Construction of double interference vector targeting both survivin and hTERT gene and its inhibitory effect on human colon cancer cell line SW480

Objective To construct the double interference vector targeting both survivin and hTERT gene and detect its silencing effects on survivin and hTERT gene in human colon carcinoma SW480 cell line.Methods Two effective targeting sequences were chosen according to hTERT and survivin cDNA sequences in Gen Bank,Then they were inserted into pGenesil-1.1 and pGenesil-1.2,pGenesil-1.1-survivin and pGenesil-1.2-hTERT were constructed.The recombinant plasmids were identified by restriction endonuclease and DNA sequencing.pGenesil-1.1-survivin and pGenesil-1.2-hTERT were digested by Hind Ⅲ and BamH Ⅰ separately,long fragment of pGenesil-1.1-survivin and small fragment of pGenesil-1.2-hTERT were collected.Then long fragment and small fragment were connected with T4 DNA ligase orientation,the double interference vector pGenesil1.1-survivin-hTERT was constructed.These siRNA recombinant expression vector separately were transfected into SW480 cell line,The effect of siRNA recombinant expression vector was detected by RT-PCR and MTT.Results By restriction endonuclease and DNA sequencing,specific RNAi expression vector targeting survivin and hTERT gene was correctly constructed.The growth rate of SW480 cells transfected with siRNA recombinant expression vector was significantly decreased.Conclusions The multiple siRNA recombinant expression vector targeting hTERT and survivin gene simultaneously has been constructed successfully.This result could provide theoretical information for the further research.