表达猪β-防御素-2成熟肽重组酵母工程菌的构建

Construction of Recombinant Yeast Expressing Porcine β-Defensin-2 Mature Peptide

作者: 专业:预防兽医学 导师:何启盖 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

porcineβ-defensin, mature peptide, Eukaryotic expression, activity in vitro

        防御素(Defensins)是抗菌肽家族中一类重要的阳离子抗菌活性肽,广泛存在于动物和植物体内。防御素的抗菌谱十分广泛,可抑制或杀灭细菌、真菌、被膜病毒等多种病原微生物,特别是哺乳动物防御素还对支原体、衣原体、螺旋体和一些肿瘤细胞有杀伤作用。同时,防御素可以调节机体免疫系统并加速免疫系统的重建,在机体的抗感染过程中发挥重要作用。猪β-防御素2(pBD-2)是猪源抗菌肽中较为重要的一种,它主要来源于上皮组织,广泛分布于皮肤、胸腺、脾、肝脏、舌、呼吸道、胃肠道等器官的组织细胞中。猪β-防御素2成熟肽是一段由37个氨基酸残基组成的多肽,富含精氨酸等带正电的氨基酸,同时含有6个保守的半胱氨酸。这些带正电的氨基酸和由半胱氨酸形成的3对二硫键赋予了猪β-防御素2成熟肽独特的作用机理,使它活性稳定且不易产生耐药性。由于猪β-防御素2成熟肽分子内二硫键的存在对其活性有很大的影响,采用传统的原核表达系统不能对表达产物进行有效的翻译后的加工修饰,并且产物对宿主菌的毒性作用也会影响表达量的提高。相比之下,真核毕赤酵母表达系统有更好的翻译后加工修饰作用和表达量高等优点,近年来,毕赤酵母表达系统应用越来越广泛,人们利用该系统已成功的表达了多种防御素蛋白。由于猪β-防御素2成熟肽在机体内分泌的量很低,体外提取天然的防御素步骤繁琐、成本高、效率低,化学合成成本更高,不能满足对其结构和活性研究的需要,因此,利用基因工程的方法表达防御素将是一条捷径。本研究根据GeneBank已公布的猪β-防御素2成熟肽基因序列,参考酵母偏爱密码子,设计3段引物序列,利用PCR技术扩增得到β-防御素2成熟肽的DNA序列。在所得到的序列两端添加EcoRⅠ、NotⅠ酶切位点,将其重组于分泌型表达载体pPIC9K和已构建好的pPIC9k-GST重组载体,得到pPIC9K-pBD-2和pPIC9k-GST-pBD-2重组载体。选取阳性克隆鉴定正确后,用SalⅠ酶将重组载体线性化,用电转化的方法将其转化到毕赤酵母KM71细胞中,经特定培养基筛选后,PCR检测验证猪β-防御素2成熟肽基因片段正确整合到酵母基因组染色体上,从而得到稳定的猪β-防御素2成熟肽的毕赤酵母表达株。筛选得到的酵母阳性克隆经甲醇诱导,通过不断调节表达条件,实现了猪β-防御素2成熟肽在BMMY培养基里的表达。
    Defensins is an important cationic antimicrobial peptides in the family of antimicrobial peptides. They widely exist in animals and plants and play an important role in natural immunity. Defensins are broad spectrum antibiotic, they can inhibit or kill bacteria, fungi, viruses and other pathogens, besides, mammals’ defensins also kill mycoplasma, chlamydia, spirochetes, and some tumor. Defensins can also regulate the immune system,play an important role in resistance to infection.Porcineβ-defensin-2 (pBD-2) is an important porcine antimicrobial peptide. It mainly come from epithelial tissue, widely distributed in the skin, thymus, spleen, liver, tongue, respiratory tract, gastrointestinal tract and other organs.Porcineβ-defensin 2 mature peptide is composed of 37 amino acids and rich in arginine and six conservative cysteines. These positively charged amino acid and three pairs of intramolecular disulfide bonds formed by six conservative cysteines give the porcineβ-defensin-2 mature peptide which have stable.activity and form resistance uneasily, a unique mechanism of action. As the presence of intramolecular disulfide bonds to porcine P-defensin-2 mature peptide has a great influence on its activity, the traditional Prokaryotic expression system have no post-translational modification to the expressed product and the toxicity from the product to the host bacteria will also effect the expression level. In contrast, Eukaryotic yeast expression system has many advantages, such as a more effective post-translational modification and the high level of expression. In recent years, Eukaryotic yeast expression system has been successfully expressed many defensins. Considering the low level of pBD-2 released by the body, the complicated extraction procedur, and the high cost of chemosynthesis, genetic engineering method seems to be a convienent way to acquired pBD-2 in large amount.This experiment based on the published sequence of pBD-2 mature peptide in GeneBank, considered yeast codon bias, designed three primers and amplified using PCR technology to get DNA sequence of pBD-2 mature peptide. One ends of the resulting sequence is added EcoRⅠrestriction site, the other NotⅠ, then clone the resulting sequence to the secretion expression vector pPIC9K and pPIC9k-GST recombinant vectors which is constructed, and obtain pPIC9K-pBD-2 and pPIC9k-GST-pBD-2 recombinant vector. Positive clones identificated correct is selected, and the pPIC9K-pBD-2 and pPIC9k-GST-pBD-2 recombinant vector are linearized with enzyme SalⅠ, then transformed them into Pichia pastoris KM71 cells by electrotransformation technology. Through screening on a specific medium and PCR detection, porcine P-defensin-2 mature peptide gene fragments is correctly integrated into genome chromosome of the yeast KM71, which is stable Pichia pastoris strains expressing porcineβ-defensin-2 mature peptide. By adjusting the conditions of expression continuously such as pH, temperature etc. Screened yeast clones are induced by methanol and porcineβ-defensin-2 mature peptide is expressed in the medium BMMY.
        

