霍乱毒素B亚基和小麦小孢子特异表达β-expansin基因的表达研究

发布时间：2018-05-31 14:40

本文选题：霍乱毒素B亚单位 + 霍乱毒素B亚单位和多角体的融合蛋白　；参考：《浙江大学》2005年硕士论文

【摘要】：1. 霍乱毒素B亚基在家蚕杆状病毒表达系统的表达与活性分析霍乱毒素B亚基(cholera toxin B submit,CTB)是霍乱毒素的无毒单位,具有很强的免疫原性,能刺激机体产生黏膜IgA和血清IgG抗体。B亚单位的主要功能是与有核细胞上的GM1-神经结苷脂结合而使得A亚单位进入细胞,这个功能使它在免疫佐剂、口服疫苗与多肽衍生疫苗的研究中具有越来越重要的作用。在本论文中,霍乱毒素B亚单位与多角体蛋白的融合基因被克隆至昆虫杆状病毒表达载体系统(BEVS)的转移载体pBacPAK8中,使其置于多角体蛋白(polyhedrin) 基因启动子控制之下,获得重组转移载体pBacPAK-mCTB。重组转移载体DNA与经 Bsu36I酶切线性化的修饰型病毒Bm-BacPAK6 DNA共转染家蚕BmN细胞,然后经空斑纯化、PCR扩增、Southern杂交等方法鉴定出含CTB基因的重组病毒 BacPAK-mCTB。将重组病毒BacPAK-mCTB感染BmN细胞及五龄幼虫, SDS-PAGE 电泳分析显示重组杆状病毒的116kD的β-半乳糖苷酶条带缺失,证实外源基因已成功取代了LacZ基因:Western blotting分析发现在14kD及70kD处有分别都有明显条带。其表达的融合蛋白产物经ELISA检测显示细胞和虫体表达分别为5.6μg/ml 54.4μg/ml.在蚕体中得到了高效表达:而GM1-ELISA说明CTB五聚体的结合GM1- 神经结苷脂的生物学功能。NOD小鼠的免疫增强实验则明显的反映了CTB的免疫试剂及佐剂功能:它能显著增强胰岛素对自身免疫疾病的治疗效果,本实验诱导免疫耐受的较佳组合为混合的100μg胰岛素和10μg CTB含量的蚕血;而且单独喂养CTB的 NOD小鼠也显示出良好的免疫增强作用,能够延缓NOD小鼠的糖尿病发作,制止进～步的胰岛的恶化。 2. 小麦小孢子特异表达β-expansin基因TaEXPB2的克隆与表达 Expansin是一种体外诱导分离的植物细胞壁伸展的蛋白,在修饰细胞壁基础上使细胞膨胀。Expansin的功能众多,除了细胞生长,还包括营养生长、形态发生、授粉受精、果实软化等,并表现出高度组织、器官和细胞特异性。本论文在小麦小孢子特异表达阶段,经基因组DNA提取、RT-PCR、Northern及 Southern技术,首次鉴定出了一个小麦小孢子特异β-expansin cDNA-TaEXPB2
[Abstract]:1. Expression and activity Analysis of Cholera toxin B Subunit in Bombyx mori baculovirus expression system Cholera toxin B submitine CTB is a nontoxic unit of cholera toxin. Strong immunogenicity can stimulate the production of mucosal IgA and serum IgG antibody. B subunit the main function of the In nucleated cells, GM1-neurodatin binds to make subunit A enter the cell, a function that causes it to enter the immune system. Agents, oral vaccines and polypeptide derived vaccines play a more and more important role in the research. In this thesis, the fusion gene of cholera toxin B subunit and polyhedrosis protein was cloned into insect baculovirus. The transfer vector pBacPAK8 of the expression vector system BEVs was placed in polyhedrin). The recombinant transfer vector pBacPAK-mCTB was obtained under the control of gene promoter. Recombinant transfer vector DNA and meridian Bsu36I restriction linearized modified virus Bm-BacPAK6 DNA was co-transfected into silkworm BmN cells and then empty. Identification of Recombinant virus containing CTB Gene by Southern blotting BacPAK-mCTB. Infection of recombinant virus BacPAK-mCTB with BmN cells and fifth instar larva, SDS-PAGE Electrophoretic analysis showed the deletion of 尾 -galactosidase band in 116kD of recombinant baculovirus, which confirmed the success of exogenous gene. Western blotting analysis, which replaced the LacZ gene, showed that there were obvious bands in 14kD and 70kD. The expression of the fusion protein was 5.6 渭 g/ml by ELISA. 54.4 渭 g / ml. Highly expressed in silkworm bodies: GM1-ELISA indicates the binding of CTB pentamers to GM1- The biological function of dadoside. The immunoenhancement test of nod mice obviously reflects the immune reagents of CTB. And adjuvant function: it can significantly enhance the efficacy of insulin in the treatment of autoimmune diseases. This experiment induces immune tolerance. The best combination was 100 渭 g insulin and 10 渭 g CTB of silkworm blood, and fed with CTB alone. NOD mice also showed a good immune enhancement effect, which can delay the onset of diabetes in NOD mice and prevent the entry of diabetes mellitus. The deterioration of islets. 2. Cloning and expression of 尾 -expansin Gene TaEXPB2 in Wheat microspore Expansin is a kind of protein that induces the cell wall extension of plant in vitro. It is based on the modification of the cell wall. Expansin has many functions, including vegetative growth, morphogenesis, pollination, in addition to cell growth. Fertilization, fruit softening, etc., and showing high tissue, organ, and cell specificity. In this paper, the genomic DNA was extracted from wheat microspore specific expression stage. A wheat microspore specific 尾 -expansin cDNA-TaEXPB2 was first identified by Southern technique.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346;Q789

【引证文献】