Recombinant Avian ?-defensin 2 And 6 Synergistically Resist Infection Of Avian Tumor Viruses

In past decades,with the rapid development of the poultry industry in China,the scale has increased,and intensive and high-density feeding has become a common feeding model.This has also led to outbreaks of tumor viruses-induced immunosuppression,such as avian leukosis virus subgroup J(ALV-J),Marek's disease virus(MDV),and reticuloendotheliosis virus(REV)and so on.Avian neoplasms viruses(ALV-J,REV,MDV)can cause tumor-associated diseases in chickens,broilers,breeders,and other strains of chickens,which has brought huge damage and economic losses to the poultry industry in China for a long time.The epidemic of poultry tumours has seriously affected the development of the poultry industry in China.However,there is no effective treatment and immunization method control these diseases.It is necessary to explore some bio-drugs to control or treat avian tumor diseases.Avian ?-defensins are a class of endogenous cationic antimicrobial peptides,rich in cysteine,and consist of three stable pairs of disulfide bonds within the molecule consisting of 6conservative cysteine residues.Avian ?-defensins play an important role in the immune system of birds and are protective films against external pathogenic microorganisms invading the body.They have broad-spectrum antibacterial,antiviral,and antifungal effects.In order to study the effect of chicken Av BD against avian tumour disease viruses,according to the characteristics of homology of chicken Av BD,tissue distribution in vivo and other characteristics,chicken Av BD2 and chicken Av BD6 were selected from 14 chicken Av BD for anti-avian tumour viruses.By PCR,chicken Av BD2 and Av BD6 genes were amplified from testis and liver tissue of SPF chickens.Two kinds of Av BD were cloned into expression vector p GEX-6p-1 to construct recombinant expression plasmid.In p GEX-Av BD,the recombinant plasmid was transformed into expression strain DH5?,and the fusion protein expressing Av BD2 and Av BD6 was induced by IPTG,and the expression product existed in the form of inclusion bodies.The expressed recombinant protein was purified and renatured to successfully obtain natural biologically active chicken Av BD2 and Av BD6.Different concentrations of Av BD2 and Av BD6 and a mix of the two proteins were then added to theCEF cells inoculated with the virus(ALV-J,MDV,REV).Real-time fluorescence quantitative PCR and Western blot were used to detect the level of viral transcription and protein levels in each experimental group.It was found that both Av BD2 and Av BD6 had inhibitory effects on ALV-J,MDV and REV,and the inhibitory effect was increased with increasing protein concentration.Enhanced,and the two avian ?-defensins have synergistic effects on the inhibition of ALV-J,MDV and REV,respectively.The ALV-J virus with the best inhibitory effect of Av BD was selected for the antiviral test in animals.The SPF chicks that had been challenged were inoculated with Av BD2 and Av BD6 and two mixed Av BD proteins.The uninfected SPF chicks were also inoculated with the corresponding Av BD.The other Av BD protein was used as a control,and was sacrificed after two consecutive instillations.The test was conducted from gross organs,histopathology,and hematology.After necropsy,the development of liver,heart,spleen,and lung in the four challenge groups was not as good as that of the non-challenge group,and in the four challenge groups,the ALV-J group was compared with other ALV-J + Av BD groups.Major organs such as liver,spleen and lung are less developed.Histopathological observation found that in the four groups of ALV-J group,lymphocytic inflammatory foci were found in the liver;lymphocyte inflammatory foci and diffuse hyperaemia occurred in the lungs;vacuolar degeneration of renal tubular epithelial cells,diffuse hyperaemia,lymphocytic infiltration;diffuse congestion of the thymus,a large loss of lymphocytes;spleen diffuse hyperemia.The lesions were most severe in the ALV-J group.The lesions of the organs in the 3 groups of Av BD+ALV-J group were followed.The lesions in the organs of the ALV-J+Av BD2+Av BD6 group weremilt.From the blood smears,the number of heterophil granulocytes in the blood of the 4 challenge groups increased,lymphocyte increased,and the number of heterophil granulocytes and lymphocytes in the blood of 3 ALV-J + Av BD groups in 4 attacks.Less than ALV-J group,from the above in vivo tests,it can be concluded that Av BD2 and Av BD6 can inhibit ALV-J virus in chickens,have a therapeutic effect,and have a certain synergistic effect.In this study,we used p GEX-6P-1 vector and DH5? expression bacteria to successfully establish the in vitro prokaryotic expression method of chicken Av BD2 and Av BD6.Real-time fluorescence quantitative PCR and Western blot were used to study the synergistic anti-avian tumour virus(ALV-J,REV,MDV)between chicken Av BD2 and Av BD6 in vitro.The synergistic anti-ALV-J in vivo of chicken Av BD2 and Av BD6 was studied by means of organ observation,histopathology,and hematology.The above research lays a foundation for the research on the expression of avian beta-defensin in vitro,the development of anti-tumor virus drug formulations or adjuvants,and the mechanism of anti-envelopment virus.