Clonging And Expression In E.Coli Of The ORF2 Main Antigen Region Gene Of Pocine Circovurus Tykpe 2

Porcine circovirus is a member of circoviridae which no envelope and as a single unit of negative chain DNA the particle diameter was only about 17 nm,so is a minimum virus we found sofar. The virus can be divided into two types, PCV-1 and PCV-2. PCV-2 can cause a disease named postweaing multisystemic wasting syndrome (PMWS), whereas PCV-1 is ubiquitous and nonpathogenic for pigs. PMWS has made a great loss in world's seine industry and aroused people's attention.Both PCV-1 and PCV-2 were knowed that they contain 2 major open reading frames (ORF) that are ORF1 and ORF2. ORF1 encodes a putative protein involved in viral replication (Rep protein) and ORF2-coding product is the major structural protein(Cap protein ).Rep protein of PCV-1shares about 85% identity with that of PCV-2,which is the major rason of PCV-1 and PCV-2 have cross-reaction of antigenicity. Cap protein shares about 68% identity between type-2 virus, however, no cross-reaction of antigenicity is observed between them. Therefore, the ORF2 gene can be used to differentiate PCV-1 and PCV-2.According to the mucleotide sequence of published porcine circovirus type 2 (PCV-2) ORF2 in GenBank,a pair of primers that were specifc to the ORF2 main areas antigen of PCV-2 were designed and synthesized in the research.The main areas antigen of ORF2 was amplified by polymerase chain reaction(PCR) from PCV-2 which was isolated with PK-15 cell line befor and had been characterized. The PCR product was cloned into the pMD18-T vector and then sequenced. By biology-software, the result of sequence analysis indicated the identity is higher between strains isolated in our country than that in other countries and the sequence cotine more areas antigen of ORF2.Using othe pair of primers, the main areas antigen of ORF2 gene, was amplified by PCR. A fragenent of 493bp of ORF2 gene was obtained and cloned into pMD18-T vector and then transgormed into pET-32a empression vector. The recombinant pET-EXP plasmid was transformed into BL21(DE3) competent cell of E.coli and induced by IPTG with a finial concertration of 0.8mmol/L 6 hours .SDS-PAGE analysis showed that the recombinant protein hand been expressed. The protein ould react with polyclonal antibody against PCV-2 Western-blot test. The result declared that the expressin protein shared a good antigencity.