本文關(guān)鍵詞： 人端粒酶逆轉(zhuǎn)錄酶 綠色熒光蛋白 質(zhì)粒構(gòu)建　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的構(gòu)建真核表達質(zhì)粒pRRL-hTERT-P2A-EGFP,探討重組質(zhì)粒在HEK293FT細胞中的表達和轉(zhuǎn)染效率。為進一步研究hTERT基因的功能及構(gòu)建永生化細胞系奠定基礎(chǔ)。方法真核質(zhì)粒pRRL-hTERT-P2A-EGFP的構(gòu)建:(1)以pBABE-puro-hTERT和pRRL-EGFP質(zhì)粒為模板利用PCR技術(shù)分別擴增目的基因hTERT、P2A和EGFP,對PCR產(chǎn)物進行膠回收純化。(2)以凝膠回收的3個目的片段為模板,采用重疊PCR法獲得目的片段hTERT-P2A-EGFP。(3)載體pRRL-與目的片段hTERT-P2A-EGFP酶切處理后進行重組反應(yīng)。(4)重組質(zhì)粒的序列測定和定點突變,設(shè)計含有突變位點正確堿基的引物,利用FastDigestDpnⅠ,進行定點突變。真核質(zhì)粒pRRL-hTERT-P2A-EGFP的鑒定:(1)復(fù)蘇凍存的HEK293FT細胞,并進行傳代培養(yǎng)。(2)經(jīng)脂質(zhì)體介導(dǎo)重組質(zhì)粒轉(zhuǎn)染到HEK293FT細胞,熒光顯微鏡觀察綠色熒光蛋白在細胞中的表達。(3)利用流式細胞儀檢測重組質(zhì)粒在HEK293FT細胞中的轉(zhuǎn)染效率。(4)四質(zhì)粒系統(tǒng)包裝病毒,利用lipo2000將已合成的所需質(zhì)粒轉(zhuǎn)染到293FT細胞系中,收集病毒進行濃縮、滴定、流式檢測,從而獲悉病毒滴度及感染力。(5)重組病毒感染人胚胎肝臟細胞后,提取細胞RNA,RT-PCR法檢測細胞hTERT基因的表達水平。結(jié)果1.PCR產(chǎn)物的瓊脂糖凝膠電泳結(jié)果顯示目的基因hTERT、P2A和EGFP的片段分別為3500、110和720bp,與預(yù)期結(jié)果一致。2.目的基因片段hTERT-P2A-EGFP的電泳結(jié)果約4300bp,與預(yù)期片段大小一致。3.測序結(jié)果顯示,目的基因1547位點發(fā)生了突變。利用定點突變技術(shù)成功誘變,再次測序后目的基因序列與GenBank公布的基因序列完全一致。4.重組質(zhì)粒pRRL-hTERT-P2A-EGFP酶切鑒定,電泳產(chǎn)生約4300bp目的條帶,質(zhì)粒構(gòu)建成功。5.重組質(zhì)粒pRRL-hTERT-P2A-EGFP轉(zhuǎn)染HEK293FT細胞24h后,在熒光顯微鏡下可以觀察到細胞中有綠色熒光產(chǎn)生。6.流式細胞檢測結(jié)果顯示重組質(zhì)粒轉(zhuǎn)染HEK293FT細胞的效率為44.8%。7.濃縮后的hTERT病毒液10μL感染細胞,流式細胞檢測293FT細胞GFP的陽性率為32.7%,經(jīng)計算重組慢病毒的滴度為1.6×107。8.hTERT慢病毒感染人胚胎肝臟細胞后,RT-PCR檢測實驗組的hTERT基因的表達較對照組有明顯升高。結(jié)論1.目的基因hTERT-P2A-EGFP的序列與GenBank公布的基因序列一致。2.經(jīng)酶切鑒定后,重組質(zhì)粒pRRL-hTERT-P2A-EGFP構(gòu)建成功。3.重組質(zhì)粒轉(zhuǎn)染HEK293FT細胞后,可在細胞內(nèi)表達。4.慢病毒感染細胞后,hTERT基因的表達量升高。
[Abstract]:Objective to construct eukaryotic expression plasmid pRRL-hTERT-P2A-EGFP. To investigate the expression and transfection efficiency of recombinant plasmid in HEK293FT cells, and to lay a foundation for further study on the function of hTERT gene and the construction of immortalized cell line. Methods the eukaryotic plasmid pRRL-hT was used to construct an immortalized cell line. Building ERT-P2A-EGFP:. (. 1) the target gene hTERT was amplified by PCR using pBABE-puro-hTERT and pRRL-EGFP plasmids as templates. P2A and EGFP, the products of PCR were recovered and purified by gel. 2) the three target fragments recovered by gel were used as templates. The target fragment hTERT-P2A-EGFP.3 was obtained by overlapping PCR method. The recombinant plasmid pRRL- was sequenced and mutated by site-directed mutagenesis after digesting with the target fragment hTERT-P2A-EGFP. A primer containing the correct base at the mutation site was designed and FastDigestDpn 鈪，

本文編號：1461271