日本血吸虫重组质粒pQE30-Sj26GST-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

Construction and expression of the recombinant plasmid pQE30-Sj26GST-Sj32 of Schistosoma japonicum in Escherichia coli BL21(DE3)

目的构建日本血吸虫重组质粒pQE30-Sj26GST-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增获Sj26GST和Sj32抗原编码基因;采用基因拼接法(gene SOEing)剪接Sj26GST和Sj32,得到Sj26GST-Sj32融合基因;将融合基因克隆至原核表达载体pQE30,构建重组质粒pQE30-Sj26GST-Sj32并转化大肠埃希菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果基因拼接法扩增出约1 991 bp的Sj26GST-Sj32融合基因;双酶切和PCR鉴定证实Sj26GST-Sj32融合基因成功插入pQE30中,SDS-PAGE显示表达产物为分子质量单位约为61 ku的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的18%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pQE30-Sj26GST-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。

Objective To construct and express the recombinant plasmid pQE30-Sj26GST-Sj32 of Schistosoma japonicum（Sj） in Escherichia coli BL21（DE3）.Methods The total RNA was extracted from Sj adult worms by ultrasound-breaking,Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA;Sj26GST-Sj32 fusion gene was obtained with gene SOEing;the fusion gene was cloned into prokaryotic expression plasmid pQE30 to construct pQE30-Sj26GST-Sj32;the recombinant plasmid was transformed into E.coli BL2（DE3）;the rBL21（pQE30-Sj26GST-Sj32） was induced with isopropyl-β-D-thiogalactopyranosid（IPTG）,the expressed products were analyzed and identified by SDS-PAGE and Western blot.Results The 1 991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pQE30 by restriction analysis and PCR identification,the recombinant plasmid pQE30-Sj26GST-Sj32 was successfully constructed;the relative molecular mass of the expressed recombinant protein was approximately 61 ku by SDS-PAGE,and the amount of the expressed protein was 18% of the total bacterial proteins;the fusion protein could be recognized by sera from rabbits infected with Sj by Western blot.Conclusion The recombinant plasmid pQE30-Sj26GST-Sj32 is successfully constructed and highly expressed in E.coli in fused form with His-tag,and the expressed fusion protein shows specific antigenicity.