可回复性永生化逆转录病毒载体pLNCTIGlox的构建与表达
陈耀凯, 李俊刚, 王宇明, 中国人民解放军第三军医大学西南医院全军感染病研究所 重庆市 400038
陈耀凯, 男,1966-12-27生, 河南省固始县人, 汉族.1998年华中科技大学工学博士, 副教授, 副主任医师.
国家自然科学基金资助, No. 30100080
通讯作者: 陈耀凯,400038, 重庆市沙坪坝区高滩岩正街29号, 中国人民解放军第三军医大学西南医院全军感染病研究所. yaokaichen@hotmail.com
电话: 023-68754475-8006 传真: 023-65461319
收稿日期: 2005-01-28 接受日期: 2005-02-16
, http://www.100md.com
Construction and expression of a retroviral vectorpLNCTIGlox for reversible immortalization
Yao-Kai Chen, Jun-Gang Li, Yu-Ming Wang
Yao-Kai Chen,Jun-Gang Li, Yu-Ming Wang, Chinese PLA Institute of InfectiousDiseases, Southwest Hospital, the Third Military Medical University,Chongqing 400038, China
Supported by National natural Science Foundation of China,No.30100080
, 百拇医药
Correspondence to: Yao-Kai Chen, Chinese PLA Institute ofInfectious Diseases, Southwest Hospital, the Third Military MedicalUniversity, Chongqing 400038, China. yaokaichen@hotmail.com
Received: 2005-01-28 Accepted: 2005-02-16
AbstractAIM: Toconstruct a retroviral vector carrying selection marker gene NeoR,reporter gene EGFP and recombination site sequence LoxP for hepatocytereversible immortalization.
, 百拇医药
METHODS: 2.1 kbSV40T gene was inserted into vector pIRES2-EGFP to obtain a 3.4 kbfragment of SV40T-IRES-EGFP by restriction endonuclease digestion. Thefragment was further subcloned into retroviral vector pLNCX2 to obtain anew vector pLNCTIG. The linearized pLNCTIG was mixed with double strandedLoxP to construct pLNCTIGlox. Escherichia coli DH5awas transformed with pLNCTIGlox and positive clones were identified bycolony polymerase chain reaction (PCR) and restriction endonucleasedigestion. PT67 cells were transfected with pLNCTIGlox and the expressionof green fluorescence protein was observed. The transfected cells werestained immunochemically to detect the expression of SV40 large T antigen.
, 百拇医药
RESULTS: Amongthe 19 randomly selected colonies, 3 showed positive DNA bands onelectrophoresis gel. One of the three clones was further analyzed byrestriction endonuclease digestion and only one 9.5 kb band was separatedelectrophoretically, demonstrating that the analyzed clone was thepositive recombinant. Twenty-four hours after transfection, PT67 cellsemitted green fluorescence under the fluorescence microscope. The nucleiof the transfected cells were positive for immunochemical staining.
, 百拇医药
CONCLUSION: Theretroviral vector pLNCTIGlox for reversible immortalization wassuccessfully constructed and expressed.
Key Words:Reversible immortalization; Hepatocytes; pLNCTIGlox
Chen YK, Li JG, Wang YM. Construction and expression of a retroviralvector pLNCTIGlox for reversible immortalization. Shijie Huaren XiaohuaZazhi 2005;13(8):975-978
摘要
, 百拇医药
目的:为建立可回复性永生化肝细胞株，构建带有筛选基因NeoR、报道基因EGFP及重组位点loxP的可回复性永生化逆转录病毒载体.
方法:将2.1kb SV40T亚克隆到pIRES2-EGFP的相应位点，再酶切下3.4kb SV40T-IRES-EGFP片段插入逆转录病毒载体pLNCX2的两个相同酶切位点之间，构成新载体pLNCTIG.最后，将线性化pLNCTIG和loxP双链片段混合连接.取连接产物转化高效感受态DH5a细胞.菌落PCR和酶切法鉴定阳性克隆.转染包装细胞PT67并在荧光显微镜下观察绿色荧光蛋白的表达.扩大培养稳定转染的细胞，免疫组化染色检测SV40T基因的表达.
结果:随机挑取19个克隆，3个电泳分离出约1.1kb阳性条带.取上述阳性克隆进行酶切鉴定，结果仅出现一条约9.5kb的条带，证明该克隆为阳性重组体.转染PT67细胞24h后，荧光显微镜下可见散在多处绿色荧光.免疫组化染色显示细胞胞核呈阳性染色.
