Prokaryotic Expression And Elisa Application Of Hemagglutinin Protein Of Influenza A(H1N1)2009 Virus

A novel influenza A(H1N1) virus emerged in spring 2009 has spread rapidly across the globe. It posed a huge threat to public health and led to enormous economic losses of countries.However, at the beginning of this research,there was no corresponding commercial kit at home and abroad. What's worse, people were susceptible to this novel influenza virus.As a result, it has a essential significance to establish a convenient rapid sensitive detective method for early diagnosis, treatment and prevention the outbreak of the epidemic.Influenza is divided into 3 major types:A, B and C according to nucleoprotein(NP) and matrix protein. Influenza A virus is further divided into various subtypes in the light of hemagglutinin(HA) and neuraminidase(NA) which are both glycoproteins expressed on the surface of influenza viruses. The 11 proteins of influenza A virus are encoded by 8 gene segments. The RNA segment 4 encoded HA which is the main glycoprotein on the surface.HA is the chief immunogenic protein, which can stimulate humoral immunity and cellular immunity and induce neutralizing antibodies against influenza virus. It mostly determines the virulence. In this research, HA was chosed to be the object of study so as to provide support for rapid diagnosis and early treatment of influenza A /H1N1/2009. The target protein were expressed in the prokaryotic expression system. The monoclonal antibodies produced by recombinant HA were used to develop the double-antiboby sandwich ELISA for detecting the influenza A /H1N1/2009 virus. In this paper,the main contents are composed of two parts:1.Expression of HA antigen: According to the relevant nucleotide sequence from GenBank, the HA gene of influenza A /H1N1/2009 virus was synthetized. The product was used for constructing a prokaryotic recombinant plasmid pET-30 Xa/LIC-HA. The constructed vector was transformed to E.coli BL21(DE3) for expression under the induction of 37℃, 0.1mM IPTG. The recombinant protein was purified by nickel affinity chromatography assay. The expressed product was identified by SDS-PAGE and Western blot analysis. The results showed that the HA genes have been expressed and the expressed protein took the form of inclusion bodies, with a relative molecular mass of about 62kD and good immunorectivity. 2.The preliminary establishment of influenza A /H1N1/2009 double-antibody sandwich ELISA detection method: On the basis of two paired strains of monoclonal antibodies originated from the recombinant HA proteins, the double-antiboby sandwich ELISA method for detecting HA of influenza A /H1N1/2009 virus were established. Also, the evalution of its specificity, sensitivity, precision and stability have been done. The results show that this assay is specific for detecting influenza A /H1N1/2009 virus and has no cross reaction with H1N2, H3N2, H5N1 and influenza B virus. The minimum detectable dose of HA is determined to be approximately 1ng/mL. This method has good precision and stability.