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

Abstract

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

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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References

[1] 吕文祥, 吕文顺, 张其才, 罗建勋, 殷宏, 窦惠芳. 我国牛蜱传性血液原虫的虫种调查和分布特征的研究. 中国兽医科技, 1995, 25(8): 13-16.

LÜ W X, LÜ W S, Zhang Q C, Luo J X, Yin H, Dou H Y. Survey of the species of tick-borne hacmoprotozoan of cattle and feature in China. Chinese Journal of Veterinary Sciences and Technology, 1995, 25(8): 13-16. (in Chinese)

[2] Tchernomorez I. Multiplication in vitro of Koch bodies of Theileria annulata. Nature, 1945, 156: 391.

[3] Hulliger L. Cultivation of there species of Theileria in lymphoid cells in vitro. Journal of Protozoology, 1965, 12: 649-655.

[4] Brown C G D, Stagg D A, Purnell R E, Kanhai G K, Payne R C. Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parva. Nature, 1973, 245: 101-103.

[5] 甘肃省兽医研究所. 环形泰勒焦虫裂殖体细胞培养及其免疫原性的研究. 动物学报, 1975, 9: 243-256.

Gansu Veterinary Research Institute. In vitro cultivation of cells infected with Theilaria annulata schizont and its immunogenecity. Acta Zoologica Sinica, 1975, 3: 243-256. (in Chinese)

[6] 吕文顺，李观娣，吕文祥. 环形泰勒焦虫裂殖体细胞培养和裂殖体胶冻细胞苗免疫力的研究. 中国农业科学, 1982, 15(4): 88-95.

LÜ W S, Li G D, LÜ W X. Studies on the cell cultures of Theileria annulata schizonts and immunity indued by the gelatin -protected, schizont -parasitized cell vaccine. Scientia Agricultura Sinica, 1982, 15(4): 88-95. (in Chinese)

[7] 罗建勋. 我国第一个兽医寄生虫病疫苗——环形泰勒虫裂殖体胶冻细胞苗. 中国兽医寄生虫病, 2003, 10: 62.

Luo J X. The first veterinary parasitic vaccine in china -- Theileria annulata gelatin -protected, schizont-parasitized cell vaccine. Chinese Journal of Veterinary Parasitology, 2003, 10: 62. (in Chinese)

[8] Seitzer U, Ahmed J. Tropical theileriosis: cytotoxic T lymphocyte response to vaccination. Vaccine, 2008, 26 (Suppl. 6): G24-G28.

[9] Preston P M, Hall F R, Glass E J, Campbell J D M, Darghouth M A, Ahmed J S, Shiels B R, Spooner R L, Jongejan F, Brown C G D. Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata. Parasitology Today, 1999, 15: 268-274.

[10] Shiels B, Langsley G, Weir W, Pain A, McKellar S. Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes. International Journal for Parasitology, 2006, 36: 9-21.

[11] Baumgartner M, Chaussepied M, Moreau M F, Werling D, Davis W C. Constitutive PI3-K activity is essential for proliferation, but not survival of Theileria parva-transformed B cells. Cellular Microbiology, 2000, 2(4): 329-339.

[12] Botteron C, Dobbelaere D. AP-1 and ATF-2 are constitutively activated via the JNK pathway in Theileria parva-transformed T-cells. Biochemical and Biophysical Research Communications, 1998, 246: 418-421.

[13] Chaussepied M, Lallemand D, Moreau M F, Adamson R, Hall R. Upregulation of Jun and Fos family members and permanent JNK activity lead to constitutive AP-1 activation in Theileria-transformed leukocytes. Molecular and Biochemical Parasitology, 1998, 94: 215-226.

[14] Dessauge F, Hilaly S, Baumgartner M, Blumen B, Werling D. c-Myc activation by Theileria parasites promotes survival of infected Blymphocytes. Oncogene, 2005, 24: 1075-1083.

[15] Seitzer U, Gerber S, Beyer D, Dobschanski J, Kullmann B. Schizonts of Theileria annulata interact with the microtubuli network of their host cell via the membrane protein TaSP. Parasitology Rresearch, 2010, 106: 1085-1102.

[16] Kyoko H, Masakazu H, Ryo N. A schizont-derived protein, TpSCOP, is involved in the activation of NF-kB in Theileria parva-infected lymphocytes. Molecular and Biochemical Parasitology, 2010, 174: 8-17.

