全新抗菌肽基因的功能及相关研究
发布时间：2018-10-04 18:33
【摘要】：根据本实验室首先克隆的mL52基因(GenBank：AY028425)序列设计引物，以人胎肝cDNA文库为模板，通过PCR扩增获得了人类同源新基因，因其富含甘氨酸而命名为—hGlyrichin。在生物信息学分析提示其为抗菌肽和初步证实该基因具有抗菌作用的基础上，为了寻找该蛋白序列的核心功能区段，进一步借助生物信息学分析结果，分段合成了系列肽段并对这些肽段的抗菌活性进行了测试，结果证实，从第42到60位氨基酸的19个氨基酸(pCM-19)为该蛋白的核心功能区。为此，我们对pCM-19的抗菌活性、抗菌作用机理及抗菌肽的普遍副作用进行了系统研究。实验结果证实，pCM-19不仅具有抗实验室工程菌的活性，而且具有抗鼠疫杆菌、伤寒沙门氏菌、绿脓杆菌和脑膜炎球菌的作用。更可喜的是该肽段具有抗携带氨苄青霉素抗性基因的实验室工程菌和抗耐甲氧西林金黄色葡萄球菌的活性，提示pCM-19具有广谱抗菌作用并为该抗菌肽的应用提供了有价值的实验数据。pCM-19的量效关系测定发现，本实验室所获得的曲线与Sunkyu Kim等人报道的gaegurin 6(GGN 6)的量效关系曲线类似，即pCM-19需达到一定浓度才能发挥抗菌作用。pCM-19抗菌活性的稳定性研究发现，，该肽段经100℃加热30min和60min及反复冻融3次，其抗菌活性完全不受影响；溶液状态在4℃放置80天，抗菌活性仍可保留98％以上；经胰蛋白酶处理60min后，抗菌活性保留30％。这些实验结果表明，pCM-19具有良好的稳定性，也为其发展成药提供了重要实验数据。 为了研究该肽段的抗菌作用机理，采用流式荧光激活细胞分析(FACScan)和扫描电子显微镜分析了pCM-19对靶细菌膜完整性的影响。两种方法所得结果一致提示，pCM-19可能作用于细菌的细胞壁和／或细胞膜，通过破坏膜的完整性并进而导致靶细菌胞质内容物外泄而发挥杀菌作用。这与目前已知阳离子抗菌肽的主要作用机制类似。 抗菌肽的溶血作用是抗菌肽常见的副作用，也是某些抗菌肽发展成药的主要障碍。为此，本文对pCM-19的溶血作用进行了体内、外测试，结果证实，pCM-19在体外无溶解人红细胞的作用，在体内无溶解鼠红细胞的作用。
[Abstract]:According to the sequence of mL52 gene (GenBank:AY028425) first cloned in our laboratory, primers were designed and human fetal liver cDNA library was used as template. A new homologous human gene was obtained by PCR amplification, which was named -hGlyrichinin because it was rich in glycine. On the basis of bioinformatics analysis and preliminary confirmation that the gene has antimicrobial effect, in order to find the core functional region of the protein sequence, the results of bioinformatics analysis were further used. A series of peptide segments were synthesized and their antibacterial activities were tested. The results showed that 19 amino acids (pCM-19) from the 42nd to 60th amino acids were the core functional regions of the protein. Therefore, we systematically studied the antimicrobial activity, antibacterial mechanism and common side effects of antimicrobial peptides of pCM-19. The results showed that PCM-19 not only had the activity of anti-engineering bacteria in laboratory, but also had anti-Yersinia pestis, Salmonella typhimurium, Pseudomonas aeruginosa and meningococci. What is more gratifying is that the peptide has the activity of anti-ampicillin resistant genes in laboratory engineering bacteria and methicillin-resistant Staphylococcus aureus. The results indicated that pCM-19 had broad spectrum antibacterial activity and provided valuable experimental data. PCM-19 showed that the curve obtained in our laboratory was similar to that of gaegurin 6 (GGN 6) reported by Sunkyu Kim et al. It was found that the antibacterial activity of the peptide was completely unaffected by heating 30min and 60min at 100 鈩
本文编号：2251439
[Abstract]:According to the sequence of mL52 gene (GenBank:AY028425) first cloned in our laboratory, primers were designed and human fetal liver cDNA library was used as template. A new homologous human gene was obtained by PCR amplification, which was named -hGlyrichinin because it was rich in glycine. On the basis of bioinformatics analysis and preliminary confirmation that the gene has antimicrobial effect, in order to find the core functional region of the protein sequence, the results of bioinformatics analysis were further used. A series of peptide segments were synthesized and their antibacterial activities were tested. The results showed that 19 amino acids (pCM-19) from the 42nd to 60th amino acids were the core functional regions of the protein. Therefore, we systematically studied the antimicrobial activity, antibacterial mechanism and common side effects of antimicrobial peptides of pCM-19. The results showed that PCM-19 not only had the activity of anti-engineering bacteria in laboratory, but also had anti-Yersinia pestis, Salmonella typhimurium, Pseudomonas aeruginosa and meningococci. What is more gratifying is that the peptide has the activity of anti-ampicillin resistant genes in laboratory engineering bacteria and methicillin-resistant Staphylococcus aureus. The results indicated that pCM-19 had broad spectrum antibacterial activity and provided valuable experimental data. PCM-19 showed that the curve obtained in our laboratory was similar to that of gaegurin 6 (GGN 6) reported by Sunkyu Kim et al. It was found that the antibacterial activity of the peptide was completely unaffected by heating 30min and 60min at 100 鈩
本文编号：2251439
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2251439.html
最近更新
教材专著
