木瓜凝乳蛋白酶基因的克隆及在大肠杆菌中的表达

目的构建木瓜凝乳蛋白酶(CP)的表达载体,并在大肠杆菌中表达。方法从生番木瓜中提取mRNA后进行反转录,得到木瓜凝乳蛋白酶的cDNA,PCR产物克隆到pET22-b(+)载体中,重组质粒转化至大肠杆菌BL21,进行诱导表达及表达产物的检测。结果表达产物的SDS-PAGE显示单一条带,相对分子质量约为26000。Western blot表明表达产物可与抗木瓜凝乳蛋白酶特异性抗体结合。结论CP基因已成功在大肠杆菌中克隆表达,为进一步纯化和动物实验奠定了基础。

Gene Cloning and Expression of Chymopapain in E. coli

Objective To construct recombinant plasmid for expression of chymopapain(CP) in E. coli.Methods The cDNA encoding CP was amplified by RT-PCR using the mRNA extracted from premature Carica papaya as a template,and inserted into vector pET22-b( ).The recombinant plasmid was transformed to E. coli BL21 for expression.The expressed product was identified by SDS-PAGE and Western blot.Results SDS-PAGE showed a single band with a relative molecular mass of(26 000).Western blot showed specific reaction band of expressed product with antibody against CP.Conclusion CP gene was successfully expressed in E. coli.It laid a foundation of further purification and animal tesu of CP.