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目的 制备抗人结肠癌单抗MC5的具功能活性的可溶性单链可变区片段 (ScFv)。方法 从分泌MC5的杂交瘤细胞分离mRNA ,RT PCR分别扩增抗体质量、轻链可变区基因 (VH和VLDNA) ,经linkerDNA连接形成ScFvDNA。将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,转化菌经辅助噬菌体M13KO7感染后 ,获得重组噬菌体。以高表达MC5结合抗原的人结肠癌细胞系SW4 80对重组噬菌体进行 2轮筛选后 ,经ELISA筛选呈现抗体ScFv片段的噬菌体单克隆。取 2个强阳性克隆的噬菌体感染大肠杆菌HB2 15 1,用于表达可溶性ScFv。经点印迹和Western印迹对可溶性ScFv进行鉴定 ,经ELISA检测可溶性ScFv的抗原结合活性。结果 所获得的VH、VL和ScFvDNA分别约为 340、32 0和 75 0bp。对重组噬菌体经 2轮亲和筛选后 ,经ELISA从 2 5个克隆中筛检出 10个抗原阳性克隆。源于 2个强阳性克隆的可溶性ScFv的分子质量为32 0 0 0u ,且浓集于细菌周质腔中 ,可溶性ScFv具有抗原结合活性 ,其结合位点与亲本单抗MC5相同。结论 针对MC5的具功能活性的可溶性ScFv的成功制备 ,为人结肠癌的体外 (乃至体内 )研究提供了新的靶向载体分子
Objective To prepare soluble functional single chain variable fragment (ScFv) of anti-human colon cancer monoclonal antibody MC5. Methods mRNAs were isolated from hybridoma cells secreting MC5. The antibody quality and light chain variable region genes (VH and VLDNA) were amplified by RT PCR. The ligation product of ScFvDNA and the phagemid vector pCANTAB5E was transformed into E. coli TG1, and the transformed bacterium was infected with helper phage M13KO7 to obtain recombinant phage. After two rounds of selection of the recombinant phage with a human colorectal cancer cell line SW480 that overexpresses the MC5-binding antigen, phage monoclonal antibody scFv fragments were screened by ELISA. Two strong positive clones were infected with E. coli HB2 15 1 to express soluble ScFv. Soluble ScFv was identified by dot blotting and Western blotting, and the antigen-binding activity of soluble ScFv was assayed by ELISA. Results The VH, VL and ScFv DNA obtained were about 340, 320 and 75 0 bp, respectively. After 2 rounds of affinity screening of recombinant phage, 10 antigen positive clones were screened out of 25 clones by ELISA. The soluble ScFv derived from two strong positive clones has a molecular mass of 3200u and is concentrated in the periplasmic space of the bacteria. The soluble ScFv has the antigen binding activity, and the binding site is the same as that of the parental monoclonal antibody MC5. Conclusions The successful preparation of functionally active soluble ScFv against MC5 provides a novel targeting vector molecule for human colon cancer in vitro (and in vivo) studies