文档介绍：TDG蛋白的表达纯化及多克隆抗体的制备
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作者:李石营,兰燕,唐波,华子春
【摘要】目的:表达纯化胸腺嘧啶糖苷酶(TDG)蛋白并制备TDG多克隆抗体。方法:PCR扩增小鼠TDG(mTDG)的基因片段并插入pET28a(+)原核表达载体,表达并纯化HismTDG融合蛋白。用纯化的HismTDG蛋白免疫兔子产生抗血清,进一步亲和纯化抗血清制备抗TDG的多克隆抗体,并检测分析制备的抗体在Western blotting和免疫荧光分析中的应用。结果:在低温(20 ℃)和低IPTG浓度( mmol·L-1)时,HismTDG融合蛋白大部分在可溶上清部分,用亲和层析法分离纯化了HismTDG蛋白,并用纯化的蛋白制备了兔抗TDG抗体,结果显示制备的抗体能够特异性地在免疫分析中使用。结论:本研究纯化了条带单一的并且具有生物学活性的TDG融合蛋白,制备了具有良好特异性的兔抗TDG抗体,为进一步研究TDG的性质、结构和功能奠定了基础。
【关键词】胸腺嘧啶糖苷酶; 原核表达; 纯化; 多克隆抗体
[Abstract] Objective: To express and purify TDG protein, and to prepare the rabbit antibody against thymine DNA glycosylase(TDG) protein. Methods: Mouse TDG(mTDG) was amplified by PCR and then was inserted into the fusion expression vector pET28a(+
). After being expressed in E. coli BL21, the HismTDG protein was purified and used to immunize antibody was obtained through affinity chromatography column,and its applications were evaluated through Western blotting and immunofluorescence : HismTDG fusion protein was almost all soluble at low temperature(20 ℃) and low IPTG condition( mmol·L-1). The fusion protein was purified and used to immunize rabbits, the antibody against TDG was obtained. The further results showed that the antibody had a good specificity in immunoassays. Conclusion: We have purified mTDG fusion protein with an obviously homogeneous band and high biology activity and prepared the rabbit antibody against TDG essfully, which lays the foundation for further study on its character, structure and function.
[Key words] thymine DNA glycosylase; prokaryotic expression; purification; polyclonal antibody
DNA***化联系着基因沉默,是重要的基因转录调控方式和常见的DNA复制后修饰形式,在生长调控和分化中扮演了重要作用[1]。然而,胞嘧啶***化也是基因组不稳定的一个根源[2],因为DNA脱***化是通过脱氨基途径先形成G
∶T错配,然后修复G ∶T错配进行[3]。***化胞嘧啶自发水解脱氨基形成的错配是基因组内源最常见的突变原因之一[4-5],而基因组的突变往往导致各种癌症等疾病[6-7]。幸运的是,所有生物都进化出了修复G ∶T错配的酶,胸腺嘧啶糖苷酶(TDG)是一个修复G ∶T错配的重要蛋白酶[8]。人源TDG蛋白包含410个氨基酸,小鼠的含421个氨基酸,然而,TDG蛋白在SDSPAGE中的条带位置大约在60 kD[9]。TDG不仅和