人β-actin蛋白原核表达及其单克隆抗体制备

Prokaryotic Expression of Human β-actin Protein and Preparation of Its Monoclonal Antibody

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。

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. coll. After cell fusion and being screened, one hybridoma cell strain with the ability to stably secrete McAb against human β3-actin was obtained, named as 2134. 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 × 10^5 , while 1 × 10^7 from as- cites 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 MeAb as normal. The titer of MeAb which was stored in 37 ℃ began to fall after 24 h. Western blot results showed that secreted McAb could specifically react with β-aetin protein of human, mice, rab-bit 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.