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目的采用哺乳动物细胞表面展示技术构建全长人源类风湿关节炎抗体库。方法分离类风湿关节炎患者的外周血淋巴细胞,提取总RNA,采用RT-PCR的方法扩增抗体全长轻链基因和重链可变区基因,分别插入哺乳动物细胞表达载体pDGB-HC-TM,电击转化感受态大肠杆菌TOPO-10,分别构建抗体轻链基因库和抗体重链基因库,然后将抗体轻链、重链基因库联合转染293T细胞,用流式细胞仪分析全长人源抗体在293T细胞表面的表达。结果成功构建了类风湿关节炎患者来源的IgG1-Kappa型抗体基因库,随机挑选单克隆经DNA序列分析显示轻链库、重链库的序列正确率分别为80%和60%,可表达的抗体库多样性为6.13×1010。结论类风湿关节炎抗体基因库转染293T细胞后能够在细胞表面表达全长人源抗体,所展示的抗体库具有良好的多样性,能够为下一步筛选特异性抗体奠定基础。
Objective To construct a full-length human rheumatoid arthritis antibody library using mammalian cell surface display technology. METHODS: Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis and the total RNA was extracted. The full-length light chain gene and heavy chain variable region of the antibody were amplified by RT-PCR and inserted into mammalian cell expression vector pDGB-HC- TM, and transformed into competent E. coli TOPO-10 by electroporation. The antibody light chain gene library and antibody heavy chain gene library were respectively constructed. Then 293T cells were transfected with antibody light chain and heavy chain gene library, and the total length of the 293T cells was analyzed by flow cytometry Expression of Human Antibodies on 293T Cell Surface. Results The IgG1-Kappa antibody gene library from patients with rheumatoid arthritis was successfully constructed. The results of DNA sequencing showed that the sequence accuracy of light chain library and heavy chain library were 80% and 60%, respectively. Antibody pool diversity was 6.13 × 1010. Conclusion The full-length humanized antibody can be expressed on 293T cells after transfection with 293T cells. The displayed antibody library has good diversity and can lay the foundation for the further screening of specific antibodies.