中华绒螯蟹螺原体重要功能基因的筛选和研究及螺原体与WSSV的多重PCR检测技术

【作者】 梁廷明；

【导师】 王文；

【作者基本信息】 南京师范大学 ， 水生生物学， 2011， 博士

【摘要】 中华绒螯蟹螺原体（Spiroplasma eriocheiris）是首次在水生甲壳动物中发现的螺原体类病原微生物,是一种新型的水生甲壳动物病原体,归属于柔膜体纲（Mollicutes）、虫原体目（Entomoplasmatales）、螺原体科（Spiroplasmataceae）、螺原体属（Spiroplasma）;在螺原体属内属于第XLⅢ血清族。它无细胞壁,具典型的螺旋形态,能透过0.22μm的滤膜,具运动性,还能在R2或M1D培养基中生长。该病原具有广泛的宿主范围,能引起中华绒螯蟹颤抖病,也能侵染克氏原螯虾、南美白对虾和罗氏沼虾并引发大规模的死亡,给水产养殖业造成重大经济损失。本课题组前期已经对其生理生化性质、分类地位、病原检测、敏感药物的筛选和有效治疗药物的药代动力学进行了研究并且完成了全基因组的测序和注释,但是其分子生物学研究还有待于深入,尤其是那些与该病原感染宿主相关的重要功能基因的研究。因此,本博士学位论文在前期中华绒螯蟹螺原体研究的基础上,开展了中华绒螯蟹血细胞的原代培养,并运用选择性捕获转录序列（SCOTS）技术在中华绒螯蟹整体水平和细胞水平上开展了螺原体感染宿主过程中的重要功能基因的筛选和研究,开展了中华绒螯蟹螺原体诱导刺激后,原代培养的血细胞的病变效应和免疫相关基因表达分析的研究。此外,还选取中华绒螯蟹螺原体特异性的重要功能蛋白类螺旋蛋白SLP31进行了基因的克隆表达分析。最后构建了利用多重PCR技术同时快速检测克氏原螯虾体内中华绒螯蟹螺原体和白斑综合症病毒的方法。本论文的主要研究结果包括以下4个方面：1.中华绒螯蟹血细胞原代培养体系的建立为研究中华绒螯蟹螺原体与中华绒螯蟹在细胞水平上的相互作用,更好地阐述中华绒螯蟹颤抖病的病理以及螺原体的分子致病机理,本文建立了一种体外培养中华绒螯蟹血细胞的方法。在实验中选用抗凝剂anticoagulant citrate dextrose solution B （ACD-B）和phosphate buffered saline （PBS）,结果显示ACD-B的抗凝效果优于PBS。实验中选择了在甲壳动物组织培养中最常用到的两种培养基（改良的L-15培养基和M199培养基）。结果显示,改良的L-15培养基能更有效地促进河蟹血细胞的生长和细胞的存活。中华绒螯蟹血细胞培养的合适生长温度是25-28℃高于其它水生无脊椎动物细胞的合适生长温度（16-20℃）。优化的血细胞原代培养体系最佳条件为：改良的L-15培养基（15%胎牛血清,青霉素和链霉素均为100 U ml-1, pH 7.2-7.4）,5%CO₂,28℃孵育。血细胞在培养箱中生长良好,存活且保持完整形态可达5周以上。此外,所培养细胞的细胞膜完整性和细胞大小也得到了荧光染色实验的证实。2.中华绒螯蟹螺原体诱导刺激下中华绒螯蟹血细胞数量的变化及原代培养血细胞的免疫反应分析根据以前建立的中华绒螯蟹颤抖病模型,本文研究了中华绒螯蟹颤抖病发病过程中血细胞数量的变化。从症状上来看,在回感前期没有明显的变化,有的中华绒螯蟹在回感前期的活力甚至还要比对照组强。在回感中期,实验组的中华绒螯蟹活力明显下降；到了回感后期,中华绒螫蟹的活力下降更加明显,绝大多数都趴在原地,少有活动,基本都表现出了明显的颤抖症状,有些实验组开始出现集中死亡的现象。在血细胞的变化上,阴性对照组中血细胞的数量基本保持稳定在1.13×107m1-1。回感2天时,血细胞的数量出现了一定的下降（3.65×106m1-1）。回感4天时,血细胞的数量出现了显著的上升（10.8×106m1-1）。回感8天时,血细胞的数量又出现了一定程度的下降（8.75×106m1-1）。回感10天时,血细胞的数量出现了大幅下降（6.25×105ml-1）。回感12天时,血细胞的数量继续下降（5.0×105ml-1）,此时实验组中出现了明显的颤抖症状和集中性的死亡本文用中华绒螯蟹螺原体诱导刺激原代培养的中华绒螫蟹血细胞,探讨了中华绒螯蟹免疫相关因子表达的影响。当中华绒螯蟹螺原体诱导刺激原代培养的血细胞之后,anti-lipopolysaccharide factor （ALF） mRNA的相对表达丰度在2-6 h时上调,并且在12h的时候达到峰值（10-fold, P< 0.05）; Peroxinectin （Pox） mRNA的相对表达丰度在2-12h之间逐渐下调,随后迅速上升并在24h的时候达到峰值（36-fold, P<0.05）,24 h之后又开始了下调。而对于clip domain serine protease （cSP） mRNA的相对表达丰度变化,首先在2h的时候出现下调,然后开始逐渐上调并且在48h的时候达到峰值（2.4-fold, P<0.05）,随后在72 h的时候出现了明显的下调。实时定量PCR结果显示ALF, Pox和cSP的表达量都发生了不同程度的变化,特别是Pox基因的变化最为显著,我们推测在中华绒螯蟹螺原体感染血细胞的过程中Pox基因起到了比ALF和cSP更为重要的作用。3.基于选择性捕获转录序列技术的中华绒螯蟹螺原体重要功能基因的筛选及类螺旋蛋白SLP31的原核表达通过利用SCOTS技术,从中华绒螯蟹螺原体感染的不同样品中（整体水平和细胞水平）捕获到了57条有效序列,经过比对在基因组中找到了27个有功能注释的基因。在这些基因编码的蛋白中,有一些是与代谢相关的蛋白,如各种酶类等参与细菌的脂代谢,糖代谢以及核酸代谢。有一些蛋白是与病原的致病性相关的,如类黏附蛋白和EF-Tu与病原的粘附侵染相关。硫醇过氧化物酶、铁蛋白、甘油吸收促进蛋白、核酸内切酶、丝氨酸/苏氨酸蛋白磷酸酶、NAD依赖的甘油三磷酸脱氢酶、N-乙酰葡糖糖胺酶等与病原的代谢相关并通过相关的代谢调控实现毒力效应。此外还捕获到了螺原体的特有蛋白-螺旋蛋白。本论文基于选择性捕获的结果对中华绒螯蟹螺原体的独特蛋白类螺旋蛋白基因（SLP31）进行了序列分析和克隆表达。该序列全长837bp,编码一个279个氨基酸的蛋白,预测的蛋白分子量和等电点分别是31kDa和PI7.72。SLP31的氨基酸序列与其它物种螺旋蛋白的序列相似性提示它可能是螺旋蛋白家族中的一员。本文从中华绒螯蟹螺原体基因组中克隆出了SLP31蛋白基因,我们也试图利用相同的引物序列从S. mirnm的基因组中克隆到该蛋白基因,然而却没克隆出来。这些结果显示SLP31蛋白基因是一个特异的基因,可以用来区分亲缘关系很近的中华绒螯蟹螺原体和非凡螺原体。SLP31被克隆后,经过从TGA到TGG的点突变,在大肠杆菌中进行了全长基因的表达。Western blotting实验显示,纯化的重组蛋白具有一定的免疫原性。SLP31还可以在中华绒螯蟹颤抖病的免疫诊断中作为一个很好的抗原应用。4.利用多重PCR技术同时检测水生甲壳动物螺原体和白斑综合症病毒检测方法的建立中华绒螯蟹螺原体和白斑综合症病毒是重要的水生甲壳动物病原微生物,近年来这两种病原都能感染克氏原螯虾并引起严重的疾病进而造成重大经济损失。本实验的目的是建立一种利用多重PCR同时检测克氏原螯虾体内中华绒螯蟹螺原体和白斑综合症病毒的方法。在一个PCR反应体系中,3对引物按照1：3：1比例混合分别扩增中华绒螯蟹螺原体、白斑综合症病毒和克氏原螯虾的特异性DNA片段。利用两种病原体攻击实验组中的克氏原螯虾,提取DNA模板并用多重PCR进行检测,出现四种不同的结果。克氏原螯虾的特异引物扩增出1195 bp特异条带作为内部参照,衡量整个PCR体系的稳定性。（1）阴性对照组只检测到1195 bp特异条带说明没有这两种病原体感染。（2）中华绒螯蟹螺原体感染组检测到1195 bp和271 bp两条条带。（3）白斑综合症病毒感染组检测到1195 bp和530 bp两条条带。（4）两种病原体同时感染组检测到1195 bp、271 bp和530 bp三条条带。这种方法可以同时检测两种病原微生物,这对克氏原螯虾的养殖监控很重要。实验证明直接利用多重PCR对这两种病原进行同时检测是可行的。本文提供了一种同时检测中华绒螯蟹螺原体和WSSV的新颖而实用的方法,这种方法高效、快捷,可以广泛应用于水产养殖业和出入境检验检疫的检测。更多还原

