人类β-防御素-2基因转染293细胞产物抗菌活性的研究
发布时间：2018-10-31 19:42
【摘要】： 研究背景：内源性抗菌肽(endogenous peptides)是天然免疫的重要介质，是炎症反应起保护作用的重要分子基础，，它是一种小分子阳离子多肽类物质，具有广谱抗微生物活性。人类防御素根据半胱氨酸残基位置及二硫键连接方式的不同，可分为α和β防御素两类。人β-防御素(human beta-defensin，HBD)是一类由34-42个氨基酸残基组成的阳离子小肽，分子量较小(3.5-4.5 kDa)，分子内含6个保守的半胱氨酸残基形成3个特征性的链内二硫键，具有广谱抗菌活性，是呼吸道、消化道、泌尿生殖道等粘膜防御系统中的重要介质。目前已发现四种β-防御素，其中hBD-2是1997年在人的银屑病损皮肤中发现，它由41个氨基酸组成，富含阳离子氨基酸和半胱氨酸。通常情况下正常上皮细胞hBD-2不表达或者表达水平很低，但当感染时，HBD-2的表达可以被TNF-a、IL-1β、LPS及细菌等多种原炎剂(Proinflammatory agents)所诱导。HBD-2是呼吸道粘膜抗感染的重要分子基础。肺囊性纤维性病变(Cysitc fibrosis)的病人很容易发生肺部感染，其发病机制与气道上皮细胞离子通道调节蛋白(CFTR)基因缺陷，导致HBD-2的杀菌作用受到抑制有关。在后抗生素时代的今天，耐药性微生物给人类健康带来了新的威胁。人防御素独特的作用机理，可能避免致病微生物对其产生耐药性，因此具有良好的药用前景。目前随着基因工程的进展，已有学者利用原核、真核微生物、植物、COS-7细胞等研究防御素的表达。 研究目的：本实验研究目的评估HBD-2基因转染293细胞的培养产物蛋白质的抗菌活性。为进一步研究HBD-2基因转染支气管上皮细胞提供依据。 研究方法：本实验利用分子生物学技术构建HBD-2真核表达重组质粒pcDNA3.1-zeo-HBD-2，经脂质体介导转染293细胞，收集培养上清，观察它对多种细菌的抗菌活性。 研究结果：本实验通过RT-PCR检测到转染细胞中HBD-2基因表达；并且重组质粒转染293细胞的培养上清的抗菌活性强于对照组。 研究结论：HBD-2基因转染293细胞后培养上清有一定的抗菌活性。
[Abstract]:Background: endogenous antimicrobial peptide (endogenous peptides) is an important medium for innate immunity and an important molecular basis for the protection of inflammatory reaction. It is a small molecular cationic polypeptide with broad spectrum antimicrobial activity. Human defensins can be classified into 伪 and 尾 defensins according to the position of cysteine residues and the disulfide bond connection. Human 尾 -defensin (human beta-defensin,HBD) is a class of small cationic peptides consisting of 34-42 amino acid residues with a relatively small molecular weight (3.5-4.5 kDa),). There are six conserved cysteine residues in the molecule to form three characteristic disulfide bonds in the chain, which have broad spectrum antibacterial activity and are important mediators in mucosal defense system such as respiratory tract, digestive tract, urogenital tract and so on. Four 尾 -defensins have been found, among which hBD-2 was found in human psoriatic lesions in 1997. It consists of 41 amino acids and is rich in cationic amino acids and cysteine. In general, normal epithelial cells do not express hBD-2 or the expression level is very low, but when infected, the expression of HBD-2 can be expressed by TNF-a,IL-1 尾. HBD-2 is an important molecular basis for anti-infection of respiratory tract mucosa induced by LPS and (Proinflammatory agents), a variety of proinflammatory agents. Pulmonary infection is easy in (Cysitc fibrosis) patients with cystic pulmonary fibrosis, and its pathogenesis is related to the deficiency of (CFTR) gene in airway epithelial cells, which results in the inhibition of bactericidal effect of HBD-2. In the post-antibiotic era, drug-resistant microorganisms pose a new threat to human health. The unique mechanism of human defensins may avoid the resistance of pathogenic microorganisms to them, so it has a good medicinal prospect. At present, with the development of genetic engineering, researchers have used prokaryotes, eukaryotes, plants, COS-7 cells to study the expression of defensins. Objective: to evaluate the antimicrobial activity of protein in 293 cells transfected with HBD-2 gene. It provides the basis for further study of HBD-2 gene transfection into bronchial epithelial cells. Methods: the eukaryotic expression plasmid pcDNA3.1-zeo-HBD-2, of HBD-2 was constructed by molecular biology technique and transfected into 293 cells by liposome. The supernatant was collected and cultured to observe its antibacterial activity against a variety of bacteria. Results: the expression of HBD-2 gene in the transfected cells was detected by RT-PCR, and the antibacterial activity of the supernatant of the recombinant plasmid transfected 293 cells was stronger than that of the control group. Conclusion: the supernatant of HBD-2 gene transfected 293 cells has certain antibacterial activity.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2303338
[Abstract]:Background: endogenous antimicrobial peptide (endogenous peptides) is an important medium for innate immunity and an important molecular basis for the protection of inflammatory reaction. It is a small molecular cationic polypeptide with broad spectrum antimicrobial activity. Human defensins can be classified into 伪 and 尾 defensins according to the position of cysteine residues and the disulfide bond connection. Human 尾 -defensin (human beta-defensin,HBD) is a class of small cationic peptides consisting of 34-42 amino acid residues with a relatively small molecular weight (3.5-4.5 kDa),). There are six conserved cysteine residues in the molecule to form three characteristic disulfide bonds in the chain, which have broad spectrum antibacterial activity and are important mediators in mucosal defense system such as respiratory tract, digestive tract, urogenital tract and so on. Four 尾 -defensins have been found, among which hBD-2 was found in human psoriatic lesions in 1997. It consists of 41 amino acids and is rich in cationic amino acids and cysteine. In general, normal epithelial cells do not express hBD-2 or the expression level is very low, but when infected, the expression of HBD-2 can be expressed by TNF-a,IL-1 尾. HBD-2 is an important molecular basis for anti-infection of respiratory tract mucosa induced by LPS and (Proinflammatory agents), a variety of proinflammatory agents. Pulmonary infection is easy in (Cysitc fibrosis) patients with cystic pulmonary fibrosis, and its pathogenesis is related to the deficiency of (CFTR) gene in airway epithelial cells, which results in the inhibition of bactericidal effect of HBD-2. In the post-antibiotic era, drug-resistant microorganisms pose a new threat to human health. The unique mechanism of human defensins may avoid the resistance of pathogenic microorganisms to them, so it has a good medicinal prospect. At present, with the development of genetic engineering, researchers have used prokaryotes, eukaryotes, plants, COS-7 cells to study the expression of defensins. Objective: to evaluate the antimicrobial activity of protein in 293 cells transfected with HBD-2 gene. It provides the basis for further study of HBD-2 gene transfection into bronchial epithelial cells. Methods: the eukaryotic expression plasmid pcDNA3.1-zeo-HBD-2, of HBD-2 was constructed by molecular biology technique and transfected into 293 cells by liposome. The supernatant was collected and cultured to observe its antibacterial activity against a variety of bacteria. Results: the expression of HBD-2 gene in the transfected cells was detected by RT-PCR, and the antibacterial activity of the supernatant of the recombinant plasmid transfected 293 cells was stronger than that of the control group. Conclusion: the supernatant of HBD-2 gene transfected 293 cells has certain antibacterial activity.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
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