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

作　　者：李琳钰 姚新欣 刘俊伶 陶倩倩 杨娟 LI Linyu;YAO Xinxin;LIU Junling;TAO Qianqian;YANG Juan(Gannan Innovation and Translational Medicine Reserch Institute,Gannan Medical University,Ganzhou 341000,China)

机构地区：[1]赣南医科大学赣南创新与转化医学研究院,赣州341000

出　　处：《实验动物科学》2024年第3期55-64,共10页Laboratory Animal Science

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R~2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。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 

分 类 号：Q95-3[生物学—动物学]