华支睾吸虫ATP合酶b亚基的组织和亚细胞定位  

Tissular and Subcellular Localization of the ATP Synthase B Subunit in Clonorchis sinensis

作　　者：胡旭初[1] 周红娟[1,2] 胡凤玉[1] 马长玲[1,3] 赵俊红[1] 黄灿[1] 郑小凌[1] 徐劲[1] 余新炳[1] 

机构地区：[1]中山大学中山医学院寄生虫教研室,广州510080 [2]杭州市红十字会医院风湿免疫实验室,杭州310003 [3]广州医学院病原生物学教研室.广州510182

出　　处：《中国寄生虫学与寄生虫病杂志》2009年第2期95-101,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家高技术研究发展计划(863计划)项目(No.2006AA02Z422);广东省重大科技专项(No.2004A30801004);广州市科技计划项目(No.2006Z3-E4011)~~

摘　　要：目的了解华支睾吸虫ATP合酶b亚基(CsATP-synt_B)的虫体组织定位,并以HeLa细胞为替代模型观察其亚细胞定位。方法用CsATP-synt_B重组蛋白免疫SD大鼠,取其抗血清对华支睾吸虫成虫石蜡切片进行间接免疫荧光染色,观察CsATP-synt_B蛋白在华支睾吸虫成虫组织中的分布。根据生物信息学预测的CsATP-synt_B线粒体转运序列(MTS序列)和核定位序列(NLS序列)位置,设计4组引物[CsATP-synt_B全长序列,以及3个缺失突变体(MTS-缺失线粒体转运序列、NLS-缺失核定位序列、MTS-NLS-线粒体-核定位双缺失)引物],扩增出全长基因和3个突变体基因,构建重组质粒pEGFP-N1-CsATP-synt_B1-300、pEGFP-N1-CsATP-synt_B30-300、pEGFP-N1-CsATP-synt_B(1-238)+(257-300)及pEGFP-N1-CsATP-synt_B(30-238)+(257-300)。将这些重组质粒用阳离子脂质体转染HeLa细胞,48h后用激光共聚焦显微镜观察CsATP-synt_B全长基因和3个突变体在HeLa细胞的表达和亚细胞定位。结果CsATP-synt_B蛋白在华支睾吸虫成虫中分布较为广泛,主要分布在腹吸盘、卵巢、卵黄腺和皮层。绿色荧光蛋白GFP表明CsATP-synt_B全长序列表达于线粒体或细胞核,线粒体转运序列缺失突变体表达于细胞核,核定位序列缺失突变体表达于线粒体,双缺失突变体仅表达于胞浆。结论CsATP-synt_B蛋白在华支睾吸虫的分布与线粒体的分布一致,主要集中在能量代谢较旺盛的组织部位。CsATP-synt_B蛋白既可进入线粒体,也可进入细胞核,理论预测的定位序列得到证实。Objective To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. Methods With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence（MTS）and probable Bipartite nuclear localization signals（NLS_BP）in CsATP-synt_B sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by LipofectamineTM 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. Results The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. Conclusion The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organsand the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

关 键 词：华支睾吸虫 ATP合酶b亚基 免疫组织化学 亚细胞定位 靶向序列 

分 类 号：R383.22[医药卫生—医学寄生虫学]