Study On The Interaction Between Chicken Viperin Protein And ALV-J

Avian leukaemia(AL)is a worldwide disease.It is caused by avian leukosis virus(ALV)in poultry.It is a second class animal epidemic in China.This disease has caused many harm to poultry industry,which can cause immunosuppression,slow development of multi-tissue organs,decrease of immunity of infected chickens and cause secondary infection of various diseases of infected birds,seriously affecting its production performance,and also can spread the harmful offspring vertically.Some studies have shown that ALV can also pollute the weak virus vaccine for birds,which poses a great threat to the breeding farm.At present,there is no effective vaccine or drug for the treatment of the disease,strict monitoring and purification measures are the main ways to prevent and control this disease.Viperin is an interferon stimulated genes ISGS,which can be induced by many viruses and bacteria.It has been proved to have the functions of inhibiting virus replication,immune regulation and metabolism regulation.At present,no reports of the inhibition about ALV replication by Viperin protein have been found.In order to study the interaction between the replication of chicken Viperin protein and ALV-J,a real-time fluorescence quantitative PCR method was established to detect the expression level of Viperin gene in chickens.The whole length CDs plasmid of Viperin was constructed by RT-PCR.Then the reaction conditions were optimized to verify the sensitivity and repeatability of the method.The established real-time fluorescence quantitative PCR method showed a good linear relationship in the range of 10~7-10~2copy/μL,the minimum detection limit was 100 copies/μL,and the repeatability was good,and the coefficient of variation was less than 2%in and between groups.The results showed that Viperin had a wide distribution of tissue.This study provides an accurate and effective method for the study of the function of chicken Viperin.Because chicken Viperin and mammals Viperin gene homology differences,to facilitate subsequent tests,this research expresses the chicken Viperin protein and immune mice.Chicken Viperin gene cloned to pET32a(+)carrier in the construction of prokaryotic expression plasmid and transformed into e.coli BL21,under the condition of 30℃add IPTG inducing expression,SDS-PAGE analysis showed that Viperin recombinant proteins expressed in the form of inclusion body.BALB/c mice were emulsified with Freund adjuvant and subcutaneously injected into the back of the neck.Serum was collected after three immunization.The specificity of the polyclonal antibody was determined by Western-blot method,and it was found that the antibody could specifically recognize the eukaryotic expression protein of Viperin.The establishment of real-time fluorescence quantitative PCR method for detecting chicken Viperin gene and the preparation of chicken Viperin protein polyclonal antibody provided the material basis for the subsequent experiments.In order to study the effect of ALV-J infection on the expression of Viperin gene in chickens,the CEF cells and 1-day-old SPF chickens were inoculated with ALV-J SDAU1005strains respectively.The transcription level of Viperin gene was detected by the established real-time fluorescence quantitative PCR.The cells were collected 12h,24h,36h,48h,72h,96h,120h and 144h after the infection,extracted total RNA and the expression of Viperin gene after ALV-J infection was analyzed.The results showed that transcription level of Viperin gene in the blood of CEF cells infected with virus and SPF chickens increased significantly.At 96h,the level of Viperin transcription reached the peak,and the corresponding ALV-J transcription level decreased significantly.In order to explore the effect of Viperin on ALV-J replication,the cell level of ALV-J was detected by overexpression and RNA interference.The eukaryotic expression plasmid pcDNA Viperin with V5 label was constructed and transfected into the cells.It was found that overexpression of Viperin could effectively interfere with the replication of the virus.Then,the specific si RNA for Viperin was designed and synthesized and transfected into cells showed that interfering with the expression of Viperin could promote the replication and release of virus.The result is Viperin could inhibit the replication of ALV-J on the cells cultured in vitro.In order to clarify the region of Viperin,primers were designed to amplify the gene fragments of three active regions,connect to the pEGFP-N1 vector,construct the eukaryotes of PEGFP-N,pegfp-sam and pEGFP-C,and observe the specific green fluorescence under fluorescence microscope after transfection of CEF cells,indicating that the transfection effect of the plasminogen was good and the corresponding protein could be expressed.The CEF cells were transfected with ALV-J and the role of Viperin in ALV-J replication was evaluated.It was found that in the cells transfected with pEGFP-N plasmid,the virus replication was inhibited,while the eukaryotes of pEGFP-SAM and pEGFP-C did not affect the replication of ALV-J,which proved that the active region of Viperin-N played an important role in anti ALV-J replication.In order to further screen the proteins interacting with Viperin,we designed primers to amplify the gene fragments corresponding to ALV-J proteins,and then connect them to pC-flag vector,and construct the eukaryotic expression plasmids of each gene.Co-transfered pcDNA-Viperin plasmid to cells,used laser confocal and immunocoprecipitation to detected the interaction between Viperin and virus genes.The results showed that p19 protein and Viperin co-located,and immunocoprecipitation test confirmed the same result,which confirmed that p19 protein and Viperin could interact.In summary,Viperin is an innate immune factor that inhibitions the virus,and ALV-J infection can induce the expression of Viperin in vivo and in vitro.Viperin inhibits the replication of ALV-J by interacting with p19 protein,and the N domain is the key regional in which Viperin plays an antiviral role.The study in this paper has enhanced the understanding of the anti-virus mechanism of Viperin,expanded the range of its antiviral spectrum,and provided a basis for further exploring the anti-ALV-J mechanism of Viperin protein.