摘要
目的 利用蛋白自剪切功能原核表达系统构建肿瘤坏死因子相关凋亡诱导配体（TRAIL）胞膜外段融合蛋白表达载体，并进行表达条件优化和初步纯化。方法 设计基因拼接引物，合成TRAIL基因胞膜外段双链cDNA序列，将其连接于克隆载体pMD18-T中；将酶切、纯化的TRAIL基因与经相同处理的载体pTWIN1相连接，转化感受态大肠杆菌JM109，筛选阳性重组子。将鉴定（酶切及序列分析）阳性的重组子质粒转人大肠埃希菌ER2566表达菌中，采用蛋白质SDS—PAGE电泳方法分析不同浓度诱导剂异丙基硫代-β-D-半乳糖苷（IPTG）、不同诱导温度、不同诱导时间下目的蛋白表达情况。结果 成功获得了包含TRAIL基因胞膜外段的双链cDNA序列，酶切及序列分析证实成功构建了TRAIL胞膜外段蛋白自剪切功能原核表达载体。采用0．3mmol／LIPTG、15℃诱导过夜（14～16h），目的蛋白获得较高表达量，且大部分为可溶性蛋白。结论 采用大肠埃希氏菌ER2566，TRAIL胞膜外段融合蛋白获得高表达，经简单后续处理即可获得不含任何额外氨基酸的可溶性目的蛋白。
Objective To purify an active extraeellular region of the TNF related apoptosis inducing ligand （TRAIL） protein. Methods According to the high-usage codons in Escherichia coli and the multiple cloning site of expression vector pTWIN1 of a self-splicing prokaryotic expression system, the extracellular region of TRAIL gene was designed and synthesized, which was cloned into pMD18-T vector. After pMD18-T/TRAIL and pTWIN1 were digested by Nru I and EcoR I . the target fragment purified was linked to the expression vector pTWIN1, which was transferred into the competent cell JM109, and positive recombination was screened. After positive recombination vector pTWIN1/TRAIL （identified with restrictive endonuclease digesting and sequencing） was transferred into the ER2566. the expression was induced by different IPTG concentration at different temperature and culture time. The expression products were analyzed by 150 g/L SDS-PAGE. Results The extraeellular region of TRAIL gene was obtained by PCR, and was constructed successfully in a self-splicing prokaryotic expression vector pTWIN1/TRAIL. By 0.3 mmol/L IPTG at 15 ℃ for 14 to 16 hours, the soluble target protein was expressed efficiently. Conclusion High-expression level of the extracellular region of TRAIL fusion protein was attained by use of E. coli ER2566, and the soluble target protein without any additional amino acid was successfully purified by simple treatment.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第5期670-674,678,共6页
Journal of Sichuan University(Medical Sciences)
