重组人颗粒溶素的纯化及其单克隆抗体的制备

发布时间：2018-03-17 20:51

本文选题：颗粒溶素　切入点：纯化　出处：《兰州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：纯化原核表达的重组人颗粒溶素(GNLY),并制备其单克隆抗体(mAb),为GNLY相关疾病的诊断提供一种新指标。方法：Ni离子亲和层析法纯化重组人GNLY,用其免疫BALB/c小鼠,采用常规杂交瘤技术制备相应mAb。间接ELISA法测杂交瘤细胞培养上清和腹水mAb的效价,ELISA相加试验测定抗原表位,硫氰酸盐洗脱法测定相对亲和力指数,并行Ig亚类、特异性及杂交瘤细胞分泌mAb的稳定性检测。结果：纯化的重组GNLY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GNLY mAb的杂交瘤细胞4株,分别命名为5E5、5G7、6C8和9C6。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(0.2-8)×10-4。5E5杂交瘤细胞株分泌mAb的Ig亚类为IgM,其余3株mAb为IgG1。4株杂交瘤细胞体外连续培养2个月及液氮冻存6个月后复苏,分泌mAb的水平仍能达到之前的水平。4株mAb的相对亲和力指数为1.5-3.0mol/L；5G7、6C8和9C63株杂交瘤细胞株分泌mAb的相加指数(AI)＜50%,而5E5分泌mAb与上述3株细胞分泌mAb的AI＞50%。4株纯化后的mAb与GNLY抗原有较高的吸光度值,而与其他包被的抗原不反应。结论：利用Ni离子亲和层析法获得可溶性重组GNLY;通过杂交瘤技术,获得4株能分泌特异性强、效价高的GNLY mAb杂交瘤细胞株。
[Abstract]:Objective: to purify the recombinant human granulysin (GNLY) expressed in prokaryotic expression and prepare its monoclonal antibody (McAb) to provide a new index for the diagnosis of GNLY related diseases. Methods the recombinant human GNLYY mice were immunized with the purified recombinant human GNLYY by affinity chromatography. The corresponding mAbs were prepared by conventional hybridoma technique. Indirect ELISA assay was used to detect the antigenic epitopes in the supernatant of hybridoma cell culture and ascites mAb. The relative affinity index, Ig subclass, specificity and stability of mAb secreted by hybridoma cells were determined by thiocyanate elution method. Results: the purity and content of purified recombinant GNLY fusion protein were 95% and 0.8 g / L, respectively. Four hybridoma cells secreting anti-human GNLY mAb stably were screened. The titers of mAb in the supernatant and ascites of the hybridoma cell lines named 5E5, 5G7C8 and 9C6.4 were 1: 100-1: 3200 and 0.2-8 respectively) 脳 10-4.5E5 hybridoma cell lines secreting mAb. The Ig subclasses of mAb secreted by the hybridoma cells of the other three strains of mAb were IgG1.4 hybridoma cells in vitro for 2 months, respectively. Liquid nitrogen was frozen for six months and then recovered. The relative affinity index of mAb was 1.5-3.0mol / L 5G7C8 and 9C63 hybridoma cell lines secreted mAb (AI < 50), while 5E5 secreted mAb and the AI of mAb secreted by the above three strains were higher than 50.4%. GNLY antigen has high absorbance value. It does not react with other coated antigens. Conclusion: the soluble recombinant GNLY was obtained by Ni ion affinity chromatography, and four GNLY mAb hybridoma cell lines with strong specificity and high titer were obtained by hybridoma technique.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11

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