白喉毒素无毒变异体CRM_(197)的表达及其载体作用

目的表达白喉毒素无毒变异体CRM197,并考察其载体作用。方法利用基因工程技术在大肠杆菌BL21(DE3)中表达CRM197,用金属镍离子亲和层析纯化;以重组CRM197为蛋白载体,在EDAC的作用下,与活化的A群脑膜炎球菌荚膜多糖(GAMP)结合,制备GAMP-rCRM197结合物,免疫BALB/c小鼠,间接ELISA法测定血清中A群脑膜炎球菌多糖特异性IgG抗体,并分析其免疫原性。结果重组CRM197在大肠杆菌中主要以包涵体形式表达,表达量占菌体总蛋白的25%左右;Western blot证明重组CRM197具有良好的反应原性;各针接种后,结合物诱生的多糖特异性IgG水平均显著高于GAMP组和GAMP+rCRM197混合物组,具有较强的免疫原性;多次接种产生了免疫增强效应,重组CRM197具有载体蛋白的作用。结论已在大肠杆菌BL21(DE3)中成功表达了重组CRM197,以纯化的重组CRM197作为载体制备的GAMP-rCRM197结合物具有良好的免疫原性,为以重组CRM197为蛋白载体制备其他结合疫苗奠定了实验基础。

Expression of Nontoxic Diphtheria Toxin Mutant CRM197 as A Carrier Protein

Objective To express the nontoxic mutant CRM197 of diphtheria toxin and study its effect as a carrier protein. Methods Express CRM197 in E. coli BL21(DE3) by gene engineering technique and purify the expressed product by nickel ion affinity chromatography. Link the purified CRM197 as a carrier protein to activated group A meningococcal polysaccharide (GAMP), using EDAC as linker. Immunize BALB / c mice with the prepared GAMP- CRM197 conjugate and determine the specific IgG against GAMP in sera by indirect ELISA. Results Recombinant CRM197 was expressed in E. coli mainly in a form of inclusion body. The expressed product contained about 25% of total somatic protein and showed good reactogenicity as proved by Western blot. The IgG level against GAMP induced by GAMP- CRM197 conjugate was significantly higher than those by GAMP and by the mixture of GAMP and rCRM197. Repeated immunization with the conjugate induced immunopotentiation, indicating the effect of CRM197 as a carrier protein. Conclusion Recombinant CRM197 was successfully expressed in E. coli, and the GAMP-rCRM197 conjugate prepared with the purified expressed product showed good immunogenicity. It laid an experimental foundation of preparation of other conjugate vaccine using CRM197 as a carrier protein.