[1]柴丹丹),李庆华),阎赟梦),等.杜氏盐藻S腺苷高半胱氨酸水解酶酵母双杂交诱饵载体的构建及自激活和毒性检测*[J].郑州大学学报(医学版),2012,(01):16.
 CHAI Dandan,LI Qinghua,YAN Yunmeng,et al.Construction of a bait vector for SAHH of Dunaliella salina and evaluation of its selfactivation in yeast twohybrid system[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(01):16.
点击复制

杜氏盐藻S腺苷高半胱氨酸水解酶酵母双杂交诱饵载体的构建及自激活和毒性检测*
分享到:

《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2012年01期
页码:
16
栏目:
论著
出版日期:
2012-01-20

文章信息/Info

Title:
Construction of a bait vector for SAHH of Dunaliella salina and evaluation of its selfactivation in yeast twohybrid system
作者:
柴丹丹1)李庆华1)阎赟梦1)毛丽红1)李靓1)朱立强2)薛乐勋1)#
1)郑州大学生物工程系细胞生物学研究室 郑州 450001 2)郑州大学第二附属医院检验科 郑州 450014
Author(s):
CHAI Dandan1)LI Qinghua1)YAN Yunmeng1)MAO Lihong1)LI Liang1)ZHU Liqiang2)XUE Lexun1)
1)Laboratory for Cell Biology,Department of Bioengineering, Zhengzhou University, Zhengzhou 450001 2)Clinical Laboratory,the Second Affiliated Hospital,Zhengzhou University, Zhengzhou 450014
关键词:
S腺苷高半胱氨酸水解酶酵母双杂交诱饵载体自激活杜氏盐藻
Keywords:
Sadenosyhomocystine hydrolaseyeast twohybridbait vectorselfactivationDunaliella salina
分类号:
Q782
摘要:
目的:构建杜氏盐藻S腺苷高半胱氨酸水解酶(SAHH)的酵母双杂交诱饵载体pGBKT7SAHH,并检测其对酵母细胞的毒性和自激活作用。方法:应用RTPCR扩增杜氏盐藻的SAHH cDNA,测序分析后与酵母双杂交诱饵载体pGBKT7连接,构建诱饵载体pGBKT7SAHH。用PEG/LiAc法将诱饵载体转入AH109和Y187酵母中,通过表型筛选检测诱饵蛋白对酵母有无毒性和自激活作用。结果:成功构建了诱饵载体pGBKT7SAHH并成功转化到酵母细胞AH109和Y187中,表达的融合蛋白对宿主酵母细胞无毒性。在AH109和Y187酵母细胞中诱饵载体均未激活报告基因HIS3和ADE2,但是激活了报告基因MEL1。结论:诱饵载体pGBKT7SAHH可用酵母双杂交方法筛选与SAHH相互作用的蛋白。
Abstract:
Aim:To construct a bait vector for Sadenosyhomocystine hydrolase(SAHH) of Dunaliella salina and to evaluate its selfactivation activity and toxic effects in yeast twohybrid system.Methods:The fulllength SAHH gene was amplified by RTPCR and confirmed by sequencing. A fragment of the gene was then subcloned into the vector SAHH of pGBKT7 to construct the bait vector. The constructed bait vector pGBKT7SAHH was transformed into yeast strains AH109 and Y187 by PEG/LiAc method and its selfactivation was tested by the phenotype assay.Results:The constructed bait vector of pGBKT7SAHH was successfully transformed into yeast strains Y187 and AH109, and the fusion proteins were not toxic to yeast cells;the reporter genes HIS3 and ADE2 were not selfactivated, but MEL1 was selfactivated.Conclusion:The pGBKTSAHH could act as a bait to screen interaction proteins of SAHH in yeast twohybrid system.

参考文献/References:

