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Issue:ISSN 1000-7083
          CN 51-1193/Q
Director:Sichuan Association for Science and Technology
Sponsored by:Sichuan Society of Zoologists; Chengdu Giant Panda Breeding Research Foundation; Sichuan Association of Wildlife Conservation; Sichuan University
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Your Position :Home->Past Journals Catalog->2016 Vol.35 No.5

The Prokaryotic Expression of Canine Zona Pellucida 3 Protein and Preparation of its Antiserum
Author of the article:ZHANG Beibei, WANG Ying, ZHANG Ji, ZHANG Fuchun*
Author's Workplace:College of Life Sciences and Technology, Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University, Urumqi 830046, China
Key Words:dog; zona pellucida 3; prokaryotic expression; fusion protein; antiserum
Abstract:To develop the canine zona pellucida 3 (CZP3) contraceptive vaccine that could be used to control the birth rate of stray dogs, antiserum is required to carry out immune detection. In this study, the full length cDNA of CZP3 (1 281 bp) and the core fragment of CZP3 (CZP3C, 984 bp) without the transmembrane domain and the signal peptide were cloned, respectively. CZP3C was ligated to pET28a vector to generate the recombinant plasmid pET28a-CZP3C, and then the plasmid was transferred into Escherichia coli BL21(DE3). The expression of CZP3C fusion protein was induced with 0.6 mmol·L-1 isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28℃ for 4 h. The specific antiserum of CZP3C fusion protein was obtained from the rabbit after immunization, and the specificity and titer of the prepared antiserum were evaluated. The results of SDS-PAGE showed that CZP3C mainly existed in the form of inclusion bodies. High purity of the fusion protein was obtained after nickel column affinity purification and 8 mmol·L-1 urea renaturation. Two Newzealand rabbits were immunized with CZP3C of 0.8 μg·μ L-1, respectively, the generated polyclonal antiserum was detected with Western blot and indirect immunofluorescence. The results showed that the anti-CZP3C rabbit serum had high specificity, and the titer was 1:409 600 as determined by the enzyme linked immunosorbent assay (ELISA). The prepared antiserum can provide testing materials for the development of stray dog contraceptive vaccines.
2016,35(5): 697-702 收稿日期:2016-05-31
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