环形泰勒虫感染细胞抑制性消减文库的构建及ESTs分析

doi:10.3864/j.issn.0578-1752.2015.22.015

中国农业科学 ›› 2015, Vol. 48 ›› Issue (22): 4551-4563.doi: 10.3864/j.issn.0578-1752.2015.22.015

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

环形泰勒虫感染细胞抑制性消减文库的构建及ESTs分析

赵洪喜^1,3，刘军龙¹，李有全¹, 杨聪山¹，赵帅阳¹，刘娟¹, 刘爱红¹，谢俊仁¹，田占成¹，刘志杰¹，刘光远¹, 殷宏^1，2，关贵全¹，罗建勋¹

¹中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室，兰州 730046
²江苏省动物重要疫病与人兽共患病防控协同创新中心，江苏扬州225009
³宁夏大学农学院，银川 750021

收稿日期:2015-03-19 出版日期:2015-11-16 发布日期:2015-11-16
通讯作者: 罗建勋，E-mail：ljxbn@163.com。关贵全，E-mail：guanguiquan@163.com
作者简介:赵洪喜，E-mail：zhaohongxi2006@163.com
基金资助:
国家“973”计划（2015CB150300）、国家自然科学基金（31372432，31402189）、PiroVAC（KBBE-3-245145）

Construction and ESTs Analysis of SSH Library from Host Cells Infected by Theileria annulata

ZHAO Hong-xi^1,3, LIU Jun-long¹, LI You-quan¹, YANG Cong-shan¹, ZHAO Shuai-yang¹, LIU Juan¹, LIU Ai-hong¹, XIE Jun-ren¹, TIAN Zhan-cheng¹, LIU Zhi-jie¹, LIU Guang-yuan¹, YIN Hong¹^，2, GUAN Gui-quan¹, LUO Jian-xun¹

^1.State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Lanzhou 730046
²Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou 225009, Jiangsu
³Agricultural College of Ningxia University, Yinchuan 750021

Received:2015-03-19 Online:2015-11-16 Published:2015-11-16

摘要/Abstract

摘要： 【目的】环形泰勒虫（Theileria annulata）可以感染牛B淋巴细胞、树突状细胞和巨噬细胞，并可使被感染细胞发生转化，体外培养时可获得类似肿瘤样细胞的无限增殖能力。本研究通过构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库，筛选环形泰勒虫裂殖体基因及转化细胞与宿主正常细胞之间的差异表达基因。【方法】以环形泰勒虫裂殖体转化细胞和非感染牛外周血单个核细胞（即非转化宿主细胞）为试验材料，提取总RNA和mRNA，反转录合成cDNA；然后分别作为tester和driver，进行抑制性消减杂交（SSH），构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库；对随机挑取的阳性克隆进行测序及生物信息学分析，并利用实时荧光定量PCR对文库部分差异基因进行验证。【结果】通过对文库454个阳性克隆进行测序，共获得了有效表达序列标签（ESTs）364条，大小主要分布在350—1 200 bp之间；将这些ESTs序列在GenBank中进行BLASTn网上序列比对，其匹配于192条基因序列；其中包括环形泰勒虫序列60条(11条为虫体蛋白酶基因序列、10条为膜蛋白基因序列、7条为假设蛋白基因序列、5条为凋亡相关虫体蛋白基因序列、5条为核糖体蛋白基因序列、4条为细胞周期蛋白基因序列、4条为虫体抗原基因序列和虫体其它基因序列14条)、牛基因序列131条（其中19条为肿瘤相关蛋白基因序列）和1条未知序列；其中已有功能注释的ESTs 285条，再将已知功能的ESTs通过WEGO软件从生物学过程（biological process）、细胞组成（cellular component）和分子功能（molecular function）3个部分进行GO聚类分析，结果表明，宿主细胞差异基因主要集中在细胞、细胞进程、结合、催化、代谢进程、生物调节等功能方面。另外，通过对部分环形泰勒虫裂殖体转化宿主细胞差异基因进行荧光定量PCR分析，结果表明，肿瘤相关基因HCLS1和SENP5在转化宿主细胞中转录水平分别是非感染牛PBMCs(非转化宿主细胞)的2.06和1.32倍。【结论】利用SSH技术，成功构建了环形泰勒虫感染宿主细胞的抑制性消减文库，筛选获得了转化宿主细胞和环形泰勒虫裂殖体的表达基因，并通过荧光定量PCR分析获得了2个可能参与宿主细胞转化的基因，为进步探索环形泰勒虫与宿主细胞相互作用的分子机制奠定了基础。

