Expression Of Capsid Gene Of Chinese Isolate Of Rabbit Hemorrhagic Disease Virus And Preliminary Study On The Immunogenicity Of Recombinant Protein

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease characterized by a hemorrhagic syndrome that affects domesticated and wild rabbits of the genus Oryctolagus cuniculus. RHD has a high mortality rate and is responsible for large economic losses to rabbit producers. The disease was first described in China in 1984 and is currently endemic in most parts of the world. Morphological, biochemical, protein and genomic studies have shown that the virus is a calicivirus, an icosahedral non-enveloped positive-sense singlestranded RNA virus with a diameter of about 40 nm. RHDV was recently designated as the type species of the genus Lagovirus, which includes the European brown hare syndrome virus (EBHSV), a highly pathogenic virus of hares (European brown hare Lepus europaeus and "varying hares" L timidus). In 1996, a non-pathogenic virus preliminarily called Rabbit calicivirus (RCV), which is more closely related to RHDV than to EBHSV was described.Until now, the attempts at in vitro propagation of RHDV failed. So at the moment, there are several commercially available vaccines against rabbit hemorrhagic disease (RHD) on the market, but all are elaborated from tissues collected from experimentally infected rabbits. Although they have proved to be effective tools for prevention of the disease, manufacturing this kind of killed vaccines gives rise to many problems resultingfrom the use of infectious virus and the risk of its dissemination from vaccine factories. Thus, it is important to develop alternative approaches for producing vaccines. To this aim, current research is dedicated to avoid such limitations by expression of VP60, the major structural protein of RDHV using recombinant bacteria, baculovirus-infected insect cells and Pichia pastor yeast to look for alternative procedures to produce useful vaccines.First of all, the capsid protein (VP60) gene of rabbit hemorrhagic disease virus(RHDV) isolate WX84 was amplified by reverse transcript polymerase chain reaction ( RT-PCR ) technique and cloned into pGEM-T vector for sequence. The sequence analysis showed that the nucleotide sequence of the VP60 gene of WX84 was 1740 bp and encoding a protein of 580 ammo acids. The accession number of this sequence to GENEBANK is AF402614. Sequence comparison with other published RHDV VP60 genes showed that the homology of the nucleotide was between 98.2% and 99.0%. This result showed that VP60 gene was a highly conserved gene.The RHDV capsid protein (VP60) gene was subcloned into the expressing plasmid vector pGEX-6p-1. The recombinant was verified by restriction endonuclease analysis and nucleotide sequencing. Then it was transformed into E.coli strain BL21 for VP60 expression. The expression of VP60 gene was identified by SDS-PAGE and Western-blot. A specific protein band of 87Kd was found as a fusion protein with glutathione transferase(GST) protein. The specific band of expression was excised from the gel and used to immunize the mice. The antisera was collected from the immunized mice and detected by ELISA in 96-well plates coated with RHDV. The positive results showed that the in vitro expressed protein of VP60 gene as a GST fusion protein maintains some antigenicity of native RHDV.To investigate the impact of the length of fusion protein on the expressed protein, the capsid protein (VP60) gene was subcloned into the expressing plasmid vector PET 5a, which only has an eleven amino acid fusion expression tag derived from the amino terminus of the T7 gene 10 protein under the transcriptional control of a bacteriophage T7 promoter, much shorter than that of pGEX-6p-l. The recombinant was initiallytransformed into a E. coli strain JM109 which does not carry the gene for T7 RNA polymerase. Following verification of the construct by restriction endonuclease analysis, the recombinant plasmid was then transferred into a DE3 host BL21(DE3) pLysS which carry a copy of the T7 RNA polymerase gene integrated into the host chromosome under the control of the inducible lacUVS promoter for VP6...