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目的:纯化原核系统表达的颗粒溶素(GNLY),并制备其单克隆抗体(mAb)。方法:用Ni亲和层析纯化重组人GNLY,用其免疫BALB/c小鼠,采用常规杂交瘤技术制备相应的mAb。mAb纯化后,用间接ELISA法测mAb的效价,ELISA相加试验测定抗原表位,硫氰酸盐洗脱法测定mAb的相对亲和力指数,并进行Ig亚类、特异性及杂交瘤细胞分泌mAb的稳定性检测。结果:经Ni亲和层析纯化的可溶性GN-LY融合蛋白,其纯度和含量分别为95%和0.8g/L。共筛选出能稳定分泌抗人GNLYmAb的杂交瘤细胞4株,分别为6C8、9C6、5G7和5E5。4株杂交瘤细胞培养上清及腹水中mAb的效价分别为1∶100～1∶3200和(0.1～8)×10-4,5E5mAb的Ig亚类为IgM,其余3株mAb均为IgG1。结论:成功地纯化人GNLY融合蛋白。以其为免疫原制备的鼠抗人GN-LYmAb,为实验室及临床研究奠定了一定的基础。
OBJECTIVE: To purify the GNLY expressed by prokaryotic system and to prepare its monoclonal antibody (mAb). METHODS: Recombinant human GNLY was purified by Ni affinity chromatography and used to immunize BALB / c mice. The corresponding mAbs were prepared by conventional hybridoma technique. After the mAb was purified, the titer of mAb was measured by indirect ELISA, the antigen epitope was determined by ELISA and the relative affinity index of mAb was determined by thiocyanate elution method, and the Ig subclass, specificity and secretion of hybridoma cells mAb stability test. Results: The purity and content of soluble GN-LY fusion protein purified by Ni affinity chromatography were 95% and 0.8 g / L, respectively. A total of 4 hybridoma cells secreting anti-human GNLY mAb were screened, respectively. The titer of mAbs in supernatant and ascites of 6C8, 9C6, 5G7 and 5E5.4 hybridoma cells were 1: 100 ~ 1: 3200 And (0.1 ~ 8) × 10-4, 5E5mAb Ig subclass IgM, the remaining three mAbs are IgG1. Conclusion: Human GNLY fusion protein was successfully purified. The mouse anti-human GN-LYmAb prepared by using it as an immunogen lays a solid foundation for laboratory and clinical research.