人β-actin蛋白原核表达及其单克隆抗体制备

为获得分泌抗人β-actin蛋白单克隆抗体(McAb)的杂交瘤细胞, 通过在大肠杆菌中原核表达人β-actin蛋白, 以纯化的人β-actin蛋白作为抗原免疫BALB/c小鼠。经过细胞的融合及筛选获得1株能稳定分泌抗人β-actin蛋白McAb的杂交瘤细胞, 命名为2B4。采用间接ELISA和Western blot方法对McAb的特异性、稳定性和适用范围进行鉴定。结果显示: 蛋白的相对分子质量为43 kDa, 可溶于8 mol/L尿素; 杂交瘤细胞上清的抗体效价为1×105, 腹水的抗体效价为1×107; 间接ELISA结果表明, 杂交瘤细胞在体外传20代或液氮冻存3个月后, 分泌的抗体效价不变; 37 ℃保存24 h后, 抗体的效价开始下降。Western blot结果显示, 单克隆抗体识别人、鼠、兔和鱼的β-actin蛋白, 与其发生特异性反应。2B4分泌的单克隆抗体可以广泛的应用于细胞生物学和免疫学试验, 具有良好的应用价值。

Abstract

In order to obtain hybirdoma cells secreting monoclonal antibody (McAb) directly against human β-actin, BALB/c mice were immunized with purified human β-actin protein which prokaryotic expressed from E.coli. After cell fusion and being screened, one hybridoma cell strain with the ability to stably secrete McAb against human β-actin was obtained, named as 2B4. The specificity, stability and the applicable scope of McAb were determined by indirect ELISA and western blot. The results indicated: The molecular weight of recombinant protein which could resolve in 8 mol/L urea was about 43kDa. The titers of secreting antibody from cell culture supernatant were 1×105, while 1×107 from ascites of the mice. ELISA results showed that hybridoma cells which in vitro after 20 generations and freeze-stored 3 months in liquid nitrogen could secret McAb as normal. The titer of McAb which was stored in 37 ℃ began to fall after 24 h. Western blot results showed that secreted McAb could specifically react with β-actin protein of human, mice, rabbit and fish. Hybridoma strain that secrete McAb specific against β-actin was produced. McAb will be wildly used in cell biology and immunity experiment and has a good application value.

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吴俊文, 许凤, 熊璎珞, 鲁健, 邱义兰, 刘胜姿, 李烨, 刘如石. 人β-actin蛋白原核表达及其单克隆抗体制备[J]. 激光生物学报, 2012, 21(3): 238. WU Juwen, XU Feng, XIONG Yingluo, LU Jian, QIU Yilan, LIU Shengzi, LI Ye, LIU Rushi. Prokaryotic Expression of Human β-actin Protein and Preparation of Its Monoclonal Antibody[J]. Acta Laser Biology Sinica, 2012, 21(3): 238.

人β-actin蛋白原核表达及其单克隆抗体制备

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