Cloning And Sequencing Of Complete Genome Of PCV-2 Isolates From Shangdong And Establishment Of Digoxigenin-Labeled Nucleic Acid Probe For PCV-2

Porcine Circovirus Type 2 (PCV-2) is a small non-enveloped single-stranded circular DNA virus classified in the Circoviridae family. PCV-2 was identified to be the pathogeny of PMWS and associated diseases and caused significant economic losses to pig industry. To understand the hereditary diversity of the PCV-2 strains in the different areas and period, developing a simple and rapid diagnostic method had great significance to prevent and control the prevalence of the PCV-2. In this study the samples in different districts of Shandong province were collected and the complete sequences of PCV-2 were cloned. According to the epidemiology investigation of the PCV-2 in our province, the study revealed the origin and diversity of PCV-2 strains, analyzed the hereditary diversity and evolution among the different PCV-2 strains and developed the detection of Digoxigenin-Labeled Nucleic Acid Probe for PCV-2 in order to offering the academic basis to the vaccine research and the establishment of diagnostic technique. This study included two parts.PartⅠ: Cloning and sequencing of complete genome of porcine circovirus type 2 (PCV-2) Shandong strains.The complete genomes of PCV-2 were cloned and sequenced from the tissues of the suspicious PMWS sampled in different districts of Shandong province isolated from 2002 to 2004. The results of sequencing showed that the complete genome of every strain was consisted of 1767bp. The nucleotide sequence homology among the eight PCV-2 isolated strains were found to be 97.3～99.8% respectively. The homologies were 95.6～99.8% compared with the sequences of PCV-2 published in Genbank. The results of phylogenetic tree showed that nucleotide sequence homology were near among the eight PCV-2 isolated strains with United Kingdom,French and Australia strains.The study of ORF1 and ORF2 for the eight PCV-2 isolated strains showed that the nucleotide sequence homology which coding Rep protein and Cap protein were 98.4%～99.7% and 96.6～99.9% respectively, thus the diversity of PCV-2 mainly concentrated on the ORF2. Through analyzing the amino acid sequence of constitutive protein,the PCV-2 strains diversity was found from 53th to 91th position of amino-acid residue, which corresponded with immunological reaction area, and it may be useful for virus avoiding immunologic response.According to the sequence comparison of complete genomes, PCV-2 had two genotypes: the length of the complete genome,one was 1767bp, the other was 1768bp. All of 1767bp strains were lack of basic T in the 1039th position and basic T in 1035th mutated to base A. TTA was changed for TAA, produced termination codon and led to the end of ORF5 .PartⅡ: Establishment and application of Digoxigenin-labeled nucleic acid probe for PCV-2In this study the detection of Digoxigenin- labeled nucleic acid probe for PCV-2 was successfully developed. According to the genomic sequence of PCV-2 published in Genbank, one pair of primers were designed in the ORF2 for amplifying the 371bp fragment by PCR. The PCR product was labeled with digoxigenin as DNA probe for detection of PCV-2. The result of hybridization assay showed that the eight recombinant bacterias of PCV-2 were positive and no cross-hybridization was detected with DNAs of PCV-1,PRRS,PPV and PRV. The sensitivity result showed that as few as 10pg DNA of PCV-2 could be detected by the probe. The DIG-labeled probe was proved to be a specific, sensitive, rapid and simple method and could be used in research of epidemiology and clinical diagnosis of PCV-2 in molecular level.