CAJ | 학술논문

目的：构建重组人C反应蛋白（rhCRP）甲醇酵母分泌表达菌株，表达、纯化rhCRP并鉴定其免疫反应性。方法将人工设计合成的rhCRP基因克隆到甲醇酵母表达载体pPICZαA上，经SacⅠ线性化得到重组质粒pPICZαA/rhCRP后转化至甲醇酵母X-33中，利用甲醇诱导进行分泌表达，并用组氨酸标签进行亲和层析纯化 rhCRP。采用SDS-PAGE、Western blotting检测表达、纯化的目的蛋白，通过间接酶联免疫吸附试验（ELISA）鉴定其免疫反应性和稳定性。结果成功构建人CRP的重组甲醇酵母表达载体pPICZαA/rhCRP ，重组酵母菌株成功诱导分泌表达23×103的 rhCRP ；一步纯化即得纯度为90．42％的目的蛋白，间接ELISA法检测表明，纯化产物rhCRP具有特异的免疫反应性和一定的稳定性。结论利用甲醇酵母表达系统，成功获得了较高纯度的具有免疫反应性的rhCRP ，为下一步制备抗人CRP抗体及自主研发人CRP检测试剂提供重要的实验基础。
목적：구건중조인C반응단백（rhCRP）갑순효모분비표체균주，표체、순화rhCRP병감정기면역반응성。방법장인공설계합성적rhCRP기인극륭도갑순효모표체재체pPICZαA상，경SacⅠ선성화득도중조질립pPICZαA/rhCRP후전화지갑순효모X-33중，이용갑순유도진행분비표체，병용조안산표첨진행친화층석순화 rhCRP。채용SDS-PAGE、Western blotting검측표체、순화적목적단백，통과간접매련면역흡부시험（ELISA）감정기면역반응성화은정성。결과성공구건인CRP적중조갑순효모표체재체pPICZαA/rhCRP ，중조효모균주성공유도분비표체23×103적 rhCRP ；일보순화즉득순도위90．42％적목적단백，간접ELISA법검측표명，순화산물rhCRP구유특이적면역반응성화일정적은정성。결론이용갑순효모표체계통，성공획득료교고순도적구유면역반응성적rhCRP ，위하일보제비항인CRP항체급자주연발인CRP검측시제제공중요적실험기출。
Objective To construct the secretory expression vector of recombinant human C-reactive protein(rhCRP) for its se-cretory expression in Pichia pastoris ,rhCRP was expressed as a secretory protein and purified ,and the immunity reactivity of the purified protein was identified .Methods The DNA fragment of rhCRP which was designed and synthesized was cloned into pPICZαA vector .Recombinant plasmid pPICZαA/rhCRP was linearized by SacⅠand transformed into Pichia pastoris X-33 by elec-trotransformation .The rhCRP was secreted into the medium under the methanol induction .RhCRP was purified by Histamine affin-ity chromatography .The purified rhCRP was identified by SDS-PAGE and Western blotting ,and its immunity reactivity and stabili-ty was identified by indirect ELISA .Results The pPICZαA/rhCRP expression vector was successfully constructed .The rhCRP of 23 × 103 was inducted and successfully expressed as a secretory protein by the recombinant Pichia pastoris strains .The rhCRP was purified by one step up to 90 .42% purity ,and it was showed good immunity and stability by indirect ELISA .Conclusion The rh-CRP with higher purity and immunoreactivity was successfully obtained by using the Pichia pastoris expression system ,which pro-vided an important experimental basis for producing anti-human CRP antibodies and developing testing CRP reagent .