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Screening of Classical Swine Fever Virus NS2 Interaetive Proteins in Swine Umbilical Vein Endothelial Cell Line by Yeast Two-hybrid

Author: KangZuo
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus NS2 protein Yeast two-hybrid Pig vascular endothelial cells
CLC: S858.28
Type: Master's thesis
Year: 2011
Downloads: 59
Quote: 0
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Abstract


Swine fever virus (Classical swine fever virus, CSFV) cause of classical swine fever (Classical swine fever, CSF) is classified by the World Organization for Animal Health (OIE) animal infectious diseases that must be reported . Due to the low titer of the CSFV cultured in vitro proliferation without causing cell lesions , at home and abroad on the CSFV mostly focused on the detection of the virus as well as the research and development of new vaccines , and the pathogenic mechanism of CSFV relatively small . The NS2 protein is a non-structural protein of CSFV . Available information indicates that the NS2 protein CSFV replication through interaction with the host cell have some regulatory role and produce protease activity , cutting NS2-3 complex release NS3 monomer NS3 monomer accumulation causes the inoculation culture cells cell lesions ( CPE ) . NS2 protein and host cell interactions as well as adjust the mechanism of viral RNA replication is still not very clear. Target to research NS2 protein and host cell protein interactions using the yeast two-hybrid technology , CSFV host cell pig vascular endothelial cells (SUVEC) NS2 protein - protein interaction screening . Obtained the following findings : (1) from pGFP-NS2 green fluorescent amplified a 360 bp coding region of the C-terminus of NS2 gene expression vector , and built into pGBKT7-NS2-C360 bait expression vectors of yeast cells without toxicity and no self- activation of the expression vector pGBKT7 bait . (2) was successfully isolated , purified porcine vascular endothelial cells in double-stranded cDNA is used to express the fusion protein of the prey library . (3 ) pGBKT7-NS2-C360 carrier , double-stranded cDNA prey vector pGADT7-Rec transformed into yeast competent cells , 16 positive clones were obtained after screening . Comparison analysis can be obtained by PCR, sequenced , 8 kinds of known proteins that interact with CSFV having , respectively, cytosol nonspecific - peptidase ( CNDP2 ) , heat shock protein 40 ( Hsp40 ) , the CFTR associated with the body ( CAL ) , E3 ligase Pan hormones ATP synthase 6 ( ATPase6 ) is NRAS related proteins , apoptosis regulation gene (MOAP1) and ribonucleoprotein H1 . The test screening of known protein Hsp40 protein repeatability is relatively high, and the available data indicate that , Hsp40 involved in the regulation of viral RNA replication . So Hsp40 focus of further study as a screening of protein .

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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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