【作者】 杨德英；

【导师】 杨光友；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2012， 博士

【摘要】 豆状带绦虫呈世界性分布,成虫寄生于犬、狐狸等终末宿主的小肠,中绦期幼虫----豆状囊尾蚴寄生于家兔等兔形目动物的肝脏被膜、胃大网膜和肠系膜等部位,可引起家兔消瘦和抗病力减弱,甚至死亡。中国是世界养兔大国,兔豆状囊尾蚴病在我国广泛流行,给养兔业带来了巨大的经济损失。由于豆状囊尾蚴病无明显的临床症状,生前诊断较困难,其防治仍以药物为主。化学药物的长期使用容易导致虫株产生耐药性和兔产品中药物的残留,这不仅潜在地威胁着人类健康,同时也给豆状囊尾蚴病的防治带来困难。目前,对豆状带绦虫及其囊尾蚴病的研究多集中于病原学、流行病学调查和药物治疗,而该虫种的生前诊断和免疫防治研究未见相关报道。因此,本论文对豆状带绦虫种群遗传结构、转录组及功能基因进行了系统分析和研究,期望为豆状囊尾蚴病的诊断和免疫防治提供基础资料。主要研究工作和结果如下：1.基于线粒体基因cox2和nad4对四川地区豆状带绦虫种群遗传多样性的分析对采自中国四川7个不同地理区域(雅安、攀枝花、乐山、广元、泸州、广安和阿坝州)的23株兔豆状囊尾蚴进行了种群遗传多样性分析。PCR扩增了获得的cox2(468bp)序列中存在21个变异位点,核苷酸序列的变异率为0-3.0%,检测到5个单倍型,单倍型多样性(Hd)和核酸多样性(Pi)分别为0.451和0.00536。nad4(1,077bp)基因部分序列中有32个变异位点,核苷酸序列的分歧度为0-2.9%,检测到7个单倍型,单倍型间的Hd值和Pi值分别为0.625和0.00538。构建的NJ/MP进化树表明7个种群的系统发生与地域分布没有相关性,遗传多样性较低。2.基于线粒体基因cytb对四川地区豆状带绦虫种群遗传结构的分析采用PCR技术获得了四川8个不同地理区域(雅安、成都、攀枝花、乐山、广元、泸州、广安和阿坝州)的53株兔豆状囊尾蚴的cytb基因部分序列(922bp),并对其种群遗传结构进行了分析。cytb序列中共检测到12个单倍型,地域内分离株的变异占总变异的98.43%,地域间分离株的变异占1.57%。地域间分离株的遗传多样性和遗传分化较低,其遗传分化和基因流与它们的地理分布没有相关性,推测这8个地域株可能属于一个较大的种群。同时,NJ树和MrBayes树显示8个地域分离株均未形成明显的地理聚类,但单倍型聚类较为明显。种群动态分析结果表明,四川不同地理区域兔豆状囊尾蚴在过去的传播过程中群体规模可能保持稳定,推测相邻单倍型间的突变时间约开始于更新世晚期。3.基于线粒体基因coxl和nadl对四川地区豆状带绦虫种群结构的分析为探讨四川不同地理区域(雅安、成都、攀枝花、乐山、广元、泸州、广安和阿坝州)的61株兔豆状囊尾蚴的种群遗传结构,本文采用PCR技术获得了coxl (1,427bp)和nadl (738bp)基因的部分序列,并将两个基因串联后进行分析。结果表明：串联序列中共检测到5个单倍型,推测相邻单倍型间的突变时间约开始于更新世晚期,地域内分离株的分子遗传变异是变异的主要来源。除泸州分离株不存在变异外,其余地域内的分离株均存在较低的遗传多样性。地域间分离株遗传分化中等,地理分布特点与其遗传分化和系统发生没有相关性,推测8个地域分离株可能来源于一个较大的种群。核苷酸序列不配对差异分布曲线呈现多峰,推测四川不同地理区域兔豆状囊尾蚴在过去传播过程中群体规模保持稳定。4.豆状带绦虫转录组的研究采用第二代测序技术(Illumina sequencing)获得了豆状带绦虫成虫转录组数据库,将获得的267万条clean reads组装后得到72,957条unigeneso与四个蛋白质数据库(nr, Swiss-Prot, COG和KEGG)的相似性分析结果表明：26,012条unigenes(去冗余)与已知蛋白具有同源性,其中15,920条unigenes匹配到203个KEGG代谢通路。豆状带绦虫转录组数据库中的72,957条unigenes与猪带绦虫的30,700条ESTs以及细粒棘球绦虫和多房棘球绦虫间保守的1,058条ESTs比较分析后,得到了4种绦虫共有功能基因的分布特征。豆状带绦虫转录组中大量保守基因的鉴定,为豆状囊尾蚴病的生前诊断和免疫防治抗原的筛选奠定了基础,同时为绦虫功能基因组学、免疫学和基因表达谱的研究提供了基础资料。5.豆状带绦虫TpFABP基因的克隆表达及重组抗原Dot-ELISA诊断方法的建立根据已知带科绦虫的FABP抗原基因的序列设计特异性引物,通过PCR扩增获得了豆状囊尾蚴FABP基因的开放阅读框402bp。将TpFABP基因片段插入pET32a原核表达载体,经过IPTG诱导在BL21(DE3)型大肠杆菌中表达,获得约36.1kDa的目的蛋白。免疫印迹结果表明该重组蛋白能与兔豆状囊尾蚴病阳性血清特异结合。免疫组化结果显示,TpFABP蛋白在成虫的核周胞质和豆状囊尾蚴的囊壁层组织中表达。此外,通过TpFABP重组抗原建立了家兔豆状囊尾蚴病Dot-ELISA诊断方法。对169份家兔血清的检测结果表明,该方法具有较高的敏感性(93.22%)和特异性(94.21%)。6.豆状带绦虫Tp18基因的克隆表达与重组蛋白对家兔的免疫保护效果研究从豆状带绦虫转录组数据库中筛选到Tpl8基因,以激活的豆状带绦虫六钩蚴的cDNA为模板,采用RACE技术扩增得到378bp的cDNA序列。构建的pET32a/Tp18原核表达载体,经IPTG诱导获得分子量约为33kDa的重组蛋白。重组蛋白能与兔豆状囊尾蚴病阳性血清特异性结合。利用纯化后的Tpl8重组蛋白进行家兔豆状囊尾蚴病免疫保护实验,重组蛋白免疫组的减虫率为95.59%和97.38%,并产生了特异性的体液免疫应答抗体IgG。7.豆状带绦虫TpcCl基因的克隆表达及重组抗原Dot-ELISA诊断方法的建立以豆状带绦虫转录组数据库中TpcC1基因的unigene为模板设计特异引物,采用RACE技术从激活六钩蚴的cDNA中获得了TpcCl基因的序列。TpcC1基因的编码区为1,044bp,编码347个氨基酸。通过构建的原核表达载体pET32a/TpcCl,获得了分子量为58.3kDa的重组蛋白。经免疫印迹检测,该重组蛋白能与兔豆状囊尾蚴病阳性血清特异性结合。利用该重组蛋白建立了家兔豆状囊尾蚴病重组蛋白Dot-ELISA诊断方法,对169份家兔血清进行检测结果显示具有较高的敏感性(94.55%)和特异性(96.49%)。更多还原

【Abstract】 The larval of the tapeworm Taenia pisiformis (Cestoda: Taeniidae) is the causative agent of cysticercosis, and widely distributed in the world. Cysticercosis usually parasitizes in the liver capsule, the gastric omentum majus and the mesentery of rabbits, while adult T. pisiformis establish and mature in the small intestine of dogs and foxes. China is the largest producer of rabbits, and the T. pisiformis has become one of the most common parasites, which severely affecting rabbit breeding. It mainly causes emaciation, weakens resistance against other diseases of the host, and what is worse may cause death. There are no obvious clinical symptoms of Taenia pisiformis cysticercosis. So far, the pharmacotherapy is generally useful for controlling cysticercosis, but which may cause drug resistance and drug residues. Furthermor, the cost of pharmacotherapy is relatively high, and which were harmful for human health. Most current reports mainly pay