[1]Haba GD,Cantoni GL. The enzymatic synthesis of SadelosylLhomoeysteine from adenosine and homoeysteine[J].J Biol Chem,1959,234(3):603 [2]Chiang PK.Biological effects of inhibitors of Sadenosylhomocysteine hydrolase[J].Pharmacol Ther,1998,77(2):115 [3]Pritchard PH, Chiang PK, Cantoni GL, et al. Inhibition of phosphatidylethanolamine Nmethylation by 3deazaadenosine stimulates synthesis the of phosphatidylcholine via the CDPeholine pathway [J]. J Biol Chem,1982,257(11):6362 [4]Boissier F,Bardou F,Guillet V,et al.Further insight into Sadenosylmethioninedependent methyltransferases[J].J Biol Chem,2006,281(7):4434 [5]Wang C,Leffler S,Thompson DH, et al. A general flurescencebased coupled assay for Sadenosylmethioninedependent methyltransferases[J].Biochem Biophys Res Co mmun,2005,331(1):351 [6]Cai SM, Li QS, Fang JW, et al. The rationale for targeting the NAD/NADH cofactor binding site of parasitic SadenosylLhomocysteine hydrolase for the design of antiparasitic drugs[J].Nucleosides Nucleotides Nucleic Acids,2009,28(5):485 [7]Castro R,Rivera I,Struys EA,et al.Increased homocysteine and Sadenosylhomocysteine concentrations and DNA hypomethylation in vascular disease[J].Clin Chem,2003,49(8):1292 [8]Hermes M, Osswald H, Kloor D. Role of Sadenosylhomocysteine hydrolase in adenosineinduced apoptosis in HepG2 cell[J].Exp Cell Res,2007,313(2):264 [9]Malanovic N,Streith I,Wolinski H,et al. SadenosylLhomocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast: implications for homocysteine as a risk factor of atherosclerosis[J].J Biol Chem,2008,283(35):23989 [10]Fields S, Song O.A novel genetic system to detect proteinprotein interactions[J].Nature,1989,340(6230):245 [11]马生秀,邓璐霞,罗林,等.防御素HNP3成熟肽素酵母双杂交诱饵质粒的构建及自激活和毒性检测[J].航天医学与医学工程,2007,20(2):151 [12]阎赟梦,李庆华,李杰,等.杜氏盐藻S腺苷高半胱氨酸水解酶基因的克隆及功能分析[J].郑州大学学报:医学版,2011,46(4):517

相似文献/References:

[1]马晶),赵国强),朱含),等.酵母双杂交系统检测SK2与RyR2蛋白的相互作用[J].郑州大学学报(医学版),2011,(01):45.
 [J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2011,(01):45.
[2]阎赟梦,李庆华,李杰,等.杜氏盐藻S腺苷高半胱氨酸水解酶基因的克隆及功能分析*[J].郑州大学学报(医学版),2011,(04):517.
 YAN Yunmeng,LI Qinghua,LI Jie,et al.Cloning and functional analysis of SAdenosyl homocysteine hydrolase gene of Dunaliella salina[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2011,(01):517.
[3]侯永杰),李杰),李庆华),等.杜氏盐藻寡糖基转移酶亚基stt3a酵母双杂交诱饵载体的构建及自激活和毒性检测*[J].郑州大学学报(医学版),2011,(06):835.
 HOU Yongjie),LI Jie),LI Qinghua,et al.Construction of yeast twohybrid bait plasmid for stt3a of oligosaccharyltransferase from Dunaliella salina and detection of selfactivation and toxicity of stt3a[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2011,(01):835.
[4]李靓),崔柳青),王瑞莉),等.与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的新蛋白的酵母双杂交筛选*[J].郑州大学学报(医学版),2013,(01):24.
 LI Liang,CUI Liuqing,WANG Ruili,et al.Screening of novel proteins that interact with FLA8C terminal from Dunaliella salina using yeast twohybrid system[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2013,(01):24.
[5]王伟琼,王冲,刘延方#,等.应用酵母双杂交系统筛选与TRF1相互作用的蛋白质*[J].郑州大学学报(医学版),2014,(02):188.
 WANG Weiqiong,WANG Chong,LIU Yanfang,et al.Screening proteins that interact with telomeric repeat binding factor 1 via yeast twohybrid system[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2014,(01):188.
[6]许尧,张楠楠,张彦婷,等.杜氏盐藻环盒子1相互作用蛋白的酵母双杂交法筛选*[J].郑州大学学报(医学版),2015,(03):326.
 XU Yao,ZHANG Nannan,ZHANG Yanting,et al.Screening of proteins that interact with RBX1 from Dunaliella salina using yeast twohybrid system[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2015,(01):326.
[7]李庆华,张彦婷,朱立强,等.酵母双杂交技术筛选杜氏盐藻cDNA文库中与S-腺苷高半胱氨酸水解酶相互作用的 蛋白[J].郑州大学学报(医学版),2016,(01):5.
 LI Qinghua,ZHANG Yanting,ZHU Liqiang,et al.Screening of proteins that interact with Sadenosyhomocystine hydrolase by yeast twohybrid assay in Dunaliella salina cDNA library[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2016,(01):5.

备注/Memo

备注/Memo:
*国家自然科学基金资助项目30700014;科技部国际科技合作基金资助项目2007DFA01240 doi:10.3969/j.issn.16716825.2012.01.006 #通讯作者,男,1944年2月生,教授,博士研究生导师,研究方向:肿瘤标志物与基因工程,Email:xuelx@zzu.edu.cn
更新日期/Last Update: 1900-01-01