, 百拇医药
结论:成功构建并表达可回复性永生化逆转录病毒载体pLNCTIGlox.
关键词:可回复性永生化;肝细胞;pLNCTIGlox
陈耀凯,李俊刚,王宇明.可回复性永生化逆转录病毒载体pLNCTIGlox的构建与表达.世界华人消化杂志 2005;13(8):975-978
(PDF) 以egfpPa和loxPas为引物，菌落PCR法鉴定pLNCTIGlox重组阳性克隆. N: 阴性对照; M:DNA分子量标准; 1-19: pLNCTIGlox克隆1-19的菌落PCR产物，其中克隆2，14和19为阳性.
图2 (PDF) 酶切电泳鉴定pLNCTIGlox逆转录病毒载体. 1: 未酶切pLNCTIGlox载体质粒; 2: BamHⅠ单酶切pLNCTIGlox质粒; 3: BamHⅠ和XbaⅠ双酶切pLNCTIGlox质粒; 4: 未酶切pLNCTIG质粒; 5: BamHⅠ单酶切pLNCTIG对照; 6: BamHⅠ和XbaⅠ双酶切pLNCTIG对照; M: DNA分子量标准.
, http://www.100md.com
2.2EGFP基因的表达 转染后24h在荧光显微镜下观察，可见散在多处绿色荧光(图3).
2.3SV40大T抗原的免疫组化染色 经扩大培养的转染细胞胞核呈阳性染色(图4)，而作为阴性对照的未转染细胞则未见阳性反应.
图3 转染pLNCTIGlox载体后24 h 的PT67细胞, IF×100.
图4 转染pLNCTIGlox载体后的PT67细胞核呈阳性反应, SABC×400.
3 讨论采用两种限制性内切酶消化外源片段和质粒载体后进行连接，是质粒载体构建过程中最容易、最有效的克隆策略.我们将pLLTSN上的2.1kb SV40T插入到pIRES2-EGFP的EcoRⅠ和BamHⅠ位点之间即是采用这一方法.然而质粒与外源片段的酶切位点之间并非总是能找到“门当户对”的理想搭配关系，此时必须采取不同的策略.在本研究中我们拟在逆转录病毒载体pLNCTIG的3’长末端重复(LTR)序列的U3区插入loxP特异重组位点，但该处仅有一个XbaⅠ单酶切位点可供利用，因此只能采用单酶切连接策略插入loxP片段.用XbaⅠ酶切载体pLNCTIG后产生2个相同的粘端，通常要将其5’端去磷酸化，以减少载体自身环化导致较高的非重组体菌落背景.但若将载体pLNCTIG5’端去磷酸化，将无法与loxP双链的匹配末端连接，原因是loxP作为合成的DNA是缺少5’磷酸基团的.此时有两种选择可以采用:(1)将载体pLNCTIG5’端去磷酸化，再使合成的loxP双链DNA5’端磷酸化，然后进行连接.该方式的缺点是会使loxP片段在重组质粒中形成串联拷贝，不利于单拷贝重组体的筛选.(2)不将载体pLNCTIG5’端去磷酸化，直接与缺少5’磷酸基团的loxP双链DNA进行连接反应，在此过程中loxP片段在重组质粒中不会形成串联拷贝，阳性重组质粒只能是插入单拷贝loxP序列的克隆.我们采用第2种方式，为了减少载体自身环化，我们将载体与外源loxP片段的摩尔比提高到1∶8，以减少非重组体菌落背景.结果成功，并已筛选出正向插入XbaⅠ酶切位点的阳性克隆.由于loxP双链DNA粘端与XbaⅠ酶切粘端连接后，改变了原来的酶切位点序列，XbaⅠ酶切位点不复存在，用XbaⅠ酶不能切开重组质粒.用BamHⅠ和XbaⅠ酶切重组质粒和pLNCTIG，前者只能被BamHⅠ酶切线性化，而后者可被BamHⅠ和XbaⅠ酶切为约1.7kb和7.8kb大小的两个片段.实际结果与理论预期完全符合.
, 百拇医药
Cre/LoxP是最常用的基因位点重组系统，最早在噬菌体P1的研究中被发现[8].LoxP位于噬菌体P1染色体交叉部位，由两个反向重叠序列(revertedrepeat)和一个8bp间隔区组成.Cre是催化位点重组的酶，与反向重叠序列结合，剪切两个LoxP之间的DNA序列.本研究成功构建带有目的基因、筛选基因及LoxP重组位点的质粒载体，并进行蛋白表达与鉴定，为下一步进行细胞永生化奠定了良好的基础.
