Construct And Screen Artificial MiRNA Based On The Gene Of Ccr5, Vif And Pol

Backgroud and Objective: RNAi is commonly useful in the research of HIV,constrcting effective and safe anti-hiv elments is the base. siRNAs or miRNAs can attactthe target mRNA specificlly, cause its degradation or the translation inhibition. CCR5orCXCR4is essencial when HIV enter the cell. CCR5deletion32decrease or resist theinfection of HIV, and the body is normal. Vif is the infection core, and maybe influentthe effecion of HIV, the production of HIV, and the propagation. Pol code many enzymes,such as reverse transcriptase enzyme, protein enzyme, is important to copy of HIV. Thestudy choose ccr5, vif, pol as target gene, and consruct miRNA, screen the better silentmiRNAs.Method: First, key sequences were selected based on the conserved regions in theHIV-1ccr5, vif, pol gene. Synthesize the sequence, transfer on the carrier, transfect the293T cells and choose highly expressing CCR5, Vif, or Pol ones respectively. Second,according to the gene sequence design the primer and synthesize four pairs of miRNAtargeting ccr5, vif, and pol, respectively. Third, The target sequence in the stem-loopstructure of the well-characterized native miR-155was replaced with ccr5, vif, polsequences, and the artificial miRNA expression vectors were co-transfected with theHIV-1clone, results showed that HIV-1can be effectively inhibited by miRNA. TargetmiRNA was confirmed by co-transfection assay with reporter plasmids containingdifferent target sequences. screen the most silent ccr5-miRNA, vif-miRNA andpol-miRNA by the method of qPCR.Result：After the interference effect evaluation we have obtained the most silentccr5-miRNA, vif-miRNA and pol-miRNA, and the interference are72%,68%and76%.Conclusion：First we get HEK293highly expressed ccr5,vif or pol, then screen themost silent miRNA of ccr5, vif, and pol, the silence are72%,68%and76%respectively.