丁香假单胞菌极毛蛋白hrpA基因的克隆与表达

摘要

将丁香假单胞菌番茄变种DC3000(P.syringae pv.tomato DC3000)极毛蛋白hrpA基因克隆到pET32a(+)载体上,获得重组表达载体pET32a(+ )-hrpA.将重组质粒转入宿主E.coli BL21( DE3)溶源菌中,通过SDS-PAGE分析表明,在1.0mmol·L-1异丙基硫代-β-D半乳糖苷(IPTG)的诱导下,重组子成功表达了分子质量约为28ku的融合蛋白(目标蛋白基因与硫氧还蛋白基因的融合表达产物).并利用Ni2+-NTA柱亲和层析分离获得纯化的HrpA蛋白,质量浓度为91.8 g·L-1.%HrpA gene was obtained from Pseudomonas syringae pv. Tomato DC3000 by PCR and the protein expression system of Escherichia coli BL21 (DE3) for hrpA was constructed with vector pET32a( + ). SDS-PAGE analysis showed that the transformant with recombinant expression plasmid pET32a ( + ) - hrpA produced a fusion protein of TrxA-HrpA with corresponding molecular weight of 28 ku. Furthermore, HrpA recombination protein was purified through Ni2 + -NTA and its concentration was measured to be 91.8 g·L-1.