家蝇幼虫抗菌肽Defensins基因的克隆与序列分析

目的克隆家蝇幼虫的抗菌肽defensins基因并对其进行序列分析。方法提取家蝇幼虫总RNA,根据家蝇成虫defensins基因cDNA序列设计一对引物,RT-PCR扩增目的基因片段,T-A克隆后测序,应用相关生物信息学软件对该序列进行分析。结果RT-PCR扩增出一条约310bp的目的片段,序列分析表明,其与家蝇成虫防御素基因同源性高达97%,核酸序列变异主要发生在信号肽区域。结论成功克隆及分析了家蝇幼虫防御素基因全长编码区cDNA序列,为其下一步的重组表达和生物活性研究奠定了基础。

大劣按蚊感染约氏疟原虫defensins基因的克隆及生物信息学分析

目的为探讨大劣按蚊对约氏疟原虫侵入的免疫反应,克隆大劣按蚊的抗菌肽defensins基因并对基因序列进行生物信息学分析。方法分别提取感染疟原虫的大劣按蚊的蚊胃总RNA,根据冈比亚按蚊defensins基因设计、合成简并引物,进行RT-PCR扩增目的片段,TA克隆后测序并进行序列分析。结果用简并引物的RT-PCR扩增出约氏疟原虫感染的大劣按蚊的189bp目的片段,序列分析表明与冈比亚按蚊defensins有87%同源性,与白蚊伊蚊defensinsD有88%同源性,为大劣按蚊的defensins基因。结论成功克隆并分析了大劣按蚊抗菌肽defensins基因,为进一步的重组表达和研究按蚊抗菌肽的生物活性奠定基础。

Defensins couple dysbiosis to primary immunodeficiency in Crohn's disease

Antimicrobial peptides,including defensins,are essential effectors in host defence and in the maintenance of immune homeostasis.Clinical studies have linked the defective expression of both α-and β-defensin to the reduced killing of certain microorganisms by the intestinal mucosa of patients suffering from ileal and colonic Crohn's disease(CD),respectively.Only recently have the events leading to defective expression of defensins in CD been further elucidated,and are discussed herein.These events may account for CD-associated alterations in the microbiome and may subsequently precipitate the development of granulomatous inflammatory lesions in genetically-predisposed patients.We also address how these discoveries may pave the way for the development of a molecular medicine aimed at restoring gut barrier function in CD.

Defensins：动物细胞的内源性抗菌肽──一种极具开发潜力的眼科抗感染药物

中性粒细胞重要组成成分之一的Ｄｅｆｅｎｓｉｎｓ，在体外呈现了强烈的抗细菌、病毒、真菌和肿瘤细胞等多种生物学特性。本文综述了Ｄｅｆｅｎｓｉｎｓ自被分离纯化以来人们对其结构、功能、作用机理、体内调节机制等方面的研究现状。

Regulatory role of defensins in inflammatory bowel disease

Defensins are endogenous antimicrobial peptides (AMPs) produced by professional phago- cytes, Paneth cells, and epithelial cells at mucosal surfaces, which mediate innate immunity through their potent antimicrobial activity in the intestinal tract. In addition, defensins also regulate the function of diverse host immune cells, thereby play an important role in both innate and adaptive immune responses against pathogenic microbes. Abundant evidences have proved that attenuated changes in defensins expression are observed in inflammatory bowel disease (IBD). Further studies have discovered the concentration of different defensins subgroups has the relationship with various clinical characteristics of IBD and that mucosal surface destruction causes defensins deficiency as a result of in- flammatory damage. This article is to review new current approaches on defensins expression in the intestinal mucosa, particularly in IBD and their potential roles in immune responses in the gut mucosa.

Human Beta Defensins 1,2 and 3 Produced by Amniochorion Membranes Is Similar in Term and Preterm Delivery

Amniochorion membranes were collected from 25 pregnant women at preterm labor, in the presence or not of Preterm Premature Rupture of Membranes (PPROM) and 27 pregnant women at term in the presence at labor, in order to quantify the expression and to evaluate the immunoreactivity of human beta defensins (HBD)1, HBD2, HBD3 and HBD4 in amniochorion membranes from pregnancies complicated by spontaneous prematurity. The HBDs were evaluated by immunohistochemistry, real time quantitative PCR and ELISA. Statistical analyses were performed using Chi-squared and Mann Whitney tests. There was no significant difference in HBDs expression between study and control groups: HBD1 (Md = 0.62 (0.0 - 105.0) vs Md = 0.80 (0.02 - 25.0);p = 0.85), HBD2 (Md = 0.17 (0.0 - 5.2) vs Md = 0.0 (0.0 - 43.2);p = 0.16), HBD3 (Md = 0.11 (0.0 - 140.5) vs Md = 0.06 (0.0 - 972.1);p = 0.91). Also, HBD1, HBD2 and HBD3 protein expression was not significant different between the groups: HBD1 (1.32 pg/mL (0.0 - 1.85) vs 1.08 pg/mL (0.04 - 2.22);p = 0.67), HBD2 (0.00 pg/mL (0.0 - 1.74) vs 0.02 pg/mL (0.0 - 1.24);p = 0.69), HBD3 (0.04 pg/mL (0.0 - 1.05) vs 0.09 pg/mL (0.0 - 1.05);p = 0.63). The immunoreactivity of HBD1, HBD2 and HBD3 was observed in amnion, chorion and decidua cells from preterm and term pregnancies. Amniochorion membranes are sources of HBD1, HBD2 and HBD3 and their expressions are similar in term and preterm pregnancies.

