Bacterial Expression Of Porcine Circovirus Type 2 Genes And Preparation Of Monoclonal Antibody And Animal Experiment With Infectious Molecular Clone

One porcine circovirus type 2 (PCV2), originally isolated from a pig affected with postweaning multisystemic wasting syndrome (PMWS), was propagated through porcine kidney cell line PK15. The virus genome amplified by polymerase chain reaction (PCR) was inserted into pMD-18T vector as pMD-PCV. The genomic nucleotide sequence was .found to have homology with other PCV2s deposited in the GenBank database from 93% to 99%. The isolated strain was named PCV2 JXL and registered to GenBank with number AY491310.To benefit understanding the pathogenesis and the etiological role of PCV2, a biologically pure PCV2 infectious clone was generated by transfection of PK15 with pMD-PCV molecular clone. PCV2 virions produced by in vitro transfection were infectious as the propagated progeny were successfully detected by indirect immunofluorescence assay (IFA) at 4-passage transfected PK15 cell. Furthermore, in vivo transfection of piglets was performed by injected into the superficial iliac lymph nodes with 500ng of pMD-PCV DNA molecular. There was no remarkable clinical signs in inoculated pigs while mild gross lesions were characterized by systemically enlarged lymphodes. Histopathological lesions and PCV2-specific antigen were detected in numerous tissues including lymph nodes, kidney, spleen, lung and tonsil using polyclonal antiserum by IFA.PCV2 contains two major genes of ORF1 and ORF2 which code for Rep protein and Cap protein correspondingly. Rep protein is known as replicase-related, while Cap is the mere capsid protein. Three pairs of primers were designed and the genes of ORF1 and ORF2 as well as two fragments overlying ORF2, i.e. N-end fragment of 737-421nt and C-end fragment of 421-37nt, were a mplified from JX L b y PCR with t he 1 ength were 0.9bp, 0.34bp and 0.4bp, respectively. Each was inserted into pMD-18T, resulted in three kinds of recombinant clones named pMD-ORFl, pMD-PCV737-421 or pMD-PCV421-37, respectively.Using prokaryocyte expression system pPROEXHTb which harbors 6 His residues and restricted endonuclease sites of BamHI/Hind, subcloned ORF1 from pMD-ORFl into pPROEXHTb, resulted in recombinant plasmid of pPRO-Rep. In the same way, using prokaryocyte expression system pGEX-6p-l designed for espression of gene fused with GST and restricted endonuclease sites of BamHI/Sal., subcloned PCV737-421 from pMD-PCV737-421 into pGEX-6p-l to get pGEX-PCV737-421, also subcloned PCV421-37 to get pGEX-PCV421-37 using sites of EcoRI/Sal.According to the product protocol, the recombinant genes of pPRO-Rep and pGEX-PCV737-421 as well as pGEX-PCV421-37 were induced by isopropylthio-p-D-galactoside (IPTG) in E.coli, respectively. The fuse expressed foreign proteins of His-Rep, GST-PCV737-421 and GST-PCV421-37 were discerned among total bacterial proteins through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the molecular weights were all about 40ku when fused with Hisor29kuofGST.In order to identify whether both GST-PCV737-421 and GST-PCV421-37 have biological reactivity or not, the gel containing recombinant proteins purified through SDS-PAGE was ground and inoculated BALB/c mice intraperitoneally. The blood was collected after the 3rd inoculation. Serum antibody were detected by indirect IFA with PCV2-infected PK15 suggests that PCV737-421 and PCV421-37 contain PCV antigen epitopes and could be used to construct serum diagnostic reagent.Additionally, a quantity competitive PCR assay was constructed by inserting part of somatostain (SS) gene of 180bp into recombinant plasmid of pET-Cap containing total PCV2 ORF2 gene to obtain competitive template pET-Cap-SS. Two different sizes of PCR amplified fragment, i.e. 880bp and 700bp, could be differentiated through agarose gel electrophoresis when dyed with ethidium bromide.A series of reagents and approach obtained here might be very advantageous for future PCV2 studies.