幽门螺杆菌抗生素耐药基因的分子检测及分子对接分析
Abstract 摘要
Aim:
To explore the mutation characteristics of H. pylori resistance-related genes to antibiotics of clarithromycin (CAM), levofloxacin (LVX) and metronidazole.
目的：探讨幽门螺杆菌对克拉霉素(CAM)、左氧氟沙星(LVX)和甲硝唑等抗生素耐药相关基因的突变特点。Methods:23S rRNA, gyrA, gyrB, rdxA, and frxA genes were amplified and sequenced, respectively. Molecular docking study was performed to explore molecular interactions between chemotherapeutic agents and target proteins. What's more, we performed natural transformation with some of these mutated DNA fragments to see if they really impact susceptibility to antibiotics.方法：分别对23S rRNA、gyrA、gyrB、rdxA和frxA基因进行扩增和测序。使用分子对接探讨抗生素药物与靶蛋白之间的分子相互作用。更重要的是，本研究用其中一些突变的DNA片段进行了自然转化实验，看看它们是否真的会影响对抗生素的敏感性。
Results: In the CAM-resistant strains, the mutation rate in site A2143G was 74.2% (n = 23). The interactions in sites of G1949A, C1953T, and G2211T with CAM were weaker after mutation. In the LVX-resistant strains, the mutation rates in 87 (N to K/I) and 91 (D to N/Y/G) of gyrA were 28.6% (n = 16) and 12.5% (n = 7), respectively. We could infer by docking studies that N87 K/I, D91Y/G/N, D99N, and D143E mutations in GyrA protein all had weakened interaction with LVX. The mutation types of RdxA protein consisted of protein truncation caused by premature stop codons (n = 26, 33.3%), frameshift mutations (n = 8, 10.3%), FMN-binding sites (n = 16, 20.5%), and the others (n = 11, 14.1%). Docking analysis showed that some mutations in those four types of RdxA protein could reduce interaction with MNZ, and play a significant role in antibiotic resistance. 结果：在CAM耐药菌株中，A2143G位点的突变率为74.2%（n=23）。G1949A、C1953T和G2211T的位点突变后与CAM的相互作用较弱。在LVX耐药株中，gyrA的87（N～K/I）和91（D～N/Y/G）的突变率分别为28.6%（n＝16）和12.5%（n＝7）。我们通过分子对接研究推断，GyrA蛋白的N87 K/I、D91Y/G/N、D99N和D143E突变都与LVX的相互作用减弱。RdxA蛋白的突变类型包括过早出现终止密码子引起的蛋白截断（n = 26，33.3%）、框移突变（n = 8，10.3%）、FMN结合位点（n = 16，20.5%）和其他（n = 11，14.1%）。分子对接分析表明，这4种类型的RdxA蛋白的一些突变可以减少与MNZ的相互作用，并在抗生素耐药性中发挥重要作用。
Conclusion: Our study suggested that in addition to the reported mutation sites, H. pylori antibiotic resistance in this region may also be associated with changes in some new sites. Our study provides novel ideas and methods for the identification of H. pylori resistancerelated mutations, and also clues and basis for further investigation on specific molecular mechanism.结论：我们的研究表明，除了已报道的突变位点外，幽门螺杆菌抗生素耐药性还可能与一些新位点的变化有关。我们的研究为幽门螺杆菌耐药相关突变的鉴定提供了新的思路和方法，也为进一步研究具体的分子机制提供了线索和依据。
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