Study On Transgenic Animal Of Antisense RNA Of Pseudorabies Virus IE180 Gene

IE 180 gene (Immediate-early 180 gene) is the only immediate early gnen found up to now in PRV(Pseudorabies virus, PRV) genome. This gene is critical to the replication of PRV. Only does the IE 180 gene begin to transcript and translate into IE180 protein, the down stream genes can be transcripted by the activation of IE180. If IE 180 gene of PRV is inhibited, the replication of the PRV will not be completed. In this study the genome DNA of PRV was extracted and digested with Bamlll, then cloned 4.8kbs fragment into plasmid pBluescript SK(+). The 4.8kbs fragment contains the whole 4383bps coding sequence of IE180 gene from ATG to TGA in addition to 215bp regulatory sequence and 269bp polyA sequence. Digested the 4.8kbs fragment with SacI, Xhol, Ncol and got DNA bands exactly identical to expectation. This proved that the 4.8kb fragment was IE180 gene. Since the DNA sequence of PRV is coliner with IE180 mRNA, this means there is no intron in the mRNA of PRV IE180 gene. According to this experimental data, 4 antisense fragments were designed with length 2.0kbs, 0.50kb, 0.28kb and 0.12kb respectively. At the two ends of the fragments of 0.5kb, 0.28kb, 0.12kb, XhoI and Xbai site were added. 4 antisense plasmids (APCIE1.85, APCIE0.50, APCIE0.28 and APCIE0.12) were obtained through clone and/or PCR, Tranfectcd the 4 antisense plasmids to BHK-21 cell and obtained 4 antisense ceII lines after the selection of G418. Collected the cells after expanding culture and extracted RNA from the cells with "one -step"method. To prove whether the antisense fragment express into antisense RNA, RT-PCR method was carried out. The results suggested that all the antisense cell lines expressed the antisense RNA. Measured the TCID50 value of the cell lines to evaluated the anti-virus effect of antisense RNA. Among the 4 plasmids APCIE0.12 had the best result and its inhibit rate to virus reached as high as 99.13%. APCIE0.28, APCIE1.85 and APCIE0.5 were 92.41%, 89.77% and 85.87% respectively.Through the analysis of the ceJJ lines we got the best antisense KNA fragment which inhibit virus replication. This fragment includes ATG site and part of activation region. The antisense RNA of this fragment can effectively inhibit the translation of IE180 and stops the replication of PRV.Adult female mice were chosen to be the donors of fertilized egg and the recipients. Superovulated the females with PMSG and HCG and mated with males immediately after the injection of HCG. Fetilized eggs were got from the ovaduct of the females and cumulus cells were digested with hyaluronidase. APCIE0.12 was digested with BgIII and BamHI and 3.5kbs fragment was recovered and diluted. Fixed egg by a holding pipette under the microscope and injected 2-3pl DNA into the malepronuclear with a fine(0.5-lum) glass needle.Culturcd the eggs in the incubator at 38 5%CO2 and then transferred the eggs to ovaducts of the recipients. 38 offspring were obtained. Extracted DNA from mice tails and checked the integration of the foreign fragment in the transgenic mice. Through PCR, RT-PCR and Southern blot, 2 transgenic mice were proved to be positive.Transgenic mice and control group were attacked by PRV with 10 LD50(LD50=10" 2.5/0.1ml). One of the 2 transgenic mice lived 3 days longer than the control group. The other could servival to the virus. The results indicated that the antisense RNA expressed by the foreign fragment in transgenic mice had the obvious ability of anti-virus. This study supplys the experiment data for application of the antisense RNA in domestic animals and the model of anti-PRV transgenic stock.