Construction Of Expression Vector Of SiRNA Specific For Tim-1 And Study On The Expression Of Tim Family Moleculein Hepatitis Model Mice

Aim:To construct Tim-1HA eukaryotic expression vector and siRNA expression vectors which could inhibit the expression of Tim-1 and study the expression of Tim family molecule in concanavalin A-treated mice and HBV transgenic mice. Methods:1. Expression of Tim-IHA in HepG2 cell lineTo construct the expression plasmid of Tim-IHA, cDNA fragment encoding mouse Tim-1 was amplified by RT-PCR with the designed primer containing HAtag at 3' end from BALB/C spleen cells. The product was cloned into pTARGE-T vector. Recombinant plasmid pTARGETim-1HA was digested and sequenced, and was delivered into HepG2 cell line by liposome-mediated transfection. The expression of Tim-IHA was determined by RT-PCR and Western blot.2. Construction of Tim-1 siRNA expression vectors and its inhibitory effect on Tim-IHA expressionFour pairs of oligonucleotide targeting to Tim-1were designed and synthesized. After annealing, the oligonucleotides were cloned into pAVU6+27 vector under U6 promoter. The recombinants were double digested and sequenced. To detect the inhibitory effect of these recombinant siRNA expression vector on the expression of Tim-1, Tim-IHA expression plasmid and siRNA expression vectors were cotransfected into hepatomocyte HepG2 cell line. The inhibitory effect of siRNA expression vectors was assayed by RT-PCR and Western blot 72 hours post transfection.3. Concanavalin A -induced hepatitis in mice...