Construction of recombinant adenoviral vectors carrying antisense RNA to dualª²target chemokine receptors CCR5 and CXCR4

    LI Wenª²Gang, SHAO Yi, CHEN Hong, WANG Yanª²Bin, YU Min, XU Xiaoª²Yuan

    1Department of Infectious Disease, First Hospital, Lanzhou Medical College, Lanzhou 730000, China, 2Department of Infectious Disease, First Hospital, Peking University, Beijing 100034, China, 3Beijing Ditan Hospital, Beijing 100038, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant adenoviral vector carrying antisense RNA to chemokine receptors CCR5 and CXCR4 and to obtain recombinant adenovirus, which will be used to resist HIVª²1 infection. METHODS: The 653 bp and 636 bp DNA fragments targeted to the initial part of CCR5 and CXCR4 mRNA 5¡ä sides translation were obtained by RTª²PCR from peripheral blood mononuclear cells (PBMCs) and were conversely inserted into adenoviral vector pAdTrackª²CMV. The homologous recombination with adenovirus backbone plasmid pAdEasyª²1 was performed in BJ5183 bacteria and the recombinant vector was selected by antiª²kanamycin plate. The recombinant vector was packaged and amplified in 293 cells and the expression of GFP was observed by fluorescence microscope. The recombinant adenovirus was detected by PCR and purified by CsCl density gradient centrifugation. RESULTS: The recombinant adenoviral vector carrying antisense RNA to CCR5 and CXCR4 had been constructed and recombinant adenovirus had been obtained and accurately detected. Its tier was 7.2¡Á1012 PFU/mL. CONCLUSION: The construction of the recombinant adenovirus carrying antisense RNA to CCR5 and CXCR4 lays the basis for the study of its inhibitive effect on HIVª²1 infection. ......