摘要 目的真核表达华支睾吸虫乳酸脱氢酶基因并研究CsLDH的亚细胞及虫体组织定位。方法构建pEGFP-N1-CsLDH真核表达质粒,转染HeLa细胞,通过绿色荧光蛋白标签GFP的示踪来揭示CsLDH在HeLa细胞中的亚细胞定位;免疫组织化学方法进行CsLDH的虫体组织定位。结果双酶切和测序均表明真核表达重组质粒pEGFP-N1-CsLDH构建成功,经RT-PCR鉴定转染正确的HeLa细胞能稳定表达,部分HeLa细胞的细胞膜出现强烈的绿色荧光;免疫组织化学的结果显示CsLDH主要定位成虫的表膜,口、腹吸盘,咽及肠支处。结论华支睾吸虫乳酸脱氢酶为定位于虫体皮层和消化道界面的膜蛋白。
关键词 ：
华支睾吸虫 ,
乳酸脱氢酶 ,
免疫组织化学
Abstract ：To comfirm the predicted membrane-subcellular location of lactate dehydrogenase of Clonorchis sinensis(CsLDH)and its localization in adult,the coding sequence of CsLDH was inserted in vector pEGFP and then transfected into HeLa cells to observe the distribution of fluorenscene of the tranfected cells;the localization of the protein in adult was demonstrated by immunohistochemical method.It was found that the CsLDH protein was expressed in HeLa cells transfected with pEGFP-CsLDH and green fluorenscene was observed on the cellomembrane of some HeLa cells.The protein was found in tegument,oral and ventral sucker,pharynx and intestinal cecum of adult C.sinensis.These results imply that CsLDH protein was one of the membrame proteins,which might locate at the surface of digestive trace and tegument in adult worm.
Key words ：
Clonorchis sinensis
lactate dehydrogenase
收稿日期: 2008-10-20
[1]梁子良,曹晓鸥,钟诚,等.佛山市南海区人群华支睾吸虫感染现况分析[J].热带医学杂志,2007,7:979-987.
[2]方悦怡,吴军,柳青,等.广东省华支睾吸虫病流行现状调查和分析[J].病原生物学杂志,2007,2:54-56.
[3]Komuniecki R,AGM.Tielens.Carbohydrate and energy metabo-lism in parasitic hel minths[J].Mol Medl Parasitol,2003,339-358.
[4]Lee KW,Shalaby KA,Medhat AM,et al.Cloning of the gene for phosphoglycerate kinase fromSchistosoma mansoniand charac-terization of its gene product[J].Mol Biochem Parasitol,1995,71:221-231.
[5]Goudot-Crozel V,Caillol D,Djabali M,et al.The major parasite surface antigen associated with human resistance to schistosomia-sis is a37-kDa glyceraldhyde3P-dehydrogenase[J].J Exp Med,1989;170:2065-2080.
[6]Shoemaker C,Gross A,Gebremichael A,et al.cDNAcloning and functional expression of theSchistosoma mansoniprotective anti-gen triose-phosphate isomerase[J].Proc Natl Acad Sci USA,1992;89:1842-1846.
[7]Hong SJ,Seong KY,Sohn WM,et al.Molecular cloning andim-munological characterization of phosphoglycerate kinase from Clonorchis sinensis[J].Molecular and Biochemical Parasitology.2000,108:207-216.
[8]Kang IK,Lee SH,Seo BS.Study on the14C-glucose metabolism byClonorchissinensis.Paper chromatographic analysesin combi-nation with autoradiography[J].Korean J Parasitol,1969,7:43-52.
[9]徐劲,胡旭初,余新炳等.华支睾吸虫成虫全长基因表达文库的构建和基因表达谱的建立[J].中国人兽共患病杂志,2004,20:383-386.
[10]YangG,Jing CX,Zhu PX,et al.Molecular cloning and charac-terization of a novel lactate dehydrogenase gene fromClonorchis sinensis[J].Parasitol Res,2006,99(1):55-64.
[11]胡旭初,徐劲,吕刚,等.生物信息学分析华支睾吸虫乳酸脱氢酶(CsLDH)基因及其蛋白的结构与特性[J]热带医学杂志,2007,7(12):1145-1148.
[12]吕刚,余新炳,胡旭初,等.日本血吸虫乳酸脱氢酶(SjLDH)结构与功能的生物信息学分析[J],中国寄生虫学与寄生虫病杂志,2007,25:202-205.
[13]Mukamolova GV,Kaprelyants AS,Young DI,et al.Afamily of autocrine growth factors inMycobacteriumtuberculosis[J].Mol Microbiol,2002,46(3):623-635.
[14]Chalfie M,Tu Y,Euskirchen,G,et al,Prasher,D.C.,Green fluorescent protein as a marker for gene expression[J].Sci-ence,1994,263:802-805.
[15]Kwak KH,Cheon EW,Kim CH.Localization and characteris-tiCsof lactate andmalate dehydrogenasein the Sparganumand a-dult worm ofSpirometra erinacei[J].Korean J Parasito,l1996,34(1):59-68.
[16]BORK S,OKAMURA M,BOONCHIT S,et al.Identification ofBabesia bovisL-lactate dehydrogenase as a potential chemo-therapeutical target against bovine babesiosis[J].Mol Biochem Parasitol,2004,136(2):165-172.
[17]吕刚,胡旭初,余新炳等.棉籽酚、吡喹酮、蒿甲醚对重组日本血吸虫乳酸脱氢酶的作用,中国寄生虫学与寄生虫病杂志,2007,25(5):401-405.
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