more attention to the epidemiology, pathogenicity, prevention and treatment of T. pisiformis. However, the study of prenatal diagnosis and immune prevention of T. pisiformis do not have reported Therefore, it is essential for the study of the population genetic structure, transcriptome, and functional genes of T. pisiformis. The main research work is as follows:1. Population genetic diversity based on cox2and nad4genes in Taenia pisiformis from SichuanIn order to determine the level of genetic variation and population genetic diversity of T. pisiformis of rabbit from seven different regions (Ya’an, Panzhihua, Leshan, Guangyuan, Luzhou, Guang’an, and Aba) in Sichuan, China, the partial sequences of the mitochondrial cytochrome oxidase subunit2(cox2,468bp) and NADH dehydrogenase subunit4(nad4,1,077bp) gene of23isolates of T. pisiformis were obtained by PCR. The analysis results showed that there were21variable sites in cox2sequences, and23variable sites in nad4sequences. The sequence divergence of cox2and nad4were0-3.0%and0-2.9%, respectively. Five haplotypes with0.451of Hd value and0.00536of Pi value was presented in cox2nucleotide sequences, and seven haplotypes with0.625of Hd value and0.00538of Pi value was presented in nad4nucleotide sequences. NJ/MP trees showed that there was no direct correlation between phylogeny of seven populations and geographical regions. The results indicate that the lower genetic variation and heredity diversity presented in seven geographical populations among intrapopulation and intraspecific T. pisiformis.2. Genetic population structure analysis of Taenia pisiformis from Sichuanby using the mitochondrial cytochrome b geneThe genetic population structure of T. pisiformis from eight separate regions in Sichuan province were analyzed based on the sequences of mitochondrial cytochrome b (cytb). The fragments of cytb (922bp) gene were amplified from53isolates in eight districts of T. pisiformis.12haplotypes were found in the53cytb sequences. Molecular genetic variation intra-districts included98.43%of intra-district and1.57%of inter-districts. Lower genetic diversity was presented in district, and genetic differentiation of inter-district was not obvious. There was no correlation between genetic differentiation, gene flow and geographic distribution. Eight districts did not form apparent geographical clusters, but the clustering of their haplotypes was obvious in NJ and MrBayes trees. Thus, eight districts may come from a larger population.The analysis of population dynamics presented that size of populations may not experienced expansion in process of transmission history. And mutational time adjacent haplotype began with late pleistocene.3. Genetic population structure of Taenia pisiformis based on mitochondrial coxl and nadl genes61isolates of T. pisiformis from eight separated regions in Sichuan province, China, were used for determining their genetic population structure.based on thepartial sequences of the mitochondrial cytochrome c oxidase subunit I (coxl,1,427bp) and NADH dehydrogenase1(nadl,738bp). Five haplotypes were found, with molecular genetic variation of intra-district as the main source. There was no genetic variation in Luzhou; the other districts had the lower genetic diversity. No correlation between genetic differentiation, phylogeny and geographic distribution was observed, and mutational time adjacent haplotype begun with late pleistocene. Eight districts may belong to a larger population. And the analysis of population dynamics presented that size of populations may not experenced expansion in process of transmission history.4. Transcriptomic analysis of Taenia pisiformisIn present study,2.67million sequencing clean