Isolation And Identification Of J-avian Leukosis Virus And Establishment Of Indirect ELISA Method

The avian leukemia (AL) is caused by avian leukemia virus and avian sarcoma virus, whichgive rise to the poultry varieties of neoplastic diseases collectively. The virus can cause manykinds of infectious benign and malignant tumors. Subgroup J avian leukosis(AL-J), which is a kindof neoplastic disease mainly outbreaks in broilers is caused by a subgroup J avian leukosisvirus(ALV-J).In this study, the clinically suspected infection chickens were necropised and observedsystematically. There was found that enlargement of the liver with white nodules, subcapsular clotat and under surface. Kidneys showed moderate to severe enlargement with diffuse gray-whitenodules. Histopathological observation demonstrated that identified focal and diffuse infiltrationof tumor cells of myeloid with moderate to severe parenchymatous degeneration and necrosisinliver and kidney. IFA demonstrade the virus infected DF-1cells had specific green fluorescence,and PCR showed correct results. All above, it was successfully isolated and identified of an ALV-Jsubsets virus strain.According to the the NCBI published avian leukemia virus gene sequence, primers weredesigned to amplify the gp85gene of ALV-J. Proviral DNA was extracted from diseased material.The gp85was successfully amplified with the size of924bp using PCR method. With theprokaryotic expression vector pET-30a(+), the recombinant plasmid pET-gp85was constructed andidentified, it was then transformed into E. coli Rosetta competent cells and induced with1mmol/LIPTG. SDS-PAGE analysis showed that there was a band of40kDa of size and in the form ofinclusion bodies. It was characterized with Western blot analysis, the results illustrated that therecombinant gp85protein had good reactogenicity.pET-gp85purified protein was used as coating antigen, the indirect ELISA method fordetection of ALV-J-gp85antibodies was established and optimized. Working conditions of indirectELISA method were as followed:1%BSA of blocking solution;1μg/mL of coating antigen;1h ofincubation time and1:400dilution of clinic serum;1h of incubation time and1:4000dilution forsecondary antibody; and the reaction time for substrate was10min. The established indirectELISA had good repeatability, and had no cross reactions with other reference viral and bacterialdisease positive serum, based on the clinical a lot of samples testing, is a science of positive. It could provide to be a specific and sensitive tool for the clinical detection and diagnosis of ALV-J.