Sequencening And Analysis Of Genome Of Porcine Circovirus Type 2 Strains And Construction Of The PCV2 Virus's DNA Vaccine And Its Immune Effect To Mice

According to the sequence of porcine circovirus 2(PCV2)published in GenBank, a pair of primers were designed and local PCV2complete sequences were amplified by reverse PCR. The results showedthat the complete genome of the nine strains was 1768bp or 1767bpin length. There was a 95.6％～100％identity of the complete genomicnucleotides among the nine isolates and 95.1～99.8％identity withthe PCV2 isolates from GenBank. Comparison of nucleotide sequenceconfirmed that the nine isolates were close to the isolates fromFrance and Newsland. The nine isolates ORF2 had 93.1％～100％nucleotide sequence identidy and 91.8％～99.6％amino acid identidy.In this research, we analyzed nucleotide sequences of 26 strainsof PCV1 and 262 strains of PCV2, also, we analyzed the ORF1 and ORF2nucleotide sequences and deduced amino acid sequences. At thenucleotide level, the results show that PCV1 sequences and PCV2sequences are divided into two different branchs, obviously, and among the PCV1 sequences there haven't any rules, but among PCV2sequences, there are divided into two different sub-gene types. Thesub-gene typeⅠis mainly consis of sequences from American, Canadaand Australia, and the sub-gene typeⅡis mainly consist of sequencesfrom Franch and Newzealand. At the amino acid level, the phylogenetictree is very complexity, and doesn't have any rules. 97 strainssequences from china were analyzed, we find that 9 strains sequencesfrom GuangXi,ZhanJiang,HaiNan are part of sub-gene typeⅡand 11strains sequences from TAIWANG are part of sub-gene typeⅠ. But thedistributing of sequences from the other provinces of china are verycomplex, most of sequences are part of sub-gene typeⅡ, remains arepart of sub-gene typeⅠ. By statistic, we discover that, althoughnearby ten years have been pasted, the sequences homology of PCV don't have large changes, the results showthat, PCV2 is very conserved.After analyzed the phylogenetic tree, we discover that thedistributing of PCV2 don't have geographic and time restriction.Also, we analyzed the mutant dots of the PCV2 nucleotide sequencesand deduced amino acid sequences, we discover that, PCV2 nucleotidesequences are easy to transversion, but don't have any tropism tobases or amino acids.Porcine circovirus type 2(PCV2) recently emerged as an importantinfectious pathogen for pigs in the world, moreover, PCV2 can induceimmunosuppression in postweaning pigs. But unfortunately, there is noefficient method or vaccine to control PCV2 infection until now. Inthis study, recombined plasmids PCDNA3-ORF2,PCDNA3-V1,PCDNA3-V2, PCDNA3-V3 and PCDNA3-V1-V2 were constructed by using PCDNA3, PCV2 ORF2, and variation regions of PCV2 ORF2.Production and purification of these plasmids, then immune the 5-weeks-old female mice, and detected the antibody in serum by usingELISA. The results show that all these plasmids can induce antibodyproduction in mice, but the level of antibody is difference, theplasmid PCDNA3-V1-V2 can induce higher level antibody and may enduremore long.