摘要
目的制备抗RecQ解旋酶单克隆抗体(m Ab),鉴定其生物学特性并开展其对肿瘤细胞中RecQ解旋酶的检测。方法以纯化鉴定后的重组大肠杆菌RecQ解旋酶免疫BALB/c小鼠,采用杂交瘤技术制备抗RecQ解旋酶m Ab。用秋水仙素阻断法对杂交瘤细胞进行染色体核型分析,用间接ELISA检测m Ab的免疫球蛋白(Ig)亚型与效价,用Western blot法和荧光偏振技术鉴定m Ab的生物学特性,使用间接免疫荧光技术检测m Ab与MDA-MB-231乳腺癌细胞中BLM、RecQ4和RecQ5的作用。使用免疫组织化学染色法分析该m Ab与K562肿瘤细胞及其干细胞中RecQ解旋酶的结合。结果获得能稳定分泌抗RecQ解旋酶m Ab的杂交瘤细胞株6H5,染色体数目在94~104,抗体亚型为Ig G1型,腹水效价为1×10-7。该m Ab能特异性结合大肠杆菌RecQ解旋酶,抑制其与DNA的结合,并能识别BLM、RecQ4和RecQ5解旋酶,还可敏锐检测K562肿瘤细胞与肿瘤干细胞中RecQ解旋酶的表达。结论成功制备获得效价高、特异性强的抗RecQ解旋酶m Ab。
Objective To prepare monoclonal antibodies （mAb） against RecQ helicase, characterize their biological properties, and then investigate their activity against RecQ helicase in tumor cells. Methods BALB/c mice were immunized with purified recombinant RecQ helicase from E. coil Hybddoma technique was used to generate the mAb against RecQ helicase. The chromosome karyotype analysis of the hybridoma cells was performed by colchicine blocking. The Ig subtype and titer of mAb in ascitic fluid were determined by ELISA. The biological properties of mAb were detected via Western blotting and fluorescence polarization. Immunofluorescence was used to detect the interactions of mAb with BLM, RecQ4 and RecQ5 helicases in human breast cancer MDA-MB-231 cells. Immunohistochemistry was applied to detect the binding of mAb and RecQ helicase in KA62 tumor cells and the corresponding stem cells. Results One stable hybridoma cell line expressing anti-RecQ mAb was obtained and named 6H5. The number of chromosomes was from 94 to 104, and Ig subtype was IgGl. The titer of 6H5 in ascitic fluid was 1× l0^-7. The mAb could specifically recognize E. coil RecQ helicase and thus inhibit its DNA binding activity. Besides, the mAb could also recognize BLM, RecQ4 and RecQ5 in MDA-MB-231 cells, and sensitively detect the expression of the RecQ helicase in 1〈562 tumor cells and the corresponding stem cells. Conclusion The mAb against RecQ helicase was successfully prepared with a high titer and specificity.
作者
张荣红
贾铁文
廉芳
刘杰麟
周孟
ZHANG Ronghong;JIA Tiewen;LIAN Fang;LIU Jielin;ZHOU Meng(Tissue Engineering and Stem Cell Research Center;Department of Immunology;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM, Ministry of Education ＆ State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550004, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2018年第2期169-174,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81360349)
贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2006)57]
贵州省国际科技合作计划[黔科合外G字(2011)7012]
关键词
RecQ解旋酶
单克隆抗体
制备
鉴定
应用
RecQ helicase
monoclonal antibody
preparation
identification
application
