细胞分选蛋白17多克隆抗体的制备和鉴定及在小鼠体内的表达  

Preparation and characterization of sorting nexin 17(SNX17)polyclonal antibody and detecting its expression in mice

作　　者：程媛[1] 牛苗苗[1] 马博[1] 唐岩[1] 李枫[1] 战军[1] 

机构地区：[1]北京大学基础医学院组织学与胚胎学教研室,北京100191

出　　处：《解剖学报》2012年第6期756-761,共6页Acta Anatomica Sinica

基　　金：教育部博士点基金资助项目(20100001110046);国家自然科学基金资助项目(30830048);北京市与中央在京高校共建项目

摘　　要：目的制备细胞分选蛋白家族成员细胞分选蛋白17(SNX17)的多克隆抗体,为深入研究SNX17奠定基础。方法 PCR扩增编码人SNX17C端270个氨基酸的cDNA片段,DNA重组入原核表达载体pGEX-4T-1和pMal-C2x,转化大肠杆菌BL-21(DE3)菌株,异丙基-β-D-硫代半乳糖苷(IPTG)诱导分别表达GST-SNX17C端和MBP-SNX17C端融合蛋白。采用电泳纯化的GST-SNX17C端融合蛋白于第1天,第28天,第42天3次免疫新西兰白兔。采用亲和层析和分子筛纯化的MBP-SNX17C端融合蛋白,酶联免疫吸附(ELISA)法检测血清中多克隆抗体的效价,于第56天取兔全血,析出血清,纯化IgG。免疫印迹法检测SNX17兔多克隆抗体的有效性和特异性,免疫印迹法检测小鼠30种器官中SNX17的表达。结果成功构建了pGEX-4T-1/SNX17C端和pMal-C2x/SNX17C端的表达质粒,成功诱导表达纯化了GST-SNX17C端和MBP-SNX17C端融合蛋白,纯化后的IgG可识别COS-7细胞中外源转入的GFP-SNX17,并与GFP抗体识别的条带在同一位置上,通过SNX17C端多克隆抗体检测了SNX17在小鼠子宫、卵巢和乳腺中表达高而在睾丸、前列腺和输精管中表达低。结论成功制备特异而有效的兔抗人SNX17C端多克隆抗体,此抗体可用于免疫印迹法分析,初步确定了SNX17在小鼠器官中的表达谱。Objective To prepare a polyclonal antibody against SNX17, one of cell sorting nexin protein family members, and to provide a basis for future functional studies of the protein. Methods The DNA sequence encoding C- terminal 270 amino acid of Human SNX17 was obtained by PCR from eDNA library of human blood cells and was cloned into PGEM-4H plasmid expressing GST and pMal-C2x plasmid expressing MBP. The GST-SNX17-C-terminal fusion protein and MBP-SNX17-C-terminal fusion protein were expressed by E. coli BL21 （DE3） after IPTG induction. The New Zealand rabbit was immunized with the purified electrophoresis GST-SNX17-C-terminal fusion protein at the 1st day, the 28th day and the 42ed day for three times. We used the ELISA assay to determine the titer of the antiserum by MBP- SNX17-C-terminal fusion protein which was purified by Affinity chromatography and Gel filtration chromatography. The rabbit blood was collected at the 56th day and IgG was purified. Effectiveness and specificity of the antiserum were identified by Western blotting. The SNX17 expression in 30 kinds of mouse organs was detected by Western blotting. Results We constructed the pGEX-4T-1/SNX17-C-terminal plasmid and pMal-C2x/SNX17-C-terminal plasmids successfully. The GST-SNX17-C-terminal fusion protein and MBP-SNX17-C-terminal fusion protein were induced and expressed successfully. The purified antibody specifically recognized GFP-SNX17 and the band was in the same position as that recognized by GFP antibody. We demonstrated that SNX17 expressed at a high level in the mouse uterus, ovary and breast, while expressed at a low level in the testis, prostate and vas deferens. Conclusion Rabbit anti-human SNX17-C- terminal polyclonal antibody was prepared successfully. This polyclonal antibody can be used for Western blotting analysis. The expression of the SNX17 in mouse organs was tested initially.

关 键 词：细胞分选蛋白17 蛋白纯化 多克隆抗体 免疫印迹法 小鼠 新西兰大白兔 

分 类 号：R392.11[医药卫生—免疫学]