重组Trail蛋白在大肠杆菌中的表达及纯化条件的优化  

Expression and purification of recombinant Trail protein in E.coli and the optimal conditions for Trail purification

作　　者：蔡媛媛[1] 李雪燕[1] 吴玉[1] 杜晶春[1] 徐霞[1] 

机构地区：[1]广州医学院检验系,广东广州510182

出　　处：《细胞与分子免疫学杂志》2012年第6期596-600,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：第三轮广州市属高校重点学科建设经费资助(穗教高教[2011]31号)

摘　　要：目的:构建肿瘤坏死因子相关凋亡配体胞外功能区的原核表达质粒,优化诱导蛋白表达的相关条件,检测切胶纯化所得蛋白的抗原结合活性,以及亲和层析纯化蛋白的促凋亡功能。方法:扩增Trail胞外功能区第114-281个氨基酸基因序列,插入融合表达载体pET-28α(+)的多克隆位点,构建重组表达质粒pET-28α(+)-Trial114-281。以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组子,调整诱导前菌群的密度(A600)值,诱导时IPTG的浓度、温度及诱导时间,确定最佳诱导条件,同时比较超声、渗透冲击和IP裂解三种破碎细菌方法以及切胶回收和Ni-NTA亲和层析方法对纯化目的蛋白的影响。Western blot鉴定切胶纯化蛋白的抗原结合活性,用Ni-NTA亲和层析方法得到的蛋白作用于A549细胞,采用流式细胞术检测检测该细胞的凋亡率。结果:成功扩增了Trail胞外区基因序列,经测序证实其正确插入到表达载体pET-28α(+)中,经IPTG诱导,在37℃时呈包涵体表达,25℃时为可溶性表达,经软件分析确定A600=0.6,IPTG终浓度为0.6 mmol/L,诱导4 h为包涵体表达的最佳条件;A600=1.0,IPTG 1.0 mmol/L,诱导4 h为可溶性目的蛋白表达的最佳诱导条件。三种破菌方法的比较,超声法获得的蛋白最多。切胶和Ni-NTA亲和柱纯化的方法都得到了目的蛋白,Westernblot分析显示,切胶纯化的蛋白有较好的抗原结合活性,亲和层析得到的可溶性蛋白可以促使A549细胞发生凋亡。结论:成功构建了重组表达载体pET28α-Trial114-281,在A600值、IPTG以及诱导时间都相同的情况下,37℃出现包涵体表达,而25℃则出现了可溶性表达。切胶纯化获得蛋白有较好的抗原结合活性,亲和层析纯化的可溶性蛋白能保持其原有的功能不被破坏,促使肿瘤细胞A549的凋亡。AIM： To construct the expression vector pET-28α-Trail114-281 and find the optimal conditions for target gene expression, host bacteria lysis, and protein purification, and to detect the apoptosis function of the recombinant protein. METHODS： The functional domain of Trail,4281 was amplified by PCR and cloned into the expression vector pET-28c（（＋）. After confirmed by DNA sequencing, the Trail.4 281 was expressed in E. coil BL21 under the condition of different Am, IPTG concentration and temperature. Host bacteria were lysed using three different ways, including ultrasonication, osmotic shock and IP lysis, and the target protein was purified using Ni-NTA affinity chromatography or cutting-gel purification. The advantages and shortcomings of these methods were compared to find the most efficient ways for expression and purification of the recombinant pro- tein. The immunocompetence of Trail protein from cuttinggel purification was analyzed by Western blotting, A549 cell apoptosis induced by purified protein from Ni-NTA chromatography was detected by flow cytometry. RESULTS： The 516 bp Trai114-281 gene was cloned, and expressed in E. coil BL21. When A600= 0. 6, recombinant host bacteria were induced by 1.0 mmol/L IPTG at 3700 for 4 h, which was the optimal condition for the expression of inclusion body, and the soluble protein was expressed stably on the condition of 25℃, A600 = 1.0, IPTG1.0 mmol/L. Ultrasonication could get maximal protein compared to the other methods. The two purification ways both could purify taget protein successfully. Western blot analysis showed that the protein purified by cutting-gel has a good immunologic activity. Protein from Ni-NTA affinity chromatography caused cell apoptosis. CONCLUSION： The expression vector pET-28α-Trai114-281 can be constructed and expressed in E. coil BL2I success- fully. Temperature is a more important effect factor of Trai114-281 expression in host bacteria compared with other factors. Cutting-gel protein has immunogenicity, and Ni-NTA protei

关 键 词：pET-28α-Trail114-281 优化条件 目的蛋白 纯化 

分 类 号：Q78[生物学—分子生物学]