捻转血矛线虫ES15/ES24在秀丽隐杆线虫体内的表达特性研究
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摘要
捻转血矛线虫病是危害牛、羊等反刍动物的主要寄生虫病,其病原是捻转血矛线虫,成虫以宿主的血液和粘膜为食,导致动物贫血、消瘦、发育不良、生长缓慢甚至死亡,给畜牧业带来重大损失。目前捻转血矛线虫的控制主要靠广谱驱虫药物,化学药物过度和长期使用导致抗药虫株不断出现,同时也造成了肉制品药物残留和环境污染,寻找新的控制方法尤其是疫苗的开发显得十分迫切。捻转血矛线虫在宿主体内的寄生过程或体外培养的过程中可以向体外分泌产生多种分子,这些分子被命名为分泌排泄产物(Excretory/Secretory product, ESP),由于分泌排泄抗原(ES抗原)直接暴露于宿主免疫系统,是最有可能激发宿主产生保护性免疫的靶抗原之一,成为寄生虫抗原成分研制的热点。本研究以捻转血矛线虫寄生阶段释放的ES15／24抗原为研究对象,以秀丽隐杆线虫(Caenorhabditis elegans)作为模式生物,进行捻转血矛线虫分泌排泄抗原ES15／24转基因表达的可行性研究,以为寄生性线虫的疫苗研制提供技术支持。
利用PCR技术从C. elegans基因组中克隆得到C. elegans肠道表达基因：sre-49、acdh-7、cpr-1的5’侧翼区作为表达外源基因的候选启动子筛选对象,用以在C. elegans体内表达ES抗原,将sre-49、acdh-7、cpr-1的5’侧翼区序列克隆到表达载体pPD-95.77, GFP基因的上游,利用显微注射技术转入C. elegans的性腺,通过检测报告基因EGFP的表达情况分析其作为启动子转录外源基因的可行性。结果发现：sre-49基因的5’侧翼区启动功能较弱,GFP表达量低；在acdh-7基因的5’侧翼区的转录下,GFP能够在虫体的咽部和肠道特异性表达,但表达呈区段性,即咽部、咽肠交接部及肠道末端呈现较高强度的表达,表达量和表达强度因虫体个体不同有一定差异；而cpr-1基因的5’侧翼区的转录功能则强而稳定,GFP可以在除胚胎期外所有阶段虫体的整个肠道表达,尤其4期幼虫(larve 4, L4)和成虫阶段表达量特别高。根据GFP表达的组织定位、L4幼虫和成虫阶段GFP表达的强度和稳定性,最后选择cpr-1基因的5’侧翼区作为C. elegans表达ES蛋白的启动子。
根据已发表的分泌排泄抗原基因序列设计两对引物,以H. contortus,总RNA为模板,用RT-PCR方法分别扩增出大小为400bp和600bp的产物。序列分析结果表明：与已发表的的序列一致。将目的基因插入到重组质粒Cpr1-pPD-95.77中,构建重组真核表达质粒cprl-ES15/24-pPD-95.77。通过显微注射技术将cpr1-ES15-pPD-95.77与marker质粒PRF4共注射到C. elegans的性腺,通过紫外辐照的方法获得可稳定遗传虫株。通过对转基因C. elegans进行单虫PCR、RT-PCR及western-blot分析发现,ES15基因能够在C. elegans体内转录,可以在ES15转基因C. elegans肠道内检测到绿色荧光信号；而ES24转基因虫体不能检测明显的荧光信号,可能由于ES24基因或其转录产物对虫体有毒性,具体原因有待进一步的研究。
为分离纯化秀丽隐杆线虫表达的ES15重组蛋白,在ES15抗原成功的在秀丽隐杆线虫表达的基础上,进一步对重组载体cpr-15’侧翼区：：GFP进行了改造,即添加了His标签或将ES15基因下游的GFP终止,实验结果表明,改造后的ES15的表达强度和表达量与改造前基本一致。但有关虫体转基因目的蛋白的纯化和提取条件,仍需要进一步的摸索和优化。
综上,本实验首次利用显微注射技术研究了捻转血矛线虫ES15／24抗原在秀丽隐杆线虫体内的表达情况,成功实现了ES15抗原在秀丽隐杆线虫体内的表达,为今后捻转血矛线虫抗原在秀丽隐杆线虫体内的表达、纯化研究奠定了理论基础。
Haemonchosis is a major parasitic disease of ruminants such as cattle and sheep, whose pathogen is Haemonchus contortus, which feed on the mucous membrane and blood of the host. Its infection may be manifested as anemia, weight lost, stunted growth or even death in some case, which cause significant losses to livestock industry. The control of haemonchosis is depandent on the use of broad-spectrum deworming drugs currently. However, alternative methods of control are required, such as vaccine, with the emergence of anthemintic resistant strains of H. contortus and drug residues in meat products. H. contortus in host body during parasitic process or in vitro secreted a variety of molecules named excretory product (ESP). As ES antigen directly exposed to the host immune system, it is most likely to stimulate host protective immunity as a target antigen. ES antigens have become a hot spot in parasite research. Therefore, the studies carried were focused on proteins released during parasitic pHase of infection-ES15/24. Caenorhabditis elegans was choosen as an alternative expression system to try to express ES antigens. My research aimed to take C. elegans as a heterologous system, analyze the transcriptional activity of ES15/24 and make sure the feasibility of expression of ES15/24 using C. elegans, based on established transgenic platform, which would provide technical supports for the vaccination against parasitic nematodes.
