| As a member of the family Circoviridae, Porcine Circovirus(PCV) is a small ,nonenveloped, icosahedral mammalian virus containing a circular single-stranded DNA genome. PCV type 2(PCV2) is closed associated with Postweaning multisystemic wasting syndrome(PMWS), The clinical mainfeastations of PMWS include poor body condition with varying degrees of muscle wasting,dyspnea and enlarged lymph nodes;pallor,diarrhea.the diversity of the incidence and the death rate of the diease is biggness.when it breaks urgently,the death rate can reach 15%,PMWS has caused great economy loss all over the world. Two types of PCV all contain two open reading frames: ORFland ORF2.ORF1 gene exeperssed the Rep proteins which was associated with replicating of the viruse.Recombinant protein of the ORF2 was a major structural protein .The ORF1 of the two types of PCV shows only minor variation with a homology of 85%,and the variation of ORF2 sequences of PCVl and PCV2 is higher and the major structural protein can form viral capsid-like particles in insevt cells. Blanchard approved that the major structural protein can diagnose PCV2 from PCVl by ELISA.therefore the study of the two types of PCV is very necessary.The PCV2-ORF2 fragment was exepressed as glutathione-s-tansferase fusion protein in escherichia coil by Isoprophlthio-B-D-galactoside(IPTG) inducing after the recombinant plasmid PGEX-ORF2 was transformed into BL21 .Two hybridoma cell strains against PCV2-ORF2 fusion protein named 3B2F4 and 9C3D2 respectively,were developed by indirect enzyme-linked immunosorbent assay method after fusion between sp2/0 myeloma cells and spleen cellsBalb/c mice immunized with redentatured and purified fusion protein .The ELISA titer of culture supernatant werel:1024 and.l:512, and ascites were 1:204800 and l:102400.The results of ELISA indicated that the two monoclonal antibodies were only against PCV-ORF2 fusion protein but not reacted with PGEX-KG protein ,PPV and PRRSV which showed high specificity.And the results of IFA made known that the two monoclonal antibodies only can combine with PCV2 but not with PCVl which proved them to be specific to PCV2.Our research provided a powerful tools for the research of PCV2-ORF2 gene and eastablishing some methods for discriminating and diagnosing two sero-type of PCV.In order to amplify ORF2 gene of Porcine Circovirus I,a pair of primer was designed according to the Genbank sequence .The ORF2 gene was obtained by polymerase chain reaction(PCR) and the plate was the plassmid SK-PCVl which wasestablished by our laboratory.Then the gene was cloned into PMD18-T veter and identified by BamHI and Hindlll digestion.The nucleotide sequence of PCV1-0RF2 gene was compared with the counterpart sequence of PCV1-ORF2(Accession NO.AF071879) and PCV2-ORF2(Accession No.AF027217),The nucleotide homology of 0RF2 between two PCV1 share of 97%;when compared with PCV2-ORF2,two regions were lack for each other. When compared with the animo acid of PCV2-ORF2,There existed several conseral conserved founctional domains,including potential N-liked glycosylation sequence,nuclear localization sequence and tyrosine phosphorylation sites.After the analysis of the amino acid sequence of PCVl-ORF2,a domiain of the amino acid sequence of the protein that has the character of nuclear localization sequences was found.deletion of this domain from PCVl-ORF2,the remainder domain wassubcloned and fused to Enhanced Freen Fluorescence Protein(EGFP).Then the fusion gene construct was transfected to Hela cells.The green fluorescence was observed around the whole cytosol.Meanwhile ,a fusion gene construct which can express the fusion protein of EGFP and PCV1-ORF2 in eukaryotic cells was transfected into Hela cells ,too.The green fluorescence could be seen to locate mainly in the nuclei.This means that the basic amino acids region near the N end is very important for PCV1-ORF2 fusion protein nuclear localization. |
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