【作者】 郭瑞娟；

【导师】 李淑红；

【作者基本信息】 吉林大学 ， 病原生物学， 2007， 硕士

【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。更多还原

【Abstract】 Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. The patients run a six-fold higher risk of HIV infection. Trichomonas vaginalis virus(TVV),is a kind of ds RNA virus parasitized in Trichomonas vaginalis,the prior research shows that the existence of TVV has relationship with the invasiveness and Metronidazole resistance of Trichomonas vaginalis. the discovery of Trichomonas vaginalis virus(TVV) shows a new way for T.vaginalis study in molecular biology. At present, the vector system reconstructed from GLV has been used in several area ,for example : expression of exogenous gene, structure and function analysis of G.. lamblia gene. It has been a significant tool for the study of G.. lamblia in molecular biology.While the study about TVV vector is relatively lagged. Chimera of TVV cDNA and enhanced green fluorescent protein (EGFP) were constructed and their in vitro transcript were electroporated into TVV-infected T. vaginalis trophozoites, the control signals of the TVV genome required for expression of foreign gene were preliminary determined according to the expression level of EGFP in the transfected parasites. This transfection system should provide a valuable tool for genetic study of T. vaginalis.Construction of GCV Transfer Vector According to feature of T. vaginalis virus (TVV) genome in China,a system was developed here for expression of a foreign gene in this organism by flanking the enhanced green fluorescent protein(EGFP) gene with the fragments of TVV positive-strand RNA . Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS were constructed.Expression of EGFP mediated by TVV Transcript of the Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS were synthesized in vitro with T7 RNA polymerase and used to transfect T. vaginalistrophozoites already infected with TVV. The results indicated that chimera pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS appeared fluorescent signal under fluorescence microscope at 488nm, and without attenuating after the fifth passage .We detected the EGFP mRNA by RT-PCR in transfected T. vaginalis and the results of SDS-PAGE demonstrated that EGFP was expressed in T. vaginalis and mainly secreted to medium supernatant. while chimera pTVVL289/EGFP did not appear fluorescent signal under fluorescence microscope, it shows that the 3’UTRs of TVV play a part in replication,translation and/or package of virus particle.更多还原