Cys C的原核表达载体构建和鉴定

Construction and identification of prokaryotic expression vector of human cystatin C

目的:构建人胱抑素C(Cys C)的原核表达载体,为进一步表达纯化Cys C重组蛋白奠定基础。方法:从HL-60细胞中提取总RNA,RT-PCR扩增Cys C基因,再将目的基因克隆至PET32a(+)表达载体中,构建人Cys C原核表达质粒PET32a(+) /Cys c;再将此重组子进行双酶切电泳鉴定和测序鉴定。结果:酶切电泳鉴定结果显示Cys C基因克隆入载体中,测序结果证实克隆的基因序列与GeneBank中的Cys C序列相符。结论:获得了人Cys C原核表达质粒PET32a(+)/Cys C,为进一步表达和纯化Cys C重组蛋白奠定了工作基础。

Objective：To Construct a prokaryotic expression vector of cystatin C（Cys C ） to provide a basis of purify Cys＇C protein produced by the expression system. Methods：Total RNA was isolated from HL-60 cells, and human Cys C gene was amplified with RT-PCR. The cDNA fragment was cloned into pET32a（＋ ） vector,which was confirmed with sequencing and electrophoresis. Resu/ts：The result of electrophoresis showed that Cys C had been cloned into pET32a（＋） vector,and the result of. DNA sequence analysis also showed the cloned Cys C gene sequence was completely correspondent to GeneBank data. Conclusion：The prokaryotic expression vector of cystatin C（Cys C） was obtained,providing a basis of purification of Cys C protein produced by the expression system.