【摘要】 目的:研究人类Tudor-SN蛋白TSN结构域4个亚片段的功能,构建重组质粒pEGFP-C2-Tudor-SN-TSN(Ⅰ~Ⅳ)。方法:以重组质粒pSG5-Tudor-SN-flag为模板,聚合酶链反应(PCR)扩增出目的基因,将双酶切后带有粘性末端的目的片段和pEGFP-C2载体连接,从而构建重组质粒pEGFP-C2-Tudor-SN-TSN(Ⅰ~Ⅳ)。将构建成功的重组质粒用脂质体法转染HeLa细胞,并在荧光显微镜下观察融合蛋白的荧光表达情况,Western印迹检测融合蛋白的表达。结果:对重组质粒进行双酶切鉴定可见Tudor-SN-TSN(Ⅰ~Ⅳ)的cDNA片段;脂质体转染重组质粒后可观察到绿色荧光蛋白的表达。Western印迹后可在相应位置检测到融合蛋白pEGFP-C2-Tudor-SN-TSN(Ⅰ~Ⅳ)。结论:真核pEGFP-C2-Tudor-SN-TSN(Ⅰ~Ⅳ)重组质粒构建及表达成功。更多还原

【Abstract】 Objective: To Construct recombinant plasmids pEGFP-C2-Tudor-SN-TSN (Ⅰ-Ⅳ) for the further functional research of human Tudor-SN TSN domain’s 4 sub-fragments. Methods: The genes of Tudor-SN-TSN fragments were amplified by PCR from the recombinant pSG5-Tudor-SN-flag plasmid, then the EcoRI/Sal I digested fragments were combined with pEGFP-C2 vector. The recombinant expression plasmid pEGFP-C2-TSN(Ⅰ-Ⅳ) was transfected into HeLa cells. The expression of fusion proteins were examined by fluorescent microscopy and Western blot. Results: The sub-fragments of TSN domain can be detected in products of the restriction double enzyme digestion. The green fluorescent fusion proteins were observed in HeLa cells after transfection. The fusion proteins pEGFP-C2-TSN(Ⅰ-Ⅳ) can be detected from the lysate of transfected HeLa cells by Western blot. Conclusion: These recombinant eukaryotic plasmids,pEGFP-C2-Tudor-SN-TSN(Ⅰ-Ⅳ),were constructed successfully and expressed effectively.更多还原