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利用DNA重组技术分离人补体C3α链cDNA的3个重叠(overlapping)克隆并在大肠杆菌中表达出不同的人C3α链片段。Western 印迹法表明,多克隆和单克隆抗人C3抗体H215与它们均呈阳性反应。为进行人C3α链的亚克隆化,构建了新的表达质粒载体pECL1-3,获ReC3-H2cDNA亚克隆。并在大肠杆菌中表达出小片段人C3。多克隆抗人C3抗体与ReC3H2呈阳性反应,而单抗H215则阴性。结合酶图谱分析和免疫学测定,证明H215的抗原识别点位于C3α链C末端约120个氨基酸的区域内。
Three overlapping clones of human complement C3 alpha chain cDNA were isolated using DNA recombination technology and different human C3 alpha chain fragments were expressed in E. coli. Western blotting showed that both polyclonal and monoclonal anti-human C3 antibody H215 reacted positively with them. For subcloning human C3α chain, a new expression plasmid vector pECL1-3 was constructed and subcloned by ReC3-H2 cDNA. And expressed small fragments of human C3 in E. coli. Polyclonal anti-human C3 antibodies reacted positively with ReC3H2, whereas mAb H215 was negative. Combined with enzyme mapping analysis and immunological assay, it was demonstrated that the antigen recognition site of H215 was located in the region of about 120 amino acids at the C-terminal of C3α chain.