摘要
目的克隆植物乳杆菌中胆盐水解酶(BSH)基因,原核表达并纯化重组蛋白。方法利用PCR技术扩增植物乳杆菌BSH基因,克隆至表达载体pET-30a,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行纯化及Western blot鉴定。结果重组表达质粒pET-30a-BSH经双酶切鉴定,可见961bp的目的基因条带。表达的重组蛋白相对分子质量约为40000(含6个His标签),主要以包涵体形式表达,表达量约占菌体总蛋白的43%,纯化后,纯度达90%以上,且可与植物乳杆菌多克隆抗血清发生特异性反应。结论已成功克隆了植物乳杆菌中BSH基因,并在大肠杆菌中获得高效表达。纯化的重组蛋白纯度较高,为高效降解胆固醇的基因工程菌株的研究奠定了基础。
Objective To clone the bile salt hydrolase （BSH） gene of Lactobacillus plantarum for prokaryotic expression, and purify the expressed protein. Methods The BSH gene of Lactobacillus plantarum was amplified by PCR and cloned into expression vector pET-30a. The constructed recombinant plasmid pET-30a-BSH was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was purified then identified by Western blot. Results A target gene band at length of 961 bp was observed on restriction map of recombinant plasmid pET-30a-BSH. The expressed protein, with a relative molecular mass of about 40 000 （with 6 His tag）, mainly existed in a form of inclusion body and contained about 43% of total somatic protein. After purification, the recombinant protein reached a purity of more than 90%, and showed specific reaction with polyclonal antibody against Lactobacillus plantarum. Conclusion The BSH gene of Lactobacillus plantarum was successfully cloned and expressed in E. coli, which laid a foundation of study on recombinant bacterial strain for high degradation of cholesterol.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第9期876-879,共4页
Chinese Journal of Biologicals
基金
大庆市科技局攻关项目(SGG2007-031)
大庆市高新区创新基金资助项目(DQGX08YF036)
黑龙江省研究生创新科研资金项目(YJSCX2008-066HLJ)
关键词
植物乳杆菌
胆盐水解酶
原核表达
纯化
Lactobacillus plantarum
Bile salt hydrolase （BSH）
Prokaryotic expression
Purification
