Prokaryotic Expression Of Deleted ORF2 Capsid Protein Of PCV2 And The Eastishment Of The Agar Immunodiffusion Test To Detect Antibody Of PCV2

Porcine circovirus (PCV), classified in the family Circoviridae, is small nonenveloped DNA virus with a circular genome. This isometric virus is 17~20 nm. PCV1 is nonpathogenic in swine, a new disease, named postweaning multisystemic wasting sydrome (PMWS), has emerged in pigs. A genetic variant strain of PCV1, designed PCV2. Genetic and pathogenesis studies revealed that the nonpathogenic PCV1 and PCV2 belong to two different geno types. The genomic DNA of PCV2 consisit of two major open reading frames,ORF1 and ORF2, ORF2 encodes a major capsid protein. ORF2 sequence identity between PCV1 and PCV2 is about 66% at the nucleotide.Addition of 41 amino acid at the N terminus of PCV2 ORF2. Expression, purification and application of the NLS deleted ORF2 capsid protein was demonstrated in experiment.The primes was designed according to the strain of PCV2-871. The gene was amplified and cloned into the expression vector pQE-32 by BamH I/ HindⅢnamed pQE-PCV581. The recombinant plasmid pQE-PCV581 was transformed into E.coli M15, then induced by IPTG at 37℃.The recombinant protein was detected by analysis of SDS-PAGE, confirmed that the protein was expressed abundantly. furthermore the recombinant protein was purified and detected by Western blot and ELISA. The recombinant protein had good antigenicity.The agar diffusion reaction assay method was eastablished in experiment. Solubility and its antibody was mixed in agarose gel by electrolyte, presented precipitation reaction.We determined that disposal of agarose gel is optimal, at the same time we made replication, specificity tests. The results showed possesed good replication, specificity. The range of Concentration of recombinant protein was 0.91-1.83 mg/mL.The detection method possesed handling is convenient, quickly, was fit for applying to basement layer.