乳酸乳球菌Lactococcus lactis 1.2472 usp 45基因的克隆与原核表达

Cloning and Prokaryotic Expression of usp 45 gene of Lactococcus lactis 1.2472

从乳酸乳球菌Lactococcus lactis 1.2472基因组中PCR扩增usp 45基因,与pMD-18T载体连接获pT-usp 45质粒;酶切鉴定并测序正确后,将usp 45基因克隆入原核表达载体pET-28a,获重组质粒pET-28a-usp 45,转入E.coli BL21(DE3);经IPTG诱导表达及镍亲和层析纯化,由SDS-PAGE和Westren blot进行鉴定。结果表明,本试验获得1 371 bp长度的usp 45基因,构建pET-28a-usp 45质粒,诱导表达并纯化分子量约50 kDa的目的蛋白,为进一步研究usp 45蛋白奠定基础。

Usp45 gene was amplified by PCR and cloned into pMD-18T Simple Vector. The sequence of cloned usp 45 was confirmed by restriction analysis and DNA sequencing. An usp 45 prokaryotic expressing plasmid, pET-28a-usp 45 was then constructed . pET-28a-usp 45 was transformed into E.coli BL21（DE3） and induced by IPTG. The expression of Usp45 protein was purified by nickel affinity chromatography, and analyzed by SDS-PAGE and Western blot. The results showed that usp 45 gene in size of 1371 bp was cloned successfully and the recombinant pET-28a-usp 45 was constructed. A Mr 50×103 protein was expressed in E.coli BL21（DE3） induced by IPTG and purified successfully, and confirmed to be Usp45 protein. Our study established a solid basis of further studying the biological function of Usp45 protein.