摘要
单线态氧产生者(mini singlet oxygen generator,miniSOG)是改造于拟南芥向光素2蛋白而获得的小分子荧光黄素蛋白,由106个氨基酸残基组成。miniSOG被广泛用于电镜及荧光显微镜,并被广泛用于发色团辅助的光灭活(Chromophore-assisted light inactivation,CALI)蛋白、DNA或RNA,并结合免疫光敏用于癌症的治疗,以及结合细胞消融用于神经科学、免疫生物学等。但是目前国内并无商品化的miniSOG抗体,为了辅助课题研究,制备抗miniSOG蛋白的单克隆抗体,本研究根据miniSOG基因的ORF区序列构建miniSOG-GST重组蛋白的原核表达载体PGEX-4T-1-miniSOG,然后将该重组质粒转化于BL21感受态大肠杆菌中,异丙基硫代半乳糖苷(Isopropylβ-DThiogalactoside,IPTG)诱导融合蛋白的表达,并经亲和层析方法进行纯化。将纯化后的重组蛋白免疫BALB/c小鼠,利用杂交瘤技术制备单克隆抗体。筛选获得稳定分泌抗miniSOG的单克隆抗体细胞株,最终选取性能最优的4F9细胞株进行小鼠腹水制备并纯化。单抗4F9亚型为IgG1/Kappa,腹水滴度达到1∶200 000,纯化抗体灵敏度达到5 ng/mL。并通过Western Blot、免疫荧光及免疫组化方法进行特异性鉴定,该单克隆抗体能特异性识别转染细胞和转基因小鼠脑组织中的miniSOG蛋白。本研究在国内首次制备了miniSOG单克隆抗体,将丰富其在其它方面的应用。
MiniSOG(mini singlet oxygen generator)is a small-molecule fluorescein protein modified from the phototropin-2 protein of Arabidopsis thaliana. It has 106 amino acids,is a singlet-oxygen producer,and is used widely in electron microscopy, fluorescence microscopy, and chromophore-assisted photoinactivation of proteins, DNA or RNA. MiniSOG is used in the cancer treatment, cell ablation, neuroscience and immunobiology. However,a commercial miniSOG antibody has not been developed. We prepared a monoclonal antibody against miniSOG protein and applied it. Prokaryotic expression of miniSOG-GST recombinant protein containing pGEX-4 T-1-miniSOG was constructed according to the ORF-region sequence of miniSOG. Then,the recombinant plasmid was transformed into BL21. IPTG-induced fusion protein underwent affinity purification. The purified recombinant protein was used to immunize BALB/c mice,and hybridoma technology was employed to prepare monoclonal antibodies. Cell lines with stable secretion of monoclonal antibody against miniSOG were screened, and the best cell line(4 F9) was selected for preparation and purification of monoclonal antibodies in mice ascites. The subtype of 4 F9 cells was IgG1/Kappa, the ascites titer reached 1∶ 200,000, and the sensitivity of purified antibodies reached 0.005 μg/mL. Western Blot,immunofluorescence and immunohistochemistry revealed that the monoclonal antibody could identify miniSOG protein specifically in transfected cells and transgenic mouse-brain tissues. We firstly prepared miniSOG monoclonal antibody in China,which will enrich its application in other medical fields.
作者
高利萍
武月章
杨雪花
陈冬冬
肖康
董小平
石琦
GAO Liping;WU Yuezhang;YANG Xuehua;CHEN Dongdong;XIAO Kang;DONG Xiaoping;SHI Qi(State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2019年第6期880-887,共8页
Chinese Journal of Virology
基金
国家科技重大专项(项目号：2018ZX10711001),题目：病毒感染高通量快速检测与应急筛检技术研究
国家自然科学基金(项目号：81572048),题目：线粒体损伤致朊病毒病分子机制的研究
国家自然科学基金(项目号：81630062),题目：朊病毒持续感染机制的研究
传染病预防控制国家重点实验室“创新青年基金”(项目号：2015SKLID503),题目：朊病毒病蛋白组学的研究
传染病预防控制国家重点实验室“杰青申报资助”课题(项目号：2019SKLID501)：遗传型朊病毒病发病机制的研究~~
