| Autor: |
Xie C; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Wang R; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Saeed AFUH; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Yang Q; Fuzhou Maixin Biotech.Co., Ltd, Fuzhou, 350100, China., Chen H; Fuzhou Maixin Biotech.Co., Ltd, Fuzhou, 350100, China., Ling S; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Xiao S; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Zeng L; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China., Wang S; Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China. wshyyl@sina.com. |
| Abstrakt: |
Podoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene encoding the extracellular part of PDPN (ePDPN) was synthesized and used to expressed fusion protein ePDPN-His and GST-ePDPN, respectively, in E.coli. The purified GST-ePDPN fusion protein was mixed with QuickAntibody-Mouse5W adjuvant to immune mice, and the antiserum titer was determined by indirect ELISA. A stable cell line named 5B3 generating anti-PDPN monoclonal antibody (mAb) was obtained by hybridoma technology. The isotype of 5B3 cell line was IgG 2b , and the chromosome number was 102 ± 4. The 5B3 mAb was purified successfully from ascites fluid through Protein G column, and its affinity constant was 2.94 × 10 8 L/mol. Besides, excellent specificity of the 5B3 mAb was further demonstrated in ELISA, western blot and immunohistochemistry experiments, suggesting that 5B3 mAb displays similar application value to D2-40, a commercial available antibody. Hence, the current study provides conclusive guidelines for preparation of other mAbs and their applications in immunohistochemistry diagnosis. |
