Tudor-SN蛋白一级结构间断的SN5结构域质粒的拼接构建

目的 实现Tudor-SN蛋白TSN结构域内间断的SN5基因片段（SN5α、SN5β）的拼接以及与绿色荧光蛋白在HeLa细胞中的融合表达.方法 利用Geno 3D对拼接的SN5进行结构预测.以重组质粒pSG5-Tudor-SN-flag为模板,PCR分别扩增出SN5α和SN5β的基因,双酶切并纯化后,先将SN5β引入pEGFP-C2,完成重组质粒pEGFP-C2-SN5β,再将SN5α引入pEGFP-C2-SN5β,完成重组质粒pEGFP-C2-SN5.将pEGFP-C2-SN5β/ SN5脂质体法转染HeLa细胞,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 ① 拼接的SN5结构预测显示与TSN完整结构中的SN5高度重合;②对重组质粒进行双酶切鉴定可见SN5α、SN5β、SN5的cDNA片段;③ 转染重组质粒后可观察到绿色荧光蛋白的表达;④ Western印迹后可在相应位置检测到融合蛋白.结论 pEGFP-C2-SN5/SN5β重组质粒构建成功,SN5α和SN5β在pEGFP-C2中实现了顺序拼接;目的片段可与绿色荧光蛋白在HeLa细胞中融合表达,融合蛋白可与抗GFP抗体结合用于蛋白检测.

Splicing Construction of Recombinant Eukaryotic Plasmids Containing Primary Structure Intermittent SN5 Domain of Tudor-SN Protein

Objective To construct eukaryotic green fluorescent protein （GFP） expressing recombinant plasmid, pEGFP-C2-Tudor-SN-SN5, implement the splicing of the two intermittent parts of SN5 （SN5α, SN5β） . Methods Prediction of SN5 （SN5α ＋ SN5 β） structure was carried outby Geno 3D. Tudor-SN-SN5α/β gene fragments were amplified by PCR from the recombinant pSGS- Tudor-SN-flag plasmid. The SN5 β fragments were combined with pEGFP-C2 vector to form pEGFP- C2-SN513. The SN5ct fragments were then inserted into Tudor-SN-SN5β to form pEGFP-C2- SN5. Transfection of the recombinant pEGFP-C2-SN5β/SN5 plasmid was performed in HeLa cells. Expression of the fusion proteins was examined by fluorescent microscopy and Western blot. Results （1） Structural predication of spliced SN5 showed high resemblance to that in the TSN domain. （2） Double enzyme digestion displayed the expected SN5a, SN513, SN5 fragments. （3） GFP fluorescent was observed in HeLa cells after transfection. （4） The fusion protein pEGFP-C2-SNSβ/ SN5 was detected from the lysate of transfected HeLa cells by Western blot. Conclusion The re- combinant eukaryotic expression plasmid pEGFP-C2-SN5β/SN5 was successfully constructed to ex- press SN5 fragment fusion protein that can be detected by anti-GFP antibody.