| Infectious haematopoietic necrosis(IHN)is caused by infectious haematopoietic necrosis virus(IHNV)infection of the Novirhabdovirus genus in the Rhabdoviridae family of viruses,which can infect all mites Fish is an acute and cause highly contagious viral diseases.The highly pathogenic strain of IHNV has a mortality rate of more than 90% for juveniles.The disease caused by IHNV has become a worldwide salmon disease,which has brought major economic losses for the aquaculture industry.However,there is currently no effective drug for the treatment of IHN,and vaccine is still one of the most effective means of preventing IHNV.The major antigenic protein of IHNV is the surface glycoprotein G,which contains neutralizing epitopes that induce cells to produce immune neutralizing antibodies.In this experiment,we analyzed the signal peptide region,the hydrophobic transmembrane region and the epitope region of the G protein gene sequence of the IHNV HLJ-09 strain using the bioinformatics principle.And we truncated the G protein gene fragment using the E.coli expression system and analyzed the immunogenicity of the expressed protein and the immune protection for the iridescent juvenile.The results as follow:1.The prokaryotic expression vector p ET-28a-d G was constructed and induced by IPTG to obtain the target protein of 53 k Da.After purification of the target protein by Western blotting,the target protein can be specifically recognized by the mouse anti-IHNV whole virus serum,and has good immunogenicity.2.The mouse antiserum was prepared by using purified protein of interest(d G)and could interact with the recombinant protein and IHNV HLJ-09 strain by enzyme-linked immunosorbent assay(ELISA)and the titers were 1:25600 and 1:1600,respectively.The virus neutralization test was used by the prepared mouse antiserum.According to the Reed-Muench method,the neutralization titer was 1:112,indicating that the mouse antiserum can recognize IHNV and has a certain neutral effect,which can produce corresponding neutralizing antibodies.3.In order to analyze the immunoprotective effect of IHNV d G-induced rainbow trout juvenile fish,the purified target protein was emulsified with Freund’s incomplete adjuvant and inoculated into rainbow trout juveniles.The experiment was divided into three groups,namely vaccine group,recombinant protein group and PBS as the control group and a dose of 2 μg/g of fish body weightper tail was administered intraperitoneally.Fish serum was collected at 0,1,2,3,4,5,and 6 weeks after immunization,and was measured for the specific antibody Ig M level.The results showed that the antibody level of the experimental group was significantly increased after immunization(P<0.01),and significantly higher than the control group(P<0.01),peaked in the fourth week after immunization,and then decreased.The results of the challenge with IHNV in the fourth week after immunization showed that the relative protection rates of the inactivated vaccine group and the recombinant protein group were 62% and 60%,respectively.The above results indicate that the truncated IHNV G protein expressed by E.coli prokaryotic expression system can be used as a candidate immunogen for IHNV subunit vaccine,which can induce neutralizing antibodies against IHNV in rainbow trout juveniles,and laid a certain theoretical foundation for the subunit vaccine. |
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