【作者】 杨银凤；

【导师】 范光丽；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2004， 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。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。已有研究报道在哺乳动物—牛、羊、猪、鼠、猴体内有β-防御素的表达。本研究中我们在骆驼体内又发现了一种新的β-防御素，命名为骆驼β-防御素-1(camelβ-defensin-1，caBD-1)。从骆驼舌黏膜上皮组织中提取总RNA，根据反刍动物—牛和羊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caBD-1的cDNA,并重组到pBlueselect 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caBD-1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。获得全长cDNA序列是研究基因结构、基因表达和基因功能的重要前提。我们根据已获得的骆驼β-防御素caBD-1 cDNA的已知序列设计了一条序列特异性引物作为上游引物，反转录引物中的部分序列即3ˊ接合器引物作为下游引物，成功地克隆了骆驼β-防御素caBD-1 cDNA的3ˊ末端序列。另外，采用反向嵌套 PCR 5ˊRACE法，根据caBD-1 cDNA 的已知序列，设计了一条5ˊ末端磷酸化的特异性反转录引物和两对特异性反向嵌套PCR 引物，首先进行反转录（Reverse Transcription，RT）, 然后将mRNA反转录成的cDNA进行环化，最后进行反向嵌套巢式PCR，成功地克隆了骆驼β-防御素caBD-1 cDNA的5ˊ末端序列。与锚定PCR法相比，反向嵌套PCR法具有特异性强、扩增效率高等优点，是一种有效扩增cDNA 5ˊ末端序列的方法。caBD-1 cDNA 序列全长322bp，其中192bp组成的ORF编码64个氨基酸残基的前原caBD-1肽，通过计算机软件预测该前原肽包含20个氨基酸残基的信号肽、6个氨基酸残基的前片段和38个氨基酸残基的成熟肽。在预测的38个氨基酸残基的成熟肽中有6个在特定位置上的不变的半胱氨酸残基，9个带正电荷的氨基酸残基（2个精氨酸Arginine－R，7个赖氨酸Lysine－K），没有带负电荷的氨基酸残基，因此caBD-1是一个阳离子肽。其分子量为4007.94Da , 等电点pI为9.71。通过计算机软件将caBD-1和其他哺乳动物、人及禽类的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：caBD-1与猪的β-防御素pBD-1<WP=8>的同源性最高（78.1%，76.6%），与牛和羊的β-防御素之间的同源性次之（67.7%～74.9%，51.6%～60.9%），与人、猴、鼠和禽类的β-防御素的同源性最低（大部分在30%以下）。由此可以看出caBD-1无论是cDNA序列还是前原肽序列与猪的β-防御素pBD-1的亲缘关系比与其他反刍动物—牛、羊的β-防御素近，这说明防御素进化的多样性。为了检测caBD-1 mRNA在骆驼体内的可能表达器官，我们又根据已知的caBD-1 cDNA序列设计了一对预计扩增产物为203bp的引物，通过RT-PCR检测了骆驼的整个消化管（舌、食管、前胃、皱胃、十二指肠、空肠、回肠、结肠前段、结肠后段、直肠）黏膜、肝脏、胰腺、气管黏膜、肺脏、肾脏、膀胱黏膜、卵巢、子宫内膜、脾脏、淋巴结、心脏等器官内caBD-1 mRNA的表达。结果显示：caBD-1 mRNA在整个消化道、气管、膀胱、子宫等管状器官内的黏膜层内有表达，而在实质性器官如心脏、肝脏、胰腺、肺脏、脾脏、淋巴结、卵巢、肾脏中无caBD-1 mRNA表达。caBD-1 mRNA的这种表达形式提示我们骆驼体内的这种内源性抗微生物肽有助于骆驼的黏膜宿主防御。为了进一步确定caBD-1在消化道内的表达部位，我们选择了被覆有复层扁平上皮的舌和被覆有单层柱状上皮的回肠作为组织代表，根据已克隆的骆驼β-防御素caBD-1 cDNA的序列设计并合成了长为42bp的反意核酸探针，探针经地高辛标记后应用原位杂交技术检测了caBD-1 mRNA的表达部位。结果表明：caBD-1 mRNA表达于骆驼回肠隐窝的柱状上皮以及舌背表面复层扁平上皮的中层和深层上皮内，而在舌的表层角化上皮中不表达。caBD-1的发现为我们更好地理解骆驼黏膜防御机制提供了有力的依据。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. 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. It has been reported that β-defensins express extensively in the epithelial tissues of many organs in mammals (cow, sheep, goat, pig, mouse, rat, human, monkey) and aves (chicken, turkey). In this study, we have identified in camels a novel β-defensin, named camel β-defensin-1 (caBD-1). Total RNA was extracted from the tougue epithelial tissue of a camel and the cDNA encoding caBD-1 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, sheep and goat )β-defensins. The purified RT-PCR product was cloned in pBlueselect T vector. After the restriction endonuclease pattern analysis of reombinant plasmid, the cDNA was sequenced and the result of cDNA sequencing demonstrated that the caBD-1 is belong to the family of β-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 the β-defensin consensus sequence of six invariantly spaced cysteine residues. Obstaining full-length cDNA is the essential foundation of the research on gene structures, gene expressions and gene functions. Based on the known cDNA sequence of caBD-1, we successfully cloned the 3ˊcDNA end of caBD-1 using anchored PCR RACE with a sequence specific primer as up primer and 3ˊsites adaptor primer as down primer. In addition, inverse nestd PCR can rapidly amplify 5ˊcDNA end by using a specific RT primer whose 5ˊend is phosphated and two pairs specific inverse nested PCR primers, which are designed basing on the known cDNA sequence of caBD-1. The caBD-1 cDNA which come from caBD-1 mRNA by RT is encircled, then inverse nestd PCR is performed. Contrasting with the anchored PCR, inverse nestd PCR has better specificity and higher amplification effects. It is a very effective method of amplifying 5ˊcDNA ends. The full-length caBD-1 cDNA is 322bp and includes a ORF of 192bp which encode the prepro-caBD-1 of 64 amino acid residues. With computer software we predict that the prepro-caBD-1 contain a signal peptide of 20 amino acid residues, a propiece of 6 amino acid <WP=10>residues, a muture peptide of 38 amino acid residues. In the muture peptide there are six invariantly spaced cysteine residues and 9 positive amino acid residues ( 2 Arginine-R, 7 Lysine-K ) , there is no negative amino acid residue in the muture peptide. Therefore the caBD-1 is a positive peptide which molecular weight is 4007.94 dalton and pI is 9.71. The results of pair distances of the cDNA and prepro-peptide sequences of caBD-1 and otherβ-defensins in human, other mammals and aves by computer software indicate that the relative relationship of caBD-1 to pBD-1 is highest (78.1%, 76.6%), to the β-defensins of cow, sheep and goat is middle (67.7%～74.9%, 51.6%～60.9%), to the β-defensins of human, monkey, mouse, rat, chicken and turkey is lowest (mostly less than 30%). Therefore, we can conclude that the relationship of caBD-1 to pBD-1 is closer than caBD-1 to other ruminant’s (cow, sheep and goat) β-defensins with the sequences of cDNA and prepro-peptide. The result demonstrates the diversity of defensins evolution In order to examine the organs that express possibly caBD-1 mRNA in camels, we designed again another pair of primers, which expecte更多还原