作　　者： ; ; ; ; ; ; ;

机构地区： 江苏大学基础医学与医学技术学院

出　　处： 《临床检验杂志》 2013年第9期687-690,共4页

摘　　要： 目的筛选抗原性强的烟曲霉二肽基肽酶Ⅴ(dipeptidyl peptidasesⅤ,DPPⅤ)肽段并进行原核细胞表达。方法通过生物信息学分析技术,从DPPⅤ全长序列中选取抗原决定簇密集的片段,采用生物合成和PCR技术,获得其肽段相应的DNA序列,并克隆至测序载体;DNA测序验证后构建重组表达质粒,转染原核表达宿主菌BL21(DE3),IPTG诱导表达后用Talon亲和层析柱纯化;western blot分析产物免疫反应性。结果 DPPⅤ19～144p及DPPⅤ145～447p在大肠埃希菌中均以包涵体形式表达,DPPⅤ19～447p以可溶性蛋白质形式表达。western blot结果表明筛选出的DPPⅤ肽段均可与侵袭性曲霉病(IA)患者血清发生抗原抗体反应。结论成功表达烟曲霉二肽基肽酶不同抗原决定簇分布的3个肽段,且重组片段均具有良好免疫反应性。 Objective To screen the dipeptidyl peptidases V （DPP V） fragment of Aspergillus fumigatus with strong antigenicity, and express it by a prokaryotic system. Methods The antigenicity of DPP V was predicted by the bioinformatics applications, and two re-gions with dense epitopes were selected and synthesized. Then, the two fragments were amplified by PCR, and cloned into the pMD18-T vector for DNA sequencing, respectively. After the sequence verified, it was inserted into prokaryotic expression vector pET28a（ ＋ ） to construct the recombinant plasmid. The plasmid was introduced into E. coli BL21 （ DE3 ） for expressing the recombinant protein with the induction of IPTG. The recombinant protein was purified with Talon metal affinity resins and identified by western blot. Re-sults The recombinant proteins DPPV 19-144p and DPP V 145-447p were expressed as inclusion bodies in E. coli, but the DPP V 19 - 447p as a soluble form. Western blot demonstrated that these recombinant proteins could react with the serum from the invasive as-pergillosis （IA） patients. Conclusion The recombinant proteins DPPV19-144p, DPPV145-447p and DPPV19-447p with different epitopes were successfully prepared, and they all had strong immunoreactivity.

关 键 词： 烟曲霉 二肽基肽酶 肽段 原核表达

领　　域： [生物学]