| Pigeon adenovirus disease is a viral infectious disease characterized by anorexia,vomiting,diarrhea and liver damage caused by avian adenovirus.In recent years,the epidemic range has increased,and novel adenovirus variants have been reported,so the diagnosis and prevention of adenovirus disease in pigeons have brought certain risks.In 2021,suspected cases of adenovirus infection of pigeons will occur in pigeon flocks in the pigeon farms in Liuhe District and Lishui District of Nanjing.Through pigeon farm sampling and laboratory research,it was determined that pigeon adenovirus was the main pathogen,and epidemic of novel pigeon adenovirus was detected.Subsequently,novel pigeon adenovirus hexon protein(Hexon)was studied,including the cloning and prokaryotic expression of Hexon protein and the preparation of monoclonal antibodies,paving the way for the development of effective vaccines in the future.This test consists of the following four parts:Experiment 1 Epidemiological investigation of PiAdV infection in Nanjing areaA total of 248 throat swabs and liver samples were collected from the diseased pigeons and the same group of pigeons in the diseased pigeon farm.Two pairs of primers were designed to establish a PCR method for simultaneous detection of pigeon adenovirus type ? and novel pigeon adenovirus.The collected clinical samples were detected.Hexon gene was purified,cloned and sequenced,and DNAStar software was used for bioinformatics analysis.The results showed that the detection rate of pigeon adenovirus was 15.7%,among which the pigeon adenovirus type ? accounted for 71.8%and the novel pigeon adenovirus accounted for 28.2%.The detected pigeon adenovirus was mainly young pigeons less than 1 month old,and the detection rate reached 40%of the total number of positives.The observation of pathological sections of the diseased tissue revealed typical basophilic inclusion bodies.The clinical samples were also tested for the infection of pigeon circovirus and pigeon herpes virus,and the detection rates of the two viruses were 19.7%and 8.4%,respectively.The above results show that the infection rate of pigeon adenovirus in Nanjing area is high,the pathogen type is no longer single but has a mixed infection trend,and the pathogen has obvious variation.Experiment 2 Cloning and prokaryotic expression of novel pigeon adenovirus Hexon proteinIn order to explore the immunological characteristics of the main structural protein Hexon of the novel pigeon adenovirus,referring to the gene sequence of the novel pigeon adenovirus Hexon,DNAstar and other software were used to analyze its hydrophilicity,antigenicity,and antigenic epitopes,and a section of sequences with rich antigenic determinants was intercepted.A pair of specific primers were designed to amplify part of the Hexon sequence by PCR,and a single product with a size of about 1038bp was obtained.Part of the Hexon gene was cloned into the pET-32a vector,and the pET32a-Hexon recombinant plasmid was successfully constructed.After the recombinant plasmid is transformed into BL21 competent cells,the recombinant protein can be expressed after induction by IPTG.After identification by SDS-PAGE electrophoresis,a band with a size of about 57kDa can be obtained,which is in line with the expected size,and the protein is expressed in the form of inclusion bodies.After the recombinant protein was purified,SDS-PAGE electrophoresis showed that there was only a band of about 57kDa,indicating that the protein was expressed correctly and a prokaryotic expression system was successfully established.Experiment 3 Preparation of monoclonal antibody against Hexon protein of novel pigeon adenovirusThe monoclonal antibody against novel pigeon adenovirus was further developed.The purified recombinant protein was used as an immunogen,emulsified with adjuvant and inoculated into 6-8 week-old female Balb/c mice.After the third immunization,serum antibody levels were determined,and mice that reached the positive standard were selected,and their spleen cells were fused with SP2/0 cells.A mAb-2B3 that can stably secrete anti-Hexon protein was prepared by using monoclonal antibody technology.Through the monoclonal antibody subtype identification test,it was found that the mAb-2B3 was IgG1 subtype,? light chain.Immunohistochemical experiments showed that mAb-2B3 could bind to the novel pigeon adenovirus Hexon protein,and the protein was expressed in the cytoplasm.Hybridoma cell 2B3 was intraperitoneally injected into 8-week-old female Balb/c mice,and a week later,a very high titer of mouse ascites could be collected,and the titer of mouse ascites was identified by ELISA test up to 1:204800.Experiment 4 Epitope identification of anti-Hexon protein monoclonal antibodyThe epitope of the mAb-2B3 of a novel pigeon adenovirus was explored.The truncated Hexon protein expressed in prokaryotic cells was used as the antigen,and the mAb-2B3 was used as the primary antibody for Western blot analysis to identify the antigenic epitope.After the initial positioning,the same method was used to precisely locate the bound antigen region,and finally the antigen recognition epitope was screened to 386SGVPQG391.Comparing the homology of the antigenic epitopes of the monoclonal antibody,the results showed that the antigenic epitopes of mAb-2B3 were poorly conserved and could only bind to the novel pigeon adenovirus,but not to other types of adenoviruses.In conclusion,this study established a PCR detection method that can simultaneously detect pigeon adenovirus ? and novel pigeon adenovirus.The results showed that pigeon adenovirus was prevalent in Nanjing area and showed a trend of constant variation,and there was mixed infection with other pathogens.For the Hexon sequence of the novel pigeon adenovirus,a prokaryotic expression system was successfully established,and then a monoclonal antibody against the Hexon gene was prepared and the corresponding epitope was identified,which laid a good foundation for the subsequent establishment of a serological diagnostic method. |
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