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

Effects of Different Induced Culture Conditions on Expression of the Second Extracellular Loop of Sheep β_2-adrenoceptor in E. coli

【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。

【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 β2-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 β2-adrenoceptor in E.coli BL21（DE3） when the recombinant bacteria grow to OD600 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.