关键词: 环形泰勒虫, 抑制性消减杂交, ESTs

Abstract: 【Objective】Theileria annulata can transform B lymphocytes、dendritic cells and macrophages it infects. Parasitized cells can be immortalized and acquire the ability to proliferate continuously as tumor cells in vitro culture. This study was carried out to construct a suppression subtractive hybridization (SSH) library in host cells transformed and non-transformed by T. annulata and to screen for schizont genes of T. annulata and differential genes of host cells from T. annulata schizont transformed cells and PBMCs of uninfected calves .【Method】 The total RNA and mRNA samples were extracted from T. annulata schizont transformed cells and PBMCs of uninfected calves, and the cDNA were synthesized by reverse transcription respectively. The cDNA subtracted library of host cells transformed and non-transformed by T. annulata was constructed using SSH technology, with cDNA from T. annulata transformed cells as the “tester” and cDNA from PBMCs of uninfected calves as the “driver”. Positive clones randomly chosen were sequenced and analyzed by bioinformatics. Furthermore, real-time PCR was employed to detect significant changes of part of differential genes from the cDNA subtracted library.【Result】A total of 364 effective differentially Expressed Sequence Tag (ESTs) were obtained via DNA sequencing from 454 positive clones, ranging from 350 to 1200 bp insert size. After comparing the sequences with BLASTn in the GenBank, these ESTs belonged to 192 gene sequences. Sixty of ESTs matched to T. annulata genes (11 ESTs were T. annulata protease genes, 10 T. annulata membrane protein genes, 7 T. annulata hypothetical protein genes, 5 apoptosis related T. annulata protein genes, 5 T. annulata ribosomal protein genes, 4 T. annulata cell-cycle protein genes, 4 T. annulata antigen protein genes and 14 T. annulata other genes), 131 bovine genes (19 were bovine tumor related genes) and one unknown gene. A total of 285 ESTs gene functions were annotated with Gene Ontology (GO) on the basis of biological process, cellular component, molecular function. Most of these were associated with cell, cellular processes, binding, catalytic, metabolic processes and biological regulation. Differential expressed genes from T. annulata transformed host cells were verified by real-time PCR, The results showed that the mRNA abundance of HCLS1 and SENP5 from bovine in transformed cells was 2.06 and 1.32 times that of non-transformed cells.【Conclusion】A SSH cDNA library of bovine cells infected by T. annulata was successfully constructed, and gene sequences of T. annulata schizonts and host cells transformed by T. annulata were obtained in this study. We acquire two bovine tumor related genes which possibly influence the host cell immortalized by real-time PCR. Our observations establish a foundation for better understanding of the molecular mechanisms of interactions between T. annulata and host cells.

Key words: Theileria annulata, suppression subtractive hybridzation, expressed sequence tag

赵洪喜，刘军龙，李有全, 杨聪山，赵帅阳，刘娟, 刘爱红，谢俊仁，田占成，刘志杰，刘光远，殷宏，关贵全，罗建勋. 环形泰勒虫感染细胞抑制性消减文库的构建及ESTs分析[J]. 中国农业科学, 2015, 48(22): 4551-4563.

ZHAO Hong-xi, LIU Jun-long, LI You-quan1, YANG Cong-shan, ZHAO Shuai-yang, LIU Juan, LIU Ai-hong, XIE Jun-ren, TIAN Zhan-cheng, LIU Zhi-jie, LIU Guang-yuan, YIN Hong, GUAN Gui-quan, LUO Jian-xun. Construction and ESTs Analysis of SSH Library from Host Cells Infected by Theileria annulata[J]. Scientia Agricultura Sinica, 2015, 48(22): 4551-4563.

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环形泰勒虫感染细胞抑制性消减文库的构建及ESTs分析

Construction and ESTs Analysis of SSH Library from Host Cells Infected by Theileria annulata

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