Prokaryotic Expression The Capsid Protein And Establishment Of An Indirect ELISA Method For Duck Circovirus

According to the the DNA sequence of duck circoviru (DuCV) GHO1 strain submitted by our laboratory, the specific primers were designed to amplify DuCV Capsid (Cap) gene and the templates were isolated from tissue samples which were collected in Sichuan, and carried out:the characteristics of Cap gene and Cap protein of DuCV GHO1 strain, prokaryotic expression of the truncated Cap gene, purification of recombinant proteins by affinity chromatography, establishment and application of the indirect enzyme linked immunosorbent assay (ELISA) for DuCV antiserum by using Cap protein as coating antigen. The results are shown below:1. Clone and molecular characteristics of DuCV Cap gene The full-length of DuCV GHOl strain Cap gene was amplified by PCR, and recombinant plasmid was constructed, after restriction enzyme digestion and sequencing, the Cap gene and Cap protein characteristics were analyzed. The results as follows:the complete Cap gene with a length of 774bp, the T cloning plasmid successfully constructed, which named pMD18-cap; encoding the Cap protein with 257 amino acids and an isoelectric point (PI) of 10.79, and its molecular weight was 29.7kD; there was a conserved domain in Cap protein which belongs to Circovirus capsid superfamilies; it predicted that Cap protein contained 3 glycosylation sites,21 phosphorylation sites, and 5 possible antigen epitope domains, located in 1-39,60-85,96-116,129-180 and 211-243 regions. There was no signal peptide and transmembrane domain, the secondary structure of Cap protein possessed many random coil, and tertiary structure prediction showed it had only a suitable model for homology modeling, it was porcine circovirus Cap protein; the phylogenetic tree revealed that DuCV GH01 strain had the closest genetic relationship with duck circovirus FJFQ315 strain, while a closed genetic relationship with goose and swan circovirus in same genus, but the relationship between DuCV GH01 and Chicken anemia virus was farthest; codon usage analysis showed that the DuCV GH01 Cap gene has the codon bias tropism, the frequency of GGG, CAA, AAG, TTA and TCA was zero in codon usage; by performing subcelluar location experiment, it showed Cap protein mainly located in nucleus.2. Cloning, prokaryotic expression and protein purification of DuCV truncated Cap gene Based on the sequence of DuCV GH01 full-length Cap gene, a pair of primers was designed that lack of the nuclear localization signals (NLS), and the material saved in our laboratory as template for the to amplify the truncated Cap gene, then, the Cap gene cloned into pMD18-T vector, which was confirmed by restriction enzyme digestion and sequencing. Subsequently, the Cap gene fragment was diagested and then connected to expression vector pET-32a (+), resulting in the recombinant expression plasmid was transformed into Rosetta. After optimalization of temperature, concentration of IPTG and induction time, the best conditions of expression was founded. Recombinant protein was purified by Ni-NTA affinity chromatography, the eluent was collected and determination of the protein concentration, and analyzed the purification effect by SDS-PAGE. The results showed that the truncated Cap gene was amplified successfully and the size was 567bp, identical with expectation size; the recombinant expression plasmid pET-32a-cap was constructed after Cap gene and expression vector connection; highest level of protein expression was obtained when the recombinant bacteria were induced by IPTG with the concentration of 0.8mmol/L at 30℃ for 16h; recombinant Cap protein was 39kDa and expression in the form of inclusion bodies, purified recombinant protein was about 1.5mg/mL and the band was clearer. The successful expression of DuCV Cap protein has laid a firm foundation on subsequent test.3. Establishment and application of indirect ELISA for the detection of DuCV antiserum by using recombinant Cap protein as coating antigen The indirect ELISA conditions was optimized as follows:antigen concentration, serum dilution, package conditions, closed conditions and secondary antibody dilution, the best operating conditions was determined and the cut-off values was calculated, the specificity, sensitivity and repeatability of indirect ELISA was detected, and finally, the indirect ELISA methods established in this study was compared with conventional PCR detection method for detection of clinical samples. The results showed that the optimal conditions of this indirect ELISA were as follows:the concentration of coating antigen was 4μg/well, the dilution of the examined serum was 1:40, the coated condition was 37℃ for 1h followed by incubation at 4℃ overnight, the blocking time in 1% BSA was 1h at 37℃ and the second antibody dilution was 1 : 800, calculated the cut-off value was 0.352. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DuCV anti-sera detection. The sensitivity of indirect ELISA reached 1:2560, and the coefficient of variation between intra-assay and inter-assay were less than 10%, which indicated that the establishment of indirect ELISA had a good specificity, sensitivity and repeatability. Compared the compliance rate of PCR and indirect ELISA established in study was 95.6% on detecting 59 tissue samples collected.