Construction and evaluation of short hairpin RNA interference vector targeting CP> gene of Epinephelus coioides nervous necrosis virus

The short hairpin RNA interference sequences targeting CP gene of red-spotted grouper nervous necrosis virus were designed by using online designing software of Invitrogen Co.,Ltd.The structure consisted of 19 nt sense strand,-4 nt loop and -19 nt antisense strand.These sequences are synthesized,annealed,and cloned into pENTR^TM/U6 to construct the shRNA interference vectors named pshRNA-124,pshRNA-896 and pshRNA-NNV,respectively.Then the expression vector of pEGFP-CP and each of the three shRNA interference vectors are co-transfected into FHM cells by using Lipofectamine 2000.Then the expression and inhibition of EGFP are observed by using fluorescent microscope.The mRNA expression level of CP gene is anaylised by using real time RT-PCR.The result shows that pshRNA-124,pshRNA-896 and pshRNA-NNV all suppress the EGFP expression with inhibitory efficiency of 47%,68% and 51%,respectively.The pEGFP-CP gene silencing efficiencies of pshRNA-124,pshRNA-896 and pshRNA-NNV at mRNA level are 60%,96% and 55%,respectively,significantly higher than that of control(P<0.05).It demonstrates that all of the three shRNAi vectors have interference effects and can interfere with the expression of EGFP,of which pshRNA-896 is the best one.The result above suggests that shRNAi vectors targeting CP gene of red-spotted grouper nervous necrosis virus has been successfully constructed,which will be a basis for further study of the function of CP gene with RNAi technique.