链霉菌FJS31-2 abx R2基因克隆表达及蛋白纯化  

Cloning and Expression of Streptomyces sp.FJS31-2 abx R2 and Protein Purification

作　　者：李泽鑫 王兰林 栗午娟 杨逸 张悦萌 高佩舒 田红英 岳昌武 LI Zexin;WANG Lanlin;LI Wujuan;YANG Yi;ZHANG Yuemeng;GAO Peishu;TIAN Hongying;YUE Changwu(Yan′an Key Laboratory of Microbial Drug Innovation and Transformation,School of Basic Medicine,Yan′an University,Yan′an 716000,China)

机构地区：[1]延安大学基础医学院、延安市微生物药物创新及转化重点实验室,陕西延安716000

出　　处：《化学与生物工程》2023年第12期34-38,共5页Chemistry & Bioengineering

基　　金：陕西省自然科学基础研究计划项目(2021JM-416),大学生创新创业训练计划项目(S202110719117)。

摘　　要：利用原核表达系统获得链霉菌FJS31-2 AbxR2重组蛋白,并进行纯化。通过PCR扩增abx R2基因,将扩增产物克隆至pET32a质粒上构建重组质粒pET32a-abx R2;将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行IPTG诱导表达,并通过尿素洗涤方式对AbxR2重组蛋白进行纯化。酶切和测序结果均证实重组质粒pET32a-abx R2构建成功;在菌液OD 600值为0.6、28℃、110 r·min^(-1)、1.0 mmol·L^(-1) IPTG诱导4 h时,AbxR2重组蛋白的表达效果较好;采用尿素洗涤方式纯化AbxR2重组蛋白,可得到单一特异蛋白条带,效果较好。为研究AbxR2蛋白的结构和功能及提高Zunyimycins产量奠定了基础。In this paper,we obtained the recombinant AbxR2 protein of Streptomyces sp.FJS31-2 by prokaryotic expression system and purified the recombinant AbxR2 protein.Firstly,we constructed the recombinant plasmid pET32a-abx R2 by amplifying the abx R2 gene through PCR and cloning the amplified product onto the pET32a plasmid.Then,we transformed the recombinant plasmid into Escherichia coli Rosetta(DE3)competent cells for IPTG-induced expression,and purified the recombinant AbxR2 protein by urea washing.The results of enzyme digestion and sequencing confirm that the recombinant plasmid pET32a-abx R2 is successfully constructed.When the OD 600 value of the bacterial solution is 0.6,the expression effect of the recombinant AbxR2 protein is the best with 1.0 mmol·L^(-1) IPTG induction for 4 h at 28℃and 110 r·min^(-1).The effect of urea washing method for purification of the recombinant AbxR2 protein is the better with a single specific protein band.This study lays a foundation for studying the structure and function of AbxR2 protein and improving the yield of Zunyimycins.

关 键 词：链霉菌 AbxR2 基因簇 原核表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]