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一种与糖基化CD7抗原结合的纳米抗体的表达和鉴定
发表时间：2018-06-15 浏览量：1413 下载量：230
| 全部作者： | 吴超，胡思怡，周伟，于远，汤金乐，安钢力，杨林 |
| 作者单位： | 苏州大学唐仲英医学研究院血液学研究中心；安徽安科生物工程（集团）股份有限公司；博生吉医药科技（苏州）有限公司 |
| 摘 要： | 目的：研究CD7纳米抗体VHH6和人源化纳米抗体huVHH6与抗原的结合是否依赖于抗原的糖基化。方法：将CD7抗原蛋白胞外区序列、纳米抗体VHH6序列和人源化纳米抗体huVHH6序列分别构建于pET28a+原核表达载体上，然后用大肠杆菌BL21异丙基硫代半乳糖苷（isopropyl thiogalactoside，IPTG）低温诱导表达，收集细菌后超声破碎。CD7抗原取菌液超声破碎后的沉淀部分，通过包涵体复性的方法纯化。VHH6和huVHH6抗体直接使用超声破碎后的上清液纯化。为避免酶联免疫吸附测定（enzyme linked immunosorbent assay，ELISA）亲和力检测时纳米抗体与CD7抗原的His标签（His-Tag）冲突，在纳米抗体序列和His-Tag之间引入一个TeV蛋白酶切位点，待纯化抗体后进行酶切和再纯化。将最终不带标签的纳米抗体分别与糖基化和非糖基化CD7抗原复合，ELISA检测亲和力大小。结果：成功构建并表达了足量的纳米抗体，对其进行酶切后再纯化，获得了高纯度不含标签的纳米抗体，通过与糖基化和非糖基化CD7抗原复合后发现，VHH6和huVHH6并不能与非糖基化CD7抗原结合，而只能与糖基化CD7抗原结合。结论：CD7纳米抗体VHH6和人源化纳米抗体huVHH6与抗原的结合依赖于抗原的糖基化修饰。 |
| 关 键 词： | 人体免疫学；纳米抗体纯化；酶联免疫吸附测定（ELISA）；CD7；糖基化 |
| Title： | Expression and identification of a nanobody combined to glycosylated CD7 antigens |
| Author： | WU Chao, HU Siyi, ZHOU Wei, YU Yuan, TANG Jinle, AN Gangli, YANG Lin |
| Organization： | Hematology Center, Cyrus Tang Medical Institute, Soochow University; Anhui Anke Biotechnology (Group) Co., Ltd.; PersonGen Biomedicine (Suzhou) Co., Ltd. |
| Abstract： | Objective: To investigate whether combining CD7 nanobody VHH6 or humanized nanobody huVHH6 with CD7 antigen is glycosylated antigen-dependent or not. Methods: The sequences of CD7 antigen extracellular domain, CD7 nanobody VHH6 and humanized CD7 nanobody huVHH6 were respectively constructed to the same prokaryotic expression vector pET28a+. We prepared nanobodies with Escherichia coli BL21 and isopropyl thiogalactoside (IPTG) induction under a low temperature, and then collected bacteria before ultrasonication. CD7 antigen was derived from precipitates after ultrasonication by inclusion body renaturation, while VHH6 and huVHH6 were derived from supernate after ultrasonication by purification. To avoid the reduplication between His-Tag from both nanobodies and CD7 antigen by enzyme linked immunosorbent assay (ELISA) affinity test, we added a sequence of TeV enzyme cutting site between nanobody sequence and His-Tag, to cut the His-Tag off before the second purification. Finally, we detected the affinity between nanobodies without any tags and glycosylated or unglycosylated CD7 antigen by ELISA. Results: We successfully constructed and prepared sufficient recombined nanobodies. And we digested nanobodies before purifying to achieve a high level of purification without any His-Tag. After the combination between nanobodies and glycosylated or unglycosylated CD7 antigen, we found that VHH6 and huVHH6 were only combined to glycosylated instead of unglycosylated CD7 antigen. Conclusion: The incorporation between CD7 antigen and CD7 nanobody VHH6 was or humanized nanobody huVHH6 relied on glycosylation. |
| Key words： | human immunology; nanobody purification; enzyme linked immunosorbent assay (ELISA); CD7; glycosylation |
| 发表期数： | 2018年6月第11期 |
| 引用格式： | 吴超，胡思怡，周伟，等. 一种与糖基化CD7抗原结合的纳米抗体的表达和鉴定[J]. 中国科技论文在线精品论文，2018，11（11）：1114-1122. |
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