| Since1997, Goose paramyxovirus disease had broken out and spread in themajority of our region, and was identified that the pathogen was paramyxovirusserotypeâ… , the disease was named Goose paramyxovirus disease, also maybe calledNewcastle disease of goose. All ages goose are susceptible to the disease, the gaggleincidence rate of27.67%on average, the mortality rate can be as high as100%individual, an average rate of18.19%. This disease cause serious economic losses tothe goose industry in China. At present, the prevention of Goose paramyxovirus diseasewas mainly injected vaccine, although a variety of the attenuated vaccine andinactivated vaccine for immunization prevention, but still can not control theoccurrence of Goose paramyxovirus disease. The results showed that Newcastledisease virus virulent vaccine or inactivated vaccine even traditional LaSotaattenuated vaccine to prevent Goose Newcastle disease virus disease also can not playcompletely immune protective effect. Therefore, we need to find a new preventionideas and technical means for Goose Newcastle disease virus.With the development of molecular biology and its technical methods, the geneblocking technology has also been a great deal of development. The siRNA inducedRNAi effect in mammalian or other animal cells, to achieve effective inhibition ofspecific genes, this method has become an important tool for studies of gene function.Lentiviral vector-mediated gene transduction is a new technology, its advantages werethe high efficiency of transfection and can effectively infect quiescent cells andexpress the target gene. In this study, the use of genomic integration features and highinfection capacity of lentiviral vector and mediated RNA interference technology,research the effect of proliferative capacity of NA-1strains of Goose Newcastledisease virus in vitro and in vivo, and thus established the foundation for anti-virus research of poultry.First, three pairs shRNA sequences were designed for the conserved regionsequences of NP, M gene of Goose Newcastle disease virus NA-1strain respectivelywhile a pair negative control shRNA sequence was designed, then synthesized andannealing to form double-stranded, and connected to the lentivirus vector(pLKD-GFP)with Pol â…¢ U6promoter which was linearized by double digestion. The recombinantvectors were named pLKD-shNP268, pLKD-shNP437, pLKD-shNP710,pLKD-shM168, pLKD-shM646, pLKD-shM1062and pLKD-control (negative)respectively. These recombinant lentiviral vector plasmid were confirmed by PCR andsequencing. The different recombinant lentivirus vector plasmids were transientlytransfected Vero cells, after48h, the cells were super-infected the NA-1strain ofGoose Newcastle disease virus. To determine whether NP and M gene expression ofthe NDV strain NA-1could be inhibited by lentivirus vector-mediated siRNA, therelative expression levels of the NP and M genes were evaluated by real-time RT-PCR.The results of the real-time RT-PCR indicated a significant inhibitory effect in therelative expression level of NP and M gene transcripts by85.8%and84.6%,respectively, when plasmids pLKD-shNP710and pLKD-M646were used, ascompared to the mock and pLKD-control transfected cells at48h p.i. PlasmidspLKD-shNP268and pLKD-shNP437down-regulated mRNA expression levels of theNP gene by55.6%and64.2%, while pLKD-shM168and pLKD-shM1062down-regulated mRNA expression levels of the M gene by56.5%and69.0%,respectively. To demonstrate the inhibitory effect of the different recombinantinterference plasmids further, the TCID50assay was used to titrate the NA-1virus inthe cell supernatants at48h p.i. The results show that the TCID50was10-4.16/0.1mLand10-5.15/0.1mL when cells were transfected with pLKD-shNP710and pLKD-M646,respectively. These values indicate significant viral titer reduction when compared tothe mock group, which had a TCID50/0.1mL of10-9.37/0.1mL. The TCID50values forthe other plasmids were as follows:10-7.83/0.1mL for pLKD-shNP268,10-7.12/0.1mLfor pLKD-shNP437,10-6.97/0.1mL for pLKD-shM168, and10-7.1/0.1mL forpLKD-shM1062. We next used an indirect immunocytochemistry assay to detect theexpression of NA-1in Vero cells. The results indicated that the expression of NA-1 was significantly lower in cells transfected with pLKD-shNP710and pLKD-M646than in the Mock Vero and pLKD-control groups. Indeed, the replication of NA-1wassignificantly inhibited in vitro.Based on the promising results above, we generated recombinant lentivirus thatcontained a U6Pol III promoter and the NP and M gene siRNA cassette targeting the710-731nt (NP gene) and646-667nt (M gene) regions. The recombinant-defectivepseudotype lentiviruses carried an EGFP reporter gene for quantification by FACSanalysis. These lentiviruses containing the NP and M gene siRNAs were titrated inVero cells, which were found to contain5×107TU/mL. We transduced Vero cells withrecombinant lentivirus stocks LV-NP710, LV-M646, LV-GFP, LV-control andscreening them by puromycin, then we can obtain the resistant cells. These resistantcells were passaged to the20th generations. And extracted the genomic of5th,10th,15th,20thgeneration cell and detected the shRNA sequences by PCR and DNAsequencing methods, the results show that each generation of the cell genomecontaining the shRNA and GFP gene sequences, and the green fluorescent proteinexpression to a higher level. These results proved that it is successful to establish astable cell lines, the cell lines were named that NP710-Vero, M646-Vero, GFP-Veroand control-Vero.The different Vero cell lines which can stably expressing shRNA were infectedwith NA-1strain of Goose Newcastle disease virus (1000TCID50/0.1mL). The relativeexpression levels of the NP and M genes were evaluated by real-time RT-PCR. Theresults of the real-time RT-PCR indicated a significant inhibitory effect in the relativeexpression level of NP and M gene transcripts by86.8%and85.9%, respectively inNP710-Vero and M646-Vero cell lines. TCID50assay was used to titrate the NA-1virus in the cell supernatants at48h p.i. The results showed that the TCID50/0.1mLvalues were10-4.21/0.1mL and10-5.17/0.1mL when cells were transduced withLV-NP710and LV-M646, respectively. The mock Vero TCID50/0.1mL was10-9.43/0.1mL, and the LV-control TCID50/0.1mL was10-9.18/0.1mL. The results were similar tothe results of Vero cell transfected the recombinant lentivirus plasmids. We also nextused an indirect immunocytochemistry assay to detect the expression of NA-1indifferent stable Vero cell lines. The results indicated that the expression of NA-1was significantly lower in cells transduced with LV-NP710and LV-M646than in the MockVero and LV-control groups. Indeed, the replication of NA-1was significantlyinhibited in vitro.To further research the inhibitive effect of recombinant lentivirus expressingshRNA for Goose Newcastle disease virus in vivo, we take the fresh goose fertilizedegg, recombinant lentivirus solution (titer to106TU/mL) injected into the fresh goosefertilized egg embryo inferior vena, and capped with sterilized the eggshell with shellmembrane, and fixed with medical tape, and then hatched the embryo in accordancewith the standard incubator program. Extracted the genomic DNA of embryomic, andidentified the integration of the shRNA sequence, the results showed that the embryois a chimera embryo. |
