Construction and identification of recombinant adenovirus containing apM1

LI Bing-rong; et al

doi:10.13406/j.cnki.cyxb.2010.11.003

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Construction and identification of recombinant adenovirus containing apM1

更新时间：2024-01-17

- Construction and identification of recombinant adenovirus containing apM1
- Journal of Chongqing Medical University Vol. 35, Issue 11, Pages: 1676-1680(2010)
- 作者机构：
  
  重庆医科大学附属第一医院内分泌科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.11.003
  CLC： Q781
- Published：2010，
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[1]李丙蓉,郑宏庭,邓华聪,郑丹,刘金波,兰丽珍,程伟.人脂联素基因重组腺病毒的构建与鉴定[J].重庆医科大学学报,2010,35(11):1676-1680.

LI Bing-rong, et al. Construction and identification of recombinant adenovirus containing apM1. [J]. Journal of Chongqing Medical University 35(11):1676-1680(2010)
[1]李丙蓉,郑宏庭,邓华聪,郑丹,刘金波,兰丽珍,程伟.人脂联素基因重组腺病毒的构建与鉴定[J].重庆医科大学学报,2010,35(11):1676-1680. DOI： 10.13406/j.cnki.cyxb.2010.11.003.

LI Bing-rong, et al. Construction and identification of recombinant adenovirus containing apM1. [J]. Journal of Chongqing Medical University 35(11):1676-1680(2010) DOI： 10.13406/j.cnki.cyxb.2010.11.003.

摘要

目的:构建含有人脂联素基因apM1的重组腺病毒载体

制备能表达人脂联素蛋白的重组腺病毒

为脂联素用于体内外干预动脉粥样硬化奠定基础。方法:自质粒pGEM-T-apM1上扩增两端带有SalⅠ和Hind Ⅲ酶切位点的全长apM1基因

将PCR产物和穿梭质粒pShuttle-CMV经SalⅠ和Hind Ⅲ双酶切后

连接、转化、筛选

进行PCR鉴定后送双向测序。将鉴定正确的重组质粒pShuttle-CMV-apM1用PmeⅠ酶切使其线性化后与腺病毒骨架质粒pAdEasy-1共转BJ5183菌

进行细菌内同源重组

筛选、鉴定、扩增

PacⅠ酶切后用LipofectamineTM2000转染低代数的HEK293包装细胞

进行包装出毒。结果:重组穿梭质粒pShuttle-CMV-apM1经PCR和测序鉴定正确

与腺病毒骨架质粒pAdEasy-1同源重组成功

转染293细胞进行包装

产生的病毒经鉴定正确

并且无具有复制能力的腺病毒存在

生物安全性高。结论:成功的制备了apM1基因重组腺病毒

可进一步用于脂联素干预动脉粥样硬化的研究。

Abstract

Objective:To construct recombinant replication-deficient adenovirus containing apM1 gene for the gene therapy research on atherosclerosis.Methods:The shuttle plasmid pShuttle-CMV-apM1 in which apM1 cDNA was inserted into the downstream of CMV promoter was established by DNA recombination.Then the linearized shuttle plasmid was cotransformed into colibacillus BJ5183 with adenovirus vector pAdEasy-1 to generate the recombinant replication-deficient adenovirus plasmid pAd-apM1 through homologous recombination.The linearized pAd-apM1 was transferred into HEK 293 cells with lipofectamine2000 for packing

and the recombinant replication-deficient adenovirus

Ad-apM1

was finally obtained.Results:The recombinant shuttle plasmid pShuttle-CMV-apM1 was confirmed by specific PCR and sequencing.CPE(cytopathic effect) could be observed on the eighth day after packing the linearized pAd-apM1 in HEK293 cells.The recombinant adenovirus generated had no replication capability. Conclusion:The replication-deficient recombinant adenovirus containing apM1 gene was successfully constructed

which can be further studied for gene therapy.

关键词

apM1基因腺病毒同源重组

Keywords

apM1 geneadenovirusHomogenous recombination

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