| Duck circovirus (DuCV) is a new member of the circovirus genus and the circovirus family, which is nonenveloped and icosahedral viruses, with a circular, single-stranded DNA genome. DuCV is Mainly encroaching on the host immune system, and make the host hypoimmunity, which easily causes secondary infections by other bacterial and viral pathogens, and bring huge economic loss to the duck-raising industry.However, as the cuture method in vitro for DuCV is currently unavailable, there are still many difficulties in researched on the pathogenicity and mechanism of DuCV infection. This study aims to investigate the genetic diversity and variation of DuCV in Guangxi, and to prokaryotic expression of Cap protein of DuCV as well.1. The genetic variation analysis of DuCV in Guangxi2569tissue samples from ducks collected in Guangxi markets during2010-2012were detected by PCR in this study.194samples were positive, and the positive rate was7.55%.14representive DuCV were isolated, and their fulllength genomic sequences were determined. All of them were 1993nt～1995nt in length, the same as other DuCV strain’s genome sizes that had been reported. Their nucleotide similarities were93.5%～99.8%to each other. They share a relative high similarity of92.2%～99.6%with the Geman strain, the USA strain,4Korean isolates and the24isolates from Chinese mainland. When compared whith the other18isolates from Chinese mainland and4isolates from Taiwan, however, the homology was only82.3%～86.2%.No phenomenon of nucleotide insertion or deletion in the genomic coding region was found in all14Guangxi isolates. The Cap genes (ORF C1) of the14Guangxi isolates in this study were774nt in length, encoding257amino acids. The nucleotide sequence homology and the amino sequence homology were89.5%～99.9%and95.3%～100.0%, respectively. The ORF C2were237nt in length, encoding78amino acids.The nucleotide sequence homology was96.6%～100.0%, and the amino sequence homology was91.1%～100.0%.The Rep gene were879nt in length, encoding292amino acids.The nucleotide sequence homology was95.9%～99.7%, and the amino sequence homology was98.3%～100.0%.The phylogenetic tree analysis based on all DuCV genome sequences showed that the two distinct branches were existed, the genotype1and the genotype2. And genotype1can be further divide into gene subtype1.1, gene subtype1.2and gene subtype1.3. The subtype1.1is comprised of22Chinese mainland isolates and the German strain. Subtype1.2included14Chinese mainland isolates, the USA strain (33753-52) and4Korean isolates. Subtype 1.3has only two Chinese mainland strains, LJ07and LJ33. Similarlly, there were also three subtypes in the genotype2, subtype2.1, subtype2.2and subtype2.3. The subtype2.1had only4Taiwan China strains, subtype2.2had15Chinese isolates, and only3Chinese mainland isolates, MH02-07, MH11and HZ09belong to subtype1.3. The14Guangxi isolates obtained in this study belong to genotype1. In perticular, QZ37-2012, QZ04-2011, LZ11-09, FC35-2012, QZ36-2012, D41-09, FC33-2012, FC34-2012, FC32-2012, NN13-2012belong to subtype1.1, and LZ01-2011, NN11-2012, NN12-2012, PX08-2011belong to subtype1.2.2. Prokaryotic expression of DuCV Cap proteinUsing software Gene Designer2(DNA2inc), a codon optimized Cap gene was designed, with BamH.Ⅰ and Sal Ⅰ enzyme sites were introduced to the upperstream and downstream, respectively. The Cap gene fragment was obtained by enzyme digestion using BamH Ⅰ and Sal Ⅰ, and subcloned into the expression vector pET-32a(+) to generate the recombinant plasmid pET-32a-Cap. The pET-32a-Cap was transferred to E. coli BL21(DH3) competent cells. Following induced by0.1mmol/L IPTG, the bacteria were culture in37℃,250r/min shake, for4h. The result of SDS-PAGE analysis showed that the fusion Cap protein with about48Ku was successfully expressed mainly in the form of inclusion body. Western blot proved that the fusion Cap protein could be specifically recognized by the His tag antibody. Finally, recombinant Cap protein was futher pufified by using the Ni-ion affinity purification column. |
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