Expression Of The Main Region Nucleoprotein Of H5 Subtype Avian Influenza Virus And Development Of Elisa Based On Recombinant Protein

The avian influenza virus (AIV) is the the most serious disease of poultry industry currently. It is widely spreed in many countries and aeeas throughout the wor -ld,and great economic losses in poultry .The nucleoprotein (NP) is one of the most conservative constructive protein and has higer homology with nucleoprotein of influenza A viruses.It is one of the main determinants of species specificity of influenza A viruses. AIV nucleoprotein is a multifunctional RNA-binding protein,It emergs arlier than other structual protein ,and it is antigen of cell-mediated immunity;it can induce cytotoxicity T lymphocyte(CTL) and stimulates specific cytotoxicity. Therefore,it is very important for the early diagnosis of AI that to development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to AIV.In the current study,the full length of NP gene (1497bp) was cloned from pGEM-T-NP plasmid ,and inserted the prokaryotic expression vector pGEX-6p-1 without modifica -tion.The recombinant plasmids containing NP was indentified by restriction endonucl -eases analysis,PCR analysis and the sequence analysis. The results showed that the prokaryotic expression vectors were constructed successfully. Then the recombinants was transformed into BL21 (DE3) for gene expression with IPTG inducing. The expressed proteins were measured by SDS-PAGE.For some unknoww reason,the the full length of NP gene could not be successfully expressed in the pokaryotic express system.Subsequently ,the gene of the main antigenic domains of NP was subcloned and inserted the prokaryotic expression vector pET-28a(+).After indentified ,the recombinant plasmids was transformed into BL21(DE3).The recombinant was highly expressed induced by 1 .0mmol/L IPTG in the form of inclusion bodies. Westen-blot analysis with AIV positive serum against H5 subtype showed the recombinant protein shared goof antigencity and specifity.The enginering bacteria were cultivate and induced enlarged, he inclusion bodies were obtained by sonication of the bacterial cells, from which the purified activative expressed recombinant NP protein were got by urea and Triton X-100 washing and by...