华支睾吸虫乳酸脱氢酶(CsLDH)亚细胞及虫体组织定位

Subcellular and immunohistochemistrical localization of Clonorchis sinensis lactate dehydrogenase

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。

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.