【摘要】 目的以噬菌体展示具有天然结构的人CD28分子胞外区同二聚体,检测其抗原性和生物活性,为CD28拮抗性类肽的筛选奠定基础。方法RTPCR从Jurkat细胞扩增CD28胞外区,将其克隆入噬菌粒载体pComb3HSS,构建的重组载体电转入受体菌XL1blue,并加辅助噬菌体VCSM13,使CD28胞外区展示在噬菌体表面。以ELISA、流式细胞仪检测噬菌体展示CD28的抗原性和与B71的结合力。MTT法检测噬菌体展示CD28的生物活性。结果成功构建表达人CD28分子胞外区同二聚体的重组噬菌粒载体pComb3HCD28,并以噬菌体展示系统展示CD28分子。ELISA和FCM结果显示,噬菌体展示CD28可与抗CD28抗体特异性结合,也可与Jurkat细胞表面CD28竞争结合抗CD28抗体,具有CD28抗原性。噬菌体展示CD28可与B71特异性结合,也可与Raji细胞表面CD80结合,具有与配体结合能力。体外生物活性实验显示,噬菌体展示CD28可抑制抗CD28抗体刺激的人外周血淋巴细胞增殖反应。结论利用噬菌体展示系统成功展示CD28同源二聚体,噬菌体展示CD28具有抗原性及生物活性,为以CD28为靶点体外筛选CD28拮抗性类肽,从而防治移植排斥反应和自身免疫病奠定了基础。更多还原

【Abstract】 Objective To display human CD28 extra-cellular domain homodimer on phage using phage display technique and to detect its antigenicity and bioactivity. Methods Extra-cellular region of CD28 gene was amplified from Jurkat cell using RT-PCR and was then cloned into pComb3HSS vector. The recombinant phagemid vector pComb3H-CD28 was transfected into competent bacteria XL1-blue, then the transfected bacteria was co-infected with help phage VCSM13. CD28 extra-cellular domain homodimer was displayed on the phage surface. The antigenicity and bioactivity of CD28 displayed on phage was detected by ELISA, FCM and inhibition assay of lymphocyte proliferation(MTT). Results The recombinant phagemid vector containing human CD28 gene extra-cellular domain, pComb3H-CD28, was successfully constructed. CD28 was displayed on phage. ELISA and FCM detection showed that phage- displayed CD28 could bind to CD28Ab and could bind with CD28Ab by competing with CD28 on the surface of Jurkat cell. These data indicated that phage-displayed CD28 possessed antigenicity. Phage-displayed CD28 could also bind to rhB7-1 and CD80 on the surface of Raji cell, demonstrating that phage-displayed CD28 not only maintains its antigenicity but also possesses its native structure of binding to its ligand. In further, MTT inhibition assay showed that lymphocytes proliferation stimulated by PHA and CD28Ab could be inhibited by phage-displayed CD28, indicating that it has bioactivity of CD28. Conclusion The phage display system can display human CD28 extra-cellular domain homodimer with antigenicity and bioactivity.更多还原