【Abstract】 Spiroplasma eriocheiris is a first found and isolated spiroplasma pathogenic microorganisms from aquatic crustaceans. It is a novel aquatic crustacean pathogen. Taxonomically, S. eriocheiris belong to the domain Bacteria, phylum Firmicutes, class Mollicutes, order Entomoplasmatales, family Spiroplasmataceae and genus Spiroplasma. It belongs to a new Spiroplasma group XLIII. S. eriocheiris possess wall-less, helical morphology and motility, it also be able to pass through the 0.22μm millipore filter and grow in MID or R2 liquid media at temperatures between 20℃and 40℃(optimum growth occurred at 30℃) in vitro. S. eriocheiris has the extensive host range, which could infect Eriocheir sinensis, Procambarus clarku, Macrobrachium rosenbergii and Penaeus vannamei, and caused serious diseases and catastrophic economic losses in aquaculture. Furthermore, S. eriocheiris also can infect suckling mice and cause cataract. The prevenient studies on the physiological and biochemical properties, evolutionary state, pathogen detection, screening of the effective medications, pharmacokinetic in E. sinensis and the whole genome sequencing and annotation were the base of this work. There is a limited known about the molecular biology of S. eriocheiris, especially for what function genes in the pathogen infection process. In the present study, we establish a primary cell culture system for in vitro culture of the hemocytes of E. sinensis with high viability. Selection and study of the important function genes from the infected host (on individual level and cellular level) by S. eriocheiris was performed. Meanwhile, the cytopathic effects and the direct immune relationship between S. eriocheiris and host’s hemocytes were presented. Based on the result of SCOTS, Spiralin-like protein SLP31 from S. eriocheiris was cloned and expressed which could be as a potential antigen for immunodiagnostics of tremor disease in E. sinensis. We also establish a multiplex PCR method for simultaneous and rapid detection of S. eriocheiris and white spot syndrome virus (WSSV) in P. clarkii. Main results of this dissertation included the following four parts:1. Establishing a primary cell culture system for in vitro culture of the hemocytes of E. sinensisA primary culture system for in vitro culture of the hemocytes of E. sinensis with high viability was developed. Sterile anticoagulant citrate dextrose solution B (ACD-B) and phosphate buffered saline (PBS) were employed, the results showed ACD-B had the better anticoagulant effect than PBS. We chose two kinds of common culture mediums (Modified L-15 medium and M199 medium) to culture the hemocytes. Our results demonstrated that the Modified L-15 medium were more efficiency for promoting growth and cell survival of hemocytes of E. sinensis than M199 medium. In disagreement with other crustacean cell lines culture work, the cell cultures were achieved at a temperature of 25-28℃rather than 16-20℃which has been favored for other freshwater