Putative Virulence Associated Genes of Haemophilus parasuis Type 4 Strains Identified by Suppression Subtractive Hybridization

doi:10.3969/j.issn.1000-7091.2012.01.011

Abstract

Abstract: The objective of this paper is to find putative virulent genes of Haemophilus parasuis(HPS). Suppression subtractive hybridization(SSH) was used to construct the subtracted library between virulent strain SW124(HPS serovar 4 reference strain) and non-virulent strain H465(HPS serovar 11 reference strain). Using Southern hybridization and PCR to screen the differentially expressed genes. Seven clones were identified containing fragments that were present in the SW124 genome while absent in the H465 genome and the DNA sequences of these fragments were determined. These DNA fragments involved putative virulence associated genes encoded 7 proteins, including transport systems periplasmic components protein, phage recombination protein, phosphoribosylformylglycinamidine synthase, ribosomal-protein-alanine N-acetyltransferase, ABC-type nitrate/sulfonate/bicarbonate transportsystems periplasmic components protein, hypothetical protein and unknown. Based on PCR analysis of the 9 HPS serovar strains, these DNA fragments were found in most virulent serovars(4, 5, 12, 14, 15), but absent in the nonvirulent serovars(3, 11). The subtracted library of HPS SW124 and H465 was successfully constructed using SSH technique and seven specific genomic fragments were obtained. In additon, most of these fragments could be detected in other serotypes HPS virulent strains with different distribution.

Key words: Haemophilus parasuis, Suppression subtractive hybridization(SSH), Virulence associated gene

CLC Number:

LI Miao, LI Chun-ling, SONG Shuai, YANG Dong-xia. Putative Virulence Associated Genes of Haemophilus parasuis Type 4 Strains Identified by Suppression Subtractive Hybridization[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(1): 57-62. doi: 10.3969/j.issn.1000-7091.2012.01.011.

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References

[1] 蔡旭旺.副猪嗜血杆菌的分离鉴定及诊断方法与灭活疫苗的研究[D] 武汉:华中农业大学,2006.
[2] Hill C E,Metcalf D S,MacInnes J I.A search for virulence genes of Haemophilus parasuis using differential display RT-PCR[J] Veterinary Microbiology,2003,96(2):189-202.
[3] Sack M,Baltes N.Identification of novel potential virulence associated factors in Haemophilus parasuis[J] Veterinary Microbiology,2009,136(3-4):382-386.
[4] Zhou M,Guo Y,Zhao J,et al.Identification and characterization of novel immunogenic outer membrane protein of Haemophilus parasuis serovar 5[J] Vaccine,2009,27(38):5271-5277.
[5] Zhou M,Zhang A,Guo Y,et al.A comprehensive proteome map of the Haemophilus parasuis serovar 5[J] Proteomics,2009,9(10):2722-2739.
[6] Diatchenko L,Lau Y F,Campbell A P,et al,Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J] Proceedings of the National Academy of Sciences of the USA,1996,93:6025-6030.
[7] Gurskaya N G,Diatchenko L,Chenchik A,et al.Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction:cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate[J] Analytical Biochemistry,1996,240(1):90-97.
[8] 李会勇,黄素华,赵久然,等.应用抑制差减杂交法分离玉米幼苗期叶片土壤干旱诱导的基因[J] 中国农业科学,2007,40(5):882-888.
[9] 李星,于秀梅,李亚宁,等.叶锈菌诱导的小麦叶片抑制差减杂交文库构建及其分析[J] 中国农业科学,2008,41(12):4069-4076.
[10] Radnedge L,Gamez-Chin S,McCready P M,et al.Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis[J] Applied and Environmental Microbiology,2001,67(8):3759-37621.
[11] Janke B,Dobrindt U,Hacker J,et al A subtractive hybridization analysis of genomic differences between the uropathogenic E.coli strain 536 and the E.coli K-12 strain MG16551[J] FEMS Microbiology Letters,2001,199(1):61-661.
[12] Sawada K,Kokeguchi S,Hongyo H,et al.Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of Porphyromonas gingivalis[J] Infection and Immunity,1999,67(11):5621-56251.
[13] Parsons Y N,Panagea S,Smart C H,et al.Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa[J] Journal of Clinical Microbiology,2002,40(12):4607-46111.
[14] Zhou H,Yang B,Xu F,et al.Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization[J] Veterinary Microbiology,2010,26,144(3-4):377-383.
[15] Kielstein P,Rapp-Gabrielson V J.Designation of 15 serovars of Haemophilus parasuis on the basis of irnmunodiffusion using heat-stable antigen extracts[J] Journal of Clinical Microbiology,1992,30(4):862-865.
[16] Whitfield C.Biosynthesis and assembly of capsular polysaccharides in Escherichia col[J] Annual Review of Biochemistry,2006,75:39-68.
[17] Neidhardt F C,Curtiss R.Escherichia coli and Salmonella:Cellullar and Molecular Biology[M] 2nd Ed.Washington D C:American Society for Microbiology Press,1996:104-122.
[18] Sussman M.Escherichia coli:Mechanisms of Virulence[M] Cambridge:Cambridge University Press,1997:113-143.
[19] Metcalf D S,MacInnes J I.Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid[J] Canadian Journal of Veterinary Research,2007,71(3):181-188.
[20] Waldor M K,Friedman D I.Phage regulatory circuits and virulence gene expression[J] Current Opinion of Microbiology,2005,8(4):459-465.

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