【作者】 杨毅；

【导师】 高荣；

【作者基本信息】 四川大学 ， 遗传学， 2006， 硕士

【摘要】 近年来由于抗生素的不规范使用等多种原因，细菌耐药问题日益严重，人们正努力从各个方面来解决，其中机体天然产生的抗菌肽(antibacterial peptide)由于其对耐药菌的强大抗菌作用而受到人们的关注。抗菌肽是一种阳离子小分子多肽，在天然免疫和获得性免疫中都发挥着重要作用。防御素(defensin)是抗菌肽中较为重要的一种，主要来源于皮肤、呼吸道等的上皮组织，是正常机体抵抗外界病原微生物入侵的重要防线。 为了构建具有广谱高效的抗菌肽基因，获得新型抗菌融合蛋白，本实验采用了改进的重叠区基因拼接法对牛的β防御素3’(BNBD3)cDNA和人的α防御素3(HNP3)cDNA分别加以改造。在去除BNBD3的终止密码子和HNP3的起始密码子的基础上，在BNBD3的5’端引入BamHI酶切位点，并在HNP3的3’端引入EcoRI酶切位点，同时在二者之间加上Ser-Ser-Gly-Ser-Gly-Ser柔性连接肽序列。利用多次PCR扩增出目标片段，并构建了pGEX-4T-1-BNBD3／HNP3原核表达载体，经质粒PCR以及BamHI／EcoRI双酶切鉴定，测序结果证实BNBD3／HNP3融合基因已成功克隆至原核表达载体pGEX-4T-1。 将已构建的BNBD3／HNP3融合蛋白表达载体质粒转化大肠杆菌BL21(DE3)，用2×YT培养基培养，以IPTG诱导融合蛋白表达，经SDS-PAGE分析表达蛋白分子量符合预期的34.9KDa。融合蛋白的表达产物在细胞破碎的上清中以可溶形式存在，将带有GST标签的融合蛋白用亲和层析法纯化。融合蛋白在体外抗菌实验中表现出了明显的抑制大肠杆菌，绿脓杆菌，金黄色葡萄球菌和肺炎球菌生长的抗菌活性(P＜0.05)，表明我们所构建的BNBD3／HNP3融更多还原

【Abstract】 As a result of the abuse of antibiotics, the problem of drug-resistant bacteria has become deteriorated. The natural antibacterial peptide has been received many concerns for its strong effect on drug resistant bacteria. Antibacterial peptides are cationic molecules that play an important role in innate immunity and acquired immunity. Defensin is a major member of the antibacterial peptide family, secreted in skin, respiratory tract and other epithelial tissues, and a vital defense against the invasion of exoteric microorganism.In order to construct novel antibacterial fusion gene that has broader or enhanced bioactivity over the original donors, we spliced the genes of bovine neutrophil beta defensin 3 (BNBD3) and human alpha defensin 3 (HNP3) by overlap extension. Meanwhile, the stop codon of BNBD3 and the start codon of HNP3 were deleted. The restriction sites of BamHI and EcoRI were introduced at the two ends of this fusion gene. The reconstructed BNBD3 and HNP3 genes were connected together via a linker sequence encoding Ser-Ser-Gly-Ser-Gly-Ser amino acid residues. The PCR amplified product of the fusion gene was cut by BamHI and EcoRI endonucleases and then cloned into the pGEX-4T-1 expression plasmid更多还原