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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。
OBJECTIVE: To express a full-length fusion protein of human immune related guanosine triphosphatase gene (IRGM) a to prepare high quality rabbit anti-human IRGM polyclonal antibody. Methods: The full-length cDNA fragment of IRGMa was cloned into the prokaryotic expression vector pGEX-4T-2 and the eukaryotic expression vector pCMV-myc respectively, and the sequence was verified by sequencing. The IRGMa / GST fusion protein was expressed in Escherichia coli Ecoli BL21, and the purified anti-IRGMa / GST fusion protein was used to immunize New Zealand white rabbits to prepare rabbit anti-human IRGM antibody. The anti-IRGM antibody was purified by ELISA, Western blot and competitive inhibition Sensitivity and specificity. Results: The specificity and sensitivity of homemade rabbit anti-human IRGM polyclonal antibody were higher than those of the commercially available antibody. Conclusion: The rabbit anti-human IRGM polyclonal antibody was successfully prepared, which laid the foundation for further functional studies.