invertebrates. In brief, the modified Leibovitz’s L-15 medium consisting of 15% fetal bovine serum,100 U ml-1 penicillin, and 100 U ml-1 streptomycin was suitable for hemocytes culture from the E. sinensis adjusting pH 7.2-7.4, and incubated at 28℃with 5% carbondioxide. The cells survival lasted over 35 days in Modified L-15 medium in the optimization condition.2. Variations in the number of hemocytes in E. sinensis and the expression levels of ALF, Pox and cSP genes in primary culture of hemocytes from E. sinensis after S. eriocheiris challengedIn the former study, we have established the tremor diseased model of E. sinensis. In the study, we used the model to research the variations in the number of hemocytes in E. sinensis after challenged by S. eriocheiris. From the symptoms, there were no significant changes at the early infection stage; the vitality of E. sinensis in experiment groups was declined obviously at the middle infection stage; most of E. sinensis in the experiment groups showed signs of weakness and began to tremor. Some experiment groups began to appear the concentrated death at the late stage. For the variations in the number of hemocytes, the numbers retained stable (1.13×107 ml-1) in the negative group. The hemocytes numbers appear declined (3.65×106 ml-1) after infected two days; the hemocytes numbers appear significant increase (10.8×106 ml-1) after infected four days; the hemocytes numbers appear declined (8.75×106 ml-1 and 6.25×105 ml-1) again after infected eight and ten days; the hemocytes numbers continued to decline (5.0×105 ml-1) after infected twelve days, and the experiment groups appear the concentrated death.The effects of S. eriocheiris stimulation on the expression levels of anti-lipopolysaccharide factor (ALF), Peroxinectin (Pox) and clip domain serine protease (cSP) genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced. After S. eriocheiris stimulation, the relative abundance of the ALF mRNA was up-regulated and peaked at 12 h (10-fold, P<0.05). The expression of Pox dropped gradually, after receiving S. eriocheiris. As time progressed, Pox transcripts significantly increased and peaked at 24 h (36-fold, P<0.05), and then after 24 h, the Pox mRNA was down-regulated. In contrast, cSP transcripts were first down-regulated in 2 h, whereafter the relative abundance of cSP was up-regulated and peaked at 48 h (2.4-fold, P <0.05), and then significant reduced at 72 h. The Pox transcripts greatly increased and peaked at 24 h (36-fold, P< 0.05); the fold was outweighing the other two immune genes’ greatly (ALF and cSP) which indicated that the Pox gene played more important role after S. eriocheiris challenge.3. Selection and study of the important function genes from the infected host by S. eriocheiris based on SCOTS and prokaryotic expression of Spiralin-like protein SLP31 from S. eriocheirisFifty-seven effective sequences were captured from the different infected samples (on individual level and cellular level) by SCOTS. There were 27 effective sequences with the annotation information in the S. eriocheiris genome. Some of these proteins were related with metabolic of S. eriocheiris, such as lipid metabolism, sugar metabolism and nucleic acid metabolism. Some proteins