Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (7): 1406-1417.doi: 10.3864/j.issn.0578-1752.2012.07.020

• VETERINARY SCIENCE • Previous Articles     Next Articles

Construction of Subtractive Hybridization cDNA Library to Screen Differentially Expressed Genes from the Spleen of C57BL/6 and A/J Mouse and Their Functional Analysis

 XIE  Zheng-Lu, SHEN  Xue-Huai, LIU  Lin, YIN  Fu-Jian, MA  Hai-Tian, FAN  Hong-Jie   

  1. 1.南京农业大学动物医学院/农业部动物生理生化重点开放实验室，南京210095
    2.南京农业大学动物医学院/农业部动物疫病诊断与免疫重点开放实验室，江苏南京210095
  • Online:2012-04-01 Published:2012-04-01

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Abstract: 【Objective】 The aim of this study was to discuss different mechanisms of infecting Streptococcus suis type 2 in C57BL/6 and A/J mouse strains by detecting the immunization parameters and constructed of cDNA library to screen different expressed genes from the spleen of C57BL/6 and A/J mouse strains. 【Method】 Immunization parameters was detected by ELISA. The substracted cDNA library was constructed from the 8-week-old age spleen of C57BL/6 and A/J mouse strains by suppression subtraction hybridization (SSH). 【Result】 The result showed that, the content of IgA was significantly higher in A/J mouse than C57BL/6 mouse,  but there was no significantly differences in IgG and IFN between A/J and C57BL/6 mouse stains. A total of 149 positive clones were screened by PCR and sequenced. After dislodging, clustering and splicing the redundant cDNA sequence, total of 56 differentially expressed sequence tags (ESTs) were obtained in the whole subtractive cDNA library. After comparisons with GenBank using online software of the BLAST, total of 26 specific gene fragment and two unknow sequences were found. 【Conclusion】 Many of those ESTs were highly homologous with the important functional genes with related to signal transduction, apoptosis and immunity and so on, and the results would provide useful baseline for screening and cloning specific resistant genes and understanding the molecular mechanism of Streptococcus suis.

Key words: C57BL/6 mouse, A/J mouse, SSH, Differentially expressed genes, Subtractive cDNA library

[1]Dom?´nguez-Punaro M C, Segura M, Radzioch D, Rivest S, Gottschalk M. Comparison of the susceptibilities of C57BL/6 and A/J mouse strains to Streptococcus suis serotype 2 infection. Infection and Immunity, 2008, 76(9): 3901-3910.

[2]Higgins R, Gottschalk M. Distribution of Streptococcus suis capsular type in 2000. Canada Veterinary Journal, 2001, 42(3): 223.

[3]Hynes W L, Walton S L. Hyaluronidases of gram-positive bacteria. FEMS Microbiol Lett, 2000, 183(2): 201-207.

[4]万遂如. 我国猪链球菌II型流行情况与人类健康. 动物科学与动物医学, 2005, 22(4): 38-39.

Wan S R. Streptococcus suis serotype 2 epidemic in China and human health. Animal Science and Animal Medical Science, 2005, 22(4): 38-39. (in Chinese)

[5]姚火春, 陈国强, 陆承平. 猪链球菌1998分离株病原特性鉴定. 南京农业大学学报, 1999, 22(2): 67-70.

Yao H C, Chen G Q, Lu C P. Identification of isolates of swine Streptococcus in Jiangsu Province during 1998. Journal of Nanjing Agricultural University, 1999, 22(2): 67-70. (in Chinese)

[6]陆承平, 姚火春, 范红结. 猪链球菌致病性研究及其公共卫生意义.中国预防医学杂志, 2006, 7(4): 361-362.

Lu C P, Yao H C, Fan H J. Study and research Streptococcus pathogenicity and purpose of public health. China Preventive Medicine, 2006, 7(4): 361-362. (in Chinese)

[7]Li R, Zhang A, Chen B, Teng L, Wang Y, Chen H, Jin M. Response of swine spleen to Streptococcus suis infection revealed by transcription analysis. BMC Genomics, 2010, 11:556.

[8]De Jonge W J, van der Zanden E P, The F O, Bijlsma M F, van Westerloo D J, Bennink R J, Berthoud H R, Uematsu S, Akira S, van den Wijngaard R M, Boeckxstaens G E. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nature Immunology, 2005, 37(8): 844-852.

[9]甘肃农业大学. 兽医微生物学实验指导. 北京: 中国农业出版社, 1980. 11.

Gansu Agricultural University. Veterinary Microbiology Experimental Guide. Beijing: China Agriculture Press. 1980, 11. (in Chinese)

[10]Munir S, Kapur V. Transcriptional analysis of the response of poultry species to respiratory. Pathogens Poultry Science, 2003, 82(6): 885-892.

[11]Moens L N, Soetaert A, van der Ven K, Del-Favero J, De Coen W M. Use of suppression subtractive hybridization PCR for the development of cDNA arrays for the detection of endocrine disruption in carp (Cyprinus carpio). Comparative Biochemistry and Physiology, 2007, 2(1): 18-33.

[12]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.

[13]Akopyants N S, Fradkov A, Diatchenko L, Hill J E, Siebert P D, Lukyanov S A, Sverdlov E D, Berg D E. PCR-based subtractive hybridization and differences in gene content among strains of Helicobactor pylori. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(22): 13108-13113.

[14]Larsen A N, Solstad T, Svineng G, Seppola M, Jørgensen T Ø. Molecular characterization of a goose-type lysozyme gene in Atlantic cod (Gadus morhua L.). Fish & Shell?sh Immunology, 2009, 26(1): 122-132.

