论文标题:人β防御素3（hβD-3）的cDNA克隆与融合蛋白的表达
Cloning of Human Beta-Defensin 3 and Expression of Its Fusion Protein
论文作者 陈姗
论文导师 董燕麟；何凤田,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 第三军医大学,点击次数 124,论文页数 61页File Size2689k
2002-10-01论文网 http://www.lw23.com/lunwen_143620222/ hβD-3;抗菌肽;克隆;融合表达
hβD-3;antimicrobial peptide;cDNA cloning;prokaryotic expression;fusion protein
抗生素的耐药问题日益严重，人们正努力从各个方面来解决，其中包括抗菌肽(antimicrobial peptide)的研究和开发。防御素(defensin)是抗菌肽中较为重要的一种，是一种生物体产生的具有强大的抗菌功能的阳离子小分子多肽，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。人β-防御素3(human beta-defensin 3，hβD-3)是新近被克隆出来的第三种人源性β-防御素，它表现出了与其它几种人β-防御素不同的特性和优越性，被认为具有独特的研究和开发价值。本试验利用基因工程技术，利用大肠杆菌获得具有抗菌活性的重组hβD-3融合蛋白，为进一步研究和开发hβD-3打下了基础。 目的：扩增编码hβD-3成熟肽的cDNA，将其构建于原核表达载体后利用大肠杆菌诱导表达，最终获得具有抗菌活性的hβD-3融合蛋白。 材料和方法：1．克隆编码hβD-3成熟肽的cDNA。自人扁桃体组织提取细胞总RNA后，根据GenBank中编码hβD-3成熟肽45个氨基酸的cDNA序列，设计特异引物进行RT-PCR。2％琼脂糖凝胶中电泳鉴定扩增产物，观察并记录结果。PCR产物连于pUC18质粒，重组质粒命名为pUC18／hβD-3，送上海生工公司测序。2．构建表达融合蛋白的重组质粒和对照质粒。将自pUC18／hβD-3回收的目的片段连同表达运载蛋白DHFR的基因片断一起连接于原核高效表达载体pQE-80L，经酶切鉴定，得到重组原核表达载体pQE-80L／DHFR／hβD-3。同时还将表达运载蛋白DHFR的基因单独连接于表达载体pQE-80L，得到对照质粒pQE-80L／DHFR。3．诱导表达融合蛋白。根据优化确定的IPTG诱导浓度、诱导温度和时间进行诱导表达。5％的积层胶，15％的分离胶，变性聚丙烯酰胺凝胶电泳(SDS-PAGE)检查表达情况。蛋白质免疫印迹(Westem B10tting)检测蛋白的表达。4.分离、纯化表达蛋白。表达目的蛋白于SOml菌液中，收集细菌并鉴定融合蛋白的表达方式。溶解包涵体，将包涵体溶解物上Ni一TA slurry，利用该融合蛋白表达时带有的His一tag与Ni+的亲和作用分离、纯化表达蛋白。最后通过多步透析法将表达蛋白复性，冷冻抽干保存。5.体外抗菌试验鉴定表达蛋白的抗菌活性。 结果:(l)编码hpD一3成熟肤的cDNA的克隆。RT一PCR扩增片断克隆于pUC18质粒进行序列鉴定，结果显示所扩增cDNA片断长1 3 sbp，序列分析与GenBank中报道的编码h pD一3成熟肤的cDNA序列完全一致。本序列作为自中国人克隆的hpD一3被GenBank登录，登录号为AF516673。 (2)hpD一3融合蛋白的原核表达载体pQE一80L/DHFR月lpD一3和相应的对照质粒pQE一80L/DHFR的构建。pQE一soL/DHF侧h pD一3重组质粒经KpnI+Hindm双酶切后，可见大小约为1 35bp的片段。经BamHI+KPnl和BamHI+Hindlll双酶切后，可分别见到大小约为550bp和大小约为7oobp的片断。pQE一80L/ DHFR重组子经BamHI十KPnl双酶切后，可见大小约为550bp的片断。酶切结果表明两质粒构建成功。 (3)融合蛋白的诱导表达。经过对诱导表达条件的优化和筛选，确定最佳诱导表达条件为终浓度lmmol/L的IPTG在37℃诱导表达4小时。SDS一队GE分析可见重组原核表达载体pQE一80L/ DHF侧h pD一3表达的目的蛋白带和对照质粒pQE一soL/ DHFR表达的蛋白带，大小分别约为31kD和26kD。其中前者的表达量经软件分析，达到了细菌表达蛋白总量的约40%。经W亡Stern Blot证实，在大小分别约为3 IkD和26kD的位置有两条清楚的蛋白带。 (4)表达蛋白的分离、纯化。经SDS一队GE检测，表达蛋白主要是以包涵体的形式存在。包涵体溶解物过Ni一NTA柱纯化，目的蛋白洗脱液经多步透析法复性后冷冻抽千，50ml菌液中得到约5一10mg的表达蛋白。 (5)对复性后目的融合蛋白的抗菌生物学活性的检测。通过琼脂糖扩散试验我们可以看到表达的目的融合蛋白表现出了对金黄色葡萄球菌和多重耐药金黄色葡萄球菌的抗菌活性，而用于对照的DHFR融合蛋白未表现出任何的抗菌活性。此外，在较大剂量时，目的融合蛋白还表现出了对白色念珠菌的抗菌活性。但是在我们的试验中，提取的目的融合蛋白没有表现出对大肠杆菌的抗菌活性。 结论:本试验利用RT一PCR技术，通过使用所设计的针对h pD一3的特异引物，成功地自人扁桃体克隆了编码hpD一3成熟肤的cDNA，并将其与表达运载蛋白DHFR的基因一起成功地构建于新型高效原核表达载体pQE一80L，得到重组原核表达质粒pQE一SOL/ DHF刃h pD一3。同时构建了重组对照质粒pQE一80L/ DHFR。上述质粒分别转化M巧大肠杆菌，经IPTG诱导均分别表达了hpD一3融合蛋白和DHFR融合蛋白，大小分别为3IkD和26kD。重组h pD一3融合蛋白的表达量达到了细菌表达蛋白总量的约40%。分离、提纯和活化后的h母D一3融合蛋白表现出了对金黄色葡萄球菌等细菌的抗菌活性，而经过同样分离、提纯和活化的DHFR融合蛋白没有表现出任何抗菌活性，从而证明了hpD一3融合蛋白的抗菌活性完全是hpD一3本身作用的结果。也就是说，我们成功地利用原核表达系统高效表达出了具有抗菌生物学活性的重组hpD一3融合蛋白。
In recent years, for the irrational use of antibiotics produces resistant strains and other reasons, bacterial resistance is more and more serious. