新孢子虫NcSAG4基因克隆及原核表达

目的构建新孢子虫NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中表达。方法根据新孢子虫NcSAG4基因序列设计特异性引物,以新孢子虫总核酸为模板PCR扩增目的片段,与pMD-18-T连接,构建克隆载体pMD-NcSAG4,经双酶切后回收目的片段,与表达载体pGEX-T连接,构建原核表达载体pGEX-NcSAG4,用IPTG诱导,通过SDS-PAGE及Western blot进行鉴定。结果成功构建了新孢子虫NcSAG4基因原核表达载体pGEX-Nc-SAG4;SDS-PAGE电泳显示,在IPTG诱导下阳性菌高效表达了分子质量单位为18.79ku的蛋白质;Western blot显示表达产物可被抗新孢子虫的多克隆血清识别。结论成功构建了NcSAG4基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达,为建立经济、实用的诊断新孢子虫病的ELISA方法奠定了基础。

Cloning and prokaryotic expression of the Neospora caninum NcSAG4 gene in Escherichia coli

Objective To construct a prokaryotic expression vector of the Neospora caninum NcSAG4 gene and express it in Escherichia coli.Methods Special primers were designed on the basis of the reported Neosporosis NcSAG4 gene.The NcSAG4 gene was amplified by PCR from the total DNA of N.caninum and was cloned into pMD-18-T to construct pMD-NcSAG4.The plasmid pMD-NcSAG4 was then digested with restriction ribozymes and subcloned into prokaryotic expression plasmid pGEX-T to construct pGEX-NcSAG4.It was then 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 NcSAG4 gene was constructed and expressed in E.coli.Induced with IPTG,the expressed recombinant protein was detected as a band of 18.79 ku by SDS-PAGE.A special reaction band to anti-NcSAG4 sera was observed in Western blot.Conclusion The fusion protein of NcSAG4 gene was successfully expressed in prokaryotic cells and it has the potential to facilitate diagnosis via ELISA.