山梨糖脱氢酶在脱氮副球菌中的表达  

Expression of Sorbose Dehydrogenase in Paracoccus denitrificans

作　　者：朱斌[1,2] 熊向华[1] 汪建华[1] 何建勇[2] 张惟材[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学,辽宁沈阳110016

出　　处：《生物技术通讯》2012年第2期204-206,共3页Letters in Biotechnology

基　　金：国家自然科学基金(31100024);国家高技术研究发展计划(2006AA020303);国家科技支撑计划(2007BAI46B01)

摘　　要：目的:在脱氮副球菌PD1222中表达山梨糖脱氢酶(SDH)。方法:从质粒pMD-18T上复制氨苄西林抗性基因Ampr,从酮古龙酸菌中复制SDH基因sdh,先后酶切连接到pIND4质粒上,构建pIND4-Ampr-sdh穿梭质粒;再把pIND4-Ampr-sdh电转入大肠杆菌S17-1λpir作为供体菌,脱氮副球菌PD1222为受体菌进行双亲本接合转移;挑取壮观霉素和氨苄西林双抗平板上的接合子进行培养,菌液PCR复筛接合子,测序鉴定,通过DCIP法和非变性聚丙烯酰胺凝胶电泳法检测阳性克隆的SDH活性。结果:构建的质粒pIND4-Ampr-sdh成功转入脱氮副球菌PD1222中,SDH获得表达并检测到其蛋白活性。结论:实现了SDH在脱氮副球菌中的表达,为在脱氮副球菌中研究SDH的下游电子传递链奠定了基础。Objective： To express the sorbose dehydrogenase（SDH） in Paracoccus denitrificans. Methods： The plasmid plND4-Ampr-sdh was constructed by ligating the Ampr gene cloned from the pMD18-T plasmid and the sdh gene from Ketogulonigenium vulgare. The plasmid plND4-Ampr-sdh was electroporated into E.coli S17-1pir as the donor strain, then it was transferred into P.denitrificans strain PD1222 by diparental conjugal transfer. The transformed clone was identified by PCR and plasmid retransformation after which the inserted fragment was se- quenced.The SDH activity in PD1222 was detected by DCIP and native electrophoresis. Results： The pIND4-Am- pr-sdh plasmid was successfully constructed and tranferred into PD1222. The transformed clone was proved to be positive in SDH activity by DCIP and active electrophoresis. Conclusion： The SDH was successfully expression in PD1222, which will be the foundation for the further research of the electron transport chain of the SDH in P.denitrificans.

关 键 词：山梨糖脱氢酶 脱氮副球菌 接合转移 酮古龙酸菌 

分 类 号：Q78[生物学—分子生物学]