华支睾吸虫成虫和囊蚴cDNA文库表达序列标签（EST）测序及分析
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摘要
华支睾吸虫病是华支睾吸虫寄生在人类或者其他的哺乳动物肝胆管内而引起的一种重要的人兽共患寄生虫病,不仅给人类健康造成了威胁,而且对渔业生产造成了不小的经济损失。全世界约有3,500万人口感染此病,而在中国就高达1,200万左右。尽管华支睾吸虫自发现(1875年)以来已有130多年的历史,国内外众多学者对该病也做了大量的研究工作,然而至今仍未找到对该病的理想诊断抗原,华支睾吸虫病的感染率依旧呈现明显的上升趋势。目前,华支睾吸虫病的诊断仍主要采用漏诊率较高的粪便虫卵检查法,而免疫学诊断方法则因所使用抗原的非特异性或低敏感性而无法应用于临床的实际诊断中。因此,寻找高特异性及高敏感性的抗原以建立有效的免疫学诊断方法是目前控制该病广泛流行的有效可行手段。本实验通过构建成虫cDNA文库和利用已构建好的囊蚴文库进行表达序列标签(EST)测序分析,在分泌蛋白/膜蛋白和高表达蛋白中筛选出潜在的抗原,同时也为了获得华支睾吸虫整个转录组的信息,这就为进一步探索华支睾吸虫病的诊断抗原基因及保护性基因的深入研究提供了宝贵的材料与研究基础。我们希望本研究,能够加速华支睾吸虫的分子研究,并且能够发现华支睾吸虫病较好的诊断抗原。
Discovery of novel parasite genes by customary methods are tedious and time-consuming. In the current research field, large scale transcriptome sequencing identifying new genes might serve as little time and more information for various studies. Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on randomLy selected clones from cDNA libraries. To date, over 45million ESTs have been generated fromover 1,400 different species of eukaryotes.
Adult C. sinensis were collected from infected dogs’bile duct in Jilin province which is epidemic area of Northeast China. The liver and gall bladder were obtained from this dog and bile ducts were perfused with normal saline after sniped off the edge of liver. C. sinensis adult would run out from bile ducts and would be collected after washed by RNase-free water. The collected C. sinensis adult and excysted metacercariae can be prepared for library construction.Total RNA of C. sinensis adult worm was extracted by Trizol reagent (Invitrogen, USA) and mRNA was further purified with Oligotex mRNA Purification Kit (QIAGEN, USA). A cDNA expression library from adult worms of C. sinensis was constructed with lambda ZAP-cDNA GigapackⅢGold Cloning Kit (Stratagene, USA). The first strand cDNA was synthesized by using MMLV reverse transcriptase. After the synthesis of the second strand, the cDNA were ligated with EcoRⅠa daptors to the blunt ends and digested with XhoⅠrestriction enzyme. Then, the cDNA were purified by CHROMA SPIN-400 kit and ligated with lambda ZAP Express vector (uni-ZAP XR), then packaged in vitro and amplified by transfecting into Escherichia coli XL1-Blue cells. The capacity and the recombination rate of the cDNA library were measured. After capacity measurement, the remnant cDNA library was entirely coated on the agar plates and amplified. The original C. sinensis adult library contained 1.5×106 pfu cDNA clones, the titer of amplified library reached 1.5×1010 pfu/mL, in which about 99% clones were recombinants and most of insert DNA fragments were 0.4~2.0 Kb. The library was then stored at -80°C with addition of DMSO.
