Cloning And Sequence Analysis Of The ORF2 Gene Of Henan PCV2 Strain And Its Expression In Eukaryotic

Porcine circovirus type 2(PCV-2)is the main pathogens of Postweaning multisystemic wasting syndrome(PMWS) and Porcine circovirus-associated disease(PCVAD). The ORF2 gene of PCV-2 is the main region encoding the structural protein, which has good antigenicity and can be used to differentiate PCV-1 and PCV-2. In this paper, the ORF2 gene of PDSH and LY strain were aquired by PCR(polymerase chain reaction), then cloned to Eukaryocytic expression vector. After transformation to BHK-21(baby hamster kidney-21) cell, the ORF2 protein was found successful expression, then the protein was identified preliminarily.According to the sequence of PCV-2 gene, a pair of specific primers was designed to amplify the ORF2 gene of PCV-2 from PDSH/LY. The ORF2 gene, a fragment of 719bp, was amplified by polymerse chain reaction(PCR), then cloned to pMD18-T easy vector and identified by BamH I and Hind III digestion. After sequencing, the homology comparison was conducted with the published ORF2 sequence of PCV-2 and the phylogenetic tree was drawn. The aquired ORF2 gene of PCV-2 of 719 bp encodes 233 amino acids with a molecular weight of 27 995.93D.The sequence homology of ORF2 gene of PCV-2 of Henan isolates ranges from 97.0% to 99.0%, the deduced amino acid sequence is between 95.3 and 99.1%. The highest nucleotide and deduced amino acid homology was found between XX and LY strains, which is up to 98.9% and 99.1% respectively. Compared with the virus strains in China, the strains of PDSH and HZ, and LY and HANGZHOU have the highest homology of 99.3%;Compared with the strains outside, the strains of LY and Denmark have the highest homology of up to 99.6%;and the strain of PDSH has homology of up to 99.3% with of Brazil and Denmark strains. LY strains has the highest amino acid homology of 99.6% with of Denmark strain, and PDSH strain has the highest amino acid homology of 98.7% with Brazil strain. The lowest was found between JZ and PDSH strains.A pair of primers with Restriciton enzyme site(REs) was designed to amplify the main antigenic plot of 566bp from the cloning plasmid pMD18-T-ORF2, then the amplicon of 566bp was digested by BamH I and Hindâ…¢, cloned to Eukaryotic expression vector pcDNA3.0. After identified by RE digestion and sequencing, a recombinant expression plasmid named pcDNA3.0-ORF2 was successfully constructed, and transiently transinfected to BHK-21 cell. The mRNA can be detected by reverse transcription polymerase chain reaction(RT-PCR) from the transinfected cell. Indirect immunofluorescence assay(IFA) shows the recombinant protein can be recognized by PCV-2 specific antiserum that has good antigenicity.The result shows the plasmids of pMD18-T-ORF2 and pcDNA3.0-ORF2 were successfully constructed. The Cap protein was transiently expressed in the cytoplasm of BHK-21 cells and displays specific immune responses to PCV-2 antiserum.In this paper, a clone plasmids of pMD18-T-ORF2 and a expression plamid pcDNA3.0-ORF2 were successfully constructed. The Cap protein was transiently expressed in the cytoplasm of BHK-21 cells containing the recombinant plasmid, which has biologic activity. This research may lay a foundation for the study of PCV-2 DNA vaccine and monoclonal antibody(McAb).