[1]肖航,王换换,王凯,等.猪血管紧张素转化酶2重组蛋白诱导表达和纯化条件的优化[J].南京农业大学学报,2019,42(2):316-321.[doi:10.7685/jnau.201802018]
 XIAO Hang,WANG Huanhuan,WANG Kai,et al.The expression and optimization of purifying recombinant conditions of the recombinant ACE2 protein of porcine[J].Journal of Nanjing Agricultural University,2019,42(2):316-321.[doi:10.7685/jnau.201802018]
点击复制

猪血管紧张素转化酶2重组蛋白诱导表达和纯化条件的优化()
分享到:

《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
42卷
期数:
2019年2期
页码:
316-321
栏目:
动物科学
出版日期:
2019-03-19

文章信息/Info

Title:
The expression and optimization of purifying recombinant conditions of the recombinant ACE2 protein of porcine
作者:
肖航 王换换 王凯 张源淑
南京农业大学农业农村部动物生理生化重点开放实验室, 江苏 南京 210095
Author(s):
XIAO Hang WANG Huanhuan WANG Kai ZHANG Yuanshu
Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture and Rural Affairs, Nanjing Agricultural University, Nanjing 210095, China
关键词:
ACE2重组蛋白包涵体KCl染色切胶Ni2+-NTA亲和层析
Keywords:
recombinant protein ACE2inclusion bodiespurification by KCl gel slicesNi2+-NTA chromatography purification
分类号:
S852.2
DOI:
10.7685/jnau.201802018
摘要:
[目的] 本文旨在探索猪血管紧张素转化酶2(ACE2)在大肠杆菌中最佳表达条件和纯化蛋白的最佳方法。[方法] 将已构建好的猪重组表达载体pET-32a-ACE2转染至大肠杆菌BL21(DE3),通过改变IPTG诱导浓度以及诱导时间来对诱导表达条件进行筛选,实现目的蛋白高效表达后选用Ni2+-NTA亲和层析和KCl染色切胶2种方法,对获得的重组猪pET-32a-ACE2融合蛋白进行纯化,SDS-PAGE和免疫印迹等分析鉴定表达产物。[结果] 当IPTG浓度为1 mmol·mL-1、诱导时间为10 h时,ACE2蛋白在BL21(DE3)中表达量最高,以包涵体形式存在。表达产物的相对分子质量约为100×103,与预期目的蛋白大小相符。选用的Ni2+-NTA亲和层析和KCl染色切胶2种方法纯化重组pET-32a-ACE2融合蛋白包涵体,均能得到可溶性的重组蛋白,但KCl染色切胶法纯化获得的可溶性重组蛋白浓度及纯度都更佳,纯化蛋白经Western blot鉴定具有良好的抗原性。[结论] 本试验建立猪pET-32a-ACE2重组蛋白诱导表达的最佳条件:IPTG浓度1 mmol·mL-1,诱导时间为10 h。并且以KCl染色切胶法纯化该重组pET-32a-ACE2融合蛋白包涵体效果更好。
Abstract:
[Objectivs] The paper aims to optimize the condition for expression of ACE2 of porcine in Escherichia coli,this study compare the effect of purification of recombinant pET-32a-ACE2 fusion protein from inclusion bodies by KCl gel slices and Ni2+-NTA chromatography,and explore a better method for purifying recombinant protein.[Methods] The recombinant plasmid pET-32a-ACE2 of porcine was transformed into E.coli BL21(DE3) and in order to improve the expression level of target protein,temperature for induction,and concentration of IPTG were optimized. Inclusion bodies of recombinant pET-32a-ACE2 fusion protein obtained were purified by Ni2+-NTA chromatography and KCl gel slices,respectively. Antigenicity of soluble recombinant protein obtained was identified by SDS-PAGE and Western blot.[Results] The SDS-PAGE result showed that the ACE2 fusion protein expressed under the induction with 1.0 mmol·mL-1 IPTG for 10 h of relative molecular mass approximately 100×103 was mainly expressed in inclusion body. Two methods both could purify recombinant pET-32a-ACE2 fusion protein from inclusion bodies,but the purity and yield of soluble recombinant which was obtained by KCl gel slices were better. Western blot confirmed that soluble recombinant pET-32a-ACE2 fusion proteins had satisfactory antigenicity.[Conclusions] When the IPTG concentration was 1.0 mmol·mL-1 and the induction time was 10 h,the expression level of pET-32a-ACE2 recombinant protein was the highest,and the purification of the recombinant pET-32a-ACE2 fusion protein from inclusion bodies in KCl gel slices was a relatively simple,effective method.

