本文選題：陰道毛滴蟲 + 轉(zhuǎn)染載體��； 參考：《吉林大學(xué)》2008年碩士論文

【摘要】： 本研究構(gòu)建了陰道毛滴蟲病毒轉(zhuǎn)染載體和陰道毛滴蟲DNA轉(zhuǎn)染載體。 陰道毛滴蟲病毒轉(zhuǎn)染載體的構(gòu)建:根據(jù)我國(guó)陰道毛滴蟲病毒基因組的序列特征,在本室構(gòu)建陰道毛滴蟲病毒瞬時(shí)表達(dá)載體的基礎(chǔ)上,用新霉素磷酸轉(zhuǎn)移酶(NEO)編碼基因替換TVV全部編碼區(qū),同時(shí)引入多克隆酶切位點(diǎn)(MCS),構(gòu)建陰道毛滴蟲病毒轉(zhuǎn)染載體。以增強(qiáng)型綠色熒光蛋白(EGFP)作為報(bào)告基因,經(jīng)T7 RNA聚合酶體外轉(zhuǎn)錄成mRNA后,經(jīng)電穿孔法轉(zhuǎn)染陰道毛滴蟲攜病毒株細(xì)胞內(nèi),進(jìn)行G418篩選,連續(xù)傳代24次,熒光顯微鏡都可以觀察到EGFP的表達(dá),同時(shí)提取轉(zhuǎn)染后蟲體的總核酸,用RT-PCR方法檢測(cè)到EGFP的mRNA,結(jié)果顯示EGFP在陰道毛滴蟲轉(zhuǎn)染細(xì)胞內(nèi)得到了穩(wěn)定表達(dá)。 陰道毛滴蟲DNA轉(zhuǎn)染載體構(gòu)建:首先克隆陰道毛滴蟲α-琥珀酰輔酶A合成酶(α-SCSB)全序列,并對(duì)其序列進(jìn)行了分析;利用陰道毛滴蟲α-琥珀酰輔酶A合成酶啟動(dòng)子序列,構(gòu)建EGFP表達(dá)盒pα-SCSB5/EGFP/Eα-SCSB3,對(duì)α-SCSB轉(zhuǎn)錄活性進(jìn)行了鑒定;構(gòu)建NEO抗性盒pα-SCSB5/NEO /Nα-SCSB3,通過G418抗性篩選建立G418抗性蟲株;最后將抗性盒和表達(dá)盒串聯(lián),構(gòu)建陰道毛滴蟲DNA穩(wěn)定轉(zhuǎn)染載體pNEO/EGFP,經(jīng)電穿孔法轉(zhuǎn)染陰道毛滴蟲細(xì)胞內(nèi),進(jìn)行G418篩選,連續(xù)傳代17次,熒光顯微鏡都可以觀察到EGFP的表達(dá),成功實(shí)現(xiàn)了EGFP在陰道毛滴蟲轉(zhuǎn)染細(xì)胞內(nèi)的穩(wěn)定表達(dá)。 本研究成功構(gòu)建了陰道毛滴蟲轉(zhuǎn)染載體,介導(dǎo)了目的基因EGFP在陰道毛滴蟲體內(nèi)的穩(wěn)定表達(dá),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]:In this study, Trichomonas vaginalis vector and Trichomonas vaginalis DNA transfection vector were constructed.
Construction of transfection vector of Trichomonas vaginalis virus: Based on the sequence characteristics of Trichomonas vaginalis virus genome in our country, on the basis of constructing the transient expression vector of Trichomonas vaginalis virus in our room, the whole coding region of TVV was replaced with neomycin phosphonase (NEO) gene encoding gene, and the polyclonal site of MCS was introduced to construct the vagina Mao Dichong The virus transfection carrier, with enhanced green fluorescent protein (EGFP) as the reporter gene, was transcribed into mRNA by T7 RNA polymerase in vitro and transfected into the virus cell of Trichomonas vaginalis by electroporation and screened by G418 for 24 successive generations. The expression of EGFP could be observed by the fluorescence microscope, and the total nucleic acid of the insect body after transfection was extracted and R was extracted with R. The mRNA of EGFP was detected by T-PCR. The results showed that EGFP was stable in the transfected cells of Trichomonas vaginalis.
Construction of DNA transfection vector of Trichomonas vaginalis: first cloned the whole sequence of Trichomonas vaginalis alpha succinyl coenzyme A synthetase (alpha -SCSB), and analyzed its sequence. Using the promoter sequence of alpha succinyl coenzyme A synthetase of Trichomonas vaginalis, the EGFP expression box P alpha -SCSB5/EGFP/E alpha -SCSB3 was constructed, and the activity of alpha -SCSB transcriptional activity was identified, and NEO was constructed for NEO. Resistance box P alpha -SCSB5/NEO /N alpha -SCSB3 was selected to establish G418 resistant strain by G418 resistance screening. Finally, the resistance box and expression box were connected in series, and DNA stable transfection carrier pNEO/EGFP of Trichomonas vaginalis was constructed and transfected into Trichomonas vaginalis cells by electrical perforation, and G418 screening was performed for 17 times. The expression of EGFP was observed by fluorescence microscopy. The stable expression of EGFP in Trichomonas vaginalis transfected cells was achieved.
This study successfully constructed the transfection vector of Trichomonas vaginalis, which mediates the stable expression of the target gene EGFP in Trichomonas vaginalis, which lays the foundation for further study of 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】：2008
【分類號(hào)】：R383

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 韓乾忠;柔嫩艾美耳球蟲病毒的鑒定及細(xì)胞內(nèi)分布[D];吉林大學(xué);2011年

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

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本文編號(hào)：2021924