摘要
[目的]将狂犬病病毒糖蛋白(RVGP)上3种针对不同类型表位的单克隆IgG抗体改造为IgM工程化抗体,对改造前后针对不同类型表位抗体的效力学差异进行探究。[方法]通过质粒构建将不同类型表位的单克隆IgG抗体进行IgM工程化改造并表达纯化。通过ELISA和RFFIT检测了改造前后抗体亲和力、中和活性以及二者变化的相关性,检测了不同病毒MOI下改造后的IgM抗体抑制病毒的效力变化。[结果]纯化出3种IgM工程化抗体,经鉴定,针对1号线性表位改造后IgM亲和力变大,EC50由0.5985μg/mL降至0.0648μg/mL,中和活性由32.43 IU/mg升至842.97 IU/mg,且在高浓度病毒下该抗体仍具备较好的中和活性。三种抗体改造后亲和力变化与中和活性变化具有相关性(P<0.05)。[结论]针对RVGP的1号线性表位IgM工程化改造能够提升抗体的生物学活性,亲和力、中和活性分别提升了9.24倍、25.99倍,且改造前后亲和力与中和活性变化呈线性相关。
[Objective]To engineer three types of monoclonal IgG antibodies targeting distinct epitopes on the rabies virus glycoprotein(RVGP)into IgM engineered antibodies and to investigate the differences in their efficacy before and after the modification.[Method]The monoclonal IgG antibodies were engineered into IgM through plasmid construction and expressed and purified.ELISA and RFFIT were conducted to assess the changes in antibody affinity and neutralizing activity before and after the engineering,as well as the correlation between these changes.The efficacy of the engineered IgM antibodies in inhibiting the virus at different MOI was evaluated.[Result]Three IgM engineered antibodies were purified.Upon identification,the IgM affinity increased,and EC_(50)for the modified linear epitope 1 decreased from 0.5985μg/mL to 0.0648μg/mL.The neutralizing activity increased from 32.43 IU/mg to 842.97 IU/mg.This antibody retained good neutralizing activity at high virus concentrations.The changes in affinity and neutralizing activity after engineering for the three antibodies were correlated(P<0.05).[Conclusion]The IgM engineering of the first linear epitope on RVGP enhanced the biological activity of the antibody.This enhancement results in a 9.24-fold increase in affinity and a 25.99-fold increase in neutralizing activity.The changes in affinity and neutralizing activity before and after the modification were linearly correlated,indicating a significant enhancement in antibody performance.
作者
陈硕
郭馨文
黄雨杭
陈越
杨欢
李静雯
马良
王红叶
朱小永
李冰香
孙明
CHEN Shuo;GUO Xinwen;HUANG Yuhang;CHEN Yue;YANG Huan;LI Jingwen;MA Liang;WANG Hongye;ZHU Xiaoyong;LI Bingxiang;SUN Ming(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,China)
出处
《生物技术》
2025年第2期152-158,共7页
Biotechnology
基金
中国医学科学院医学与健康科技创新工程重大协同创新项目(2021-I2M-1-043)
云南省治疗性中和抗体创新团队项目(202405AS350026)。