were related with S. eriocheiris pathogenicity. Adhesin-like protein and EF-Tu were related with adhere and infection. Furthermore, the putative spiralin was also be selected in the study. However, NAD-dependent glycerol-3-phosphate dehydrogenase, thiol peroxidase, ferritin, glycerol uptake facilitator protein, ferrous iron transport protein B, endonuclease I, serine-threonine protein phosphatase and N-acetylglucosaminidase were related with the S. eriocheiris metabolism, which control and realize the virulence effect through relevant metabolic. Based on the results of SCOTS, we described here the identification of a spiralin-like protein (SLP31) from S. eriocheiris and expression in Escherichia coli. Analysis of the nucleotide sequence revealed that the clone has an open reading frame of 837 bp encoding a protein of 279 amino acids. Theoretical isoelectric point and molar mass for SLP31 are 7.72 and 31 kDa, respectively. The similarity of SLP31 deduced amino acid sequence shared with the spiralin from other species indicated that the gene may be a member of spiralin family. However, spiralin-like protein gene from S. mirum could not be cloned which confirmed that this is a special gene which can be used to distinguish S. mirum and S. eriocheiris. After cloning the SLP31, the gene was site-mutated from TGA to TGG and transcribed in E. coli to full expression of SLP31. The purified recombinant protein was used to determine the immune reactivity by Western blotting which suggests that SLP31 could be a good antigen for immunodiagnostic of tremor disease in E. sinensis. 4. Simultaneous and rapid detection of S. eriocheir and white spot syndrome virus (WSSV) by multiplex polymerase chain reaction in P. clarkiiS. eriocheiris and WSSV are the important pathogenic microorganisms from aquatic crustaceans; both of them could infect crayfish and cause serious diseases and catastrophic economic losses in recent years. The aim of the study is to establish a multiplex PCR method for simultaneous and rapid detection of S. eriocheiris and white spot syndrome virus (WSSV) in P. clarkii. Three primer sets were mixed at a ratio of 1:3:1 to amplify specific fragments of the S. eriocheiris, WSSV, and P. clarkii genomes, respectively. S. eriocheiris and WSSV were used to challenge the different groups. Total DNA of the samples were purified and detected by multiplex PCR. The PCR amplified products produced four cases. One fragment of 1195 bp showing no pathogen amplified by primer set ITS-crayfish/28S-crayfish as an internal control. In samples only challenged by S. eriocheiris or WSSV, two fragments could be seen:either from S. eriocheiris (271 bp) plus the internal control or WSSV (530 bp) plus the internal control, respectively. In cases of double challenged, all three amplified products were detected simultaneously. Simultaneous and rapid detection of two pathogens in P. clarkii is important to crayfish aquaculture. The direct detection of S. eriocheiris and WSSV from P. clarkii is possible with multiplex PCR. The new method provides a novel and highly practical detection assay for S. eriocheirs and WSSV infections, and has the potential to be widely used in the aquaculture industry, entry-exit inspections and quarantines.更多还原