadly infectious disease of poultry and serious threatto the poultry industry which spreads widely in the world. Especially theepidemic and high mortality causes important economic losses to countriesand people. A highly contagious Avian influenza virus (AIV) can spreadacross species. In recent years, the human also become one of the hostsof this virus. Since the end of2003to19June,2008, it has been reportedthat383cases in15countries of Asia, Africa and Europe were laboratoryconfirmed human H5N1avian influenza and241died. AIV is threatening tohuman health and life as a new bird flu disease, yet human beings haveno immune protection to it. In early2004, the World Health Organizationwarned the world of the AIV epidemic and many countries and regions putthe top priority on the control of avian influenza. Avian flu is causedby the pathogen bird flu virus which has a very high variability. AIVsubtypes can be up to256and can restructure with other influenza viruses,then place the virus mutation and change of the host, so many scholarstake the complex AIV genome as the greatest gene pool of human influenzavirus.As the avian influenza virus is in a wide variety of poultry and wildbird bodies, the bird flu control is a long-term and arduous task. Vaccinedevelopment and use is the most practical, effective and economicalmeasure to this challenge. The ideal vaccine would be the availabilityof good safety, and not only can stimulate the body’s humoral immunityto produce antibodies, but also be able to fully help to regulate thecellular immunity to remove virus. Based on the above concept, we chose the H5N1subtype avian influenza virus which was mainly popular in Asiaas the research object, and selected a high homology HA1and NP proteinof H5N1subtype avian influenza virus strains which was used as standardreference sequence by comparing the protein sequences of Chinese AIV viruswith its neighboring countries. Through analysis of the HA1proteinsequence by computer software, we selected the major T cell epitopes andB cell epitopes. And then selected the main optimized CTL epitopes afterconducted a comprehensive analysis of the NP protein. According to thesepreferred epitopes, we designed the recombinant AIV H5N1subtype vaccine.This vaccine contains the major protective B cell epitopes and covers avariety of types of MHC II DR epitopes of H5N1virus HA1protein as themain skeleton. And at its C terminal we added the optimized CTL cellepitopes. After optimized the codon and base composition of the vaccinegenes, we obtained the genes coding the avian influenza virus subtype H5N1vaccine genes by using the synthetic method. We clone the vaccine genesinto the expression vector pRSET B in E. coli and got a good expressionthat the vaccine protein got to30%of total bacterial protein, and thetarget protein expressed in the form of inclusion bodies.The use of vaccines is the most effective means for preventing theAIV disease. So the output and cost control in the vaccine production arehighly demanded. In this study, we explored and optimized the bestexpression of the growth and pH value range, carbon source concentration,culture medium, and induction conditions for the recombinant AIV subtypeH5N1vaccine. We established the high-density fermentation of theengineering strain and ensured that the engineering bacteria densityreached54OD600and the recombinant vaccine protein theoretical yieldachieved about3.4g/L on the premise of the vaccine protein expressionwas30%. On this basis, we further explored a simple and efficient vaccineprotein purification and recovery process. After a simple washing of inclusion bodies and Ni+affinity chromatography, the recombinant AIVsubtype H5N1vaccine could reach protein purity95.5%and refolded proteinvaccine stock solution concentration was up to2.4mg/mL.To evaluate the avian influenza virus subtype specific and immuneeffects of the recombinant AIV H5N1subtype vaccine, this study dividedinto four parts. One is to establish an indirect ELISA method to determinethe specific reaction of the vaccine antigen and avian influenza H5(Re-4strain, Re-5strain) standard positive serum, H9standard positive serum,H7standard positive serum; Second, through the mouse experiments,tested the antibody levels and changes in cytokine levels.Through the miceexperimental model, we got the initial description of the vaccine antigen;Third, we selected the three recommended adjuvants of the French SEPPICcompany to emulsify with the vaccine and immuned SPF chickens. Bycomparing the antibody level, we chose the most suitable MONTANIDETMISA50V2for the best vaccine immune adjuvant; The last, we got the optimalimmunization dose by immuned SPF chickens. At the same time, we found theparallel relations between the ELISA method and the HI method. Throughthis four-part study, we completed the most primary work of the effectverifying test and ensured the smooth progress of the follow-up.