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目的构建阴道毛滴虫TPI基因原核表达载体,并在大肠埃希菌BL21(DE3)中表达。方法根据阴道毛滴虫TPI基因开放阅读框设计并合成特异性引物,以阴道毛滴虫总cDNA为模板PCR扩增目的片段,与pMD-18-T连接构建克隆载体pMD-TPI,经双酶切回收目的片段,与表达载体pGEX-T连接,构建原核表达载体pGEX-TPI,经IPTG诱导后通过SDS-PAGE及Western blot鉴定表达产物。结果成功构建了阴道毛滴虫TPI基因原核表达载体pGEX-TPI;SDS-PAGE电泳显示,在IPTG诱导下重组质粒转化菌高效表达分子质量单位为27.5ku的蛋白质;Western blot显示表达产物可被抗阴道毛滴虫的多克隆血清识别。结论成功构建了TPI基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达,为进一步研究阴道毛滴虫TPI基因功能奠定了基础。
Objective To construct a prokaryotic expression vector for Trichomonas vaginalis TPI gene and express it in Escherichia coli BL21 (DE3). Methods According to the open reading frame of Trichomonas vaginalis TPI gene, specific primers were designed and synthesized. The total fragment of Trichomonas vaginalis was used as a template to amplify the target fragment and ligated with pMD-18-T to construct the cloning vector pMD-TPI. The target fragment was cut back and ligated with the expression vector pGEX-T to construct the prokaryotic expression vector pGEX-TPI, which was induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic expression vector pGEX-TPI of TPI gene of Trichomonas vaginalis was constructed successfully. SDS-PAGE electrophoresis showed that the recombinant plasmid transformed with E.coli was induced by IPTG to efficiently express the protein of 27.5ku. Polyclonal serum identification of Trichomonas vaginalis. Conclusion The prokaryotic expression vector of TPI gene was successfully constructed and highly expressed in Escherichia coli BL21 (DE3), which laid the foundation for further study on the function of TPI gene of Trichomonas vaginalis.