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目的构建系统性红斑狼疮(SLE)儿童个体特异性哺乳动物细胞表面展示的全长人抗体基因库。方法采集SLE确诊患儿外周血,分离外周血淋巴细胞,提取外周血淋巴细胞总RNA,用RT-PCR扩增全长Kappa轻链(LCκ)和重链可变区(VH)基因、构建抗体轻链基因库和抗体重链基因库,将抗体基因库转染293T细胞,流式细胞仪分析抗体在293T细胞表面的表达。结果以0.8μg总RNA为模板,用6套引物成功扩增全长Kappa轻链和重链可变区基因,插入表达载体,构建获得的重链基因库容量为9.4×104,轻链基因库容量为8.4×104。随机挑选的10个重链克隆有8个含有正确的阅读框架,编码8个不同的氨基酸序列,10个轻链克隆7个含有正确的阅读框架,编码7个不同的氨基酸序列。流式细胞仪分析挑选的单克隆和抗体基因库,均检测到全长抗体在293T细胞表面的表达,理论上抗体库中可表达的抗体多样性可以达到109。结论以0.8μg总RNA为模板,通过RT-PCR扩增,成功构建SLE儿童特异性全长人抗体基因库,并在293T细胞表面成功展示SLE儿童个体抗体库中的全长抗体,为进一步研究SLE儿童自身抗体的特点及自身抗体在SLE发病机制中的作用及临床应用打下良好基础。
Objective To construct a full-length human antibody gene bank displayed on the surface of individual mammalian cells in systemic lupus erythematosus (SLE). Methods Peripheral blood was collected from patients with SLE. Peripheral blood lymphocytes were isolated and the total RNA of peripheral blood lymphocytes was extracted. The full length Kappa light chain (LCκ) and heavy chain variable region (VH) genes were amplified by RT-PCR to construct antibodies Light chain gene library and antibody heavy chain gene library, the antibody gene library was transfected into 293T cells, and the expression of the antibody on the 293T cell surface was analyzed by flow cytometry. Results 0.8 μg of total RNA was used as a template to amplify full-length Kappa light chain and heavy chain variable region genes with 6 sets of primers and inserted into expression vector to construct a heavy chain gene library with a capacity of 9.4 × 104 and a light chain gene library The capacity is 8.4 × 104. Eight randomly selected 10 heavy chain clones contained the correct reading frame, encoded 8 different amino acid sequences, and 7 of the 10 light chain clones contained the correct reading frame and encoded 7 different amino acid sequences. The monoclonal and antibody gene pools selected by flow cytometry analysis all detected the expression of full-length antibodies on the surface of 293T cells. In theory, the number of antibodies that can be expressed in the antibody library can reach 109. Conclusion 0.8 μg of total RNA was used as template to amplify the full-length human antibody library of SLE children by RT-PCR. The full-length antibody of SLE children antibody library was successfully displayed on the surface of 293T cells for further study The characteristics of SLE children’s autoantibodies and the role of autoantibodies in the pathogenesis of SLE and clinical application lay a good foundation.