The Effect Of Non Structural Protein 2B、3A In Foot-and-mouthdisease Virus On Cell Apoptosis And The Observation Of 3D Gene Interference In The Virus’ Inhibition

Foot and mouth disease is an acute infectious disease caused by animal spirits, mainly against foot-and-mouth disease pathogen of cloven hoofed animal tissue, it has been considered the World Animal Health(OIE) as the first A class of animal diseases, there are seven serotypes,containing more than 70 subtypes, no protective immunity among almost all types, and therefore brought great difficulties about the prevention and control of foot and mouth disease. In recent years, foot and mouth disease virus has made great progress in the relationship between the replication, cell recognition, virus structure and function. But the pathogenesis of the virus is still not very clear. Research for FMDV infection induces host cell apoptosis is relative lack at home and abroad. In order to prevent an outbreak of foot and mouth disease virus, people have started to cultivate anti-viral transgenic animal models, our laboratory has also bred five anti-FMDV transgenic pigs. To validate its anti-virus capabilities, efficiency against FMD virus were evaluated at the cellular level.1. FMDV non-structural protein 2B, 3A expression of active verification: using molecular biological methods, in accordance with FMDV non-structural protein 2B, 3A full length sequences, primers were designed. Extraction total virues RNA, reverse transcription into c DNA, c DNA regarded as template, Amplified from 2B, 3A gene’s DNA sequences by PCR. The recovered product is connected to the cloning vector, after sequence accurate, Then containing the gene 3A, 2B of the cloning vector was cut by double digestion, then connection to the eukaryotic expression vector p EGFP-N1, and by sequence analysis and double digestion verify recombinant plasmid p EGFP-N1-3A, p EGFP-N1- 2B.The plasmid was transfected into hamster kidney cells(BHK), after 48 h transfection were extracted protein from normal cells, p EGFP-N1 and the experimental group cells, Added protein lysates were lysed, after collecting sufficient protein was verified Western blot analysis and electrophoresis analysis. Experiments show that: after 24 hours green fluorescence is observed in the inverted fluorescence microscope, indicating that the plasmid was successf μLly transfected.Validated by Western blotting, in 41 k Da was seen at a bright band proved that the fusion protein 3A-EGFP, 2B-EGFP can be successfully expressed in hamster kidney cells.2. The impact of non-structural proteins of foot and mouth disease virus infected cells apoptosis: Flow cytometry detection with 3A-EGFP, 2B-EGFP fusion protein on apoptosis. The results show: after 24 h transfection of the cells under a fluorescence microscope, the green fluorescence can be observed.Through flow cytometry display: 3A-EGFP fusion protein has a role in promoting apoptosis, compared with the control group, a significant difference(P <0.01). The 2B-EGFP fusion protein apoptotic effect on cells compared with the control group was not significantly different(P> 0.05).3. The study of the effect of anti-mouth disease virus(FMDV) and separation from Transgenic pigs fibroblasts cells. The interference fragment Si RNA antiviral effect in transgenic pig intracellular, using collagenase digestion and animal tissue block to separate cells,successfully obtained anti-viral transgenic pig fibroblasts, cells are extracted genomic DNA, After PCR Verification, the sequence was contained the sh RNA and was detected a bright band at 400 bp.But normal pigs was not appeared a bright band. Sequencing resμLts showed that: transgenic pigs contained of exogenous 3D anti-FMDV of Si RNA fragments.The two fibroblasts cells was inoculated quantitative FMD virus. By pathological changes resulting cells(CPE) and real-time quantitative PCR calculated the contents of virus in the cells, the results showed that: inoculating the same dose of virus’ s, anti-FMDV transgenic pigs and same-day-old normal pig fibroblasts cell in resistancee efficiency of virus are significant differences.lesions time occur transgenic pig fibroblasts was longer than the normal pigs lesions occur in time, and by detecting the virus in both cell content, at 18 h, the virus in transgenic pig fibroblasts was lower copy number Cells with normal porcine fibroblasts, indicating that the target FMDV interference sections have good antiviral ability.