重组hGM-CSF乳酸链球菌的构建及初步验证  

Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor

作　　者：马高峰[1] 陈学清[1] 杨晓强[1] 武金宝[1] 张振书[1] 

机构地区：[1]南方医科大学南方医院消化内科,广东广州510515

出　　处：《南方医科大学学报》2008年第4期576-578,共3页Journal of Southern Medical University

基　　金：国家自然科学基金(30570839);广东省自然科学基金(05004735)

摘　　要：目的运用基因克隆技术将自行合成的人粒-巨噬细胞集落刺激因子(hGM-CSF)转化于乳酸链球菌,并筛选获得高表达重组hGM-CSF的乳酸链球菌稳定株。方法将经过优化适合在乳链菌表达的hGM-CSF基因克隆于含有P59启动子、USP45蛋白信号肽、终止密码子的pNBC1000载体,得到重组质粒pNCSF,然后将pNCSF进一步克隆于穿梭载体pTR1001c,获得乳链菌表达载体pTRCSF,通过电穿孔转化于乳链菌,并通过聚丙烯酰胺凝胶蛋白电泳验证hGM-CSF的表达。结果获得了重组质粒pNCSF并经酶切鉴定和测序证实,酶切鉴定显示构建乳链菌表达载体pTRCSF及重组hGM-CSF乳链菌成功,蛋白电泳初步验证获得了高表达重组hGM-CSF的乳链菌株LL-CSF。结论运用基因克隆技术成功构建了重组乳酸链球菌LL-CSF,为进一步研究重组hGM-CSF的生物学特性及评价其潜在临床应用价值打下了基础。Objective To transfer human granulocyte-macrophage colony stimulating factor （hGM-CSF） gene into A ctococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF （LL-CSF）. Methods The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF, pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF. Results DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE. Conelusion The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.

关 键 词：粒-巨噬细胞集落刺激因子 乳酸链球菌 基因克隆 重组质粒 蛋白表达 

分 类 号：R346[医药卫生—基础医学]