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  本文關(guān)鍵詞： 慢病毒 載體 DND1 基因　出處：《湖南師范大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的：在本研究中,將用PCR法從小鼠10.5d胚胎cDNA文庫(kù)中擴(kuò)增出的Dnd1基因引入慢病毒載體系統(tǒng),并生產(chǎn)Dnd1慢病毒顆粒。為進(jìn)一步明確Dnd1體外生物學(xué)功能奠定基礎(chǔ)。 方法： 1.Dnd1基因的獲得 根據(jù)GenBank中小鼠Dnd1基因序列(BC034897),使用DNAstar軟件設(shè)計(jì)基因的特異引物Dnd1-Age IF和Dnd1-AgeI-R,并且引入Age I酶切位點(diǎn)。用PCR法從小鼠10.5d胚胎cDNA文庫(kù)中擴(kuò)增出Dndl基因。PCR法篩選陽(yáng)性克隆。 2.重組慢病毒載體轉(zhuǎn)移質(zhì)粒pGC-FU-Dndl的構(gòu)建采用In-Fusion技術(shù),將Age I酶切回收后的PCR產(chǎn)物交換連接入Age I酶切的pGC-FU真核表達(dá)載體,產(chǎn)生pGC-FU-Dnd1質(zhì)粒。連接產(chǎn)物質(zhì)粒轉(zhuǎn)化大腸桿菌E.coli DH5a感受態(tài)細(xì)胞,擴(kuò)增質(zhì)粒。 3連接產(chǎn)物pGC-FU-Dnd1的酶切鑒定 收集部分?jǐn)U增后連接產(chǎn)物質(zhì)粒,酶切后經(jīng)電泳證實(shí)所得片段長(zhǎng)度均與預(yù)期相符,說(shuō)明Dnd1基因已克隆成功�；驕y(cè)序結(jié)果亦證實(shí)成功克隆Dnd1基因。 4連接產(chǎn)物pGC-FU-Dnd1質(zhì)粒轉(zhuǎn)染293T細(xì)胞 用脂質(zhì)體Lipofectamine 2000包裹構(gòu)建的pGC-FU-dnd1和輔助包裝載體,共轉(zhuǎn)染293T細(xì)胞,產(chǎn)生含有表達(dá)Dnd1蛋白的Lentivirus病毒顆粒。 結(jié)果： 1成功獲得Dnd1目的基因片段。通過(guò)PCR篩選得到Dnd1目的基因片段。 2連接產(chǎn)物pGC-FU-Dnd1的測(cè)序結(jié)果與GenBank中dnd1基因序列(BC034897)比較,表明Dnd1基因序列及讀碼框均正確。 3三質(zhì)粒轉(zhuǎn)染293 T細(xì)胞后熒光顯微鏡觀察結(jié)果及Western-blot檢測(cè)鑒定表明成功建立Dnd1慢病毒表達(dá)載體系統(tǒng)。 結(jié)論： 1成功克隆小鼠Dndl基因和成功構(gòu)建pGC-FU-Dnd1質(zhì)粒 2 pGC-FU-Dnd1表達(dá)載體經(jīng)過(guò)轉(zhuǎn)染293T細(xì)胞,48小時(shí)后觀察到綠色熒光蛋白的表達(dá)；經(jīng)Western bloting檢測(cè)到Dnd1蛋白在293T細(xì)胞中的表達(dá),從而證明我們成功建立了Dnd1重組慢病毒,對(duì)其進(jìn)行純化后采用逐孔稀釋法測(cè)定滴度,滴度測(cè)定結(jié)果為2E+9TU/ml。這為更深入的探討Dnd1基因抑制細(xì)胞增殖中等體外細(xì)胞生物學(xué)功能提供實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Aim: in this study, the Dnd1 gene amplified from mouse embryo cDNA library by PCR method was introduced into the lentivirus vector system to produce Dnd1 lentivirus particles, which laid a foundation for further clarifying the biological function of Dnd1 in vitro. Methods:. 1. Acquisition of Dnd1 gene. According to the mouse Dnd1 gene sequence BC034897 in GenBank, the specific primers Dnd1-Age IF and Dnd1-AgeI-Rx were designed by DNAstar software, and the AgeI restriction site was introduced. The Dndl gene was amplified by PCR from the cDNA library of mouse embryo at day 10.5. 2.Recombinant lentivirus vector transfer plasmid pGC-FU-Dndl was constructed by using In-Fusion technique. The PCR products recovered by Age I digestion were exchanged and ligated into Age I digested pGC-FU eukaryotic expression vector. PGC-FU-Dnd1 plasmids were produced. The plasmids were transformed into E. coli DH5a cells and the plasmids were amplified. Identification of pGC-FU-Dnd1 by enzyme digestion. After partial amplification, the plasmids of ligated products were collected, and the length of the fragments was confirmed by electrophoretic electrophoresis, which indicated that the Dnd1 gene had been cloned successfully, and the gene sequencing confirmed that the Dnd1 gene was cloned successfully. Transfection of pGC-FU-Dnd1 plasmid into 293T cells. The pGC-FU-dnd1 and auxiliary packaging vector were encapsulated with liposome Lipofectamine 2000 and co-transfected into 293T cells to produce Lentivirus virus particles expressing Dnd1 protein. Results:. 1 the Dnd1 target gene fragment was successfully obtained, and the Dnd1 gene fragment was obtained by PCR screening. 2Compared with the dnd1 gene sequence BC034897 in GenBank, the sequencing results of the ligated product pGC-FU-Dnd1 showed that the Dnd1 gene sequence and the reading frame were correct. 3After the transfection of the three plasmids into 293T cells, the results of fluorescence microscopy and Western-blot detection showed that the Dnd1 lentivirus expression vector system was successfully established. Conclusion:. 1 successful cloning of mouse Dndl gene and construction of pGC-FU-Dnd1 plasmid. (2) the expression of green fluorescent protein was observed in 293T cells 48 hours after transfection with pGC-FU-Dnd1 expression vector, and the expression of Dnd1 protein in 293T cells was detected by Western bloting, which proved that we successfully established Dnd1 recombinant lentivirus. After purification, the titer was determined by one-hole dilution method, and the titer was 2E9TU / ml, which provided the experimental basis for further study on the biological function of Dnd1 gene inhibiting cell proliferation in vitro.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R373

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本文編號(hào)：1524013