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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 :本实验可在大肠埃希菌体内表达可溶性的TRAIL胞膜外段融合蛋白
OBJECTIVE: To study the soluble fusion protein of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Escherichia coli and to lay the foundation for its further purification. Methods: According to the requirement of restriction endonuclease of pGEX 2T multi-cloning site in prokaryotic expression vector, TRAIL extracellular domain PCR primers were designed to amplify the TRAIL cloning vector. After the target fragment was recovered, TRAIL cell membrane was constructed by DNA ligase The outer segment prokaryotic expression vector, the application of the relevant biological software to predict the physical and chemical properties of the target protein and its solubility. After digestion and identification, the positive recombinant was transformed into Escherichia coli DH5α and analyzed by SDS PAGE under different concentrations of isopropylthiogalactopyranoside (IPTG), different induction temperature and different induction time Protein soluble expression level. Results: The DNA fragment of TRAIL extracellular domain was amplified by PCR. The result of restriction enzyme digestion confirmed that the prokaryotic expression vector of TRAIL extracellular domain was successfully constructed. The prediction showed that more than 95% of TRAIL extracellular domain fusion protein was expressed in soluble form , At 0 .1mmol / LIPTG, induced at 20 ℃ for 3 ~ 4h. The soluble protein reached the peak, accounting for 28% of the total bacterial protein. Conclusion: This experiment can express soluble TRAIL outer membrane fusion protein in Escherichia coli