Design and expression of novel zinc finger protein for the detection of the DNA of pathogenic bacteria

用于检测病原菌 DNA 的新型锌指蛋白的设计和表达

• 批准号: 13660339
• 负责人: IKEBUKURO Kazunori
• 金额: $ 2.3万
• 依托单位: Tokyo University of Agriculture & Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660339/
• 关键词: Zinc linger proiein; Detection of pathogenic bacteria; PCR product; Phage display; Detection of double stranded DNA; Fluorescence polarization; Zif 268; 有機微生物検出; PCR; 分子進化工学; 遺伝的アルゴリズム

In 2002, we succeeded in preparing the zif268 displayed phage with which we can directly detect the specific base sequence of the double stranded DNA. Zif 268 is one of the zinc finger protein and it is a translation factor of mouse. It specifically recognizes the 9 sequential base pairs, GCGTGGGCG and binds the double stranded DNA bearing this sequence with nano molar level dissociation constant. We tried to design and express the new zinc finger protein recognizing the specific sequential 9 base pairs in the invA gene specific to Salmonella which is one of the most popular pathogenic bacteria causing food poisoning in Japan by mutating the base sequence of the finger motif which recognize the base sequence, but the one showing strong and specific binding was not obtained.Then we tried to develop the rapid and simple detection method of the double stranded DNA for the PCR product using fluorescence polarization method and zif268 displayed phage. We were able to detect the fluorescein … More labeled double stranded DNA bearing the target sequence of zif268, GCGTGGGCG by using zif268 displayed phage at once. We can observe the binding between two target molecules with fluorescence polarization method in solution simultaneously without the separation of bound and free molecules so that this method is one of the most simple and rapid method lo detect binding. The detection of Salmonella requires high sensitivity since it can cause food poisoning even a few bacteria, so that the PCR amplification of the DNA is required to obtain high sensitivity. However PCR product is double stranded and denaturing was required for hybridization of the DNA probe bearing complementary sequence to the target gene. By using this method developed in this research project, PCR product can be directly detected without pretreatment, therefore we can say that this is one of the most easy and rapid method to detect pathogenic bacteria. The wide application of this method to different bacteria would be expected. Less

2002年，我们成功制备了zif268展示噬菌体，可以直接检测双链DNA的特定碱基序列。 Zif 268是锌指蛋白之一，是小鼠的翻译因子。它特异性识别 9 个连续碱基对 GCGTGGGCG，并以纳摩尔水平解离常数结合带有该序列的双链 DNA。我们试图通过突变识别沙门氏菌的invA基因中特定的顺序9个碱基对的碱基序列来设计和表达新的锌指蛋白，该蛋白识别沙门氏菌的invA基因中的特定序列9个碱基对，沙门氏菌是日本最常见的引起食物中毒的致病菌之一，但没有获得表现出强而特异性结合的锌指蛋白。然后我们尝试开发一种快速、简单的PCR产物双链DNA检测方法。 荧光偏振法和zif268展示噬菌体。通过使用 zif268 展示的噬菌体，我们能够立即检测到带有 zif268 靶序列 GCGTGGGCG 的荧光素标记的双链 DNA。我们可以用荧光偏振法在溶液中同时观察两个目标分子之间的结合，而无需分离结合分子和游离分子，因此该方法是检测结合最简单、快速的方法之一。沙门氏菌的检测需要高灵敏度，因为即使是少数细菌也会引起食物中毒，因此需要对DNA进行PCR扩增以获得高灵敏度。然而，PCR 产物是双链的，带有与靶基因互补序列的 DNA 探针杂交时需要变性。利用本研究项目开发的方法，无需预处理即可直接检测PCR产物，因此可以说这是检测病原菌最简单、最快速的方法之一。预计该方法将广泛应用于不同的细菌。较少的

项目成果

Kazunori Ikebukuro: "Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase-ayidin conjugate"Biosensors and Bioelectronics. 17(11-12). 1075-1080 (2002)

Kazunori Ikebukuro：“使用吡咯喹啉醌葡萄糖脱氢酶-ayidin 缀合物的电流 DNA 传感器”生物传感器和生物电子学。

Kazunori Ikebukuro: "Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase -avidin conjugate"Biosensors and Bioelectronics. 17(11-12). 1075-1080 (2002)

Kazunori Ikebukuro：“使用吡咯喹啉醌葡萄糖脱氢酶 - 亲和素缀合物的电流 DNA 传感器”生物传感器和生物电子学。

Kazunori Ikebukuro: "Amperometric DNA sensor using the pyrroquincline quinone glucose dehydrogenase - avidin conjugate"Biosensors and Bioelectronics. 17(11-12). 1075-1080 (2002)

Kazunori Ikebukuro：“使用吡咯喹啉醌葡萄糖脱氢酶 - 亲和素缀合物的电流 DNA 传感器”生物传感器和生物电子学。

Kazunori Ikebukuro: "Electrochemical DNA sensor using genetically engineered thermostable pyrroloquinoline quinone glucose dehydrogenase"Electrochemistry. (accepted). (2003)

Kazunori Ikebukuro：“使用基因工程耐热吡咯喹啉醌葡萄糖脱氢酶的电化学 DNA 传感器”电化学。

Kazunori Ikebukuro: "Electrochemical DNA sensor using genetically engineered thermostable pyrroloquinoline quinone glucose dehydrogenase"Electrochemistry. accepted. (2003)

Kazunori Ikebukuro：“使用基因工程耐热吡咯喹啉醌葡萄糖脱氢酶的电化学 DNA 传感器”电化学。