一种与糖基化CD7抗原结合的纳米抗体的表达和鉴定
首发时间:2018-04-03
摘要:目的:为了研究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、纳米抗体序列VHH6和人源化纳米抗体序列huVHH6构建到pET28a+原核表达载体上,然后用BL21大肠杆菌IPTG低温诱导表达,收集细菌沉淀超声破碎。CD7抗原取菌液超声破碎后的沉淀部分,通过包涵体变复性的方法纯化。VHH6和huVHH6抗体取菌液破碎后上清纯化。为了避免Elisa亲和力检测时纳米抗体与CD7抗原的His标签冲突,所以在纳米抗体序列和His-Tag之间引入了一个TeV蛋白酶切位点,待纯化好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His-Tag 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从而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: 免疫学 纳米抗体纯化 Elisa CD7 糖基化修饰
For information in English, please click here
The production and identification of nanobodies combined to glycosylated CD7 antigens
Abstract:Objective: To investigate weather combining CD7 nanobody VHH6 or humanized nanobody huVHH6 to CD7 is glycosylated antigen-dependent. Methods: The CD7 antigen extracellular domain, CD7 nanobody VHH6 and humanized CD7 nanobody huVHH6 were constructed to the same prokaryotic expression vector pET28a+. We prepared nanobodies from E.coli BL21 with IPTG induction under a low temperature, and then collected bacteria before ultrasonication. CD7 antigen was derived from precipitates after ultrasonication by renaturation, while VHH6 and huVHH6 were derived from supernate after ultrasonication by purification. To avoid the reduplicate His-Tag from both nanobodies and antigen, we added a sequence of TeV enzyme cutting site between nanobody sequence and His-Tag, 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, we found that VHH6 and huVHH6 were only combined to glycosylated CD7 instead of unglycosylated CD7 antigen. Conclusion: The incorporation between CD7 antigen and CD7 nanobody VHH6 or humanized nanobody huVHH6 relied on glycosylation.?????
Keywords: immunology nanobody purification Elisa CD7 glycosylation
引用
No.****
动态公开评议
共计0人参与
勘误表
一种与糖基化CD7抗原结合的纳米抗体的表达和鉴定
评论
全部评论0/1000