【作者】 朱峰；

【导师】 郭锡杰； 沈中元；

【作者基本信息】 江苏科技大学 ， 特种经济动物饲养， 2013， 博士

【摘要】 微孢子虫是一类专营细胞内寄生的单细胞真核生物，具有广泛的寄主域，包括脊椎动物和无脊椎动物。家蚕微孢子虫（Nosema bombycis）是引起家蚕微粒子病的病原体，可以通过胚胎垂直传播，被蚕业生产国列为唯一的法定检疫对象。目前，微粒子病依然给我国和印度两个蚕丝大国的蚕丝业造成巨大的经济损失。自19世纪中期首次发现家蚕微孢子虫以来，人们对微孢子虫的研究已有150多年的历史。成熟孢子是微孢子虫唯一在细胞外的生活阶段，其外被厚厚的孢子壁。孢子壁的存在可以使成熟微孢子虫抵抗外界任何不良环境，使其在恶劣环境中生存很长时间。微孢子虫的孢壁位于孢子的最外层，主要由高密度电子层的蛋白质成分的孢子外壁和低电子密度的蛋白质和几丁质成分的孢子内壁组成。坚硬的孢子壁不但可以保护孢子抵抗各种外界压力，而且在维持微孢子虫孢内渗透压的平衡中也起着重要作用。最近研究表明，位于微孢子虫孢壁结构的一些孢壁蛋白在微孢子虫的侵染机制和孢子黏附的过程中扮演着重要的角色。SWP26是一种C-端含有一个肝素结合基序的家蚕微孢子虫孢壁蛋白，肝素结合基序能识别并结合宿主细胞表面的葡聚糖，从而介导孢子粘附和宿主细胞的感染。免疫胶体金标记的方法发现，孢壁蛋白SWP26在成熟孢子与未成熟孢子中均有表达，在孢壁的发育过程中孢子内外壁也均有SWP26蛋白的分布。这暗示着，家蚕微孢子虫孢壁蛋白SWP26在孢子壁的形成过程以及孢子黏附和宿主细胞的侵染过程中可能扮演着重要的角色。本文通过家蚕微孢子虫孢壁蛋白SWP26的时相表达分析发现，家蚕中肠组织在感染微孢子虫后的24h就可以检测到SWP26的转录本，表明该孢壁蛋白可能在孢子裂殖体阶段就需要发挥作用，这与SWP26在家蚕微孢子虫不同发育阶段的亚细胞定位相一致。然而，目前对家蚕微孢子虫SWP26与其宿主蛋白之间的分子作用依然知之甚少。在此，我们希望通过实验去了解孢壁蛋白SWP26与宿主蛋白之间是否存在着直接的相互作用。试验中我们利用家蚕核型多角体病毒Bac-to-Bac表达系统，将家蚕微孢子虫孢壁蛋白SWP26基因和绿色荧光蛋白报告基因EGFP克隆到杆状病毒转移载体pFastBac1，并转化DHI0BmBac感受态细胞，经同源重组获得重组杆状病毒穿梭质粒vBmEGFP-SWP26，随后将其转染家蚕细胞BmN获得含有目的基因的重组杆状病毒。用SDS-PAGE和Western-blot方法在感染重组病毒的家蚕BmN细胞中检测到大小约51kDa的重组蛋白条带。结果表明，家蚕微孢子虫孢壁蛋白SWP26基因可在BmN细胞中成功表达，这为继续研究其功能以及在细胞水平通过免疫共沉淀方法筛选与之互作的宿主蛋白奠定了基础。随后，利用EGFP作为抗原决定簇通过免疫共沉淀技术筛选与之相互作用的宿主细胞蛋白。通过免疫共沉淀实验，我们筛选到一个与SWP26有相互作用的家蚕类海龟蛋白（Bombyx mori turtle-like protein，BmTLP）。经酵母双杂交实验结果证实，家蚕微孢子虫孢壁蛋白SWP26与家蚕类海龟蛋白BmTLP之间的确存在相互作用。完整家蚕类海龟蛋白开放式阅读框长1344bp，编码447个氨基酸的蛋白质，分子量大小约为49.6kDa，等电点为5.55，在其N-端有一段长为27个氨基酸残基的信号肽序列，27和28氨基酸位点之间为裂解位点。通过在线BLASTp比对分析发现，BmTLP蛋白与相应鳞翅目昆虫大红斑蝶（Danaus plexippus）海龟蛋白序列的同源性高达98%（基因登录号：EHJ71750），与膜翅目模式昆虫黑腹果蝇（Drosophila melanogaster）海龟蛋白序列的同源性也达到了84%（基因登录号：ACN43741）。经在线软件NetNGlyc1.0预测该cDNA所编码的氨基酸序列具有5个潜在的N-糖基化位点，推测BmTLP蛋白很可能是一种分泌型的糖蛋白。SMART软件分析结果表明，BmTLP蛋白具有免疫球蛋白超家族（Immunoglobulin Superfamily，IgSF）的一般特征，含有两个IG结构域（氨基酸位点：34–145和342–428）和两个IGc2结构域（氨基酸位点：161–226和252–321），属于典型的类免疫球蛋白家族成员。我们发现BmTLP蛋白与家蚕先天性免疫球蛋白–hemolin（编码序列：BGIBMGA008736-TA；蛋白序列：BGIBMGA008736-PA；基因登录号：DQ096295）拥有相同的蛋白功能结构域，但是他们的分布位置有很大差异。在BmTLP蛋白中两个IGc2结构域位于两个IG结构域的中间，而hemolin蛋白与之相反。实时荧光定量PCR结果显示，接种家蚕微孢子虫后12h，家蚕中肠组织中BmTLPmRNA表达量显著高于对照组。但是，12h以后感染中肠组织中BmTLP mRNA的表达量明显下降。推测BmTLP蛋白很可能在家蚕微孢子虫感染初期发挥着比较重要的作用。更多还原

