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潘晓, 沈萍萍, 华子春. 基于酵母双杂交的微囊藻毒素检测系统初探[J]. 水生生物学报, 2008, 32(2): 201-206.
引用本文: 潘晓, 沈萍萍, 华子春. 基于酵母双杂交的微囊藻毒素检测系统初探[J]. 水生生物学报, 2008, 32(2): 201-206.
PAN Xiao, SHEN Ping-Ping, HUA Zi-Chun. DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(2): 201-206.
Citation: PAN Xiao, SHEN Ping-Ping, HUA Zi-Chun. DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(2): 201-206.

基于酵母双杂交的微囊藻毒素检测系统初探

DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM

  • 摘要: 微囊藻毒素是由淡水微囊藻产生的次生代谢物,结构为环状七肽,分子量约1000Da,对生物机体具有多种毒理作用,是一类较强的肿瘤促进剂,目前没有比较简单便宜的藻毒素检测方法,本文探索建立利用酵母双杂交系统进行藻毒素检测的方法。前期实验已用噬菌体表面展示肽库技术从随机12和7肽库中筛选获得若干与藻毒素有相互作用的多肽。本文分别挑选其中亲和力较高的3个多肽,构建到酵母双杂交系统中,将不同的肽段分别融合在含有Ac-tive Domain和Binding Domain的表达载体中,共转化酵母菌株AH109,然后测定β-半乳糖苷酶活性来反映下游报告基因半乳糖苷酶的表达情况,从而通过反映培养基里面是否含有藻毒素,进而利用此方法来比较方便地检测藻毒素。

     

    Abstract: Microcystins (MCs) are secondary metabolites produced by toxic cyanobacteria, which belong to the family of mono-cyclic heptapeptides with molecular weight about 1000Da.MCs are claimed to possess a variety of toxic effects and considered asa strong tumor inducer.Up to now, the simpler and easier MCs detection method has not been fully established.The present studyaims to establish a novel MCs detection methodology by using the principle of yeast two-hybrid system.In previous study, severalpolypeptides interacting with MCs had been identified fromthe random 12-mer peptide phage displayed library and 7-mer peptidephage displayed library.We chose three polypeptides with high affinity forMCs and cloned them into the yeast two-hybrid system.Firstly,DNA fragments encoding for those polypeptides were constructed into the yeast two hybrid expression vectors harboringActive Domain or Biding Domain, then the recombinant plasmids were co-transfected into yeast strain AH109.TheB-Gal reporter.sactivity could be easily measured by usingONPG (O-nitropheny-l B-D-galactopyranoside) as substrate and the change in B-Gal re-porter.s activity represented the presence of MCs in culturemedium,thus this paper provided amore convenient approach to de-tect MCs.

     

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