H5N1亚型禽流感病毒NS1基因的克隆及在E.coli中的表达

Cloning of Nonstructural(NS1) Gene of H5N1 Avian Influenza Virus and Its Prokaryotic Expression in E.coli

选择一株H5N1亚型高致病性禽流感病毒株A/Goose/Guangdong/1/96(简写为GD/1/96),根据测得的NS基因序列,设计一对特异性引物NS1U/NS1L,RT-PCR方法扩增该毒株的NS1基因。将酶切处理后的基因片段定向克隆到原核表达载体PET-32a(+),并进一步酶切分析和序列测定,鉴定出NS1基因的阳性重组子。阳性质粒转化E.coliBL 21(DE3)感受态细胞,在IPTG诱导下,经SDS-PAGE和Western-blotting分析鉴定,成功的在细菌包涵体中表达出45 kD的NS1融合蛋白。重组蛋白经Ni-NTA His.Bind Resins纯化。

According to the sequence of nonstructural protein （NS1） gene of A/Goose/Guangdong/1/96, a pair of primers（NS1U/NS1L） was designed, NS1 gene was amplified by RT-PCR, was cloned directionaly into the PET-32a（＋）, the constructed recombinant plasmid was analyzed. The positive plasmids were tansformed into E. coli BL21 （DE3）, and induced with IPTG. Analysis of SDS-PAGE and Western-blotting showed that recombinant fusion protein was successfully expressed in inclusion,its moleculer weight was 45 kD. The fusion protein was purified by affinity chromatograph.