抑癌基因BCSC-1原核表达载体的构建及诱导表达

Construction of the Prokaryotic Expression Vector of Tumor Suppressor Gene BCSC-1 and Its Inducible Expression

目的 构建BCSC-1基因原核表达载体并进行诱导表达、纯化.方法 PCR方法从腺病毒穿梭载体pDC316-BCSC-1扩增BCSC-1基因,构建原核表达载体ET30a-BCSC-1,转化感受态E.coli BL21（DE3）,并用IPTG进行诱导表达.SDS-聚丙烯酰胺凝胶电泳方法检测目的 蛋白的表达,并用超声洗涤方法进行目的 蛋白洗涤纯化.结果 成功构建原核表达载体pET30a-BCSC-1;在IPTG诱导下在E.coli BL21（DE3）中高效表达相对分子质量（Mr）为86 000的重组BCSC-1蛋白,目的 蛋白主要以包涵体的形式存在于沉淀中,经超声洗涤纯化后,重组蛋白纯度可达85%以上.结论 利用构建的原核表达载体成功表达出BCSC-1蛋白,为进一步制备抗BCSC-1的单克隆抗体及在临床中的应用打下良好基础.

Objective To construct prokaryotic expression vector of breast cancer suppressor candidate 1 （ BCSC-1 ） gene and express recombinant protein BCSC-1 with prokaryotic expression system. Methods Human BCSC-1 gene was amplified by PCR from plasmid pDC316-BCSC-11cloned into pET-30a vector, and then pET-30a-BCSC-1 plasmid was transferred into E. eoli BI221 （DE3） to express recombinant protein. SDS-polyacrylamide gel electrophoresis was used to observe the expression of recombinant protein. Results The prokaryotic expression vector pET30a-BCSC-1 was successfully constructed;SDS-polyacrylamide gel electrophoresis showed that the recombinant protein was highly expressed in E. coil BL21 （ DE3 ） and it mainly resided in the inclusion body of bacteria. Purity of recombinant BCSC-1 protein was more than 85% showed by ultrasonic washing inclusion body. Conclusion Human BCSC-1 protein was successfully expressed in prokaryotic expression system. The purified BCSC-I protein can be used in the further preparation of anti-BCSC-1 monoclonal antibodies.