4 参考文献 1 Kobayashi N, Okitsu T, Nakaji S, Tanaka N.Hybrid bioartificial liver:establishing a reversibly immortalizedhuman
hepatocyte line and developing abioartificial liver for practical use. J Artif Organs 2003;6:236-244
, http://www.100md.com
2 Kobayashit N, Tanaka N. Engineering ofhuman hepatocyte lines for cell therapies in humans:prospects and
remaining hurdles. Cell Transplant 2002;11:417-420
3 Sato Y, Endo H, Ajiki T, Hakamata Y, OkadaT, Murakami T,Kobayashi E. Establishment of Cre/LoxP recombination
system in transgenic rats. BiochemBiophys Res Commun 2004;319:1197-1202
4 Watanabe N, Odagiri H, Totsuka E, Sasaki M.A new method to immortalize primary cultured rat hepatocytes.
, 百拇医药
Transplant Proc 2004;36:2457-2461
5 Wege H, Le HT, Chui MS, Liu L, Wu J, GiriR, Malhi H, Sappal BS, Kumaran V, Gupta S, Zern MA. Telomerase
reconstitution immortalizes human fetalhepatocytes without disrupting their differentiation potential.
Gastroenterology 2003;124:432-444
6 Kobayashi N, Noguchi H, Westerman KA,Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Leboulch P,Tanaka N. Cre/loxP-based reversibleimmortalization of human hepatocytes. Cell Transplant 2001;10:383-386
, 百拇医药
7 Noguchi H, Kobayashi N, Westerman KA,Sakaguchi M, Okitsu T, Totsugawa T, Watanabe T, Matsumura T,Fujiwara T, Ueda T, Miyazaki M, Tanaka N,Leboulch P. Controlled expansion of human endothelial cell populationsby
Cre-loxP-based reversible immortalization. HumGene Ther 2002;13:321-334
8 Min KJ, Young CJ, Sun KM, Chang KS. In vivoexcision and amplification of large human genomic segments using
the Cre/loxP-and large T antigen/SV40 ori-mediatedmachinery. J Biotechnol 2004;110:227-233
9 李俊刚, 陈耀凯, 王宇明. SV40T外显子的拼接及逆转录病毒载体pLLTSN的构建. 世界华人消化杂志
2004;12:1104-1108
编辑 潘伯荣 审读 张海宁, 百拇医药( 陈耀凯, 李俊刚, 王宇明)
陈耀凯, 男,1966-12-27生, 河南省固始县人, 汉族.1998年华中科技大学工学博士, 副教授, 副主任医师.
国家自然科学基金资助, No. 30100080
通讯作者: 陈耀凯,400038, 重庆市沙坪坝区高滩岩正街29号, 中国人民解放军第三军医大学西南医院全军感染病研究所. yaokaichen@hotmail.com
电话: 023-68754475-8006 传真: 023-65461319
收稿日期: 2005-01-28 接受日期: 2005-02-16
, http://www.100md.com
Construction and expression of a retroviral vectorpLNCTIGlox for reversible immortalization
Yao-Kai Chen, Jun-Gang Li, Yu-Ming Wang
Yao-Kai Chen,Jun-Gang Li, Yu-Ming Wang, Chinese PLA Institute of InfectiousDiseases, Southwest Hospital, the Third Military Medical University,Chongqing 400038, China
Supported by National natural Science Foundation of China,No.30100080
, 百拇医药
Correspondence to: Yao-Kai Chen, Chinese PLA Institute ofInfectious Diseases, Southwest Hospital, the Third Military MedicalUniversity, Chongqing 400038, China. yaokaichen@hotmail.com
Received: 2005-01-28 Accepted: 2005-02-16
AbstractAIM: Toconstruct a retroviral vector carrying selection marker gene NeoR,reporter gene EGFP and recombination site sequence LoxP for hepatocytereversible immortalization.
