菌株尼古丁降解代谢中agnH基因的克隆表达与纯化
Cloning,expression and purification of agnH gene in nicotine degradation and metabolism of Agrobacterium tumefaciens SCUEC1 strain

对从湖北省襄阳市烟草种植土壤中分离筛选得到的尼古丁降解菌根癌土壤杆菌SCUEC1菌株中agnH基因进行克隆表达，并对AgnH蛋白的表达和纯化条件进行优化，为解析agnH基因的功能和揭示根癌土壤杆菌SCUEC1菌株尼古丁降解的分子机制提供理论依据。通过PCR扩增获得agnH全长基因(585 bp)，构建重组质粒pET28a(+)agnH，转化大肠杆菌BL21(DE3)菌株进行异源表达，研究不同IPTG浓度、诱导温度和诱导时间对重组质粒pET28a(+)agnH诱导表达的影响，用不同浓度的咪唑洗脱液洗脱重组蛋白，采用SDS-PAGE检测重组蛋白。结果表明：在IPTG浓度为0.4 mmol/L、诱导温度为25℃且诱导时间为25 h条件下，AgnH蛋白表达量较高；在250 mmol/L的咪唑洗脱液洗脱下，得到浓度较高的AgnH蛋白。
The agnH gene was cloned and expressed in the nicotine degrading Agrobacterium tumefaciens SCUEC1 strain,and the conditions of expressing and purifying AgnH protein were optimized.The full length agnH gene (585 bp) was amplified by PCR,and the recombinant plasmid pET28a(+)agnH was constructed and transformed into E. coli BL21(DE3) strain for heterologous expression.The effect of different IPTG concentration,induction temperature and induction time on the expression of recombinant plasmid pET28a(+)agnHwas studied.The recombinant protein was eluted with different concentrations of imidazole eluant,and the expressed protein was detected with SDS-PAGE.The results showed that the agnH gene was cloned and the recombinant plasmid pET28a(+)agnH was constructed.The expression of AgnH protein was higher under the conditions of IPTG concentration of 0.4 mmol/L,induction temperature of 25℃ and induction time of 25 h.A higher concentration of AgnH protein was obtained by elution with a 250 mmol/L imidazole eluant