不同诱导培养条件对重组绵羊β_2-AR第二细胞外环在大肠杆菌中表达的影响

【目的和方法】研究对不同诱导时间、诱导剂IPTG终浓度、培养温度和振荡培养箱转速等4个诱导培养条件对绵羊β2-肾上腺素能受体(β2-adrenoceptor,β2-AR)第二细胞外环重组质粒pET32C-AR/Se在大肠杆菌BL21(DE3)中表达水平的影响进行了研究,以筛选其最佳培养条件。【结果】随着IPTG诱导时间的延长,重组绵羊β2-AR第二细胞外环的相对表达量逐渐增加,在2 h时相对表达量达到最大值,为菌体总蛋白的30.1%。IPTG终浓度在0.01~2 mmol/L时,重组绵羊β2-AR第二细胞外环的相对表达量水平均在27%~30%;当IPTG终浓度为0.01 mmol/L时,重组绵羊β2-AR第二细胞外环的相对表达量最高,占菌体总蛋白的30.5%。在设置的4个诱导温度中,25℃时重组绵羊β2-AR第二细胞外环的相对表达量最高,占菌体总蛋白的35.5%。在振荡培养箱不同转速条件下,重组绵羊β2-AR第二细胞外环的相对表达量在33%~40%波动;在200 r/min时,相对表达水平最高,为细菌总蛋白的40.0%。【结论】绵羊β2-AR第二细胞外环基因在大肠杆菌中的表达最佳条件为:重组菌株在LB液体培养基中生长至OD6000.5左右时,加入IPTG至终浓度0.01mmol/L,于25℃、200 r/min诱导120 min。

Effects of Different Induced Culture Conditions on Expression of the Second Extracellular Loop of Sheep β2-adrenoceptor in E.coli

Objective and Method]The aim of this paper was designed to study the effect of different induced time,induced dose IPTG final concentration,culture temperature,rotation speed of shaker on the expression level of sheep β_2-adrenoceptor gene,the second extracellular loop recombinant plasmid pET32C-AR/Se harboring in order to select and screen the best culture conditions.Result]The results showed that along with prolonging of IPTG induction time, the relative expression level of the recombinant protein increased gradually and reached the peak 2 h later, counting for 30.1% of the total bacterial protein. The expression level of the recombinant protein was between 27%～30% of total bacterial protein when the final concentration of IPTG ranged from 0.01 to 2 mmol/L. The crest value of expression level was 30.5% of total bacterial protein when the final concentration of IPTG was 0.01 mmol/L. In four culture temperatures, the expression level of the protein reached the peak, 35.5% of the total bacterial protein at 25℃. When the rotation speed of shaker ranged from 100 to 200 r/min, the expression level of the protein fluctuated between 33%～40%. The highest expression level of the foreign protein reached 200 r/min, counting for 40% of the total protein. Conclusion]From the results, the conclusions could be drawn that the optimum culture conditions for expression of the second extracellular loop of sheep β_2-adrenoceptor in E.coli BL21(DE3) when the recombinant bacteria grow to OD_(600) 0.5 in LB fluid medium, IPTG added to a final concentration of 0.01 mmol/L, and induced at 25℃ and 200 r/min for 120 min.