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Cloning and Expression of Porcine Interferon-alpha Gene and Studies on Anti-HCV Activity of Recombinant Protein

Author: WuDan
Tutor: XiaChun
School: China Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Porcine interferon-α E. coli Gene Expression Anti - viral activity Classical swine fever virus
CLC: S852.65
Type: Master's thesis
Year: 2003
Downloads: 320
Quote: 10
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Abstract


Many kinds of viral disease of pigs, hazards, so the R \u0026 D has broad-spectrum antiviral activity of porcine interferon preparations of great significance. In order to develop biological engineering recombinant porcine interferon preparations in China, the prevention of porcine viral diseases, and further development of porcine interferon molecular biology research paper porcine α interferon gene (PoIFNα), cloning and expression of its recombinant protein anti-swine fever virus. Porcine interferon-α gene sequences have been logged in the DDBJ / GenBank gene pool, a pair of primers containing Sph Ⅰ and Hind Ⅲ restriction sites, take a polymerase chain reaction (PCR) method, pig genomic DNA as a template The a PoIFNα cloning. PCR products were purified recovered, inserted into pGEM T-Easy Vector, and confirmed by sequencing cloned a new subtype swine alpha interferon. Double digested from pGEM T-Easy/PoIFNαVector will PoIFNα fragment inserted into the pQE30 expression vector recombinant expression plasmid pQE30/PoIFNα. The pQE30/PoIFNα transformed into E. coli JM109 IPTG inducible expression, SDS-PAGE analysis to filter out the efficient expression rPoIFNα strains. Followed by Western-Blot expression identified the rPoIFNα. by the partially purified full renaturation rPoIFNα the cytopathic inhibit the the method determination rPoIFNα the resistant follicular stomatitis virus (VSV) activity. PK-15 cells and further anti-classical swine fever virus (HCV) the Shimen Department (SM) virulent test. The results show that, we cloned the mature peptide gene sequence of a new subtype of porcine alpha interferon, the total length of 501bp, encoding 166 amino acids and a stop codon of the mature polypeptide; compared with the gene pool in the PoIFNα nucleotide homology of 97.8%, 94.6% amino acid homology only point substitutions, insertion or deletion variation. SDS-PAGE analysis showed that the recombinant expression plasmid pQE30/PoIFNα expressed in E. coli JM109 rPoIFNα accounted for 20 to 26% of the total bacterial proteins. Western-Blot confirmed expression rPoIFNαN 'side containing six histidine tag molecular weight 20.7KDa. attributable after rPoIFNα partially purified 80 to 90% of the total bacterial protein refolding after non-toxic to cells, the anti-viral activity of up to 1 × 10 8IU/ml. And PK-15 cells by immunofluorescence reaction detection rPoIFNα anti-SM effect display of 10 ~ 3IU and 10 ~ 5IU, rPoIFNα could significantly inhibit the infection of HCV-SM. In this study, the first in the country porcine alpha interferon gene cloning, expression and anti-classical swine fever virus, which laid the foundation for the production of genetically engineered recombinant porcine alpha interferon.

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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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