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[Preparation and application of rabbit antiserum against structural protein VP2 of canine bocavirus]
Qian Zhang1, Yan Yan1, Jing Ma1
1Department of Biochemistry and Molecular Biology, School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, China.
Objective:
To prepare the rabbit polyclonal antibody against canine bocavirus (CBV) structural protein VP2 and identify its specificity.
Methods:
The target sequence of gene fragment encoding VP2 C-terminal region (300 AA) was amplified from CBV infection clone. After the restriction enzyme digestion and nucleotide sequencing, the target gene was successfully inserted into pET32a(+) prokaryotic expression vector to form recombinant plasmid pET-32a(+)-VP2. Then pET-32a(+)-VP2 was transferred into E.coli (BL21), and VP2-his fusion protein was induced under the optimized induction of isopropyl β-D-thiogalactopyranoside (IPTG). The products were identified and analyzed by SDS-PAGE. The purified fusion protein was inoculated into adult rabbits to develop polyclonal antibody. After the titer of the antiserum was detected by ELISA, Western blotting and immunofluorescence staining were performed to evaluate the features of the prepared antiserum.
Results:
Restriction enzyme digestion and sequencing showed that prokaryotic expression vector pET-32a(+)-VP2 was successfully constructed. The soluble recombinant protein was highly expressed in E.coli BL21 as expected. After purification and inoculation into adult rabbits, the high-titer polyclonal antibody was prepared and the titer was 1:6 400 000. Western blotting and immunohistochemistry demonstrated that the specificity of the prepared polyclonal antibody was perfect.
Conclusion:
The polyclonal antibody against CBV structural protein VP2 has been successfully prepared.
