南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (11): 1552-1555.

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IP-10重组腺病毒载体的构建及其病毒制备

邵紫韫； 刘志锋； 彭毅； 徐佳； 邓鹏； 姜勇；   

  1. 南方医科大学基础医学院广东省功能蛋白质组学重点实验室； 南方医科大学基础医学院广东省功能蛋白质组学重点实验室 广东广州510515； 广东广州510515；
  • 出版日期:2006-11-20 发布日期:2006-11-20
  • 基金资助:
    广东省自然科学基金重点项目(13058);广东省科技计划项目(A1090202);广州市科技计划项目(2001-z-035-01-1)~~

Construction of a recombinant adenovirus vector for interferon-γ-inducible protein 10 and the adenovirus preparation

SHAO Zi-yun,LIU Zhi-feng,PENG Yi,XU Jia,DENG Peng,JIANG YongKey Laboratory of Functional Proteomics of Guangdong Province,Southern Medical University,Guangzhou 510515,Chin   

  1. 南方医科大学基础医学院广东省功能蛋白质组学重点实验室； 南方医科大学基础医学院广东省功能蛋白质组学重点实验室 广东广州510515； 广东广州510515；
  • Online:2006-11-20 Published:2006-11-20

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摘要： 目的利用细菌内同源重组法构建带有增强型绿色荧光蛋白(EGFP)标签的!-干扰素诱导蛋白10(IP-10)重组腺病毒载体并制备重组腺病毒。方法将IP-10编码序列克隆入带有EGFP示踪的腺病毒穿梭质粒pAdTrack-CMV中,形成转移载体pAdTrack-CMV/IP-10,将其在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组,得到重组腺病毒载体pAd/IP-10;酶切鉴定正确后转染HEK293细胞。收集病毒上清,PCR鉴定正确后,扩增并再次感染HEK293细胞检测病毒感染能力。结果重组质粒经酶切、PCR和测序鉴定正确无误,并在HEK293细胞中包装成有感染活性的病毒颗粒。结论成功地构建了表达IP-10蛋白的重组腺病毒载体并制备了重组腺病毒Ad/IP-10,为研究IP-10的蛋白功能提供了一个重要工具。 

Abstract: Objective To construct a recombinant adenovirus vector for expressing interferon-γ-inducible protein 10(IP-10) by homogenous bacterial recombination.Methods IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein(EGFP).The shuttle plasmid was then transformed into E.coli BJ5183 with pAdEasy-1 vector by chemical transformation.The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells.Results The positive clones were identified with enzyme digestion and polymerase chain reaction(PCR) and were further verified by DNA sequencing.The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells.Conclusion A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein. 

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