Supression Of Swine Influenza Virus Proliferation In Vitro Of Polygene Genetically Modified Porcine

Swine influenza(SI) is an acute and highly contagious outbreak of respiratory disease caused by the swine influenza virus(SIV) and it can cause the swine immune suppression.It brings great harm to the pig industry and pose a threat to human health. RNAi(RNA interference, RNAi) refers to highly conserved during evolution, by double-stranded RNA(double stranded RNA, dsRNA) induced efficient specificity of homology mRNA degradation phenomenon. Studies confirm that RNA interference can specifically suppress virus gene expression; Mx protein is the cells themselves key molecule of natural antiviral activity induced by type I(α/β) and type III(λ) interferon(IFN), is a broad-spectrum anti-viral protein. Studies have confirmed that Mx is present efficient resistance for RNA viruses and DNA viruses. Modern pig production, there are genetic antagonism to some extent between production traits and resistance traits, the simple pursuit of high yield usually leads to pigs to reduce resistance; Through transgenic technology reform pig genetic traits, improve immunity, may be an effective means to control infectious diseases.Our team have screened RNAi, Mx and 4shRNA which were confirmed to have resistance value at the cellular level or in vivo of mice or pigs. On the base of this study we explored to construct the 4shRNA-Mx-RNAi recombinant lentiviral vector. Packing lentivirus infected the porcine embryonic fibroblasts to prepare nuclear donor cells. We successfully produced F0 pigsby somatic cell nuclear transfer technology. Use the methods of molecular biology PCR, Southern blot, RT-PCR and Western blot respectively in DNA, RNA and protein level for F0 generation swine identification. The results show that the three F0 generation of transgenic swine are all positive transgenic pigs; Choose one and the same species of wild swine(WT) hybridization, breeding F1 generation of transgenic swine. Using the above methods of molecular biology for appraisal analysis in F1 generation transgenic swine, the results show that one in the four head F1 generation of transgenic swine have the integration and expression in exogenous genes, positive for transgenic swine. Prove that exogenous gene in the RNAi-Mx-shRNA multiple genes expression transgenic pigs can be stably inherited to the F1 generation.In order to further explore the effect of inhibiting viral replication in F1 generation of transgenic pigs fibroblasts, isolation and culture one-day-old F1 generation of transgenic pigs tail tissue fibroblasts, after 100TCID50 A/Swine/Guangdong/2004(H1N1) strain infection, by indirect immunofluorescence(IFA), real-time quantitative PCR(Real-time PCR), viral titer analysis of F1 transgenic pig cells inhibit the activity of influenza virus replication. Results show that at different time points after post-infection, compared with non-transgenic pigs and WT pigs, transgenic pig fibroblasts can inhibit the proliferation of influenza virus; At each time point after Influenza virus infection, the exogenous gene has certain inhibitory activity of influenza virus, and the inhibition efficiency reached the highest in 16 h, IFA results show that the transgenic pigs fibroblasts on the inhibition efficiency of influenza virus is higher than the non transgenic pigs and WT pigs. Cells H1N1 PB2 gene replication efficiency is reduced by 2 times than that of WT, cell supernatant virus TCID50 up to 1 × 10-2.8. Show that multiple genes expression transgenic pigs at the cellular level to inhibit the activity of influenza virus replication.This study successfully constructed the 4shRNA-Mx-RNAi recombinant lentiviral vector and producted the transgenic pigs which can resist influenza virus by somatic cell nuclear transfer technology. And confirmed that the transgenic pigs have integration and expression in exogenous gene, which can stably inherited to the F1 generation, its fibroblasts with inhibition activity of influenza virus replication, for polygenic synergistic inhibition influenza virus replication study providing a transgenic pig model, for explore new inhibition influenza virus replication strategy and the study of transgenic animal disease resistance to lay the foundation.