人血管性血友病因子裂解蛋白酶pEGFP-N1真核表达载体的构建及其在HeLa细胞中的表达

摘要

This study was aimed to construct a pEGFP-Nl vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studing its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion? High-Fidelity(NEB) ,then the PCR product was double digested with EcoR I and Xho I . After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-Nl vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-Nl vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.%本研究旨在构建人血管性血友病因子裂解蛋白酶(ADAMTS13)的pEGFP-N1真核表达载体,为进一步研究ADAMTS13在细胞内合成及其分泌提供有力工具.通过PCR方法获取目的基因片段,并在目的基因两端加上限制性酶切位点.限制性内切酶酶切后,连接至pEGPF-N1真核表达载体.连接后获得质粒进行酶切鉴定及DNA测序验证,并转染HeLa细胞.通过荧光显微镜检测绿色荧光蛋白表达,Western blot方法鉴定所得蛋白.结果表明,酶切鉴定及DNA测序确认目的基因与载体正确连接,在荧光显微镜下观察到绿色荧光.Western blot显示,转染后HeLa细胞表达ADAMTS13蛋白.结论:成功构建了ADAMTS13-pEGFP-N1真核表达载体,为进一步研究ADAMTS13合成、分泌及其代谢的生理学机制提供了研究工具.