Schlebusch H, Reinartz S, Kaiser R, Grünn U, Wagner U
Department of Clinical Chemistry and Hematology, University of Bonn, Germany.
Hybridoma. 1997 Feb;16(1):47-52. doi: 10.1089/hyb.1997.16.47.
The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.
模拟肿瘤相关抗原CA125的鼠单克隆抗独特型抗体ACA125的F(ab')2片段被用作疫苗，用于诱导卵巢癌患者的抗肿瘤免疫。我们试图生成一种单链片段（ScFv），它由ACA125重链和轻链可变域通过多肽接头连接而成，作为相应F(ab')2片段的替代物。分别扩增产生抗体的小鼠杂交瘤细胞系的重链和轻链基因，并通过聚合酶链反应（PCR）将其与接头DNA组装成ScFv基因。将ScFv基因连接到噬菌粒载体pCANTAB5E中，该载体允许产生噬菌体展示的和可溶性的ScFv。用M13K07辅助噬菌体感染转化的大肠杆菌TG1细胞以产生重组噬菌体，其在丝状噬菌体M13表面将ScFv片段展示为g3p融合蛋白。经过一轮淘选后，通过与独特型抗体OC125结合可以选择重组噬菌体，并直接用于再次感染大肠杆菌TG1细胞。用先前通过酶联免疫吸附测定（ELISA）筛选的抗原阳性噬菌体克隆感染大肠杆菌非抑制菌株HB2151，以表达可溶性ScFv片段。通过蛋白质印迹法和ELISA可以在细菌周质中检测到与独特型抗体OC125 F(ab')2结合的功能性可溶性ScFv。进一步对ACA125 ScFv片段的可变重链和轻链基因进行测序，并与已知抗体序列进行比较。