【Abstract】 Microsporidia are unicellular, obligate intracellular, eukaryotic parasites. Its hostranges from protists to invertebrates and vertebrates. The species Nosema bombycis, whichis the pathogen of pebrine disease in silkworms, can be transovarially transmitted to the nextgeneration. So it is defined as the only disease needed for quarantine in sericulture. Atpresent, Pebrine disease used to prevail in the countries and areas practicing sericulture, andstill causes serious financial loss to the sericulture industry in China and India today. Sincethe N. bombycis has been discovered in the middle of the nineteenth century, it has morethan150years of history about the research on the microsporidia. The microsporidian sporestage, not the vegetative stages, is the only stage that lives outside the host, which is animportant characteristic of the phylum. The spore coat is separated from the cell by a plasmamembrane and consists of an electron-dense proteinaceous exospore and an electron-lucentendospore that contains chitin and proteins. The microsporidian spore wall plays animportant role in protection against environmental stresses and permits the long-termsurvival of the parasite after its release from the host cell. Indeed, recent studies havesuggested that some spore wall proteins are involved in the complex infection mechanism,and are responsible for spore adherence, which is an integral part of activation and host cellinfection. Previous studies showed that SWP26of N. bombycis contains a C-terminalheparin-binding motif (HMB) which is known to interact with extracellularglycosaminoglycans (GAGs) and may be involved in modulating in vitro host celladherence and infection. Transmission immunoelectron microscopy revealed that theSWP26protein is expressed at high levels in the endospore and plasma membrane duringendospore development, and is sparsely distributed in the exospore of mature spores.Furthermore, a few particles were detected in the endospore and exospore of the spore wallduring the late spore-forming phase and were not detected on the plasma membranes,thereby suggesting that SWP26is involved in endospore formation, host cell adherence, andinfection in vitro.In the present study, analysis of expression patterns revealed that the transcripts ofSWP26can be detected at24h p.i in the infected midgut of silkworm by N. bombycis.Therefore, it has been suggested that this spore wall protein is expressed at an early stageduring the development of N. bombycis. This proposal is congruent with the results of immuno-electron microscopy. However, little is known about the molecular interactionsbetween SWP26and host proteins. So, we want to comprehend that whether SWP26couldrecognize and interact with the host cell proteins.In this study, we used Bac-to-Bac baculovirus expression system to express the SWP26fused enhanced green fluorecence protein (EGFP) in BmN cells. The SWP26gene andEGFP gene were inserted into the baculovirus transfer vector pFastBac1. The transfervector pFastBac1-SWP26-EGFP was transformed into Escherichia coli DHl0Bac toconstruct recombinant vBmSWP26-EGFP. The vBmSWP26-EGFPwas then used to transfect BmNceIIs to obtain the recombinant bacuIovirus. Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) and western blotting detected a protein band of about51kDain BmN cells infected by the recombinant virus, which was correspondent to the deducedmolecuIar weight of SWP26fused EGFP. In addition, fluorescence were observed withinthe cytoplasm and within the nucleoplasm. These results indicated that the SWP26had beensuccessfully expressed in BmN ceIIs. This study laid the foundation for screening theinteractive proteins of Bombyx mori with the SWP26of N. bombycis at the cellular level.Then, we obtained a potential interacting protein--turtle like protein of B. mori(BmTLP) with SWP26using co-immunoprecipitation method. Yeast two-hybrid analysisalso indicated that the SWP26protein interacted, in vivo, with BmTLP protein. Thecomplete open reading frame (ORF) of BmTLP was obtained using PCR with thecorresponding primers. The ORF is1.344kb in size and encodes a polypeptide of447amino acids with a calculated molecular weight of about49.6kDa and an isoelectric pointof5.55. The deduced BmTLP protein sequence has an N-terminal signal peptide of27amino acids with a cleavage site between amino acids27and28. The NCBI BLASTpsearch revealed that BmTLP has the highest similarity (98%) with Danaus plexippus(GenBank Accession No.: EHJ71750), as well as a high similarity of84%with Drosophilamelanogaster (GenBank Accession No.: ACN43741). Five N-glycosylation sites arepredicted in BmTLP using the NetNGlyc1.0server online software. It suggested thatBmTLP may be a secreted glycoprotein. In addition，the protease domain of the BmTLPprotein was identified using the SMART program to analyze the amino acid sequence.BmTLP contains the basic features of the immunoglobulin superfamily (IgSF) such as IGdomains at the residual positions34–145and342–428, and IGc2-type domains at residues161–226and252–321. Interestingly, we found that the same4domains exist in both theBmTLP protein and the hemolin protein of silkworm (coding sequence:BGIBMGA008736-TA; protein: BGIBMGA008736-PA; GenBank Accession No.: DQ096295). However, these domains are differently distributed in the2proteins. In contrastto the hemolin protein, two IGc2-type domains are located in the middle of the two IGdomains.Fluorescent quantitative real-time polymerase chain reaction (qRT-PCR) analysisrevealed that the relative expression level of BmTLP was significantly higher in the infectedmidgut than that of silkworm at12h postinfection (hpi). However, the relative expressionlevel of BmTLP was notably down-regulated in the infected midgut of silkworm after12h.The results suggested that BmTLP protein may play an important role at early stage ofinfection by N. bombycis.更多还原