贝氏柯克斯体穿梭载体构建及转化方法建立  

Shuttle Vector Construction and Transformation of Coxiella burnetii

作　　者：罗声栋[1] 卢姗姗[2] 胡燕[2] 王涛[1] 于永慧[1] 冯乐[1] 孙志会[1] 宋立华[1] 

机构地区：[1]病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所,北京100071 [2]解放军第302医院临床研究管理中心,北京100039

出　　处：《生物技术通讯》2016年第5期625-628,共4页Letters in Biotechnology

基　　金：病原微生物生物安全国家重点实验室自主课题(SKLPBS1409)

摘　　要：目的:基于大肠杆菌质粒pMMB207,构建贝氏柯克斯体穿梭载体,建立贝氏柯克斯体转化系统。方法:利用PCR分别扩增eGFP基因、Kan^R抗性基因、贝氏柯克斯体组成型表达启动子P311和P1169等4段序列,然后用融合PCR技术将4段序列融合为P311-eGFP-P1169-Kan^R串联序列,经KpnⅠ和XhoⅠ酶切后,连接至经同样双酶切的pMMB207骨架上,构建穿梭载体p207-GK;将穿梭载体电转化至贝氏柯克斯体,用ACCM-2培养基进行传代培养或感染BGM细胞;利用倒置荧光显微镜观察eGFP基因的表达,传代至eGFP基因高表达时,用半固体平板培养法克隆分纯贝氏柯克斯体转化株。结果:构建了贝氏柯克斯体穿梭载体,获得了稳定表达eGFP的贝氏柯克斯体转化株,并完成了克隆分纯。结论:建立了完整的贝氏柯克斯体转化系统,为后续用遗传学方法研究贝氏柯克斯体奠定了基础。Objective： To construct a plasmid pMMB207-based shuttle vector and to establish a transformationsystem of Coxiella burnetii. Methods： Four gene fragments, eGFP gene, Kan^Rgene, and promoters of two constitu-tively expressed genes CBU0311 and CBU1169, were amplified by PCR. Overlapping PCR was then used to pro-duce a concatenated DNA fragment, P311-eGFP-P1169-Kan^R. The concatenated fragment and the p MMB207 back-bone were digested with KpnⅠ and XhoⅠ, and were then ligated to construct shuttle vector p207-GK. The vectorp207-GK was used to transform C.burnetii by electroporation. Transformed C.burnetii was cultured in ACCM-2 me-dia or on BGM cells. Semi-solid plating was used to clone transformed C.burnetii when high eGFP expression wasobserved under fluorescence microscopy. Results： Shuttle vector p207-GK was constructed and was transformed in-to C.burnetii. Stable transformants were cloned by semi-solid plating. Conclusion： We successfully established Coxiella burnetii transformation system, which laid a foundation for future genetic studies of C.burnetii.

关 键 词：贝氏柯克斯体 Q热 穿梭载体 转化 RSF1010质粒 

分 类 号：Q78[生物学—分子生物学]