| Serum type Ⅲ duck hepatitis A virus（DHAV-3） is one of the main pathogens causing duck hepatitis in recent years,which is highly infectious and lethal.In this study,insect baculovirus expression system was used to express the important immunogen gene of DHAV-3,and the immunogenicity of the expressed recombinant protein was preliminarily studied.An indirect ELISA method was established for the detection of DHAV-3 antibody in clinical duck serum samples.The specific research contents are as follows:1.The prokaryotic expression vector p Cold-VP0 was constructed and induced by IPTG to be expressed in supernatant.The recombinant VP0 protein was purified and identified by SDS-PAGE and Western blot.The results showed that the recombinant VP0 protein could react specifically with HIS antibody and DHAV-3 polyantiserum.The results showed that the best concentration of VP0-ELISA was 4μg/mL,the best dilution of serum to be tested was 1:200,and the best working concentration of enzyme-linked antibody was 1:5000.A total of 24 duck negative serum samples were tested to determine the positive value of 0.336.The coefficients of intra and inter batch are less than 5%,which shows that the method has good repeatability.Compared with STN,the positive,negative and total coincidence rates of VP0-ELISA were 80%,100%and 94%respectively.2.The P1 and 3CD gene fragments were amplified by RT-PCR,and then inserted into PPH and Pp10 promoters of the same pFast Bac-Dual donor plasmids respectively.The correct recombinant plasmids were transformed into DH10 Bac sensitive cells.After blue and white spot screening,the white positive clones were selected and the recombinant Bacmid was extracted and identified by PCR.The recombinant Bacmid was transfected into sf9 cells by cell fusion and the recombinant viruses（rDHAV3）was obtained.The infection titer of r DHAV3 was 1.0×1011 PFU/m L.Western blot and IFA were used to identify the recombinant baculovirus.The results showed that P1 precursor protein was successfully cleaved into three structures of VP0、VP1 and VP3 by 3CD protease,and the expressed recombinant protein had good reactivity.Transmission electron microscopy（TEM）showed that the recombinant baculovirus self assembled into virus like particles（VLPs）with a diameter of about 25 nm.3.SPF ducklings were immunized with the cell infection supernatant of the P3 generation rDHAV3 as antigen to detect its immunogenicity.The experimental group was rDHAV3 with water or oil adjuvant,while the control group was Bacmid and PBS.Before and after immunization,blood samples were collected from the jugular vein of each group,serum was separated,specific antibody and neutralizing antibody of DHAV-3 were detected,and peripheral blood was separated for lymphocyte proliferation test.One week after immunization,HBGT strain was used to carry out the protection test,each 0.2 m L(LD50 was 10-6.6/0.2 m L).The results showed that the level of specific antibody and serum neutralizing antibody in the experimental group was significantly higher than that in the control group（P<0.05）,and the SI value of lymphocyte in the experimental group was significantly higher than that in the control group（P<0.05）.The results showed that the protection rate of the experimental group was 80%.In conclusion,P1 and 3CD proteins of DHAV-3 were constructed and expressed in sf9 cells.The virus-like particles can effectively induce the ducklings to produce humoral immunity and cellular immunity,and can protect the ducklings from virus attack to a certain extent.The results provide an reference for the prevention and control of DHAV-3 infection and the research of genetic engineering vaccine. |
