一种可溶性单抗ScFv片段的表达及鉴定

Expression and identification of the soluble monoclonal antibody ScFv fragment

目的 利用E .coliHB2 15 1表达结肠癌单抗MC3的单链可变区片段 (Singlechainvariablefragment,ScFv) ,纯化并鉴定其抗原结合活性 ,为结肠癌体内诊断和治疗性研究提供靶向载体分子。方法 利用呈现ScFv抗体的重组噬菌体感染大肠杆菌HB2 15 1进行可溶性抗体表达 ,经点印迹和Western印迹检测可溶性单抗ScFv的表达水平 ,经ELISA检测可溶性ScFv的抗原结合活性。用双脱氧法对组成ScFvDNA的VH和VLDNA进行序列测定。结果 可溶性MC3ScFv获得了成功表达 ,表达产物主要位于周质腔中 ,其分子量约为 3 2× 10 3。来自所获得的 3个克隆周质提取物均能抑制单抗与高水平表达MC3结合抗原的AGS细胞结合 ,抑制率分别为 41.19%、3 6.89%、3 3 .77%。对ScFv的VH和VLDNA的测序结果表明所获抗体的可变区基因属IgG1亚族 ,κ型。结论 利用大肠杆菌HB2 15 1成功表达了具有抗原结合活性的MC3ScFv ,为拓展该抗体的应用范围奠定了基础。

Objective To express soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal carcinomas in E. coli HB2151 and to purify the soluble ScFv and identify its antigen binding activities to find new target vectors for the diagnosis and therapy of colorectal carcinomas. Methods The phage clones displaying ScFv fragment of the monoclonal antibody MC3 were used to infect E. coli HB2151 to express soluble antibodies. The soluble ScFvs were identified by Dot blot and Western blot and their antigen binding activities were determined by ELISA. The VH and VL DNAs of the ScFv DNA derived were sequenced based on the dideoxy method. Results The soluble MC3 ScFvs were expressed successfully. The expression products with a proximate MW of 32×10 3 were mainly secreted into the periplasm. The soluble ScFv containing periplasmatic extracts derived from three clones could inhibit the binding of MC3 with its antigen, and the inhibition rates were 41.19%, 36.89% and 33.77% respectively. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup and κ type. Conclusion Generation of E. coli HB2151 expressed ScFv of monoclonal antibody MC3 paves the way for further use of the antibody.