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Cloning and Prokaryotic Expression of N Protein Gene of Canine Distemper Virus and Antigenicity of the Recombinant Protein

Author: ShenWeiHong
Tutor: JianZiJian
School: Xinjiang Agricultural University
Course: Basic Veterinary Science
Keywords: Canine distemper virus N protein gene Clone Prokaryotic expression Antigen
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 107
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Abstract


Distemper, canine distemper virus (Canine distemper virus, CDV) infected dogs or other carnivores caused by a highly contagious, fatal disease, biphasic fever, rash, conjunctivitis, leukopenia and central nervous system damage as the main feature, an important class of diseases to harm the development of special breeding industry. Disease is no specific drug treatment, prevention and early diagnosis of this disease is particularly important. This study include the following three parts: 1-distemper N protein gene cloning and sequence analysis of the CDV N protein gene sequences published in GenBank reference, a pair of specific primers designed using Oligo 6.0 software, dogs suffering from distemper whole blood sample, total RNA was extracted 572bp gene fragment was amplified by RT-PCR, insert it into build the pMD18-CDV N recombinant plasmid pMD18-T cloning vector, and transferred into E. coli DH5, were identified and sequenced. Sequence analysis showed expression of the CDV N protein gene from the ATG start codon to the stop codon TAA over a total length of 1572 nucleotides, compared to the homologous CDV N protein gene sequences published in GenBank rate of 93.9% -99.1%. 2 distemper N protein gene prokaryotic expression of recombinant proteins reactogenicity CDV N protein gene prokaryotic expression vector pGEX-4T-2 to construct the pGEX-4T-2-CDV N recombinant plasmid was transformed into E. coli BL21 (DE3), were identified and sequenced, IPTG inducible expression. Western blot analysis showed that the expression product of 84kDa recombinant protein can be identified distemper antiserum, having a good reactogenicity. 3 distemper N protein gene prokaryotic expression conditions optimization of recombinant protein purification and immunogenicity studies in order to improve the GST-CDV N recombinant protein in Escherichia coli soluble expression of the amount of GST-CDV N recombinant protein in E. coli various expression conditions, to select the best conditions for efficient expression and purification of GST-CDV N recombinant protein with GST-Sepharose 4B affinity chromatography, to obtain the maximum amount of soluble recombinant protein. By mice immunized with GST-CDV N recombinant protein having a good immunogenicity. This study successfully cloned, expressed the the distemper N protein gene, purified recombinant protein has a strong antigenicity, provides the possibility for the establishment of canine distemper ELISA diagnostic kits, and provides a basis for the development of subunit vaccines. Basis

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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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