, 百拇医药
METHODS: 2.1 kbSV40T gene was inserted into vector pIRES2-EGFP to obtain a 3.4 kbfragment of SV40T-IRES-EGFP by restriction endonuclease digestion. Thefragment was further subcloned into retroviral vector pLNCX2 to obtain anew vector pLNCTIG. The linearized pLNCTIG was mixed with double strandedLoxP to construct pLNCTIGlox. Escherichia coli DH5awas transformed with pLNCTIGlox and positive clones were identified bycolony polymerase chain reaction (PCR) and restriction endonucleasedigestion. PT67 cells were transfected with pLNCTIGlox and the expressionof green fluorescence protein was observed. The transfected cells werestained immunochemically to detect the expression of SV40 large T antigen.
, 百拇医药
RESULTS: Amongthe 19 randomly selected colonies, 3 showed positive DNA bands onelectrophoresis gel. One of the three clones was further analyzed byrestriction endonuclease digestion and only one 9.5 kb band was separatedelectrophoretically, demonstrating that the analyzed clone was thepositive recombinant. Twenty-four hours after transfection, PT67 cellsemitted green fluorescence under the fluorescence microscope. The nucleiof the transfected cells were positive for immunochemical staining.
, 百拇医药
CONCLUSION: Theretroviral vector pLNCTIGlox for reversible immortalization wassuccessfully constructed and expressed.
Key Words:Reversible immortalization; Hepatocytes; pLNCTIGlox
Chen YK, Li JG, Wang YM. Construction and expression of a retroviralvector pLNCTIGlox for reversible immortalization. Shijie Huaren XiaohuaZazhi 2005;13(8):975-978
摘要
, 百拇医药
目的:为建立可回复性永生化肝细胞株，构建带有筛选基因NeoR、报道基因EGFP及重组位点loxP的可回复性永生化逆转录病毒载体.
方法:将2.1kb SV40T亚克隆到pIRES2-EGFP的相应位点，再酶切下3.4kb SV40T-IRES-EGFP片段插入逆转录病毒载体pLNCX2的两个相同酶切位点之间，构成新载体pLNCTIG.最后，将线性化pLNCTIG和loxP双链片段混合连接.取连接产物转化高效感受态DH5a细胞.菌落PCR和酶切法鉴定阳性克隆.转染包装细胞PT67并在荧光显微镜下观察绿色荧光蛋白的表达.扩大培养稳定转染的细胞，免疫组化染色检测SV40T基因的表达.
结果:随机挑取19个克隆，3个电泳分离出约1.1kb阳性条带.取上述阳性克隆进行酶切鉴定，结果仅出现一条约9.5kb的条带，证明该克隆为阳性重组体.转染PT67细胞24h后，荧光显微镜下可见散在多处绿色荧光.免疫组化染色显示细胞胞核呈阳性染色.
, 百拇医药
结论:成功构建并表达可回复性永生化逆转录病毒载体pLNCTIGlox.
关键词:可回复性永生化;肝细胞;pLNCTIGlox
陈耀凯,李俊刚,王宇明.可回复性永生化逆转录病毒载体pLNCTIGlox的构建与表达.世界华人消化杂志 2005;13(8):975-978
(PDF) 以egfpPa和loxPas为引物，菌落PCR法鉴定pLNCTIGlox重组阳性克隆. N: 阴性对照; M:DNA分子量标准; 1-19: pLNCTIGlox克隆1-19的菌落PCR产物，其中克隆2，14和19为阳性.
图2 (PDF) 酶切电泳鉴定pLNCTIGlox逆转录病毒载体. 1: 未酶切pLNCTIGlox载体质粒; 2: BamHⅠ单酶切pLNCTIGlox质粒; 3: BamHⅠ和XbaⅠ双酶切pLNCTIGlox质粒; 4: 未酶切pLNCTIG质粒; 5: BamHⅠ单酶切pLNCTIG对照; 6: BamHⅠ和XbaⅠ双酶切pLNCTIG对照; M: DNA分子量标准.
, http://www.100md.com
2.2EGFP基因的表达 转染后24h在荧光显微镜下观察，可见散在多处绿色荧光(图3).
2.3SV40大T抗原的免疫组化染色 经扩大培养的转染细胞胞核呈阳性染色(图4)，而作为阴性对照的未转染细胞则未见阳性反应.
图3 转染pLNCTIGlox载体后24 h 的PT67细胞, IF×100.
图4 转染pLNCTIGlox载体后的PT67细胞核呈阳性反应, SABC×400.
