S100A10蛋白的表达及其抗体的制备

The expression and antibody preparation of S100A10 protein

目的制备S100A10蛋白及其特异性抗体。方法用PCR扩增编码S100A10的基因序列,将其克隆到原核表达载体pET28a(+)中,并转化至大肠杆菌BL21(DE3)中诱导表达,表达的蛋白产物经镍柱亲和层析纯化后,作为抗原皮内接种免疫日本大耳白兔,制备多克隆抗体,分别以ELISA,Western blot法检测抗体的滴度和抗原特异性。结果成功构建原核表达载体pET28a(+)-(S100A10)2,得到纯化的(S100A10)2蛋白,制备的多抗具有较高的抗体滴度和抗原特异性。结论建立了S100A10原核表达及纯化系统,成功制备了S100A10的多克隆抗体,为S100A10的进一步研究提供了工具。

Objective To prepare S100A10 protein and its specific polyclonal antibody. Methods The full-length gene fragment of S100A10 was amplified by PCR,and then cloned into pET28a（ ＋） prokaryotic expression vector. After transformation,the vector was induced to express the recombinant（ S100A10）2protein by IPTG in E. coli BL21（ DE3）. The recombinant（ S100A10）2was then purified by Ni-NTA resin.（ S100A10）2-specific polyclonal antibody was prepared using the purified recombinant（ S100A10）2protein as antigen to inoculate rabbit intradermally. The title and specificity of the polyclonal antibody were determined by ELISA and Western blotting. Results The study successfully constructed the prokaryotic recombinant expression vector pET28a（ ＋）-（ S100A10）2,and obtained the purified recombinant（ S100A10）2protein and polyclonal antibody with high titer and specificity. Conclusion The prokaryotic expression and purification system for S100A10 has been established and polyclonal antibody of（ S100A10）2been prepared,which provides helpful fools for further researches on S100A10.