论文部分内容阅读
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 compare the expression of luciferase reporter gene with four related sequences of Schistosoma japonicum gene promoter. Methods PCR-based amplification of four related genes of Schistosoma japonicum gene promoter and PCR products were cloned into the upstream of the luciferase reporter gene using conventional molecular biology techniques. The recombinant plasmids were transfected into HEK293 cells and Schistosoma japonicum respectively by liposome transfection and electroporation. The expression of reporter gene was detected by luciferase assay. Results The results of PCR amplification showed that Eggshell protein (ESG-2A), Elongation factor 1 alpha 1 (EF), Enolase (EN) and Gynecophornal canal protein , GCP) genes and successfully cloned them into pGlu-Basic vector. Recombinant plasmid transfection showed that all four Schistosoma japonicum promoter-related sequences could drive luciferase reporter gene expression in HEK293 cells and Schistosoma japonicum. The recombinant plasmids containing GCP and EN promoter related sequences could detect higher luciferase activity in HEK293 cells and their culture medium. The recombinant plasmids containing GCP and ESG could be detected in Schistosoma japonicum High luciferase activity. Conclusions All the four sequences of Schistosoma japonicum obtained from the promoter can drive the expression of luciferase reporter gene in HEK293 cells and Schistosoma japonicum and laid the preliminary foundation for the further study on the gene manipulation of Schistosoma japonicum by using the related sequences of these promoters.