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

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—β type I receptor 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—EgβIRI—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。

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

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.