日本血吸虫重组质粒pGEX-Sj32的构建及其在大肠埃希菌BL21中的表达

发布时间：2018-04-04 17:36

本文选题：日本血吸虫　切入点：聚合酶链反应　出处：《国际检验医学杂志》2014年23期

【摘要】：目的构建日本血吸虫(Sj)重组质粒pGEX-Sj32并研究其在大肠埃希菌BL21中的表达效率。方法从该实验室保存的BL21(pET28α-Sj32)重组菌中抽提质粒pET28α-Sj32,PCR扩增Sj32抗原编码基因,定向克隆入穿梭载体pGEX-1λT,构建重组质粒pGEX-Sj32。将重组质粒转化大肠埃希菌BL21(DE3),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 PCR成功扩增出Sj32编码基因,并成功构建了重组质粒pGEX-Sj32;SDS-PAGE显示相对分子质量约为58×103的重组蛋白,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。结论成功构建日本血吸虫重组质粒pGEX-Sj32,该重组质粒在大肠埃希菌BL21中得到了高效表达,且表达蛋白具有特异的抗原性。
[Abstract]:Objective to construct the recombinant plasmid pGEX-Sj32 of Schistosoma japonicum and study its expression efficiency in Escherichia coli BL21.Methods the Sj32 antigen coding gene was amplified from the recombinant strain BL21(pET28 伪 -Sj32 (pET28 伪 -Sj32) and cloned into the shuttle vector pGEX-1 位 T to construct the recombinant plasmid pGEX-Sj32.The recombinant plasmid was transformed into Escherichia coli BL21DDE3 and was induced by isopropylthiothio- 尾 -Dgalactoside (IPTG), and the expression product was identified by SDS-PAGE and Western blot.Results the Sj32 encoding gene was successfully amplified by PCR, and the recombinant plasmid pGEX-Sj32 SDS-PAGE showed that the relative molecular weight of the recombinant protein was about 58 脳 10 ~ 3.TLC scanning showed that the expressed protein accounted for about 21% of the total bacterial protein. Western blot showed that the recombinant protein could be recognized by the sera of rabbits infected with Schistosoma japonicum.Conclusion the recombinant plasmid pGEX-Sj32 of Schistosoma japonicum was successfully constructed. The recombinant plasmid was highly expressed in Escherichia coli BL21 and the expressed protein had specific antigenicity.
【作者单位】：重庆医科大学附属第一医院传染病寄生虫病研究所;
【基金】：重庆市科委地方病重大专项基金(2008AB5055、2008AB5008、2008AB5054)
【分类号】：R378

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