CAJ | 학술논문

背景:胰岛素样生长因子1是人体内重要的细胞调控因子,已用于治疗糖尿病等多种疾病,工程菌发酵生产工艺研究是胰岛素样生长因子1实现产业化从而扩大临床应用的关键因素.目的:探讨表达重组人胰岛素样生长因子1工程菌的高密度发酵与表达条件.设计、时间及地点:酶基因工程实验,于2007-05/2008-05在浙江树人大学生物技术实验室完成.材料:重组人胰岛素样生长因子1大肠杆菌表达菌株E. coil BL21(DE3)/pET22a-IGF-1由浙江树人大学生物技术实验室保存.高密度发酵补料培养基为:葡萄糖300 g/L,蛋白胨40 g/L,酵母粉10 g/L,Na2HPO4280 mmol/L,NaH2PO4·2H2O 120 mmol/L,MgSO4 10 mmol/L,氨苄青霉素100 mg/L.方法:菌株活化后,进行摇瓶发酵试验,分别改变培养基种类、诱导剂异丙基硫代半乳糖苷浓度、诱导时间等参数,优化摇瓶发酵条件.依据摇瓶发酵优化条件,采用自控5 L发酵罐进行分批补料发酵试验.培养分分批培养和分批补料2个阶段.采用pH反馈流加的方式补料,在对数生长中后期开始诱导,加入异丙基硫代半乳糖苷至设计终浓度,培养4-6 h.主要观察指标:重组人胰岛素样生长因子1工程菌的发酵与产物表达情况.菌体浓度与菌体干重测定,目的蛋白表达量的测定,发酵液葡萄糖浓度的测定.结果:以2×YT+5 g/L葡萄糖为发酵培养基.经0.8 mmol/L异丙基硫代半乳糖苷诱导5 h,通过控制溶解氧以及pH反馈补料方式,实现工程菌高密度发酵与目的蛋白高效表达,每升发酵液收获干菌体50.1 g/L,重组人胰岛素样生长因子1含量达5.25 g/L.结论:实验成功建立了重组人胰岛素样生长因子1工程菌优化的高密度发酵工艺,为重组人胰岛素样生长因子1的卜游纯化和工业化生产奠定了基础.
배경:이도소양생장인자1시인체내중요적세포조공인자,이용우치료당뇨병등다충질병,공정균발효생산공예연구시이도소양생장인자1실현산업화종이확대림상응용적관건인소.목적:탐토표체중조인이도소양생장인자1공정균적고밀도발효여표체조건.설계、시간급지점:매기인공정실험,우2007-05/2008-05재절강수인대학생물기술실험실완성.재료:중조인이도소양생장인자1대장간균표체균주E. coil BL21(DE3)/pET22a-IGF-1유절강수인대학생물기술실험실보존.고밀도발효보료배양기위:포도당300 g/L,단백동40 g/L,효모분10 g/L,Na2HPO4280 mmol/L,NaH2PO4·2H2O 120 mmol/L,MgSO4 10 mmol/L,안변청매소100 mg/L.방법:균주활화후,진행요병발효시험,분별개변배양기충류、유도제이병기류대반유당감농도、유도시간등삼수,우화요병발효조건.의거요병발효우화조건,채용자공5 L발효관진행분비보료발효시험.배양분분비배양화분비보료2개계단.채용pH반궤류가적방식보료,재대수생장중후기개시유도,가입이병기류대반유당감지설계종농도,배양4-6 h.주요관찰지표:중조인이도소양생장인자1공정균적발효여산물표체정황.균체농도여균체간중측정,목적단백표체량적측정,발효액포도당농도적측정.결과:이2×YT+5 g/L포도당위발효배양기.경0.8 mmol/L이병기류대반유당감유도5 h,통과공제용해양이급pH반궤보료방식,실현공정균고밀도발효여목적단백고효표체,매승발효액수획간균체50.1 g/L,중조인이도소양생장인자1함량체5.25 g/L.결론:실험성공건립료중조인이도소양생장인자1공정균우화적고밀도발효공예,위중조인이도소양생장인자1적복유순화화공업화생산전정료기출.
BACKGROUND:Insulin-like growth factor-1(IGF-1)is an important cell factor which plays a special role in many disease treatments such as diabetes.The research of engineering bacteria fermentation production technology is great to IGF-1 industrialization and to clinical application.OBJECTIVE:To investigate the high cell-density fermentation and expression condition of recombinant human insulin-like growth factor-1(IGF-1) engineering bacteria.DESIGN,TIME AND SETTING:The enzyme,gene engineering,study was performed at the Biotechnological Laboratory of Zhejiang Shuren University from May 2007 to May 2008.MATERIALS:Recombinant E coli strain for IGF-1 expression as BL21 (DE3)/pET22a-IGF-1 was reserved in the Biotechnological Laboratory of Zhejiang Shuren University.Nutrient feed was composed of:glucose(300 g/L),peptone(40 g/L),yeast powder(10 g/L),Na2HPO4(280 mmol/L),Na2HPO4-2H2O2(120 mmol/L),MgSO4(10 mmol/L),and ampicillin(100 mg/L).METHODS:The strains were activated and then cultured in orbitaI shakers.Parameters such as types of media,isopropyl-β-D-thiogalactopyranoside(IPTG)concentration and induction time have been analyzed to explore optimaI fermentation conditions for expressing the recombinant protein.According to the optimal fermentation condition of orbitaI shakers.batch fermentation was carried on with 5 L-autocontroI fermentor.The process contained two stages:batch culture and fed-bafch through pH-stat solution.JPTG was added to jnduce the expression of protein in the middle and latter of the logarithmic growth phase for 4-βhours.MAIN OUTCOME MEASURES:Following described parameters were measured:fermentation and protein expression of the recombinant human IGF-1 engineering bacteria,cell concentration,cell dry weigh,objective protein,glucose concentration.RESULTS:Cells were cultured in 2×YT+5 g,L glucose medium,induced by 0.8 mmol/L IPTG for 5 hours.By controlling dissolved oxygen and by pH-stat feeding solution,high cell-density and high protein expression were achieved.Under the establishedconditions.50.1 g dry cell weight(DCW)/L could be obtained,and 5.25 g/L IGF-1 was achieved.CONCLUSION:The established high cell-density fermentative procedure of recombinant human IGF-1 engineering bacteda is the useful bases for further pudfication and large scale production of IGF-1.