日本血吸虫大陆株14-3-3信号转导蛋白epsilon亚型基因的表达及鉴定  

EXPRESSION AND IDENTIFICATION OF 14-3-3 SIGNAL TRANSDUCTION PROTEIN EPSILON ISOFORMS GENE FROM SCHISTOSOMA JAPONICUM(CHINESE STRAIN)

作　　者：唐小牛[1] 汪学龙[2] 沈继龙[2] 

机构地区：[1]皖南医学院寄生虫学教研室,安徽芜湖241001 [2]安徽医科大学病原生物学教研室,安徽合肥230032

出　　处：《中国寄生虫病防治杂志》2003年第6期358-361,共4页Chinese Journal of Parasitic Disease Control

基　　金：国家自然科学基金资助项目 (No .30 1 70 841 ) ;皖南医学院中青年科研基金资助 (No .WK2 0 0 2 0 9)

摘　　要：目的　构建日本血吸虫大陆株 14 3 3信号转导蛋白epsilon亚型基因真核表达重组质粒pBK Sj14 3 3 ,并进一步在大肠杆菌中表达和鉴定 ,为其进一步保护性免疫的研究提供条件。　方法　根据日本血吸虫 14 3 3蛋白的核苷酸序列 ,设计合成 1对引物 ,以日本血吸虫大陆株成虫总RNA为模板 ,用RT PCR法合成日本血吸虫大陆株 14 3 3蛋白epsilon亚型基因cDNA片段。将其克隆入pGEM T载体 ,经双酶切及PCR鉴定后 ,再亚克隆入 pBK CMV真核表达质粒 ,构建重组质粒pBK Sj14 3 3 ,转染到大肠杆菌BL2 1,双酶切鉴定后 ,通过IPTG的诱导在大肠杆菌BL2 1中进行表达及Western blot鉴定。　结果　RT PCR产物、pGEM T Sj14 3 3及pBK Sj14 3 3分别经双酶切均获得一特异性基因片段 ,其表达产物经SDS PAGE及Western blot鉴定后其分子质量为 3 2ku ,并能被抗 14 3 3多克隆抗体识别。　结论真核表达重组质粒 pBK Sj14 3 3在大肠杆菌中的表达产物是一分子质量为 3 2ku融合蛋白 ,并能被抗 14 3 3多克隆抗体识别。Objective To construct an eukaryotic expression plasmid pBK-Sj14-3-3 of 14-3-3 signal transduction protein(epsilon isoforms) gene from Schistosoma japonicum (Chinese strain) and to express and identify the fusion protein, so as to provide conditions for further studies on protective immunity. Methods A pair of primers were designed and synthesized according to the known nucleotide sequence of Sj14-3-3 protein. Using adult worms total RNA of S. japonicum as template, a cDNA first strand synthesis was driven by RT-PCR technique. The product was cloned into pGEM-T vector and identified by double endonuclease digestion and PCR. The fragment was subcloned into an eukaryotic expression vector pBK-CMV again, a recombinant pBK-Sj14-3-3 was constructed and transferred into E. coli BL21, after identification expressed in E. coli BL21. Results The same specific gene fragment was obtained by RT-PCR, endonuclease digestion of pGEM-T-Sj14-3-3 and pBK-Sj14-3-3, SDS-PAGE revealed that the molecular weight of this expressed product was around 32 ku. Western-blot showed that the recombinant protein was identified by 14-3-3 polyclonal antibody. Conclusion Using an eukaryotic expression plasmid pBK-Sj14-3-3, a 32 ku fusion protein was expressed in E. coli BL21 and identified by 14-3-3ε polyclonal antibody.

关 键 词：日本血吸虫 epsilon亚型 基因表达 Western-blot鉴定 大肠杆菌 

分 类 号：R383.24[医药卫生—医学寄生虫学]