Prokaryotic And Eucaryotic Expression Of H9N2 Avian Influenza Virus NS1 Gene And The Effect Of NS1 Protein On Host Cells' Apoptosis

Avian Influenza (AI) is an infection or syndrome caused by influenza virus type A, a member of Orthomyxoviridae. Highly pathogenic avian influenza (HPAI) is an infectious diseases by World Organization for Animal Health (OIE). Avian Influenza H9N2 virus continues to threaten the poultry population worldwide. H9N2 virus infections in chickens result in mild respiratory signs and egg production losses. The virus has been reported to cause flu-like disease in humans. The NS1 protein of AIV plays an important role in virus replication and the interaction between virus and cells. Meanwhile, because of its antigenicity and conservatism NS1 protein has very important uses in some fields such as differential diagnosis between vaccine immune and natural infection, epidemic survey and forecast of influenza. Therefore, The study used molecular biology methods to express NS1 gene in the prokaryotic and eucaryotic expression systems, which provided base materials to research the function of NS1 gene. This study including:1. The NS1 gene of H9N2 AIV isolates (WN) was amplified by PCR with specific primers, and cloned into pMD18-T simple vector and sequenced. Then, the gene were inserted between BamHI and HindIII sites of pET-32a(+) after cleavage by corresponding enzymes. The recombinant plasmid was named pET-NS1. SDS-PAGE and western-blotting showed an expected protein of 44 ku in size was successfully expressed in E. coli BL21(DE3) induced by IPTG.2. The NS1 gene of H9N2 AIV isolates (WN) was amplified by PCR with specific primers, and cloned into pMD18-T simple vector and sequenced. It was, then, subcloned into eukaryotic vector pEGFP-C1. The recombinant plasmid pEGFP-NS1 was transfected into 293 cells using LipofectamineTM 2000. The H9N2 AIV NS1 protein expression in 293 cells was detected by Western-blotting and The special band of NS1 protein expressed in 293 cells were identified. The result showed that the NS1 gene was successfully expressed in 293 cells and had very good antigenicity.3. The recombinant plasmid pEGFP-NS1 were transfected into MDCK,Vero and 293 cells with the liposome mediated method.The typical morphological changes such as decreased cell size, cytomorphosis, cell shrinkage and detaching were observed by inverted microscope. Apoptosis was observed with flow cytometer. Results showed that NS1 protein could induce the early apoptosis of MDCK and 293 cells, but NS1 couldn't induce the early aopotosis of Vero cells.