目的：构建白喉毒素(Diphtheria toxin)无毒突变体CRM197(Cross-reacting materials 197)的原核表达载体,并在大肠杆菌中表达重组蛋白。方法以白喉杆菌(ATCC39255)基因组DNA为模版,采用聚合酶链式反应(Poly-merase chain reaction, PCR)扩增 CRM197基因,插入表达载体 pET11b 中,构建重组原核表达质粒 pET11b-CRM197。经双酶切及测序鉴定正确后,重组质粒被转化入大肠杆菌Rosetta 2(DE3) pLysS, IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切及测序鉴定,结果表明与预期一致；表达的重组蛋白相对分子质量约58000,并可与鼠抗CRM197单克隆抗体特异性结合。结论已成功构建了重组原核表达载体pET11b-CRM197,重组的CRM197蛋白在大肠杆菌中得到了表达,为以该重组突变体作蛋白载体制备结合疫苗奠定了基础。%Objective To construct the prokaryotic expression vector for nontoxic mutant CRM197 of diphtheria toxin and express it in E. coli. Methods CRM197 gene was amplified by PCR using the genomic DNA of diphtheria bacillus strain as a template, and then amplified fragment was inserted into expression vector pET11b. The recombinant plasmid pET11b-CRM197 was identified by the analysis of restriction endonuclease digestion and sequencing, then transformed into E. coli Rosetta2(DE3)pLysS for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results It is demonstrated that recombinant plasmid pET11b- CRM197 was constructed successfully. The expressed recombinant protein, with a relative molecular mass of about 58000, showed specific binding to mouse mono-clonal antibody against CRM197. Conclusion Recombinant plasmid pET11b- CRM197 was successfully constructed and expressed in E. coli, which laid a foundation for further study on the preparation of conjugate vaccine using recombinant CRM197 as protein carrier.
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