[15]Chuang T H, Ulevitch R J. Cloning and characterization of a sub-family of human Toll-like receptors: hTLR7, hTLR8 and hTLR9. Europe Cytokine Network, 2000, 11(3): 372-378.

[16]Town T, Bai F, Wang T, Kaplan A T, Qian F, Montgomery R R, Anderson J F, Flavell1 R A, Fikrig E. Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin 23-depen-dent immune cell infiltration and homing. Immunity, 2009, 30(2): 242-253.

[17]Takami K, Takuwa N, Okazaki H, Kobayashi M, Ohtoshi T, Kawasaki S, Dohi M, Yamamoto K, Nakamura T, Tanaka M, Nakahara K, Takuwa Y, Takizawa H. Interferon- γ inhibits hepatocyte growth factor stimulated cell proliferation of human bronchial epithelial cells Upregulation of p27kip1 Cyclin-Dependent Kinase Inhibitor. American Journal of Respiratory Cell and Molecular Biology, 2002, 26 (8): 231- 238.

[18]Lawson J K, Yu B, Thomas L L, Landay A, Moy JN. Toll- like receptor 8 (TLR8) mediated inhibition of Th2 cytokine response to ragweed antigen in peripheral blood mononuclear cells (PBMC) of ragweed allergic individuals. Journal of Allergy and Clinical Immunology, 2008, 121 (2): 261.

[19]Mcfarland W C, Stocker B A. Effect of different purine auxotrophic mutations on mouse-virulence of a Vi-positive strain of Salmonella dublin and of two strains of Salmonella typhimurium. Microbial Pathogenesis, 1987, 3(2): 129-141.

[20]Mahanm J, Slauch J M, Mekalanos J J. Selection of bacterial virulence genes that are specifically induced in host tissues. Science, 1993, 259(5095): 686-688.

[21]Noriega F R, Losonsky G, Lauderbaugh C, Liao F M, Wang J Y, Levine M M. Engineered deltagua B-A deltavir G Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity and potential efficacy as a mucosal vaccine. Infection and Immunity, 1996, 64 (8):3055-3061.

[22]唐胜建, 王小柯, 陈建华, 王雪耘, 梁伟中, 梁晓琴.无瘢痕愈合中核糖体蛋白s29基因表达产物对皮肤成纤维细胞增生的作用. 中国临床康复, 2004, 23(8): 4762-4764.

Tang S J, Wang X K, Chen J H, Wang X Y, Liang W Z, Liang X Q. Effect of ribosomal protein s29 gene expression-yan Wang product on the proliferation of skin fibroblasts in non-scar healing. Chinese Journal of Clinical Rehabilitation, 2004, 23(8): 4762-4764. (in Chinese)

[23]Khanna N, Reddy V G, Tuteja N, Singh N. Differential gene expression in apoptosis: identification of ribosomal protein S29 as an apoptotic inducer. Biochemical and Biophysical Research Communications, 2000, 277(2): 476-486.

[24]杨　帆, 刘卫平. 核糖体蛋白基因及其与人类疾病的研究进展. 临床与实验病理学杂志, 2005, 20(3): 351-356.

Yang F, Liu W D. Ribosomal protein gene and human disease to study and research progression. Journal of Clinical and Experimental Pathology, 2005, 20(3): 351-356. (in Chinese)

[25]Kondoh N, Noda M, Fisher R J, Schweinfest C W. The S29 ribosomal protein increases tumor suppressor activity of K rev-1 gene on v-K ras-transformed NIH3T3 cells. Biochemistry and Biophysics Acta, 1996, 1313(1): 41-46.

[26]Height S E, Swansbury G J, Matutes E, Treleaven J G, Catovsky D, Dyer M J S. Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia. Blood, 1996, 87(12):5242-5250.

[27]Duke V M, Gandini D, Sherrington P D, Lin K, Heelan B, Amlor P, Mehta A B, Hoffbrand V, Foroni L. V(H) gene usage differs in germline and mutated B-cell chronic lymphocytic leukemia. Haematologica, 2003, 88(11): 1259-1271.

[28]Chu H W. The mechanism of globin gene regulation by effective concentrations of transcription factors[D]. Taibei: National Yang- Ming University Taiwan of China , 2004.

[29]Stambolic V, Suzuki A, de la Pompa J L, Brothers G M, Mirtsos C, Sasaki T, Ruland J, Penninger J M, Siderovski P, Mak T W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell, 1998, 95(1):29-39.

[30]Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, Gavrilova N, Mueller B, Liu X, Wu H. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(11): 6199-6204.

[31]Qiu F Y, Song X X, Zheng H, Zhao Y B, Fu G S. Thymosin beta4 induces endothelial progenitor cell migration via PI3K/Akt/eNOS signal transduction pathway. Journal of Cardiovascular Pharmacology, 2009, 53(3): 209-214.

[32]Rossdeutsch A, Smart N, Riley P R. Thymosin β4 and Ac-SDKP: Tools to mend a broken heart. Journal of Molecular Medicine (Berlin, Germany), 2008, 86(1): 29-35.

[33]Wyczo´?kowska J, Walczak-Drzewiecka A, Wagner W, Dastych J. Thymosin b4 and thymosin b4-derived peptides induce mast cell exocytosis. Peptides, 2007, 28(4): 752-759.

[34]Sosne G, Chan C C, Thai K, Kennedy M, Szliter E A, Hazlett L D, Kleinman H K. Thymosin beta 4 promotes corneal wound healing and modulates inflammatory mediators in vivo. Experimental Eye Research, 2001, 72(5): 605-608.
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