Abstract:

Objective To construct the recombinant lentivirus carrying chemokine receptor 5 (CCR5)-short hairpin RNA (shRNA), and investigate its effects on the expression of CCR5 mRNA of CD34+ hematopoietic stem cells (CD34+ cells) in peripheral blood of mice. Methods RNA interference (RNAi) target sequence was designed by Ambion RNAi target sequence and references. The target sequence was amplified after transduction into plasmid pBSHH1, and was transducted into FG12 lentiviral vector containing enhanced green fluorescent protein (EGFP). The recombinant lentivirus of CCR5-shRNA was packaged, and the virus titer was determined. The recombinant lentivirus was transducted into CD34+ hematopoietic stem cells in peripheral blood of mice, and the transduction efficiency was measured. Then, CD34+ cells transfected with CCR5-shRNA lentivirus were injected into mice, and the expression of CCR5 mRNA was detected by Real-Time PCR after one week. Results The lentivirus was verified to carry both RNAi target sequence and H1 RNA polymerase III gene. CCR5-shRNA lentiviral vector was successfully constructed. The lentiviral infection titer was 5×10⁷ TU/mL. The efficiency of CCR5shRNA lentivirus in transfection of CD34+ hematopoietic stem cells in peripheral blood of mice was 97.9%. Real-Time PCR revealed that the expression of CCR5 mRNA in CD34+ cells in peripheral blood of mice significantly decreased after CD34+ cells transfected with CCR5-shRNA lentivirus were injected into mice. Conclusion The recombinant CCR5-shRNA lentivirus of high titer is successfully constructed, which effectively reduces the expression of CCR5 mRNA in CD34+ cells in peripheral blood of mice and lays a foundation for the treatment of rejection after organ transplantation.

Key words: lentiviral vector, RNA interference, organ transplant, chemokine receptor 5