【摘要】 目的用含有人Sy2酪氨酸磷酸酶基因的pOTB7质粒,构建Sy2原核表达重组子,并用该重组子转化原核细胞,表达出含Sy2的融合蛋白,为将来以此融合蛋白为抗原免疫Balb/c小鼠,制备单克隆抗体,进一步研究该基因的功能打好基础。方法用PCR扩增Sy2的PP2C结构域序列,限制性内切酶双酶切,构建到表达载体pET28a(+)中,酶切、测序等鉴定后,重组子pET28a(+)-Sy2转染原核细胞,用载体标签His抗体经SDS-PAGE、Western blot方法检测Sy2重组蛋白的表达。结果成功构建出pET28a(+)-Sy2重组质粒,Western blot方法检测出融合蛋白的表达。结论成功构建原核表达载体并在原核细胞中表达成包涵体,这种包涵体可以作为抗原制备Sy2的单克隆抗体。更多还原

【Abstract】 Objective To construct and express the recombination vector of pET28a(+)-Sy2(a novel Ser/Thr protein phosphatase,named Sy2) plasmid and ready to make Sy2 monoclonal antibody for study its biologic function. Methods Sy2 phosphatase domain cDNA was amplified by PCR,The fragment was digested and recombined into pET28a(+) vector.The prokaryotic vector transfected into BL21(DE3) cell line,and then induced its expression by IPTG.The production of inducing expression was identified by Blotting with anti-tag-his Ab.Results The recombination plasmid was constructed and the fusion protein was expressed successfully.Conclusions pET28a(+)-Sy2 plasmid,constructed as a prokaryotic expression vector,could express the Sy2 fusion protein.This fusion protein could be used as antigen to development monoclonal antibody against Sy2.It provided a powerful tool for the future study of biological function of a novel Sy2 phosphatase.更多还原