细粒棘球蚴转化生长因子βⅠ型受体胞内域原核表达载体的构建及蛋白纯化  

Construction of a prokaryotic expression vector containing the intracellular domain of the transforming growth factor beta type I receptor of Echinococcus granulosus and purification of the fusion protein

作　　者：杨乐[1,2] 王丽敏[1] 张传山[1] 李亮[1] 王俊华[1] 吕国栋[1] 王慧[1] 温浩[1] 林仁勇[1] 

机构地区：[1]新疆医科大学第一附属医院,新疆重大疾病医学重点实验室-省部共建国家重点实验室培育基地,新疆包虫病基础医学重点实验室,新疆乌鲁木齐830054 [2]新疆医科大学第一附属医院肝胆腔镜外科

出　　处：《中国病原生物学杂志》2013年第12期1089-1092,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81260252,81101271);长江学者和创新团队发展计划(No.IRT1181)

摘　　要：目的构建细粒棘球蚴(Echinococcus granulosus,Eg)转化生长因子βⅠ型受体胞内域(transforming growth factor-βtypeⅠreceptor intracellular domain,TβRⅠ-Ⅰ)原核表达载体,诱导表达并纯化EgTβRⅠ-Ⅰ蛋白。方法采集感染Eg的羊源原头蚴,Trizol法提取原头蚴总RNA,RT-PCR扩增EgTβRⅠ-Ⅰ基因片段,克隆至原核表达载体pET28a(+),经限制性内切酶双酶切和序列鉴定正确的重组质粒转化至大肠埃希菌感受态细胞BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,经镍柱亲和层析纯化蛋白,SDS-PAGE检测目的蛋白的表达。结果 pET28a-EgTβRⅠ-Ⅰ原核表达载体构建成功,经IPTG诱导可表达EgTβRⅠ-Ⅰ蛋白(分子质量单位为48ku),纯化后获得大量纯度较高的可溶性蛋白。结论成功构建pET28a-EgTβRⅠ-Ⅰ原核表达载体,并获得纯化的可溶性目的蛋白,为研究EgTβRⅠ-Ⅰ在Eg体内的生物学作用及其作用方式奠定了基础。Objectives To construct a recombinant prokaryotic expression vector containing the intracellular domain of the transforming growth factor-β type I receptor （TβRI） of Echinococcus granulosus and to express and purify the fusion protein. Methods A gene fragment of TβRI from E. granulosus RNA was amplified with RT PCR and then cloned into a pET28a（＋） prokaryotic expression vector. After identification using restriction endonuclease analysis and sequence de- termination, the correct recombinant vector was transferred into BL21 competent E. coli cells. Various concentrations of IPTG were then used to induce the expression of a TβRI fusion protein. The protein was purified by Ni2＋ affinity chromatography and tested with SDS-PAGE. Results A pET28a-Eg TβRI prokaryotic expression vector was successfully constructed. The EgTβRI fusion protein was expressed via induction with IPTG. The fusion protein was successfully purified with an Ni-NTA purification column. Conclusion A pET28a- EgTβRI prokaryotic expression vector was successfully constructed, and purified soluble protein was obtained. This research has laid the foundation for further study of the biological role and mode of action of TβRI in E. granulosus.

关 键 词：细粒棘球绦虫 转化生长因子受体I 基因表达 蛋白纯化 

分 类 号：R383.33[医药卫生—医学寄生虫学]