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作者：段明珠集美大学

学位级别：硕士

抗菌肽(antimicrobial peptide,AMP)是具有抗微生物活性的多肽,也是生物体重要先天免疫因子之一,具有重要的理论研究价值及应用前景。本文从日本鳗鲡(Anguilla japonica)中克隆鉴定了3类共5条AMP基因序列,并研究了在正常养殖的日本鳗鲡... 详细信息

抗菌肽(antimicrobial peptide,AMP)是具有抗微生物活性的多肽,也是生物体重要先天免疫因子之一,具有重要的理论研究价值及应用前景。本文从日本鳗鲡(Anguilla japonica)中克隆鉴定了3类共5条AMP基因序列,并研究了在正常养殖的日本鳗鲡体内的表达模式,及受外源物LPS、PolyI:C和迟缓爱德华氏菌(Edwardsiella tarda)的刺激下,该5个基因在多个组织器官中的转录表达量的变化。该研究将为揭示日本鳗鲡抗菌先天免疫机制积累有益资料。1、日本鳗鲡肝脏表达抗菌肽-2(AjLEAP-2):本研究应用RACE方法克隆获得AjLEAP-2基因。其成熟肽含有LEAP-2保守基序“C-X5-C-X4-C-X4-C”,基因组结构与其他脊椎动物LEAP-2相同,都包含有三个外显子。该基因在黑仔鳗的肝脏中转录表达量最高,其次是肠,但是,后者的表达量仅为肝脏的1/130;在玻璃鳗中AjLEAP-2基因的转录表达量约为黑仔鳗肠道的2倍。LPS和***刺激能显著上调鳗鲡血液中AjLEAP-2基因的表达,刺激16 h后分别上调86倍和12倍(Pdefensin):本研究克隆获得2个β-defensin基因,Ajdefensin1和Aj-defensin2,两者氨基酸相似度为25.3%。其成熟肽部分都含有脊椎动物β-defensin高度保守的基序C-X5-C-X3-C-X11-C-X5-CC。Ajdefensin基因组结构与其他鱼类β-defensin相同,都具有三个外显子。该两基因的转录表达模式不同,Ajdefensin1主要在黑仔鳗的肝脏中转录表达,Ajdefensin2则在鳔中的转录表达量最高。此外,Ajdefensin1在玻璃鳗中的转录表达量仅次于黑仔鳗的肝脏,而Ajdefensin2的表达量约为Ajdefensin1表达量的一半,***浸泡感染玻璃鳗后,Aj-defensin 2在12 h显著性下调(Pdefensin1基因的表达,分别在8 h和16 h显著上调5倍(Pdefensin1基因7倍;LPS刺激后,在肠中8 h和16 h,胃中72 h显著下调(Pdefensin2经Poly I:C诱导后在皮肤中显著上调。据实验结果推测,Ajdefensin基因在日本鳗鲡抗细菌和病毒感染过程中起有十分重要的作用。3、日本鳗鲡杀菌/渗透增强蛋白(AjBPI):本研究克隆获得2个BPI基因,AjBPI1和AjBPI2,两者具有44.7%的相似度。AjBPI1不具有信号肽,AjBPI2具有19个氨基酸的信号肽。两者从N-端到C-端均含有脊椎动物中保守的结构域,依次是:LPS结合结构域;一对二硫键;脯氨酸富集中心结构域。检测AjBPI基因在日本鳗鲡中的组织分布,发现两者在日本鳗鲡的组织/器官中表达模式相似,具有组织特异性,均在储存血液多的器官中高表达,其中AjBPI1在黑仔鳗组织/器官中表达量由高到低依次:肝脏,中肾,脾脏,头肾,皮肤,血液,肠,鳃,心脏,胃,鳔,性腺。AjBPI2在中肾,头肾,血液中的表达量远高于其他组织。检测AjBPI基因在玻璃鳗阶段的转录表达水平,结果显示两者表达量都较低。LPS刺激能诱导AjBPI1在中肾中显著性上调,头肾中显著下调(P<0.05),诱导AjBPI2在鳃和中肾的显著性上调;Poly I:C刺激能诱导AjBPI1脾脏、中肾中显著性上调,诱导AjBPI2在血液、鳃、脾脏、中肾中显著上调(P<0.05);***刺激能诱导AjBPI1在中肾和脾脏中显著上调,诱导AjBPI2在脾脏,血液和鳃中显著上调(P<0.05)。体外刺激外周血白细胞实验显示,PolyI:C刺激4 h和6 h后AjBPI1上调表达近200倍,AjBPI2上调近20倍,据实验结果推测,AjBPI基因在日本鳗鲡抗细菌和病毒感染过程中起有十分重要的作用。

