阴道毛滴虫病毒转染载体的构建及EGFP在阴道毛滴虫内的表达

发布时间：2018-02-12 06:16

本文关键词： 阴道毛滴虫阴道毛滴虫病毒病毒载体 EGFP　出处：《吉林大学》2007年硕士论文　论文类型：学位论文

【摘要】： 本研究根据我国阴道毛滴虫病毒基因组的序列特征,借鉴以往原虫病毒载体构建的研究经验,用绿色荧光蛋白编码基因替换TVV全部或部分编码区,构建阴道毛滴虫病毒转染载体pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS。阴道毛滴虫病毒转染载体经T7RNA聚合酶体外转录成mRNA后,经电穿孔法转染含TVV的阴道毛滴虫细胞内,用荧光显微镜观察转染后不同时间内EGFP的表达情况。结果表明:载体pTVVL289/EGFP的体外转录体转染阴道毛滴虫后未观察到荧光信号,表明在TVV的3’末端UTR存在有与病毒复制和/或包装有关的序列。载体pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS体外转录体转染后可检测到绿色荧光信号,并在持续传代培养5代后仍不减弱;提取转染后虫体的总核酸,用RT-PCR检测到EGFP的mRNA的存在;SDS-PAGE分析转染虫体的培养上清和虫体中EGFP的表达,检测到大小为27KD的蛋白。说明我们构建的阴道毛滴虫病毒载体可以应用于介导外源基因在阴道毛滴虫体内的表达。本研究构建了阴道毛滴虫病毒转染载体,介导了目的基因EGFP在阴道毛滴虫体内的表达,为深入研究我国阴道毛滴虫病毒与虫体及其宿主之间的关系提供了新的研究工具,为进一步研究阴道毛滴虫的分子生物学特性奠定基础。
[Abstract]:According to the sequence characteristics of trichomonas vaginalis virus genome and the previous experience of protozoa virus vector construction, the whole or part of TVV coding region was replaced by green fluorescent protein encoding gene. The vector pTVVL289 / EGFPN pTVVL289 / EGTV VL559 / EGFP / TVVS was constructed to construct the vector pTVVL289 pTVVL559 / EGFP / TVVS. Trichomonas vaginalis virus transfection vector was transcribed into mRNA by T7 RNA polymerase in vitro, and transfected into Trichomonas vaginalis cells containing TVV by electroporation. Fluorescence microscope was used to observe the expression of EGFP in different time after transfection. The results showed that no fluorescence signal was observed after transfection of the vector pTVVL289/EGFP transcript into Trichomonas vaginalis. The results showed that there was a sequence related to viral replication and / or packaging in the 3'terminal UTR of TVV. The vector pTVVL289 / EGFP / TVVSN pTVVL559 / EGFP / TVVS could detect green fluorescent signal after transfection, and the green fluorescence signal was not weakened after 5 generations of continuous passage culture. The total nucleic acid of the transfected insect was extracted. The presence of mRNA of EGFP was detected by RT-PCR. The culture supernatant of the transfected insect and the expression of EGFP in the transfected insect were analyzed by SDS-PAGE. A 27KD protein was detected, indicating that the constructed Trichomonas vaginalis virus vector could be used to mediate the expression of foreign genes in Trichomonas vaginalis. In this study, the transfection vector of trichomonas vaginalis virus was constructed, and the expression of the target gene EGFP in Trichomonas vaginalis was mediated, which provided a new tool for further study on the relationship between trichomonas vaginalis virus and trichomonas vaginalis virus and its host. It will lay a foundation for further studying the molecular biological characteristics of Trichomonas vaginalis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

【引证文献】