葡萄糖碳源对组成型表达重组人C―反应蛋白的影响.docVIP

葡萄糖碳源对组成型表达重组人C―反应蛋白的影响 　　[摘要] 目的 探讨不同浓度的葡萄糖对Pichia pastoris组成型表达重组人C-反应蛋白（rhCRP）的影响并筛选出最优葡萄糖碳源。 方法 分别以浓度为0.5%、1.0%、1.5%、2.0%、2.5%、3.0%、3.5%（w/v）的葡萄糖作为碳源，利用毕赤酵母菌株组成型表达rhCRP。取不同表达时间的培养基上清进行Western blot检测及灰度比较分析。 结果 毕赤酵母菌株X-33（pGAPZαA/rhCRP）以葡萄糖作碳源组成型表达rhCRP时，1.0%（w/v）的葡萄糖为最优碳源。 结论 葡萄糖碳源的浓度可影响Pichia pastoris组成型表达rhCRP的水平。本研究结果将为rhCRP的进一步优化表达及大规模生产提供有力的实验依据。 　　[关键词] C-反应蛋白；葡萄糖；碳源；组成型表达；Pichia pastoris 　　[中图分类号] Q819 [文献标识码] A [文章编号] 1673-7210（2017）10（c）-0018-04 　　Influence of glucose carbon source on the constitutive expression of recombinant human C-reactive protein 　　LI Junming YANG Liping WANG Chenyu ZHAO Qi ZHENG Qing SUN Chengming▲ 　　Department of Clinical Laboratory， Yantai Yuhuangding Hospital， Shandong Province， Yantai 264000， China 　　[Abstract] Objective To investigate the effect of different concentrations of glucose on the constitutive expression of recombinant human C-reactive protein （rhCRP） in Pichia pastoris， and to screen out the optimum concentration of the glucose carbon source. Methods The different concentrations [0.5%， 1.0%， 1.5%， 2.0%， 2.5%， 3.0% and 3.5% （w/v）] of glucose were used to constitutively express rhCRP in Pichia pastoris. Samples were taken from individual tubes of the cultures at the desired time points and the expression of rhCRP in the culture supernatant was analyzed by Western blot and gray scale. Results When rhCRP was constitutively expressed in Pichia pastoris X-33 using glucose as carbon source， 1.0% glucose （w/v） was selected as the best carbon source. Conclusion The concentration of glucose carbon source plays an important role in the constitutive expression of rhCRP in Pichia pastoris. The results of this study will provide a strong experimental basis for further optimized expression and large-scale production of rhCRP. 　　[Key words] C-reactive protein； Glucose； Carbon source； Constitutive expression； Pichia pastoris 　　C-反??蛋白（C-reactive protein，CRP）是一种系统发育上十分古老、结构上高度保守的典型的急性时相反应蛋白[1]。人们对它的研究已有80余年的历史，发现其生物作用似乎相当复