Construction and purification of the recombinant adenoviral vector carrying antisense RNA to chemokine receptor CCR5

Objective To construct the recombinant adenoviral vector carrying antisense RNA to chemokine receptor CCR5 and obtain recombinant adenovirus which will be used to resist HIV-1 infection. Methods The 653pb DNA fragment targeted to the initial part of CCR5 mRNA5' side translation obtained by RT-PCR from peripheral blood mononuclear cells(PBMCs) was inserted into adenoviral vector pAdTrack-CMV in a converse way.Then its homologous recombination with adenovirus backbone plasmid pAdEasy-1 was performed in BJ5183 bacteria,and recombinant vector was selected by anti-kanamycin 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 gradien centrifugation. Result The recombinant adenoviral vector carrying antisense RNA to CCR5 had been constructed and recombinant adenovirus had been obtained.Its tier was 5×10 11 PFU/ml. Conclusion The recombinant adenovirus carrying antisense RNA to CCR5 has laid down a good foundation for studying its inhibiting effect on HIV-1 infection.