人PTP1B真核载体在COS-7细胞中的表达及其体外抑制剂实验  

Human PTP1B expressed in the COS-7 cell and inhibitors experiment in vitro

作　　者：张吕钊[1] 鲁云霞[2] 李俊[1] 

机构地区：[1]安徽医科大学药学院,合肥230032 [2]安徽医科大学生化教研室,合肥230032

出　　处：《安徽医科大学学报》2007年第5期477-481,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省教育厅自然科学基金资助项目(编号:2006kj371B)

摘　　要：目的构建人蛋白酪氨酸磷酸酶1B(PTP1B)真核表达载体pcDNA3.1-hPTP1B并在猴肾成纤维细胞COS-7中稳定表达,分离纯化表达的重组融合蛋白,进行酶活性测定及抑制剂的初步实验。方法应用RT-PCR从2型糖尿病患者的外周血总RNA中扩增出PTP1BcDNA全长序列,并在基因上下游分别引入BamHⅠ、EcoRⅠ两个酶切位点,酶切后定向导入pcDNA3.1/Hismyc-B多克隆位点,构建真核表达载体pcDNA3.1-hPTP1B,转染COS-7细胞,G-418筛选2周后取细胞裂解上清液经Ni2+亲和层析柱纯化后,测定了其酶活性及小分子抑制剂钒酸钠的IC50。结果从2型糖尿病患者外周血中扩增得到1301bpPTP1BcDNA全长序列,经双酶切鉴定及测序分析,表明hPTP1B已克隆到pcD-NA3.1/Hismyc-B中。RT-PCR和免疫印迹表明转染成功。酶活性实验初步显示rhPTP1B的活性随酶浓度增加而增加,随PTP1B的特异性抑制剂钒酸钠的浓度增加而减少。结论获得具有酶活性的融合蛋白rhPTP1B,为进一步筛选其特异性抑制剂奠定基础。Objective To clone hPTP1B cDNA into the pcDNA3. 1/Hismyc-B vetor and construct pcDNA3. 1- hPTP1 B, transfect the recombinant plasmid-hPTP1B into the COS-7 eukaryotic system, to test the enzyme activity of the expressed fusion protein and Na3VO4 inhibition. Methods PTP1B full length cDNA was amplified using RT- PCR from the total RNA of the peropheral blood of the T2DM patient（ BMI 〉 28 kg/m^-2） ,then subcloned into pcDNA3.1/Hismyc-B, transfected into the COS-7 cells. After the two weeks screening with 400 mg/L G-418, we got the purified recombinant PTP1 B through the Ni^2＋ affinity chromatography and tested the processed protein with the enzyme activity and the IC50 of PTP1B specific inhibitor. Results The whole sequence PTP1 B was obtained and testified by DNA sequencing and BamH I and EcoR I digestion. RT - PCR and Western blotting showed the suc -cessful transfection into COS-7 cells. Enzyme activity test showed the PTP1 B activity enhanced with the increase of the enzyme concentration, and decreased with the increase concentration of a specifiec inhibitor （Na3VO4）. Conclusion The construction and eukaryotic expression of the pcDNA3. 1-rhPTP1 B has been successfully established, and the purified rhPTP1 B fusion protein could be used for the inhibitor research

关 键 词：蛋白质酪氨酸磷酸酶/拮抗剂和抑制剂 COS细胞 基因表达 遗传载体 

分 类 号：R345.61[医药卫生—基础医学] R329.24