Construction and identification of recombinant adenovirus with GFP labeled protein Ⅸ
10.3760/cma.j.issn.1003-9279.2016.02.023
- VernacularTitle:GFP标记Ⅸ蛋白的重组腺病毒的构建与鉴定
- Author:
Xiaohui ZOU
1
;
Xiaojuan GUO
;
Min WANG
;
Jianguo QU
;
Zhuozhuang LU
;
Tao HONG
Author Information
1. 中国疾病预防控制中心病毒病预防控制所
- Keywords:
Adenovirus;
capsid protein Ⅸ;
GFP;
fluorescent labeling
- From:
Chinese Journal of Experimental and Clinical Virology
2016;30(2):204-207
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the role of protein Ⅸ(pⅨ) in the interaction between adenovirus and host cell dynamically,we attempt to construct a recombinant human adenovirus with the capsid protein pⅨ being labelled with Green Fluorescent Protein (GFP).Methods GFP gene was inserted into the downstream of pⅨ genein the shuttle plasmid to form pShuttle-IXGFP;and adenoviral plasmid pAd5-IXGFP was generated by homologous recombination of pShuttle-IXGFP with the backbone plasmid (pAdeasy-1) in E.coli BJ5183 strain.Recombinant adenovirus (Ad5-IXGFP) was rescued by transfecting 293 cells with the linearized pAd5-IXGFP.Results After 4 rounds of propagation in 293 cells,viruses were purified with CsCl ultracentrifugation method.Ad5-IXGFP in 1.0 ml with a particle titer of 6.9 × 1011 vp/ml was finally obtained.PCR was performed to confirm the fusion of Ⅸ with GFP gene in the genome of Ad5-IXGFP.Virions were incubated with suspended HEp-2 cells at 4 C for 5 h,and the attachment of Ad5-IXGFP on the cellular membrane could be observed under fluorescence microscope.Conclusion GFP-labelled recombinant adenovirus was successfully constructed,which could be a valuable tool for subcellular localization fo pⅨ after virus entry real-time single adenovirus tracking by confocal fluorescence microscopy.
