Cloning And Expression Of DuCV ORF3 Gene, And Preparation For Its Monoclonal Antibodies And Application

Duck circovirus (DuCV) belonging to Circoviridae circovirus, the functions of DuCV 0RF3 protein are still not clear by now. In order to preparation for antibody against the protein, further to explore its function, series researches on the DuCV ORF3 gene has been carried out in this paper, which including bioinformatics analysis, expression and purification of ORF3 gene, preparation for monoclonal antibodies of ORF3, and establishment and application of competitive ELISA method. The results as follows.1 Bioinformatics analysis of DuCV 0RF3The DuCV GH01 isolate has a full length of 1988 bp, and ORF3 gene is 297 bp. The content of A, T, G, C in ORF3 is 21.89%,25.25%,24.58%,28.28%respectively. This gene locates genome in 399th to 103th bases, and encodes 98 amino acids. Forthermore, the molecular weight of ORF3 is about 11.6 KD. Random coil in the protein secondary structure is about 62.3%, α-helix and β-fold structure account for 31.6%and 6.1%respectively. No conserved domain, signal peptide and transmembrane domain are contained in this protein. It is prompted that ORF3 may be one of the key protein to the virus evolution, neither a secretory protein nor transmembrane protein. There is no glycosylation sites in ORF3 gene, but exsists three potential phosphorylation sites. Antigen epitopes mainly concentrated in 2th-12th,35th-40th,49th-53th,62th-65th,72th-75th,77th-84th amino acids. And so many antigen epitopes may be suggested that ORF3 is good for antigenicity, and can be used as an immune protein.2 Expression and purification of DuCV ORF3 proteinThe ORF3 gene was cloned into T-vector after PCR amplification with specific primers. Subsequently, it was inserted to the expression vector by digesting (EcoR I and Xho I), and connection. And pET-32a-ORP3 and pGEX-6p-ORF3, which are prokaryotic expression plasmids, were constructed successfully. The recombinant plasmids were transformed into Rosetta (DE3) E.coli bacterium, the fusion proteins of ORF3, which with 30 KD and 35 KD, were expressed inducing with IPTG. The optimal IPTG concentration, used for inducing expression fusioned ORF3 protein in pET-32a-ORF3 was 0.8 mM, and that in pGEX-6p-ORF3 was 0.2 mM. These two kinds of fusioned ORF3 proteins mainly exists in the form of inclusion body in host bacterials. Ni2+ affinity chromatography, inclusion body wash and SDS-PAGE gel recycle methods were used for purification of the two fusion proteins respectively, which had a high level of concentration and purity. To detecte the fusion proteins whether or not ORF3 proteins, western blot analysis was carried out and the result showed that the two fusion proteins could link to serum with duck circovirus positive serum specifically. All of thos results were indicated that the two fusion ORF3 proteins had immunological activity.3 Preparation of Monoclonal Antibodies against DuCV ORF3After refolded, the purified ORF3 fusion protein which is resouce from pET-32a-ORF3 plasmid, immunization of Balb/c female mice by intraperitoneal injection with 100 μg. When antibody titer attained to 104, spleen cells were prepared, and fused with SP2/0 myeloma cells in condition of the presence of PEG4000. Positive hybridoma were screened using the established indirect ELISA (optimal concentration for antigen coating was 1 μg/well) by my own, and the fusion rate was 25%. According to three times limited dilution subcloning, hybridoma purity reached 100%, and four strains of hybridoma cells were obtained (named 1,2,3,4), which secreting ORF3 antibody stably. Ascites antibodies were prepared which were resource from ORF3 positive hybridoma cells, and antibody titers were detected with ranging from 1:25600 to 1:102400. All of them were Ig M subclasses, and could be combined with the ORF3 protein specifically.4 Establishment an indirect competitive ELISA method for DuCV detection and its applicationTo develop a stable and reliable method for DuCV ORF3 diagnosis, an indirect competitive ELISA method has been established, which was based on purified pET-32a-ORF3 fusion protein. Checkerboard titration was performed to determine the optimal concentration of coating antigen and ascites of monoclonal antibodies. Then the serum dilution, the competition time, and the cut- off decision value were optimized. The results showed the optimum conditions of this method as following, coated pET-32a-ORF3 protein at the concentration 1 μg/well, and closed with 5% BSA, incubation 30 min in DuCV positive serum with dilution 1:800, incubation 40 min in ascites of monoclonal antibodies with dilution 1:4000, incubation 30 min in HRR-goat anti-duck antibody, then collor by TMB. The method of the cut-off determine value was 10.9%, reacted with DuCV positive serum specifically, and the sensitivity was 1:1600 of serum dilution.45 unknown samples were detected by this method and PCR, the results showed that, there were 25 positive samples in this experiment method, while PCR detection had 37 positive samples which contains this 25 positive samples, and the consistent positive rate of the two methods was 100%.