论文部分内容阅读
目的利用原核表达系统制备2型猪链球菌细胞分裂起始因子DivIVA重组蛋白并进行纯化,为研究其在调控细胞分裂中的作用奠定基础。方法以2型猪链球菌05ZYH33全基因组为模板,PCR扩增divIVA基因片段,经BamHⅠ和XholⅠ双酶切后将目的片段连接至表达载体pET28a,构建重组表达质粒pET28a-divIVA,经测序鉴定后将重组质粒转化进入表达菌E.coli BL21,以终浓度为0.1mmol/L的IPTG于37℃培养4h,诱导DivIVA蛋白表达。用Ni离子亲和层析柱分离纯化目的蛋白,SDS-PAGE和Western blot进行检测和验证。结果成功构建重组表达质粒pET28a-divIVA,并在E.coli BL21中表达主要以包涵体形式存在的DivIVA蛋白。包涵体蛋白经8mol/L脲变性溶解后经Ni离子亲和层析柱纯化,获得的目的蛋白经SDS-PAGE检测分子质量为36ku,与预期大小相符;经Western blot检测,该蛋白能被相应抗体特异性识别。结论在E.coli BL21中成功表达并纯化了DivIVA蛋白,为研究该蛋白在2型猪链球菌的细胞分裂机制奠定了基础。
Objective To prepare and purify the DivIVA recombinant protein of Streptococcus suis of type 2 using prokaryotic expression system and lay the foundation for the study of its role in the regulation of cell division. Methods The full-length genomic DNA of Streptococcus suis type 2 was used as template to amplify the divIVA gene fragment. After digested with BamHⅠ and XholⅠ, the fragment was ligated into expression vector pET28a to construct the recombinant plasmid pET28a-divIVA. After sequencing, The recombinant plasmids were transformed into E. coli BL21 and induced with IPTG at a final concentration of 0.1mmol / L for 4h at 37 ℃ to induce DivIVA protein expression. Purification of the target protein by Ni ion affinity chromatography, SDS-PAGE and Western blot detection and verification. Results The recombinant plasmid pET28a-divIVA was successfully constructed and expressed in E. coli BL21 as a DivIVA protein mainly in the form of inclusion bodies. The inclusion body proteins were purified by Ni ion affinity chromatography after being denatured by 8 mol / L urea. The molecular weight of the target protein was 36 kuu by SDS-PAGE, which was in accordance with the expected size. Western blot analysis showed that the protein was correspondingly Antibody specific recognition. Conclusion The DivIVA protein was successfully expressed and purified in E. coli BL21, which laid the foundation for the study of the cell division mechanism of Streptococcus suis type 2.