2型猪链球菌FtsZ重组蛋白的制备及其酶活性测定

Purification and enzyme activity assay of filamentous temperature-sensitive protein Z in Streptococcus suis serotype 2

目的制备2型猪链球菌丝状温度敏感蛋白(Filamentous temperature-sensitive protein Z,FtsZ)重组蛋白并测定其水解三磷酸鸟苷(GTP)的酶活性。方法从2型猪链球菌中国强毒株05ZYH33基因组中PCR扩增目的基因ftsz,构建重组表达质粒pET28a-ftsz,转化E.coli BL21(DE3)感受态细胞,筛选阳性转化子进行IPTG诱导表达,SDS-PAGE鉴定表达产物,Ni 2+亲和层析柱纯化重组蛋白,Western blot分析。重组蛋白免疫新西兰兔后收集多抗血清,间接ELISA法检测抗体效价,用孔雀绿-磷酸检测法测定FtsZ重组蛋白的GTP酶活性。结果成功构建pET28a-ftsz重组表达质粒;纯化获得大量纯度较高的FtsZ重组蛋白;ELISA检测兔多抗血清效价达1∶13 107 200;Western blot显示FtsZ重组蛋白能够与His-Tag单抗和兔多抗血清发生反应;以GTP为底物检测重组蛋白具有GTP酶活性。结论成功制备了具有GTP酶活性的2型猪链球菌FtsZ重组蛋白并以重组蛋白为抗原制备出高效价的多抗血清,为进一步研究2型猪链球菌FtsZ在细胞分裂调控过程中的作用奠定基础。

We conducted purification of filamentous temperature-sensitive protein Z of Streptococcus suis serotype 2 （S. suis 2） and measured its GTPase activity. The ftsz gene in the genome of the Chinese 05ZYH33 strain of S. suis 2 was success- fully amplified using PCR, and then the ftsz gene was cloned into prokaryotic expression plasmid pET28a, and the recombinant plasmid pET28a-ftsz was transformed into E. coli BL21. After induction by IPTG, the isolated FtsZ protein was analyzed with SDS-PAGE. Then the recombinant protein was purified by Ni^2＋-NTA affinity chromatography. The rabbit serum was harvested after immunization with recombinant FtsZ protein, and was analyzed by indirect ELISA and Western blotting. The GTPase activity of FtsZ was measured with the malachite green method. Results showed that successfully constructed recombinant plasmid pET28a-ftsz and the recombinant protein with high purity was obtained; Western blot result indicated that FtsZ could react with the His-tag antibody and the rabbit serum- the polyelonal antibody titer of the rabbit serum reached 1 ： 13 107 200; FtsZ have GTPase activity. We successfully prepared S. suis 2 recombinant protein FtsZ having GTPase activity and high titer antiserum would be useful for the further study of S. suis 2 cell division mechanism.