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目的构建日本血吸虫重组质粒pET32α-Sj26GST-Sj32,分析该质粒在大肠埃希菌BL21(DE3)中的表达情况。方法超声粉碎日本血吸虫成虫提取总RNA,通过RT-PCR扩增获得Sj26GST和Sj32抗原编码基因,然后采用基因拼接法(gene SOEing)剪接Sj26GST和Sj32,得到Sj26GST-Sj32融合基因,克隆至原核表达载体pET32α(+),构建重组质粒pET32α-Sj26GST-Sj32,转化入大肠埃希菌BL21(DE3),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1991bp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pET32α(+)中,SDS-PAGE分析显示表达产物为分子质量约82000Mr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22%;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pET32α-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特异的抗原性。
Objective To construct the recombinant plasmid pET32α-Sj26GST-Sj32 of Schistosoma japonicum and analyze its expression in Escherichia coli BL21 (DE3). Methods Sj26GST and Sj32 antigen genes were amplified by RT-PCR after sonicating the adult worms of Schistosoma japonicum. Sj26GST and Sj32 were spliced by gene SOEing to obtain Sj26GST-Sj32 fusion gene and cloned into prokaryotic expression vector pET32α-Sj26GST-Sj32 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant plasmid pET32α-Sj26GST-Sj32 was induced by IPTG and then expressed by SDS-PAGE and Western-blot analysis of expression products and identification. Results Sj26GST-Sj32 fusion gene of about 1991bp was amplified by gene splicing. Double-digestion and PCR proved that Sj26GST-Sj32 fusion gene was successfully inserted into pET32α (+). SDS-PAGE analysis showed that the recombinant product was a recombinant with a molecular mass of about 82000Mr The protein was consistent with the expected result. The expressed protein accounted for about 22% of the total bacterial proteins. The recombinant protein was identified by Western-blot in rabbits infected with Schistosoma japonicum. Conclusion The recombinant plasmid pET32α-Sj26GST-Sj32 of Schistosoma japonicum was constructed successfully. The fusion protein was highly expressed in Escherichia coli BL21 and the expressed fusion protein had specific antigenicity.