胸膜肺炎放线杆菌Apxll毒素的基因序列特征

Sequence characterization of Apxll toxin of Actinobacillus pleuropneumoniae

为了深入地了解胸膜肺炎放线杆菌(APP)分泌的ApxⅡ毒素的分子结构及序列特征,本实验以一株APP 7型现地分离株L25-4株为实验材料,对其分泌的ApXⅡ毒素的结构基因apxⅡA及其上游启动子区进行了PCR扩增和测序。结果表明APP7型L25-4株apxⅡA基因与其它血清型apxⅡA基因核苷酸序列同源性达99．5%以上,氨基酸序列同源性达98．5%以上。在ApxⅡA蛋白的N末端有3个大的疏水结构域,分布在240～422位氨基酸之间。在ApxⅡA蛋白的C末端有富含甘氨酸(Gly)和天门冬氨酸(Asp)的九肽(nonapeptides)重复序列Leu/Ile/Phe-Xaa-Gly-Gly-Xaa-Gly-Asm/Ssp-Asp-Xaa,串连重复9次。这些结构域的起始氨基酸的位置分别为374、503、630、735、753、762、771、780和802。推定的赖氨酸酰基化作用位点为557位氨基酸,与E．coli的HlyA相比,在688位氨基酸处少了一个赖氨酸酰基化作用位点。apxⅡA基因启动子-10区序列为AATAAT,-35区序列为TTAAT,-10区与-35区间隔序列为11个碱基。

In order to deeply understand the structure and molecular pathogenesis of Apx Ⅱ toxin of Actinobacillus pleuropneumoniae, the stucture gene apx Ⅱ A of Apx Ⅱ toxin of Actinobacillus pleuropneumoniae serotype 7 strain L25-4 was amplified by PCR and sequenced as well as the promoter gene upstream. The result showed that the nucleotide and amino acid homogeneity of serotype 7 strain L25-4 with other serotypes are 99.5 % and 98.5 %, respectively. There are three large hydrophobic domains at N＇-terminal part of Apx Ⅱ A, they located between 240-422 amino acid. A typical nonapeptides repeats domain was found at C＇-terminal part of Apx Ⅱ A which riches in Gly and Asp, Leu/I1e/Phe-Xaa-Gly-Gly-Xaa-Gly-Asm/Ssp- Asp-Xaa repeats for 9 times within Apx Ⅱ A. The initial sites of the repeated domains are 374, 503, 630, 735, 753, 762, 771, 780 and 802, respectively. The putative Lys acylation site （GK） of ApxⅡA is 557 amino acid, however, there is a deletion of GK at 688 amino acid compared with E.coli H1yA. The sequence of -10 region of apx Ⅱ A promoter is AATAAT, -35 region is TTAAT, there are 11 bases within the spacer between -10 and -35 region.