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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 polyclonal antibody against cell sorting protein 17 (SNX17) of cell sorting protein family, which lays the foundation for further study of SNX17. Methods A 270 amino acid cDNA fragment encoding human SNX17C was amplified by PCR. The recombinant plasmid pGEX-4T-1 and pMal-C2x were transformed into E. coli BL-21 (DE3) Thiogalactoside (IPTG) induced the expression of GST-SNX17C and MBP-SNX17C fusion proteins, respectively. New Zealand white rabbits were immunized with GST-SNX17C fusion protein purified three times on day 1, day 28 and day 42. MBP-SNX17C fusion protein was purified by affinity chromatography and molecular sieve. The titer of polyclonal antibody in serum was detected by enzyme-linked immunosorbent assay (ELISA). Total serum was collected on day 56, serum was precipitated and IgG was purified. The efficiency and specificity of polyclonal antibody against SNX17 rabbit were detected by Western blotting. The expression of SNX17 in 30 organs of mice was detected by Western blotting. Results The expression plasmids of pGEX-4T-1 / SNX17C and pMal-C2x / SNX17C were successfully constructed, and the fusion protein GST-SNX17C and MBP-SNX17C were successfully induced and purified. The purified IgG could recognize COS-7 cells both in vitro and in vivo Derived from GFP-SNX17 and co-located with the GFP antibody-recognizing band. SNX17 was detected by SNX17C polyclonal antibody expression in mouse uterus, ovary and mammary gland but was highly expressed in testis, prostate and vas deferens low. Conclusion A specific and effective rabbit anti-human SNX17C polyclonal antibody was successfully prepared. This antibody can be used in western blot analysis to determine the SNX17 expression profile in mouse organs.