一株新蛋白磷酸酶基因(Sy2)的原核表达载体构建及鉴定  

Construction and identification a prokaryotic expression vector of a novel protein phosphatase

作　　者：马广文[1] 陈兵[2] 鲁云霞[2] 李菲菲[3] 倪芳[3] 郑红[3] 汪思应[3] 

机构地区：[1]淮北市人民医院骨科,安徽淮北235000 [2]安徽医科大学基础医学院生化教研室,安徽合肥230032 [3]安徽医科大学基础医学院病理生理教研室,安徽合肥230032

出　　处：《疾病控制杂志》2006年第4期339-342,共4页Chinese Journal of Disease Control and Prevention

基　　金：国家自然科学基金(30572206);安徽省自然科学基金(00044202;No01043709);安徽省人才开发基金(2002Z035)

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。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.

关 键 词：蛋白磷酸酶 遗传载体 聚合酶链反应 

分 类 号：R342[医药卫生—基础医学]