| In this study, the CQW1 strain (GenBank NO. KM233707.1) of duck tembusu virus (DTMUV) was purified by sucrose density gradient centrifugation, and the morphological structure of the virus particle was observed by transmission electron microscope. Then, the purified virus that inactivated by formaldehyde was used to immunize ducklings to obtain antisera. As a template, the nucleic acid of this virus was used for E gene amplification by RT-PCR, and the target segments were inserted in vector for expression in the prokaryotic system. Based on the recombinant protein that expressed by E gene, a colloidal gold immune chromatography test strip was developed for rapid detection of DTMUV serum antibody. The main research contents are shown as follows.1. Purification, morphological observation of DTMUV, and preparation of immune serum against DTMUVHere, we inoculated 9-day-old duck embryos with the CQW1 strain of DTMUV and then collected allantoic fluid of the duck embryos which died after inoculated for 48 hours. Virus suspension was purified through refrigerated ultracentrifugation with the sucrose of 60%,45% and 30% density gradient. The morphological structure of purified virus particle was detected by transmission electron microscope. The results showed that the virus was mainly distributed between 45% and 60% sucrose concentration. The virus particle with about 50 nm in size has the structures including core, envelope and protrusion. After inactivated by formaldehyde, the purified DTMUV was used to immunize ducklings for obtaining antiserum. The results of double agar gel immune diffusion test showed that the titer of the duck hyperimmune serum was 1:32. This study has laid the foundation for the following works.2. Amplification and expression of DTMUV truncated E geneBased on E gene structure of the CQW1 isolate, the transmembrane region was removed and amino acid residues with well-immunogenicity were used to design specific primers for E gene amplification by RT-PCR. The E gene fragment was cloned into pET-32a(+) vector, and then inserted into E. coli Rosetta (DE3) for expression. Sequencing data showed that the E gene fragment was about 1206 bp, consistent with the expected size. The truncated E gene was successfully expressed in E. coli Rosetta (DE3) after induced with IPTG. The recombinant E protein was approximately 65 kDa. Western blotting showed that the protein could specifically bind to the antiserum of DTMUV, thus indicating that the recombinant E protein has the immunological activity.3. Development and application of an immunochromatographic strip for detecting antibody against DTMUVIn this study, an immunochromatographic test strip for detection antibody against DTMUV was successfully developed. The truncated recombinant E protein and goat anti-mouse IgG were conjugated with colloidal gold particles, respectively. The E protein was coated in the test line (T line) and the mouse IgG was coated in the control line (C line) of the test strip. Comparison of the experimental conditions showed that the most suitable labeling pH of colloidal gold particles with recombinant E protein and goat anti-mouse IgG was 6.8 and 6.7, respectively. The optimal labeling concentration of E protein and goat anti-mouse IgG were 5.5μg and 17.0μg in 1 mL colloidal gold solution, respectively. The antisera of DTMUV, Duck circovirus (DuCV), Duck hepatitis virus type 3 (DHAV-3), Duck hepatitis B virus (DHBV), Duck plague virus (DPV), Riemerella anatipestifer (RA), Escherichia coli (E. coli), and Salmonella enteritidis (SE) were detected by this strip, and only the strip that detected antisea of DTMUV showed positive results. The strip was used to detect the antiserum of DTMUV that diluted to 1:21,1:22.....1:210 respectively, and the result showed that it can detected most in 1:2 diluted serum. Here,217 duck sera collected from clinical samples were tested by the trip and enzyme-linked immunosorbent assay (ELISA) in the same time. The agreement of these two methods was 97.24%. |
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