猪表皮生长因子真核表达产物的体外活性试验

Study on Activity of Eukaryotic Expression Products of Porcine Epidermal Growth Factor in Vitro

为建立一种快速检测猪表皮生长因子(pEGF)活性的方法,用本实验室构建的pEGF酵母表达载体进行摇瓶培养,通过鉴定为所需要的目的蛋白,用pEGF为添加物培养Balbc/3T3细胞,以pEGF浓度为横坐标,吸光度为纵坐标建立曲线。结果表明,理想的接种细胞的个数为5 000个/孔,细胞计数试剂盒(CCK-8)孵育的最佳时间为1.5 h,pEGF和人表皮生长因子(hEGF)标准品,在1 ng/mL^100 ng/mL范围内对细胞的增值作用相当,表明pEGF和hEGF有相同的生物学活性。建立了用CCK-8快速测定pEGF活性的方法。

To estabilish a method of rapid detecting the bioactivity of pEGF, porcine epidermal growth factor expression vector in Pichia pastoris is constructed, which is produced in shake flask. It is identified as target protein, with cultured Balbc/3T3 cell as additive. The curve was estabilished as abscissa was the concentration of pEGF, while y-axis was absorptance. The result showed that optimal cell numbers planted were 5 000 unit per well, the optimal incubation time was 1.5 h. pEGF and standard hEGF had the same prolific effect with the concentration ranging from 1 ng/mL-100 ng/mL, which showed pEGF and hEGF had the same bioactivity. The bioactivity of pEGF was measured rapidly through CCK-8 kit method being established.