摘要
目的探讨重组丝氨酸蛋白酶抑制剂(Vaspin)蛋白的原核表达条件,为研究其功能提供实验依据。方法通过基因重组技术,将编码Vaspin蛋白的密码子优化后插入带有His标签的pET-28a载体中,构建原核表达载体pET-28a-Vaspin,转化BL21感受态细菌,分别给予不同诱导时间(6、8、10、12、15、20 h)、不同诱导温度(32、37、42℃)、不同浓度诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)(0.1、0.2、0.5、1.0、2.0 mmoL·L^-1)进行培养,诱导产物经十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行鉴定,表达出来的Vaspin蛋白进行镍离子亲和层柱纯化。结果成功构建重组Vaspin蛋白的原核表达载体,经IPTG诱导、电泳分析,在IPTG浓度为1 mmol·L^-1、诱导温度37℃、诱导时间为15 h且转化优化密码子的菌体沉淀中出现相对分子质量为48000的特异条带,与Vaspin相对分子质量一致,经His标签纯化,灰度扫描分析显示,Vaspin蛋白的纯度为95%。结论Vaspins蛋白在IPTG终浓度为1 mmol·L^-1、37℃、培养时间15 h条件下可成功表达并纯化,且该条件下实验具有重复性。
Objective To investigate the prokaryotic expression conditions of visceral adipose tissue-derived protease inhibitro(Vaspin)recombinant protein and provide the experimental basis for the study of its function.Methods The codon-optimized human Vaspin protein was inserted into pET-28a vector with His label by gene recombination technology to construct pET-28a-Vaspin vector.PET-28a-Vaspin was transferred into BL21 competent bacteria.Then,the transformed BL21 were cultured under the different induction time(6,8,10,12,15,20 h),induction temperature(32,37,42℃),concentration of isopropyl-β-D-thiogalac toside(IPTG)(0.1,0.2,0.5,1.0,2.0 mmoL·L^-1).The expression of Vaspin protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified by Nickel ion affinity chromatography.Results Prokaryotic expression vector of recombinant protein Vaspin was successfully constructed,and the target band appeared at molecular weight of 48000 in codon-optimized bacterial expression strains with IPTG concentration of 1.0 mmol·L^-1,induction temperature of 37℃and induction time of 15 h,and the purity of the protein was 95%after purification by His tag.Conclusion Vaspins protein can be successfully expressed and purified under the conditions of IPTG final concentration of 1.0 mmol·L^-1,induction temperature of 37℃and induction time of 15 h,and the experiment is reproducible under this condition.
作者
陈雪利
胡婧婧
王天云
赵春澎
王小引
CHEN Xueli;HU Jingjing;WANG Tianyun;ZHAO Chunpeng;WANG Xiaoyin(Grade 2016,School of Basic Medicine,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;International Joint Research Laboratory for Recombinant Protein Expression,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《新乡医学院学报》
CAS
2020年第12期1126-1129,共4页
Journal of Xinxiang Medical University
基金
河南省高校创新团队科研项目(编号:18IRTSTHN027)
新乡医学院团委大学生科研创新课题。
关键词
丝氨酸蛋白酶抑制剂
密码子优化
原核表达
蛋白纯化
visceral adipose tissue-serine protease inhibitro
codon optimization
prokaryotic expression
protein purification
