重组人心肌肌钙蛋白I单克隆抗体的制备及鉴定

发布时间：2018-04-22 03:22

本文选题：人心肌肌钙蛋白I + 单克隆抗体　；参考：《中国协和医科大学》2009年硕士论文

【摘要】： 肌钙蛋白复合物由肌钙蛋白C、肌钙蛋白I和肌钙蛋白T三个不同的亚基组成,在Ca~(2+)作用下,主要参与肌肉的收缩调节。肌钙蛋白I作为一个亚单位主要抑制肌球蛋白ATP酶的活性。心肌肌钙蛋白I不在任何骨骼肌组织中表达,具有心肌组织特异性,这使得其成为检测心肌损伤最好的生化标志物之一。研究表明急性心肌梗死患者胸痛发生的早期心肌肌钙蛋白I就被释放入血流,14～36小时其浓度达到最高峰。心肌损伤后的3～7天仍能检测到心肌肌钙蛋白I水平的升高。因此,心肌损伤的检测可通过检测血清中心肌肌钙蛋白I的水平来进行,并且具有灵敏度高、特异性强和诊断窗口期长的特点。进一步研究表明急性冠脉综合症死亡风险与心肌肌钙蛋白I的水平具有相关性。在一些非致死的心肌损伤、充血性心衰等心脏疾病中也检测到心肌肌钙蛋白I水平的升高。目前,心肌肌钙蛋白I已成为临床上诊断心肌损伤的“金标准”。本研究的目的是制备重组人心肌肌钙蛋白I(recombinant human cardiactroponinI,rhcTnI)的单克隆抗体,并对其特性进行鉴定,为cTnI诊断试剂盒的研制奠定基础。利用已构建的基因工程菌株,大量表达、纯化重组目的蛋白。纯化后的目的蛋白作为抗原常规免疫Balb/c小鼠,取其脾细胞与同系骨髓瘤细胞Sp2/0进行常规融合,甲基纤维素半固体培养基法筛选杂交瘤细胞株,利用ELISA、Western blotting等方法鉴定单克隆抗体的性质。筛选出3株稳定分泌抗重组目的蛋白抗体的杂交瘤细胞株,分别命名为4B_3、7D_5和3E_6,所产生单克隆抗体的免疫球蛋白类及亚类分别为IgGl(48_3、7D_5)和IgG2b(3E_6)。单克隆抗体与重组目的蛋白发生特异性结合,而与空白菌、LDH、CK、CK-MB无特异性交叉反应,单克隆抗体的亲和常数分别为1.76×10~9L/mol、1.43×10~9L/mol和1.04×10~9L/mol。综上所述,成功获得了亲和力高、特异性好的单克隆抗体。
[Abstract]:The troponin complex is composed of three different subunits, troponin C, troponin I and troponin T. It is mainly involved in the contractile regulation of muscle under the action of Ca~ (2+). The troponin I as a subunit mainly inhibits the activity of myosin ATP enzyme. Cardiac troponin I is not expressed in any skeletal muscle tissue and has myocardial tissue. Specificity, which makes it one of the best biochemical markers for detecting myocardial damage, has shown that early cardiac troponin I in acute myocardial infarction is released into the blood flow and its concentration reaches its peak at 14~36 hours. The cardiac troponin I level can still be detected at the 3~7 day after myocardial injury. The detection of muscle damage can be carried out by detecting the level of cardiac troponin I in serum, with high sensitivity, specificity, and long period of diagnosis. Further studies show that the risk of acute coronary syndrome is associated with the level of cardiac troponin I. In some nonfatal myocardial injuries, congestive heart failure Cardiac troponin I levels have also been detected in heart diseases. At present, cardiac troponin I has become the "gold standard" for clinical diagnosis of myocardial injury.
The purpose of this study is to prepare monoclonal antibodies against recombinant human cardiac troponin I (recombinant human cardiactroponinI (rhcTnI)), and to identify the properties of the recombinant human cardiac troponin cardiactroponinI (rhcTnI), which lays the foundation for the development of cTnI diagnostic kit.
Using the constructed genetic engineering strain to express and purify the recombinant target protein, the purified target protein was used as the antigen routine immunization Balb/c mice, and the spleen cells were fused with the homologous myeloma cell Sp2/0, the methylation semisolid culture method was used to screen the hybridoma cell lines, and the methods of ELISA, Western blotting and other methods were used. Identify the properties of the monoclonal antibody.
3 hybridoma cell lines that secrete anti recombinant target protein antibodies were selected, named 4B_3,7D_5 and 3E_6 respectively. The immunoglobulins and subclasses of monoclonal antibodies were IgGl (48_3,7D_5) and IgG2b (3E_6) respectively. The monoclonal antibody combined with the recombinant target protein, and was not specific to the blank bacteria, LDH, CK and CK-MB. The affinity constants of the monoclonal antibodies were 1.76 x 10~9L/mol, 1.43 x 10~9L/mol and 1.04 x 10~9L/mol., respectively.
In conclusion, the monoclonal antibody with high affinity and specificity has been successfully obtained.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】