BCR/ABL癌蛋白片段的表达、纯化及其抗血清的制备  

Expression and purification of BCR/ABL fusion protein and the production of its antiserum

作　　者：梁英民[1] 蒋姗姗[1] 孙强[2] 吴绒丽[1] 陈萍[2] 李荣[2] 周鹏[2] 王键[2] 韩骅[2] 

机构地区：[1]第四军医大学唐都医院血液科,陕西西安710038 [2]第四军医大学基础部遗传与发育生物学教研室

出　　处：《第四军医大学学报》2001年第14期1315-1317,共3页Journal of the Fourth Military Medical University

摘　　要：目的　克隆并表达 bcr/ abl融合基因片段 ,并制备其抗血清 .方法　 PCR扩增包含蛋白融合点的 bcr/ abl癌基因片段 ,序列测定后克隆入带有 His标签的原核表达载体 p ET-16 b,转化宿主菌 BL2 1,IPTG诱导表达 ,SDS- PAGE分析表达结果 ,Ni- NTA树脂亲和纯化 ,皮下多点注射免疫小鼠 ,EL ISA确认抗血清的滴度 .结果　 PCR扩增得到大小约 5 2 3bp的目的片段 ,序列测定表明与发表序列完全一致 ;得到了一种新的癌基因 bcr/ abl片段的原核表达载体 p ET- 16 b- bcr/abl,在 BL 2 1中表达可见于 Mr 2 1× 10 3处有一蛋白新生带 ,表达的 BCR/ ABL蛋白免疫小鼠后 ,EL ESA确认抗血清的滴度 >1∶ 2 0 0 0 .结论　成功的克隆、表达了 bcr/ abl融合基因片段 ,并制备了相应的抗血清 .AIM To clone and to express bcr/abl fusion gene fragment and to produce immune serum against the expressed fusion protein. METHODS PCR amplified bcr/abl gene fragment was inserted into plasmid pET 16b with His tag to form a new plasmid pET 16 bcr/abl (pEb). The BCR/ABL fusion protein was expressed in E.coli BL21 transformed with pEb and induced with IPTG. The expressed protein was purified by affinity resin Ni NTA and analyzed by SDS PAGE. In order to produce anti serum, Mice were immunized by subcutaneously multi site injection with BCR/ABL protein. The anti serum was assayed by ELISA. RESULTS Molecular weight of the PCR aimplified bcr/abl gene fragment which was consistent with the published sequence was about 523 bp. Plasmid pEb was constructed. The molecular size of BCR/ABL protein expression was about Mr 21×10 3. The titer of the BCR/ABL protein anti serum was more than 1∶2000. CONCLUSION Fragment of bcr/abl fusion gene is successfully cloned and expressed. The antiserum of BCR/ABL protein fragment has been produced.

关 键 词：BCR/ABL 基因表达 融合蛋白 抗血清 慢性粒细胞白血病 

分 类 号：R733.72[医药卫生—肿瘤] R73－36[医药卫生—临床医学]