摘要
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 express avirulent diphtheria toxin（DT）mutant H21 G protein in a secretory form in prokaryotic cells, and lay a foundation of further development of H21G-based vaccine. Methods H21 G gene was amplified by PCR using plasmid p ET16a-H21 G as a template and subcloned to vector p ET22 b, and the constructed recombinant plasmid p ET22b-H21 G was transformed to competent E. coli BL21（DE3） for expression under induction of IPTG. The expressed protein was purified by osmotic shock treatment, DEAE Sepharose Fast Flow ion exchange chromatography and Phenyl Sepharose 6FF（high sub） hydrophobic chromatography, and determined for purity by reduced / non-reduced SDS-PAGE and HPLC, for isoelectric point by isoelectric focusing（IEF）, for relative molecular mass by MALDI-TOF, and for Nterminal amino acid sequence by N-terminal sequencing. Results Restriction analysis（NcoⅠand XhoⅠ）and sequencing proved that recombinant secretory expression plasmid p ET22b-H21 G was constructed correctly. Expressed H21 G protein existed in periplasmic space of host cell in a soluble form, and contained about 10% -15% of total somatic protein.Purified H21 G protein reached a purity of more than 95%, of which the isoelectric point was 4. 55 - 6. 20, and the relative molecular mass and N-terminal amino acid sequence were consistent with those of DT. Conclusion Avirulent recombinant DT mutant H21 G protein was successfully expressed in a secretory form, which laid a foundation of further development of H21G-based vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第12期1262-1266,共5页
Chinese Journal of Biologicals
关键词
白喉毒素
无毒突变体H21G
分泌性表达
纯化
Diphtheria toxin（DT）
Avirulent mutant H21G
Secretory expression
Purification
