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A Thesis Submitted to Zhejiang Sci-Tech University for Degree of Master of Science

Author: HuangChunMei
Tutor: ZhangYaoZhou
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: PPRV H Bac-to-Bac Systerm Baculovirus/Silkworm Surface Display System Recombinant BmNPV-SHT Immunogenicity Detecting
CLC: S855.3
Type: Master's thesis
Year: 2011
Downloads: 33
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Abstract


Peste des petits ruminants (PPR) is an A type contagious disease caused by Peste des petits ruminants virus (PPRV), has the characteristic of drastic and acute. It mainly infects petits ruminants, especially goat, using vaccine for PPR prophylaxis is the most effective way at present. H glycoprotein is one of PPRV`s chief antigen proteins, the full length of the gene which codes PPRV H protein is 1852 bp, and the length of it`s open reading frame is 1827 bp, coding 609 amino acid residues, molecular mass is about 70 kD., can induce body produce neutralizing antibody.Baculovirus/Silkworm Surface Display System is a new Baculovirus Expression System, it can display foreign proteins by inserting interest peptides into the baculovirus` coat protein GP64. GP64, as well as GP64-fusion proteins, can be detected on the surface of virally infected insect cells. In this system, recombinant baculovirus can be used as immunogen to immune animals to obtain monoclonal antibody, avoid traditional tedious protein purify procedure.Our experiment using Baculovirus/Silkworm Surface Display System to express H glycoprotein, displayed it on the surface of baculovirus, for preparing vaccine for PPR prophylaxis. We obtained a fusion sequence of PPRV H gene, which has fused BmNPV GP64 signal peptide and transmembrane sequence by using reverse transcription-PCR, named it SHT sequence. Then cloned it into pFastBac1 vector and successfully constructed the recombinant vector. Transformed recombinant vector to E. coli DH10Bac, used blue/white selection acquired the recombinant plasmid Bacmid-SHT, PCR detection found them contained PPRV H gene. Transfected recombinant plasmid to silkworm BmN cells to produce recombinant baculovirus BmNPV-SHT. From RT-PCR and Western blotting results,we found PPRV H gene had been transcribed and expressed not only in BmN cells but also in silkworm pupa, which had been transfected byrecombinant baculovirus BmNPV-SHT. We purifed recombinant baculovirus to immune Balb/c mice, determined antibody titer of immuned mice serum was 1:7000, neutralization activity was 1:32, T cell stimulation index was 6.1 while control group stimulation index is 1.8. The results showed that recombinant baculovirus BmNPV-SHT can arouse body produce antibody and immune cells, had good immunogenicity and immunoreactivity, gave us a rudimentary proof that the BmNPV display PPRV H protein on its surface has great potentiality to be a new vaccine against PPR.

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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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