人YB-1的高效原核表达及其标准蛋白与抗血清的制备  

Preparations of prokaryotic expression system,standard protein and antiserum of human Y-box binding protein 1

作　　者：李朴[1] 史静[1] 郭变琴[2] 钟梁[1] 梁勤东[1] 涂植光[1] 

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016 [2]重庆市肿瘤医院检验科,重庆400030

出　　处：《激光杂志》2011年第5期67-69,共3页Laser Journal

基　　金：国家自然科学基金资助项目(30872417)

摘　　要：目的:构建YB1-GST表达系统,建立经济高效YB-1蛋白制备方法并制备其多抗。方法:将YB-1编码序列亚克隆至表达载体pGEX-6P-1;转化表达菌并确定可溶性表达最佳条件;采用GST亲和层析与层析柱上PSP酶切融合蛋白获取无标签蛋白的YB-1,经超滤浓缩及Western blot鉴定后,进一步真空冷冻干燥,制备YB-1标准蛋白;采用大剂量YB-1长程免疫方案免疫家兔以制备其抗体。结果:成功构建了表达载体pGEX-YB1;SDS-PAGE结果显示,YB1-GST融合蛋白以可溶性表达为主;Western blot结果证实,表达产物经GST亲和层析、PSP酶切以及真空冷冻干燥后获得了纯度较高的YB-1标准蛋白,将该蛋白免疫家兔获得了较高效价与特异性的抗体。结论:建立了YB-1-GST表达系统与经济高效的YB-1蛋白纯化方法;获得了质量较高的YB-1多抗,为进一步制备YB-1单抗、深入探讨其在肿瘤细胞中的生物学功能以及YB-1定量检测方法的建立奠定了基础。Aim： To construct a GST- expression system of human Y - box binding protein 1 （YB - 1）, prepare the YB - 1 standard protein and its antiserum. Methods ： The code sequence of YB - 1 was subcloned to the expression vector pGEX - 6P - 1. The recombinant vector was transformed into E. coli BL21 to express fusion protein GST- YB1. SDS- PAGE was applied to analyze the expression level and form of fusion protein. Then, YB- 1 standard protein was obtained by the means of GST - affinity chromatography and column - protease - digestion. The rabbit was immunized with YB - 1 protein to prepare the anti - YB1 polyclonal antibody. Results： The recombinant expression vector was constructed. SDS - PAGE and Western blot results showed that the GST - fusion protein was high- level expressed with soluble - form, and YB - 1 standard protein and its antisermn were obtained successfully. Conclusions： An economical, rapid method to prepare YB - 1 standard protein is established, and further obtained the high titer and affinity YB - 1 polyclonal antibody, which lay foundations for preparation of YB - 1 monoclonal antibody and development of YB - 1 quantitative analysis methods.

关 键 词：YB-1 GST亲和层析 柱上酶切 多抗制备 

分 类 号：TN248.1[电子电信—物理电子学]