人類β-防御素-2基因轉(zhuǎn)染293細(xì)胞產(chǎn)物抗菌活性的研究
發(fā)布時(shí)間：2018-10-31 19:42
【摘要】： 研究背景：內(nèi)源性抗菌肽(endogenous peptides)是天然免疫的重要介質(zhì)，是炎癥反應(yīng)起保護(hù)作用的重要分子基礎(chǔ)，，它是一種小分子陽離子多肽類物質(zhì)，具有廣譜抗微生物活性。人類防御素根據(jù)半胱氨酸殘基位置及二硫鍵連接方式的不同，可分為α和β防御素兩類。人β-防御素(human beta-defensin，HBD)是一類由34-42個(gè)氨基酸殘基組成的陽離子小肽，分子量較小(3.5-4.5 kDa)，分子內(nèi)含6個(gè)保守的半胱氨酸殘基形成3個(gè)特征性的鏈內(nèi)二硫鍵，具有廣譜抗菌活性，是呼吸道、消化道、泌尿生殖道等粘膜防御系統(tǒng)中的重要介質(zhì)。目前已發(fā)現(xiàn)四種β-防御素，其中hBD-2是1997年在人的銀屑病損皮膚中發(fā)現(xiàn)，它由41個(gè)氨基酸組成，富含陽離子氨基酸和半胱氨酸。通常情況下正常上皮細(xì)胞hBD-2不表達(dá)或者表達(dá)水平很低，但當(dāng)感染時(shí)，HBD-2的表達(dá)可以被TNF-a、IL-1β、LPS及細(xì)菌等多種原炎劑(Proinflammatory agents)所誘導(dǎo)。HBD-2是呼吸道粘膜抗感染的重要分子基礎(chǔ)。肺囊性纖維性病變(Cysitc fibrosis)的病人很容易發(fā)生肺部感染，其發(fā)病機(jī)制與氣道上皮細(xì)胞離子通道調(diào)節(jié)蛋白(CFTR)基因缺陷，導(dǎo)致HBD-2的殺菌作用受到抑制有關(guān)。在后抗生素時(shí)代的今天，耐藥性微生物給人類健康帶來了新的威脅。人防御素獨(dú)特的作用機(jī)理，可能避免致病微生物對(duì)其產(chǎn)生耐藥性，因此具有良好的藥用前景。目前隨著基因工程的進(jìn)展，已有學(xué)者利用原核、真核微生物、植物、COS-7細(xì)胞等研究防御素的表達(dá)。 研究目的：本實(shí)驗(yàn)研究目的評(píng)估HBD-2基因轉(zhuǎn)染293細(xì)胞的培養(yǎng)產(chǎn)物蛋白質(zhì)的抗菌活性。為進(jìn)一步研究HBD-2基因轉(zhuǎn)染支氣管上皮細(xì)胞提供依據(jù)。 研究方法：本實(shí)驗(yàn)利用分子生物學(xué)技術(shù)構(gòu)建HBD-2真核表達(dá)重組質(zhì)粒pcDNA3.1-zeo-HBD-2，經(jīng)脂質(zhì)體介導(dǎo)轉(zhuǎn)染293細(xì)胞，收集培養(yǎng)上清，觀察它對(duì)多種細(xì)菌的抗菌活性。 研究結(jié)果：本實(shí)驗(yàn)通過RT-PCR檢測到轉(zhuǎn)染細(xì)胞中HBD-2基因表達(dá)；并且重組質(zhì)粒轉(zhuǎn)染293細(xì)胞的培養(yǎng)上清的抗菌活性強(qiáng)于對(duì)照組。 研究結(jié)論：HBD-2基因轉(zhuǎn)染293細(xì)胞后培養(yǎng)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 偽 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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346
本文編號(hào)：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 偽 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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
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本文編號(hào)：2303338
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