摘要
目的 :研究在大肠埃希菌体内可溶性表达肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)胞膜外段融合蛋白 ,为其下一步的分离纯化奠定基础。方法 :依据原核表达载体 pGEX 2T多克隆位点限制性内切酶要求 ,设计TRAIL胞膜外段PCR引物 ,对TRAIL的克隆载体进行PCR扩增 ,回收目的片段后利用DNA连接酶构建TRAIL胞膜外段原核表达载体 ,应用相关的生物学软件对目的蛋白的理化性质及其溶解性进行预测。酶切鉴定后将阳性重组体转入大肠埃希菌DH5α体内 ,分别在不同浓度异丙基硫代 β D 半乳糖苷 (IPTG)、不同诱导温度、不同诱导时间作用下 ,利用SDS PAGE分析目的蛋白可溶性表达量。结果 :PCR扩增得到TRAIL胞膜外段DNA片段 ,酶切结果证实已成功构建了TRAIL胞膜外段原核表达载体 ,预测表明 95 %以上的TRAIL胞膜外段融合蛋白是以可溶性的形式表达 ,在 0 .1mmol/LIPTG、2 0℃诱导 3～ 4h ,目的蛋白可溶性表达量达峰值 ,占菌体蛋白的 2 8%。结论
Objective:To investigate soluble expression of the extracellular region of the TNF related apoptosis induced ligand(TRAIL) protein in E.coli .Methods:According to expression vector pGEX 2T,we designed a pair of primers with recognized sites by BamHI and EcoRI for amplifying the extracellular region of TRAIL gene.After amplified by PCR,the destination fragment reclaimed was linked into the expression vector pGEX 2T,which digested by BamHI and EcoRI .Meanwhile,we predicted the physical and chemical property and the dissolution of the destination protein.After identified with digested by the same endonuclease,the recombination vector was transferred into the DH5α,the positive recombination E.coli were induced by different concentration IPTG at different temperature on different period of time.The expression products were analysed by 12% SDS PAGE.Results:The extracellular region of TRAIL gene was obtained by PCR,we successfully constructed expression vector pGEX/TRAILex and got the positive recombination E.coli .The prediction results proved that the fusion protein is soluble.We found that the destination protein was at the leak of expression in E.coli under the condition of that the recombination bacterium was induced by 0.1 mmol/L IPTG at 20℃ for 3 to 4 hours.Conclusions:We obtained soluble expression of the extracellular region of the TRAIL fusion protein with GST which expressed in E.coli in our study.
出处
《蚌埠医学院学报》
CAS
2003年第2期98-101,共4页
Journal of Bengbu Medical College
基金
安徽省教育厅自然科学研究资助项目 ( 2 0 0 2KJ2 0 7)