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides.Defensins are cationic peptides, isolated from mammals, insets and plants and they serve as effect molecules of innate immunity, providing and efficient defense against infections pathogens. A novel antimicrobial peptide, human β-defensin 3(), was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. In order to study in advanced, we need a lot of hβD-3. In our experiments, the active recombinant fusion protein was expressed in Escherichia coli system. Advanced study could be continued based on our experiments.Objectives: To amplify the cDNA encoding the 45 amino acid containing natural form of human P-defensin 3 and clone it into the prokaryotic expression vector. Then express the recombinant protein in E.coli and purify it in order to get active recombinant hβD-3 fusion protein.Materials and Methods: 1. To clone the cDNA encoding the 45 amino acid containing natural form of hβD-3. Extract the total RNA from human tonsil and amplify the hβD-3 specific cDNA sequence using RT-PCR with two special primers based on hβD-3 amino acid sequence in GenBank . Then the amplified product was analyzed on a 2% agarose gel. The RT-PCR product was inserted into pUC18 and the recombinant plasmid pUC18/hβD-3 was sent tosequence (By Sagon company).2. To construct the prokaryotic expression vector and the control vector. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vector pQE-80L/DHFR/hpD-3 was identified by restriction digestion while we also construct the recombinant control vector pQE80L/DHFR with the same strategy.3. To induce the expression of the fusion protein. The recombinant vectors were transformed into E.coli M15 respectively and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. Analyze the expression by SDS-PAGE with 15% SDS- polyacrylamide gel. Identify the expressed target protein by Western Blotting.4. Isolate and purify the target protein. Prepare 50ml E.coli expressing the interesting proteins and harvest the cells and determinate the solubility of the target protein. Completely solubilize inclusion bodies with 8M urea. Add 1ml of 50% Ni-NTA slurry to 4ml lysate and mix gently and load the mixture into an empty column. Then wash and elute the column with different buffer in order to purify the target fusion protein with the interaction of its 6 X His-tag and Ni-NTA matrices . Renature the purified target fusion protein through multistep dialysis. Then unwater the frozen protein in order to store.5. Check and measure the antimicrobial properties of the recombinant hpD-3 fusion protein.Results: (1) The amplification of the cDNA encoding the 45 ammo acid containing natural form of hpD-3. After RT-PCR with special primers, an about 140 bps fragment could be seen on 2% agarose stained with EB. This fragment was sequenced after inserted into pUC18 . The sequencing result show that the cloned cDNA was 135 bps and consistent with the cDNA sequence encoding the hpD-3 mature peptide reported in GenBank. This cDNAsequence has been accepted by GenBank as hpD-3 cloned from Chinese. The number is AF516673.(2) The construction of the recombinant prorarylic expression vector pQE80L/DHFR/hpD-3 and the control vector pQE80L/DHFR. An about 135 bps fragment could be seen after pQE80L/DHFR/hpD-3 vector was digested by Kpn I +HindIII. An about 550 bps and an about 700 bps fragment could be seen after the vector pQE80L/DHFR/hpD-3 was digested by BamH I +Kpn I and by BamH I +HindIII r
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