心肌α肌球蛋白重鏈啟動(dòng)子驅(qū)動(dòng)的綠色熒光蛋白表達(dá)載體的構(gòu)建及鑒

1、    心肌肌球蛋白重鏈啟動(dòng)子驅(qū)動(dòng)的綠色熒光蛋白表達(dá)載體的構(gòu)建及鑒 醫(yī)學(xué)論文                                         關(guān)鍵詞】   心作者：

2、潘國(guó)棟 黃永章， 郭凌鄖， 唐俊明， 孔 霞， YANG Qinglin， 王家寧 本文由中國(guó)論文范文收集整理。肌特異性；肌球蛋白重鏈；啟動(dòng)子；增強(qiáng)型綠色熒光蛋白；干細(xì)胞          目的： 構(gòu)建并鑒定心肌肌球蛋白重鏈啟動(dòng)子驅(qū)動(dòng)的綠色熒光蛋白表達(dá)載體（MHC-EGFP），該基因只在心肌中特異性表達(dá)。 方法 ： 設(shè)計(jì)5和3分別具有SalI/Hind酶切位點(diǎn)的EGFP引物，聚合酶鏈反應(yīng)法（PCR）從pLEGFP質(zhì)粒中擴(kuò)增增強(qiáng)型綠色熒光蛋白（EGFP）的cDNA，插入pGEM-T Easy載體表達(dá)盒，構(gòu)建pGEM-EGFP，

3、SalI/Hind雙酶切后回收729 kb片斷，插入經(jīng)相同酶切的含5.5 kb心肌特異性肌球蛋白重鏈啟動(dòng)子的pNC26質(zhì)粒，構(gòu)建pMHC-EGFP。連接產(chǎn)物轉(zhuǎn)化感受態(tài)DH5，挑選克隆，PCR擴(kuò)增鑒定，重組質(zhì)粒經(jīng)NotI酶切，回收7.2 kb MHC-EGFP表達(dá)盒。分離培養(yǎng)小鼠心肌細(xì)胞與骨骼肌成肌細(xì)胞。MHC-EGFP表達(dá)盒通過(guò)脂質(zhì)體法分別轉(zhuǎn)染心肌與骨骼肌成肌細(xì)胞。熒光顯微鏡觀察轉(zhuǎn)染細(xì)胞是否出現(xiàn)綠色熒光，以鑒定MHC-EGFP表達(dá)盒是否具有表達(dá)特異性。結(jié)果： EGFP cDNA正確插入pNC26質(zhì)粒中MHC啟動(dòng)子3端。成功轉(zhuǎn)染MHC-EGFP表達(dá)盒的心肌細(xì)胞出現(xiàn)綠色熒光，而轉(zhuǎn)染的骨骼肌成肌細(xì)胞

4、無(wú)熒光出現(xiàn)。結(jié)論： MHC-EGFP表達(dá)盒具有心肌特異性表達(dá)特性，為進(jìn)一步監(jiān)測(cè)干細(xì)胞向心肌分化 研究 奠定基礎(chǔ)。      心肌特異性；肌球蛋白重鏈；啟動(dòng)子；增強(qiáng)型綠色熒光蛋白；干細(xì)胞    Abstract: Objective To construct and identify the cardiac  -myosin heavy chain promoter (MHC) -driven enhanced green fluorescent protein (EGFP) expression vector (p

5、MHC-EGFP), which solely expresses EGFP in cardiomyocytes. Methods EGFP cDNA primers were synthesized with forward primer and reverse primer containing SalI and Hind, respectively. EGFP cDNA was amplified by polymerase chain reaction (PCR), added single deoxyadenosine at 3 ends, and subcloned into pG

6、EM-T easy vector to construct pGEM-EGFP. The fragment of 729 bp from SalI/Hind-digested pGEM-EGFP was subcloned into pNC26, a plasmid carried 5.5 kb MHC promoter, to construct pMHC-EGFP. The expression cassette of 7.2 kb MHC-EGFP was recovered with low melting point agarose electrophoresis. Mouse ca

7、rdiomyocytes and skeletal muscle myoblasts were isolated and cultured in vitro. MHC-EGFP expression cassette  was transfected into cardiomyocytes and myoblasts, respectively. EGFP expression was observed under fluorescent microscope. Results EGFP cDNA was successfully inserted into the 3 ends o

8、f MHC promoter. The green fluorescence could be observed in transfected cardiomyocytes, but not in transfected myoblasts. Conclusion  MHC-EGFP expression cassette can be specifically expressed within cardiomyocytes, which provides a basis to monitor the differentiation of stem cells into cardio

9、myocytes.    Key words:Cardiac-specific; Myosin heavy chain; Promoter; Enhanced green fluorescent protein; Stem cells   近年來(lái)日益興起的大量干細(xì)胞研究證實(shí)，胚胎或成體間充質(zhì)干細(xì)胞在一定條件下可以分化為心肌樣細(xì)胞，為心臟疾病的 治療 帶來(lái)了希望。然而這些心肌樣分化的干細(xì)胞是否具有成年心肌的收縮和傳導(dǎo)功能， 目前 研究還相當(dāng)缺乏。這是因?yàn)橥ㄟ^(guò)免疫組織化學(xué)或細(xì)胞化學(xué)染色判斷干細(xì)胞分化，需要先對(duì)細(xì)胞進(jìn)行固定處理，不能在活細(xì)胞

10、狀態(tài)下觀察干細(xì)胞的心肌分化。本研究將心肌特異性肌球蛋白重鏈（alpha-myosin heavy chain, MHC）啟動(dòng)子與增強(qiáng)型綠色熒光蛋白（enhanced green fluorescent protein, EGFP）連接，構(gòu)建在心肌中特異表達(dá)的報(bào)告基因系統(tǒng)，使之用于在活細(xì)胞狀態(tài)下觀測(cè)判斷干細(xì)胞的心肌分化，為深入進(jìn)行干細(xì)胞的心肌分化研究奠定基礎(chǔ)。     1  材料與方法     1.1  材料限制性內(nèi)切酶SalI、Hind、NotI以及200 bp DNA ladder和Lambda DNA/Hind

11、 markers購(gòu)自華美生物工程公司；Taq聚合酶、T4 DNA連接酶、dNTP和dATP購(gòu)自Promega公司；pfu DNA聚合酶購(gòu)自New England Biolabs公司；Lipofectamine 2000陽(yáng)離子脂質(zhì)體購(gòu)自Invitrogen公司，DMEM培養(yǎng)基購(gòu)自Gibco公司；新生牛血清購(gòu)自四季青生物材料有限公司；包含5.5 kb MHC啟動(dòng)子的pNC26質(zhì)粒由Morehouse School of Medicine提供；包含EGFP cDNA的pLEGFP-C1質(zhì)粒購(gòu)自Clontech公司；pGEM-T Easy Vector系統(tǒng)購(gòu)自Promega公司；5端加有SalI和Hind酶切位點(diǎn)的EGFP cDNA PCR上下游引物（上游：5-GTC GAC ATG GTG AGC AAG GGC GAG GAG CTG TTC -3，下游：5-AAG CTT TTA CTT GTA CAG CTC GTC CAT GCC GAG -3）由北京賽百盛基因技術(shù)有限公司合成。 關(guān)鍵詞:蛋白,表達(dá),載體,構(gòu)建,綠色,啟動(dòng),醫(yī)學(xué)論文,心肌肌球蛋白重