金黄色葡萄球菌肠毒素C基因的克隆与原核表达  

Cloning and prokaryotic expression of Staphylococcal enterotoxin C

作　　者：江学斌[1] 成雪鸿 马苗鹏[2] JIANG Xuebin;CHENG Xuehong;MA Miaopeng(School of Life Science,Guangzhou University,Guangzhou 510006;Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms,College of Life Sciences,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]广州大学生命科学学院,广东广州510006 [2]华南农业大学生命科学学院,广东省农业生物发育与环境适应重点实验室,广东广州510642

出　　处：《生物技术》2025年第1期1-5,49,共6页Biotechnology

摘　　要：[目的]制备金黄色葡萄球菌肠毒素C。[方法]利用PCR技术从金黄色葡萄球菌的基因组中克隆肠毒素C(SEC)基因,PCR产物与pET-28a(+)载体连接,构建表达SEC的原核表达载体(pET-28a(+)-SEC),转入大肠杆菌BL21(DE3)中进行诱导表达,并利用Ni-NTA纯化SEC目的蛋白。[结果]成功克隆了SEC基因,构建了SEC的原核表达载体pET-28a(+)-SEC,并在大肠杆菌BL21(DE3)中被成功表达,在不同浓度IPTG(0.4 mmol/L、1 mmol/L)、不同温度(30℃、37℃)下,SEC蛋白表达量相近,占菌体蛋白总量的50%以上;重组菌株在1 mmol/L IPTG诱导物37℃诱导6 h,可溶性的SEC目的蛋白占SEC蛋白总表达量的30%以上;可溶性的SEC经Ni-NTA亲和层析纯化,纯度近90%。[结论]经克隆、表达和纯化的较高纯度的SEC蛋白为SEC蛋白来源提供一种新选择。[Objective]Prepare Enterotoxin C from Staphylococcus aureus.[Method]The Staphylococcal enterotoxin C(SEC)gene was cloned from the genomic DNA of Staphylococcus aureus by PCR and ligated with the pET-28a(+)vector.The expression vector pET-28a(+)-SEC was transferred into E.coli BL21(DE3)for expression.The target protein SEC was purified by Ni-NTA.[Result]The SEC gene was successfully cloned,the prokaryotic expression vector of SEC was constructed and successfully expressed in E.coli BL21(DE3).The expression of SEC protein was similar at different concentrations of IPTG(0.4 mmol/L or 1 mmol/L)and different temperatures(30℃or 37℃),accounting for more than 50%of the total bacterial protein.When the recombinant strain was induced at 1 mmol/L IPTG at 37℃for 6 hours,the soluble SEC target protein accounted for more than 30%of the total expression of SEC protein.Soluble SEC protein was purified by Ni-NTA with a purity of nearly 90%.[Conclusion]The recombinant SEC protein with higher purity was obtained by cloning,expression and purification,which provided a new choice for the source of SEC protein.

关 键 词：金黄色葡萄球菌 肠毒素C 基因克隆 原核表达 可溶性蛋白 蛋白纯化 

分 类 号：Q786[生物学—分子生物学]