Guan Xiaoyu, Xu Zhichao, Wang Yongqiang, Li Xiaoqi, Cao Hong, Zheng Shijun
State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jan 25;33(1):44-54. doi: 10.13345/j.cjb.160221.
To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.
为了制备抗鸡白细胞介素4（chIL-4）的单克隆抗体（McAbs），我们将成熟的chIL-4基因亚克隆到原核表达载体pET-28a和pGEX-6P-1中，然后表达并纯化重组蛋白。我们用纯化的His-chIL-4蛋白免疫BALB/c小鼠，并在4次免疫后将小鼠脾细胞与SP2/0细胞融合。我们使用GST-chIL-4蛋白作为包被抗原来建立间接ELISA以筛选阳性克隆。经过筛选和3轮克隆过程，我们获得了3株稳定分泌抗chIL-4单克隆抗体的杂交瘤，分别命名为1G11-3B、2E5-3D和1G11-5H。这些单克隆抗体的亚型均为IgG1，其解离常数（Kd）分别为1.79×10⁻⁹、1.61×10⁻⁹和2.36×10⁻⁹。通过蛋白质印迹法和间接免疫荧光测定法确定，这些单克隆抗体能特异性结合原核或真核系统表达的chIL-4。通过蛋白质印迹法确定，1G11-3B、2E5-3D和1G11-5H识别的chIL-4结合域分别位于第1-40、80-112和40-80氨基酸之间。这些单克隆抗体将有助于检测chIL-4并阐明chIL-4在免疫反应中的生物学作用。
