Wei Jingyan, Liu Yang, Yang Songchuan, Xu Junjie, Kong Hangtian, Han Bing, Bao Yongli, Wu Yin, Yin Weitian, Li Wei, Yan Ganglin, Luo Guimin, Xu Hao-Peng, Li Yuxin, Yang Bai
College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.
J Biomol Screen. 2006 Aug;11(5):546-52. doi: 10.1177/1087057106287901. Epub 2006 Apr 28.
A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.
一种新基因——睾丸特异性蛋白酶50（TSP50），在大多数乳腺癌患者中异常激活且差异表达，提示其可作为该疾病的一种新型生物标志物。目前正在研究TSP50可能作为癌基因的可能性。在本研究中，以TSP50特异性肽pep-50作为靶抗原，从噬菌体展示抗体库中淘选针对TSP50的单链可变片段（scFv）。经过4轮淘选后，通过酶联免疫吸附测定（ELISA）验证文库中的3个克隆（A1、A11和C8）对TSP50具有强结合亲和力，测序显示它们含有scFv抗体轻链和重链的可变区基因，但互补决定区不同。将scFv-A1和scFv-A11的基因克隆到表达载体pPELB中，并在大肠杆菌Rosetta中成功表达为可溶性蛋白。从1升培养物中表达产物的产量约为4.0至5.0毫克蛋白质。通过两步法纯化表达的蛋白质，第一步是离子交换色谱，然后是固定化金属亲和色谱。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，纯化的蛋白质在31 kDa位置显示为单一条带。夹心ELISA表明，表达的scFv蛋白能够与pep-50特异性反应，为研究TSP50在乳腺癌发生发展和治疗中的功能奠定了基础。
