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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.将目的基因克隆人原核表达载体pET28a,转化E.coli BL21(DE3)后采用IPTG诱导表达日的蛋白,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.suis2致病过程中的作用奠定了基础.“,”Objectives To determine the immunogenicity of aspartokinase (AK) from virulent strain 05ZYH33 of Streptococcus suis serotype 2 (S.suis 2).Methods A gene encoding AK (05SSU1811) was analyzed bioinformatically and primers were designed.The ORF encoding AK was amplified with PCR using a pair of specific primers and subcloned into a pET 28a expression vector.After the recombinant plasmid was identified with DNA sequencing,it was then transformed into E.coli BL21 (DE3).Protein expression was induced with IPTG,and the recombinant AK protein was harvested using centrifugation and analyzed with SDS-PAGE.The recombinant protein was purified with Ni2+-NTA affinity chromatography and analyzed with Western blotting,and the purified protein was then used to immunize mice four times to prepare polyclonal antibodies against the recombinant AK protein.Blood was then collected and serum was isolated.The titer and specificity of the polyclonal antibodies were determined with ELISA and Western blotting.Results In this study,an AK gene was cloned from the 05ZYH33 strain of S.suis 2 and a recombinant AK protein of about 45 ku was obtained with a pET28a:ak/BL21 prokaryotic expression system.The soluble supernatant was purified with a His affinity chromatography column,and a highly pure recombinant protein was obtained.The recombinant AK protein was detected using ASP as a substrate.The titer of the serum antibody was 1∶102,400 according to ELISA,indicating that antiserum with a high titer was obtained.Western blotting indicated that the antiserum reacted with the recombinant AK protein.Conclusion The purified recombinant AK protein was highly immunogenic.This study has laid the foundation for further research on the role of AK in the pathogenesis of S.suis 2.