关键词：日本鳗鲡抗菌肽 LEAP-2 β-defensin BPI

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爬行纲β-defensin基因多态性分析及基于真核表达的生物学功能验证

爬行纲β-defensin基因多态性分析及基于真核表达的生物学功能验证

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作者：刘雪莲大连理工大学

学位级别：硕士

defensin是宿主防御肽(hose defense peptides,HDPs)家族的重要成员之一,广泛存在于几乎所有物种,包括脊椎动物、无脊椎动物、昆虫、植物甚至微生物。defensins除了具有直接的杀菌功能外,还具有免疫调节功能,是连接宿主自然免疫和获得... 详细信息

defensin是宿主防御肽(hose defense peptides,HDPs)家族的重要成员之一,广泛存在于几乎所有物种,包括脊椎动物、无脊椎动物、昆虫、植物甚至微生物。defensins除了具有直接的杀菌功能外,还具有免疫调节功能,是连接宿主自然免疫和获得性免疫的桥梁。目前,defensin成为少数几类进入临床研究的肽类抗生素之一,该家族的PMX-30063已经完成了系统性感染适应症的Ⅰ期临床,金葡菌引起的急性细菌性皮肤感染适应症也已完成临床Ⅱ期。爬行纲物种的肽类分子由于活性强、效价高,成为了先导模板发掘的重要来源。而爬行纲defensin却鲜有报道,且仅限于基因克隆,缺少相应的功能验证。本研究在NCBI爬行纲数据库中定向发掘得到了26条全新的β-defensins序列,属于爬行纲龟鳖目鳖科中华鳖属(Pelodiscus sinensis),分别命名为Ps-BD16。Ps-BDs具有β-defensin家族共有的特征:其前体由高度保守的信号肽区域,极短的中间间隔区以及成熟肽区域组成。尽管Ps-BDs成熟肽区是高度变异的,但仍含有β-defensin家族6个保守的Cys残基以及GXC基序。通过构建的系统发育树分析得出β-defensin是一类高速率序列替换的进化速率较快的基因,而多重基因复制是导致其成熟肽区域高度差异性的原因。随后对Ps-BDs的组织表达谱及结构进行研究,试图解析β-defensin在宿主体内可能参与的防御反应及行使生物学功能的结构基础。通过RT-PCR获得了Ps-BD2、Ps-BD6、Ps-BD11、Ps-BD16以及Ps-BD21的组织表达谱,结果显示:在与外源性物质发生直接接触的器官(皮肤,肺,胃)以及与机体免疫相关的器官(肝脏,肾脏,小肠)中Ps-BDs的表达量均较高。利用Rosetta ab initio对5条代表性Ps-BDs同源建模,揭示了Ps-BDs主要含有β-片层结构,少数还含有两亲性的α-helix结构,而这两种二级结构恰是β-defensin家族行使生物学功能的结构基础。最后,我们构建了毕赤酵母真核表达载体,对Ps-BD2进行了高效表达,纯化后得到重组蛋白rPs-BD2,并进行了生物学功能验证。结果表明rPs-BD2具有广谱抗菌活性,相较于革兰氏阴性菌以及真菌,其对革兰氏阳性菌的活性更强。同时rPs-BDs对正常细胞几乎没有毒性,对红细胞无溶血性。进一步证明了defensin在宿主对抗外界病原微生物感染起重要作用,同时rPs-BD2在未来的临床应用上也具有一定的指导意义。

关键词：爬行纲 β-defensin 基因多态性真核表达生物学功能

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鳜鱼β-defensin成熟肽在毕赤酵母中的重组DNA表达

鳜鱼β-defensin成熟肽在毕赤酵母中的重组DNA表达

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2018年中国水产学会学术年会

作者：吕星星陶妍上海海洋大学食品学院

defensin主要分布于水生生物的脾脏中,是一种富含半胱氨基酸的阳离子小分子抗菌肽,具有独特的抗菌机制和广谱的抗菌、抗病毒活性以及免疫调节功能,因此,被广泛应用于新药研发和免疫疗法。根据defensin的空间结构可以将其分为不同的类型... 详细信息

defensin主要分布于水生生物的脾脏中,是一种富含半胱氨基酸的阳离子小分子抗菌肽,具有独特的抗菌机制和广谱的抗菌、抗病毒活性以及免疫调节功能,因此,被广泛应用于新药研发和免疫疗法。根据defensin的空间结构可以将其分为不同的类型,本研究以鳜鱼（Siniperca chuatsi）的成熟肽Sc BD为研究对象,通过RT-PCR从鳜鱼脾脏中克隆到编码ScBD的c DNA,将其与表达载体pPICZαA连接,并构建重组表达载体pPICZαA-ScBD,然后电转至毕赤酵母（Pichia pastoris）X-33中,得到的阳性转化子在28℃、250 r/m的条件下,经1.0%甲醇诱导表达96h后,获得的表达产物经Tricine-SDS-PAGE分析,重组蛋白的分子量约为6.345 kDa;经MALDI-TOF-TOF质谱鉴定显示,该重组体蛋白为预期的目的蛋白。

