Avian ?-Defensin 7 And 10 Resist Infection Of Three Viruses

Avian leukosis virus subgroup J(ALV-J),Marek's disease virus(MDV),Porcine circovirus 2(PCV2)all three viruses can cause immunosuppression in infected animals,causes the animal's immune system to decline,which is conducive to the invasion of other pathogens,causing concurrent infection of two or more diseases,increased difficulty in diagnosis and treatment.At present,MDV and PCV2 are mainly controlled by vaccination,although the disease is reduced to a certain extent,it also increases the speed of mutation.ALV-J is mainly purified by elimination,but in 2018,a myeloma disease broke out caused by ALV-J almost simultaneously in several provinces in China,making the ALV-J's purification path even more difficult.The use of antibiotic additives seriously damages the microecological balance of the animal's intestines,and drug residues also affect the quality of animal products and human health.Therefore,there is an urgent need to develop a drug that has no toxic side effects and does not cause resistance to pathogenic bacteria to solve this problem.Defensins have broad-spectrum anti-microbial activity and can effectively kill viruses,bacteria,fungi,spirochetes,etc,and also have cytotoxic effects on tumor cells,to date,no pathogenic strain has been found to be resistant to it.There is much evidence that defensins have a significant inhibitory effect on enveloped viruses.?-defensins are the only defensins in birds,and more than 25 avian beta-defensins have been discovered,and there are14 chicken beta-defensins.Chicken ?-defensins are widely distributed in chickens and are found in almost all tissues and organs of chickens.To study the antiviral effects of chicken?-defensins,we selected chicken ?-defensins 7(Av BD7)? chicken ?-defensins 10(Av BD10)from 14 chicken ?-defensins for anti-ALV-J,MDV,and PCV2 effects studies based on the distribution of chicken ?-defensins in chickens and their characteristics.In this study,the gene cloning method was used to amplify the AvBD7 and AvBD10 genes from the liver and testis tissues of chickens,the two genes were subcloned into the expression vector p ET-32 a to construct a recombinant plasmid,and the recombinant plasmid was transformed into BL21 expressing bacteria,the expression was induced by IPTG,and the cells were induced by ultrasonic disruption,and the supernatant and the precipitate were centrifuged for detection.The results showed that the presence of the expressed product was observed in both the supernatant and the precipitate of the disrupted cells.The expressed recombinant protein was purified by a nickel column method to obtain Av BD7 and Av BD10 proteins.First,the antiviral effects of these two proteins were verified in vitro by: the DF-1/PK-15 cells were cultured,and the ALV-J and MDV/PCV2 viruses were inoculated.After the cells were infected with the virus,the appropriate concentration of Av BD7 and Av BD10 protein solution was added,after culture for a period of time,q PCR and western blot were used to detect changes in viral load and protein expression in different experimental groups.The results showed that Av BD7 and Av BD10 protein had a good inhibitory effect on MDV,but the inhibitory effect on ALV-J and PCV2 was not obvious.To further validate the antiMDV effects of Av BD7 and Av BD10 proteins,we designed and performed in vivo experiments,in vivo experiments were divided into 6 groups,namely MDV,MDV+ Av BD7,MDV+ Av BD10,Av BD7,Av BD10 and Normal,in which the Normal group was given the same dose of imidazole as the defensin protein.the first three groups were vaccinated with MDV at 3 days of age,and the latter three groups were not treated.After confirming the success of the poisoning,the corresponding protein solution and imidazole solution were injected according to the group.Finally,the unified sectioning and killing treatment was performed,and the symptoms were analyzed in different groups from gross necropsy observation,hematology observation and histopathological observation.The results of gross necropsy showed that the spleen,thymus and bursa of the MDV-infected group had a certain degree of atrophy,mild fibrinous exudation in the glandular stomach,and white spotted foci in the liver,and other tissues and organs were relatively normal;the other three groups did not show obvious eye lesions.Hematological observations showed an increase in the number of inflammatory cells in the blood of the virus-infected group,mainly lymphocytosis,and a small amount of inflammatory cells were present in the blood of the two defensin-treated groups.Histopathological observation showed that there were inflammatory lesions in the liver of the three mice infected with MDV,and the MDV group was the most serious;Inflammatory cell infiltration occurred between the glandular ducts of the glandular stomach in the MDV-infected group,and the other groups had mild inflammatory reactions;The kidneys of the MDV-infected group had large inflammatory lesions,and the renal tubular epithelial cells in the MDV+Av BD7 and MDV+Av BD7 groups were mildly vacuolated.In summary,in vivo injection of Av BD7/Av BD10 can alleviate the pathological damage caused by MDV infection.In this study,a large-scale expression method of Av BD7 and Av BD10 in vitro was successfully established using the prokaryotic expression vector p ET-32 a.In vitro experiments were conducted to investigate the resistance of Av BD7 and Av BD10 to three immunosuppressive viruses(ALV-J,MDV,PCV2),and in vivo experiments to explore the resistance to MDV.The results of this study enriched the understanding of chicken ?-defensin function and laid a foundation for the development of new antiviral drugs.