Thanatin突变体在大肠杆菌中的表达及纯化-东南大学学报医学版

Thanatin突变体在大肠杆菌中的表达及纯化

单位：1.中国药科大学,生物技术中心,江苏,南京,210009； 2.东南大学附属中大医院,检验科,江苏,南京,210009

分类号：Q786

出版年·卷·期（页码）：2007·26·第三期（220-224）

摘要：

目的:在大肠杆菌中克隆表达thanatin突变体(Th-T)蛋白并纯化. 方法:PCR合成Th-T基因并克隆到pET32a载体的BamHⅠ和XhoⅠ酶切位点之间,构建Th-T原核表达载体(pET32a-Th-T)并转化到大肠杆菌BL21融合表达,通过正交实验优化表达条件,His-Bind介质纯化.考察经酶切纯化后的重组Th-T对4种供试菌的最低抑制浓度.结果:菌体在LB培养基(pH 6.5)培养4.5 h,IPTG 0.4 mmol·L-1诱导7 h,Th-T融合蛋白大量可溶性表达,经药敏试验证明纯化的重组Th-T具有预期的抗菌活性.结论:本研究为通过大肠杆菌体系表达重组Th-T抗菌药物奠定了基础.

Objective To clone and express thanatin mutant(ThT) in Escherichia coli(E.coli) and purify it.Methods Th-T cDNA synthesized by PCR was inserted into the BamHⅠ-XhoⅠsite of pET32a to construct Th-T prokaryotic expression vector(pET32a-Th-T) in E.coli.The vector was transformed into BL21 to express fusion protein.Through orthogonal experimental design,the condition of expression was optimized,and fusion protein was purified with His-Bind resin.After enzyme digestion,the minimal inhibitory concentration of recombinant Th-T was tested in four kinds of bacteria.Results The bacteria were induced with 0.4 mmol·L-1 IPTG for 7 hours to express fusion protein after cultivating in LB culture for 4.5 hours.Through chemosensitivity,recombinant Th-T was expected to have antimicrobial activities.Conclusion The study provides a foundation for future development of Th-T as antimicrobial medicines.

参考文献：

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