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Construction of Peste Des Petits Ruminants Virus-like Particles and Evaluation of Their Immune Effects in Mice

Author: LiuFuXiao
Tutor: LiuZengShan
School: Jilin University
Course: Preventive Veterinary Medicine
Keywords: Peste des petits ruminants virus Baculovirus expression system Co-expression Virus-like particle Codon-optimization Immunization Neutralizing antibody Neutralization antibody titer
CLC: S855.3
Type: PhD thesis
Year: 2013
Downloads: 89
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Abstract

Peste des petits ruminants (PPR) is an acute or subacute and highly contagiousdisease of small ruminants, caused by peste des petits ruminants virus (PPRV). PPRwas included in the single OIE (Office International des Epizooties) list of notifiableterrestrial animal diseases. PPRV is classified into the genus Morbillivirus in thefamily Paramyxoviridae. Structurally, mature PPRVs are morphologicallypleomorphic particles (400～500nm) enveloped by lipid membrane with viralglycoproteins, namely haemagglutinin (H) and fusion (F) proteins, seen as peplomersprotruding from the envelope. The matrix (M) protein lies beneath the virion envelopeand interacts with the internal nucleocapsid and the cytoplasmic tails of the surfaceglycoproteins. The nucleocapsid (N), revealing a herringbone structure, contains theviral genome, a single strand of RNA of negative polarity.VLPs are composed of viral structural proteins that self-assemble intoparticulates closely resembling the natural virions from which they derive. They arereplication and infection incompetent, due to lacking infectious genetic materials. Aclose resemblance to native viruses in molecular scaffolds makes VLPs effectivelyelicit not only humoral but likely cell-mediated immune responses even with norequirement of adjuvant for vaccines. The baculovirus expression system (BES) hasbeen a versatile platform for the production of recombinant proteins requiringmultiple post-translational modifications, such as folding, glycosylation, disulfidebond formation and proteolytic cleavage. In addition, a relatively large genome sizemakes a baculovirus accommodate more exogenous genes without physiologicalchanges, and very late promoters of baculovirus can efficiently express exogenousproteins. More importantly, baculoviruses generally only infect arthropods and do notthreaten human and animals. Therefore, the BES has widely been used for theproduction of recombinant proteins and the construction of VLP.In this study, the PPRV M protein was split into two truncated forms to besuccessfully expressed in E. coli by analyses of SDS-PAGE, Western blot andMALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with0.2mM IPTG at28°C for12h, whereby bothproteins nevertheless were expressed in the insoluble form. Therefore, both proteinswere purified under the denaturing condition. Balb/c mice were immunized with thecomplex of purified proteins and then effectively produced M polyclonal antibodies,which in sera reached to a relatively high titer after the third immunization by theanalysis of ELISA. The specificity of the prepared polyclonal antibodies was checkedby Western blot and immunofluorescence, revealing them with the desirablespecificity against both non-denatured and denatured M proteins.In this study, quad-cistronic recombinant baculoviruses containing PPRV M, H,N and F open reading frames (ORF) were constructed. A RT-PCR analysis showedthat M, H, N and F mRNAs could be transcribed in infected Spodoptera frugiperda9(Sf9) cells, and however, a Western blot analysis demonstrated that only the M and Nproteins were successfully expressed. Subsequently, the codons of the M and H ORFswere optimized based on insect cells using an OptimumGeneTMsoftware, andmono-cistronic recombinant baculoviruses containing codon-optimized H and MORFs, respectively, were constructed. Analyses of Western blot and indirectimmunofluorescence showed that both the H and M proteins could be expressed ininsect cells, but at a relatively low level.Using the ORFs of codon-optimized H and M as well as native M and N, threebaculoviruses were constructed, namely BV-M-N co-expressing M and N proteins,BV-M*-H*co-expressing M and H proteins and BV-M*-H*-N co-expressing M, Hand N proteins (BV: baculovirus,*: codon-optimization). Analyses of Western blot,indirect immunofluorescence, flow cytometry and confocal microscope confirmedtheir co-expression in Sf9cells. Subsequent analyses of ultrathin section, immunogoldlabeling and transmission electron microscopy showed that VLPs were generatedthrough self-assembly of foreign proteins expressed by three recombinantbaculoviruses in Sf9cells, further budding into supernatant. The VLPs resulting fromBV-M-N were spikeless, and the VLPs resulting from BV-M*-H*and BV-M*-H*-Nwere with spikes. Herringbone-like capsids were occasionally observed in the VLPsresulting from BV-M*-H*-N.BV-M*-H*-and BV-M*-H*-N-infected cell culture supernatants (SM*H*and SM*H*N) were collected and then subjected to sucrose density gradient centrifugationto purify the VLPs (VLPM*H*and VLPM*H*N). Subsequently, SM*H*, SM*H*N, VLPM*H*and VLPM*H*Nwere used for immunization of Balb/c mice. Sera were collected threeweeks post primary immunization and two weeks post secondary immunization,respectively, for analyses of ELISA and virus neutralizing test. The results showedthat SM*H*, SM*H*N, VLPM*H*and VLPM*H*Ncould induce not only specific H and Nantibodies, but also virus neutralizing antibodies, regardless of use of adjuvant.However, the four samples were difficult to stimulate production of interferon-α,interferon-γ, interleukin-4and interleukin-10in the mice.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Infectious Diseases > Virus disease
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