Construction of double siRNA expressing vector of Coxsackie virus B4 / 吉林大学学报(医学版)

ABSTRACT

Objective To construct the double hairpin siRNA and green fluorescent protein(GFP) expressing vector pU6/double-siRNA/Neo/GFP/1A/2A to interfere 1A and 2A gene of Coxsackie virus B4.Methods 21 bp fragments of the Coxsackie virus B4 2A and 1A gene were chosen as the targets and 65 bp complimentary fragments were synthesized,then the target gene fragments were cloned into pSilencer2.1U6 Neo and pGCsi-U6/Neo/GFP/siNeGative,respectively,then the double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A /2A was constructed by restrict endonuclease digestion,elctrophoresis isolation and reclaimer,ligatied by T4 DNA ligase;then the double siRNA expressing plasmid was transfected into Hela cells,and the GFP was observed under fluorescent microscope.Results The correct results showed that the recombinant plasmid had the correct special fragments and DNA sequence detected by restrict endonuclease digestion, electrophoresis and DNA sequencing;and GFP was also observed in Hela cells tansfected with pU6/double-siRNA/Neo/GFP/1A /2A under fluorescent microscope more than 15 d after transfection.Conclusion The double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A/2A is constructed successfully;it has the correct target viral gene sequences and can express GFP gene in Hela cells more than 15 d after transfection.