2型猪链球菌天冬氨酸激酶的原核表达及抗原性检测

发布时间：2018-05-02 11:27

本文选题：型猪链球菌 + 原核表达　；参考：《中国病原生物学杂志》2017年04期

【摘要】：目的原核表达2型猪链球菌(Streptococcus suis 2)天冬氨酸激酶(aspartokinase,AK)并检测其免疫原性。方法对S.suis 2中国强毒株05ZYH33的AK编码基因05SSU1811进行生物信息学分析并设计引物,通过PCR从05ZYH33基因组中扩增目的片段。将目的基因克隆入原核表达载体pET28a,转化E.coli BL21(DE3)后采用IPTG诱导表达目的蛋白,用His亲和层析柱纯化重组蛋白,并进行SDS-PAGE和Western blot鉴定。用纯化的重组AK免疫BALB/c小鼠4次,采血,分离血清,采用间接ELISA检测抗体效价,并进行Western blot分析。结果构建的重组质粒在宿主菌中高效表达目的蛋白,His亲和层析柱纯化的重组蛋白,能与感染05ZYH33全菌的猪恢复期血清反应。ELISA检测重组AK免疫鼠血清抗体效价为1∶102 400,Western blot显示该抗血清能与重组蛋白反应。结论成功制备重组AK蛋白,该蛋白具有抗原性,为进一步研究AK在S.suis 2致病过程中的作用奠定了基础。
[Abstract]:Objective to express and detect the immunogenicity of Streptococcus suis 2 (Streptococcus suis 2). Methods the AK encoding gene 05SSU1811 of S.suis 2 Chinese virulent strain 05ZYH33 was analyzed by bioinformatics and primers were designed. The target fragment was amplified from the 05ZYH33 genome by PCR. The target gene was cloned into prokaryotic expression vector pET28a and transformed into E.coli BL21DE3. The target protein was induced by IPTG. The recombinant protein was purified by His affinity chromatography and identified by SDS-PAGE and Western blot. BALB/c mice were immunized with purified recombinant AK for 4 times, blood samples were collected, serum was isolated, antibody titers were detected by indirect ELISA, and Western blot analysis was performed. Results the recombinant plasmid was highly expressed in the host strain and purified by his affinity chromatography. The antibody titer of recombinant AK immunized mice was 1: 102 400 g / blot, which showed that the antiserum could react with the recombinant protein. Conclusion Recombinant AK protein was successfully prepared, which laid a foundation for further study on the role of AK in the pathogenesis of S.suis 2.
【作者单位】：温州医科大学检验医学院生命科学学院;南京军区军事医学研究所;
【基金】：国家自然科学基金项目(No.81571965,81172794,81071317,81471920) 江苏省自然科学基金项目(No.BK20151091) 江苏省333工程科研资助项目(No.BRA2014363)
【分类号】：S852.611

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