张静 李玉静 张二敬 李紫然 刘静静 李春生

摘 要：为制备猪骨骼肌肌钙蛋白T单克隆抗体，利用生物化学方法提取猪骨骼肌肌钙蛋白并免疫小鼠，通过细胞融合制备稳定分泌单克隆抗体的杂交瘤细胞株，采取体内诱生腹水法批量制备单克隆抗体，并对其免疫学特征进行鉴定。结果表明：最终筛选获得1 株杂交瘤细胞株，将其命名为2E8，其分泌的抗体为免疫球蛋白G 2a亚型，效价为1∶4.1×105，亲和力常数为5.14×106 L/mol;采用间接酶联免疫吸附测定检测单克隆抗体2E8特异性，结果表明，单克隆抗体2E8与鸡、鸭检测原的交叉反应率均为25%;与牛、羊检测原的交叉反应率分别为1.7%、6.3%;免疫印迹检测结果显示，单克隆抗体2E8与猪检测原反应条带清晰，与羊、鸡、鸭检测原反应条带轻微，与牛检测原几乎不发生反应。

关键词：猪骨骼肌肌钙蛋白T;单克隆抗体;肉类掺假;间接酶联免疫吸附测定;免疫学特征

Preparation and Characterization of Monoclonal Antibody against Porcine Skeletal Muscle Troponin T

ZHANG Jing1， LI Yujing2， ZHANG Erjing3， LI Ziran3， LIU Jingjing1， LI Chunsheng1，*

（1. Institute of Biology， Hebei Academy of Science， Shijiazhuang 050081， China; 2. Hebei Jiaotong Vocational and Technical College， Shijiazhuang 050035， China; 3. College of Life Sciences， Hebei Normal University， Shijiazhuang 050024， China）

Abstract： In order to prepare monoclonal antibodies against porcine skeletal muscle troponin T （sTnT）， porcine skeletal muscle troponins were extracted by biochemical methods and used to immunize mice. By means of cell fusion， we acquired hybridoma cell strains that can secrete monoclonal antibodies （mAbs） stably. Ascites was induced in vivo to further prepare and characterize monoclonal antibodies against sTnT. Finally， we screened out one hybridoma cell strain that secretes mAb stably and named it 2E8. The monoclonal antibody 2E8 belonged to the IgG2a subtype， whose titer and affinity constant were 1 ： 4.1 × 105 and 5.14 × 106 L/mol， respectively. The specificity of 2E8 was evaluated by indirect enzyme-linked immunosorbent assay （iELISA）. The cross-reactivity with chicken sTnT and duck sTnT were both 25%. The cross-reactivity with bovine sTnT and goat sTnT were 1.7% and 6.3% respectively. Immunoblotting data showed that the monoclonal antibody 2E8 exhibited a clear band after reaction with the porcine antigen， but only a slight band after reaction with the goat， chicken， or duck antigen. In addition， the monoclonal antibody hardly reacted with the bovine antigen.

Keywords： porcine skeletal muscle troponin T; monoclonal antibody; meat adulteration; indirect enzyme-linked immunosorbent assay; immunological characteristics

DOI：10.7506/rlyj1001-8123-20210417-102

中圖分类号：TS251.1                                      文献标志码：A 文章编号：1001-8123（2021）09-0033-05

引文格式：

张静， 李玉静， 张二敬， 等. 猪骨骼肌肌钙蛋白T单克隆抗体的制备及鉴定[J]. 肉类研究， 2021， 35（9）： 33-37. DOI：10.7506/rlyj1001-8123-20210417-102.    http：//www.rlyj.net.cn

ZHANG Jing， LI Yujing， ZHANG Erjing， et al. Preparation and characterization of monoclonal antibody against porcine skeletal muscle troponin T[J]. Meat Research， 2021， 35（9）： 33-37. DOI：10.7506/rlyj1001-8123-20210417-102.

http：//www.rlyj.net.cn

肉类及其制品是百姓餐桌上不可或缺的美味，市场对肉类的需求逐年增加，肉类产品的质量问题随之成为重要的民生问题，在利益驱使下，肉类及其制品掺假问题频繁发生。例如，猪肉、鸡肉或鸭肉部分代替牛肉或羊肉[1-2]、马肉充当牛肉[3]、马肉冒充驴肉[4]等。目前，对肉类及其制品掺假检测技术主要有感官鉴别[5]、聚合酶链式反应[6-12]、红外光谱[13-17]、色谱-质谱联用[18-22]、酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）[23-27]等方法。……