家蚕微孢子虫水通道蛋白NbAQP基因克

发布时间：2018-05-21 14:42

本文选题：家蚕微孢子虫 + 水通道蛋白　；参考：《江苏科技大学》2016年硕士论文

【摘要】：微孢子虫(Microsporidia)是一类寄主域广泛的真核生物,从脊椎动物到无脊椎动物都会被微孢子虫感染。微孢子虫作为鱼类、甲壳类和许多经济类昆虫的病原体,常给农业生产带来巨大损失。由家蚕微孢子虫(Nosema bombycis)引起的家蚕微粒子病是蚕业生产中一种重要的蚕病,不仅能够水平传播,还能经由卵垂直传播给后代,有时会给蚕业生产造成严重影响。迄今为止,虽然在家蚕微孢子虫的传染规律和防治技术方面取得了不少研究进展,但在其侵染机制方面还没有完全弄清楚。微孢子虫对宿主细胞的侵染,往往是通过孢子萌发将孢子内的孢原质注射到宿主细胞中来实现的。目前普遍认为的微孢子虫萌发感染宿主经历了以下几个事件:(1)孢子与宿主细胞的粘附;(2)孢子被激活;(3)孢子内的渗透压升高;(4)极丝外翻弹出;(5)孢原质通过弹出的极丝进入宿主细胞。孢子的发芽是微孢子虫感染宿主体的首要前提,研究其发芽机制对防治微孢子虫病有着重要的作用。有观点认为微孢子虫中可能有水通道蛋白的存在,将孢外的水分运输到孢子内部,造成孢子内部渗透压的升高,这种升高的内压便成为孢子发芽的动力。因此,本文希望通过研究,探索家蚕微孢子虫中是否存在水通道蛋白以及该蛋白参与孢子发芽的机制。本研究根据微孢子虫数据库和MIP数据库交叉比较分析筛选出可能的家蚕微孢子虫水通道蛋白基因,成功克隆出家蚕微孢子虫NbAQP序列,该序列具有完整的开放阅读框ORF,长750bp,共编码249个氨基酸,蛋白质分子质量约为26.66kD,等电点为5.12,无信号肽。NbAQP氨基酸序列与其他5种微孢子虫的水通道蛋白序列同源性为50%以上,含有6个跨膜结构域,三级结构预测分析显示有6个长螺旋和2个短螺旋,分别以两个螺旋为起点,各自延伸出一条氨基酸链,氨基酸链在外部环绕后返回起点处。用半定量PCR的方法检测家蚕微孢子虫感染家蚕后NbAQP的转录情况。利用酿酒酵母真核表达系统对家蚕微孢子虫NbAQP蛋白的体外表达进行了研究。首先设计带有酶切位点的引物,将NbAQP基因克隆到真核表达载体pYES2-NTC中,构建重组表达质粒并转化酵母感受态细胞。随后在INVSc1菌株中使用β-半乳糖诱导带His标签的融合蛋白的表达以及通过Western blot免疫印迹鉴定,免疫印迹检测到大小为31kD的His-NbAQP融合蛋白条带,说明家蚕微孢子虫NbAQP蛋白可在真核表达系统中表达。预测蛋白抗原表位并合成多肽,以该条多肽作为抗原免疫新西兰兔制备多克隆抗体,利用免疫胶体金电镜技术对NbAQP蛋白在家蚕微孢子虫中进行定位分析。免疫电镜结果显示,在家蚕微孢子虫的孢子壁上能够看到胶体金颗粒分布,而对照组中相应位置则没有胶体金的分布,说明家蚕微孢子虫NbAQP蛋白主要定位于家蚕微孢子虫的孢子壁上,对其功能的行使有着重要意义。为了研究家蚕微孢子虫NbAQP蛋白是否能够参与孢子发芽的过程,进而在微孢子虫感染家蚕的事件中发挥作用,对NbAQP蛋白做初步功能研究。通过抗体封闭实验检测抗体封闭处理后孢子发芽率的变化。结果显示,使用多克隆抗体孵育封闭1h的处理组的发芽率为16.58%,与空白对照组的发芽率(23.05%)相比,差异性显著(P0.05)。抗体封闭对孢子发芽的抑制率约为28%。说明封闭NbAQP蛋白后能够降低孢子的发芽率,NbAQP蛋白可能参与了家蚕微孢子虫发芽的过程。本研究结果表明,家蚕微孢子虫中存在水通道蛋白,该蛋白主要存在于孢子壁和原生质膜,在微孢子虫感染家蚕、增殖以及孢子形成的整个生活史均有转录表达,且在孢子的发芽过程中起到重要作用。
[Abstract]:Microspore (Microsporidia) is a wide range of eukaryotes in the host region. Both vertebrates and invertebrates are infected by microspore. Microspore, as the pathogen of fish, crustaceans and many economic insects, often causes huge losses in agricultural production. The silkworm particles caused by the Nosema bombycis of the silkworm (silkworm, microspore) Disease is an important sericulture disease in the production of sericulture, which not only can spread horizontally, but also propagate vertically to the offspring, sometimes causing serious influence on the production of sericulture. So far, although a lot of research progress has been made in the infection laws and control techniques of the silkworm microspore worm, the infection mechanism has not yet been completely made. It is clear that the infection of microspore to host cells is often achieved by injecting the sporozoite into the host cell by spore germination. At present, the commonly believed microspore germinating host has experienced several events: (1) the conidium and host cell adhesion; (2) the spores are activated; (3) the osmotic pressure in the spores is increased; (4) Polar filament echinovalgus pops out; (5) spores enter the host cells through the pop-up pole. Spore germination is the primary prerequisite for microspore infection, and its germination mechanism plays an important role in the prevention and control of microspore disease. It is believed that the presence of water channel protein in microspore is considered to be transported to the spores. The internal pressure of the internal osmotic pressure of the spores is increased, and the internal pressure of this increase becomes the motive force of spore germination. Therefore, this paper hopes to explore the existence of aquaporin in the microspore of silkworm and the mechanism of its participation in spore germination. This study is based on the cross comparison and analysis of microspore