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

2002年，我们成功地制备了展示噬菌体zif268，利用它可以直接检测双链DNA的特异性碱基序列。ZIF 268是一种锌指蛋白，是小鼠的一种翻译因子。它特异性识别9个连续碱基对GCGTGGGCG，并以纳摩尔水平的解离常数结合带有该序列的双链DNA。我们试图设计并表达新的锌指蛋白，其识别沙门氏菌（Salmonella）特异性的invA基因中的特异性连续9个碱基对，沙门氏菌是在日本引起食物中毒的最流行的病原菌之一，通过突变识别碱基序列的指基序的碱基序列，在此基础上，我们尝试建立了一种快速、简便的双链DNA荧光检测方法极化法和ZIF 268展示的噬菌体。我们检测到了荧光素 ...更多信息 利用zif268展示的噬菌体，一次性标记了带有zif268靶序列的双链DNA，GCGTGGGCG。利用荧光偏振法可以在溶液中同时观察到两个靶分子之间的结合，而不需要分离结合分子和游离分子，是检测结合的最简单、快速的方法之一。沙门氏菌的检测需要高灵敏度，因为即使是少量细菌也会引起食物中毒，因此需要PCR扩增DNA以获得高灵敏度。然而，PCR产物是双链的，并且携带互补序列的DNA探针与靶基因杂交需要变性。本研究所建立的方法不需要对PCR产物进行预处理，可以直接对PCR产物进行检测，是目前检测病原菌最简便、快速的方法之一。该方法有望广泛应用于不同的细菌。少

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