Characterization and functions of beta defensins in the epididymis

The epididymal β-defensins have evolved by repeated gene duplication and divergence to encode a family of proteins that provide direct protection against pathogens and also support the male reproductive tract in its primary function. Male tract defensins also facilitate recovery from pathogen attack. The β-defensins possess ancient conserved sequence and structural features widespread in multi-cellular organisms, suggesting fundamental roles in species survival. Primate SPAG11, the functional fusion of two ancestrally independent β-defensin genes, produces a large family of alternatively spliced transcripts that are expressed according to tissue-specific and species-specific constraints. The complexity of SPAG11 varies in different branches of mammalian evolution. Interactions of human SPAG11D with host proteins indicate involvement in multiple signaling pathways. （Asian J Andro12007 July; 9： 453- 462）

Role of defensins in diabetic wound healing

The adverse consequences resulting from diabetes are often presented as severe complications.Diabetic wounds are one of the most commonly occurring complications in diabetes,and the control and treatment of this is costly.Due to a series of pathophysiological mechanisms,diabetic wounds remain in the inflammatory phase for a prolonged period of time,and face difficulty in entering the proliferative phase,thus leading to chronic non-healing wounds.The current consensus on the treatment of diabetic wounds is through multidisciplinary comprehensive management,however,standard wound treatment methods are still limited and therefore,more effective methods are required.In recent years,defensins have been found to play diverse roles in a variety of diseases;however,the molecular mechanisms underlying these activities are still largely unknown.Defensins can be constitutively or inductively produced in the skin,therefore,their local distribution is affected by the microenvironment of these diabetic wounds.Current evidence suggests that defensins are involved in the diabetic wound pathogenesis,and can potentially promote the early completion of each stage,thus making research on defensins a promising area for developing novel treatments for diabetic wounds.In this review,we describe the complex function of human defensins in the development of diabetic wounds,and suggest potential therapeutic benefits.

Defensins as a promising class of tick antimicrobial peptides: a scoping review

Background: Ticks are hematophagous parasites that transmit an extensive range of pathogens to their vertebrate hosts.Ticks can destroy invading microorganisms or alleviate infection via their rudimentary but orchestrated innate immune system.Antimicrobial peptides(AMPs)are important components of tick innate immunity.Among these humoral effector molecules,defensins are well-studied and widely identified in various species of Ixodidae(hard ticks)and Argasidae(soft ticks).This review was aimed at presenting the characterization of tick defensins from structure-based taxonomic status to antimicrobial function.Main text: All published papers written in English from 2001 to May 2022 were searched through PubMed and Web of Science databases with the combination of relevant terms on tick defensins.Reports on identification and characterization of tick defensins were included.Of the 329 entries retrieved,57 articles were finally eligible for our scoping review.Tick defensins mainly belong to the antibacterial ancient invertebrate-type defensins of the cis-defensins superfamily.They are generally small,cationic,and amphipathic,with six cysteine residues forming three intra-molecular disulfide bonds.Tick defensins primarily target membranes of a variety of pathogens,including Gram-positive and Gram-negative bacteria,fungi,viruses,and protozoa.Since tick defensins have a high degree of variability,we summarize their common biological properties and enumerate representative peptides.Along with the various and potent antimicrobial activities,the role of tick defensins in determining vector competence is discussed.Conclusions: Due to their broad-spectrum antimicrobial activities,tick defensins are considered novel candidates or targets for controlling infectious diseases.

