肺炎链球菌SpxA蛋白的原核表达、纯化及多克隆抗体的制备

Expression and Purification of Streptococcus Pneumoniae SpxA and Preparation of Its Polyclonal Antibody

目的 构建肺炎链球菌SpxA蛋白的原核表达系统,制备其多克隆抗体.方法 设计引物,利用PCR技术扩增肺炎链球菌D39菌株的spxA基因,并插入表达载体pET-28a（＋）内,测序鉴定.重组质粒转化至大肠埃希菌BL21（DE3）中,以IPTG诱导表达含6个组氨酸标签的SpxA重组蛋白,经Ni-NTA亲和层析柱纯化后,以其为抗原免疫BALB/c小鼠制备多克隆抗体.用ELISA及Western印迹方法分别检测多克隆抗体的效价及特异性.结果 从大肠埃希菌中诱导出高表达的SpxA重组蛋白,纯化后免疫小鼠获得抗血清,ELISA测定其效价可达1：2 560 000以上,Western印迹结果显示其能特异性地作用于肺炎链球菌SpxA.结论 成功构建了pET-28a（＋）-spxA原核表达质粒,获得了高纯度的目的 蛋白和高滴度、高特异性的多克隆抗体.

Objective To construct the prokaryotic expression system of Streptococcus pneumoniae （S. pn） SpxA protein and prepare polyclonal antibody against SpxA protein. Methods The full-length spxA gene of S. pn D39 was amplified by PCR with specific primers, and then inserted into pET28a （ ＋ ） expression vector, followed by sequencing analysis. The recombinant plasmid was transfbrmed into E. coil BL21 （DE3） to express 6 His-tagged SpxA protein under IPTG induc- tion. After purified by Ni-NTA affinity chromatography, the recombinant protein was introduced to BALB/c mice as an antigen to obtain polyclonal antibody. Antibody titers and specificity were determined by ELISA and Western blot respectively. Results The SpxA recombinant protein was highly expressed in E. coli. Antiserum was obtained from immunized mice with purified SpxA protein. ELISA showed the of the purified antibody up to 1 ： 2 560 000. The antibody specificity to S. pn SpxA protein was proved by Western Blotting. Conclusion The prokaryotic expression plasmid pET-28a （ ＋ ） -spxA was successfully constructed. High purity of SpxA protein and its polyclonal antibody with high titer and high specificity were obtained.