To product highly purified diphtheria toxin mutant cross reacting material 197(CRM197),it can be used as a carrier protein for the development of bacterial conjugate vaccines.In this current work,the CRM197 gene was codon-optimized in Escherichia coli and cloned into pET28a(+),and the correctly identified recombinant plasmid pET28a-CRM197 was transformed into Escherichia coli BL21(DE3).The recombinant plasmid was induced by IPTG,and its expression form was analyzed and optimized expression conditions.Then the expressed recombinant CRM197 protein was purified,and finally purified CRM197 was analyzed by western blotting WB,purity,molecular weight and circular dichroism(CD).The results showed that the recombinant plasmid pET28a-CRM197 was successfully constructed by PCR digestion and sequencing,and it was transformed into Escherichia coli BL21(DE3)to obtain recombinant engineering bacteria E.coli(DE3/p28a/197).The optimal inoculum amount for fermentation was 5%-10%(v/v),and it was mainly expressed in a form of inclusion body.The harvested cells were crushed under the condition of crushing pressure of 1000 bar,and then dissolved with 6mol/L guanidine hydrochloride,refolded and ultrafiltered by centrifugal filters(30 kD),and then purified by AKTA pure150 M system,and purified by one-step anion exchange chromatography.The purity of CRM197 was more than 98%,and the identification results of WB,molecular weight and circular dichroism are all consistent with the standard product. The results indicated that CRM197 expression system was established,which has no label,high yield and purity,and can be used for the large-scale production of the protein.

目的 建立反相高效液相色谱法(reverse phase high performance liquid chromatography,RP-HPLC)检测白喉毒素无毒突变体CRM197蛋白纯度.方法 利用Agilent AdvanceBio RP-mAb SB-C8(100 mm×2.1 mm)分析柱和Agilent1260高效液相色谱系统,以含0.1％三氟乙酸水溶液-异丙醇(98∶2)为流动相A,以含0.1％三氟乙酸乙腈溶液为流动相B,进行梯度洗脱,体积流速为0.5 mL/min,检测波长为280 nm,柱温为65℃,进样体积为10 μL,采用面积归一法检测CRM197蛋白纯度,并对方法的适用性、专属性、重复性、中间精密度、线性、灵敏度和耐用性指标进行考察.用建立的方法检测CRM197蛋白酸处理供试品溶液、碱处理供试品溶液及3批原液的纯度.结果 建立的方法系统适用性良好;专属性验证表明空白溶液在目标峰积分范围内无干扰峰,热处理CRM197蛋白目标峰与其他杂质峰分离度＞1.5;6次重复进样目标峰面积相对标准偏差(relative standard deviation,RSD)为1.14％,保留时间RSD为0.81％;中间精密度分析目标峰面积RSD为1.62％,保留时间RSD为0.06％;CRM197蛋白含量在20～2 000 μg/mL范围内,与峰面积存在良好的线性关系(R2=0.999 8);最低检测限为10 μg/mL,定量限为20 μg/mL;在改变柱温色谱条件时,目标峰面积百分比与保留时间均RSD≤5.00％,耐用性研究合格.对酸处理、碱处理供试品溶液及3批原液进行纯度检测,纯度在76.5％～96.9％,结果准确.结论 建立的RP-HPLC检测CRM197蛋白纯度的方法,经验证各项指标均符合要求,可用于CRM197蛋白的纯度检测.

目的 基于紫外分光光度法,建立一种测定白喉毒素无毒突变体CRM197蛋白含量的方法.方法 在天然折叠状态和非折叠状态(CRM197蛋白经盐酸胍变性)下,分别测定相同含量的CRM197蛋白A280nm值,再根据朗伯-比尔定律c=A/εL计算其折叠状态消光系数.同时,使用当前建立的方法与BCA法分别测定CRM197蛋白的含量,并对2种方法的差异和检测特点进行比较分析.结果 根据CRM197蛋白的氨基酸一级序列与相对分子质量计算出其理论消光系数为0.934 mL/(mg·cm),在280 nm处的实验消光系数为(0.959±0.006)mL/(mg·cm).2种方法测定同一蛋白样本的结果一致,分别为(2.17±0.02)mg/mL和(2.14±0.09)mg/mL;本方法检测值的Cv为0.70％,BCA法检测值的CV为4.15％.结论 本研究建立的CRM197蛋白含量测定方法准确性高、线性范围宽、获取数据快,可用于CRM197蛋白含量的测定,同时为其他载体蛋白的含量测定提供了参考.