炭疽芽孢杆菌的分离鉴定及其与疫苗株的鉴别诊断

Isolation and identification of Bacillus anthracis strains and the differential diagnosis of vaccine strains

目的建立炭疽芽孢杆菌临床分离株和疫苗株的鉴别诊断方法,为炭疽疫情判断和疾病控制提供依据。方法以炭疽芽孢外壁胶原样蛋白的编码基因bclA和pXO1质粒上的aat序列为靶点,设计引物,通过PCR扩增片段多态性和测序结果分析可变串联重复序列(VNTR)。在生物安全3级实验室分离炭疽菌体蛋白,使用MALDL-TOF/TOF质谱仪进行检测,BioTyper软件分析图谱。结果炭疽分离株与Ⅱ号、无荚膜疫苗株,VNTR分析结果均有不同。2个分离株测定bclA基因序列全长均为1 113bp,编码的蛋白包含66个GXX重复和5个BclA重复。质谱分析发现,2个分离株菌体蛋白表达峰图基本一致,而2个疫苗株与之相比有很大的差异,许多蛋白的表达明显下调。结论两种方法都能够有效鉴别炭疽临床分离株和疫苗株。

The differential diagnosis methods were developed to identify the Bacillus anthracis clinical strains and vaccine strains.Firstly,the variable-number tandem repeat（VNTR）analysis method was created basing on the bclAgene of exosporium collagen-like protein.It was founded that the PCR amplification fragment length of the two isolated strains was different from the II vaccine strain and non-capsulated vaccine strain.The BclA protein of isolated strain contain 66 copies of GXX repeat and five copies of（GPT）5GDTGTT（BclA repeat）.Secondly,the bacterial protein expression level of isolated strain was also different from the vaccine strains by MALDI-TOF/TOF mass spectrometry analysis.Both of the two methods can effectively identify the Bacillus anthracis clinical isolated strains and vaccine strains.