通过融合表达COMP48自折叠肽提高纳米抗体与CD47抗原的亲和力  

Enhance the affinity of nanobodies to CD47 antigen by fusion expression of COMP48 self-folding peptide

作　　者：张琪 秦瑞坪 范利华 马晓玲 李江伟[1] Zhang Qi;Qin Ruiping;Fan Lihua;Ma Xiaoling;Li Jiangwei(Xin Jiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xin Jiang University,Urumqi 830046,China;College of Biological and Geographical Sciences,Yili Normal University,Yining 835000,China;Urumqi Hengkang Zhiyuan Biological Technology Limited Company,Urumqi 830011,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,乌鲁木齐830046 [2]伊犁师范学院生物与地理科学学院,伊宁835000 [3]乌鲁木齐恒康致远生物技术有限公司,830011

出　　处：《国际生物医学工程杂志》2019年第4期288-293,共6页International Journal of Biomedical Engineering

基　　金：国家自然科学基金(31570935).

摘　　要：目的利用自折叠肽人软骨寡聚基质蛋白(COMP48)改造CD47纳米抗体,以提高其与CD47抗原的亲和力.方法设计和合成COMP48与CD47纳米抗体(VHHB1)DNA的融合序列,构建重组质粒pET22b-VHHB1-COMP48,并将其转化至大肠杆菌BL21(DE3)中,诱导表达融合蛋白.采用蛋白质印迹法、间接酶联免疫吸附测定(ELISA)和非竞争ELISA法检测融合蛋白和抗原CD47的结合特异性和亲和力.结果采用1 mmol/L异丙基-β-D-硫代半乳糖苷诱导得到重组蛋白VHHB1-COMP48,经固定化金属离子亲和层析纯化后得到纯度为90%的重组蛋白.蛋白质印迹结果显示,重组蛋白VHHB1-COMP48可特异性结合抗原CD47,但不结合无关蛋白.间接ELISA和非竞争ELISA结果显示,偶联COMP48的纳米抗体VHHB1-COMP48与未偶联的纳米抗体VHHB1相比亲和力增加,差异具有统计学意义(P<0.01).通过非竞争ELISA测得融合COMP48的纳米抗体的结合常数为6.97×10^7L/mol,解离常数为1.434×10^-8 mol/L.结论在纳米抗体后偶联COMP48可提高纳米抗体与抗原的亲和力.Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90%homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant ( P<0 . 01 ). Through non-competitive ELISA , the constants of affinity and dissociation constants were 6.97 ×10^7L/mol and 1.434 ×10^-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody.

关 键 词：纳米抗体 人软骨寡聚基质蛋白 亲和力成熟 自折叠肽 CD47 

分 类 号：R392-33[医药卫生—免疫学]