本文關(guān)鍵詞： 重組腺病毒載體 Gateway技術(shù) 限制性酶切和連接 Brcc3　出處：《揚(yáng)州大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的： 構(gòu)建能夠在體外真核細(xì)胞中過(guò)表達(dá)目標(biāo)基因Brcc3的重組腺病毒真核表達(dá)載體,并進(jìn)而使用HEK293細(xì)胞包裝重組腺病毒,從而為后續(xù)轉(zhuǎn)染小鼠心臟成纖維細(xì)胞(cardiofibroblasts, cFs)探討細(xì)胞內(nèi)Brcc3基因過(guò)表達(dá)對(duì)TGF-β信號(hào)通路各環(huán)節(jié)的影響做準(zhǔn)備。 方法： 1、目的基因片段的擴(kuò)增(擴(kuò)增包括雙酶切位點(diǎn)的目標(biāo)基因序列) 分別以Ndel-Sacl-Brcc3-F和Brcc3-R為上下游引物擴(kuò)增Sacl-Brcc3;以Brcc3-GST-F和Xhol-Spel-GST-R擴(kuò)增GST-Xhol及以Ndel-Sacl-Brcc3-F和Xhol-Spel-GST-R擴(kuò)增Sacl-Brcc3-GST-Xhol。 2、利用酶切連接構(gòu)建pDown-Brcc3/GST/IRES/EGFP并測(cè)序鑒定 Sacl/Xhol雙酶切骨架載體pDown-MCS-IRES/EGFP和PCR回收產(chǎn)物Sacl-Brcc3-GST-Xhol,并將骨架片段pDown-MCS-IRES/EGFP和目的基因Brcc3-GST連接,接著轉(zhuǎn)化連接產(chǎn)物到感受態(tài)細(xì)胞大腸桿菌stbl3,進(jìn)行菌落PCR鑒定后挑取單克隆搖菌提取質(zhì)粒pDown-Brcc3/GST/IRES/EGFP送測(cè)序。 3、利用Gateway Technology構(gòu)建pAV.Ex1d-CMVBrcc3/GST/IRES/EGFP pDown-Brcc3/GST/IRES/EGFP和pAV.Des1d進(jìn)行LR反應(yīng),將目標(biāo)基因克隆到腺病毒載體pAV.Des1d,于(反應(yīng)產(chǎn)物轉(zhuǎn)化到)大腸桿菌感受態(tài)細(xì)胞stbl3中進(jìn)行(復(fù)制),PCR鑒定陽(yáng)性克隆,挑單克隆搖菌提取質(zhì)粒送檢測(cè)序。 4、將重組腺病毒轉(zhuǎn)染293A細(xì)胞,進(jìn)行病毒的包裝和擴(kuò)增,并提取轉(zhuǎn)染的CFs細(xì)胞蛋白行Western blot檢測(cè)Brcc36蛋白表達(dá)水平。 主要結(jié)果： 1、經(jīng)瓊脂糖電泳鑒定表明目的片段Sacl-Brcc3-GST-Xhol擴(kuò)增成功。 2、經(jīng)PCR檢測(cè)、瓊脂糖電泳及基因測(cè)序表明載體pDown-Brcc3/GST/IRES/EGFP構(gòu)建成功。 3、經(jīng)PCR檢測(cè)、瓊脂糖電泳、基因測(cè)序及western blot分析表明重組腺病毒真核表達(dá)載體pAV.Exld-CMVBrcc3/GST/IRES/EGFP構(gòu)建成功。 主要結(jié)論： 本研究利用Gateway技術(shù)成功構(gòu)建重組腺病毒真核表達(dá)載體pAV.Exld-CMVBrcc3/GST/IRES/EGFP,并成功包裝獲得過(guò)表達(dá)Brcc3基因的腺病毒。
[Abstract]:Objective: To construct the recombinant adenovirus eukaryotic expression vector which can over-express the target gene Brcc3 in eukaryotic cells in vitro, and then use HEK293 cells to package the recombinant adenovirus. Therefore, it can be used to transfect the cardiac fibroblasts of mouse heart fibroblasts. CFS) to investigate the effect of overexpression of Brcc3 gene on TGF- 尾 signaling pathway. Methods: 1. Amplification of target gene fragments (amplification of target gene sequences including double restriction sites) Sacl-Brcc3 was amplified with Ndel-Sacl-Brcc3-F and Brcc3-R as upstream and downstream primers, respectively. Amplification of GST-Xhol with Brcc3-GST-F and Xhol-Spel-GST-R and Ndel-Sacl-Brcc3-F and Xhol-Spel-GS with Ndel-Sacl-Brcc3-F. T-R amplification of Sacl-Brcc3-GST-Xhol. 2. PDown-Brcc3/GST/IRES/EGFP was constructed by restriction endonuclease ligation and identified by sequencing Sacl/Xhol double enzyme digestion framework carrier pDown-MCS-IRES/EGFP and PCR recovery product Sacl-Brcc3-GST-Xhol. The skeleton fragment pDown-MCS-IRES/EGFP was ligated with the target gene Brcc3-GST, and then the ligation product was transformed into the competent Escherichia coli stbl3. After the colony PCR identification, the clone pDown-Brcc3/GST/IRES/EGFP was selected and sequenced. Using Gateway Technology to build pAV.Ex1d-CMVBrcc3/GST/IRES/EGFP The target gene was cloned into adenovirus vector pAV.Des1d by LR reaction with pDown-Brcc3/GST/IRES/EGFP and pAV.Des1d. The positive clones were identified by PCR in the stbl3 of E. coli competent cells, and the plasmids extracted from monoclonal bacteria were selected and sequenced. 4Recombinant adenovirus was transfected into 293A cells for packaging and amplification. The expression of Brcc36 protein was detected by Western blot. Main results: 1. The fragments of the epidermis were amplified successfully by agarose electrophoresis. 2. By PCR detection, agarose electrophoresis and gene sequencing showed that the vector pDown-Brcc3/GST/IRES/EGFP was successfully constructed. 3. Agarose electrophoresis was performed by PCR. Gene sequencing and western blot analysis showed that the recombinant adenovirus eukaryotic expression vector pAV.Exld-CMVBrcc3/GST/IRES/EGFP was successfully constructed. Main conclusions: In this study, the recombinant adenovirus eukaryotic expression vector pAV.Exld-CMVBrcc3/GST/IRES/EGFP was successfully constructed by using Gateway technique. The adenovirus expressing Brcc3 gene was successfully packaged.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R3416

