小鼠抗人腺病毒55型六邻体(HAdV55 Hexon)蛋白单克隆抗体的制备  

Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon(HAdV55 Hexon)protein

作　　者：袁若栋 董阳超 武福星 段甜 薛潘 张剑 袁明城 薛智丰 张海军 张欠欠[1] 高小朋[1] 雷迎峰 YUAN Ruodong;DDONG Yangchao;WU Fuxing;DUAN Tian;XUE Pan;ZHANG Jian;YUAN Mingcheng;XUE Zhifeng;ZHANG Hajun;ZHANG Qianqian;GAO Xiaopeng;LEI Yingfeng(School of Life Science,Yan'an University,Yan'an 716000;Department of Microbiology and Pathogenic Biology,Basic Medical Science Academy,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]延安大学生命科学学院,陕西延安716000 [2]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2023年第6期544-551,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：空军军医大学军事医学珠峰工程人才计划(zfrclyf);陕西省重点研发计划(2021ZDLSF01-05)。

摘　　要：目的制备特异性小鼠抗人腺病毒55型六邻体蛋白(HAdV55 Hexon)单克隆抗体(mAb)。方法以化学合成HAdV 55、3、4、7、16、21的Hexon基因为模板,PCR扩增后分别构建原核表达质粒pET28a-HAdV55 Hexon与真核表达质粒pCAGGS-HAdV3、4、7、16、21、55 Hexon;将pET28a-HAdV55 Hexon质粒转化至大肠杆菌(E.coli)感受态细胞BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,将纯化的包涵体经过变性与复性后利用切向流膜过滤系统获得纯化蛋白;将pCAGGS-HAdV55 Hexon用于拔罐免疫BALB/c小鼠,HAdV55 Hexon蛋白用于加强免疫,通过杂交瘤技术制备抗HAdV55 Hexon的mAb,并进行抗体效价测定、抗体亚类鉴定;对上述抗体利用pCAGGS-HAdV55 Hexon转染HEK293T细胞和BHK细胞分别进行Western blot法和免疫荧光法(IFA)以鉴定其特异性;选择其中效价高的两株抗体,进一步利用pCAGGS-HAdV3、4、7、16、21、55 Hexon转染细胞分别进行Western blot法和IFA做交叉反应性分析。结果成功构建pET28a-HAdV55 Hexon和pCAGGS-HAdV55 Hexon、3、4、7、16、21表达质粒;IPTG诱导pET28a-HAdV55 Hexon转化的BL21细菌,HAdV55 Hexon蛋白主要以包涵体形式表达,经过变性及复性后超滤得到纯化的HAdV55 Hexon蛋白;筛选得到6株可分泌HAdV55 Hexon mAb的杂交瘤细胞株,抗体亚类分析显示2株为IgG2a亚型,4株为IgG2b;获得与HAdV3 Hexon、HAdV4 Hexon、HAdV7 Hexon、HAdV16 Hexon、HAdV21 Hexon无交叉反应性的2株高效价的特异性HAdV55 Hexon抗体。结论获得的特异性小鼠抗HAdV55 Hexon的mAb为建立其抗原检测方法提供了实验基础。Objective To prepare specific mouse monoclonal antibody(mAb)against human adenovirus type 55 Hexon protein(HAdV55 Hexon).Methods The Hexon genes of HAdV55,3,4,7,16 and 21 were chemically synthesized as templates for PCR amplification.The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3,4,7,16,21 and 55 Hexon were constructed respectively.The pET28a-HAdV55 Hexon plasmid was transformed into E.coli competent cell BL21(DE3)and was induced by IPTG.After the purified inclusion body was denatured and renatured,Hexon55 protein was purified by tangential flow filtration system.pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping,and HAdV55 Hexon protein was used to booster immunization.The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined.The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay(IFA)using BHK cells transfected with pCAGGS-HAdV55 Hexon.Both clones with high titer were selected,and the cross-reactivity of pCAGGS-HAdV3,4,7,16,21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA.Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon,3,4,7,16 and 21 expression plasmids were successfully constructed.BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG.The HAdV55 Hexon protein was mainly expressed in the form of inclusion body.After denaturation and renaturation,the purified HAdV55 Hexon protein was obtained by ultrafiltration.Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained.The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b.Two specific HAdV55 Hexon antibodies with high titer were obtained,and there was no cross-reactivity with HAdV3,4,7,16,21 Hexon.Conclusion The specific micemAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

关 键 词：人腺病毒55型(HAdV55) 六邻体蛋白(Hexon) 单克隆抗体(mAb) 

分 类 号：Q813.2[生物学—生物工程] R392.11[医药卫生—免疫学] R392.33[医药卫生—基础医学]