[17] Swan D G, Phillips K, Tait A. Evidence for localisation of a Theileria parasite AT hook DNA-binding protein to the nucleus of immortalised bovine host cells. Molecular and Biochemical Parasitology, 1999, 101(1-2): 117-129.

[18] Schmuckli-Maurer J, Casanova C, Schmied S. Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family. PLoS One, 2009, 4(3): 1371.

[19] 代小梅, 程晓霞, 曾会明, 韩烈保. 日本结缕草总RNA提取和mRNA分离方法的优化. 生物技术通报，2010, 12: 132-136.

Dai X M, Cheng X X, Zeng H M, Han L B. The Optimization of total RNA extraction and mRNA isolation from zoysiagrass (Zoysiajaponica). Biotechnology Bulltin, 2010, 12: 132-136. (in Chinese)

[20] Hubank M, Schatz D G. Identifying differences in Mrna expression by representational difference analysis of Cdna. Nucleic Acids Research, 1994, (25): 5640-5648.

[21] Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E D, Siebert P D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(12): 6025-6030.

[22] Duggan D J, Bittner M, Chen Y, Meitzer R, Trent J M. Expression profiling using cDNA microarrays. Nature Genetics, 1999, 21: 10-14.

[23] Surachetpong W, Singh N, Cheung K W, Luckhart S. MAPK ERK Signaling Regulates the TGF-b1-Dependent Mosquito Response to Plasmodium falciparum. PLoS Pathogen, 2009, 5(4): e1000366.

[24] Bermudez O, Pagès G, Gimond C.The dual-specificity MAP kinase phosphatases: critical roles in development and cancer. American Journal of Physiology Cell Physiology, 2010, 299: 189-202.

[25] Jacqueline SM, Jane K, Sreerekha P, Pascal H, Sue M, William W, Dirk D, Brian S. Modulation of NF-kB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton. Cellular Microbiology, 2010, 12(2): 158-173.

[26] Baumgartner M. Theileria annulata promotes Src kinase-dependent host cell polarization by manipulating actin dynamics in podosomes and lamellipodia.Cellular Microbiology, 2011: 13(4): 538-553.

[27] Chou K C, Shen H B. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PloS One, 2010, 5: e1335.

[28] Schnittger L, Katzer F, Biermann R, Shayan P, Boguslawski K, McKellar S. Characterization of a polymorphic Theileria annulata surface protein (TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines. Molecular and Biochemical Parasitologyl, 2002, 20: 247-256.

[29] Dessauge F, Lizundia R, Baumgartner M. Taking the Myc is bad for Theileria. Trends Parasitol, 2005, 21(8): 377-385.

[30] Zha J P, Harada H, Yang E, Jockel J, Korsmeyer S J. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell, 1996, 87: 619-628.

[31] Leto D, Uhm M, Williams A, Chen XW, Saltiel AR. Negative Regulation of the RalGAP Complex by 14-3-3. The Journal of Biological Chemistry, 2013,3: 9272-9283.

[32] Seimiya H, Sawada H, Muramatsu Y, Shimizu M, Ohko K, Yamane K, Tsuruo T. Involvement of 14-3-3 proteins in nuclear localization of telomerase. Embo Journal, 2000, 19: 2652-2661.

[33] Taniuchi I，Kitamiira D，Maekawa Y，Fukuda T，Kishi H, Watanabe T. Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HSl protein, a substrate of the antigen-receptor-coupled tyros inekina ses. Embo Journal, 1995, 14: 3664-3678.

[34] 葛娟. HCLS1参与调控B淋巴瘤细胞分化机制的初步研究[D]. 广州：南方医科大学，2014.

Ge J. Preliminary study on the mechanisms of HCLS1 involves in the differentiation of B Lymphocytes [D]. Guangzhou : Southern Medical University, 2014. (in Chinese)

[35] Di Bacco A, Ouyang J, Lee H Y, Catic A，Ploegh H, Gill G. The SUMO-specific protease SENP5 is required for cell division. Molecular and Cellular Biology, 2006, 26: 4489-4498.

[36] Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride H M. The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. Journal of Cell Science, 2007, 120: 1178-1188.

[37] Rivki C, Helit C, Rotem B H, Alona Z , Sol E. SENP5 mediates breast cancer invasion via a TGFβRI SUMO ylation ascade. Oncotarget, 2014, 5( 4): 1071-1082.

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