毛竹EMF2类基因原核表达条件优化

在对毛竹EMF2基因功能研究的过程中,需要制备其抗体用于蛋白表达的检测。由于PheEMF2原核表达量低,纯化到的蛋白量不利于抗体制备,所以本研究用正交设计的方法对PheEMF2原核表达诱导条件进行优化,提高其原核蛋白表达量。根据L25(56)正交设计表设计诱导时间、诱导温度、IPTG终浓度、菌液OD值四因素五水平的融合蛋白表达诱导条件。用凝胶图像光密度分析系统分析各诱导条件下融合蛋白的表达量,发现菌液OD值和诱导温度对蛋白表达量有显著影响,而IPTG终浓度和诱导时间对蛋白表达量影响不显著。根据正交设计的效应曲线和方差分析结果,选择在菌株生长值OD600为1.1时,加入IPTG至终浓度1.0mmol/L,放入25℃摇床中振荡8 h诱导融合蛋白表达,融合蛋白的表达量约提升了10倍,占细菌总蛋白量的21.4%。

Optimization of Prokaryotic Expression Condition of EMF2-like Gene from Moso Bamboo

During the functional study of PheEMF2 gene,the PheEMF2 antibody was required to be prepared for the detection of protein expression.But the expression level of PheEMF2 was so low in prokaryocyte expression system,that the amount of purified protein was unfavorable for antibody preparation.In this study,through orthogonal design method,the induction conditions of prokaryocyte expression system was optimized to improve expression level.According to L25(56) orthogonal design table,the induction conditions of fusion protein expression of four factors including induction time,induction temperature,final IPTG concentration and concentration of bacterium solution,as well as five levels,were designed.The expression level of fusion protein in different induction condition was analyzed through the gel documentation analysis system.The results showed that both of the concentration of bacterium solution and induction temperature influence on the expression of fusion protein significantly,but the effect of final IPTG concentration and induction time was insignificant.According to the results of effect curve and variance analysis from orthogonal design,when the concentration of bacterium solution was 1.1(OD600) and final IPTG concentration 1.0 mmol/L was added to be oscillated for eight hours at 25℃,the expression level of fusion protein increased approximately by 10 times,and its amount accounted for 21.4% of the total proteins of the bacterium.