日本血吸虫4个基因启动子相关序列的克隆和初步研究

Preliminary evaluation of promoter regions of four Schistosoma japonicum genes for expressing a luciferase reporter

目的比较4个日本血吸虫基因启动子相关序列对荧光素报告基因的表达情况。方法利用PCR技术扩增4个日本血吸虫基因启动子的相关序列,并利用常规分子生物学技术将PCR产物克隆到荧光素酶报告基因的上游。利用脂质体转染和电穿孔技术将纯化的重组质粒分别转染到HEK293细胞和日本血吸虫体内,并以荧光素酶检测系统测定了报告基因表达情况。结果PCR扩增获得了日本血吸虫卵壳蛋白(Eggshell protein,ESG-2A)、延伸因子1(Elongation factor 1 alpha 1,EF)、烯醇酶(Enolase,EN)和抱雌沟蛋白(Gynecophornal canal protein,GCP)基因的启动子相关序列,并将它们成功地克隆到pGlu-Basic载体。重组质粒转染表明4个日本血吸虫启动子相关序列均可驱动荧光素酶报告基因在HEK293细胞和日本血吸虫虫体表达。其中含有GCP和EN启动子相关序列的重组质粒可在HEK293细胞及其培养基中检测到较高的荧光素酶活性,含有GCP和ESG的重组质粒可在日本血吸虫虫体培养基中检测到较高的荧光素酶活性。结论获得的4个日本血吸虫启动子相关序列均可驱动荧光素酶报告基因在HEK293细胞和日本血吸虫虫体表达,为进一步利用这些启动子相关序列开展血吸虫基因操作研究奠定了初步基础。

Objective To evaluate the relevant promoter regions of four Schistosoma japonicum genes for expressing a lucifer ase reporter.Methods The polymerase chain reaction（PCR）was used to amplify the promoter regions of four S.japonicum genes and then each PCR product was cloned into a pGlu-Basic vector according to the standard molecular procedures.These recombi nant plasmids were either transfected into human HEK293 cells by using lipofectamine or introduced into cultured schistosomes by electroporation.Then,the luciferase activities were measured by using a dual luciferase reporter system in a luminometer.Re sults Each promoter region of four S.japonicum genes was obtained and the corresponding recombinant vector containing the pro moter region was successfully constructed.The transfection of the recombinant plasmids into the human HEK293 cells and cul tured schistosomes resulted in a significant elevation of the luciferase reporter activity.Conclusions The promoter regions of four S.japonicum genes are obtained and the luciferase reporter genes driven by the four promoter regions are preliminarily evaluated.The study provides a foundation for the usage of these promoters for genetic manipulation in S.japonicum.