database and MIP database. The NbAQP sequence of microspore microsporworm, silkworm, was successfully cloned. The sequence has a complete open reading frame ORF, long 750bp, a total of 249 amino acids, protein molecular mass of about 26.66kD, isoelectric point 5.12, no signal peptide.NbAQP amino acid sequence and water channel eggs of other 5 microspore insects. The homology of the white sequence is over 50% and contains 6 transmembrane domains. The three stage structure prediction analysis shows that there are 6 long helix and 2 short helix, respectively, with two helices, respectively extending an amino acid chain, and returning the amino acid chain to the starting point after the external surround. A semi quantitative PCR method is used to detect the NbAQP infection of silkworm after silkworm infection. By using the eukaryotic expression system of Saccharomyces cerevisiae, the expression of NbAQP protein in the silkworm microspore was studied in vitro. First, the primers with the enzyme cut site were designed, the NbAQP gene was cloned into the eukaryotic expression vector pYES2-NTC, and the recombinant expression plasmid was constructed and transformed into the yeast susceptible cell. Then the beta strain was used in the INVSc1 strain. The expression of fusion protein with His label with galactose was induced and identified by Western blot immunoblotting. The His-NbAQP fusion protein band of 31kD was detected by Western blot. It indicated that the NbAQP protein of the silkworm microspore can be expressed in the eukaryotic expression system. The epitope of the protein antigen was predicted and the polypeptide was synthesized, and the polypeptide was used as the antigen free of the antigen. The polyclonal antibody of New Zealand rabbits was prepared. The NbAQP protein was located in the microspore of silkworm by immuno colloid gold electron microscopy. The results of immuno electron microscopy showed that the colloid gold particles could be found on the spore wall of the microspore worm, while the distribution of colloid gold was not found in the corresponding position of the control group, indicating the microspore of the silkworm. The insect NbAQP protein is mainly located on the spore wall of the microspore worm of the silkworm, which plays an important role in the exercise of its function. In order to study whether the NbAQP protein of the microspore worm can participate in the germination of the spores, and then play a role in the events of the microspore worm infection of the silkworm, the preliminary function of the NbAQP protein is studied. The results showed that the germination rate of the treated group which was incubated with 1H with polyclonal antibody was 16.58%, and the difference was significant compared with that of the blank control group (23.05%). The inhibition rate of antibody closure on spore germination was about 28%. indicating that the spores could reduce the spores after the closed NbAQP protein was closed. The germination rate, NbAQP protein may be involved in the process of germination of microspore of silkworm. The results of this study showed that there was aquaporin in the microspore of silkworm, which mainly existed in the spore wall and protoplasm membrane. The whole life history of microspore infecting silkworm, proliferation and spores formation were all transcriptional and germinated in the spores. It plays an important role in the process.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S884

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