Construction and identification of RNA interference lentiviral vector targeting CD40 gene in rats / 中华实用儿科临床杂志

ABSTRACT

Objective To construct the RNA interference (RNAi) vector targeting the CD40 gene in rats.Methods Effective target sequences that target at CD40 gene were designed,then the oligonucleotide sequences after annealing of the complementary strands were synthetized,the DNA fragments were connected to the GV118 vectors by double digestion with HpaI and XhoI,and the lentiviral vectors which expressed short hairpin RNA were constructed.The lentiviral vectors were identified by PCR and DNA sequencing.Western blot was employed to sort the best interfering targets exogenously,and lentiviral packaging as well as assaying viral titer were also accomplished.Results CD40 siRNA was successfully inserted into the lentiviral vectors and the lentiviral vector was packaged into 293T cells.The determination of virus titer was 2.0 × 1012 TU/L.Conclusions The RNA interference lentiviral vector targeting the CD40 gene in rats was constructed successfully,which will provide the foundation for further exploring the pathogenesis of viral myocarditis and novel therapies in future.