贝氏柯克斯体九里株Ⅱ相突变体的克隆与鉴定

Cloning and Identification of Coxiella burnetii Nine Mile Phase Ⅱ Mutant

目的:克隆分离国内贝氏柯克斯体九里株Ⅱ相突变体,并利用基因组测序方法进行鉴定,为贝氏柯克斯体研究提供参考依据。方法:用半固体平板法克隆分离贝氏柯克斯体,对随机选择的克隆株CB01进行全基因组测序分析,将测序结果与国外贝氏柯克斯体不同克隆株的基因组进行比较分析。结果:CB01的基因组全长2.024 Mb,包含1个1.987 Mb的环状染色体和1个37.321 kb的质粒。与九里株Ⅰ相菌(RSA 493克隆)基因组相比,CB01多出5个重复序列,有1个25 997 bp的删除区,23个单核苷酸多态性位点(SNP)差异。与九里株Ⅱ相菌(RSA 439克隆)基因组相比,CB01同样多出5个重复序列,但仅有6个SNP差异。CB01与RSA 439的CBU_0533基因序列完全相同,可解释CB01缺失O抗原从而成为Ⅱ相菌。结论:贝氏柯克斯体九里株Ⅱ相菌克隆CB01与国外Ⅱ相菌RSA 439高度同源,CB01可用做国内贝氏柯克斯体研究用参考菌株。

Objective:To clone and purify Coxiella burnetii Nine Mile phaseⅡstrain and to characterize the clone by genome sequencing,so as to facilitate future C.burnetii research.Methods:C.burnetii was cloned by semi-solid plating.The whole genome of the randomly selected CB01 clone was resolved by next generation sequencing.The CB01 genome was compared with reported genomes of different C.burnetii Nine Mile clones.Results:The 2.024 Mb CB01 genome contains a 1.987 Mb circular chromosome and a 37.321 kb plasmid.Compared with the genome of phaseⅠNine Mile RSA 493 clone,CB01 has extra five repetitive sequences,a deletion region of 25 997 bp,and a minimum of 23 single nucleotide polymorphisms(SNP).Compared with the genome of phaseⅡNine Mile RSA 439 clone,CB01 has only 6 variations regarding SNP.CB01 and RSA439 share identical sequences of the CBU_0533 gene,which genetically explains the phase variation of CB01 due to the absence of O-antigen.Conclusion:The genome of C.burnetii Nine Mile CB01 clone is highly homologous with that of phaseⅡNine Mile RSA 439 clone.CB01 can be used as a reference clone for C.burnetii research in China.