人乳头瘤病毒16/18型E7双特异亲和体融合颗粒酶B的免疫亲和毒素制备和鉴定  

Preparation and Identification of Immunoaffitoxin Based on Bispecific Affibody Fused with Granzyme B Targeting to Human Papillomavirus 16/18 E7

作　　者：余丽君 李妍姮 黄智羡 杨佳妮 谢自新[1] 张丽芳[1] 李文姝[1] YU Lijun;LI Yanheng;HUANG Zhixian;YANG Jiani;XIE Zixin;ZHANG Lifang;LI Wenshu(Department of Microbiology and Immunology,Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学微生物学与免疫学教研室,温州325000

出　　处：《病毒学报》2023年第5期1326-1335,共10页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81973216),题目:负载GrB双靶向HPV16/18亲和毒素抗宫颈癌生物学效应研究;浙江省基础公益研究计划项目(项目号:LY23C010003),题目:HPV16E7特异性免疫亲和毒素抗宫颈癌双功能作用及机制研究~~。

摘　　要：制备人乳头瘤病毒(Human papilloma virus,HPV)16/18型双特异性亲和体(Affibody,Z_(HPV16-18E7))融合颗粒酶B(GrB)的免疫亲和毒素,并鉴定其诱导靶细胞凋亡效应。于实验室前期已构建的Z_(HPV16-18E7)的N-端、中间、及C-端,偶联GrB构建不同融合形式的免疫亲和毒素,并在原核表达体系中制备与纯化;在线网站分析免疫亲和毒素基本理化性质和空间结构预测;酶联免疫吸附实验(ELISA)检测免疫亲和毒素分别对靶蛋白HPV16E7、HPV18E7的特异性结合力;流式细胞分析术检测免疫亲和毒素分别诱导人宫颈癌细胞SiHa(HPV16阳性)、HeLa(HPV18阳性)的细胞凋亡效应。成功构建三种不同组合形式的免疫亲和毒素,各免疫亲和毒素空间结构符合理论预测,且理化特性趋于一致;融合于Z_(HPV16-18E7)中间和C-端的两种免疫亲和毒素组合形式可在原核表达体系中表达出预期目的蛋白;制备的免疫亲和毒素可特异性结合相应的靶蛋白,结合效价均可达到1∶5625与1∶78125之间,且可分别诱导靶细胞产生显著的细胞凋亡效应。本研究成功构建与制备了基于亲和体的免疫亲和毒素,为HPV感染宫颈癌靶向治疗研究提供了新思路。To construct and prepare the immunoaffitoxin with human papillomavirus(HPV)16/18 E7 bispecific Affibody(Z_(16-18))fused to granzyme B(GrB),and evaluate its ability to induce target cell apoptosis.Based on the Z_(HPV16-18E7)constructed in our previous work,GrB was inserted into N-terminal,middle,or C-terminal of Z_(HPV16-18E7),respectively,to prepare three conjugates of immunoaffitoxin by molecular cloning technique.Immunoaffitoxins were prepared and purified using prokaryotic expression system.Protein physicochemical properties and spatial structure of immunoaffitoxins were analyzed and predicted on line.The specific binding ability of immunoaffitoxins to target protein HPV16E7 or HPV18E7 were detected by enzyme linked immunosorbent assay(ELISA).The induced apoptotic effects of immunoaffitoxins on target cells of SiHa(HPV16 positive),or HeLa(HPV18 positive)were evaluated by flow cytometry.Three immunoaffitoxin conjugates were successfully constructed.Their spatial structures were consistent with theoretical predictions and their physicochemical properties tend to be consistent.Two immunoaffitoxin conjugates of Z_(16)-GrB-Z_(18)(GrB fused into Z_(16-18)middle terminal)and Z_(16-18)-GrB(GrB fused into Z_(16-18)C-terminal)could express the desired proteins in the prokaryotic expression system.The prepared immunoaffitoxins could specifically bind to the corresponding target proteins binding with potency between 1∶5625 and 1∶78125,and induce significant apoptosis on target cells.In this study,a novel immunoaffitoxin based on affibody fused with GrB was successfully constructed and prepared,and this work provide a new approach for targeted treatment of HPV associated cervical cancer.

关 键 词：人乳头瘤病毒 HPV16/18型 AFFIBODY 颗粒酶B 双靶向 细胞凋亡 

分 类 号：R373.9[医药卫生—病原生物学]