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目的原核分泌性表达白喉毒素无毒突变体H21G蛋白并进行纯化,为下一步开发以重组H21G蛋白为基础的疫苗奠定基础。方法以p ET16a-H21G质粒为模板,PCR扩增H21G基因,亚克隆至p ET22b载体中,构建重组原核表达质粒p ET22b-H21G,转化感受态大肠埃希菌BL21(DE3),IPTG诱导重组H21G蛋白分泌性表达。重组蛋白经渗透压休克,DEAE Sepharose Fast Flow阴离子交换层析和Phenyl Sepharose 6FF(high sub)疏水层析纯化后,使用还原/非还原SDS-PAGE和HPLC法分析纯化蛋白的纯度,窄范围等电聚焦分析其等电点,MALDI-TOF质谱法分析其相对分子质量,N-末端测序法鉴定N-末端氨基酸序列。结果重组原核分泌性表达质粒p ET22b-H21G经双酶切(NcoⅠ和XhoⅠ)和测序证明构建正确;目标蛋白以可溶形式存在于宿主菌周质间隙,表达量为10%~15%;经纯化后,重组蛋白纯度达95%以上,等电点在4.55~6.20之间,相对分子质量和N-末端氨基酸序列均与白喉毒素相符合。结论成功分泌性表达并纯化了重组白喉毒素无毒突变体H21G蛋白,为下一步开发以重组H21G蛋白为基础的疫苗奠定了基础。
Objective To prokaryotically express and purify H21G protein of non-toxic mutant of diphtheria toxin and lay the foundation for further development of a recombinant H21G protein-based vaccine. Methods The plasmid p ET22a-H21G was used as a template to amplify the H21G gene by PCR and subcloned into p ET22b vector to construct recombinant prokaryotic expression plasmid pET22b-H21G. The recombinant plasmid was transformed into competent E. coli BL21 (DE3), IPTG induced recombinant H21G Protein secretory expression. The recombinant protein was purified by osmotic shock, DEAE Sepharose Fast Flow anion exchange chromatography and Phenyl Sepharose 6FF (high sub) hydrophobic chromatography. Purity and narrow range isoelectric point of the purified protein were analyzed by reduction / non-reducing SDS-PAGE and HPLC The isoelectric point was focused on, the relative molecular mass was analyzed by MALDI-TOF mass spectrometry, and the N-terminal amino acid sequence was identified by N-terminal sequencing. Results The recombinant prokaryotic expression plasmid p ET22b-H21G was verified by double enzyme digestion (NcoⅠand XhoⅠ) and sequencing. The target protein was soluble in the periplasmic space of the host bacteria and the expression level was 10% ~ 15% After purification, the purity of the recombinant protein was above 95%, and the isoelectric point was between 4.55 and 6.20. The relative molecular mass and N-terminal amino acid sequence were consistent with diphtheria toxin. Conclusion The successful expression and purification of the recombinant diphtheria toxin non-toxic mutant H21G protein laid the foundation for the next step to develop a recombinant H21G protein-based vaccine.