CGD-1, a defensin-related peptide derived from marine Chinese medicine Ostreae concha, inhibits the Gram-negative bacteria by membrane attack

There is an increasing interest in discovering new antibacterial agents derived from nature to enhance the treatment of various bacterial infections.Defensins and their derived peptide fragments exhibit significant antibacterial activity without any cytotoxic effects,making them attractive features for potential novel antibacterial therapeutics.Crassostrea gigas,a traditional seafood that has been used worldwide for centuries,has its shells applied in Chinese medicine as Ostreae concha.In this study,bioinformatics analysis was used to obtain a novel antibacterial peptide,CGD-1,derived from marine Chinese medicine Ostreae concha.The physicochemical characterization and circular dichroism analysis results demonstrated that CGD-1 assembled into anα-helical structure in a simulated membrane environment,and it displayed antibacterial action against Gram-negative bacteria.The minimal inhibitory concentrations against both Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC25922 were 25μM.CGD-1 was able to efficiently permeate the cell membrane.Changes in bacterial cell morphology were evaluated using a field emission scanning electron microscope.The results suggested that CGD-1 exerted its antibacterial activity through permeabilizing and disrupting the bacterial cell membrane.Therefore,CGD-1 may have potential applications in fighting against pathogenic bacteria such as P.aeruginosa and E.coli.

家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定

目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。

家蝇抗菌肽defensin对人肝癌细胞HepG2的增殖、凋亡和周期的影响

采用四甲基偶氮噻唑蓝（MTT）比色法，分析家蝇抗菌肽defensin对人肝癌细胞HepG2体外生长增殖与凋亡的影响，用倒置光学显微镜检测defensin对人肝癌细胞HepG2和人正常心肌细胞H9C2生长增殖情况的影响，并采用流式细胞术检测defensin对人肝癌细胞HepG2细胞凋亡及细胞周期的影响。结果显示，家蝇抗菌肽defensin对人肝癌细胞HepG2的生长有抑制作用，对人正常心肌细胞H9C2无抑制作用。通过显微镜观察HepG2细胞凋亡的形态变化，MTT检测发现defensin对肿瘤细胞的增殖抑制率呈剂量反应关系和时间反应关系。defensin能诱导人肝癌细胞HepG2发生凋亡，并且影响人肝癌细胞HepG2的细胞周期。家蝇抗菌肽defensin对人肝癌细胞HepG2具有增殖抑制作用及凋亡作用，对正常人心肌细胞H9C2基本无抑制作用。

白纹伊蚊和埃及伊蚊defensin A基因克隆及序列分析

应用PCR技术从白纹伊蚊和埃及伊蚊基因组中扩增出defensinA基因 ,并与文献报道的defensinA的5个型的cDNA序列进行同源性比较 ,发现此两序列中存在内元 ;从埃及伊蚊体内扩增的片段为蚊虫defen sinAl的前体AaDefAl;从白纹伊蚊体内扩增的片段为defensinA的 1个新型 ,命名为DefA6。

家蝇抗菌肽Defensin在毕赤酵母中的表达及活性鉴定

拟通过酵母系统表达家蝇抗菌肽Defensin基因,并初步鉴定其抗菌活性。采用限制性内切酶SacⅠ将分泌型重组表达质粒pPIC9K/Defensin线性化后,运用氯化锂转化法将其转入巴斯德毕赤酵母GS115中,行G418加压筛选和PCR鉴定;所得阳性转化子转至摇瓶,28℃条件下0.5%甲醇诱导表达,连续诱导培养4 d后,上清液进行Tricine-SDS-PAGE检测表达效果,并采用琼脂糖孔穴扩散法检测表达产物对大肠杆菌E.coliK12D31的抑制作用。结果显示,家蝇抗菌肽Defensin在毕赤酵母中得到了成功表达,表达产物对大肠杆菌E.coliK12D31的生长有抑制作用。说明酵母系统适合抗菌肽的表达。

贻贝抗菌肽的研究进展

贻贝中存在多种抗菌肽 ,根据一级结构的不同分为 4种 :defensin(防卫肽 )、mytilin、myticin和mytimycin。抗菌肽以前体的形式存在 ,在缺乏感染等刺激物的情况下 ,成熟前体以活性方式储存在血液中 ,当受到感染后便参与组织反应。它们的抗感染反应与哺乳动物吞噬细胞的作用相似 ,不同的抗菌肽具有不同的抗菌谱。抗菌肽是机体在进化过程中非特异性免疫系统中的一种。

Defensin expression in chronic pouchitis in patients with ulcerative colitis or familial adenomatous polyposis coli

AIM. Pouchitis develops in ileoanal pouches in up to 50% of patients with ulcerative colitis during the first 10 years after pouch surgery while being rare in patients after proctocolectomy for familial adenomatous polyposis coil （FAP） syndrome. Defensins are major components of the innate immune system and play a significant role in gastrointestinal microbial homeostasis. Pouch defensin and cytokine expression were correlated with states of pouch inflammation to study their role in pouchitis.METHODS： Patients with ulcerative colitis and FAP syndrome were stratified into groups with pouches after surgery, pouches without or with pouchitis. Biopsies from terminal ileum from a healthy intestine or from normal terminal ileum of patients with ulcerative colitis served as controls, mRNA from pouches and controls was analysed for defensin and cytokine expression.RESULTS： Expression of defensins was increased in all pouches immediately after surgery, compared to ileum of controls. Initially, pouches in ulcerative colitis revealed higher defensin expression than FAP pouches. Defensin expression declined in both patient groups and increased again slightly in pouchitis in patients with ulcerative colitis. FAP pouches without pouchitis had strong expression of β-defensin hBD-1, while all other defensins remained at low levels. Cytokine expression in ulcerative colitis pouches was high, while FAP pouches showed moderately elevated cytokines only after surgery.CONCLUSION： Development of pouchitis correlates with decreased defensin expression in ulcerative colitis in addition to high expression of cytokines. The low incidence of pouchitis in FAP pouches correlates with increased expression of hBD-1 β- defensin in association with low cytokine levels.