The cDNA libraries were converted into phagemid form by mass in vivo excision using helper phage and transfected into E. coli SOLR strain. The pellet of SOLR bacteria in fresh LB broth was preserved with ampicillin and 50% glycerol, and then was sequenced. The total cDNA sequences were read once from its 5’-end using T3 primer. Downstream of the sequencing reaction, a number of sophisticated bioinformatics pipelines have been developed that process the raw sequence read to remove low-quality sequence information and contaminating vector sequence. High-quality ESTs (>100 bp) were then assembled and clustered into contiguous sequences (contigs). The total clone of cDNA sequences is 41,541 ESTs(Of which C. sinensis adult sequences are 21,560 ESTs and metacercariae sequences are 19,981 ESTs). After vector sequences trimmed off and low quality sequences (<100 bp) removed, 37,442 ESTs (19,398 ESTs for adult and 18,044 ESTs for metacercariae) with an average length of 470 bases were recovered. Using Blast tools, unigenes comprising contigs and singletons were compared to the NCBI nonredundant (nr) protein and nucleotide (nt) databases for annotation, respectively. The cut-off value for sequence similarity was E < 10-5. Of these, only 1,441 unigenes (10.97%) were shared in two cDNA resources. This indicates that most genes related to developmental stages could be isolated form C. sinensis. The two BLAST methods demonstrated that the top three matched species of adult stage annotated were Schistosoma, Trichoderma and Plantago and of metacercariae annotated were Salicornia herbacea, Schistosoma and Trichoderma. On the whole, 3,545 clusters (27.00%) had homologies in Blastx NCBI Nr database and 3,703 clusters (28.20%) had matches with Blastn NCBI Nt database. Using these two databases, blast searches exhibited 5,882 clusters (44.80%) have no homologous sequences from other species, which possibly represents new genes for unknown functions of C. sinensis. For the sake of obtaining the maximum amount of functional information from the quickly collected genome sequences, all conserved clusters need to be assigned gene ontology (GO) classifications and 4,942 clusters could be assigned. Annotation of unigenes are used for GO classification including three categories:“cellular component,”“molecular function,”and“biological process.”KEGG(Kyoto Encyclopedia of Genes and Genomes) is a database for systematic analysis of gene function, which linked gene products information with cells metabolic pathways, predicted the function of these gene products and studied further the complex biological behavior. 1,470 unigenes (11.20% ) of C. sinensis two stages were assigned into specific pathways by the KEGG database. We utilize the tools named Signal P and TMHMM to screen the secretory or transmembrane proteins. There are 1,847 (14.07%) proteins included signal peptides/anchor and 1,464(11.15%)proteins included transmembrane domains from C. sinensis two stages. At last, we screened some important antigen such as Cysteine family, Calcium-binding EF-hand, Glutathione-S-transferase, Tropomyosin, Heat shock proteins, Annexin, antigen with tandem amino acid repeats. It should be pointed out that gene discovery of expressed sequence tags from C. sinensis isn’t totally secreted and trans-membrane protein-coding genes, also including highly expressed genes from interpro database.In addition, we compared some representative genes with Korean, Vietnam and other places in China which have published in NCBI and would like to find some distinction in different places.
This study should facilitate a more fundamental understanding of the gene expression in C. sinensis and provide valuable data not only for geneome, annotation, gene discovery of C. sinensis but also for its biology, evolution, and the host-parasite interplay. This genomic approach is having significant impact on many aspects of C. sinensis research, with contributions to both understanding the pathophysiology of C. sinensis and the development of improved interventions for disease control included discovery of novel antigens, drug and vaccine targets. The present study lays foundations for further recombination of genes that encode high-reactionogenicity antigens, for antigen recombination through combined gene expression, for the discovery of sensitive and specific methods for immunological detection of Clonorchis sinensis, and for the development of effective vaccines against Clonorchis sinensis.
Discovery of novel parasite genes by customary methods are tedious and time-consuming. In the current research field, large scale transcriptome sequencing identifying new genes might serve as little time and more information for various studies. Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on randomLy selected clones from cDNA libraries. To date, over 45million ESTs have been generated fromover 1,400 different species of eukaryotes.
Adult C. sinensis were collected from infected dogs’bile duct in Jilin province which is epidemic area of Northeast China. The liver and gall bladder were obtained from this dog and bile ducts were perfused with normal saline after sniped off the edge of liver. C. sinensis adult would run out from bile ducts and would be collected after washed by RNase-free water. The collected C. sinensis adult and excysted metacercariae can be prepared for library construction.Total RNA of C. sinensis adult worm was extracted by Trizol reagent (Invitrogen, USA) and mRNA was further purified with Oligotex mRNA Purification Kit (QIAGEN, USA). A cDNA expression library from adult worms of C. sinensis was constructed with lambda ZAP-cDNA GigapackⅢGold Cloning Kit (Stratagene, USA). The first strand cDNA was synthesized by using MMLV reverse transcriptase. After the synthesis of the second strand, the cDNA were ligated with EcoRⅠa daptors to the blunt ends and digested with XhoⅠrestriction enzyme. Then, the cDNA were purified by CHROMA SPIN-400 kit and ligated with lambda ZAP Express vector (uni-ZAP XR), then packaged in vitro and amplified by transfecting into Escherichia coli XL1-Blue cells. The capacity and the recombination rate of the cDNA library were measured. After capacity measurement, the remnant cDNA library was entirely coated on the agar plates and amplified. The original C. sinensis adult library contained 1.5×106 pfu cDNA clones, the titer of amplified library reached 1.5×1010 pfu/mL, in which about 99% clones were recombinants and most of insert DNA fragments were 0.4~2.0 Kb. The library was then stored at -80°C with addition of DMSO.
