表皮葡萄球菌8-32-B 株aap基因domain B的原核表达

为获得表皮葡萄球菌aap基因domain B片段的原核表达产物,采用PCR方法扩增引起新疆南疆地区奶牛乳房炎的表皮葡萄球菌株aap基因domain B片段,并克隆到原核表达载体pET-32a(+)中,构建重组质粒pET-32a(+)-aap,将重组质粒转化至宿主菌E.coli BL21中诱导表达。结果表明,克隆得到aap基因domain B片段为2 316bp,Aap蛋白分子质量184.5ku。在宿主菌E.coli BL21中表达的最佳诱导时间为4h,IPTG最佳诱导浓度为0.75mmol/L;纯化时洗杂缓冲液中咪唑浓度为40mmol/L,洗脱缓冲液中咪唑浓度为200mmol/L时可以获得单一Aap蛋白。

Prokaryotic Expression of Domain B in aap Gene of Staphylococcus epidermidis Strain 8-32-B

To obtain prokaryotic expression poroduct of domain B in aap gene of Staphylococcus epidermidis, the domain B of aap gene was amplified by PCR and cloned into a plasmid vector pET-32a(+). The recombinant plasmid, pET-32a(+)-aap, was transformed into E.coli BL21 cells, by wich, expression of domain B in aap gene was induced. The results showed that the size of domain B in aap gene is 2 316 bp, and the molecular weight of expressed Aap protein is about 184.5 ku. The Aap protein was massively expressed after induced for 4 h with 0.75 mmol/L IPTG. The targeted protein was purified under the conditions of 40 mmol/L imidazole in washing buffer and 200 mmol/L imidazole in the elution buffer.