1株青海牦牛腹泻产气荚膜梭菌基因分型及cpa基因生物信息学分析  

Molecular typing of a Qinghai yak-origin Clostridium perfringens and bioinformatics analysis of its cpa gene

作　　者：吴丹 罗润波 黄家旗 蔡重振 郭欣冉 郭晓庆 丹增卓嘎 李彬 宋仁德 索朗斯珠[1] WU Dan;LUO Runbo;HUANG Jiaqi;CAI Chongzhen;GUO Xinran;GUO Xiaoqing;DANZENG Zhuoga;LI Bin;SONG Rende;SUOLANG Sizhu(Academy of Animal Sciences,Tibet College of Agriculture and Animal Husbandry,Nyingchi,Tibet 860000,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Yushu Comprehensive Experiment Station,National Beef Cattle and Yak Industrial Technology System,Yushu,Qinghai 815000,China)

机构地区：[1]西藏农牧学院、动物科学院,西藏林芝860000 [2]江苏农业科学研究院、兽医研究所,江苏南京210014 [3]国家肉牛牦牛产业技术体系、玉树综合试验站,青海玉树815000

出　　处：《中国兽医学报》2023年第1期123-131,共9页Chinese Journal of Veterinary Science

基　　金：财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-37);西藏自治区科技厅2019年度重点资助项目(201901);西藏农牧学院研究生创新计划资助项目(YJS2021-11);国家重点研发计划资助项目(2021YFD1801100)。

摘　　要：为了解1株从青海玉树地区牦牛腹泻中分离的牦牛源产气荚膜梭菌Qinghai-1的分子型和cpa基因编码蛋白的生物信息,首先利用16S rDNA扩增、毒素基因检测、MLST分型、基因克隆等分子生物学方法对菌株毒素基因和管家基因进行分析,随后通过对cpa基因双向高通量测序、生物软件分析等生物信息学方法对其编码的α毒素蛋白的一级结构、二级结构、三级结构、蛋白特殊区域、B细胞线性抗原表位及蛋白互作关系进行分析。结果显示,Qinghai-1菌株分型毒素仅含有α毒素,各管家基因的等位基因序号依次为colA:9、groEL:96、sodA:91、plc:44、gyrB:67、sigK:55、pgk:8、nadA:16;cpa基因全长1197 bp,共编码398个氨基酸,与丹麦鸡源(EU839784.1)分离株plc基因差异性较小;其编码的α毒素蛋白分子式为C2036H3061N533O631S12,相对分子质量为45.5 ku,pI=5.68,脂肪指数:61.36,不稳定指数:19.88,亲水性总平均值-0.756,拥有1个跨膜螺旋和43个潜在磷酸化位点,B细胞线性抗原表位存在的区域为:25~36,81~121,194~202,312~324,356~364,389~394;其主要作用蛋白磷脂酶C(plc)与其他10个蛋白共形成23条相互作用连线。综上,Qinghai-1菌株为A型产气荚膜梭菌,是1株新的MLST型菌株,为ST-414型;其α毒素蛋白为含跨膜结构、含信号肽、含磷酸化位点、含多个B细胞线性抗原表位和能与多个蛋白相互作用的、稳定的亲水性蛋白。研究结果可为进一步了解牦牛源产气荚膜梭菌分子型以及通过深入的细胞毒性作用研究为后续进一步研究α毒素致病机理提供重要理论依据。The aim of this study is to understand the molecular type of a strain of Clostridium perfringens Qinghai-1 from yak origin and the biological information of the protein encoded by the cpa gene.Molecular biology methods such as 16S rDNA amplification,toxin gene detection,MLST typing,and gene cloning were used to analyze bacterial toxin genes and housekeeping genes,and then bioinformatics such as bidirectional high-throughput sequencing of cpa gene and biological software analysis were carried out.The primary structure,secondary structure,tertiary structure,special protein regions,linear epitopes of B cells and protein interactions of the encoded alpha toxin protein were analyzed.The results showed that the Qinghai-1 strain typing toxin only contains alpha toxin,and the allele numbers of each housekeeping gene were colA:9,groEL:96,sodA:91,plc:44,gyrB:67,sigK:55,pgk:8,nadA:16.The cpa gene was 1197 bp in length,encoding a total of 398 amino acids,which was less different from the plc gene of the Danish chicken source(EU839784.1)isolate.The molecular formula of the alpha toxin protein encoded by it was C2036H3061N533O631S12,the molecular weight was 45.5 ku,pI=5.68,fat index was 61.36,instability index was 19.88,total average hydrophilicity was-0.756,with one transmembrane helix and 43 potential phosphorylation sites,B cell linear epitope existing regions were:25-36,81-121,194-202,312-324,356-364,389-394.Its main role protein phospholipase C(plc)and other 10 proteins have gene co-expression.The phenomena of collaboration,gene fusion,and the field of genes formed a total of 23 interaction lines.In summary,Qinghai-1 strain is a type A Clostridium perfringens,a new MLST type strain,type ST-414;its alpha toxin protein contains transmembrane structure,signal peptide,and phosphoric acid A stable hydrophilic protein containing multiple B cell linear epitopes and is capable of interacting with multiple proteins.The research results can provide references for further understanding of the molecular type of Clostridium perfringen

关 键 词：牦牛 产气荚膜梭菌 分子分型 cpa基因 生物信息学分析 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]