应用抑制性差减杂交筛选副猪嗜血杆菌4型菌株可能毒力基因  

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

作　　者：李淼[1,2] 李春玲[1,2] 宋帅[1,2] 杨冬霞[1,2] 

机构地区：[1]广东省农业科学院兽医研究所,广东广州510640 [2]广东省兽医公共卫生公共实验室,广东广州510640

出　　处：《华北农学报》2012年第1期57-62,共6页Acta Agriculturae Boreali-Sinica

基　　金：广东省农业科技重大专项(2009A020101006);广州市农业科技重大专项(2009A1-E041);广东省农科院兽医研究所所长基金

摘　　要：为了寻找副猪嗜血杆菌可能毒力基因,采用抑制性差减杂交技术,以HPS有毒力菌株SW124(血清4型)和无毒力菌株H465(血清11型)为研究对象,构建差减杂交文库,利用Southern Blot分析和PCR鉴定进行差异基因片段的筛选。从构建的文库中共筛选得到7个特异性差异基因片段,经同源性分析,其中5个片段分别与噬菌体重组蛋白、糖基转移酶/荚膜多糖磷酸转移酶、磷酸核糖甲酰甘氨脒合酶、核糖体蛋白丙氨酸转移酶和ABC型硝酸盐/磺酸盐/碳酸氢盐转运系统周质蛋白等编码的基因有同源性;2个差异基因片段与功能未知蛋白或假定蛋白编码基因有关。利用PCR对上述可能与毒力相关的差异基因片段在9种不同血清型HPS中的分布进行了检测,结果表明,7个片段存在于多数的有毒力菌株(血清4,5,12,14,15型)中,而在无毒力菌株(血清3,11型)中均未检测到。利用SSH技术成功构建了HPS有毒力菌株SW124和无毒力菌株H465的差异表达基因差减文库,筛选获得了7个差异基因片段,且上述片段在HPS不同血清型有毒力菌株中分布广泛。The objective of this paper is to find putative virulent genes of Haemophilus parasuis （HPS）. Sup- pression 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 frag- ments 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, phosphoribosylformylglyci- namidine synthase, ribosomal-protein-alanine N-acetyltransferase, ABC-type nitrate/sulfonate/bicarbonate trans- portsystems periplasmie 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 non- virulent 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.

关 键 词：副猪嗜血杆菌 抑制性差减杂交 毒力基因 

分 类 号：Q78[生物学—分子生物学]