乳糖诱导的重组人血管内皮抑素(rhEndostatin)蛋白的表达

研究用乳糖替代IPTG作为诱导剂进行重组蛋白的表达,观察乳糖对乳糖操纵子调控的基因工程菌发酵及重组血管内皮抑素表达的影响,从而选取最佳诱导表达条件。以重组人血管内皮抑素表达工程菌pETrhEN/BL21(DE3)作为研究对象,分别用IPTG和乳糖作为诱导剂,在摇瓶中进行表达实验。并对重组蛋白质表达量进行分析。然后在5 L发酵罐中进行验证。在摇瓶培养条件下,乳糖浓度大于0.5 g/L即可以诱导目的蛋白的表达。乳糖浓度1 g/L时诱导目的蛋白表达量与1 mmol/L的IPTG相当,当乳糖浓度为10 g/L,目的蛋白表达量达到最大。在发酵罐培养条件下,补料4 h后葡萄糖浓度基本耗尽,此时开始加入乳糖。诱导后1 h,即有重组蛋白表达,在诱导后4 h达到高峰(占菌体可溶性蛋白的56%),与此同时,诱导后5 h菌体浓度也达到最高值。在以乳糖操纵子为调控手段的工程菌表达系统中,可以使用乳糖作为诱导剂,诱导应在葡萄糖消耗完后进行。

Expression of recombinant endostatin gene in E.coli using lactose as inducer

The factors influencing the fermentation of recombinant E. coli strain using lactose as an inducer instead of IPTG were investigated. The recombinant E. coli BL21 （DE3） strain expressing the rhEndostatin was incubated using glucose as carbon source and the lactose as inducer at various concentrations. Then, the test result was further verified in a 5 L fermentor. In shake culture, the lactose at a concentration of more than 0.5 g/L could effectively induce the expression of target protein, and the 1 g/L of lactose was equivalent to that induced by IPTG with the concentration of 1 mmol/L The best concentration of lactose was 10 g/L. In fermentor, the rhEndostatin was expressed after 1 h of induction, and got the highest lever with 56 % volume after induction for 4 h. The recornbined bacteria kept growing to the highest lever after induction for 5 h. In the expression system of recombinant bacterial strain using lactose operon, lactose could be successfully used as an inducer. However, the induction might not be done until the glucose was consumed.