Constructi
Construction and identification of a recombinant adenovirus vector expressing His-tag-ICP47 fusion gene
WANG Peng1 , KAN Quan-cheng2, YU Zu-jiang2, LI Ling3 , ZHANG Zhenxiang1 and PAN Xue1
1.Nursing College, Zhengzhou University, Zhengzhou, Henan 450052, China (Wang P, ZHANG ZX and Pan X)
2.Clinical Pharmacology Base (Kan QC), Department of Infectious Disease (Yu ZJ), First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan 450052, China
3.Department of Palliative Care and Hospice Care, the Ninth People’s Hospital of Zhengzhou, Zhengzhou, Henan 450053, China (Li L)
Abstract: Background: The response of host immune systems against gene products expressed by genetically modified cells is a major obstacle to successful gene therapy. Major histocompatibility complex (MHC) classⅠantigen presenting pathway is very important in acute allograft rejection and blocking MHCⅠantigen expression is becoming a research hotspot of inducting immune tolerance. Infected cell protein 47 (ICP47) expressesd by herpes simplex virus type 1 (HSV-1), inhibits MHCⅠantigen presentation pathway by binding to host transporter associated with antigen presentation (TAP), and thereby reduces the rate of cytolysis of infected cells by sensitized CTL and evades the host immune clearance. The aim of this study is to construct and identify a recombinant adenovirus expressing His-tag-ICP47 fusion gene for the following studies of its immunological activities. Methods: The adenoviral vector system of AdEasy-1was used to prepare the recombinant adenovirus expressing His-tag-ICP47 fusion gene or the control empty recombinant adenovirus r-Track. His-tag-ICP47 fusion gene was firstly cloned into the pAdTrack-CMV vector. The gene fragments digested by PmeⅠwere co-transformed with adenoviral backbone vector pAdEasy-1 in E.coli BJ5183 cells to produce recombinant adenoviral vector pAdEasy-H-ICP47. Linearized with PacⅠ, recombinant adenoviral vector was subsequently transfected into 293 cells to product r-H-ICP47. Meanwhile, the control empty recombinant adenovirus r-Track was built in the same way. Finally, The viruses were amplified and virus particle titres were determined. Purified virus was analyzed for the presence and expression of His-tag-ICP47 fusion gene by PCR and Western-blot analysis.Results: The recombinant adenoviruses of r-H-ICP47 and r-Track were successfully constructed and successfully transfected into 293 cells and virus granules appeared. The virus particle titres of r-H-ICP47 and r-Track were determined with the resulting of 3.7×1010efu/mL and 4.4×1010efu/mL The proteins producted by recombinant adenovirus-infected cells were confirmed by PCR and Western-blot analysis. Conclusion: The replication-defective recombinant adenoviruses of r-H-ICP47 and r-Track are successfully constructed, and the virus particle titres are highly enough to infect and express relevant genes in cells at a desired level, respectively.
[WANG Peng , KAN Quan-cheng, YU Zu-jiang, LI Ling , ZHANG Zhenxiang and PAN Xue. Construction and identification of a recombinant adenovirus vector expressing His-tag-ICP47 fusion gene. Life Sci J 2012;9(1):756-763] (ISSN:1097-8135). http://www.lifesciencesite.com. 109
Key Words: recombinant adenovirus; infected cell protein 47; fusion
