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Construction and Immunogenicity to Piglets of Chimeric Porcine Circovirus

Author: ShaoXiaoXue
Tutor: ZhouShuangHai；RanDuoLiang；WangZhiCai
School: Xinjiang Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Porcine circovirus type 1 Porcine circovirus type 2 Infectious DNA clone Chimeric porcine circovirus Immunogenicity
CLC: S858.28
Type: Master's thesis
Year: 2010
Downloads: 75
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Abstract


Porcine circovirus (PCV) has PCV1 and PCV2 two genotypes are not pathogenic, PCV1, PCV2 can cause postweaning multisystemic wasting syndrome and other diseases. This study by cloning the PCV1 full genome sequence, to construct a PCV1 infectious DNA clone; infectious DNA clone by chimeric PCV (PCV1-2), to obtain biologically pure PCV1-2, and analyzed through experimental inoculation piglets PCV1-2 pairs The immunogenicity of the piglets. Was amplified by PCR and cloned a PCV1 genome, named the BJ-1 strains. The full genome sequence results showed that the strains with other PCV1 isolates 99.4% nucleotide homology sequence of the open reading frame (ORF) results show more than 92.7% nucleotide homology. Although the whole genome between each PCV1 isolates with the main ORF nucleotide homology is high, but still showing a certain geographical relevance. The the PCV1 full genome cloned into the vector pBluescript II sk () construct PCV1 DNA cloning, transfection of PK-15 cell line, was rescued viruses PCV1. The will save PCV1 in PK-15 cell lines cultured for 5 consecutive generations, its the TCID50 reach 106.55/mL, showed higher titers of infection. Inverse PCR technology deleted from PCV1 ORF2, and replace the entry of PCV2 ORF2 constructed chimeric porcine circovirus (PCV1-2) DNA cloning, transfection of PK-15 cell line the bio pure PCV1-2. PCV1-2 after 5 generations of continuous culture in the PK-15 cell line, which TCID50 to reach 106.05/mL showed the obtained having a high titer of PCV1-2. PCV1-2 inoculated 35-day-old healthy piglets, observed the piglet clinical changes the PCV2 ORF2 antibody in serum was measured by ELISA using conventional and fluorescence quantitative PCR PCV1-2 DNA content was measured in serum and tissue. The results show no visible clinical symptoms and pathologic changes piglets inoculated with PCV1-2, piglet weight gain and feed conversion efficiency is unaffected; PCV2 ORF2 antibodies in the serum 14 d after inoculation seroconversion, 28 d. antibody positive rate of 100%, indicating that the PCV1-2 on piglet has good immunogenicity; PCV1-2 DNA content in serum increased gradually after inoculation, the highest 28 d after the rapid decline; slaughter in the first 35 d, The highest concentrations of virus in piglets hilar lymph nodes, heart virus lowest content; conventional PCR techniques are difficult to detect PCV1-2 DNA, that PCV1-2 inoculated piglets only cause minor infections. Comprehensive analysis shows that this test successfully constructed with higher infectious titer and good immunogenicity of chimeric porcine circovirus, PCV2 attenuated vaccine research and development has laid a good foundation.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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