【作者】 董开忠；

【导师】 余四九；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2008， 博士

【摘要】 内源性抗微生物肽是生物体内先天性免疫的重要组成成分,防御素(defensins)是广泛分布于动物和植物界的一类富含半胱氨酸的阳离子内源性抗微生物肽,是内源性抗微生物肽中的一个大家族。是由生物细胞特定基因编码,经特定外界条件诱导产生的一类多肽,具有抵抗外界微生物侵害、消除体内突变细胞的作用。根据防御素分子内半胱氨酸的位置和连接方式、前体性质及表达位置的差异可分为α-防御素、β-防御素、θ-防御素、昆虫防御素和植物防御素五种类型。β-防御素主要分布于哺乳动物的黏膜上皮细胞内,所以,β-防御素被确认为黏膜表面抗微生物屏障的组成成分。本试验以牦牛为实验材料,无菌采血,用40%聚蔗糖和76%复方泛影葡胺按一定比例配制成细胞分层液,以确定牦牛中性粒细胞及其他主要血细胞的漂浮密度。然后配制Percoll分层液通过密度梯度离心法分离纯化牦牛中性粒细胞,用5%的冰乙酸抽提结合高速离心获得中性粒细胞粗提物,并进行了体外抑菌活性、溶血和对血细胞凝集的活性测定。活性检测结果表明,牦牛中性粒细胞粗提物对三种测试菌有很强的抑菌活性,对兔和绵羊红细胞没有发生溶血现象,对兔血细胞稍有凝集(+)而对绵羊红细胞无凝集。对牦牛中性粒细胞粗提物进行了分离纯化。通过Bio-Gel P-10凝胶过滤和反相高效液相色谱(RP-HPLC),收集到牦牛中性粒细胞提取物样品共22个峰,其中检测出9个抑菌活性峰。选取有抑菌活性的9个峰进行了质谱分析。在试验中我们以牦牛为材料,从牦牛舌黏膜上皮组织中提取总RNA,根据反刍动物—牛β-防御素cDNA的保守序列设计了一对引物,采用RT-PCR技术扩增出yLAP的cDNA,并重组到pGM-T载体,经限制性内切酶图谱分析后进行DNA序列测定,测序结果证实所克隆的yLAP的cDNA为β-防御素,因为该cDNA包含一个由192个碱基组成的开放读码框(Open Reading Frame,ORF),该ORF编码64个氨基酸残基的前原防御素肽,该前原防御素肽含有β-防御素的特征性结构,即6个在特定位置上的保守半胱氨酸残基。通过计算机软件分析其碱基分布、核苷酸和氨基酸同源性等。结果表明,yLAP基因与GenBank中已报道序列的同源性为99%;将yLAP和其他哺乳动物β-防御素进行cDNA碱基序列和前原肽氨基酸序列的同源性比较分析,结果显示:yLAP与驯鹿、山羊和水牛的β-防御素序列一致性最高(91.7%,88.5%,93.1%),与猪、驴和马的β-防御素之间的次之(64.5%～73.3%),与犬和恒河猴的β-防御素的最低(在30%以下)。由此可以看出yLAP的cDNA序列与驯鹿、山羊和水牛的β-防御素亲缘关系较近,这说明防御素进化的多样性。再以重组质粒为模板,用特异性表达引物扩增得到目的基因:即编码yLAP的CDS片段(207bp),将其定向克隆到pET-28a(+)表达载体中,构建的原核表达质粒pET-yLAP转化大肠杆菌BL21(DE3),结果表明,成功表达出17KDa左右的目的蛋白。经优化实验,确定最佳诱导表达条件为终浓1mmol/L的IPTG在37℃,pH值7.2条件下诱导表达6h。SDS-PAGE进行检测,表达量平均占菌体总蛋白的31.5%,以包涵体的形式存在。琼脂扩散实验检测表达产物的体外抑菌活性,在大肠杆菌中表达的牦牛β-防御素具有体外抑菌活性。牦牛β-防御素的发现有利于对牦牛黏膜的防御机制进行进一步的研究,同时丰富了牦牛功能基因的研究。更多还原

