摘要
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 of the Trichomonas vaginalis TPI gene and express it in Escherichia coli（BL21（DE3）.Methods Special primers were designed on the basis of the reported Trichomonas vaginalis TPI gene.The TPI gene was amplified by PCR from the total cDNA of T.vaginalis and was cloned into pMD-18-T to construct pMD-TPI.The plasmid pMD-TPI was then digested with restriction ribozymes and subcloned into the prokaryotic expression plasmid pGEX-T to construct pGEX-TPI.It was expressed in E.coli BL21（DE3） induced with IPTG.The fusion product was identified by SDS-PAGE and Western blot.Results A prokaryotic expression vector of the TPI gene was constructed and expressed in E.coli.Induced with IPTG,the expressed recombinant protein was detected as a band of 27.5 ku by SDS-PAGE.A special reaction band to anti-TPI sera was observed in Western blot.Conclusion The fusion protein of the TPI gene was successfully expressed in prokaryotic cells and has provided a basis to further study the function of the T.vaginalis TPI gene.
出处
《中国病原生物学杂志》
CSCD
北大核心
2012年第7期516-518,共3页
Journal of Pathogen Biology
基金
"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(No.2008ZX10401-B)
关键词
阴道毛滴虫
TPI基因
克隆
原核表达
Trichomonas vaginalis
TPI gene
clone
prokaryotic expression
