卡介苗生产用菌株的遗传稳定性研究  

Genetic stability of BCG strain for vaccine production in China

作　　者：程鹏飞[1] 刘朝阳[1] 陈艳红[1] 徐帆洪[1] 马相虎[1] 

机构地区：[1]上海生物制品研究所有限责任公司质量检定室,上海200052

出　　处：《中国生物制品学杂志》2013年第10期1361-1365,共5页Chinese Journal of Biologicals

摘　　要：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 BCG China strain D2PB302 （referred to as BCG Chinese strain） for vaccine production in China. Methods Genomic DNAs of passages 4, 7, 10, 13 and 15 of working seeds of BCG Chinese strain were extracted and used as a template, with which 16S ribosomal RNA（referred to as 16S rRNA） was am- plified by PCR and sequenced. The regions of deletion （RD） of BCG Chinese strain, i. e. RD1, RD2, RD8, RD14 and RD16, as well as SenX3-RegX3 repeat tandem were tested by multiplex PCR, based on which the RD1 and Esat6 gene were further tested by multiplex PCR, and the gene locus SenX3-RegX3 was sequenced. Results The 16S rRNA se- quences of various passages of BCG Chinese strain showed no variation, of which the homology was 100% to that of Pas- teur 1173P2 strain （AM408590. 1 ） reported in GenBank. ＂Hie locations of bands on characteristic gel electrophoretic pro- file of BCG Chinese strain of various passages were in agreement, while three repeats were observed at SenX3-Regx3 lo- cus. The repeats, each started with ATG and ended with TG, were linked together. No Esat6 gene encoding virulence-as- sociated protein was detected in working seeds of various passages of BCG Chinese strain. Conclusion BCG Chinese strain showed characteristic genetics as compared with routine international BCG substrain, of which the genetic stability was high within 15 passages. The genetic characters of vaccine prepared with BCG Chinese strain within 12 passages as specified in Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition） was stable.

关 键 词：卡介苗 遗传学 质量控制 传代 稳定性 

分 类 号：R392.2[医药卫生—免疫学] R392-33[医药卫生—基础医学]