日本血吸虫重组质粒pGEX-Sj14-3-3的构建及其在大肠埃希菌BL21(DE3)中的表达

Construction and Expression of the Recombinant Plasmid pGEX-Sj14-3-3 of Schistosoma japonicum in BL21(DE3)

目的构建并研究日本血吸虫重组质粒pGEX-Sj14-3-3在大肠埃希菌BL21(DE3)中的表达。方法从日本血吸虫成虫组织提取总RNA;RT-PCR扩增Sj14-3-3编码基因;将此基因克隆至载体pGEX-1λT,构建重组质粒pGEX-Sj14-3-3;转化入DE3中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达;表达产物用SDS-PAGE和Western blot进行鉴定。结果 RT-PCR扩增出399bp的Sj14-3-3编码基因;并成功将其插入pGEX-1λT构建了重组质粒pGEX-Sj14-3-3;SDS-PAGE显示相对分子质量约为40 000的重组蛋白,与预期结果相符,薄层扫描分析显示表达蛋白约占菌体总蛋白的21%;Western blot显示重组蛋白可被日本血吸虫感染兔血清识别。结论日本血吸虫重组质粒pGEX-Sj14-3-3构建成功,重组质粒在大肠埃希菌BL21中可较高效表达,且表达蛋白具有特异的抗原性。

Objective To construct and research the expression of the recombinant plasmid pGEX-Sj14-3-3 in Escherichia coli BL21（DE3）.Methods Sj14-3-3 gene was amplified by RT-PCR from template of the total RNA extracted from adult worms of S.japonicum,and then cloned into the vector pGEX-1λT to construct pGEX-Sj14-3-3.The recombinant plasmid pGEX-Sj14-3-3 was transformed into E.coli BL21（DE3）.BL21（pGEX-Sj14-3-3） was induced with isopropyl-β-D-thiogalactopyranosid（IPTG）,and the expressed products were identified by SDS-PAGE and Western blot.Results A 399 bp fragment of Sj14-3-3 coding gene was successfully amplified by RT-PCR and cloned into the vector pGEX-1λT,and the recombinant plasmid pGEX-Sj14-3-3 was constructded successfully.The molecular mass of the expressed recombinant protein was proximately 40 000 Dolton as detected by SDS-PAGE.The amount of the expressed protein was about 21% of the total bacterial protein.Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.Conclusion The recombinant plasmid pGEX-Sj14-3-3 was successfully constructed.The Sj14-3-3 protein was highly expressed in E.coli and the expressed recombinant protein possessed specific antigenicity.