The core promoter region of sre-49 gene and acdh-7 gene chosen as candidate genes of target promoter for the expression of ES antigen in C. elegans were amplified from the genomic DNA of C. elegans using PCR technology. The 5'flanking region of sre-49, acdh-7 and cpr-1 were cloned into the expression vector pPD-95.77. After microinjected into C. elegans' gonad, worms were picked up to detect the expression of EGFP reporter gene. The results showed that fluorescence directed by 5' flanking region of sre-49 gene was poor. The fluorescence directed by 5'flanking region of acdh-7 gene was detected in the pHarynx, anterior intestine and posterior intestine of transgenic-worms, but the expressions were segmental and there were some differences in the expression's patterns and intensities of GFP between different individuals. The transcriptional function of the 5'flanking region of cpr-1 gene was not only strong but also stable compared to the former two genes, GFP could expressed intensively in the whole intestine of worms in all stages expect embryos. So 5'flanking region of cpr-1 gene was determined to direct the expressions of ES15/24 eventually, referred to the location of GFP, the expression intensity in L4 and adult C. elegans, and also stability between different individuals.
RT-PCR was carried out to amplify the cDNA of ES15/24 from total RNA of H. contortus, referred to the sequence of ES15/24 from H. contortus reported.400bp and 600bp product fragments were amplified and inserted into plasmid cprl-pPD-95.77, aimed to construct the recombinant plasmid cpr-ES15/24-pPD-95.77 which were injected into C. elegans'gonad respectively together with a marker plasmid--PRF4 by micro injection. Through ultraviolet irradiation, stably inherited transgenic C. elegans strains had been obtained. The transcripts of ES15/24 could be found in transgenic C. elegans by single-worm PCR and RT-PCR analysis. With fluorescence microscopy, green fluorescence could be found in the guts of ES15 transgenic C. elegans, while green fluorescence could not be found in the bodies of ES24 transgenic worms. Maybe ES24 gene was toxic to C. elegans, which still requires further research.
In order to obtain recombinant protein of ES 15 expressed by C. elegans, some changes were made to the recombinant vector-cpr-1 5'flanking region::GFP with the method of adding His tag or ending GFP gene in the downstream of ES15. Experimental results showed that the intensity and pattern of expression of ES15 were consistent with the transformation of the former results. As for the conditions for purification of recombinant protein from transgenic worms, those still need us to explore and optimize.
