Isolation,Identification And Preparation Of Monoclonal Antibody Against Goose Astrovirus

Goose gout is one of the major infectious diseases endangering the healthy development of goose rearing industry of our country in recent years.During the initial outbreak of this disease,reports attributed the disease to feeding too much high protein and high nutrient feed.Researchers eventually found that the disease was caused by Goose Astroviruses(GAstV).The main symptoms of the infected geese were decreased feed intake,depression,loose stool,dehydration,emaciation and paralysis,and pathological changes such as urate adhesion on the surface of each organ were found at autopsy.Goose astrovirus belongs to the family Astrovirus.It is a single-stranded plus strand RNA virus with a diameter of 28-35 nm,no membrane and a total genome length of 6.9-7.9 kb.GAstV infection has a wide range of hosts,and Lande geese,Magang geese,Sichuan white geese,Wanxi white geese,Shigutou geese,seed geese,Wulong geese and Taizhou geese are all susceptible to Gast V infection.At present,there is still a lack of rapid and effective clinical diagnosis of GAstV,and no specific drugs or vaccines to prevent the disease.Therefore,the establishment of rapid and accurate immunological detection methods is still an important step in GAstV research.In this study,positive GAstV nucleic acid was detected by RT-PCR from goose tissue samples suspected to have died from goose astrovirus infection.A strain of goose astrovirus was successfully isolated by goose embryo and named as Gastv-NJ2021 strain.Sequence analysis showed that the virus belonged to GAstV type 1.The one-step growth of the virus was determined by three limited dilution and purification of chicken hepatocellular carcinoma cells(LMH).The results of animal regression showed that GAstV could infect goslings by subcutaneous injection or intramuscular injection,and was detected in the heart,liver,spleen,lung,kidney and other organs of infected geese,presenting systemic infection.To prepare a monoclonal cell line of GAstV,LMH cells were used to amplify the virus in large quantities.After the cell debris was removed by centrifugation,the virus was concentrated by polyethylene glycol(PEG)chemical deposition and purified by chromatography column to obtain a purified antigen for mouse immunization.Monoclonal cells were prepared by hybridoma cell fusion technique and immunized Balb/c mice with the purified antigen mixed with Freustal adjuvant after emulsification.After 3 times of immunization,serum antibody levels of the immunized mice were detected by Enzyme linked immunosorbent assay(ELISA).Mouse spleen cells with high serum antibody levels were fused with myeloma cells(SP20).The fused cells were screened with limited dilution,and the monoclonal antibodies were verified by Immunofluorescent assay(IFA).After three subclones,8monoclonal cells with IFA activity were obtained.In this study,GAstV-NJ2021 was successfully isolated and identified to establish an animal infection model.The virus was purified by PEG chemical deposition,and 8monoclonal cell lines with IFA activity were screened out by immunizing mice.An indirect immunofluorescence assay for GAstV was established,which laid a foundation for the study and clinical diagnosis of GAstV’s pathogenicity.