2型猪链球菌FtsZ重组蛋白的制备及其酶活性测定

发布时间：2018-06-04 13:03

本文选题：型猪链球菌 + 丝状温度敏感蛋白　；参考：《中国人兽共患病学报》2017年03期

【摘要】：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。
[Abstract]:Objective to prepare the recombinant protein of Streptococcus suis type 2 filamentous temperature-sensitive protein zFtsZ and to determine the enzyme activity of its hydrolyzed guanosine triphosphate (GTP). Methods the target gene ftsz was amplified by PCR from the 05ZYH33 genome of Streptococcus suis type 2 Chinese virulent strain. The recombinant expression plasmid pET28a-ftsz was constructed and transformed into E.coli BL21DE3. The recombinant protein was purified by IPTG induced SDS-PAGE and purified by affinity chromatography with Ni2 affinity chromatography. After the recombinant protein was immunized with New Zealand rabbits, the polyantiserum was collected and the antibody titer was detected by indirect ELISA method. The GTP enzyme activity of FtsZ recombinant protein was determined by malachite green phosphoric acid assay. Results the recombinant expression plasmid of pET28a-ftsz was successfully constructed, and a large number of purified FtsZ recombinant proteins were purified. The titer of rabbit polyclonal antiserum detected by Elisa was 107,200 tons. The results showed that FtsZ recombinant protein could react with His-Tag monoclonal antibody and rabbit polyclonal antibody serum. GTP was used as the substrate to detect the GTP activity of the recombinant protein. Conclusion the recombinant protein of Streptococcus suis type 2 FtsZ with GTP enzyme activity was successfully prepared and the polyclonal antibody serum with high titer was prepared by using the recombinant protein as antigen, which laid a foundation for further study on the role of FtsZ type 2 in the regulation of cell division.
【作者单位】：中国药科大学中药学院;南京军区军事医学研究所;
【基金】：国家自然科学基金(No.81571965,No.81471920) 江苏省自然科学基金(No.BK20151091) 江苏省333工程科研资助项目(No.BRA2014363)联合资助~~
【分类号】：S852.611

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