Zhang Yong-xia, Wang Bao-cheng, Yu Xin, Dai Yun-jian, He Yong-zhi, Cong Cong, Xia Yong, Wang Ming-rong
Chengdu Institute of Biological Products Co., Ltd., Chengdu, China.
Yao Xue Xue Bao. 2012 Oct;47(10):1329-35.
Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
从过敏患者外周血中分离出淋巴细胞，提取总mRNA，然后通过RT-PCR构建重链可变区（VH）和轻链可变区（VL）cDNA文库。使用引物和接头肽（Gly4Ser）3组装用于兔网织红细胞裂解物核糖体展示的人scFv模板。使用原位单引物RT-PCR回收抗体-核糖体-mRNA复合物中结合的mRNA，富集三轮抗IgE scFv DNA。将目标DNA片段进行双酶切并连接到质粒pET22b（+）中，随后转化到大肠杆菌Rosseta（DE3）中。使用克隆PCR、斑点印迹和抗原ELISA筛选阳性克隆。获得了正确长度的VH（400 bp）和VL（710 bp）PCR产物。在琼脂糖凝胶电泳中也观察到了预期的1000 bp核糖体展示模板。经过三轮核糖体展示，目标序列得到有效富集，形成了一个包含10（13）个成员的文库。在菌株pET-IgE-6中产生了对IgE具有最高ELISA值的抗体。本文成功构建了人抗IgE scFv文库。使用原位单引物RT-PCR作为回收程序的核糖体展示有效地富集了目标序列。鉴定出了具有高亲和力和特异性的抗IgE scFv。制备的人抗IgE scFv片段可能自行开发成为治疗哮喘的先导药物。我们的研究为快速发现具有治疗重要性的人抗体提供了一种替代方法。
