DNA的G四链体抗体的制备和鉴定  

Preparation and identification of DNA G-quadruplex antibody

作　　者：张毅强[1,2] 兰晓瑜 王爽[1] 李美宁[1] 刘志荣[1] 申金雁[1] 师帅帅[3] 张悦红[1] 解军[1] 程牛亮[1] 

机构地区：[1]山西医科大学生物化学与分子生物学教研室,山西太原030001 [2]长治医学院生化教研室,山西长治046000 [3]长治医学院附属和济医院肾内科,山西长治046011

出　　处：《细胞与分子免疫学杂志》2015年第7期977-981,共5页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的构建DNA的G四链体抗体的原核表达质粒，利用BL21（DE3）菌表达此抗体，并进行纯化及鉴定。方法化学合成DNA的G四链体抗体的基因BC,4，插入以pET-26b（＋）为骨架构建的载体pSANGl0中，构建DNA的G四链体抗体表达载体pSANGl0-BG4。以大肠杆菌B121（DE3）为宿主菌进行该抗体的自诱导表达，渗透压裂解法收集此抗体，并经His亲和层析纯化，用SDS-PAGE及Western blot法鉴定此抗体，并在SW480结肠癌细胞中验证此抗体功能。结果DNA的G四链体抗体表达载体经双酶切及基因测序鉴定构建成功。该抗体相对分子质量（M1）为30000～37000，以可溶性的形式表达于BL21菌细胞间质，表达产物与目的蛋白大小一致。结论成功制备了DNA的G四链体抗体。Objective To construct a prokaryotic expression plasmid of DNA G-quadruplex antibody, express it in E, coil BL21 （DE3） bacterial expression system, purify and identify the antibody. Methods Chemically synthesized BG4 gene of DNA G-quadruplex antibodies was inserted into pSANGIO plasmid to construct DNA G-quadruplex antibody expression vector pSANG10-BG4. BL21 （DE3） as the host strain was utilized for self-induced expression of the protein. Osmotic lysis method was used for collecting this protein. Thereafter, the protein was purified by histidine tag affinity chromatography and identified by SDS-PAGE and Western blotting. The function of this protein was verified in SW480 colon cancer cells. Results Double enzyme digestion and gene sequencing confirmed that DNA G-quadruplex antibody expression vector was successfully constructed. The relative molecular mass （Mr） of this protein was 30 000 to 37 000. The protein in a soluble form was expressed in the periplasm of BL21. The protein was of the same size as expected. Conclusion The DNA G-quadruplex antibody has been successfully prepared,

关 键 词：G四链体 原核表达载体 组氨酸标签 

分 类 号：R392.11[医药卫生—免疫学] R392.33[医药卫生—基础医学]