Overall, this experiment is the first study about transgenic expression of ES15/24 in C. elegans using microinjection techniques. We successfully achieved the expression of ES15 antigen in C. elegans taking the advantage of established transgenic platform, which laid a theoretical foundation for the expression and purification of antigens of H. contortus using C. elegans in the future.
利用PCR技术从C. elegans基因组中克隆得到C. elegans肠道表达基因：sre-49、acdh-7、cpr-1的5’侧翼区作为表达外源基因的候选启动子筛选对象,用以在C. elegans体内表达ES抗原,将sre-49、acdh-7、cpr-1的5’侧翼区序列克隆到表达载体pPD-95.77, GFP基因的上游,利用显微注射技术转入C. elegans的性腺,通过检测报告基因EGFP的表达情况分析其作为启动子转录外源基因的可行性。结果发现：sre-49基因的5’侧翼区启动功能较弱,GFP表达量低；在acdh-7基因的5’侧翼区的转录下,GFP能够在虫体的咽部和肠道特异性表达,但表达呈区段性,即咽部、咽肠交接部及肠道末端呈现较高强度的表达,表达量和表达强度因虫体个体不同有一定差异；而cpr-1基因的5’侧翼区的转录功能则强而稳定,GFP可以在除胚胎期外所有阶段虫体的整个肠道表达,尤其4期幼虫(larve 4, L4)和成虫阶段表达量特别高。根据GFP表达的组织定位、L4幼虫和成虫阶段GFP表达的强度和稳定性,最后选择cpr-1基因的5’侧翼区作为C. elegans表达ES蛋白的启动子。
根据已发表的分泌排泄抗原基因序列设计两对引物,以H. contortus,总RNA为模板,用RT-PCR方法分别扩增出大小为400bp和600bp的产物。序列分析结果表明：与已发表的的序列一致。将目的基因插入到重组质粒Cpr1-pPD-95.77中,构建重组真核表达质粒cprl-ES15/24-pPD-95.77。通过显微注射技术将cpr1-ES15-pPD-95.77与marker质粒PRF4共注射到C. elegans的性腺,通过紫外辐照的方法获得可稳定遗传虫株。通过对转基因C. elegans进行单虫PCR、RT-PCR及western-blot分析发现,ES15基因能够在C. elegans体内转录,可以在ES15转基因C. elegans肠道内检测到绿色荧光信号；而ES24转基因虫体不能检测明显的荧光信号,可能由于ES24基因或其转录产物对虫体有毒性,具体原因有待进一步的研究。
为分离纯化秀丽隐杆线虫表达的ES15重组蛋白,在ES15抗原成功的在秀丽隐杆线虫表达的基础上,进一步对重组载体cpr-15’侧翼区：：GFP进行了改造,即添加了His标签或将ES15基因下游的GFP终止,实验结果表明,改造后的ES15的表达强度和表达量与改造前基本一致。但有关虫体转基因目的蛋白的纯化和提取条件,仍需要进一步的摸索和优化。
综上,本实验首次利用显微注射技术研究了捻转血矛线虫ES15／24抗原在秀丽隐杆线虫体内的表达情况,成功实现了ES15抗原在秀丽隐杆线虫体内的表达,为今后捻转血矛线虫抗原在秀丽隐杆线虫体内的表达、纯化研究奠定了理论基础。
Haemonchosis is a major parasitic disease of ruminants such as cattle and sheep, whose pathogen is Haemonchus contortus, which feed on the mucous membrane and blood of the host. Its infection may be manifested as anemia, weight lost, stunted growth or even death in some case, which cause significant losses to livestock industry. The control of haemonchosis is depandent on the use of broad-spectrum deworming drugs currently. However, alternative methods of control are required, such as vaccine, with the emergence of anthemintic resistant strains of H. contortus and drug residues in meat products. H. contortus in host body during parasitic process or in vitro secreted a variety of molecules named excretory product (ESP). As ES antigen directly exposed to the host immune system, it is most likely to stimulate host protective immunity as a target antigen. ES antigens have become a hot spot in parasite research. Therefore, the studies carried were focused on proteins released during parasitic pHase of infection-ES15/24. Caenorhabditis elegans was choosen as an alternative expression system to try to express ES antigens. My research aimed to take C. elegans as a heterologous system, analyze the transcriptional activity of ES15/24 and make sure the feasibility of expression of ES15/24 using C. elegans, based on established transgenic platform, which would provide technical supports for the vaccination against parasitic nematodes.
