The Role Of Toll Like Receptor7on Apoptosis Of Ndv-Induced Chicken Embryo Fibroblasts

The TLR7can specifically recognize single-stranded ribonucleic acid (ssRNA) and activate its signal transduction pathway. The research is to reveal mechanism of TLR7on apoptosis of chicken embryo fibroblasts (CEF) infected with NDV which belongs to ssRNA virus.Firstly, total RNA was extracted from the NDV-infected CEF and the control CEF by Trizol method and reversed into cDNA. According to the Gallus gallus gene sequences of TLR7, MyD88, IFN-a, IL-2and β-actin available in GenBank, the respective specific primers were designed in the conserved region. Expression of chTLR7, chMyD88, chlFN-α and chIL-2were detected by real-time fluorescence quantitative PCR technique while β-actin gene was used as an internal control. The results showed that expression of chTLR7, compared with those of the control group (P<0.01) on hour12,24,36and48post infection(PI), were significantly higher than the control except24hour. The expression of chMyD88and chIL-2were significantly increased than the control group (P<0.05or P<0.01). The expression of chIFN-a were significantly increased (P<0.01) than the control group (P<0.01) on hour12,36and48PI, except24h (P>0.05). The dynamic expression of chTLR7and associated signaling molecules in CEF infected with NDV suggested that chTLR7may be involved in the early process of NDV infection in CEF.Secondly, specific primers were designed according to sequences of chTLR7gene available in GenBank. The truncated chTLR7was amplified by reverse transcription PCR (RT-PCR), which was1122bp in length. At the same time, the prokaryotic expression plasmid pGEX-6P-1-chTLR7was constructed successfully and expressed the protein with MW of63KDa in E. coli Rosetta system. The recombination protein was injected into rabbits to prepare the polyclonal antibody. The polyclonal antibody had good reactivity by the technic of western blotting and transient expression. Then, apoptosis in NDV infected CEF were detected by TUNEL method, after the polyclonal antibody of chTLR7was added to CEF. The results showed that apoptosis of NDV-induced CEF were very significantly reduced than the control group (P<0.01or P<0.05) with the25and28fold dilution concentration of chTLR7antibody on48h PI. The numbers of apoptosis was no statistics differences in the NDV infected CEF and control group (P>0.05) with the211fold dilution concentration of chTLR7antibody on48h PI, The results suggested that the apoptosis of CEF induced by NDV might be mediated through chTLR7.The research provided the foundation for investigation the role of chTLR7in NDV pathogenic molecular mechanism and provides new scientific approaches for ND control.