抗CD71单抗与蓖麻毒蛋白A链免疫毒素的制备及其体外抗肝癌细胞效应的研究

Preparation of anti-CD71 monoclonal antibody with the ricin A chain immunotoxin and it's effect of anti-hepatocellular carcinoma cells in vitro

目的因抗CD71单抗具有靶向定位特性,及利用蓖麻毒蛋白A链(RTA)的细胞毒性,将两者进行偶联,构建免疫毒素CD71 Mab-RTA,并研究其体外抗肝癌细胞效应。方法从蓖麻籽中提纯RTA,以N-琥珀酰胺-3-(2-吡啶二硫)丙酸酯[N-succinimidyl-3-(2-pyridyldithio)-propionate,SPDP]作为偶联剂,偶联抗CD71单抗,构建免疫毒素(IT),以SDS-PAGE检测IT,以间接ELISA方法测定IT的免疫反应性,以WST-1法测定IT的体外抗肝癌细胞效应。结果蓖麻籽经过研磨、过滤、亲和层析、凝胶层析、离子交换层析等步骤成功提纯RTA。提纯的RTA与CD71 mab进行偶联成功构建免疫毒素CD71 Mab-RTA,SDS-PAGE结果显示,在非还原状态下,为一条分子量>116kD的单一电泳条带,在还原状态下,为三条电泳条带,分别位于约55 kD处、约32 kD处及约28 kD处。间接ELISA结果显示,免疫毒素与单抗的曲线走势一致,但各浓度下OD值均较单抗组略低。WST-1结果显示,IT在各浓度梯度下均表现出对HepG_2细胞和LO_2细胞不同的杀伤作用(P<0.05)。结论 CD71 mab-RTA基本保留了免疫反应性,及表现出对HepG_2细胞和LO_2细胞的不同体外细胞毒效应,提示对肝癌细胞具有较好的靶向细胞毒作用,有望成为新型的抗肝癌药物。

Objective According to the cytotoxicity of ricin A chain(RTA) and the target localization feature of CD71 monoclonal antibody(Mab), to research the effect of anti-hepatoma carcinoma cell in vitro of the immunotoxin(IT) composed by RTA and CD71 Mab. Methods Taking SPDP as the coupling agent, RTA purified from castor bean was linked with CD71 Mab. After further purification, the construction of IT was detected by SDS-PAGE. The immunoreactivity was detected by indirect ELISA.The cytotoxicity in vitro was detected by WST-1 method. Results RTA was successfully purified by series of steps from ricin crude, such as grinding, filtration, affinity chromatography, gel chromatography, ion exchange chromatography and so on. The immunotoxin was constructed successfully,and there were two experimental results in the electropherogram of SDS-PAGE. Under non-reducing conditions, it migrated as a molecular weight exceeded about 116 k D; under reducing state, it migrated as three electrophoretic bands, which located at approximately 55 k D, 32 k D and 28 k D.The experimental result of indirect ELISA showed that the curves of IT and Mab had the same trend, but the absorbance of IT was lower than that of Mab in a series of concentration gradient. By WST-1 method, in a series of concentration gradient, IT displayed a different cytotoxicity between Hep G_2 cell and LO_2cell( < 0.05). Conclusions IT retained a major part of the immunoreactivity. IT displayed a different cytotoxicity between Hep G_2 and LO_2cells. IT played a specific role in suppressing the multiplication of the hepatoma carcinoma cell and had a good prospect in liver cancer treatment.