Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology

Prokaryotic and Eukaryotic Expression of VP60 Capsid Protein Gene Fragments of Rabbit Hemorrhagic Disease Virus

Author Mudasser Habib
Tutor Weihuan Fang
School Zhejiang University
Course Molecular Microbiology and Biotechnology
Keywords Rabbit hemorrhagic disease virus VP60 capsid protein porokaryotic and eukaryotic expression ELISA
CLC S852.65
Type PhD thesis
Year 2006
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Rabbit hemorrhagic disease (RHD) is a highly contagious and often fatal disease of rabbits (Oryctolagus cuniculus) with broad economic and ecologic importance, caused by rabbit haemorrhagic disease virus (RHDV). The disease is often associated with liver necrosis, hemorrhages, and high mortality. RHDV is a member of the family Caliciviridae, 30-40 nm in diameter and has a single major capsid polypeptide (60 kDa), a positively stranded RNA genome of approximately 7.5 kb and a sub-genomic RNA of 2.2 kb.So far Rabbit hemorrhagic disease virus cannot be propagated in tissue culture cells. Thus, recombinant DNA technology is substantial for the production and characterization of viral proteins. The capsid protein has been expressed in several heterologous systems including E. coli, baculoviruses, yeasts and plants etc. The recombinant VP60 obtained in all these systems has been shown to induce protection of rabbits against a lethal challenge with RHDV. However, there is a need for an antibody response assay system to test the efficacy of RHDV vaccine preparations in the field. Therefore, we sought to explore the recombinant RHDV capsid protein as the coating antigen for ELISA.In the present study cDNA fragment of full-length VP60 capsid protein gene of RHDV, designated as VP60-A, was synthesized by RT-PCR of total RNA isolated from RHDV-infected rabbit livers. This full-length cDNA was cloned into pUC19 plasmid vector and sequenced. Nucleotide sequence analysis revealed that it was over 93% homologous with other Chinese isolates, this homology even increased to over 96% with these isolates when compared at the amino acid level. Furthermore, two shorter cDNA fragments from nucleotide position 706 to 1740 and from 850 to 1365 of the capsid gene open reading frame (ORF), designated as VP60-B and VP60-C, were generated and cloned into pUC19 plasmids, likewise. These VP60 gene fragments were then cloned into prokaryotic expression vector pET-30a and recombinant plasmids pET-VP60A, pET-VP60B and pET-VP60C were thus generated. All the three protein fragments were expressed in E. coli as visualized by SDS-PAGE. Fragments A and B were reactive to positive serum against RHDV, but fragment C was not reactive as shown by Western blotting.The reactivity of the recombinant VP60 proteins expressed in E. coli was tested against RHDV anti-serum by an ELISA methodology. The results showed that the full-length capsid protein A was more immunoreactive (higher OD492 values) than proteins B and C to the positive serum, when tested at the same protein concentration. Thus VP60-A was suitable as the coating antigen for ELISA. The optimized ELISA conditions were 30μg/ml protein for coating and 100-fold dilution of the serum samples. The maternal antibody exhibited sharp yet linear decrease during the first 10 days after birth, and the decrease continued at a slower rate until day 20, after which the antibody titer approached the basal level. The antibody titers from all vaccination groups started to increase from day 20 post-vaccination, reached a plateau from days 35 to 40 and tended to decrease from day 45. Vaccination with a dose of 1 ml per rabbit generated higher antibody responses than that with 0.5 ml or 1.5 ml.Full and partial-length VP60 proteins were also expressed in baculovirus expression system. For this purpose VP60-A gene fragment was digested from previously generated pET-VP60A vector and cloned into baculovirus expression vector pFastBac HT A, whereas VP60-B and VP60-C fragments were PCR amplified from pUC-VP60B and pUC-VP60C vectors respectively, and inserted into pFastBac HT A vector. These recombinant pFastBac HT A vectors were then transformed into E. coli DH10Bac cells to generate recombinant bacmids, which were later used to transfect insect cells. The recombinant baculoviruses expressing these three recombinant VP60 proteins were generated. Recombinant VP60 proteins were produced by infecting the monolayers of Sf9 cells with recombinant baculoviruses and incubating at 27℃for 120 hr. Expression of proteins was analyzed by SDS-PAGE analysis. Due to low level expression of recombinant proteins, the specific bands of over expression of these proteins were undetectable, but Western blot analysis of cell lysates and culture supernatants revealed an 80 kDa band of VP60-A protein in the track corresponding to the Bac-VP60A infected cell lysate, depicting that VP60-A protein was highly antigenic. This finding was further confirmed on Immunofluorescent assay of Sf9 cells infected with recombinant viruses, using RHDV anti-serum. Only the cells infected with Bac-VP60A baculovirus showed fluorescence.VP60 capsid protein is the major structural polypeptide of RHDV. It was expected to self-assemble to form virus-like particles (VLPs). For this purpose, the culture supernatants of infected cells and cell lysates were clarified and then ultracentrifuged; resulting pellets were suspended in PBS, spotted onto a coated electron micrograph grid, which was stained with uranyl acetate solution (2%) and examined by transmission electron microscope. We observed VP60-VLPs in the preparation originated from Bac-VP60A infected cells. No VLPs were seen in preparations from Sf9 cells infected with Bac-VP60B, Bac-VP60C or non- recombinant baculoviruses.Based on our studies, we conclude that the antigenicity of capsid protein fragments was dependent on their lengths and the full-length VP60 capsid protein expressed in E. coli or in baculoviruses was more antigenic and immunoreactive than its partial fragments B and C. The full-length VP60 could be used to characterize and titrate the RHDV-specific antibodies arising from natural infection or from immunization. Use of recombinant full-length VP60 protein in antibody detection ELISA can replace the use of viral antigen collected from experimentally infected rabbit livers for ELISA assay, which is more laborious and difficult. Although the baculovirus-based system could express the full-length VP60 in the form of VLPs, the expression level was low. Therefore, further research is needed to improve its yield before it could be considered for application as coating antigen for ELISA or subunit vaccine.

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