|本期目录/Table of Contents|

[1]李晶,李芳娜,林雄杰,等.牛樟芝se原核表达载体构建及重组蛋白的表达、纯化[J].江苏农业科学,2017,45(16):50-53.
 Li Jing,et al.Construction of se prokaryotic expression vector and expression and purification of recombinant protein of Antrodia cinnamomea[J].Jiangsu Agricultural Sciences,2017,45(16):50-53.
点击复制

牛樟芝se原核表达载体构建及重组蛋白的表达、纯化(PDF)
分享到:

《江苏农业科学》[ISSN:1002-1302/CN:32-1214/S]

卷:
第45卷
期数:
2017年16期
页码:
50-53
栏目:
生物技术
出版日期:
2017-08-20

文章信息/Info

Title:
Construction of se prokaryotic expression vector and expression and purification of recombinant protein of Antrodia cinnamomea
作者:
李晶13 李芳娜1 林雄杰2 王锦锋13 林占熺13
1.国家菌草工程技术研究中心,福建福州 350002; 2.福建省农业科学院果树研究所,福建福州 350003;
3.福建农林大学菌草研究所,福建福州 350002
Author(s):
Li Jinget al
关键词:
牛樟芝鲨烯环氧酶原核表达正交设计
Keywords:
-
分类号:
S567.3+90.1
DOI:
-
文献标志码:
A
摘要:
鲨烯环氧酶(squalene epoxidase,SE)是多数中草药植物、药用菌中三萜合成途径中的限速酶,能影响三萜含量的积累。构建了牛樟芝鲨烯环氧酶基因(se)原核表达载体,并进行其重组蛋白诱导表达条件优化。以牛樟芝cDNA为模板扩增se,纯化后将其克隆到pMD18-T载体上,筛选阳性克隆并将其连接到原核表达载体pET28a,构建重组质粒pET-28a-se,经酶切验证后转化E.coil BL21(DE3)感受态细胞进行诱导表达,利用L9(34)正交试验优化目的蛋白诱导表达条件及纯化。结果表明,不同正交处理间的蛋白表达差异显著(P<0.05),其诱导因素的主次为时间>温度>IPTG浓度,诱导时间对重组蛋白表达影响最显著(P<0.05)。而IPTG浓度和温度对重组蛋白表达影响并不显著(P>0.05),且当IPTG终浓度为0.2 mmol/L,诱导时间为7 h,诱导温度为26 ℃时,对SE重组蛋白的诱导效果最佳。
Abstract:
-

参考文献/References:

[1]赵云生,万德光,陈新,等. 五环三萜皂苷生物合成与调控的研究进展[J]. 中草药,2009(2):327-330.
[2]Jandrositz A,Turnowsky F,Hgenauer G. The gene encoding squalene epoxidase from Saccharomyces cerevisiae:cloning and characterization[J]. Gene,1991,107(1):155-160.
[3]Kajikawa M,Kinohira S,Ando A,et al. Accumulation of squalene in a microalga Chlamydomonas reinhardtii by genetic modification of squalene synthase and squalene epoxidase genes[J]. PLoS One,2015,10(3):e0120446.
[4]Spanova M,Daum G. Squalene-biochemistry,molecular biology,process biotechnology,and applications[J]. European Journal of Lipid Science and Technology,2011,113(11):1299-1320.
[5]王森,张建国. 五味子的研究概况及其开发前景[J]. 经济林研究,2003,21(4):126-127.
[6]Han J Y,In J G,Kwon Y S,et al. Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J]. Phytochemistry,2010,71(1):36-46.
[7]胡薇,刘宁,田玉华,等. 人参鲨烯环氧酶基因的克隆与原核表达[J]. 西北农林科技大学学报(自然科学版),2012,40(10):207-212.
[8]Chang T,Chou W. Antrodia cinnamomea sp. Nov. on Cinnamomum kanehirai in Taiwan[J]. Mycological Research,1995,99(6):756-758.
[9]Lu M C,El-Shazly M,Wu T Y,et al. Recent research and development of Antrodia cinnamomea[J]. Pharmacology & Therapeutics,2013,139(2):124-156.
[10]Lu M Y,Fan W L,Wang W F,et al. Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development[J]. Proceedings of the National Academy of Sciences of the United States of America,2014,111(44):E4743-E4752.
[11]Turunen O,Winqvist R,Pakkanen R,et al. Cytovillin,a microvillar Mr 75,000 protein. cDNA sequence,prokaryotic expression,and chromosomal localization[J]. Journal of Biological Chemistry,1989,264(28):16727-16732.
[12]Rio D C,Ares M,Hannon G J,et al. Purification of RNA using TRIzol (TRI reagent)[J]. Cold Spring Harbor Protocols,2010(6):pdb.prot5439.
[13]林雄杰,王琴秋,陈少华,等. 稻瘟病菌Rab5蛋白的表达及互作蛋白的初步筛选[J]. 热带作物学报,2011,32(7):1352-1355.
[14]Wu M D,Cheng M J,Wang B C,et al. Maleimide and maleic anhydride derivatives from the mycelia of Antrodia cinnamomea and their nitric oxide inhibitory activities in macrophages[J]. Journal of Natural Products,2008,71(7):1258-1261.
[15]Lu M K,Cheng J J,Lai W L,et al. Fermented Antrodia cinnamomea extract protects rat PC12 cells from serum deprivation-induced apoptosis:the role of the MAPK family[J]. Journal of Agricultural and Food Chemistry,2008,56(3):865-874.
[16]侯双利,刘翠晶,杨利民,等. 人参鲨烯环氧酶基因的克隆与序列分析[J]. 吉林农业大学学报,2014,36(2):149-152,170.
[17]邢朝斌,曹蕾,陈龙,等. 刺五加鲨烯环氧酶基因cDNA的克隆及序列分析[J]. 中国中药杂志,2012,37(2):172-175.
[18]蒋军富,李雄英,吴耀生,等. 绞股蓝鲨烯环氧酶基因的克隆与序列分析[J]. 西北植物学报,2010,30(8):1520-1526.
[19]李宝财,修乐山,周秘,等. 刺五加鲨烯环氧酶基因的表达及其与刺五加皂苷含量的相关性分析[J]. 中药材,2013,44(7):1063-1066.
[20]蒋世翠,刘伟灿,王义,等. 西洋参不同器官中皂苷量与鲨烯合成酶和鲨烯环氧酶基因表达的相关性[J]. 中草药,2011,42(3):579-584.
[21]Wang R,Shen W B,Liu L L,et al. Prokaryotic expression,purification and characterization of a novel rice seed lipoxygenase gene OsLOX1[J]. Rice Science,2008,15(2):88-94.
[22]Bunger M K,Cargile B J,Sevinsky J R,et al. Detection and validation of non-synonymous coding SNPs from orthogonal analysis of shotgun proteomics data[J]. Journal of Proteome Research,2007,6(6):2331-2340.

相似文献/References:

[1]张知晓,付文,刘凌,等.牛樟芝药用化学及其应用研究进展[J].江苏农业科学,2019,47(18):10.
 Zhang Zhixiao,et al.Research progress on medicinal chemistry and application of Antrodia camphorate[J].Jiangsu Agricultural Sciences,2019,47(16):10.
[2]张知晓,季梅,刘凌,等.外源茉莉酸甲酯对牛樟芝产总三萜及多糖含量的影响[J].江苏农业科学,2019,47(19):133.
 Zhang Zhixiao,et al.Impact of exogenous methyl jasmonate on total triterpenoids and polysaccharide contents of Antrodia camphorate[J].Jiangsu Agricultural Sciences,2019,47(16):133.

备注/Memo

备注/Memo:
收稿日期:2016-04-10
基金项目:福建农林大学发展研究基金(编号:KF2015111);福建省2011计划(编号:K80ND8002)。
作者简介:李晶(1985—),女,湖北武汉人,博士,助理研究员,主要从事食用菌研究。E-mail:13959197195@163.com。
通信作者:林占熺,研究员,主要从事菌草技术推广研究。E-mail:lzxjuncao@163.com。
更新日期/Last Update: 2017-08-20