小鼠Prune蛋白DHH结构域的原核表达及多克隆抗体的制备

Prokaryotic expression of mouse Prune DHH domain and preparation of polyclonal antibody

[目的]表达、纯化小鼠Prune蛋白DHH结构域(m-Prune D),并制备多克隆抗体。[方法]生物信息学方法分析m-Prune D氨基酸序列;PCR扩增目的基因m-Prune D,克隆入原核表达载体p ET28a(+);IPTG诱导目的基因表达,SDS-PAGE和Western Blot鉴定蛋白表达,亲和层析法纯化蛋白;用纯化的重组m-Prune D免疫小鼠制备多克隆抗体;Western Blot检测多克隆抗体特异性。[结果]PCR成功扩增m-Prune D基因,双酶切及测序结果表明成功构建m-Prune D原核表达载体,SDS-PAGE和Western Blot鉴定表明成功表达约25 k Da的重组蛋白。纯化蛋白免疫小鼠后抗体滴度最高可达1∶25 600,所制备的多克隆抗体可特异性识别原核和真核细胞中DHH结构域蛋白。[结论]在E.coli中成功表达小鼠Prune蛋白DHH结构域,制备了多克隆抗体血清,可用于Prune蛋白生物学功能的进一步研究。

[Objective] To express and purify mouse exopolyphosphatase Prune protein DHH domain（ m-Prune D） and to prepare the polyclonal antibody. [Methods]The m-Prune D amino acids sequence was analyzed by bioinformatics methods. m-Prune D gene was amplified by PCR and cloned into the prokaryotic expression vector p ET28 a（ ＋）. The expression of m-Prune D was induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western Blot,and purified by affinity chromatography. The polyclonal antibody was prepared from mice immunized with purified protein,and the specificity of polyclonal antibody was detected by Western Blot. [Results]The m-Prune D gene was successfully amplified by PCR. The restriction endonuclease digestion and sequencing analysis indicated that the prokaryotic expression vector of m-Prune D was successfully constructed. SDS-PAGE and Western Blot showed that the recombinant protein m-Prune D was about 25 k Da. Mice were immunized with the purified recombinant protein and antibody titer was up to 1： 25 600. The prepared polyclonal antibody can specifically recognize DHH domain proteins from prokaryotic and eukaryotic cells. [Conclusion] The DHH domain of mouse Prune protein was successfully expressed in E. coli and the polyclonal antibody prepared from immunized mice,which can be used for further research of Prune protein