山羊化脓隐秘杆菌分离鉴定及其神经氨酸酶遗传进化分析

Identification of Arcanobacterium pyogenes from goats and phylogenetic analysis of neuraminidase(NA)

为确诊重庆涪陵地区一山羊养殖场发生山羊皮下脓肿的病原，采集病料分离病原、进行药敏试验。通过生化试验和16SrRNA分子方法鉴定病原茵；PCR扩增分离株的神经氨酸酶基因（neuraminidase，NA），并利用细菌神经氨酸酶的隐马尔科夫模型（Pfam编号为PF13088）搜索自建的本地蛋白质组数据库，通过MEGA7．0构建系统进化树，从而分析神经氨酸酶基因在脊椎动物、寄生虫、真菌、细菌和病毒中的遗传进化特征，并利用PredictProtein进行分离株神经氨酸酶的点突变分析。结果表明，分离株的16SrRNA基因与JQ975932．1（中国新疆）株化脓隐秘杆菌序列同源性高达99．7％，结合生化特性确诊分离株为化脓隐秘杆菌；利用模型进行检索，共检索到214条神经氨酸酶氨基酸序列。神经氨酸酶遗传系统进化树显示，细菌神经氨酸酶主要集中在放线菌门中，脊椎动物的神经氨酸酶主要聚集在GroupⅠ和GroupⅡ，寄生虫的神经氨酸酶主要集中于GroupⅠ，真菌神经氨酸酶分布较广，存在GroupⅠ、GroupⅡ以及GroupⅣ中，而病毒神经氨酸酶主要分布于GroupⅠ和GroupⅡ。点突变分析表明，细菌神经氨酸酶不同位点的氨基酸突变对神经氨酸酶功能的影响不同。

To confirm the pathogens caused subcutaneous abscess of goats in a goat farm in Chongqing Fuling. The pathogens were isolated and identified by biochemical tests and 16S rRNA analysis. Drug sensitivity test was performed. The neuraminidase（NA） gene of this isolate was amplified by PCR,and local bacterial proteome database was first built based on the bacterial pro rein sequences,and Hidden Markov Model（HMM,Pfam Number. PF1308813088）was used to find NA. The genetic differentiation characteristics of NA from vertebrates, parasites, fungi and bacteri- a were analyzed by a phylogenetic tree with MEGA7.0. Then we used the PredictProtein to study the isolate point mutation. The results showed that the 16S rRNA gene of the isolate shared 99.7 ~/00 homology with JQ975932. 1（Xinjiang, China） strain, and combined with biochemical char acteristics,the strain was A. pyogenes. In total 214 NA sequences were retrieved by the model. The NA phylogenetic tree revealed that the bacterial NA are gathered in Actinobacteria phylum ,verte- brate NA can be found in Group I and Group II ,fungal NA are differently located in Group I , GroupII and Group IV, and the viral NA are mainly distributed in Group I and Group II. In addition,the point mutation of bacterial NA has different funcations depend on positions.