仅具有CDR3的单域抗体NBL42作为纳米抗体亲和力转移的移植框架研究  

Affinity transfer of nanobodies to a CDR3 only single-domain antibody fragment NBL42

作　　者：刘娅菲 冯亚宁 张鑫 谢媛 李江伟[1] Liu Yafei;Feng Yaning;Zhang Xin;Xie Yuan;Li Jiangwei(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;The Affiliated Tumor Hospital,Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源与基因工程重点实验室,乌鲁木齐830046 [2]新疆医科大学附属肿瘤医院,乌鲁木齐830011

出　　处：《国际生物医学工程杂志》2022年第3期200-206,219,共8页International Journal of Biomedical Engineering

基　　金：国家自然科学基金(31370933)。

摘　　要：目的:评价前期获得的1株仅具有互补决定区3(CDR3)的单域抗体(sdAbs)NBL42作为纳米抗体亲和力移植通用框架的潜力。方法:采用亲和力转移的方法将分别结合蒜氨酸酶、程序性死亡受体-1(PD-1)、溶菌酶和CD47的4种抗体VHH-A4、VHH-H5、cAb-Lys3和B6H12的CDR3序列移植到NBL42对应的CDR3区。DNA合成获得4种仅具有CDR3的重组sdAbs,镍柱纯化并获得目的蛋白,间接ELISA法检测4种重组纳米抗体与抗原的结合及其热稳定性。用Swiss Model三维建模分析移植前后sdAbs的空间结构。结果:纯化后的4种重组蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)中呈现单一条带,相对分子质量与预期相符。移植后的重组sdAbs NBL42-A4CDR3和NBL42-H5CDR3获得了与蒜氨酸酶和PD-1的特异结合能力,但结合活性降低了约50%,并转变为可溶性表达形式。移植后的重组sdAbs NBL42-L3CDR3和NBL42-B6H12CDR3完全丧失了对原抗原溶菌酶和CD47的结合能力。在热稳定性分析中,移植后的重组sdAbs NBL42-A4CDR3和NBL42-H5/CDR3保留了约50%的剩余结合活性。根据C88Y突变实验和Swiss Model三维建模分析,提示NBL42-FR3中88位半胱氨酸对于亲和力转移可能具有重要作用。结论:NBL42具有作为CDR3移植和亲和力转移框架的潜力。Objective To evaluate the potential of a previously identified CDR3 only single-domain antibodies(sdAbs)fragment,NBL42,as a general framework for affinity transfer.Methods The H3 loops of VHH-A4(A4),VHH-H5(H5),cAb-Lys3(L3)and B6H12 which bind with alliinase,PD-1,lysozyme and CD47,respectively,were grafted into the corresponding loop of NBL42.The genes of the reconstituted CDR3 only sdAbs were synthesized,expressed in E.coliand purified with Ni2+column affinity chromatography.The antigen binding and stability of the recombinant CDR3 only sdAbs were assayed by ELISA.Results The recombinant NBL42-A4CDR3,NBL42-H5CDR3,NBL42-L3CDR3 and NBL42-B6H12CDR3 ran as a single peak at 15,15,28 and 16 kDa,respectively,in SDS-PAGE as expected molecular weight.Grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 expressed in a soluble form and specifically bind with alliinase and PD-1,respectively,but lost about 50%of their binding activity.In contrast,the grafted sdAbs NBL42-Lys3CDR3 and NBL42-B6H12CDR3 completely lost their antigen binding capacity.NBL42 sdAbs and grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 retain roughly half of their binding activity after 90℃heat treatment,indicating high stability.The C88Y mutation in NBL42 and the Swiss Mode 3D model predicted that the C88Y residue in FR3 may play a key role in NBL42 stability and CDR3 affinity transfer.Conclusions The structure of NBL42 has potential as a framework for CDR3 transplantation and affinity transfer.

关 键 词：CDR3移植 亲和力转移 纳米抗体 抗原结合 通用框架 

分 类 号：R392[医药卫生—免疫学]