肌酸激酶同工酶CK-MB的构建表达、抗体制备及初步鉴定
Constructive Expression of Creatine Kinase Isoenzyme CK-MB and Antibody Production and Preliminary Validation
李琳钰 1姚新欣 1刘俊伶 1陶倩倩 1杨娟1
作者信息
- 1. 赣南医科大学赣南创新与转化医学研究院,赣州 341000
- 折叠
摘要
目的 构建异源二聚体 CK-MB重组蛋白并制备其特异性基因工程抗体,以此作为 CK-MB 定量检测试剂盒的诊断原料,从而辅助临床急性心肌梗死的早期诊断,提高诊断的准确性.方法 通过共表达载体获取异源二聚体 CK-MB并进行动物免疫,从而获取小鼠浆细胞;进一步使用单细胞光导系统分离抗原特异性小鼠单个 B 细胞,并通过单细胞 PCR获取抗体轻重链的可变区基因序列后构建基因工程抗体.基于初步的功能验证筛选出候选抗体,并进一步通过棋盘滴定法探究最佳的配对方案,并利用双抗夹心 ELISA 原理初步组装 CK-MB 定量检测试剂盒.结果 成功获取 CK-MB免疫原;通过单 B细胞光导技术筛选出 CK-MB抗原特异性小鼠单个浆细胞并制备出2 株小鼠源基因工程抗体,且均具有识别天然 CK-MB的能力;经由双抗夹心 ELISA法筛选出两种配对方案,其拟合曲线的 R2 均大于 0.99.结论 成功制备出靶向 CK-MB的小鼠源基因工程抗体,经由 ELISA初步验证抗体的特异性和亲和力,为进一步开发高特异性、高灵敏度的 CK-MB新型定量检测试剂盒奠定了坚实基础.
Abstract
Objective Construct heterodimeric CK-MB recombinant protein and prepare its specific gene-engineered antibody as the diagnostic raw material of CK-MB quantitative detection kit,so as to assist the early diagnosis of acute myocardial infarction in clinic,and to improve the accuracy of diagnosis.Method Mouse plasma cells were obtained by obtaining heterodimeric CK-MB through co-expression vector and animal immunization,further,single mouse B cells specific for antigen were isolated using single-cell photoconductive system,and genetically engineered antibodies were constructed after obtaining the gene sequences of the variable regions of the light and heavy chains of the antibodies through single-cell PCR.Candidate antibodies were screened based on preliminary functional validation,and the optimal pairing scheme was further explored by checkerboard titration,and a CK-MB quantitative detection kit was initially assembled using the principle of double-antibody sandwich ELISA.Result CK-MB immunogen was successfully obtained;single plasma cells specific for CK-MB antigen were screened by single B-cell photoconductivity and two strains of mouse-derived genetically engineered antibodies were prepared,and both of them had the ability of recognizing the natural CK-MB;two pairwise protocols were screened by double-antibody sandwich ELISA,and the R2 of the fitted curves were both greater than 0.99.Conclusion Mouse-derived genetically engineered antibody targeting CK-MB was successfully prepared,and the specificity and affinity of the antibody was preliminarily verified by ELISA,laying a solid foundation for the further development of a new type of CK-MB quantitative detection kit with high specificity and high sensitivity.
关键词
肌酸激酶同工酶CK-MB/急性心肌梗死/基因工程抗体Key words
creatine kinase isoenzyme CK-MB/acute myocardial infarction/genetically engineered antibody引用本文复制引用
出版年
2024
NETL
NSTL
万方数据