关键词：鳜鱼 β-defensin 重组DNA表达毕赤酵母

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Isoleucine, an Essential Amino Acid, Induces the Expression of Human <i>β</i>defensin 2 through the Activation of the G-Protein Coupled Receptor-ERK Pathway in the Intestinal Epithelia

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Food and Nutrition Sciences 2012年第4期3卷 548-555页

作者： Youkou Konno Toshifumi Ashida Yuhei Inaba Takahiro Ito Hiroki Tanabe Atsuo Maemoto Tokiyoshi Ayabe Yusuke Mizukami Mikihiro Fujiya Yutaka Kohgo Division of Gastroenterology and Hematology/Oncology Department of Medicine Asahikawa Medical University Asahikawa Japan IBD Center Sapporo Higashi Tokushukai Hospital Sapporo Japan Innate Immunity Laboratory Department of Cellular Life Science Faculty of Advanced Life Science Hokkaido University Sapporo Japan

Anti-microbial peptides are essential for the intestinal innate immunity that protects the intestinal epithelia from attacks by foreign pathogens. Human β-defensin (HBD) is one of the pivotal anti-microbial peptides that are expressed in the colonic epithelia. This study investigated the effect and the signaling mechanism of inducible β-defensin HBD2 by an essential amino acid, isoleucine (Ile) in colonic epithelial cells. Here we examined the expression level of HBD2 on induction of Ile in epithelial cells, and checked this pathway. HBD2 mRNA was induced by co-incubation with IL-1α and Ile in Caco2 cells, but not by Ile alone. An inhibitor of either ERK or Gi, a subunit of G-proteins, reduced the induction of HBD2 mRNA by Ile. The treatment with Ile also increased the intracellular calcium ion concentration, thus suggesting that the GPCR and ERK signaling pathway mediate the effects of Ile. These results indicate that an essential amino acid, Ile, enhances the expression of an inducible β-defensin, namely HBD2, by IL-1α through the activation of GPCRs and ERK signaling pathway. The administration of Ile may therefore represent a possible option to safely treat intestinal inflammation.

关键词： Isoleucine Human β-defensin G-Protein Coupled Receptor Extracellular Signal-Regulated Kinases Pathway Inflammatory Bowel Disease Crohn’s Disease

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An Effective Method for Raising Antisera Against β-defensins: Double-copy Protein Expression of mBin1b in E. coli

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 571-576页

作者： Li-Qing XIAO, Ai-Hua LIU, and Yong-Lian ZHANG State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

Bin1b is a rat epididymis specific β-defensin which may have fertility related functions in addition to its antimicrobial activity. β-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new β-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of β-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the β-defensin gene family.

关键词： Bin1b epididymis β-defensin double-copy protein polyclonal antiserum

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DICER1 regulates antibacterial function of epididymis by modulating transcription of p-defensins

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Journal of Molecular Cell Biology 2019年第5期11卷 408-420页

作者： Chunhua Tang Minjie Ni Shengsong Xie Yao Zhang Chaobao Zhang Zimei Ni Chen Chu Ligang Wu Yuchuan Zhou Yonglian Zhang State Key Laboratory of Molecular Biology Shanghai Key Laboratory of Molecular Andrology CAS Center for Excellence in Molecular Cell Science Shanghai Institute of Biochemistry and Cell Biology Chinese Academy of Sciences University of Chinese Academy of Sciences Shanghai 200031 China

DICER1 is a key enzyme responsible for the maturation of microRNAs. Recent evidences suggested that DICER1 and microRNAs expressed in epididymis were involved in the control of male fertility. However, the exact mechanism remains to be elucidated. Here, we created a mouse line by targeted disruption of Dicerl gene in the principal cells of distal caput epididymis. Our data indicated that a set of β-defensin genes were downregulated by DICER1 rather than by microRNAs. Moreover, DICER1 was significantly enriched in the promoter of β-defensin gene and controlled transcription. Besides, the antibacterial ability of the adult epididymis significantly declined upon Dicerl deletion both in vitro and in vivo. And a higher incidence of reproductive defect was observed in middle-aged Dicerl^-|- males. These results suggest that DICER1 plays an important role in transcription of β-defensin genes, which are associated with the natural antibacterial properties in a microRNA-independent manner, and further impacts the male fertility.

