The Mechanism Of Avian Leucosis Subgroup J Inhibiting Autophagy And The Development Of Anti-ALV-J DNA Vaccine

Subgroup J Avian leucosis(ALV-J)is kind of neoplastic disease caused by Subgroup J Avian leukosis virus(ALV),which induces clinic symptoms such as malignant proliferation of hematopoietic cells.It causes immunodepression of the organism,and is the one of the major poultry diseases that have been undermining the poultry industry in China and even in the rest of the world.At present,there are no effective medicine and vaccine to prevent it,and it is merely controlled by eliminating the infected chickens.As such,it is imperative to probe into the pathogenesis of ALV-J and further develop an effective anti-ALJ-J vaccine.1.In this research we have studied the impact ALV-J has on the autophagy steady-state level of DF-1 cells through Western Blot and laser confocal immunofluorescence.What We have found is that ALV-J down regulates the autophagy steady-state level of DF-1.2.Through Western Blot,we analyzed the expression of the autophagy protein substrates p62/SQSTM after ALV-J infection.It is found that during the infection,p62/SQSTM in the infected groups accumulates when compared with that in the blank group.This indicates that the down-regulation of autophagy steady-state level of DF-1 attributes to the upstream block of autophagy.3.To identify autophagy-related protein,in this research,the eukaryotic expression vectors of ALV-J's genes was constructed.By using Western Blot and laser confocal immunofluorescence.As a result,the transformation of LC3 in the transfected p RK5flag-gp37 group was inhibited,and the fluorescent spot in this group was smaller than that in the blank group.This shows that ALV-J gp37 can down-regulate the autophagy steady-state level.4.To screen the host protein interacting with ALV-J gp37,we transfected p RK5 flag-gp37 and conducted pull down using flag antibody,carried out SDS-PAGE and had it stained,and excised five target bands to carry out mass spectrometric detection.As a result we obtained 5 kinds of host protein that interact with ALV-J gp37,which are TCP-1?CDK1?ANAXA1?HSP60 and LLP.5.We has tested the function of the 5 kinds of host protein that possibly interact with ALV-J gp37.Through Western Blot and laser confocal immunofluorescence,tested the autophagy steady-state level after these vectors were transfected.Immunoprecipitation assay was adopted to verify the interaction between ALV-J gp37 protein and TCP-1 protein.The result of Western Blot and laser confocal immunofluorescence demonstrated a decrease of autophagy steady-state level in cells in the transfected p RK5 flag-TCP-1 group,indicating that TCP-1 inhibits autophagy.The result of immunoprecipitation assay has verified that ALV-J gp37 protein can interact with TCP-1 protein.6.In this research we have developed autophagy-target DNA vaccine which can be inducted into organism together with rapamycin as an effectiveness booster after electrical stimulation.According to the result,the anti-ALV-J DNA vaccine we have developed is capable of inducing chicken to produce effective antibodies against ALV-J,and increasing the number of CD3+CD8+ positive T cells in peripheral blood;when testing cytokine,we found the expression of IL-2?IL-10 and IFN-gamma in the p VAXI-gp85-LC3B+Rap group is greater that in other control groups.This is a further evidence proving the effectiveness of the anti-ALV-J DNA vaccine we have developed.This research is to probe into the mechanism of ALV-J inhibiting autophagy and the pathogenesis of ALV-J so as to develop a new anti-ALV-J DNA vaccine with rapamycin as a booster of its effectiveness.The vaccine plays a significant role in our efforts of controlling and preventing ALV-J,and further enhancing the ongoing development of the quality chicken industry in China.