大肠杆菌内同源重组法构建含mIκBα基因的重组腺病毒及其在Hep G2细胞中的表达

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (03): 308-312.

大肠杆菌内同源重组法构建含mIκBα基因的重组腺病毒及其在Hep G2细胞中的表达

金吴东¹, 陈龙华¹, 穆峰²

1. 南方医科大学南方医院放疗科, 广东, 广州, 510515;
2. 南方医科大学南方医院胸心外科, 广东, 广州, 510515

出版日期:2005-03-20 发布日期:2005-03-20
基金资助:
收稿日期:2004-9-17。
基金项目:广东省医学科学技术研究基金(A437)
作者简介:金吴东(1962- ),博士,讲师,电话:020-61642136,E-mail:jwd18@21.com

Construction of recombinant adenovirus containing mIκBα gene by homogenous recombination in E.coli. and its expression in Hep G2 cells

JIN Wu-dong¹, CHEN Long-hua¹, MU Feng²

1. 南方医科大学南方医院放疗科, 广东, 广州, 510515;
2. 南方医科大学南方医院胸心外科, 广东, 广州, 510515

Online:2005-03-20 Published:2005-03-20

摘要/Abstract

摘要： 目的通过在大肠杆菌内同源重组,快速、有效地制备含人mIκBα基因的腺病毒重组体的方法,并观察其在肝癌细胞株Hep G2细胞中的表达。方法首先将目的基因mIκBα克隆进结合有绿色荧光蛋白报告基因的穿梭质粒-pAdTrack-CMV中,将新形成的质粒pAdTrack-CMV-mIκBα用限制性内切酶Pme I线性化后,再与腺病毒骨架质粒-pAdEasy-1一同转化进大肠杆菌株BJ5183细胞中。用对卡那霉素抗性挑选重组体质粒-pAd-mIκBα,并通过限制性内切酶分析,确认重组。最后将线性化后的重组体质粒转染进腺病毒包装细胞株293细胞。腺病毒重组体Ad-mIκBα在7~10 d内产生。借助GFP的表达,测定和观察在293细胞中重组腺病毒的滴度及其在Hep G2细胞中的表达。结果经PCR检测提示重组体腺病毒含有mIκBα基因。Ad-mIκBα的滴度是2.7×109 PFU/ml。当MOI等于10时,在Hep G2细胞中,Ad-mIκBα病毒可有效和稳定地表达,其感染效率24 h为57%,48 h达100%。结论运用在大肠杆菌内同源重组法能有效和方便地构建含目的基因mIκBα的腺病毒重组体Ad-mIκBα。该重组体能在293细胞中扩增,并有效地感染Hep G2细胞。为进一步研究mIκBα基因的功能和该基因治疗肝癌提供了一个良好的基因转导载体。

Abstract: Objective To develop a rapid and efficient method for preparing recombinant adenovirus containing human mIκBα gene by homogenous recombination in E.coli. and detect its expression in Hep G2 cells.Methods The mIκBα gene was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene, followed by linearization of the resultant plasmid pAdTrack-CMV- mIκBα by Pme I digestion and subsequent cotransformation into E.coli BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-mIκBα was selected for kanamycin resistance and confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into 293 cells, in which Ad- mIκBα were generated within 7 to 10 days. The virus titer in 293 cells and its infection efficiency in Hep G2 cells were detected and calculated with the aid of GFP expression.Results PCR indicated that the recombinant adenovirus contained mIκBα gene and the titer of Ad-mIκBα was 2.7×109 PFU/ml. With a multiplicity of infection (MOI) of 10, Ad-mIκBα could be expressed stably and efficiently in Hep G2 cells and the infection efficiency was 57% at 24h and 100% at 48h after transfection.Conclusions Homogenous recombination in E.coli can efficiently and conveniently construct recombinant adenovirus containing mIκBα gene capable of amplification in 293 cells and efficient infection of Hep G2 cells. The recombinant adenovirus may serve as a good gene transfer vector for study the function of mIκBα gene and therapy for hepatocarcinoma.

中图分类号:

R373.2
R-33

金吴东¹, 陈龙华¹, 穆峰². 大肠杆菌内同源重组法构建含mIκBα基因的重组腺病毒及其在Hep G2细胞中的表达[J]. 南方医科大学学报, 2005, 25(03): 308-312.

JIN Wu-dong¹, CHEN Long-hua¹, MU Feng². Construction of recombinant adenovirus containing mIκBα gene by homogenous recombination in E.coli. and its expression in Hep G2 cells[J]. Journal of Southern Medical University, 2005, 25(03): 308-312.

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