重组人降钙素原的制备及发酵优化  

Preparation and fermentation optimization of recombinant human procalcitonin

作　　者：霍景瑞 孙文杰 张晶晶 张国安 张艳[1] 杨晓晖[1] 刘英富 HUO Jingrui;SUN Wenjie;ZHANG Jingjing;ZHANG Guoan;ZHANG Yan;YANG Xiaohui;LIU Yingfu(Cangzhou Medical College/Cangzhou Nanobody Technology Innovation Center,Cangzhou,Hebei 061001,China)

机构地区：[1]沧州医学高等专科学校/沧州纳米抗体技术创新中心,河北沧州061000

出　　处：《国际检验医学杂志》2022年第18期2207-2212,共6页International Journal of Laboratory Medicine

基　　金：河北省重点研发计划项目(19272404D);河北省沧州市重点研发计划指导项目(172301001)。

摘　　要：目的 制备人降钙素原(PCT)蛋白,用于抗体制备及检测试剂盒的研制。方法 通过分子克隆技术构建人PCT重组表达质粒(pET28a-PCT),以大肠杆菌BL21(DE3)为宿主细胞,进行PCT的原核表达,利用发酵罐进行大肠杆菌的高密度培养并优化发酵条件,镍柱纯化重组蛋白。标定重组蛋白水平后,采用酶联免疫吸附试验(ELISA)验证重组蛋白作为标准蛋白在绘制水平-吸光度标准曲线中的应用价值。结果 培养大肠杆菌BL21(DE3)/pET28a-PCT,经异丙基β-D-硫代半乳糖苷(IPTG)诱导后,重组蛋白主要表达在细菌裂解上清液中。利用发酵罐优化溶氧量、补料及温度等参数,培养温度40℃,溶氧量设定为30%,10×肉汤(LB)浓缩培养基以100 mL/h速度加入补料,以IPTG终浓度0.2 mmol/L诱导4 h, PCT产量为1 405 mg/L,镍柱亲和纯化后,纯度超过90%。用ELISA试剂盒标定重组蛋白。绘制水平-吸光度标准曲线,PCT与试剂盒提供的标准蛋白之间几乎没有差异。结论 该研究通过发酵表达及亲和纯化的方式,制备了高纯度的PCT蛋白,为PCT抗体的制备及PCT检测试剂盒的开发提供了重要原料。Objective To prepare human procalcitonin(PCT) protein for the antibody preparation and detection kit development.Methods The recombinant expression plasmid of human PCT(pET28 a-PCT) was constructed by molecular cloning technology, and Escherichia coli(E.coli) BL21(DE3) served as the host cell for conducting the PCT prokaryotic expression.The E.coli high-density culture was conducted by the fermentation tank, and the fermentation conditions were optimized.The nickel column purified recombinant protein.After calibrating the recombinant protein level, ELISA was used to verify the application value of recombinant protein as standard protein in drawing the horizental-absorbance standard curve.Results The E.coli BL21(DE3)/pET28 a-PCT was cultured.After IPTG induction, recombinant protein was mainly expressed in the supernatant of bacterial lysis.The fermentation tank was used to optimize the parameters such as the dissolved oxygen amount, supplementary material and temperature.The culture temperature was 40 ℃,dissolved oxygen amount was set at 30%,10×LB concentration medium was added with supplementary material at the rate of 100 mL/h.The final concentration of IPTG was 0.2 mmol/L for 4 h induction.The output amount of PCT was about 1 405 mg/L.After nickel column affinity purification, the purity exceeded 90%.The recombinant protein was calibrated by ELISA kit.When drawing the horizontal-absorbance curve, the standard protein provided by PCT and reagent kits almost showed no difference.Conclusion High purity PCT protein is prepared by fermentation expression and affinity purification, which provides the important material for the preparation of PCT antibody and the development of reagent kit.

关 键 词：降钙素原 脓毒症 大肠杆菌 发酵 亲和纯化 溶氧 

分 类 号：Q789[生物学—分子生物学]