STR图谱法从疫苗成品中鉴别生产用人源细胞株的研究

目的: 探讨采用短串联重复序列(STR)图谱分析法检测人二倍体细胞生产的疫苗成品,判定生产所用细胞基质种类的可能性,为疫苗的质量控制及监测等环节提供参考。方法: 研究用疫苗均以人二倍体细胞为生产基质,品种包括水痘减毒活疫苗、甲肝减毒活疫苗、麻疹风疹联合减毒活疫苗、甲肝灭活疫苗、狂犬灭活疫苗,分别来自11个厂家共22批。其中甲肝灭活疫苗均经过纯化,且均以甲醛为灭活剂。狂犬灭活疫苗来自2个厂家,1家疫苗采用了柱层析纯化工艺,2家疫苗均以β-丙内酯为病毒灭活剂。首先提取疫苗成品中的DNA,然后采用STR图谱分析方法对DNA进行检测,并对所获得的指纹图谱与标准图谱数据进行比对,判定生产所用细胞的正确性。结果: 对减毒活疫苗进行检测可准确地判定生产用细胞的种类,结果显示8家疫苗生产企业中1家使用了错误的细胞。在对灭活疫苗的检测中,无柱层析纯化工艺的狂犬灭活疫苗可得到良好的STR数据,且比对结果正确。经柱层析的狂犬灭活疫苗经酚抽提法提取的DNA检测可获得STR信号,尽管Penta E位点信号缺失也可判定出生产所用的细胞基质种类及正确性。含有铝佐剂的甲肝灭活疫苗检测均未获得STR图谱数据。进一步对2种病毒灭活剂进行初步分析,结果显示:利用β-丙内酯进行病毒灭活,对疫苗中STR检测存在干扰,甲醛灭活对检测无明显影响。结论: 利用STR图谱分析法能够直接从人二倍体细胞生产的减毒活疫苗和不含铝佐剂的灭活疫苗中鉴别生产所用的细胞基质种类,可为疫苗生产的质量控制及监测等环节提供参考。

Objective: In order to provide a reference for quality control and test of vaccine produced by human diploid cell, whether cell substrate can be authenticated from viral vaccine production by short tandem repeat (STR) was investigated. Methods: Human diploid cells were the substrates used for the production of the tested vaccines, including varicella live attenuated vaccine, HAV live attenuated vaccine, measles, rubella combined live attenuated, HAV inactivated vaccine and rabies inactivated vaccine. 22 batches of vaccines were produced by 11 manufacturers.There were purification process and formaldehyde during the production of HAV inactivated vaccines so as to inactivate HAV. In addition, all of rabies vaccines were inactivated by β-propiolactone and one of the two manufactures adopted column chromatography purification process. DNA was extracted from vaccine production and tested by STR analytic method. The correctness of cell used for production was judged by comparing the profile with standard data. Results: The kind of human diploid cell used for production of live attenuated vaccine can be identified exactly by STR, and our study found 1/8 manufacturer had used the incorrect cell. Good STR data can be acquired for rabies inactivated vaccine without column chromatography purification process, and cell was correct. Signal of DNA extracted from rabies inactivated vaccine with purification process by phenol extraction can be detected. Although signal of Penta E loci was deficient, the kind of cell used for production can be identified. No signal can be detected from hepatitis A inactivated vaccine with aluminum adjuvant. Results of two virus inactivating agents showed that inactivating virus has interference on STR detection with β-propiolactone. No interference was detected by formaldehyde inactivation. Conclusion: STR profiling analytic method can be used for identification of cell substrates from the live attenuated vaccine and inactivated vaccine without adjuvant produced by human diploid cells, and may offer a reference for quality control and test of vaccine.