兔抗人免疫相关鸟苷三磷酸酶基因多克隆抗体的制备与鉴定

Preparation and Identification of the Rabbit Anti-human Immune-related Gene Guanosine Triphosphate Polyclonal Antibody

目的:表达人免疫相关鸟苷三磷酸酶基因(IRGM)a全长融合蛋白,制备高质量的兔抗人IRGM多克隆抗体。方法:将IRGMa全长cDNA片段分别克隆到原核表达载体pGEX-4T-2和真核表达载体pCMV-myc,测序验证序列正确。在大肠埃希菌Ecoli BL21中表达IRGMa/GST融合蛋白,用纯化的可溶性IRGMa/GST融合蛋白免疫新西兰大白兔,制备兔抗人IRGM抗体,采用ELISA、免疫印迹和竞争抑制等多种方法验证其敏感度和特异度。结果:自制的兔抗人IRGM多克隆抗体的特异度和敏感度均较高,效价更高于市售抗体。结论:成功制备了兔抗人IRGM多克隆抗体,为进一步的功能研究打下基础。

Aim： To express the whole length fusion protein of human IRGMa and prepare high quality rabbit anti-human immune-related gene guanosine triphosphate（IRGM） polyclonal antibody. Methods： Clone the whole length IRGMa fragement into prokaryotic expression vector pGEX-4T-2 and eukaryotic expression vector pCMV-myc, express IRGMa/GST fusion protein in E coli.BL21, and immunize New Zealand white rabbits with IRGMa/GST to prepare rabbit anti-human IRGM polyclonal antibody. The sensitivity and specificity of the polyclonal antibody were tested by ELISA, Western Blot and competitive inhibition. Results： The polyclonal rabbit anti-human IRGM antibody was successfully obtained. The antibody is of high sensitivity and specificity. Titer of antibody made by ourselves is higher than that of antibody bought. Conclusion： The rabbit anti-human IRGM polyclonal antibody were successfully obtained.