人精胺/精眯N^1-乙酰基转移酶单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody against Human Spermine/Spermidine N^1-acetyltransferase

目的制备鼠抗人精胺/精眯N1-乙酰基转移酶(SSAT)单克隆抗体,分析该抗体的效价、抗原特异性及其在Western blot和免疫组织化学等分析技术中的可用性。方法在BL21(DE3)中原核表达带6×His标签的SSAT重组蛋白,并用Ni-NTA树脂亲和层析纯化。用SSAT重组蛋白免疫Balb/C小鼠,通过杂交瘤技术制备抗SSAT单克隆抗体,所得抗体的特异性及效价用ELISA、Western blot及免疫组织化学技术检测。结果成功建立了稳定分泌鼠抗人SSAT的单克隆抗体杂交瘤细胞株,该单克隆抗体可用于ELISA、Western blot、免疫组织化学、免疫荧光等分析技术。结论成功制备SSAT单克隆抗体,为SSAT相关的分子和病理研究奠定了基础。

Objective To prepare anti-human spermidine/spermine N1-acetyltransferase（hSSAT） monoclonal antibody and to analyze its titer,antigen specificity and usability in Western blot and immunohistochemical analysis.Methods SSAT recombinant protein with 6×His tag was expressed in BL21（DE3） and purified by Ni-NTA resin.The purified recombinant SSAT was used to immunize Balb/C mouse and the anti-SSAT monoclonal antibody was acquired by hybridoma technique.The titer and antigen specificity of this antibody were identified by ELISA,Western blot,immunohistochemical analysis,and immunofluorescence.Results A hybridoma cell line,which could stably secret anti-SSAT mAb,was successfully obtained.The mAb can be used in ELISA,Western blot,and immunohistochemical assay for SSAT.Conclusion The mouse against human SSAT monoelonal antibody with high titer and antigen specificity was successfully obtained,which laid a foundation for SSAT-related molecule biological and pathological researches.