重组人颗粒溶素的纯化及其单克隆抗体的制备  

The purification of recombinated granulysin and the preparation of its monoclonal antibody

作　　者：居军[1] 李新立[2] 王晚霞[1] 徐向红[1] 马亚茹[3] 李萍[3] 谢溱[3] 

机构地区：[1]甘肃省人民医院临床检验中心,甘肃兰州730000 [2]兰州大学第一临床医学院,甘肃兰州730000 [3]兰州生物制品研究所,甘肃兰州730000

出　　处：《细胞与分子免疫学杂志》2010年第6期563-565,共3页Chinese Journal of Cellular and Molecular Immunology

摘　　要：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。AIM：To purify recombinated GNLY which is expressed by prokaryotic expression system, and to prepare the monoclonal antibody （mAb） against it. METHODS： The solvable protein was purified by affinity chromatography. And employing the fusion protein GNLY immuned BALB/c mice, using conventional hybridoma technology prepared the mAb against human GNLY. Then purified and determined for titer by indirect ELISA method, for antigenic epitopes by additive ELISA, for relative affinity index by sulfocyanate elution method, and analyzed for subclass, specificity and stability. RESULTS： We got the soluble reactive fusion GNLY, and its purity and content were 95%, 0.8 g/L, respectively. Four cell strains secreting mAb against human GNLY were screened, 6C8, 9C6, 5G7and5E5. Their neutralizing titers were 1∶100-1∶3 200 and（0.1-8）×10-4 in supernatant and ascites. CONCLUSIONS： We successfully purified the fusion protein of GNLY, and prepared the mAb against human GNLY, lying a certain foundation for its laboratory and clinical research.

关 键 词：颗粒溶素 纯化 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]