The cDNA libraries were converted into phagemid form by mass in vivo excision using helper phage and transfected into E. coli SOLR strain. The pellet of SOLR bacteria in fresh LB broth was preserved with ampicillin and 50% glycerol, and then was sequenced. The total cDNA sequences were read once from its 5’-end using T3 primer. Downstream of the sequencing reaction, a number of sophisticated bioinformatics pipelines have been developed that process the raw sequence read to remove low-quality sequence information and contaminating vector sequence. High-quality ESTs (>100 bp) were then assembled and clustered into contiguous sequences (contigs). The total clone of cDNA sequences is 41,541 ESTs(Of which C. sinensis adult sequences are 21,560 ESTs and metacercariae sequences are 19,981 ESTs). After vector sequences trimmed off and low quality sequences (<100 bp) removed, 37,442 ESTs (19,398 ESTs for adult and 18,044 ESTs for metacercariae) with an average length of 470 bases were recovered. Using Blast tools, unigenes comprising contigs and singletons were compared to the NCBI nonredundant (nr) protein and nucleotide (nt) databases for annotation, respectively. The cut-off value for sequence similarity was E < 10-5. Of these, only 1,441 unigenes (10.97%) were shared in two cDNA resources. This indicates that most genes related to developmental stages could be isolated form C. sinensis. The two BLAST methods demonstrated that the top three matched species of adult stage annotated were Schistosoma, Trichoderma and Plantago and of metacercariae annotated were Salicornia herbacea, Schistosoma and Trichoderma. On the whole, 3,545 clusters (27.00%) had homologies in Blastx NCBI Nr database and 3,703 clusters (28.20%) had matches with Blastn NCBI Nt database. Using these two databases, blast searches exhibited 5,882 clusters (44.80%) have no homologous sequences from other species, which possibly represents new genes for unknown functions of C. sinensis. For the sake of obtaining the maximum amount of functional information from the quickly collected genome sequences, all conserved clusters need to be assigned gene ontology (GO) classifications and 4,942 clusters could be assigned. Annotation of unigenes are used for GO classification including three categories:“cellular component,”“molecular function,”and“biological process.”KEGG(Kyoto Encyclopedia of Genes and Genomes) is a database for systematic analysis of gene function, which linked gene products information with cells metabolic pathways, predicted the function of these gene products and studied further the complex biological behavior. 1,470 unigenes (11.20% ) of C. sinensis two stages were assigned into specific pathways by the KEGG database. We utilize the tools named Signal P and TMHMM to screen the secretory or transmembrane proteins. There are 1,847 (14.07%) proteins included signal peptides/anchor and 1,464(11.15%)proteins included transmembrane domains from C. sinensis two stages. At last, we screened some important antigen such as Cysteine family, Calcium-binding EF-hand, Glutathione-S-transferase, Tropomyosin, Heat shock proteins, Annexin, antigen with tandem amino acid repeats. It should be pointed out that gene discovery of expressed sequence tags from C. sinensis isn’t totally secreted and trans-membrane protein-coding genes, also including highly expressed genes from interpro database.In addition, we compared some representative genes with Korean, Vietnam and other places in China which have published in NCBI and would like to find some distinction in different places.
This study should facilitate a more fundamental understanding of the gene expression in C. sinensis and provide valuable data not only for geneome, annotation, gene discovery of C. sinensis but also for its biology, evolution, and the host-parasite interplay. This genomic approach is having significant impact on many aspects of C. sinensis research, with contributions to both understanding the pathophysiology of C. sinensis and the development of improved interventions for disease control included discovery of novel antigens, drug and vaccine targets. The present study lays foundations for further recombination of genes that encode high-reactionogenicity antigens, for antigen recombination through combined gene expression, for the discovery of sensitive and specific methods for immunological detection of Clonorchis sinensis, and for the development of effective vaccines against Clonorchis sinensis.
引文
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