【Abstract】 Endogenous antimicrobial peptides are an important component in innate immunity of organism and defensins are a kind of positive endogenous antimicrobial peptides,which contain cysteine-rich and distribute abroad in the kingdoms of animal and plant.Defensins are a big family among endogenous antimicrobial peptides.As a kind of polypeptide, controlled by certain genic code and induced under certain outside conditions,ABP can resist the invasion of outside microorganism and kill inner mutant cells.According to the differences of the position and connect manner of cysteines,the property of precursors,the position of expression,defensins are devided into five subfamilies:α-defensin,β-defensin,θ-defensin,insect defensin and plant defensin.β-defensins are proved to be a component of antimicrobial barrier because they distribute prominently in muscosal epithelial cells of mammals and aves.The yak was involved in the experiment.Firstly,this investgation definited the drift density of Yak PMN and others major hemocyte by using cells demixing liquor that was dispensed by definite ratio 40%ficoll and 76%cystografin.Then groped a species Segregation methods of PMN by using 67.5%and 87.5%percoll,namely,percoll gradient Centrifugation,Neutrophils were isolated from yak peripheral blood.The crude extraction from PMN was isolated respectively through a way of extractionion by 5%acetic acid. The results of it’s biologic activity detection showed that it’s has different biologic activity. As for it’s in vitro antibacterial activity,the crude extraction from PMN of yak showed a certain antibacterial activity to Bacterium.The crude extraction showed feeble hemolysis activity to red blood cell of sheep and rabbit.The crude extraction from PMN of yak showed feeble hemoblast anticoagulation activity to blood cell of rabbit,while to blood cell of sheep,they were less strong(+).In the process of extractionion and purification of the crude extraction from PMN of yak,22 apices were collected by Bio-Gel P-10 Polyacrylamide gel filtration and RP-HPLC,and 9 out of 22 apices showed antibacterial activity.9 apices showing antibacterial activity were selected to be analyzed by mass spectrograph.In this study,we have used yak as experiment.Total RNA was extracted from the tougue epithelial tissue of a yak and the cDNA encoding yLAP was amplified by the reverse transcription-PCR(RT-PCR ) with the pair of primers which were designed according to the cDNA conserve sequences of reported ruminents’(cow)β-defensins.The purified RT-PCR product was cloned in pGM-T vector.After the restriction endonuclease pattern analysis of reombinant plasmid,the cDNA was sequenced and the result of cDNA sequencing demonstrated that the yLAP is belong to the family ofβ-defensins because the cDNA contain a open reading frame(ORF) of 192 bases which encoded a 64 amino acid prepro-peptide and the prepro-peptide contained theβ-defensin consensus sequence of six invariantly spaced cysteine residues.We analysised the base pair distribution,amino acid homology by computer software.All this established base for in future research. Allignment results indication that there have highly identical with alexin antimicrobial peptide through protein level in NCBI Blast,The results of pair distances of the cDNA and prepro-peptide sequences of yLAP and otherβ-defensins in other mammals and aves by computer software indicate that the relative relationship of yLAP to tarandus,goat and bubalis is highest(91.7%,88.5%, 93.1%),to theβ-defensins of pig,ass and horse is middle(64.5%～73.3%),to theβ-defensins of canis and rhesus monkey is lowest(mostly less than 30%).Therefore,we can conclude that the relationship of yLAP is closer to tarandus,goat and bubalis’sβ-defensins with the sequences of cDNA and prepro-peptide.The result demonstrates the diversity of defensins evolution.Then target genes,that were CDS fragments of yLAP which coded the yLAP(207bp),were amplified by PCR with a pair of specifically expressive primers and the recombinant plasmid as template respectively,then cloned into pET-28a(+) expression vector,We constructed protein expression vector of yak Lingual antimicrobial peptide and transformation into Escherichia coli(BL21),established highly expression target pretion.Protein molecular weight is 17KDa in BL21.Positive plasmid was identified and transformed into the competent cell BL21(DE3) which was induced by IPTG.The optimal conditions were determined that the induction time was six hours,the temperature was37℃,the pH was 7.2 and the IPTG concentration was 1 mmol/L.Under the conditions,the expressed recombinant protein existed in the form of inclusion bodies,amounting to 31.5%of protein of the induced recombinant bacteria at the presence of IPTG.By detecting the expressed recombinant protein with the method of agar geldiffusion test,the beta-defensins from the tougue of yak,expressed in E.coli,showed antibacterial activity in vitro.The discovery of yLAP provides a powerful evidence for understanding mucosal defense mechanism of yaks.更多还原