[1]陈雪利,胡婧婧,王天云,等.重组丝氨酸蛋白酶抑制剂蛋白的密码子优化及原核表达[J].新乡医学院学报,2020,37(12):1126-1129.[doi:10.7683/xxyxyxb.2020.12.005]
　CHEN Xueli,HU Jingjing,WANG Tianyun,et al.Codon optimization and prokaryotic expression of recombinant Vaspin protein[J].Journal of Xinxiang Medical University,2020,37(12):1126-1129.[doi:10.7683/xxyxyxb.2020.12.005]
点击复制

重组丝氨酸蛋白酶抑制剂蛋白的密码子优化及原核表达
分享到：

《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
37
期数:
2020年12
页码:
1126-1129
栏目:
基础研究
出版日期:
2020-12-05

文章信息/Info

Title:
Codon optimization and prokaryotic expression of recombinant Vaspin protein
作者:
陈雪利1; 胡婧婧1; 王天云2; 赵春澎2; 王小引2
（1.新乡医学院基础医学院2016级，河南　新乡　453003；2.新乡医学院基础医学院重组蛋白表达实验室，河南　新乡　453003）
Author(s):
CHEN Xueli1; HU Jingjing1; WANG Tianyun2; ZHAO Chunpeng2; WANG Xiaoyin2
(1.Grade 2016,School of Basic Medicine,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;2.International Joint Research Laboratory for Recombinant Protein Expression,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
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
Keywords:
visceral adipose tissue-serine protease inhibitro; codon optimization; prokaryotic expression; protein purification
分类号:
Q51
DOI:
10.7683/xxyxyxb.2020.12.005
文献标志码:
A
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48 000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。
Abstract:
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-thiogalactoside (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 48 000 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.

参考文献/References:

［1］　刘希龙,杜振元,刘日旭,等.糖尿病肾病(DN)患者血清脂肪特异性丝氨酸蛋白酶抑制剂（Vaspin）水平的变化及其与炎症因子的关系研究［J］.现代生物医学进展,2020,20(12):2321-2324.
［2］　黄晶,白华,张明铭.多囊卵巢综合征患者血清Vaspin、CTRP9水平及相关影响因素分析［J］.中国医学前沿杂志,2018,10(6):188-191.
［3］　林立中,吴建华,夏先考.血清ET、重组人内脏脂肪特异性丝氨酸蛋白酶抑制剂检测评估冠心病动脉血管硬化病变程度［J］.中国卫生标准管理,2020,11(12):103-105.
［4］　李海英,安仲武,冯小娟,等.糖尿病肾病患者血清丝氨酸蛋白酶抑制剂水平及意义［J］.山西医药杂志,2020,49(10):1242-1243.
［5］　柯旋,李宾公,郝艳琴.Vaspin在糖尿病心肌病中的作用及机制研究［J］.中国药理学通报,2018，34（10）：1403-1408.
［6］　赵庆杰,尤军.血清LP(a)、Vaspin、copeptin水平变化与急性冠脉综合征患者Gensini评分的相关性［J］.河南医学研究,2020,29(3):529-530.
［7］　CIMEN T,EFE T H,AKYEL A,et al.Human endothelial cell-specific molecule-1 (endocan) and coronary artery disease and microvascular angina［J］.Angiology,2016,67(9):846-853.
［8］　GAO W,YIN J,BAO L,et al.Engineering extracellular expression systems in Escherichia coli based on transcriptome analysis and cell growth state［J］.ACS Synth Biol,2018,7(5):1291-1302.
［9］　HAN Y,GUO W,SU B,et al.High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag［J］.Protein Expr Purif,2018,142:25-31.
［10］　YANG Y P,MA X Y,HUO Y X,et al.Application of codon optimization strategy in heterologous protein expression［J］.Sheng Wu Gong Cheng Xue Bao,2019,35(12):2227-2237.
［11］　窦媛媛,林艳,高向征,等.重组人bFGF的原核表达及功能分析［J］.中国细胞生物学,2019,41(7):1365-1370.
［12］　RASSOULI H,TABE BORDBAR M S,REZAEI LARIJANI M,et al.Cloning,expression and functional characterization of in-house prepared human basic fibroblast growth factor［J］.Cell J,2013,14(4):282-921.
［13］　孟平平,李媛媛,高蕊,等.Neuritin原核表达载体构建及高表达菌株筛选［J］.石河子大学学报(自然科学版),2020,38(4):491-495.
［14］　MIZUKAMI M,ONISHI H,HANAGATA H,et al.Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system［J］.Protein Expr Purif,2018,150:109-118.
［15］　PARK W J,YOU S H,CHOI H A,et al.Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli［J］.Acta Biochim Biophys Sin(Shanghai),2015,47(7):488-495.
［16］　LIAO Y H,CHEN Y R.A novel method for expression and purification of authentic amyloid-β with and without 15N labels［J］.Protein Expr Purif,2015,113:63-71.

更新日期/Last Update: 2020-12-05