Abstract：The non-structural protein gene (NS) of influenza A viruses is separated into 2 alleles, A and B, with less than 70% sequence homology between each other. The NS1 is one of the major virulent factors that are able to antagonize the host immune system. In this study, on the basis of a systemic phylogenetic analysis, the NS1 protein of 4 Avian influenza viruses (AIV) strains (A/duck/Hunan/S1256/12(H3N8), A/environment/Hunan/S4484/11(H12N7), A/duck/Guangdong/07/00(H5N1) and A/duck/Shanghai/08/01(H5N1)) with NS gene of allele B prokaryotically expressed and were purified, and their thermal stability were further examined. Viral RNA (vRNA) was extracted from virus-infected allantoic fluid, followed by cDNA synthesis by reverse transcription PCR with the Uni12 primer. The full-length of NS1 fragments were then amplified with specific primers and cloned into the pGEX6P-1 vector for expression in the BL21 bacterial strain. Since the wild-type NS1 proteins were unstable and precipitated during purification, we determined to generate truncation mutants of NS1 protein containing amino acids 1M-202A as well as two mutations, R38A/K41A. The truncation mutant constructs expressed in BL21 bacterial strain, and progressively purified by glutathione-sepharose 4B affinity chromatography, cation exchange chromatography, and Hitrap Superdex75 column chromatography. The phylogenetic analysis revealed that the NS1 protein of all 4 viruses belonged to allele B. The expression and purification condition of NS1 protein was sequentially optimized from wild type, full length mutant to truncated mutant. High quality of purified NS1 protein with purity over 90% was finally obtained from the truncated mutant. In addition, the NS1 protein of A/duck/Hunan/S1256/12(H3N8) exhibited the highest heat stability. This study provides a foundation for further studying the structure and biological function of the allele B NS1 protein.
