新一代高通量RNA测序在黄热减毒活疫苗(鸡胚细胞)毒种库质控中的应用  

Application of new generation high-throughput RNA sequencing in quality control of live attenuated yellow fever vaccine(chicken embryo cell)virus seed bank

作　　者：徐宏山[1] 黄艳秋 刘欣玉[1] 贾丽丽[1] 李玉华[1] XU Hongshan;HUANG Yanqiu;LIU Xinyu;JIA Lili;LI Yuhua(National Institutes for Food and Drug Control,Bejing 102629,China)

机构地区：[1]中国食品药品检定研究院,北京102629

出　　处：《中国生物制品学杂志》2023年第11期1335-1340,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09738003)。

摘　　要：目的采用新一代Illumina/Solexa测序平台对黄热减毒活疫苗(鸡胚细胞)毒种在基因组水平进行质量控制。方法将黄热减毒活疫苗YF17D-204毒株接种原代鸡胚细胞,筛选鸡胚细胞黄热减毒活疫苗适应株,建立YFV17D-CEC三级毒种库;提取各级毒种病毒基因组RNA,制备RNA文库,采用新一代Illumina/Solexa测序平台进行高通量RNA测序,采用FastQC、Trimmomatic、SPAdes、GapFiller、PrInSeS-G、Prokka、RepeatMasker、CRT、NCBI Blast^(+)、KAAS、HMMER3、TMHMM,SignalP,LipoP、ProtCamp、MegAlign等生物学软件,对黄热病毒全基因组核酸序列进行基因组系统化分析。结果YFV17D-CEC三级毒种库全基因组全长为10862个核苷酸,其中119~10354位(10236 bp)为1个开放阅读框,编码3412个氨基酸。序列比对分析显示,YFV17D-CEC三级毒种库序列与YFV17D RKI(JN628-279.1)、YF/Vaccine/USA/Sanofi-Pasteur-17D-204/UF795AA/YFVax(JX503529.1)YFV17D-204(KF769015.1)同源性均为100%,三级毒种库全基因组未发生突变。比较黄热减毒活疫苗不同疫苗株序列发现,黄热病毒具有多个多态性位点。结论YFV17D-CEC在原代鸡胚细胞上具有良好的遗传稳定性。利用高通量RNA测序技术可快速检测YFV17D-CEC三级毒种库全基因组信息,序列分析数据可对黄热疫苗毒种库进行基因水平的质量控制。Objective To perform quality control in live attenuated yellow fever vaccine(chicken embryo cell)virus seed bank at the genomic level using the new generation Illumina/Solexa sequencing platform.Methods The live attenuated yellow fever vaccine strain YF17D-204 was inoculated into primary chicken embryo cells,and the chicken embryo cell adapted strains of live attenuated yellow fever vaccine were screened to establish YFV17D-CEC tertiary virus seed bank.The genome RNA of virus seeds was extracted,and the RNA library was prepared.The new generation Illumina/Solexa sequencing platform was used for high-throughput RNA sequencing.The whole genome nucleic acid sequence of yellow fever virus was systematically analyzed by using biological softwares such as FastQC,Trimmomatic,SPAdes,GapFiller,PrInSeS-G,Prokka,RepeatMasker,CRT,NCBI Blast^(+),KAAS,HMMER3,TMHMM,SignalP,LipoP,ProtCamp and MegAlign.Results The whole genome of YFV17D-CEC tertiary virus seed bank contained 10862 nucleotides,including an open reading frame(ORF)from 119 to 10354(10236 bp),encoding 3412 amino acids.Sequence alignment analysis showed that the sequence of YF17D-CEC tertiary virus seed bank was 100%identical with YFV17D RKI(JN628279.1),YF/Vaccine/USA/Sanofi-Pasteur-17D-204/UF795AA/YFVax(JX503529.1)and YFV17D-204(KF769015.1),and no mutation occurred in the whole genome of the tertiary virus seed bank.Comparison of the sequences of different live attenuated yellow fever vaccine strains showed that yellow fever virus had multiple polymorphic sites.Conclusion YFV17DCEC has good genetic stability in primary chicken embryo cells.High-throughput RNA sequencing technology can quickly detect the whole genome information of YF17D-CEC virus seed bank,and the sequence analysis data can be used in the gene level quality control of yellow fever vaccine virus seed banks.

关 键 词：高通量RNA测序 黄热减毒活疫苗 基因表达 毒种库 质量控制 

分 类 号：R373.3[医药卫生—病原生物学]