H5N1亚型禽流感病毒NS1基因在大肠杆菌和昆虫细胞中的表达

目的构建大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,将H5N1亚型禽流感病毒NS1基因分别在大肠杆菌和昆虫细胞中进行表达,表达产物用Western blot进行检测分析。方法酶切含有NS1基因的质粒pUC-NS1,分别克隆进大肠杆菌表达载体pET-28a(+)和杆状病毒转移载体pFastBac HT A中,分别获得表达载体pET-NS1和pFast-NS1。将pET-NS1转化大肠杆菌BL21,以异丙基硫代半乳糖苷(IPTG)进行诱导表达;将pFast-NS1转化DH10Bac感受态细胞,提取重组Bac-NS1DNA,以M13为通用引物作PCR鉴定,阳性Bac-NS1用脂质体转染sf9细胞,72h后收集感染细胞。大肠杆菌表达产物和细胞表达产物分别裂解后作SDS-PAGE和Western blot分析。结果成功构建了大肠杆菌表达载体pET-NS1和昆虫杆状病毒转移载体pFast-NS1,大肠杆菌和昆虫细胞中表达的融合蛋白Western blot都能检测到特异性条带。结论NS1基因在大肠杆菌和昆虫细胞中得到成功表达,为获得大量NS1蛋白进行功能研究及抗体制备奠定了基础。

Expression of the NS1 gene of H5N1 avian influenza virus in E.coli and insect cells

To construct the E.coli expression vector pET-NS1 and the baculovirus transfer vector pFast-NS1 for the expression of the NS1 gene of H5N1 avian influenza virus in E.coli and insect cells,the NS1 gene was inserted to the E.coli expression vector pET-28a( )or baculovirus transfer vector pFast-NS1 after digestion of the plasmid pUC-NS1 to construct the recombinant plasmids pET-NS1 and pFast-NS1 respectively.E.coli BL21 strain was transformed with pET-NS1 by induction through IPTG,whereas the buculovirus-competent cell DH10 was transfected with pFast-NS1,and then the recombinant bacmid Bac-NS1 DNA was extracted and identified by PCR with M13 universal primers.The Sf9 cells were transfected with screened Bac-NS1 by lipofectin,and the transfected cells were collected 72 hours after transfection.Meanwhile,the expressed product was analyzed with SDS-PAGE and Western blotting.In these ways,the E.coli expression vector pET-NS1 and the baculovirus transfer vector pFast-NS1 were successfully constructed,and specific bands of fusion proteins could be demonstrated in Western blot analysis.It is apparent that the NS1 gene can be expressed correctly in E.coli and insect cells with production of specific NS1 protein,thus laying a foundation to obtain large amount of NS1 protein for the preparation of specific antibodies.