3 讨论采用两种限制性内切酶消化外源片段和质粒载体后进行连接，是质粒载体构建过程中最容易、最有效的克隆策略.我们将pLLTSN上的2.1kb SV40T插入到pIRES2-EGFP的EcoRⅠ和BamHⅠ位点之间即是采用这一方法.然而质粒与外源片段的酶切位点之间并非总是能找到“门当户对”的理想搭配关系，此时必须采取不同的策略.在本研究中我们拟在逆转录病毒载体pLNCTIG的3’长末端重复(LTR)序列的U3区插入loxP特异重组位点，但该处仅有一个XbaⅠ单酶切位点可供利用，因此只能采用单酶切连接策略插入loxP片段.用XbaⅠ酶切载体pLNCTIG后产生2个相同的粘端，通常要将其5’端去磷酸化，以减少载体自身环化导致较高的非重组体菌落背景.但若将载体pLNCTIG5’端去磷酸化，将无法与loxP双链的匹配末端连接，原因是loxP作为合成的DNA是缺少5’磷酸基团的.此时有两种选择可以采用:(1)将载体pLNCTIG5’端去磷酸化，再使合成的loxP双链DNA5’端磷酸化，然后进行连接.该方式的缺点是会使loxP片段在重组质粒中形成串联拷贝，不利于单拷贝重组体的筛选.(2)不将载体pLNCTIG5’端去磷酸化，直接与缺少5’磷酸基团的loxP双链DNA进行连接反应，在此过程中loxP片段在重组质粒中不会形成串联拷贝，阳性重组质粒只能是插入单拷贝loxP序列的克隆.我们采用第2种方式，为了减少载体自身环化，我们将载体与外源loxP片段的摩尔比提高到1∶8，以减少非重组体菌落背景.结果成功，并已筛选出正向插入XbaⅠ酶切位点的阳性克隆.由于loxP双链DNA粘端与XbaⅠ酶切粘端连接后，改变了原来的酶切位点序列，XbaⅠ酶切位点不复存在，用XbaⅠ酶不能切开重组质粒.用BamHⅠ和XbaⅠ酶切重组质粒和pLNCTIG，前者只能被BamHⅠ酶切线性化，而后者可被BamHⅠ和XbaⅠ酶切为约1.7kb和7.8kb大小的两个片段.实际结果与理论预期完全符合.
, 百拇医药
Cre/LoxP是最常用的基因位点重组系统，最早在噬菌体P1的研究中被发现[8].LoxP位于噬菌体P1染色体交叉部位，由两个反向重叠序列(revertedrepeat)和一个8bp间隔区组成.Cre是催化位点重组的酶，与反向重叠序列结合，剪切两个LoxP之间的DNA序列.本研究成功构建带有目的基因、筛选基因及LoxP重组位点的质粒载体，并进行蛋白表达与鉴定，为下一步进行细胞永生化奠定了良好的基础.
4 参考文献 1 Kobayashi N, Okitsu T, Nakaji S, Tanaka N.Hybrid bioartificial liver:establishing a reversibly immortalizedhuman
hepatocyte line and developing abioartificial liver for practical use. J Artif Organs 2003;6:236-244
, http://www.100md.com
2 Kobayashit N, Tanaka N. Engineering ofhuman hepatocyte lines for cell therapies in humans:prospects and
remaining hurdles. Cell Transplant 2002;11:417-420
3 Sato Y, Endo H, Ajiki T, Hakamata Y, OkadaT, Murakami T,Kobayashi E. Establishment of Cre/LoxP recombination
system in transgenic rats. BiochemBiophys Res Commun 2004;319:1197-1202
4 Watanabe N, Odagiri H, Totsuka E, Sasaki M.A new method to immortalize primary cultured rat hepatocytes.
, 百拇医药
Transplant Proc 2004;36:2457-2461
5 Wege H, Le HT, Chui MS, Liu L, Wu J, GiriR, Malhi H, Sappal BS, Kumaran V, Gupta S, Zern MA. Telomerase
reconstitution immortalizes human fetalhepatocytes without disrupting their differentiation potential.
Gastroenterology 2003;124:432-444
6 Kobayashi N, Noguchi H, Westerman KA,Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Leboulch P,Tanaka N. Cre/loxP-based reversibleimmortalization of human hepatocytes. Cell Transplant 2001;10:383-386
, 百拇医药
7 Noguchi H, Kobayashi N, Westerman KA,Sakaguchi M, Okitsu T, Totsugawa T, Watanabe T, Matsumura T,Fujiwara T, Ueda T, Miyazaki M, Tanaka N,Leboulch P. Controlled expansion of human endothelial cell populationsby
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