猴金属硫蛋白α域(MT-α cDNA)突变体的构建、表达及工程菌对重金属的吸附

Construction and expression of metallothionein mutations and the heavy-metal adsorption capacity of the engineered bacteria

采用PCR技术和同尾酶手段,构建了猴金属硫蛋白(mMT)α域的串连体,并构建了表达MTα串连体的工程菌,获得了表达二聚串连体(MTα2)、三聚串连体(MTα3)、五聚串连体(MTα5)的工程菌.结果表明,工程菌在0.1mmol·L-1IPTG诱导3h后表达量最高.Cd2+浓度为0.5mmol·L-1时,对照菌pGEX-2T/BL21生长受到严重胁迫,工程菌表现出较强的抗性,MTαn/BL21(n=2、3、5)生长几乎不受到胁迫;Cd2+浓度为1.0mmol·L-1时,MTα5/BL21表现出较强的耐受能力.Cd2+浓度为0.5mmol·L-1时,工程菌对镉的富集能力明显高于对照,其中富集能力最强的是MTα3/BL21(76%).与对照pGEX-2T/BL21相比,MT/BL21对锌吸附能力显著提高,而转化有MTα串连体基因的工程菌对锌的吸附能力没有提高,说明工程菌对Cd2+的吸附特异性增强.结构预测显示,MTα、MTα2、MTα3的二级结构中α螺旋依次增加,推测突变蛋白的疏水性增强,三维结构稳定.MTα4、MTα5二级结构中,α螺旋数目减少,尤其是MTα5,其α螺旋几乎完全遭到破坏,疏水性大为降低.对基因工程菌吸附镉的实验表明,MTα3/BL21对镉具有较高的特异性和富集容量.

Based on PCR and isocaudamer techniques,a series of monkey metallothionein（mMT）mutations were constructed.Engineered bacteria expressing the MTα cluster were obtained as MTα/BL21,MTα2/BL21,MTα3/BL21 and MTα5/BL21.The engineered bacteria expressed the highest amount of target protein when induced with 0.1 mmol · L^-1 IPTG for 3 h.Toxicity to control was found and no toxicity of Cd^2＋ to engineered bacteria was observed for MTα2/BL21,MTα3/BL21 and MTα5/BL21 in the presence of 0.5 mmol · L Cd^2＋.As Cd^2＋ concentration increased to 1.0 mmol · L^-1,MTα5/BL21 showed higher resistance.Among the series of engineered bacteria,MTα3/BL21 removed the greatest percentage of Cd^2＋（76%）,followed by MTα2/BL21（72%）when the concentration of Cd^2＋ was 0.5 mmol · L^-1.The adsorption capacity for Zn^2＋ by MT/BL21（0.118 mmol · mg^-1 dry weight cells）was increased dramatically while no significant improvement was observed for the other engineered bacteria.The phenomenon indirectly suggests an increased specificity of the engineered bacteria for Cd^2＋ adsorption.Based on the predicted secondary structure,increased hydrophobic interaction was observed for MTα,MTα2 and MTα3.However the content of α-helix was decreased in MTα4 and MTα5,especially in MTα5.