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目的利用大肠杆菌原核表达系统表达发热伴血小板减少综合征布尼亚病毒(SFTSV)NSs重组蛋白。方法采用RT-PCR技术,从SFTSV毒株提取的病毒总RNA中,扩增NSs全长基因,将其克隆至p ET22b(+)中构建原核表达载体p ET22b-NSs,经酶切及测序鉴定后转化表达菌BL21(DE3),IPTG诱导,通过Western Blot鉴定NSs蛋白的表达。结果酶切、测序证明重组原核表达载体p ET22b-NSs构建成功,免疫印迹法可见NSs基因编码蛋白表达。结论实现了发热伴血小板减少综合征布尼亚病毒重组NSs蛋白的原核高效表达,为进一步深入研究发热伴血小板减少综合征布尼亚病毒NSs蛋白的结构与功能奠定了基础。
Objective To express prokaryotic expression vector of SFsV NSs in Escherichia coli prokaryotic expression system with fever and thrombocytopenia syndrome. Methods The full-length NSs gene was amplified from total RNA extracted from SFTSV strains by RT-PCR and cloned into pET22b (+) to construct prokaryotic expression vector p ET22b-NSs. The recombinant plasmid was identified by restriction enzyme digestion and sequencing After transformed into expression strain BL21 (DE3), induced by IPTG, the expression of NSs protein was identified by Western Blot. Results Enzyme digestion and sequencing proved that the recombinant prokaryotic expression vector p ET22b-NSs was successfully constructed. The expression of NSs gene was detected by Western blotting. Conclusion The prokaryotic expression of recombinant NSs of Bunyavirus in patients with fever and thrombocytopenia syndrome was achieved, which laid the foundation for further study on the structure and function of fever and thrombocytopenia syndromes.