抗Zhangfei蛋白单克隆抗体的制备及鉴定

发布时间：2018-06-30 16:48

  本文选题：Zhangfei + 融合蛋白　； 参考：《西北农林科技大学》2008年硕士论文

【摘要】： Zhangfei是碱性亮氨酸拉链蛋白家族成员之一,分子量为28.9ku,是参与内质网应激和未折叠蛋白反应调节的转录因子之一,一些肿瘤细胞中也存在未折叠蛋白反应,其在胚胎发育、人类疾病等生理和病理过程中具有重要的意义。为此本试验对Zhangfei融合蛋白进行了诱导表达、纯化并免疫动物,制备并鉴定了Zhangfei蛋白的单克隆抗体,试验结果如下: 1筛选了诱导表达Zhangfei融合蛋白的最佳培养条件,证明在37℃摇床培养条件下,IPTG的终浓度为3 mmol/L,诱导时间为7 h时,Zhangfei融合蛋白的表达量相对最大。 2比较了谷胱甘肽琼脂糖凝胶和电洗脱两种纯化Zhangfei融合蛋白的方法,得出电洗脱纯化方法得到纯度较高的目的蛋白。 3用电洗脱纯化的Zhangfei融合蛋白免疫Balb/c小鼠和家兔,应用间接ELISA常规方法检测血清抗体效价,免疫小鼠的血清抗体效价均超过1∶5×103,免疫兔的血清抗体效价高达1∶3×106,证明Zhangfei融合蛋白有较强的免疫原性。 4用45 %PEG(4000)融合骨髓瘤细胞和免疫脾细胞,用间接ELISA法筛选阳性孔,克隆率为98.96 %,阳性克隆率30.18 %。用有限稀释法筛选得到2株阳性杂交瘤细胞,分别命名为ZFD1和ZFD2,两株细胞株分泌单抗均为针对Zhangfei蛋白的单克隆抗体。 5应用间接ELISA常规方法检测细胞株培养上清和腹水的阴阳性及其效价,ZFD1杂交瘤细胞培养上清的效价为1∶1000,腹水单抗的效价为1∶2×105以上。ZFD2杂交瘤细胞培养上清的效价为1∶1000,腹水单抗的效价为1∶105。经冻存和传代培养证明均具有稳定分泌抗体的能力。2株细胞株分泌的抗体均属于IgG1类型。ZFD1和ZFD2分泌单抗的亲和常数分别为2.102×108,2.313×108。 上述结果为检测该蛋白在动物组织细胞中的表达,研究zhangfei蛋白在机体的生理功能提供奠定了基础。
[Abstract]:Zhangfei is a member of the alkaline leucine zipper protein family with a molecular weight of 28.9 ku.It is one of the transcription factors involved in the regulation of endoplasmic reticulum stress and unfolded protein reaction. Human diseases and other physiological and pathological processes have important significance. In this experiment, the Zhangfei fusion protein was induced and expressed, purified and immunized, and the monoclonal antibody against Zhangfei protein was prepared and identified. The results were as follows: 1 the optimal culture conditions for the induction and expression of Zhangfei fusion protein were screened. The results showed that the final concentration of IPTG was 3 mmol / L and the induction time was 7 h under the condition of 37 鈩，

本文编号：2086144