發(fā)布時(shí)間：2018-02-12 06:16

  本文關(guān)鍵詞： 陰道毛滴蟲 陰道毛滴蟲病毒 病毒載體 EGFP　出處：《吉林大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 本研究根據(jù)我國(guó)陰道毛滴蟲病毒基因組的序列特征,借鑒以往原蟲病毒載體構(gòu)建的研究經(jīng)驗(yàn),用綠色熒光蛋白編碼基因替換TVV全部或部分編碼區(qū),構(gòu)建陰道毛滴蟲病毒轉(zhuǎn)染載體pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS。 陰道毛滴蟲病毒轉(zhuǎn)染載體經(jīng)T7RNA聚合酶體外轉(zhuǎn)錄成mRNA后,經(jīng)電穿孔法轉(zhuǎn)染含TVV的陰道毛滴蟲細(xì)胞內(nèi),用熒光顯微鏡觀察轉(zhuǎn)染后不同時(shí)間內(nèi)EGFP的表達(dá)情況。結(jié)果表明:載體pTVVL289/EGFP的體外轉(zhuǎn)錄體轉(zhuǎn)染陰道毛滴蟲后未觀察到熒光信號(hào),表明在TVV的3’末端UTR存在有與病毒復(fù)制和/或包裝有關(guān)的序列。載體pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS體外轉(zhuǎn)錄體轉(zhuǎn)染后可檢測(cè)到綠色熒光信號(hào),并在持續(xù)傳代培養(yǎng)5代后仍不減弱;提取轉(zhuǎn)染后蟲體的總核酸,用RT-PCR檢測(cè)到EGFP的mRNA的存在;SDS-PAGE分析轉(zhuǎn)染蟲體的培養(yǎng)上清和蟲體中EGFP的表達(dá),檢測(cè)到大小為27KD的蛋白。說(shuō)明我們構(gòu)建的陰道毛滴蟲病毒載體可以應(yīng)用于介導(dǎo)外源基因在陰道毛滴蟲體內(nèi)的表達(dá)。 本研究構(gòu)建了陰道毛滴蟲病毒轉(zhuǎn)染載體,介導(dǎo)了目的基因EGFP在陰道毛滴蟲體內(nèi)的表達(dá),為深入研究我國(guó)陰道毛滴蟲病毒與蟲體及其宿主之間的關(guān)系提供了新的研究工具,為進(jìn)一步研究陰道毛滴蟲的分子生物學(xué)特性奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R383

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 丁鶴;陰道毛滴蟲攜病毒與無(wú)病毒株差異蛋白比較研究[D];吉林大學(xué);2011年

2 曲晗;斯氏艾美耳球蟲病毒的鑒定及特性研究[D];吉林大學(xué);2012年

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本文編號(hào)：1504969