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Sequence Analyzing and Prokaryotic Expression of ORF2 Gene in the Strain of Porcine Circovirus Type 2 Isolated from Lanzhou

Author: FuZuo
Tutor: HuYongHao；LiuZaiXin；LuZengJun
School: Gansu Agricultural University
Course: Preventive Veterinary Medicine
Keywords: PCV PMWS Isolation and identification of ORF2 Clone Expression
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 47
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Abstract


Porcine circovirus type 2 (porcine circovirus type 2, PCV2) caused by post-weaning multisystemic wasting syndrome ( post - weaning multisystemic wasting syndrome , PMWS) , reproductive disorders (sow abortion and mortality syndrome, SAMS), pig skin nephrotic syndrome ( porcine dermatitis and nephropathy syndrome, PDNS ) and respiratory syndrome and other related diseases important pathogen [ 1] [2 ] . PCV2 virus infected individuals can detect PCV1 virus , the difference between PCV1 and PCV2 weaned piglets multisystemic wasting syndrome clinical disease is correctly diagnosed . PCV2 is an immunosuppressive virus , the swine damage the immune function , causing secondary infection or co-infection . PCV virus ORF2 reading frame is highly conserved , PCV1 and PCV2 ORF2 reading frame , only 67% homology . The experimental design a pair of primers PCV1 and PCV2 shared disease material collected from the Lanzhou area farms the symptoms suspected PMWS piglets lungs , lymph nodes , PCV contamination PK-15 cells treated after inoculation , the proliferation of viruses and extraction of viral nucleic acid , PCV viral the ORF2 sequence fragments , sequenced PCR methods . The results show that , Lanzhou region PCV virus infection . Proceeds PCV virus ORF2 sequence in GenBank 8 PCV1 and PCV2 ORF2 reading frame sequence homology analysis , the Lanzhou region isolates ORF2 reading frame nucleotide homology with PCV2 virus ORF2 reading frame up to 99 % , 68% homology with PCV1 confirmed Lanzhou region of PCV2 isolates . Separate strains of gene sequencing results and the PCV2 ORF2 fragment sequences in GenBank using DNAStar analysis software to transform and optimize the resulting gene fragments , synthetic fragment of about 700bp ; using the expression vector pET-32a target gene fragment clone positive recombinant The plasmids were transformed into E. coli BL21 (DE3), IPTG induced expression ; purified by SDS-PAGE and Western blotting analysis . The results indicate that the synthetic gene fragments expressed in Escherichia coli , the coincidence of protein with a relative molecular weight of about 50KDa, Western blotting analysis confirmed the overlapping protein could PCV2 positive sera recognized .

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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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