抗草甘膦转基因大豆酶联免疫吸附测定试剂盒的研制

IF 0.7 Q4 CHEMISTRY, ANALYTICAL Methods and Objects of Chemical Analysis Pub Date : 2019-01-01 DOI:10.17721/MOCA.2019.21-29
Y. Khomenko, L. Ishchenko, V. Ishchenko, S. Midyk, D. Y. Rybalchenko, V. Ushkalov, V. Spyrydonov
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引用次数: 0

摘要

本研究的目的是开发具有竞争力的酶联免疫吸附测定试剂盒,用于检测抗草甘膦转基因植物。作为基因分离的来源,我们使用了转基因大豆的叶片,行40-3-2(孟山都)。利用聚合酶链反应分离到具有抗草甘膦功能的5-烯醇piruvilshykimat-3-磷酸合成酶(CP4 EPSPS)基因。将分离的基因克隆到质粒载体pET24(a)中,构建出遗传结构pET-CP4EPSPS。大肠杆菌(bl21 (DE3))经基因构建转化,0.5 mM IPTG诱导过表达。将分子量为45 kD的重组酶CP4 EPSPS纯化为包涵体,作为抗原进行96孔聚苯乙烯板固定。采用单克隆抗体CP4 EPSPS mAb3 (Artron BioResearch Inc., Canada)合成辣根过氧化物酶偶联物并接收诊断试剂。我们已经证明,在竞争性ELISA中,我们可以检测到至少0.1%的抗草甘膦转基因植物,例如大豆(品系RR 40-3-2),此外我们还可以检测到油菜籽(品系GT73)和甜菜(品系H7-1)。
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Development Of ELISA Kit For Detection Of Glyphosate-Resistant Genetically Modified Soybean
The goal of research was development of competitive ELISA kit for detection of genetically modified plants resistant to glyphosate. As the source for gene isolation we have used leaves of GM soybean RoundUp Ready, line 40-3-2 (Monsanto). Using polymerase chain reaction the gene encoding the enzyme 5-enolpiruvilshykimat-3-phosphate synthase (CP4 EPSPS) was isolated, which provides resistance to the herbicide glyphosate. Isolated gene has been cloned into plasmid vector pET24(a), creating genetic construct pET-CP4EPSPS. E.coli (BL 21(DE3)) has been transformed by genetic construct and over expressed after induction by 0.5 mM IPTG. Recombinant enzyme CP4 EPSPS with molecular weight 45 kD was purified as inclusion bodies and used as antigen for 96-well polystyrene plates immobilization. Monoclonal antibodies CP4 EPSPS mAb3 (Artron BioResearch Inc., Canada) was used for synthesis of horseradish peroxidase conjugate and receiving diagnostic reagent. We have shown that in the competitive ELISA we can detect at least 0.1% of GM plants resistant to glyphosate, on the example of soybean (line RR 40-3-2), moreover we can also detect rapeseed (line GT73) and sugar beet (line H7-1).
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来源期刊
CiteScore
1.00
自引率
14.30%
发文量
12
期刊介绍: The journal "Methods and objects of chemical analysis" is peer-review journal and publishes original articles of theoretical and experimental analysis on topical issues and bio-analytical chemistry, chemical and pharmaceutical analysis, as well as chemical metrology. Submitted works shall cover the results of completed studies and shall make scientific contributions to the relevant area of expertise. The journal publishes review articles, research articles and articles related to latest developments of analytical instrumentations.
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