多房棘球绦虫重组Bb-Em14-3-3疫苗的构建及其表达效率_临床医学论文.docVIP

多房棘球绦虫重组Bb-Em14-3-3疫苗的构建及其表达效率_临床医学论文 多房棘球绦虫重组Bb-Em14-3-3疫苗的构建及其表达效率_临床医学论文 【摘要】 目的 构建多房棘球绦虫(Em)重组双歧杆菌(Bb)-Em14-3-3疫苗，并研究Em14-3-3分子在大肠埃希菌BL21(DE3)中的表达效率。方法 通过PCR扩增Em14-3-3抗原编码基因；将该基因定向克隆于含有谷胱甘肽-S-转移酶(GST)基因的大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT，构建重组质粒pGEX-Em14-3-3；用电穿孔法将该质粒导入Bb，构建多房棘球绦虫重组Bb-Em14-3-3疫苗。经PCR和酶切鉴定后以IPTG诱导表达Em14-3-3/GST融合蛋白；SDS及Western blot对表达产物进行鉴定。结果 PCR成功扩增出530 bp的Em14-3-3基因；双酶切证实Em14-3-3基因插入pGEX-1λT中；PCR证实重组Bb-Em14-3-3疫苗构建成功；SDS及Western blot分析显示重组质粒转化宿主菌在IPTG诱导下高效表达了Em14-3-3/GST融合蛋白，表达效率为23%。结论 成功构建了多房棘球绦虫重组Bb-Em14-3-3疫苗，重组质粒pGEX-Em14-3-3在大肠杆菌中获得了高效表达，Western blot结果提示表达出的Em14-3-3重组蛋白具有特异抗原性。 【关键词】 多房棘球绦虫；重组Bb-Em14-3-3疫苗；构建；表达 　　ABSTRACT: Objective To construct recombinant Bb-Em14-3-3 vaccine of Echinococcus multilocularis and to investigate expression efficiency of Em14-3-3 antigen encoding gene in Escherichia coli BL21(DE3). Methods Em14-3-3 antigen gene was amplified by PCR. Then the gene was cloned into Escherichia coli-Bifidobacteria shuttle plasmid pGEX-1λ T containing gluathione-S-transferaze(GST) gene to construct pGEX-Em14-3-3. The recombinant plasmid was electroporated into Bifidobacteria bifidum (Bb) to construct rBb-Em14-3-3 vaccine. The vaccine was identified with PCR and restriction-endonuclease digestion. The expression of pGEX-Em14-3-3 was induced with isopropyl-β-D-thiogalactopyranosid (IPTG) and fusion protein Em14-3-3/GST was examined by SDSand Western blot techniques. Results 530 bp gene of Em14-3-3 was successfully amplified by PCR and cloned into pGEX-1λ T by restriction analysis. rBb-Em14-3-3 vaccine was successfully constructed by PCR and restriction-endonuclease digestion. It was demonstrated with SDSand Western blot that the fusion protein Em14-3-3/GST was expressed in E.coli, BL21. The expression efficiency is 23%. Conclusion rBb-Em14-3-3 vaccine of Echinococcus multilocularis was successfully constructed. The plasmid pGEX-Em14-3-3 was highly expressed in E.coli in fused form with GST and this kind of protein shows specific antigenicit