重组核酸酶原核表达载体的构建

Construction of recombinant nuclease prokaryotic expression vector

目的构建重组核酸酶原核表达载体,并检测其表达情况。方法根据Gen Bank公布的黏质沙雷菌胞外核酸酶(Serratia marcescens nuclease,SM nuclease)基因序列(M19495.1),去除其N-端信号肽序列,使用大肠埃希菌偏爱性密码子,设计并合成带有核酸酶基因的Ben-p GH。以核酸酶基因全长为模板,PCR法扩增该基因,将其与经NcoⅠ和XhoⅠ双酶切的A10-p ET-32a-c(+)载体连接,连接产物转入E.coli Top10中,经菌液PCR筛选阳性克隆,并进行DNA测序。将Ben-p ET-32a-c(+)转入E.coli BL21(DE3)中,经IPTG诱导表达,表达产物进行SDS-PAGE鉴定。结果 Ben-p ET-32a-c(+)中核酸酶基因与Gen Bank公布的SM nuclease基因的同源性为82%。表达的重组核酸酶以包涵体形式存在,相对分子质量为30 000,表达量约占菌体总蛋白的84.45%。结论成功构建了重组核酸酶原核表达载体,并获得稳定表达。

Objective To construct recombinant nuclease prokaryotic expression vector and determine the expressed protein. Methods Ben-p GH gene was designed and synthesized by removal of N-terminal signal peptide sequence of Serratia marcescens（SM）nuclease gene in Gen Bank and usage of E. coli-preferred codon. The target gene was amplified by PCR using the full length nuclease gene as template, and inserted into vector A10-p ET-32 a-c（＋）digested with NcoⅠand XhoⅠ. The constructed recombinant plasmid was transformed to E. coli Top10, and the positive clones were screened by PCR and sequenced. Recombinant plasmid Ben-p ET-32 a-c（＋） was transformed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE. Results The homology of nuclease gene in Ben-p ET-32 a-c（＋）was 82% to that of SM nuclease genes reported in Gen Bank. The expressed recombinant nuclease,with a relative molecular mass of 30 000, existed in a form of inclusion body, and contained about 84. 45% of total somatic protein. Conclusion The recombinant nuclease prokaryotic expression vector was successfully constructed, and target protein was stably expressed.