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳娜娜;向冬喜;鄭叢龍;;腺病毒及其研究進(jìn)展[J];大連醫(yī)科大學(xué)學(xué)報(bào);2010年05期

2 熊俊,訾曉淵,胡以平;位點(diǎn)特異性重組系統(tǒng)及其應(yīng)用[J];國(guó)外醫(yī)學(xué).遺傳學(xué)分冊(cè);2000年06期

3 徐麗,蔡俊鵬;菌落PCR方法的建立及其與常規(guī)PCR方法的比較[J];華南理工大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年05期

4 賈紹昌;陳堅(jiān);趙全年;;帶綠色熒光蛋白的米非司酮誘導(dǎo)表達(dá)腺病毒載體的構(gòu)建及表達(dá)[J];醫(yī)學(xué)研究生學(xué)報(bào);2010年01期

5 蘆春斌;馬貞麗;;重疊PCR-酶切連接法人工合成風(fēng)疹病毒E1基因及其重組質(zhì)粒構(gòu)建[J];暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版);2010年06期

6 蔣俊豪;賀修勝;;PCR-雙酶切鑒定STGC3抑癌基因重組子假陽(yáng)性研究[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2007年06期

7 金天明;武迎紅;;腺病毒及腺病毒載體的研究進(jìn)展[J];天津農(nóng)學(xué)院學(xué)報(bào);2007年02期

8 陳書(shū)霞;王曉武;房玉林;;單菌落PCR法直接快速鑒定重組克隆[J];微生物學(xué)通報(bào);2006年03期

9 顧洪生;程志安;李振宇;周文鈺;鄒小英;;利用Gateway技術(shù)構(gòu)建重組腺病毒載體pAd-LMP-1[J];中國(guó)組織工程研究與臨床康復(fù);2010年24期

10 何金生,王健偉,洪濤;腺病毒載體構(gòu)建原理與方法的研究進(jìn)展[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;2001年04期

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