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目的 制备重组抗tau抗体，探讨其在防治阿尔茨海默病(AD)中的应用效果。方法 采用重组人tau441蛋白作为免疫原，获得分泌抗tau抗体的小鼠淋巴细胞。构建鼠源免疫VH-VL基因库，将测序正确的阳性重组质粒转化至PCDNA3.1(+)。通过亲和层析纯化获得重组抗tau抗体后，分别采用ELISA法、凝胶电泳法、蛋白印迹法对其效价、纯度、特异性进行鉴定。以小鼠脑神经瘤N2a细胞为研究对象，将N2a细胞分为空白对照组(正常培养)、AD组(通过Aβ25-35诱导建立AD细胞模型)、抗tau抗体组(通过Aβ25-35诱导建立AD细胞模型后采用抗tau抗体处理)，采用CCK-8法检测细胞活力，Hoechst 33342染色分析细胞凋亡。结果 成功免疫小鼠并调取抗tau抗体VH、VL基因。通过构建VH-linker-VL载体及亲和层析纯化，获得效价高于1∶256 000、纯度为95.28%的特异性重组抗tau抗体。重组抗tau抗体能够提高Aβ25-35诱导的N2a细胞活力[AD组、抗tau抗体组细胞活力分别为(44.33±2.08)%、(68.00±2.65)%，两组相比P<0.05]，并抑制其凋亡。结论 本研究获得抗tau抗体VH、VL序列，制得高效价、高纯度、高特异性重组抗tau抗体，并证实其对Aβ25-35诱导的N2a细胞损伤具有改善作用。
Abstract：Objective To investigate the preparation of recombinant anti-tau antibody and its application in the prevention and treatment of Alzheimer's disease(AD). Methods Recombinant human tau441 protein was used as immunogen to obtain mouse lymphocytes secreting anti-tau antibody. The mouse immune VH-VL gene bank was constructed, and the correctly sequenced positive recombinant plasmid was transformed into PCDNA3. 1(+). After the recombinant anti-tau antibody was purified by affinity chromatography, its titer, purity and specificity were identified by ELISA, gel electrophoresis and Western blotting. The mouse brain neuroma N2a cells were used as the research object. The N2a cells were divided into a blank control group(normal culture), an AD group(establishing an AD cell model by inducing with Aβ25-35), and an recombinant anti-tau antibody group(treated with anti-tau antibody after establishing the AD cell model by inducing with Aβ25-35). CCK-8 assay was used to detect cell viability, and Hoechst 33342 staining was used to analyze cell apoptosis. Results Mice were successfully immunized and anti-tau antibody VH and VL genes were obtained. Through the construction of VH-linker-VL vector and purification by affinity chromatography, specific recombinant anti-tau antibody with titer higher than 1∶256 000 and purity of 95. 28% was obtained. The recombinant anti-tau antibodies can enhance the viability of N2a cells induced by Aβ25-35, and inhibit their apoptosis. The cell viability of the AD group and the recombinant anti-tau antibody group was(44. 33 ± 2. 08)% and(68. 00 ± 2. 65)% respectively. There was a statistically significant difference between the two groups(P < 0. 05). Conclusion In this study, the VH and VL sequences of anti-tau antibody are obtained and the recombinant anti-tau antibody with high titer, high purity and high specificity are prepared, and its protective effect on N2a cell injury by inducing with Aβ25-35 is confirmed.
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基本信息:
DOI：
中图分类号:R749.16
引用信息:
[1]黄宁,王升平,武彦宏,等.重组抗tau抗体的制备及其在防治阿尔茨海默病中的应用[J].老年医学研究,2025,6(04):28-32+40.
基金信息:
青岛市关键技术攻关及产业化示范类项目（23-1-6-ghgg-3-hz,体外诊断抗体试剂研发及产业化）