Cloning Of NS Gene Of H9N2 Subtype Of Avian Influenza And Construction Of Recombinant Expression Vector

Avian influenza (AI) was caused by avian influenza virus (AIV), which was first reported in 1878 in Italy. Signs of high mortality and loss of productivity characterized AI. The avian immune function was inhibited, and then was infected by other bacterium and virus. In China, the epidemic AI among poultry was reported in 1994, AIV H9N2 subtype was recently the main virus type. The AI caused by H9N2 virus was first reported in 1966, which was characterized by sign of respiratory distress in turkey. Since 1990's, H9N2 virus has made more danger to chicken. Besides the respiratory distress and significant loss of productivity, AI was responsible for significant mortality. So it's important to study the H9N2 virus in virology or veterinary medicine.In this research, the AIV (H9N2) isolate was propagated in chicken embryos. The virus was purified by ultracentrifuge. According to the NS gene sequence published by Genbank, One primer T-1 was designed to amplify the cDNA by reverse transcript. The other primers NSl-u/NS1-1 and NS2-u/NS2-1.HNS2-u were designed to amplify the NS1, NS2 and HNS2 gene. The PCR product was cloned into pMD18-T vector. The positive recombinant clone was identified by PCR and endonuclease digest. Then the inserting DNA fragment was sequenced and analysed. The NS1 gene of AIV (H9N2) isolated was 654bp, which encodes 217 amino acids, compared with the published sequence of A/chick/Hongkong/1074/ 99(H9N2) and A/chick/Hongkong/1073/99(H9N2). Sequencing analysis indicated that NS1 fragment shared homology about 94.37%, and the amino acid 89.6% and 89.1% respectively, 13 amino acids were deleted in the c-terminal. Compared with the domestic isolates, they shared the nucleotide identity with 89% to 98%, and amino acids identity with 78% to 88%. The analysis of phylogenetic tree showed that these isolates had the close relationship with HongKong isolates. The HNS2 was 336bp, encoding 111 amino acids. Compared with HongKong isolates, it shared the nucleotide identity with 94.63% and 94.33%, amino acid identity with 95.5% and 94.59%.The NS1 and HNS2 Gene from AIV (H9N2) isolate were cut from the pMD18-T-NSl vector, the NS1 gene cloned into the sites of BamH I and Xho I of prokaryotic expression vector and the HNS2 cloned into the sites of EcoR I and Xho I of prokaryotic expression vector. The recombinant vector was identified by endonuclease. the result showed that the interest gene was inserted into the expression vector. The expression vector comprised NS1 and HNS2 gene facilitate the study for the function of the nonstructural protein and interaction with host cells.