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| Prokaryotic Expression and Protective Efficacy of VP60 Truncated Gene in RHDV GS/YZ Strain |
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Abstract Abstract Rabbit haemorrhagic disease virus (RHDV) belongs to the genus Lagovirus of Caliciviridae. VP60 protein is one of the capsid proteins of RHDV, and also is main immunogenic protein which the major antigenic epitopes mainly locate in the N-terminal region. In this study, the VP60 truncated gene was cloned by employing the method RT-PCR and then subcloned to pMD20-T. After complete sequence analysis, the prokaryotic expression plasmid pET-RHDV-VP60 was successfully constructed and then inserted into the expression Escherichia coli Rosetta(DE3). The target protein was highly expressed under the induction with 1 mmol/L isopropyl β-D-1 -thiogalactopyranoside (IPTG), and the Balb/C mice were immunized with purified expression products, then the polyclonal antibody was prepared. Subsequently, the immunogenicity of recombinant protein was analyzed using indirect enzyme linked immunosorbent assay (iELISA) and Western-blot. Twenty clean rabbits were randomly divided into 4 groups and subcutaneously injected once, using commercial vaccine (5 rabbits), the RHDV GS/YZ inactivated by 0.4% formaldehyde (5 rabbits), recombinant protein mixed with adjuvant (5 rabbits) and PBS as negative controls (5 rabbits)，respectively . The dosage of recombinant protein was 500 μg mixed with equal amounts of freund's complete adjuvant per injection, with 14 days interval, the recombinant protein was 250 μg mixed with equal amounts of freund's incomplete adjuvant per injection, After 21 days, all the rabbits attacked by the RHDV GS/YZ that HA titer was 210 and observed the results after 14 days. The results showed that the homology of nucleotide sequences of VP60 truncated gene from RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 91.9% to 98.6%, and had 98.6% nucleotide sequence identity with the RHDV HB strain(accession number: KU207100.1) and RHDV WX strain (accession number: AF402614.1). The homology of amino acid sequence of RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 96.7% to 99.1%, and had 99.1% amino sequence identity with the RHDV WX strain (accession number：AF402614.1) and RHDV Mexico 89 strain (accession number：AF295785.1). The target gene was successfully expressed in Escherichia coli, and the recombinant protein expression in the form of inclusion body. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD. The titer of polyclonal antibody was 1∶32 000 by iELISA and Western-blot demonstrated that the recombinant protein could bind with antibody induced by RHDV GS/YZ and the polyclonal antibody induced by recombinant protein could also combine with vaccine of RHDV - Pasteurella multocida and wild viruses of RHDV GS/YZ. So it had good immunogenicity. The immune protection experiment showed that the rabbits in the RHDV GS/YZ inactivated by 0.4% formaldehyde group, commercial vaccine group and recombinant protein mixed with adjuvant survived facing the challenge without any clinical signs of RHD while those in the negative control group died within 96 h post-infection and exhibited typical clinical symptoms. The titer of hemagglutination inhibition of commercial vaccine was 25~28, the self-made inactivated vaccine was 25~26, and the recombinant protein mixed with equal amounts of freund's adjuvant was 24~25. The results showed that the proteins expressed by Rosetta (DE3) could be ideal candidate for RHDV vaccines.
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Received: 13 October 2017
Published: 02 May 2018
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