人CD137胞外区噬菌体展示系统的构建及鉴定

Phage display of human CD137 extra-cellular domain and its bioactivity detect

目的:以噬菌体展示具有天然结构的人CD137分子胞外区,检测其抗原性和生物活性,为以CD137为靶点体外筛选CD137拮抗性类肽药物奠定基础。方法:PCR方法从CIS质粒扩增人CD137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共感染,使CD137胞外区展示在噬菌体表面。采用ELISA法检测噬菌体展示CD137的抗原性,MTT法检测CD137噬菌体对抗CD137抗体刺激的人外周血淋巴细胞增殖作用的影响检测其生物活性。结果:成功构建表达人CD137分子胞外区的重组噬菌粒载体pComb3HCD137,并以噬菌体展示系统展示CD137分子胞外区。ELISA结果显示噬菌体展示CD137可与抗人CD137抗体特异性结合,证实CD137分子成功展示于噬菌体表面,且具有抗原性。体外生物活性实验显示,展示在噬菌体表面的CD137可抑制抗CD137抗体刺激的人外周血淋巴细胞增殖反应,证实噬菌体展示的CD137有与其配体结合的生物活性结构域。结论:利用噬菌体展示系统成功展示CD137胞外区,噬菌体展示CD137具有抗原性及生物活性。

Objective：To display human CD137 extra-cellular domain on phage using phage display technique and detect its antigenicity and bioactivity. Methods： CD137 extra-cellular gene was amplified from CIS plasmid using PCR and then clone it into pComb3HSS. The recombinant pComb3H-CD137 was transfected into competent bacteria XL1-Blue, then the transfected bacteria was co-infected with help phage VCSM13 and CD137 extra-cellular domain was displayed on the phage surface. The antigenicity and bioactivity of CD137 displayed on phage was detected by ELISA and inhibition assay of lymphocyte proliferation （MTT）. Results： CD137 PCR products was 525 bp, the recombined plasmid was named pComb3H-CD137, CD137 was displayed on phage. ELISA detection showed that phage displayed CD137 possesses antigenicity of human CD137. MTY inhibition assay showed that lymphocytes proliferation stimulated by PHA and CD137 could be inhibited by phage displayed CD137, indicating that it has bioactivity of nature CD137. Conclusion：The studies demonstrate that phage display system can display human CD137 extra-cellular domain homodimer with antigenicity and bioactivity.