抗鸡白细胞介素4单克隆抗体的制备及鉴定

Generation and characterization of monoclonal antibodies against chicken interleukin 4

摘要:

为了制备鸡白细胞介素4 (chIL-4) 单克隆抗体，将成熟的chIL-4基因亚克隆至原核表达载体pET-28a和pGEX-6P-1上，然后在大肠杆菌中分别诱导重组蛋白His-chIL-4和GST-chIL-4的表达，并纯化。将纯化后的His-chIL-4作为免疫原免疫BALB/c小鼠，经4次免疫后，取小鼠脾细胞与骨髓瘤细胞 (SP2/0) 融合。将纯化后的GST-chIL-4作为筛选抗原，利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后，获得3株稳定分泌抗chIL-4蛋白的杂交瘤细胞株，分别命名为1G11-3B、2E5-3D和1G11-5H。经ELISA检测，3株单克隆抗体的亚型均为IgG1，亲和力解离常数 (Kd) 分别为1.79×10–9、1.61×10–9和2.36×10–9。经Western blotting及间接免疫荧光试验鉴定，3株单克隆抗体均能特异性识别原核和真核表达的chIL-4蛋白。Western blotting试验证明1G11-3B、2E5-3D和1G11-5H识别的抗原表位区域分别为chIL-4蛋白N端的第1–40、80–112和40–80位氨基酸。该单克隆抗体的制备为chIL-4的检测和生物学功能研究奠定了基础。

Abstract:

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.