人胸腺素原α的克隆、表达和活性初步研究  

cDNA cloning and expression of human ProTα in E.coli

作　　者：王栋[1] 郭满盈[1] 邱磊[1] 吕岩[1] 郭葆玉[1] 

机构地区：[1]第二军医大学药学院生化药学教研室,上海200438

出　　处：《第二军医大学学报》2005年第4期415-417,共3页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30371349).

摘　　要：目的:克隆人胸腺素原αcDNA并在大肠杆菌系统内表达。方法:利用RT- PCR技术从健康男性外周血PBMC中扩增胸腺素原αcDNA,纯化后用EcoRⅠ和SalⅠ双酶切PCR 产物,然后将该基因插入经同样双酶切的原核表达载体PBV220中,在PLPR 启动子控制下,热诱导天然表达胸腺素原α,表达产物部分纯化后利用淋巴细胞玫瑰花结实验进行了初步的活性测定。结果:凝胶电泳显示PCR扩增产物的长度为300 bp左右,重组质粒测序结果表明,插入片段与人胸腺素原α的序列完全一致,DE3重组菌在热诱导下高效表达相对分子质量为12 000 的蛋白,该蛋白能提高淋巴细胞玫瑰花结形成率,表明具有一定活性。结论:成功克隆人胸腺素原α基因并在大肠杆菌中得到表达。Objective:To clone and prokaryotically express human prothymosin α(ProTα) gene. Methods: The encoding sequence of human ProTα was amplified from male PBMC total RNA by RT-PCR.The product of RT-PCR was purified and digested with EcoRⅠ/SalⅠand then was inserted into the prokaryotical expression vector PBV220.The expression of protein ProTα was induced by heat and the product was purified partially. The biological activity of rhProTα was assayed using T cell E-rosette formation test. Results: The PCR product was about 300 bp in size,which was consistent with the expected size of ProTα.The sequence of insert corresponded with the published encoding sequence of human ProTα. Plasmid PBV220-ProTα was transformed into E.coli DE3 and a new protein with a relative molecular weight of 12 000 was induced by heat,which could increase the E-rosette formation rate of human peripheral mononuclear cells. Conclusion: The encoding sequence of human ProTα gene is successfully cloned into the prokaryotical expression vector PBV220 and can be expressed efficiently in E.coli. [

关 键 词：胸腺素原α 原核表达 基因克隆 

分 类 号：Q785[生物学—分子生物学]