增强抗cd22单链抗体片段在大肠杆菌中的可溶性表达和功能。

Q2 Biochemistry, Genetics and Molecular Biology Iranian Biomedical Journal Pub Date : 2024-12-01
Monireh Gholizadeh, Zahra Sharifzadeh, Mohammad Nouri, Arash Arashkia, Shahriyar Abdoli, Farhad Riyazi Rad, Amir Ali Hamidieh
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引用次数: 0

摘要

CD22是一种主要存在于癌性b细胞谱系中的表面抗原,使其成为生物制药治疗血液b细胞恶性肿瘤的重要靶点。包括疫苗、细胞和基因疗法以及重组蛋白疗法在内的生物制药已经彻底改变了医学治疗。单克隆抗体(mab)在这一领域占有突出地位,但面临稳定性、聚集性和翻译后修饰等挑战。为了解决这些问题,研究人员开发了单链可变片段(scFvs),它是单克隆抗体的更小且可能更稳定的衍生物。方法与材料:设计合成抗cd22 - scfv并在大肠杆菌中表达。采用固定化金属亲和层析法(IMAC)对该蛋白进行了纯化。通过优化表达条件,包括诱导后温度、诱导后时间和异丙基β- d -1-硫代半乳糖苷(IPTG)浓度,使可溶性蛋白产量最大化。利用流式细胞术评估杂交纯化蛋白和天然纯化蛋白与CD22+ Raji和CD22- k562细胞的结合能力。MTT法检测抗CD22- scfv对CD22+ Raji细胞增殖的抑制作用。结果:利用pET-28a+表达载体在Rosetta (DE3)株中成功表达了抗cd22 - scfv蛋白,但在Rosetta-gami 2株中未表达。杂交纯化,12% SDS-PAGE和考马斯亮蓝染色证实蛋白表达。初始溶解度试验表明抗cd22 - scfv蛋白不溶性。优化条件为IPTG浓度为0.5 mM,诱导后温度为25℃,诱导后时间为8小时,可获得较高的溶解度。天然纯化和混合纯化的抗CD22- scfvs与CD22+ Raji细胞的结合亲和力分别为91.4%和84%。抗CD22- scfv显著降低CD22+ Raji细胞的增殖。结论和讨论:本研究证明了基于imac的一步纯化方法从重组大肠杆菌中分离抗cd22 - scfv蛋白的有效性,该方法将纯化和重折叠结合在一起,成本效益高。通过对表达菌株、诱导后温度、诱导后时间、IPTG浓度等环境参数的优化,显著提高了蛋白产量和溶解度。抗CD22- scfv在靶向和减少CD22+肿瘤细胞的增殖方面显示出良好的潜力,突出了其对b细胞恶性肿瘤的治疗潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024
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来源期刊
Iranian Biomedical Journal
Iranian Biomedical Journal Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.20
自引率
0.00%
发文量
42
审稿时长
8 weeks
期刊最新文献
25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024 25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024 25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024 25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024 25th National and 11th International Annual Congress on Research and Technology of Iranian Medical Sciences Students, Urmia, Iran, 5-7 September, 2024
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