Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.