Crohn’s disease-Defect in innate defence

Crohn’s disease may prinicipally involve the whole gastrointestinal tract. Most commonly, the inflammation occurs in the small intestine and/or in the colon with stable disease location over the years. The pathogenesis of both disease phenotypes is complex, the likely primary defect lies in the innate rather than adaptive immunity, particularly in the chemical antimicrobial barrier of the mucosa. Crohn’s ileitis is associated with a reduced expression of the Wnt signalling pathway transcription factor T-cell factor 4 (TCF4), which is regulating Paneth cell differentiation. As a result, the alpha-defensins and principal Paneth cell products HD5 and HD6 are deficiently expressed in ileal disease, independent of current inflammation. In contrast, Crohn’s colitis is typically associated with an impaired induction of the beta-defensins HBD2 and HBD3 caused by fewer gene copy numbers in the gene locus of the beta-defensins on chromosome 8. This ileal and colonic defect in innate defence mediated by a deficiency of the protective alpha- and beta- defensins may enable the luminal microbes to invade the mucosa and trigger the inflammation. A better understanding of the exact molecular mechanisms behind ileal and colonic Crohn’s disease may give rise to new therapeutic strategies based on a stimulation of the protective innate immune system.

Diagnostic utility of faecal biomarkers in patients with irritable bowel syndrome

Irritable bowel syndrome(IBS)is a common functional gastrointestinal(GI)disorder characterized by unspecific symptoms.In clinical practice it is crucial to distinguish between non-inflammatory functional problems and inflammatory,malignant or infectious diseases of the GI tract.Differentiation between these involves the use of clinical,radiological,endoscopic,histological and serological techniques,which are invasive,expensive,time-consuming and/or hindered by inaccuracies arising from subjective components.A range of faecal markers now appears to have the potential to greatly assist in the differentiation of inflammatory bowel disease(IBD)and IBS.Faecal markers of neutrophil influx into the mucosa are reliable indicators of intestinal inflammation and their role has been mainly studied in discriminating IBD from non-IBD conditions(including IBS)rather than organic from non-organic diseases.Phagocytespecific proteins of the S100 family(S100A12,calprotectin)are amongst the most promising faecal biomarkers of inflammation.Faecal leukocyte degranulation markers(lactoferrin,polymorphonuclear elastase and myeloperoxidase)have also been suggested as diagnostic tools for the differentiation of IBD and IBS.More recently,additional proteins,including granins,defensins and matrix-metalloproteases,have been discussed as differential diagnostic markers in IBD and IBS.In this review,some of the most promising faecal markers,which have the potential to differentiate IBD and IBS and to advance diagnostic practices,will be discussed.

Defensin抗菌肽的序列特性分析

对defensin抗菌肽进行了生物信息学分析.一级结构表明其具有典型的昆虫防御素特征.二级结构分析表明defensin抗菌肽中主要的结构元件为α-螺旋,但有例外.信号肽及导肽分析结果显示defensin抗菌肽均属于分泌型蛋白.通过对不同物种defensin抗菌肽的分析结果表明,非阳离子抗菌肽的作用机制可能是疏水作用力起主导作用或者是对细菌胞内酶结构与功能、对细菌胞内信号转导的影响有关.

家蝇Defensin基因原核表达条件的优化及其抑菌活性

目的优化家蝇Defensin基因原核表达条件,并初步研究其抑菌活性。方法 Defensin基因成熟肽被克隆入pGEX-4T-1原核表达载体中,构建重组质粒pGEX-4T-1/Defensin,并转化宿主菌后诱导表达,表达过程中对菌液起始浓度、IPTG浓度、诱导温度和时间进行优化。采用亲和层析法分离纯化融合蛋白,SDS-PAGE鉴定目的蛋白的表达情况和分子量。用凝血酶切除谷胱甘肽标签,并通过体外抑菌实验检测其抗菌活性。结果成功构建重组质粒pGEX-4T-1/Defensin,目的基因能够在大肠埃希菌内高效表达,分子量为4 kD,与预期值一致,表达量约达40%。抑菌实验显示其对大肠埃希菌K12D31生长有一定的抑制作用。结论本实验为家蝇Defensin基因功能的深入研究奠定了工作基础。