重组人肠道病毒71型病毒样颗粒疫苗外源DNA残留量检测方法的建立

目的建立重组人肠道病毒71型(enterovirus 71,EV71)病毒样颗粒(virus-like particles,VLPs)疫苗原液中昆虫细胞宿主DNA残留的地高辛探针杂交检测方法。方法抽提昆虫细胞sf9基因组DNA,经分子筛纯化后作为阳性对照品;分别以1 000~5 000 bp和100~1 000 bp的基因组DNA超声随机小片段为模板,制备地高辛探针,与阳性对照进行杂交试验。同时验证方法的检测范围、灵敏度和特异性,并采用该方法对3批重组EV71 VLPs疫苗原液中宿主DNA残留量进行测定。结果纯化后DNA样品浓度为47. 5 ng/μL,A260/A280值为1. 86,可作为阳性对照。以100~1 000 bp DNA超声随机小片段作为模板,探针标记效率更高,杂交显色更清晰。该法检测范围为10 pg^10 ng,灵敏度达1 pg,特异性强。3批重组EV71 VLPs疫苗原液中每人份剂量的宿主细胞DNA残留均<50 pg。结论本实验建立的方法特异性强,灵敏度高,可用于重组EV71 VLPs疫苗制备过程中的原液检定及质量控制。

Development of a method for determination of residual exogenous DNA in recombinant human enterovirus 71 virus-like particle vaccine

Objective To develop a Digoxin-probe hybridization method for determination of residual host cell DNA in the bulk of recombinant human enterovirus 71(EV71)virus-like particle(VLP)vaccine. Methods The genomic DNA of insect sf9 cells was extracted,purified by size-exclusion chromatography and used as the positive control. Digoxin probe was prepared using the random DNA fragments at lengths of 1 000 ~ 5 000 and 100 ~ 1 000 bp respectively, obtained by ultrasonication,as templates and hybridized with the positive control. The developed method was verified for detection range,sensitivity and specificity,by which the residual host DNA contents in three batches of bulk of recombinant EV71 VLPs were determined. Results The purified DNA sample,at a concentration of 47. 5 ng/μL and an A260/A280 value of1. 86,was used as the positive control. The labeling efficiency of Digoxin-probe using random DNA fragments at lengths of of 100 ~ 1 000 bp as templates was higher,while the color of hybridization bands was clearer,than those using 1 000 ~5 000 bp fragments. The method showed high specificity,of which the detection range was 10 pg ~ 10 ng,and the sensitivity was 1 pg. The residual DNA contents in three batches of bulk of recombinant EV71 VLP vaccine were less than 50 pg/dose. Conclusion The developed Digoxin-probe hybridization method is specific and sensitive,which may be applied to the quality control of bulk during preparation of recombinant EV71 VLP vaccine.