CAJ | 학술논문

本文以长效重组人胰岛素原类似物(SⅡA-0801)的大肠埃希菌工程菌为研究对象,针对采用高细胞密度培养技术表达包涵体重组融合蛋白的关键参数,重点研究了诱导培养基成份(包括碳源、氮源、无机盐、微量元素)、种子细胞密度、IPTG诱导剂的加入量及诱导时间,诱导温度等因素与包涵体表达产量的相关性.结果发现其融合蛋白的最佳表达培养基:1.5%蛋白胨,1%酵母粉,0.5%NaCl,0.8%葡萄糖和0.2%磷酸盐缓冲液(22mmol/L KH_2PO_4,40mmol/L K_2HPO_4).最佳诱导表达条件为:25%接种量,诱导温度37℃,接种培养1h后添加0.5mmol/L IPTG,继续诱导培养5h,发酵培养的总周期为6h.优化后的培养条件比传统的LB培养基及培养方法,其产量提高了6倍多.该结果为长效重组人胰岛素原类似物(SⅡA-0801)后续的生产工艺研究提供了重要的参考依据.
본문이장효중조인이도소원유사물(SⅡA-0801)적대장애희균공정균위연구대상,침대채용고세포밀도배양기술표체포함체중조융합단백적관건삼수,중점연구료유도배양기성빈(포괄탄원、담원、무궤염、미량원소)、충자세포밀도、IPTG유도제적가입량급유도시간,유도온도등인소여포함체표체산량적상관성.결과 발현기융합단백적최가표체배양기:1.5%단백동,1%효모분,0.5%NaCl,0.8%포도당화0.2%린산염완충액(22mmol/L KH_2PO_4,40mmol/L K_2HPO_4).최가유도표체조건위:25%접충량,유도온도37℃,접충배양1h후첨가0.5mmol/L IPTG,계속유도배양5h,발효배양적총주기위6h.우화후적배양조건비전통적LB배양기급배양방법,기산량제고료6배다.해결과위장효중조인이도소원유사물(SⅡA-0801)후속적생산공예연구제공료중요적삼고의거.
An recombinant E.coli expressing long-acting human insulin analogues(SⅡA-0801)was used to study the effects of various high-cell-density growth conditions.Our study has revealed that the best growth medium Was composed of 1.5%tryptone,1%yeast powder,0.5%NaCl,0.8%glucose,0.2%phosphate buffer solution(22mmol/LKH_2PO_4 ,40mmol/L K_2HPO_4)and 10μg/ml kanamycin.The optimized culture condition was the addition of 0.5mmol/LIPTG after 25%inoculation in 1h with the induction temperature at 37℃.and then continued to ferment for 5 hours.The productivity of fusion protein obtained by the optimized inducing condition and the best growth medium Was six times than the traditional induction conditions and the LB medium.All those experiential parameters obtained by the investigation provide the basis for further technology of large scale fermentation of the recombinant E.coli.