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目的观察我国生产用卡介苗上海D2PB302菌株(以下简称卡介苗上海D2株)的遗传稳定性。方法提取卡介苗上海D2株工作种子批4、7、10、13、15代单批培养物基因组DNA,以其为模板,PCR扩增16S核糖体RNA(16S ribosomal RNA,16S rRNA)基因,并进行测序鉴定;对卡介苗上海D2株缺失区RD1、RD2、RD8、RD14、RD16及双组分系统操纵子SenX3-RegX3串联重复序列进行多重PCR检测;对卡介苗上海D2株缺失区RD1及Esat6基因进行多重PCR检测;并对基因座SenX3-RegX3进行序列分析。结果卡介苗上海D2株工作种子批4、7、10、13、15代单批培养物的16S rRNA序列均未发生变异,与GenBank中登录的Pasteur 1173P2株序列(AM408590.1)相似性为100%;卡介苗上海D2株工作种子批各代次特征性凝胶电泳图谱显示条带位置均相同,其基因座SenX3-RegX3有3个串联重复序列,每个串联重复序列均以ATG起始,TG结尾,且前后重叠连接在一起;卡介苗上海D2株工作种子批各代次中均缺少编码毒力的Esat6基因。结论卡介苗上海D2株与国际常用卡介菌亚株相比具有其特征性的遗传学特性,在传代15代次以内,分子遗传学特性稳定。在《中国药典》三部(2010版)规定的12代次内制备疫苗,卡介苗遗传特性一致。
Objective To observe the genetic stability of China producing Bacillus Calmette-Guerin D2PB302 strain (hereinafter referred to as BCG Shanghai D2 strain). Methods Genomic DNA of single-batch cultures of Bacillus Calmette-Guérin (D2) seedlings from batch 4, 7, 10, 13, and 15 were extracted and used as template to amplify 16S ribosomal RNA (16S rRNA) Sequencing and identification were carried out. Multiplex PCR was carried out on RD1, RD2, RD8, RD14, RD16 and two-component system operator SenX3-RegX3 tandem repeat of BCG Shanghai D2 deletion region. Multiplex PCR was performed on RD1 and Esat6 genes of BCG- PCR detection; and locus SenX3-RegX3 sequence analysis. Results The 16S rRNA sequences of single-batch cultures of Bacillus Calmette-Guerin-Shanghai D2 strain 4,7,10,13,15 were similar to those of Pasteur 1173P2 strain (AM408590.1) registered in GenBank with 100% ; Bacillus Calmette-Guérin D2 Shanghai work seed batch batch gel electrophoresis showed that the bands were the same, the locus SenX3-RegX3 has three tandem repeats, each tandem repeat sequence ATG start, TG end , And overlapping before and after the connection together; Bacillus Calmette-Guérin D2 Shanghai work seed lots in each generation are lacking coding virulent Esat6 gene. CONCLUSION Bacillus Calmette-Guerin D2 strain has the characteristic genetic characteristics compared with the commonly used BCG strains in the world, and within 15 generations of subculture, the molecular genetic characteristics are stable. In the “Chinese Pharmacopoeia” three (2010 edition) provides 12 generations of vaccines, BCG genetic characteristics consistent.