| Since May 2016,an infectious disease with gout as the main disease has erupted in goose farms in Anhui,Jiangsu,Shandong,Yunnan and Sichuan,etc.5 to 19-day-old goslings are susceptible and the mortality rate is as high as 30%,which causes serious economic losses to the goose industry and related industries.In this study,we first conducted a retrospective analysis of laboratory-going RT-PCR Goose Astroviruses(GoAstV)positive clinical disease goose samples collected from various places in2016-2019.ORF1b(encoding RNA-dependent RNA polymerase protein),ORF2(encoding viral capsid protein)gene sequence alignment analysis.The results showed that Samples LA1605,ZJ1703,ZJ1704,ADY1708,LY1912 have 99.9%homology with the gene sequences of multiple goose astroviruses that have been published in China.The GoAstV Cap prokaryotic expression vector pET-30a-rCap has been constructed,transformed BL21(DE3),induced expression with IPTG,detected protein expression by SDS-PAGE and Western blot,expressed and purified a large amount of His-tagged recombinant protein To obtain recombinant protein with a concentration of 0.74 mg/mL.The purified protein was used as an immunogen to immunize BALB/c mice.Enzyme linked immunosorbent assay(ELISA)was used to detect antibody titers of immunized mice.Classical cell hybridoma technology was used to prepare monoclonal antibodies.The monoclonal antibodies were identified by Western blot and Immunofluorescent assay(IFA).The results showed that the antibody titers increased rapidly after immunization(titers>51200)and showed good reactivity and specificity.After three rounds of sub-cloning,five MAbs specific to Cap protein were obtained.For strains of 1C11B11,9A12B12,the heavy chain type was IgG2a while for strains of 1C11B11 and 9A12B12,the heavy chain type was IgG1.All the strains'light chain type was?.MAbs of 5B7G8 strain were able to react with the GoAstV verified by western blot while MAbs of 1C11B11,9A12B12 strain were able to react with the GoAstV verified by western blot and immunofluorescence assay.The experiment uses the conserved region at the 5'end of the viral capsid protein(Cap)sequence as a template to design fluorescent quantitative PCR primers and probes.Using 19T-Cap recombinant plasmid as a positive standard template,a 10-fold gradient dilution was used to construct a standard curve,and a GoAstV Taq-Man real-time fluorescence quantitative RT-PCR absolute quantitative detection method was established.The linear equation of the standard curve is y=-3.4469x+41.528,R~2=0.9948;this method can detect 3.98 copies/?l of virus,without cross-reaction with other viruses,and the coefficient of variation is less than 2%.Using the prepared monoclonal antibody and the established fluorescence quantitative RT-PCR detection method,the in vivo and in vitro proliferation characteristics of goose astrovirus were studied.Four different routes of oral,intramuscular,subcutaneous,and nasal drip were used to inoculate6-day-old goslings with GoA embryo-proliferated GoAstV-CH16 strain.Random necropsy and 12tissues of heart,liver,spleen,lung,kidney,brain,pancreas,gland stomach,duodenum,jejunum,ileum,and cecum were taken.The viral load of various tissue samples was tested by established methods.The test results showed that the virus content in the body reached a peak one week after the goose astrovirus infection of the goslings.The viral load of the oral challenge route in each tissue was the highest,among which the kidney viral load was the highest.GoAstV-CH16 strain was used to infect Goose embryo Kideny Cells(GKC).Western-blot and IFA tests were performed on GKC cells after GoAstV infection.The cell culture fluids at different time points after GoAstV infection were collected and detected using the established absolute quantitative detection method of GoAstV Taq-Man fluorescence quantitative PCR,and the one-step growth curve of GoAstV-sensing GKC was drawn.Further grasped the proliferation characteristics of goose astrovirus in GKC cells. |
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