Watanabe Rie, Hasegawa Atsuhiko, Miyazawa Takayuki, Kato Hirotomo, Iwata Hiroyuki
Department of Veterinary Hygiene, Yamaguchi University, Yoshida, Japan.
J Vet Med Sci. 2004 Sep;66(9):1053-7. doi: 10.1292/jvms.66.1053.
Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.
不含信号肽的猪白细胞介素-6（PIL-6）蛋白在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合蛋白表达。然而，融合蛋白在不溶性部分表达，通过使用尿素的复性程序将其溶解。从溶解的蛋白中，通过使用谷胱甘肽琼脂糖4B的批量方法和PreScission蛋白酶切割纯化重组PIL-6（rPIL-6）。通过B3B1杂交瘤细胞增殖试验，证实了纯化的rPIL-6的生物活性。以rPIL-6作为免疫原产生了三种单克隆抗体（MAb），分别命名为2B-1、5A-8和4C-3。这些单克隆抗体的免疫球蛋白亚型为IgG2a（4C-3）和IgG2b（2B-1和5A-8）。为了进行表位分析，使用PIL-6的缺失突变体进行了加成酶联免疫吸附试验和免疫印迹分析。这些实验表明，两种单克隆抗体（2B-1和5A-8）识别重叠表位，另一种（4C-3）识别不同表位，并且所有表位都位于PIL-6的aa26-64区域。
