针对断裂点集簇区-Abelson鼠白血病病毒癌基因(BCR-ABL)b3a2位点的兔多克隆抗体制备及鉴定  

Preparation and identification of rabbit polyclonal antibody against BCR-ABL b3a2 fusion protein

作　　者：李香凝 屈佳肴 蒋静 王丽 罗迪贤[1,2,3] 李佳 段丽丽[1,2] 贺荣章 胡政[1,2,3] LI Xiangning;QU Jiayao;JIANG Jing;WANG Li;LUO Dixian;LI Jia;DUAN Lili;HE Rongzhang;HU Zheng(Translational Medicine Institute,Chenzhou Hospital Affiliated to University of South China,Chenzhou 423000;National&Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology,Chenzhou Municipal First People’s Hospital,Chenzhou 423000;First School of Clinical Medicine,Southern Medical University,Guangzhou 510000;Department of Laboratory Medicine,Shenzhen Third People’s Hospital,Shenzhen 518000,China)

机构地区：[1]南华大学附属郴州医院南华大学转化医学研究所,湖南郴州423000 [2]郴州市第一人民医院高通量分子诊断技术国家地方联合工程实验室,湖南郴州423000 [3]南方医科大学第一临床学院,广东广州510000 [4]深圳市第三人民医院检验科,广东深圳518000

出　　处：《细胞与分子免疫学杂志》2021年第8期746-751,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：湖南省自然科学基金(2017JJ2004);第九批博士后科学基金特别资助项目(2016T90765);湖南省卫计委课题(C20180166)。

摘　　要：目的制备兔抗断裂点集簇区-Abelson鼠白血病病毒癌基因(BCR-ABL)b3a2位点的多克隆抗体并鉴定其特异性。方法针对BCR-ABL融合蛋白b3a2型的特异性抗原序列,设计并合成纯度高于90%的多肽。一方面通过透孔螺血蓝蛋白(KLH)偶联融合多肽用于免疫新西兰兔,多次加强免疫得到抗血清;另一方面通过仅偶联单独BCR或者ABL肽段对抗血清进行二次纯化,吸附并去除未结合融合表位的抗血清以达到进一步纯化抗体的效果。最终得到的抗血清用于ELISA及Western blot法检测。结果免疫前兔血清背景均低,免疫后抗血清效价达到1∶32000,满足实验要求。抗原亲和纯化所得抗体纯度及效价高,Western blot法检测表明抗血清能特异性识别BCR-ABL融合基因的b3a2位点,特异性好。结论制备了针对BCR-ABL基因b3a2位点的特异性兔源多克隆抗体。Objective To prepare and identify rabbit anti-breakpoint cluster region-Abelson leukemia virus oncogene(BCR-ABL) b3a2 subtype polyclonal antibody. Methods A peptide containing the fusion sequence of the b3a2 subtype BCR-ABL fusion protein was designed and synthesized with the purity higher than 90%. The fusion polypeptide was coupled to Keyhole Limpet hemocyanin(KLH) and used to immune New Zealand rabbits. Antiserum was purified after multiple immunizations, in addition to using the b3a2 subtype fusion polypeptide for affinity purification. Peptides harboring only BCR or c-ABL amino acid sequences were also synthesized and used to purify the antibody in the secondary purification. The antibody that only bound to part of the epitope was absorbed and removed. ELISA and Western blotting were performed to determine the antibody titer and specificity. Results The rabbit serum background was low before immunization. The titer of the polyclonal antibody reached 1∶32 000 after immunization, which met the experimental requirements. Western blotting showed that the antibody could specifically recognize the b3a2 subtype fusion protein of BCR-ABL. Conclusion The experiment has prepared the specific rabbit polyclonal antibody against BCR-ABL b3a2 subtype.

关 键 词：断裂点集簇区-Abelson鼠白血病病毒癌基因(BCR-ABL) b3a2 多克隆抗体 亲和纯化 

分 类 号：R392.11[医药卫生—免疫学] R733.7[医药卫生—基础医学]