CAJ | 학술논문

目的:构建并表达进一步人源化且效果显著增强的抗EGFR抗体.方法:通过计算机辅助设计的结果,合成新型抗EGFR抗体的轻链和重链可变区序列,拼接成完整的轻链和重链基因并克隆入pIRES双表达载体.瞬时转染293T细胞,用rProtein A亲和层析柱从细胞培养上清中纯化抗体,并进行SDS-PAGE和免疫印迹鉴定.采用Biacore3000技术检测抗体结合抗原的能力;细胞侵袭实验初步检测抗体的功能.结果:成功构建3种不同突变体表达载体C2、C3和C5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×10~3和50×10~3两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合.Biacore3000实验结果表明,C3抗体与抗原具有良好亲和活性(亲和力为6.13×10~(-10) mol/L);细胞侵袭实验结果表明,C2和C3抗体对肿瘤细胞生长迁移均具有一定的抑制作用.结论: 成功构建、表达了3种抗EGFR人源化抗体C2、C3和C5,其中C3具有良好的抗原结合能力和抑制肿瘤细胞生长迁移能力.
목적:구건병표체진일보인원화차효과현저증강적항EGFR항체.방법:통과계산궤보조설계적결과,합성신형항EGFR항체적경련화중련가변구서렬,병접성완정적경련화중련기인병극륭입pIRES쌍표체재체.순시전염293T세포,용rProtein A친화층석주종세포배양상청중순화항체,병진행SDS-PAGE화면역인적감정.채용Biacore3000기술검측항체결합항원적능력;세포침습실험초보검측항체적공능.결과:성공구건3충불동돌변체표체재체C2、C3화C5,순화적항체재환원SDS-PAGE중표현위상대분자질량약위25×10~3화50×10~3량조대;면역인적분석표명,해인원화항체가여양항인IgG특이성결합.Biacore3000실험결과표명,C3항체여항원구유량호친화활성(친화력위6.13×10~(-10) mol/L);세포침습실험결과표명,C2화C3항체대종류세포생장천이균구유일정적억제작용.결론: 성공구건、표체료3충항EGFR인원화항체C2、C3화C5,기중C3구유량호적항원결합능력화억제종류세포생장천이능력.
Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.