羊种布鲁氏菌Wzt基因的克隆及原核表达

Cloning and Prokaryotic Expression of Wzt Gene of Brucella melitensis

为进一步研究Wzt蛋白在光滑型脂多糖合成路径中的作用,本试验利用PCR技术,以羊种布鲁氏菌16 M株基因组为模板,扩增出大小为759bp的Wzt基因片段,将其连入pMD20-T载体,测序正确后构建重组质粒pET-28a-Wzt,转化E.coli BL21（DE3）工程菌,IPTG诱导其表达,最后用Western blotting鉴定蛋白。结果显示,扩增出的Wzt基因片段大小为759bp,与GenBank中登录的羊种布鲁氏菌16M株Wzt基因序列（登录号：AF047478.1）同源性为99.87%,证明成功克隆了Wzt基因,同时成功构建了pET-28a-Wzt原核表达载体,并在E.coli BL21（DE3）工程菌中表达了Wzt蛋白,诱导得到的融合蛋白大小约为30ku,位于25～35ku之间,与目的蛋白大小一致,结果表明成功表达了目的基因。

In order to further study the role of Wzt protein in the synthesis of smooth lipopolysaccharide, the peR technology was used to amplify the Wzt gene of 759 bp . Then it was ligated into pMD20- T vector. The recombinant plasmid pET-28a-Wzt was transformed into E. coli BL2ICDE3） for expression under induction of IPTG. The protein product was analyzed by SDS-PAGE and Western blotting. The results showed that the size of the Wzt gene was 759 bp and the sequence homology was 99. 87 % compared with the sequence of Brucella melitensis 16M strain with the access number of AF04 7478.1 in GenBank , proving that the Wzt gene was successfully cloned. Also, the prokarotic expression vector pET-28a-Wzt was successfully constructed and the Wzt protein with molecular weight of 30 ku was highly expressed in E. coli BL2ICDE3）, which all the above proved that Wzt gene was successfully expressed.