CTP-HBcAg_(18-27)-Tapasin融合表达载体的构建及其表达和纯化  

Construction,expression and purification of recombinant plasmid of fusion protein CTP-HBcAg_(18-27)-Tapasin

作　　者：刘红红[1] 陈小华[1] 周丽芹[1] 刘雪妮[1] 余永胜[1] 臧国庆[1] 汤正好[1] 

机构地区：[1]上海交通大学附属第六人民医院感染科,上海200233

出　　处：《胃肠病学和肝病学杂志》2013年第7期684-687,共4页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金青年项目(31000414);国家自然基金面上项目(81270502)

摘　　要：目的构建CTP-HBcAg18-27-Tapasin融合基因表达质粒并分离、纯化融合蛋白。方法以质粒pCMV-SPORT6中Tapasin基因为模板,设计一对引物,上游引物带有融合基因CTP-HBcAg18-27序列,进行PCR反应;PCR产物回收纯化克隆到质粒pREST-B中,双酶切及测序鉴定;将鉴定正确的质粒转化大肠埃希菌BL21(DE3)诱导表达;镍柱亲和层析法纯化融合蛋白;Western blotting鉴定融合蛋白。结果由质粒pCMV-SPORT6 PCR扩增得到1 480 bp大小的条带,为CTP-HBcAg18-27-Tapasin融合序列;将其克隆到表达质粒pREST-B后经酶切、测序结果正确;表达质粒转化DE3后成功表达,经纯化得到52.3 KD的蛋白;Western blotting鉴定为CTP-HBcAg18-27-Tapasin融合蛋白。结论成功构建了CTP-HBcAg18-27-Tapasin融合表达质粒,并成功表达,为进一步研究此融合蛋白的功能提供了实验基础。Objective To construct and identify recombinant plasmid of fusion protein CTP-HBcAg18-27ET-Tapasin which was further separated and purified. Methods A pair of primers was designed based on Tapasin gene in pCMV- SPORT6 with the forward primer having fusion gene CTP-HBcAg18-27. Then Tapasin gene was amplified by PCR, the product of which was recycled, purified and cloned into pREST-B. After identification by enzyme cutting and sequence test, the correct vector was transformed into BL21 （DE3） to express the fusion protein. Furthermore, the fusion protein was purified by Ni^2＋ -chelating colum and identified by Western blotting. Results 1 480 bp band was obtained by PCR from pCMV-SPORT6. Then the fusion gene was cloned into pREST-B and proved to be correct by enzyme cutting and sequence test. Then the expression plasmid was transformed to DE3 and expressed successfully. Finally, 52.3 KD protein was obtained by purifying the product of expression and identified to be fusion protein CTP-HBcAg18-27-Tapasin by Western blotting. Conclusion The fusion protein CTP-HBcAg18-27- Tapasin was successfully synthesized, and it is to pave way for research to the function of fusion protein CTP-HBcAg18-27-Tapasin.

关 键 词：CTP TAPASIN HBcAg18-27 融合蛋白 

分 类 号：R575.7[医药卫生—消化系统]