Construction and identification of recombinant adenovirus vector containing mouse CD200 gene

Abstract:

Objective：To construct the recombinant adenovirus vector containing mouse CD200 gene and to detect its expression in mice pancreas endothelial cells（mile sven 1，MS1）. Methods：mCD200 gene was subcloned into the shuttle plasmid pYr-adshuttle-4 and recombinant plasmid pYr-ads-4-mCD200 was acquired. After that mCD200 mRNA expression frame was transfered to pAd/PL-DEST adenovirus vector via LR（attL×attR） in vitro homologous recombination from pYr-ads-4-mCD200. Recombinant adenovirus plasmid was digested by PacⅠand transfected into HEK 293 cells and packaged out recombinant adenovirus rAd-4-mCD200，which was identified by digestion and PCR analysis. Virus titer was measured by the tissue cell infectious dosage 50（TCID50） method. Meanwhile，adenovirus was amplified in HEK 293 cells and used to infect MS1 cells. Expression of mCD200 gene was measured by fluorescence microscope and real-time PCR. Results：PCR identification demonstrated the construction of recombinant adenovirus car－rying rAd-4-mCD200 was successful and its titer was about 109-1010 pfu/ml after amplification. Through the fluorescence microscope and real-time PCR，recombinant adenoviral vector of mCD200 gene was constructed successfully in the MS1 cells. Conclusions：MS1 cells infected by constructed adenovirus vector rAd-4-mCD200 could express mCD200 successfully and may provide the foundation for the next step to carry out the CD200 gene immunosuppression regulation and other related study.