The Construction Of Rescuing GPMV Cell Lines And Inhibition Of GPMV Replication By Targeted Silence The P Gene

Goose paramyxovirus disease is a high mortality rate of infectious diseases caused by the goose paramyxovirus. The disease was susceptibility to all age of goose.Less 15-day-old goslings' morbidity and mortality can be as high as 100%. It was identified that the goose paramyxovirus is theâ… -type serum virus, so the goose paramyxovirus virus may also called the Newcastle disease of goose. In recent years, this disease cause serious economic damage to the raising goose industry in China, threatening the healthy development of goose industry. The prevention of goose paramyxovirus mainly relied on the Attenuated vaccine, so the creating of new vaccine is one of the most important links to prevent and control the disease. It's a hot research that to gain the attenuated vaccine of Newcastle disease by reverse genetic manipulation, and one of the most important basic link is that it requires a cell lines to express T7RNA polym erase stably.The replication of the Newcastle disease virus mainly depend on the RNP, and a necessary condition for the formation of RNP is the P protein needed, the complex of P protein and L protein have a RNA-dependent RNA polymerase activity, so this research select to target to science the P gene, in order to prevent the replication of the virus.To clone the T7RNA polymerase gene, and construct the eukaryotic expression vector PCI-T7. Transfect the expression vector to the cell lines V7 by the method of Liposome transfection, thus constructing a V7 cell lines which express the T7 RNA polymerase. And detect the HA protein by Western blot and indirect immunofluorescence, which confirmed that the T7 RNA polymerase gene have expressed in VT7 cell lines stably and efficiently.According to the Published P gene sequence of goose paramyxovirus, requirements of SiRNA expression vector and the selection principle of RNAi targeted sequences, selected the SiRNA sequences specific for goose paramyxovirus by BLAST tools, and design negative RNAi sites in the same time. According to the requirement of the designed target molecule and expression vector of RNAi, the target molecule was connected to the dedicated expression vector of RNAi which was after Enzyme cutting when it has annealed and purified, and be transfected into CEF and chicken embryo,which would be inoculate the virus after 6h. in order to observe the influence of RNAi to the GPMV NA-1 which was replicated and reproduced in CEF 36h post-infection, Real Time RT-PCR, detecting of virus titer, indirect immunofluorescence and cell disease had been used.And testing the inhibition of RNAi in CEF by hemagglutination test. The experimental result shows that, the two RNAi sites aiming at the P protein of GPMV NA-1 showed a strong inhibitory effect, and the inhibition efficiency of RNAi-P641 is stronger. What was proofed that the P gene is necessary to the replication of goose paramyxovirus virus.DNAzyme is the DNA molecules with the function of RNA cutting, which was selecting from random sequence library composed in vitro, it's one of the hot spot in Gene silence research at present. By the bioinformatics having been used, this test design the DZ-P-358, DZ-P-446, DZ-P-702 three kinds of 10-23 DNAzyme aiming at GPMV NA-1 P protein, the cutting experiments in vitro has shown that the DZ-P-702, DZ-P 358 has cut activity, and DZ-P-702 has higher activity. It is proofed that DZ-P-702,DZ-P-358 can inhibit the replication of the goose paramyxovirus virus by observing the cell disease, detecting of virus titer, indirect immunofluorescence in CEF level; Test indicated that DZ-P-358 and DZ-P-702 may reduce the transcription level of P protein in CEF through the method of Real-time PCR, of which, the inhibition efficiency of DZ-P-702 may reach 78.5%. By determine the Blood clots titer of allantoic fluid, it proved that DZ may inhibit the proliferation of GPMV in CEF, and shown the application prospect of DNAzyme in production practice.Summary:To have successfully constructed VT7 cell lines that can express T7 RNA polymerase steady, and forming the massy foundation for gaining the AMERVAC PRRS of Newcastle disease by reverse genetic manipulation; and have selected two interference sites which may inhibit the expression of GPMV P gene effectively.Screened out the specific DNAzyme DZ-P-702 that is aiming at the goose paramyxovirus P gene. And the screening and establishment of the effective targets provide the technical foundation for further study of antiviral therapy and resistance of disease breeding areas.