Inhibition Of Marek's Diseases Virus Replication By RNA Interference

Different DNA sequences targeted to UL49(encoding tegument protein VP22) gene of RBIB strain of Marek's diseases virus type 1 for small hairpin RNAs(shRNA) are designed by consulting the successful experience of Ambion and Nippon Bioservice companies and utilizing web-based tool. At the same time,negative shRNA is designed according to the correspounding principles for negative design.By BLAST analysis ,the selected sequences aren't the same as the other ones of chick and the ones of other viruses .The sense strand of siRNA was composed of the start 19 nucleotides of the 5' end of target sequence . An TTCAAGAGA was added to between sense strand and ansense strand,and an TTTTTT was added to the 3' end of target sequence as stop codes of the U6 promoter. Thus the target sequence was transcribed into a small hairpin RNA. Besides BamH I(GATCC) was designed at the 3'end of target sequence, Sal I (GTCGAC) and Hind III(TTCGA)were designed at the 5'end of target sequence.After these target sequences were cloned into psilence2.0-U6 respectively, pu6vp22-1, pu6vp22-2, pu6vp22-3, pu6vp22-4, pu6vp22-5, pu6vp22-6 and pu6vp(negative control) vectors expressed shRNAs were constructed.Then these vectors were digested with Sal I and were measured sequences individually .The results demonstrated the different vectors expressed shRNAs(small hairpin RNA )targeting UL49 were constructed successfully.SimultaneousIy,pu6EGFP vector (as positive control) inhibited the expression of EGFP protein was constructed according to the siRNA sequence published by Ambion company.With the help of Primer 5.0 software a pair of primers was designed accoding to the sequence of UL49 gene of Marek's diseases virus type 1 Md5 strain published in the GenBank .And UL49 (750bp)was amplified by polymerase chain reaction (PCR) with extracted total DNA of MDVtype 1 RBIB strain.And the UL49 was cloned into pMD18-T Simple Vector,PMDvp22 was constructed . Then the pMDvp22 was digested with BamHI and Hindlll, UL49 was cloned into pEGFP-Cl,accordingly pEGFP-vp22 fusion expressin vector was constructed .After the pEGFP-vp22 plasmid DNA was transfected into secondary chicken embryonic fibroblasts(CEF )cells ,the fluorescence in CEF cells was observed at 24, 48, 72 hours respectively.The result was showed the fluorescence in CEF cells was detected at 24 hours, the most at 48 hours and was gradually decreased andweakened at 72 hours. Besides the number of fluorescence in CEF cells changed with time ,they localized in both the cytoplasmic and nuclear compartments at 24 hours ,mainly in nuclear compartments at 48 hours and completely in the nuclear compartments.This differed dramatically from the cytoplasmic distribution of GFP alone after pEGFP-Cl plasmid DNA was transfected into CEF cells ,which was found diffusely throughout the cytoplasm,and the fluorescence of EGFP in CEF cells was gradually decreased and weakened with time .In order to detect inhibition effect,different shRNA expression vectors were respectively transfected into CEF cells with pEGFP-vp22, the fluorescence in CEF cells was observed at 24 , 48, 72 hours.The results were showed the fluorescence in CEF cells treated pu6vp22-l or pu6vp22-4 plasmid trended to both decrease and weaken at 48 hours after transfection,especially the fluorescence in CEF cells treated pu6vp22-4 was too few to be seen But negative control didn't show inhibition effect. Based on this, the cDNA of VP22 gene was derived from cells at 60 hours after transfection and were performed with semi-quantitative PCR.The expression VP22 gene of pu6vp22-l + pEGFP-vp22 and pu6vp22-4+ pEGFP-vp22 reduced by 69% and 93% compared to that of pEGFP-vp22. The inhibition effect of different shRNA expression were validated at the level of mRNAIn order to confirm whether shRNA expression vector could inhibiter MDV plaque formation, pu6vp22-4 vector was selected to transfect into CEF cells, 8 hours after transfection, RBIB strain of MDV type 1 was inoculated .The result demonstrated pu6vp22-4 plasmid efficiently inhibited MDV plaque formation, and the...