关键词： epididymis antibacterial property DICER1 β-defensin

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Concentrations of α-and β-defensins in gastric juice of patients with various gastroduodenal diseases

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World Journal of Gastroenterology 2005年第1期11卷 99-103页

作者： YoshitoNishi HajimeIsomoto HiroshiMukae HiroshiIshimoto Chun-YangWen AkihiroWada KenOhnita YoheiMizuta IkuoMurata ToshiyaHirayama MasamitsuNakazato ShigeruKohno SecondDepartmentofInternalMedicine NagasakiUniversitySchoolofMedicineSakamoto1-7-1NagasakiJapan DepartmentofMolecularPathology AtomicBombDiseaseInstituteNagasakiUniversitySchoolofMedicineSakamoto12-4NagasakiJapan DepartmentofBacteriology InstituteofTropicalMedicineNagasakiUnive~itySchoolofMedicineSakamoto12-4NagasakiJapan ThirdDepartmentofInternalMedicine MiyazakiMedicalCollegeKiyotakeMiyazakiJapan

AIM: To determine the concentration of α-and β-defensins in gastric juice of patients with various gastroduodenal diseases. METHODS: Concentrations of human neutrophil peptides (HNPs) 1-3, the major forms of α-defensins, and human β-defensin (HBD)-1and HBD-2were measured by radioimmunoassay in plasma and gastric juice of 84 subjects, consisting of 54 Helicobacter pylori-infected and 30 uninfected subjects. They included 33 patients with chronic gastritis (CG), 12 with gastric ulcer (GU), 11 with duodenal ulcer (DU), 11 with benign gastric polyp (BGP) and 16 with normal mucosa (N group) on upper endoscopy. Plasma pepsinogen Ⅰ and Ⅱ levels, biomarkers for gastric mucosal inflammation and atrophy, were also measured. RESULTS: Gastric juice HNPs 1-3 levels in patients with CG, GU and BGP were significantly higher than those in patients with DU and N. Gastric juice HBD-2 concentrations in patients with CG and GU were significantly higher than those in the N group, but were significantly lower in DU patients than in GU patients. Gastric juice HBD-1 levels and plasma levels of these peptides were similar in the patient groups. Concentrations of gastric juice HNPs 1-3 and HBD-2 of in H pylori-infected patients were significantly different from those in uninfected subjects. HNPs 1-3 concentrations in gastric juice correlated negatively with plasma pepsinogen I levels and Ⅰ/Ⅱ ratios. HBD-2 levels in gastric juice correlated positively and negatively with plasma pepsinogen Ⅱ concentrations and Ⅰ/Ⅱratios, respectively. CONCLUSION: HNPs 1-3 and HBD-2 levels in gastric juice are diverse among various gastrointestinal diseases, reflecting the inflammatory and atrophic events of the background gastric mucosa affected by H pylori.

关键词： Helicobacter pylori infection Gastroduodenal diseases α-defensins β-defensin

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Downregulation of epididymal β-defensins expression and inhibition of sperm motility in epididymitis of rat by lipopolysaccharide

Downregulation of epididymal β-defensins expression and inh...

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中国病理生理学会受体、肿瘤和免疫专业委员会联合学术会议

作者： Dongmei Cao~1,Yidong Li~1,Rui Yang~1,Yan Wang~1,Yuchuan Zhou~2,Hua Diao~2,Yue Zhao~2,Yonglian Zhang~(2*),Jian Lu~(1*) 1 Department of Pathophysiology,Second Military Medical University,Shanghai,China 2 State Key Laboratory of Molecular Biology,Institute of Biochemistry & Cell Biology,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Shanghai,China

Although there are more than 40β-defensins identified in rat epididymis,little is known about their regulation and their relation to male infertility caused by *** a rat model of epididymitis induced by lipopolysaccharide(LPS),we examined thirteen epididymalβ-defensins expressed in caput segment and found ten out of them were *** downregulated expression of Bin1b was accompanied with a significant decrease in both sperm binding to Bin1b and sperm motility,and supplement of the spermatozoa with recombinant Bin1b in vitro could remarkably rescued sperm motility. Further experiment in primary cultured epithelial cells from caput epididymis of rat demonstrated that expression of Bin1b mRNA was not affected by LPS,estrogen or glucocorticoid,but partially upregulated by *** findings demonstrated that LPS-induced epididymitis can decrease the expression of epididymalβ-*** them the downregulation of Bin1b expression was involved in the impairment of sperm motility.

关键词： Bin1b epididymis β-defensin gene expression LPS sperm motility

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