表达猪β-防御素-2成熟肽重组酵母工程菌的构建

摘要6-7
ABSTRACT7-8
缩略语表(Abbreviation)9-10
1 文献综述10-24
    1.1 哺乳动物防御素研究进展10-13
        1.1.1 哺乳动物防御素的分类与分布10-11
        1.1.2 哺乳动物防御素的分子结构与进化特性11-12
        1.1.3 哺乳动物防御素的作用机理和生物活性12-13
    1.2 猪β-防御素(pBD)的研究进展13-18
        1.2.1 猪β-防御素的分类13-14
        1.2.2 猪β-防御素的结构特征14-15
        1.2.3 猪β-防御素的抗菌活性与作用机理15-16
        1.2.4 猪β-防御素克隆表达16-17
        1.2.5 猪β-防御素发展前景17-18
    1.3 毕赤酵母表达系统的研究进展18-22
        1.3.1 表达宿主和载体18-19
        1.3.2 外源基因的整合19-20
        1.3.3 外源基因的表达20
        1.3.4 毕赤酵母表达系统特点20-21
        1.3.5 毕赤酵母表达系统高表达策略21-22
    1.4 研究目的与意义22-24
2 猪β-防御素-2成熟肽基因酵母工程菌的构建和表达24-49
    2.1 材料24-27
        2.1.1 质粒和菌株24
        2.1.2 主要仪器设备24
        2.1.3 主要试剂及配制24-27
        2.1.4 主要应用的软件27
        2.1.5 引物列表27
    2.2 方法27-39
        2.2.1 pBD-2成熟肽基因片段的设计27-28
        2.2.2 pBD-2成熟肽基因的PCR扩增28-29
        2.2.3 大肠杆菌DH5α感受态细胞的制备(CaCl_2法)29
        2.2.4 pPIC9k-GST重组质粒的构建和鉴定29-30
        2.2.5 pPIC9k-GST-pBD-2和pPIC9k-pBD-2重组载体的构建和鉴定30-31
        2.2.6 含有pPIC9k-GST-pBD-2和pPIC9k-pBD-2质粒的阳性菌落的筛选31-32
        2.2.7 毕赤酵母工程菌的构建32-35
        2.2.8 pBD-2基因在毕赤酵母KM71中的表达35
        2.2.9 Tricine-SDS-PAGE凝胶的制备及酵母表达上清液的蛋白电泳鉴定35-37
        2.2.10 GST-pBD-2成熟肽的WesternBlot检测37-38
        2.2.11 pBD-2表达上清的抑菌活性检测38-39
    2.3 结果39-46
        2.3.1 pBD-2基因的PCR扩增与双酶切39
        2.3.2 GST基因的PCR扩增与双酶切39
        2.3.3 pPIC9k载体的双酶切39-40
        2.3.4 pPIC9k-GST-pBD-2重组载体的鉴定40-41
        2.3.5 pPIC9k-pBD-2重组载体的鉴定41-42
        2.3.6 重组毕赤酵母工程菌的筛选与鉴定42-44
        2.3.7 重组酵母蛋白表达44-45
        2.3.8 GST-pBD-2蛋白的WesternBlot验证45
        2.3.9 pBD-2成熟肽的抑菌活性检测45-46
    2.4 讨论46-48
        2.4.1 pBD-2成熟肽基因的设计46
        2.4.2 pPIC9k-GST-pBD-2和pPIC9k-pBD-2重组载体的构建46-47
        2.4.3 毕赤酵母表达47-48
        2.4.4 pBD-2活性检测48
    2.5 结论48-49
参考文献49-55
致谢55
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