Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene |
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Authors: | GUO Xiao-ti DENG Yu-bin LU Cai-sheng LI Shu-nong |
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Institution: | 1. Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University, Guangzhou 510080, China;
2. Department of Rheumatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China |
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Abstract: | AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×1012 phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases. |
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Keywords: | T-lymphocytes cytotoxic Immunoglobulins Adenoviral vector Gene therapy |
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