汉坦病毒S基因在大肠埃希菌中的克隆及表达

目的：在大肠埃希菌中克隆和表达汉坦病毒SEO型及HTN型S基因。方法：PCR扩增汉坦病毒SEO型L99株及HTN型Z10株S基因读码区，前者经EcoRI及XhoI双酶切，后者经SacI及XhoI双酶切，将两者定向克隆入pET32a（＋），转入感受态E．coli Top10。获取重组质粒，经PCR、双酶切及序列分析鉴定后，转入E．coli BL21感受态。经IFTG诱导表达，其产物经SDS-PAGE和Western-blot检测。结果：SEO型L99株及HTN型Z10株S基因正确克隆于pET32a（＋），目的蛋白的分子质量约为67．1ku，表达量占菌体总蛋白的40％以上，Western-blot有特异性条带。结论：SEO型及HTN型汉坦病毒S基因在大肠埃希菌中获得表达，为其后的应用奠定了基础。

Cloning and Expression of Hantavirus S Gene in E.coli

Objective: To clone and express S gene of Hantavirus SEO and HTN type in E.coli. Methods: Hantavirus S gene's ORF of SEO type L99 strain and HTN type Z10 strain was amplified by PCR. The former was double enzymly cut by EcoRI and XhoI and the latter was double enzymly cut by SacI and XhoI. Both of them were cloned into vector pET32a( ) directly then transformed into competent cell E.coli TOP10. Two recombinant plasmids were transformed into competent cell E.coli BL21 after being identified by PCR, double enzyme cutting and sequence analysis which was induced by IPTG,the product was screened by SDS-PAGE and Western-blot. Results: S gene of SEO type L99 strain and HTN type Z10 strain was cloned into pET32a( ) correctly. Molecular weight of target protein was about 67.1 ku, and its yield exceeded 40% of total protein. The result of Western-blot had specified strains. Conclusion: S gene of Hantavirus SEO and HTN type in E.coli has expression and makes a primary basis on further work.