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Study on the Full-length cDNA Clone of Hog Cholera Virus Lapinized Chinese C81/WHHV Strain and Expression of Nonstructural Protein2

Author: GuoDongChun
Tutor: ChenHuanChun
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus Non-structural protein NS2 Expression
CLC: S852.65
Type: Master's thesis
Year: 2005
Downloads: 143
Quote: 3
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Abstract


Hog cholera (classical swine fever; CSF) is a classical swine fever virus (classical swine fever virus; CSFV) pigs caused by a highly contagious and fatal diseases, fever and bleeding as the main feature. Classical swine fever was distributed worldwide, the International Bureau will be listed as Class A veterinarian 16 kinds of legal infectious diseases. Reference to the Chinese classical swine fever virus strain Lapinised genome sequence, designed seven pairs of full-length genome coverage primers were amplified by RT-PCR of classical swine fever virus genome cDNA, cloning, sequencing, sequence assembly software to get the use of classical swine fever virus C81/WHHV strain genomic length cDNA sequence (GenBank: AY663656). C81/WHHV strain genome length 12310bp, contains an open reading frame encoding a 3898 amino acid polyprotein precursors. Sequence analysis showed that, C81/WHHV with classical swine fever virus strain attenuated strain ORF nucleotide sequence homology of 84.4% -99.6% amino acid sequence homology of 91.6% -99.4%. Meanwhile, compared CSFV5'-NTR characteristic sequence and predicted C81/WHHV strain polyprotein precursor functional domains. Design a set of primers to amplify different lengths NS2 gene into the expression vector pGEX-KG in different recombinant plasmids were constructed pKG-NS2, pKG-NS876, pKG-NS400, identification, transformed into E. coli BL21 (DE3 ), IPTG induction, by SDS-PAGE analysis: Only pKG-NS400 bacterial cell lysate appears at a molecular weight of about 40kDa and the expected molecular weight of the protein bands match; Western-blot analysis showed that expression products with pigs distemper virus-positive sera, a single reaction occurs with the expression products mainly in inclusion bodies. While other cell lysate protein expression without a purpose. The amplified to a full-length NS2 gene was cloned into the eukaryotic expression vector pcDNA3.1 () to construct the recombinant plasmid pCD-NS2, liposome-mediated transfection of mammalian cells, PK-15, the indirect immunofluorescence assay showed that : NS2 protein initially achieved in mammalian cells, the expression of NS2 protein mainly in the cell membrane. This next step will be to establish stable cell lines expressing NS2 protein NS2 protein complementation test and to lay the foundation for research. Yeast codon usage, using a pair of primers to amplify the NS2 gene was cloned into the yeast expression vector pPIC9K, constructed recombinant plasmid p9K-NS2, Buster into Pichia pastoris GS115, methanol is induced after The silver-stained SDS-PAGE, Western-blot test confirmed NS2 failed to achieve expression in yeast, which may be used with yeast codon preference for sex-related, although mutations in the NS2 gene partial nucleotide ends, but the genes inside codon of gene or have a big impact.

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