The core promoter region of sre-49 gene and acdh-7 gene chosen as candidate genes of target promoter for the expression of ES antigen in C. elegans were amplified from the genomic DNA of C. elegans using PCR technology. The 5'flanking region of sre-49, acdh-7 and cpr-1 were cloned into the expression vector pPD-95.77. After microinjected into C. elegans' gonad, worms were picked up to detect the expression of EGFP reporter gene. The results showed that fluorescence directed by 5' flanking region of sre-49 gene was poor. The fluorescence directed by 5'flanking region of acdh-7 gene was detected in the pHarynx, anterior intestine and posterior intestine of transgenic-worms, but the expressions were segmental and there were some differences in the expression's patterns and intensities of GFP between different individuals. The transcriptional function of the 5'flanking region of cpr-1 gene was not only strong but also stable compared to the former two genes, GFP could expressed intensively in the whole intestine of worms in all stages expect embryos. So 5'flanking region of cpr-1 gene was determined to direct the expressions of ES15/24 eventually, referred to the location of GFP, the expression intensity in L4 and adult C. elegans, and also stability between different individuals.
RT-PCR was carried out to amplify the cDNA of ES15/24 from total RNA of H. contortus, referred to the sequence of ES15/24 from H. contortus reported.400bp and 600bp product fragments were amplified and inserted into plasmid cprl-pPD-95.77, aimed to construct the recombinant plasmid cpr-ES15/24-pPD-95.77 which were injected into C. elegans'gonad respectively together with a marker plasmid--PRF4 by micro injection. Through ultraviolet irradiation, stably inherited transgenic C. elegans strains had been obtained. The transcripts of ES15/24 could be found in transgenic C. elegans by single-worm PCR and RT-PCR analysis. With fluorescence microscopy, green fluorescence could be found in the guts of ES15 transgenic C. elegans, while green fluorescence could not be found in the bodies of ES24 transgenic worms. Maybe ES24 gene was toxic to C. elegans, which still requires further research.
In order to obtain recombinant protein of ES 15 expressed by C. elegans, some changes were made to the recombinant vector-cpr-1 5'flanking region::GFP with the method of adding His tag or ending GFP gene in the downstream of ES15. Experimental results showed that the intensity and pattern of expression of ES15 were consistent with the transformation of the former results. As for the conditions for purification of recombinant protein from transgenic worms, those still need us to explore and optimize.
Overall, this experiment is the first study about transgenic expression of ES15/24 in C. elegans using microinjection techniques. We successfully achieved the expression of ES15 antigen in C. elegans taking the advantage of established transgenic platform, which laid a theoretical foundation for the expression and purification of antigens of H. contortus using C. elegans in the future.
引文
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Newton, S.E., Meeusen, E.N.,2003. Progress and new technologies for developing vaccines against gastrointestinal nematode parasites of sheep. Parasite immunology 25,283-296.
Ofran, Y., Rost, B.,2003. Analysing six types of protein-protein interfaces. Journal of molecular biology 325,377-387.
