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目的:构建组成型表达重组人C-反应蛋白(Recombinant human C-reactive protein,rhCRP)的毕赤酵母工程菌株,表达、纯化rhCRP并鉴定其免疫反应性。方法:将设计合成的rhCRP基因克隆到表达载体pGAPZαA上并转化至毕赤酵母X-33中进行组成型分泌表达,通过His亲和层析柱纯化rhCRP。分别采用SDS-PAGE、Western blot和间接ELISA法检测目的蛋白并鉴定其免疫反应性。结果:重组表达载体pGAPZαA/rhCRP经酶切及DNA测序鉴定构建成功。重组毕赤酵母工程菌株成功组成型表达23kDa的rhCRP,27h即达到最大表达水平,表达量约3mg/L。经一步分离纯化获得纯度为96.58%的rhCRP,经间接ELISA检测表明其具有免疫反应性。结论:成功构建了组成型分泌表达rhCRP的毕赤酵母菌株,为进一步自主研发人CRP检测试剂奠定了基础。
Objective: To construct a recombinant Pichia pastoris strain expressing recombinant human C-reactive protein (rhCRP), express and purify rhCRP and identify its immunoreactivity. Methods: The designed rhCRP gene was cloned into the expression vector pGAPZαA and transformed into Pichia pastoris X-33 for constitutive secretion. The rhCRP was purified by His affinity chromatography. The target proteins were detected by SDS-PAGE, Western blot and indirect ELISA, respectively, and their immunoreactivity was identified. Results: The recombinant plasmid pGAPZαA / rhCRP was successfully constructed by enzyme digestion and DNA sequencing. Recombinant Pichia pastoris strains successfully constitutively expressed 23 kDa rhCRP, reached the maximum expression level 27 h, the expression level of about 3 mg / L. Purified by one step to obtain a purity of 96.58% of rhCRP, indirect ELISA test showed that it was immunoreactive. CONCLUSION: Pichia pastoris constitutively secreted rhCRP was successfully constructed, which lays the foundation for the further independent research on human CRP detection reagent.