Cloning, Expression Of Hemagglutinin Of Avian Influenza Virus And Its Research In Diagnosis Of AIV Subtype

Avian influenza (AI) is an infectious disease caused by influenza A virus, a member of the family Orthomyxoviridae. It is the most common infectious disease which happens suddenly in the whole world. Since the disease have broken out in Italian chicken flocks for the first time in 1878, Avian Influenza Virus break out in succession in all parts of the world. Especially the Highly Pathogenic Avian Influenza Virus, which can cause up to 100% outbreak rate with/ or death rate and even can infect mankind. The threat of low pathogenic Avian Influenza Virus is equally serious, especially the H9 AIV. In particular the 1997 Hong Kong avian influenza event and the influenza in Southeast Asia countries including China at the end of 2003 as well as the beginning of 2004, which aroused serious concern of the government and people all over the world. Avian influenza of A type has many subtypes and changes to a new strand easily. To date, 15 Hemagglutinin(HA,H)subtypes and 9 neuraminidase(NA,N) subtypes have been recognized. There are not cross -reaction among the different subtypes, which make the diagnosis and prevention very difficult. Consequently, the comparatively ideal subtype diagnosis is in the great need. Hemagglutinin (HA) is the most important surface antigen of virus and has subtype speciality, which induce the peculiar antibody. Therefore, it is feasible to set up subtype diagnosis using the HA protein. In the paper, indirect ELISA to detect antiboies against H5, H9 Avian Influenza Virus was developed using the HA protein expressed in E.coli as antigen. So the following researchs were explored:1. Cloning and sequence analysis of H5 and H9 HA genesAccording to published HA genes sequence of H5 and H9 AIV, primers were designed and synthesized. A 1683bp and 1707 bp fragments were amplified from Avian Influenza virus RNA (A/Chicken/HuBei/326/2002(H5Nl) , A/Duck/HuBei/405/2003 (H9N2)) by RT-PCR and cloned into the pMD18-T vector.The recombinant plasmid was proved to be true by enzyme analysis and sequencing. The sequence analysis showed that H5 and H9 HA gene consisted of 1683bp and 1707 bp respectively.2. Subcloning and expression of H5, H9 HA gene in E.coliBased on the negative function of signal peptide of HA gene in expression,we lackedthe signal peptide of HA gene through the genetic engineering means and obtained the HA gene with the signal peptide removed. Then the fragment of HA was subcloned into prokaryotic expression vector pGEX-KG and was expressed in E.coli B121. The expressed HA-GST fusion protein in E.coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90KDa and 92KDa protein with immunogenicity.3. Development of indirect H5HA-ELISA in Diagnosis the antibodies of H5 AIV Subtype ^ H9HA-ELISA in Diagnosis the antibodies of H9 AIV SubtypeThe fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The indirect ELISA method was established by selecting the conditions with the optimal dilution of antigen of HA protein to be 1:80, the optimal dilution of serum to be 1:80. It revealed a negative reaction with positive sera of Marek's disease, Newcastle disease, Parainfluenza, infectious bronchitis, infectious laryngotracheitis, infectious bursal disease, aviadenovirus I, aviadenovirus III, standard negative serum and serum of avian uninfected by avian influenza. It revealed a positive reaction with some type of avian influenza standard positive serum and sera from avian influenza. In order to know reliability of the ELISA, a repetitive experiment was conducted too. The 8 sera samples was used. A total of 339 chicken sera samples was used in HA and ELISA to detect the specificity, sensitivity and reduplication. The above results indicated that the assay has good specificity, high sensitivity and excellent reduplication. It could be used to differently detect antibodies to H5 and H9 ATV. At present, at the same time, a total of 856 chicken sera samples from hubei province was detect and the result showed that the ELISA can be used in in Diagnosis the antibodies of H5 AIV Subtype and H9 AIV Subtype.