reads and72,957unigenes of Taenia pisiformis were obtained using the RNA-seq technique (Illumina sequencing). Based on a sequence similarity with known proteins, a total of26,012unigenes (no redundancy) were identified after quality control procedures via the alignment of four databases. Overall,15,920unigenes were mapped to203Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Functional distribution characteristics were gained through comparing four cestode species (72,957unigenes of T. pisiformis,30,700ESTs of T. solium,1,058ESTs of between Echinococcus granulosus and E. multilocularis]), with the cluster of orthologous groups (COG) and gene ontology (GO) functional classification systems. Furthermore, the conserved common genes in these four cestode species were obtained and aligned by the KEGG database. The identification of conserved genes may provide novel approaches for potential drug targets and vaccinations against cestode infections. The results of the study can provide the basic data for study of functional genomics, immunity and gene expression profiles of cestode species.5. Cloning, expression of TpFABP gene and construction of Dot-ELISA diagnostic method for Taenia pisiformis cysticercosisThe complete coding region of TpFABP gene was amplified from cDNA of cysticeri, using specific pair primers by TsFABP gene of T. solium. This sequence contain an open reading frame encoding a putative protein of133amino acids. The expression vetcor of pET32a/TpFABP was successfully constructed and obtained the36.1kDa fusion protein after IPTG induced.. The positive serum of rabbit Taenia pisiformis cysticercosis could specifically bound to TpFABP recombinant protein through Western Blotting. And TpFABP recombinant protein was expressed in perinuclear cytoplasm of tapeworm and cystic wall of cysticeri by immunohistochemistry. Furthermore dot-ELISA diagnostic method of TpFABP successfully constructed, which had higher sensibility (93.22%) and specificity (94.21%) in169rabbits serum for T. pisiformis cysticercosis. 6. Cloning, expression of Tpl8gene and immune efficacy analysis of rabbit Taenia pisiformis cysticercosisTp18gene was selected from transcriptome of adult T. pisiformis. The Tp18cDNA fragments were amplified by RACE t from the activated oncospheres. Then the Tp18gene was subcloned into pET32a vector. The vetcor of pET32a/Tpl8was transformed into Escherichia coli BL21and obtain the Tp18fusion protein after IPTG induced. The expression product was analyzed by SDS-PAGE and Western blotting. An open reading frame of Tp18gene was378bp. The recombinant Tp18protein had an approximate molecular mass of33kDa, and had postival signals with the serum from rabbit naturally infected with T. pisiformis eggs. The Reduction in numbers of metacestodes with the Tp18recombinant protein was95.59%and97.38%. Meanwhile, the specific antibody was IgG for recombinant Tp18protein.7. Cloning, expression of TpcC1gene and construction of Dot-ELISA diagnostic method for rabbit Taenia pisiformis cysticercosisThe complete coding region of TpcC1gene was amplified from the cDNA of activated oncospheres by RACE technology, and the primers were designed by TpcC1unigene from transcriptome dataset of adult T. pisiformis. The expression vetcor of pET32a/TpcCl was successfully constructed and expressed in E.coli BL21(DE3) with about58.3kDa fusion TpcCl protein. The positive serum of rabbit T. pisiformis cysticercosis could specifically bound to TpcCl recombinant protein through Western blotting. Furthermore Dot-ELISA diagnositic method for T. pisiformis cysticercosis successfully constructed with higher sensibility (94.55%) and specificity (96.49%) in169rabbits serum using TpcC1recombinant protein.更多还原