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[Construction of recombinant vectors expressing antisense RNA to CCR5 and expression in eukaryotic cells]
Huichun Xing1, Xiaoyuan Xu, Qinhuan Wang
1Department of Infectious Disease, Peking University First Hospital, Beijing 100034, China.
Background:
To construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection.
Methods:
The DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell.
Results:
The psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA.
Conclusions:
The gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.