Paolini, V., Bergeaud, J.P., Grisez, C., Prevot, F., Dorchies, P., Hoste, H.,2003. Effects of condensed tannins on goats experimentally infected with Haemonchus contortus. Veterinary parasitology 113,253-261.
Pessoa, L.M., Morais, S.M., Bevilaqua, C.M., Luciano, J.H.,2002. Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus. Veterinary parasitology 109,59-63.
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Rathore, D.K., Suchitra, S., Saini, M., Singh, B.P., Joshi, P.,2006. Identification of a 66 kDa Haemonchus contortus excretory/secretory antigen that inhibits host monocytes. Veterinary parasitology 138,291-300.
Redmond, D.L., Clucas, C., Johnstone, I.L., Knox, D.P.,2001. Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans. Molecular and biochemical parasitology 112,125-131.
Rhoads, M.L., Fetterer, R.H.,1990. Biochemical and immunochemical characterization of 1251-labeled cuticle components of Haemonchus contortus. Molecular and biochemical parasitology 42, 155-164.
Ruyssers, N.E., De Winter, B.Y., De Man, J.G., Loukas, A., Pearson, M.S., Weinstock, J.V., Van den Bossche, R.M., Martinet, W., Pelckmans, P.A., Moreels, T.G.,2009. Therapeutic potential of helminth soluble proteins in TNBS-induced colitis in mice. Inflammatory bowel diseases 15, 491-500.
Schallig, H.D., van Leeuwen, M.A., Hendrikx, W.M.,1994. Immune responses of Texel sheep to excretory/secretory products of adult Haemonchus contortus. Parasitology 108 (Pt 3),351-357.
Schallig, H.D., van Leeuwen, M.A., Cornelissen, A.W.,1997a. Protective immunity induced by vaccination with two Haemonchus contortus excretory secretory proteins in sheep. Parasite immunology 19,447-453.
Schallig, H.D., van Leeuwen, M.A., Verstrepen, B.E., Cornelissen, A.W.,1997b. Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus. Molecular and biochemical parasitology 88,203-213.
Semnani, R.T., Law, M., Kubofcik, J., Nutman, T.B.,2004. Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface. J Immunol 172,6229-6238.
Siracusano, A., Margutti, P., Delunardo, F., Profumo, E., Rigano, R., Buttari, B., Teggi, A., Ortona, E., 2008. Molecular cross-talk in host-parasite relationships:the intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis. International journal for parasitology 38, 1371-1376.
Stringfellow, F.,1986. Cultivation of Haemonchus contortus (Nematoda:Trichostrongylidae) from infective larvae to the adult male and the egg-laying female. The Journal of parasitology 72, 339-345.
Vercruysse, J., Schetters, T.P., Knox, D.P., Willadsen, P., Claerebout, E.,2007. Control of parasitic disease using vaccines:an answer to drug resistance? Revue scientifique et technique (International Office of Epizootics) 26,105-115.
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Vervelde, L., Van Leeuwen, M.A., Kruidenier, M., Kooyman, F.N., Huntley, J.F., Van Die, I., Cornelissen, A.W.,2002. Protection studies with recombinant excretory/secretory proteins of Haemonchus contortus. Parasite immunology 24,189-201.
Vuong, D., Capon, R.J., Lacey, E., Gill, J.H., Heiland, K., Friedel, T.,2001. Onnamide F:a new nematocide from a southern Australian marine sponge, Trachycladus laevispirulifer. Journal of natural products 64,640-642.
Xenarios, I., Rice, D.W., Salwinski, L., Baron, M.K., Marcotte, E.M., Eisenberg, D.,2000. DIP:the database of interacting proteins. Nucleic acids research 28,289-291.
Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W., Heck, A.J., de Vries, E.,2003. Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition. The Journal of biological chemistry 278,16941-16951.
Zhu, S., Hanneman, A., Reinhold, V.N., Spence, A.M., Schachter, H.,2004. Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:alpHa-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase Ⅰ. The Biochemical journal 382,995-1001.