| Duck Viral Hepatitis(DVH)is a highly lethal,rapidly spreading viral disease caused by Duck Hepatitis Virus(DHV),which has become one of the most serious epidemics for duck breeding in China.DHV has three serotypes:DHAV,DAstV-1,DAstV-2,and DHAV has three genotypes(DHAV-A,B,C or DHAV-1,2,3).At present,DVH in China is mainly caused by DHAV-1 and DHAV-3,DHAV-3 has even replaced DHAV-1 as the dominant serotype,and Co-infection of 2 genotype viruses occurs sometimes,which may be the reasons why many duck farms that have been vaccinated with the traditional DHAV-1 vaccine but still experiencing duck hepatitis.Currently,there is no commercial vaccine for DHAV-3,so it is of great significance to develop some reagents for diagnosis and control of DHV.The VP1 is the main capsid protein of DHAV,posses many domain determinants of neutralizing antibody,can stimulate the body to produce the protective antibodies,and its gene has highly variable regions of the DHAV genome sequence,with the greatest genetic diversity.In order to learn more about the biological characteristics of DHAV wild strains,and develop the diagnosis and prevention of DHAV-3 reagent,22 of DHAV-1 and DHAV-3 strains were isolated and identified in this study.VP1 sequences were analysed,and VP1 gene of a DHAV-3 isolate were expressed with eukaryotic and prokaryotic expression systems respectively.An indirect ELISA method was established for detecting antibodies of DHAV-1 and DHAV-3 with the purified prokaryotic expression of VP1 protein as antigen.The potential of the VP1 eukaryotic expression systems as DNA vaccine were evaluated.This study is mainly divided into three parts:1.isolation,identification and sequence analysis of VP1 gene of 22 duck hepatitis A virus strainsIn order to learn more about the characteristics of duck hepatitis A virus circulating in China,27 samples of the cases suspected in clinical were collected for pathogen isolation and identification from 2017 to 2019 in Shandong province.Then the VP1 genes of the isolates were sequenced and the phylogenetics based on these sequences were analyzed.The results showed that 8 isolates were duck hepatitis A virus type ?(DHAV-1)and 14 isolates were duck hepatitis A virus type 3(DHAV-3).Sequences analysis of VP 1 showed that nucleotide homology of 8 DHAV-1 isolates compared with 15 reference strains was 91.7%to 98.6%,amino acid homology was 93.7%to 97.5%,and nucleotide homology among 8 isolates themselves was 97.2%to 100%,amino acid homology was 97.9%to 100%.14 DHAV-3 isolates compared with 17 strains of reference strains of nucleotide homology of 89.4%to 98.9%genes,amino acid homology was 92.1%to 100%,14 strains isolates themselves nucleotide homology of 93.9%to 100%,amino acid homology was 96.7%to 100%,belong to the type ? in the gene cluster(GI).These results will provide a reference for prevention and control of the disease.2.eukaryotic expression of VP1 gene of Duck hepatitis A virus type 3The VP1 gene of DHAV-3 was amplified by RT-PCR and were used to construct recombinant plasmids pCDNA3.1-3-VP1,KpCDNA3.1-3-VP1(including kozak sequence)and pCAGGS-3-VP1,which were transfected into Vero cells.IFA and WB was used to analyze the expression products.The results showed that VP1 gene was expressed in pcDNA3.1 and pCAGGS vectors,and the size of the expressed recombinant protein was 27kDa.Then,the recombinant plasmid was used as DNA vaccine to immunize mice with 100 ?g each one,negative controls were set with empty vectors,and immunized for 3 times in total.The antibody level in mice was detected by indirect ELISA.The results showed that the mice could produce antibodies at 14 days after immunization,while no specific antibodies were detected in the control group.The results indicated that the constructed recombinant plasmids could induce the mice to produce specific antibodies.3.prokaryotic expression of VP1 gene and development of ELISA method for DHAV-3RT-PCR was used to amplify VP1 gene of DHAV-3 isolate and a recombinant plasmid PET-32a-3-VP1 with His label was constructed.The recombinant plasmid PET-32a-VP1 was transformed into host bacteria BL21(DE3),and induced by IPTG.The expression products were identified by SDS-PAGE and WB,and the results showed that the recombinant plasmid was successfully constructed,and the size of the recombinant protein was about 40.8kDa.The recombinant protein VP1 expressed by prokaryotes was purified by His-labeled protein purification column and used as antigen detection.An indirect ELISA test for antibody detection was preliminarily established.Then the optimization of the reaction were determined:the concentration of antigen package is 4.28 ?g/mL,and placed at 4? for the night,duck serum sample was diluted at the ratio of 1:20,and incubated for 90 min,the HRP labeled Goat anti-Duck IgG was diluted at the ratio of 1:400,and incubated for 90 min,TMB substrate color solution was incubated for 15 min.The tests of this ELISA showed high repeatability and specificity,and those results may lay the foundation for the detection of DHAV-3 antibodies in the future. |
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