Cloning And Sequencing Of Complete Genome Of Porcine Circovirus Type 1(PCV1) And Porcine Circovirus Type 2 (PCV2) And Study On The Reciprocal Chimeric Virus Of PCV1-2 And PCV2-1

A pair of primers (P1/P2) were designed based on the published sequence of Porcine Circovirus type 1(PCV1) to amplify the complete PCV1 genome of YA1 strain, as a 1,759-bp polymerase chain reaction(PCR) product, and cloned into pMD18-T vector and sequenced. Similarity analysis showed that YA1 strain shared 98. 8%-99. 9% nucleotide sequence identity with the other six strains in GenBank. The full-length PCV1 of YA1, containing a 938bp open reading frame 1(ORF1) encoding 312 amino acid, a glycosylated site at amino acid residues 20-22(NPS);containing a 701bp open reading frame 2(ORF2) encoding 233 amino acid, a glycosylated site at arnino acid residues 102-104(NYS). Hydrophobic prediction analysis revealed the deduced ORF1 protein sequence contain nine potential HydropHobic regions, ORF2 contain two potential hydropHobic regions. Transmembrane prediction analysis revealed the deduced 0RF1 protein sequence contain two transmembrane regions, 0RF2 contain one transmembrane regions.One strain of Porcine Circovirus type 2 (PCV2),designated as SC,isolated from clinical weanling pigs by means of ELISA and IFT.A pair of primers (P3/P4) were designed based on the published sequence of Porcine Circovirus type 2(PCV2) to amplify the complete PCV2 genome of SC strain, as a 1,767-bp polymerase chain reaction(PCR) product, and cloned into pMD18~T vector and sequenced. Similarity analysis showed that SC strain shared 93.6%-98. 6% nucleotide sequence identity with the other sixteen strains in GenBank and was more distantly related to YA1. The ORF1 gene of homology of nucleotide between SC strain and the corresponding other sixteen strains in GenBank from America, Europe and China were 94. 7. 3%-99. 4%, yet the ORF2 gene were 90. 3%-99.6%. The full-length PCV2 of SC, containing a 944bp open reading frame 1(ORF1) encoding 314 amino acid, three glycosylated site at amino acidresidues 23-25(NPS), 256-258(NQT)and 286-288(NAT);containing a 701bp open reading frame 2(ORF2) encoding 233 amino acid, a glycosylated site at amino acid residues 143-145(NYS). The corresponding homology of amino acid between SC and YA1 were 85. 3%, 65. 2% respectively. HydropHobic prediction analysis revealed the deduced ORFl protein sequence contain six potential HydropHobic regions, 0RF2 contain one potential hydropHobic regions. Transmembrane prediction analysis revealed the deduced ORFl protein sequence contain one transmembrane regions,but 0RF2 is short of transmembrane regions.To construct a reciprocal chimeric PCV1-2 DNA, fragment A (530bp) from recombinant plasmid of pMD18~T-PCV2-SC digested with restriction enzymes Ball and StuI, purified using low-melting point agarose, and cloned into vetor B(3867bp) which from recombinant plasmid of pMD18-T-PCVl-YAl digested with restriction enzymes Ball and StuI,purified using low-melting point agarose. Escherichia coli DH5 Competent cells were used for transformation. To construct a reciprocal chimeric PCV2-1 DNA, fragment B( 584bp ) from recombinant plasmid of pMD18-T-PCVl-YAl digested with restriction enzymes Ball and StuI, purified using low-melting point agarose, and cloned into vetor A (3929bp) which from recombinant plasmid of pMD18~T-PCV2-SC digested with restriction enzymes Ball and StuI, purified using low-melting point agarose. E. coli DH5ot competent cells were transformed.The reciprocal chimeric PCV1-2 and PCV2-1 DNA were showned to be accurate by digesting, PCR and sequencing. Two pairs of PCR primers were designed: the first pair(P5/P6), amplified a fragment of 1705 bp, by using the PCV1-2 DNA as the PCR template, purified using low-melting point agarose;the second pair(P7/P8), amplified a fragment of 1821 bp, by using the PCV2-1 DNA as the PCR template, purified using low-melting point agarose.By cotransfection of ligating PCR products respectively in 1BRS cells with calcium pHospHate transfection system. Results showed that the chimeric PCV1-2 and PCV2-1 DNA have replicated of IBRS cells, because which amplified a fragment of 243 bp(P9/P10),by using PCV1-2 and PCV2-1 viruses growing and gaining in IBRS cells as the PCR template. The PCV1-2 and PCV2-1 viruses were all inoculated intranasally of 4-weeks-old piglets with lml. There were no significant differences in clinical sign or in weight gain of the groups. This experimental study showed, in contrast to PCV1, the pathogenicity of PCV1-2 viruse was not influenced;however,the pathogenicity of PCV2-1 viruse induced in comparison with PCV2. The PCV1-2 and PCV2-1 viruses were all injected intramuscularly of4-weeks-old piglets with lml and at 14 days after inoculated intranasally with lml PCV2-SC strain. The immunogenicity result showed there were no differences in clinical sign but the growth was influenced in the inoculated PCV1-2 groups; however, piglets in the inoculated PCV2-1 groups had a normal growth. We deduce that piglets in the inoculated PCV2-1 could be protected after a challenge with virulet PCV2;meanwhile, piglets in the inoculated PCV1-2 could reduce clinical sign and delay disease devolpment.-To construct a recombiant expression vector pEGFP-Cl-PCV2-SC, full-length fragments of PCV2 ( 1767bp) from recombinant plasmid of pMD18~T-PCV2-SC digested with restriction enzymes Sacil, purified using low-melting point agarose, and cloned into pEGFP-Cl vetor which digested with restriction enzymes SacII, purified using low-melting point agarose. E. coli DH5oc competent cells were transformed. Recombiant expression vector pEGFP-Cl-PCV2-SC digested with restriction enzymes EcoRV, then it was digested with Exonucleaselll/Sl Nuclease through time limit, purified using low-melting point agarose, using T4 DNA Ligase.E. coli DH5a competent cells were transformed. A eukaryotic expression plasmid pEGFP-Cl-PCV2-SOA which delected a portion of ORF1 (37bp) was constructed. IBRS cells were transfected in vitro with them. The expression of PCV2(delected 37bp) was detected by IFT. Piglets were injected intramuscularly with pEGFP-Cl-PCV2-SC-A by different doses. Detecting of antibodies indicated that pEGFP-Cl-PCV2-SC-A had different immune responses by different doses by ELISA.