牦牛源C型产气荚膜梭菌动物致病性及其β毒素基因生物信息分析  

Analysis of the animal pathogenicity of a yak-derived C-type Clostridium perfringens and its β-toxin gene encoding protein

作　　者：吴丹 黄家旗 罗润波 郭欣冉 吕家煌 宋仁德 索朗斯珠 WU Dan;HUANG Jiaqi;LUO Runbo;GUO Xinran;LYU Jiahuang;SONG Rende;SUOLANG Sizhu(Department of Animal Science,Tibet Agriculture and Animal Husbandry College,Nyingchi,Tibet 860000,China;Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Qinghai Yushu Tibetan Autonomous Prefecture Animal Husbandry and Veterinary Work Station,Yushu,Qinghai 815000,China)

机构地区：[1]西藏农牧学院动物科学学院,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]青海玉树藏族自治州畜牧兽医工作站,青海玉树815000

出　　处：《中国兽医学报》2022年第6期1184-1193,共10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)。

摘　　要：复苏培养本实验室保存的牦牛源C型产气荚膜梭菌Qinghai-06(GenBank登录号:MW980095),利用振荡比浊法、动物试验和病理切片方法对菌株生长曲线及动物致病性进行研究,随后通过对β毒素基因扩增、克隆、双向高通量测序、生物软件分析等分子生物学与生物信息学方法对其编码蛋白的一级结构、二级结构、三级结构、蛋白特殊区域及B细胞线性抗原表位进行分析。结果显示,牦牛源C型产气荚膜梭菌Qinghai-06菌株按照5%体积分数接种后,2~6h为对数期;菌液D_(600 nm)值与稀释倍数的关系式为y=-0.857ln(x)+2.236;菌液D_(600 nm)值与菌落CFU的关系式为:y=0.881x-0.017;菌株对感染小鼠的LD_(50)为0.408×10^(8)CFU/m L。H.E染色镜检观察到可引起肠、脑、肾脏、肝脏、脾脏组织病理变化。β毒素基因序列长927bp,共编码309个氨基酸,分子式为C_(1535)H_(2396)N_(406)O_(502)S_(9),相对分子质量为34.9ku,p I=5.52,不稳定指数为35.91,亲水性总平均值为-0.567,含有全部20种氨基酸,为单体,含1个Zn^(2+)配体。结果表明:牦牛源C型产气荚膜梭菌Qinghai-06属强致病菌株,其β毒素蛋白为含丰富无规则卷曲结构和潜在磷酸化位点的、稳定的亲水性蛋白,有效B细胞线性抗原表位主要集中于140~290区段。Resuscitate the yak-derived C-type Clostridium perfringens Qinghai-06(GenBank accession number:MW980095) preserved in our laboratory,and use the oscillatory turbidimetric method,animal experiment and pathological section methods to analyze the growth curve and animal pathogenicity of the strain research,followed by molecular biology and bioinformatics methods such as β-toxin gene amplification,cloning,two-way high-throughput sequencing,biological software analysis and other molecular biology and bioinformatics methods for the primary structure,secondary structure,tertiary structure,and protein specificity of the encoded protein Areas and linear epitopes of B cells were analyzed.The results showed that the yak-derived C-type Clostridium perfrin gens Qinghai-06 strain was inoculated according to a 5 % volume fraction,and the log phase was 2-6 h;the relationship between the D_(600 nm) value of the bacterial solution and the dilution factor was y=-0.857 ln(x)+2.236;the relationship between the Dvalue of the bacterial solution and the CFU of the colony is:y=0.881 x-0.017;the LDof the strain to infected mice is0.408× 10^(8) CFU/mL.H.E staining microscopy observed that it can cause pathological changes in the intestine,brain,kidney,liver,and spleen.The β-toxin gene sequence is 927 bp long and encodes a total of 309 amino acids.The molecular formula is C_(1535)H_(2396)N_(406)O_(502)S_(9),the molecular weight is34.9 ku,pI=5.52,the instability index is 35.91,the total average hydrophilicity is-0.567,and it contains all 20 amino acids.The monomer contains a Zn^(2+) ligand.The results showed that:yak-derived C-type Clostridium perfringens Qinghai-06 is a strongly pathogenic strain,and the β toxin protein contained in it is rich in random coiled structure,containing potential phosphorylation sites,and stable hydrophilicity.Protein,effective B cell linear antigen epitopes are mainly concentrated in the 140-290 segment.

关 键 词：牦牛 产气荚膜梭菌 病理切片 Β毒素 生物信息分析 

分 类 号：S852.61[农业科学—基础兽医学]