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Cloning and Expression of Porcine P58IPK Gene and Regulation of Its Expression During Influenza A Virus Infection

Author: WenJunGe
Tutor: ZhangDeLi
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: P58IPK Polyclonal antibody Western blotting qRT-PCR Immunohistochemistry
CLC: S858.28
Type: Master's thesis
Year: 2011
Downloads: 15
Quote: 0
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Abstract


P58 IPK interferon induction of the dsRNA-activated protein kinase PKR intracellular inhibitor. Influenza virus infection P58 IPK in the post-translational level activation, P58 IPK activated lead PKR-mediated phosphorylation of eIF-2α reduce the level of viral protein expression levels, Advantageously on virus replication. However, knockout P58 IPK mice infected mortality was significantly elevated, and severe lung lesions and inflammatory responses. The purpose of this study is to analyze the pigs P58 IPK influenza virus infection the specific experimental content and the results are as follows: 1. Pig study in accordance with the gene sequences cloned human P58 IPK source the P58 IPK IPK . Swine the P58 IPK total length of 1 518 bp, encoding 505 amino acids, the N-terminal and C-terminal of the protein is highly conserved, derived from pigs P58 IPK Human P58 IPK protein similarity is as high as 99.21%. The P58 IPK cloned into the prokaryotic expression vector, pET-32a, was transformed into E. coli expression strain BL21 (DE3), IPTG concentration of 0.2 mmol / L, 37 ° C for 7 h induced soluble expression of the fusion protein His-P58 IPK, and purified by column chromatography HiTrapTM Chelating HP immunize New Zealand white rabbits, obtained P58 IPK of polyclonal antibodies. Detection of multi-resistant titer of 1:100,000, with atopy. Destination cloned into pEGFP-C1 and pEGFP-N1 eukaryotic expression vector use the liposomal transfection were transfected with swine umbilical vein endothelial cells (SUVECs). 24 h after transfection, fusion the protein EGFP-P58 , IPK -the C1 and P58 , IPK -EGFP-N1 fluorescent expression levels maximum. Determined according to the position of the fluorescence emission pigs the source P58 IPK protein localized in the cytoplasm, and the cells were collected 48 h after lysis, Western blotting detected fusion protein EGFP-P58 , IPK and endogenous P58 IPK protein. Influenza virus strains H1N1 and H3N2 infection in healthy pigs, 7 d after take pig lung tissue the part mentioning RNA and reverse transcribed into cDNA, using real-time quantitative PCR method to detect P58 IPK gene changes another part is stored in 4% formaldehyde solution, do lung histopathology slices and the P58 IPK protein immunohistochemical detection. The results showed that the infected pig lung tissue significant pathological changes in the body P58 IPK mRNA expression and protein expression were raised after the viral infection.

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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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