摘要
Atsttrin是颗粒蛋白前体(Progranulin,PGRN)的截断突变体,具有比PGRN更强的TNFα受体拮抗能力,从而可产生更显著的抗炎治疗效果。为了筛选出高表达Atsttrin原核表达系统,致力于为抗炎新药的开发奠定工作基础,故开展该研究。研究以人血管内皮细胞(HUVEC)的c DNA文库为模板,PCR克隆得到pgrn基因,进而通过重叠PCR成功扩增获得atsttrin目的基因。分别将atsttrin插入到p ET-22b(+)、p GEX-6p-3和p ET-40b(+) 3个不同的表达载体上,并转化大肠杆菌BL21(DE3)、Rosetta(DE3)得到6株表达菌株。通过测定各菌株蛋白表达量,E. coli BL21(DE3)/p GEX-Atsttrin菌株中目的蛋白的表达水平最高。为进一步提高表达量,对诱导温度、诱导时期、诱导剂浓度和诱导时间进行了优化。实验结果表明诱导表达最佳条件为37℃培养到A600为1. 0时加入终浓度为3 mmol/L的乳糖,30℃诱导培养12 h,在此条件下进行5 L发酵罐诱导10 h后重组蛋白产量为550 mg/L。作者成功建立了Atsttrin的大肠杆菌表达系统,并对其表达条件进行了优化,从而为相关药物的开发奠定了工作基础。
Atsttrin is a truncated mutant of granular protein precursors(Progranulin,PGRN),and it has a stronger TNFαreceptor antagonism than PGRN,which can produce more significant inflammatory therapeutic effect.In order to screen out the high expression of Atsttrin prokaryotic expression system and lay a working foundation for the development of new anti-inflammatory drugs,this study was carried out.In this study using the c DNA of HUVEC cells as PCR templates,the gene of pgrn was amplified by PCR,and the coding DNA of atsttrin was then obtained by the method of overlap PCR and cloned into three different expression vectors(p ET-22 b,p ET-40 b and p GEX-6 p-3).Then recombinant plasmids were transformed into E.coli BL21(DE3)and Rosetta(DE3)host strain and six transformants were gained.It demonstrated that the BL21(DE3)/p GEX-Atsttrin had higher expression of target protein.To achieve a higher expression level,a series of factor experiments were employed to optimize the expression conditions,in terms of temperature,induction time,concentration of lactose and post-induction time.The results showed that when the culture was propagated(37℃,200 r/min)to an optical density(A600)of 1.0,then induced by 3.0 mmol/L lactose at 30℃for 12 h and under the conditions of 5 L fermenter,the recombinant protein yield was 550 mg/L after 10 h induction.Atsttrin’s E.coli expression system was successfully established and its expression conditions were optimized,which laid a foundation for the development of related drugs.
作者
王重喜
姜雅杰
陈小颖
王楠
张同存
罗学刚
WANG Chong-xi;JIANG Ya-jie;CHEN Xiao-ying;WANG Nan;ZHANG Tong-cun;LUO Xue-gang(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,School of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Institute of Biology and Medicine,Wuhan University of Science and Technology,Wuhan 430000,China)
出处
《药物生物技术》
CAS
2019年第4期289-295,共7页
Pharmaceutical Biotechnology
基金
天津市科技支撑计划(NO.14CZDSY00052)
