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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。
Objective To construct a prokaryotic expression vector for transforming growth factor-beta type I receptor intracellular domain (Eβ) of Echinococcus granulosus (Eg) and to express and purify EgTβRⅠ-Ⅰ protein. Methods Total RNA of protoscoleces from Eg sheep infected with Eg was collected by Trizol method. EgTβRⅠ-Ⅰ gene fragment was amplified by RT-PCR and cloned into prokaryotic expression vector pET28a (+). The recombinant plasmid was digested by restriction enzyme The recombinant plasmids with the correct sequence identification were transformed into E. coli competent cells BL21 and induced by IPTG. The proteins were purified by nickel column affinity chromatography and analyzed by SDS-PAGE Detection of the expression of the target protein. Results The prokaryotic expression vector pET28a-EgTβRⅠ-Ⅰ was constructed successfully. EgTβRⅠ-Ⅰ protein (48ku) was induced by IPTG. A large amount of soluble protein was obtained after purification. Conclusion The prokaryotic expression vector pET28a-EgTβRⅠ-Ⅰ was successfully constructed and the purified soluble target protein was obtained. This study laid the foundation for the study of EgTβRI-Ⅰ in Eg and its biological function.