参考文献/References:

[1] Tipnis S R,Hooper N M,Hyde R,et al. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase[J]. J Biol Chem,2000,275(43):33238-33243.
[2] Lambert D W,Clarke N E,Turner A J. Not just angiotensinases:new roles for the angiotensin-converting enzymes[J]. Cell Mol Life Sci,2010,67:89-98.
[3] 翁智远,晋学庆,吴可贵. 肾素-血管紧张素系统的重大发现-ACE2与Ang(1~7)[J]. 高血压杂志,2004,12(3):191-193. Weng Z Y,Jin X Q,Wu K G. Major findings of the renin-angiotensin system:ACE2 and Ang(1-7)[J]. Chinese Journal of Hypertension,2004,12(3):191-193(in Chinese).
[4] Pei Z,Meng R,Li G,et al. Angiotensin-(1-7) ameliorates myocardial remodeling and interstitial fibrosis in spontaneous hypertension:role of MMPs/TIMPs[J]. Toxicol Lett,2010,199(2):173-181.
[5] Wang K,Liu X,Xiao H,et al. The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J]. Int Immunopharmacol,2017,46:23-30.
[6] Yu X B,Lin Q,Qin X,et al. ACE2 antagonizes VEGFa to reduce vascular permeability during acute lung injury[J]. Cellular Physiology and Biochemistry,2016,38(3):1055-1062.
[7] Hashimoto T,Perlot T,Rehman A,et al. ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation[J]. Nature,2012,487(7408):477-481.
[8] 于在江,马学恩,周建华. 切胶纯化表达蛋白包涵体的可行性分析[J]. 生物技术,2007,17(3):46-48. Yu Z J,Ma X E,Zhou J H. A modified method for purification of inclusion bodies proteins in gel slices[J]. Biotechnology,2007,17(3):46-48(in Chinese with English abstract).
[9] 柴燕涛,姜棋予,谢国明,等. 包涵体蛋白3种纯化方法的比较[J]. 中国医药导报,2016,13(10):4-6. Chai Y T,Jiang Q Y,Xie G M,et al. Comparison of three methods for purification of inclusion body protein[J]. China Medical Herald,2016,13(10):4-6(in Chinese with English abstract).
[10] Herget-Rosenthal S,Feldkamp T,Volbracht L,et al. Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay:precision,interferences,stability and reference range[J]. Annals of Clinical Biochemistry,2004,41(2):111-118.
[11] 肖航,王凯,王换换,等. 真核表达质粒pcDNA.3.1(+) -ACE2的构建及其在CHO细胞中的表达[J]. 畜牧与兽医,2018,50(1):54-58. Xiao H,Wang K,Wang H H,et al. Construction of eukaryotic expression plasmid pcDNA3.1(+) -ACE2 and its expressions in CHO cells[J]. Animal Husbandry & Veterinary Medicine,2018,50(1):54-58(in Chinese with English abstract).
[12] Bio-Rad Laboratories. Model 422 electro-eluter instruction manual[Z]. Hercules:Bio-Rad Laboratories,2005:1-11.
[13] 范贵荣,杨致邦,田一玲,等. 两种纯化幽门螺旋杆菌VacA-HpaA融合蛋白包涵体方法的比较[J]. 中国病原生物学杂志,2009,4(2):81-84. Fan G R,Yang Z B,Tian Y L,et al. Comparison of two methods of purifying recombinant VacA-HpaA fusion protein inclusion bodies of Helicobacter pylori[J]. Journal of Pathogen Biology,2009,4(2):81-84(in Chinese with English abstract).
[14] 高慎阳,李一经. 猪流行性腹泻病毒重组M蛋白膜外区原核表达IPTG最佳诱导条件的确定[J]. 中国畜禽种业,2008,4(7):75-76. Gao S Y,Li Y J. Determination of optimal inducing conditions for IPTG expression of recombinant M protein of porcine epidemic diarrhea virus[J]. The Chinese Livestock and Poultry Breeding,2008,4(7):75-76(in Chinese).

备注/Memo

备注/Memo:
收稿日期:2018-2-9。
基金项目:江苏省高校优势学科建设工程资助项目(PAPD)
作者简介:肖航,硕士研究生。
通信作者:张源淑,教授,博导,主要从事动物机能和营养的生物化学与分子生物学、反刍动物乳蛋白和乳脂调控机制研究,E-mail:zhangyuanshu@njau.edu.cn。
更新日期/Last Update: 1900-01-01