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目的测定狂犬病病毒(rabies virus,RV)PV-2061株全基因组序列,并分析其抗原性及免疫原性。方法 RTPCR法对RV PV-2061株基因组进行分段扩增,分别克隆至p MD18-T Simple Vector,转化感受态E.coli TOP10,挑取阳性克隆,进行测序。应用DNAstar Meg Align软件对测序结果进行拼接和分析;ELISA法检测PV-2061株的抗原性;用主种子批毒种制备的原疫苗免疫小属,检测PV-2061株的免疫原性。结果 RV PV-2061株的基因组全长11 932 bp,由3′端至5′端依次排列着N、P、M、G、L 5个结构基因,每个基因由3′端到5′端的非编码区及中间的编码区构成。与Gen Bank中已知全基因组序列的毒株比较,基因组前端3′先导序列和N基因的非编码区均无长度变化,从P基因开始,基因非编码区的长度开始出现差异,导致各毒株基因组长度不同。RV PV-2061株的抗原值为7.46 IU/ml;主种子批毒种制备原疫苗保护指数为1 585。结论确定RV PV-2061株全基因组序列为基因1型,且具有RV特有的抗原性和良好的免疫原性,为狂犬病疫苗的设计和毒株的筛选提供了实验依据。
Objective To determine the genome sequence of rabies virus (RV) PV-2061 strain and analyze its antigenicity and immunogenicity. Methods The genome of RV PV-2061 strain was amplified by RTPCR and cloned into pMD18-T Simple Vector. The recombinant plasmid was transformed into E.coli TOP10. The positive clones were selected and sequenced. The sequencing results were stitched and analyzed by using DNAstar Meg Align software. The antigenicity of PV-2061 strain was detected by ELISA. The immunogenicity of PV-2061 strain was detected by immunoprecipitation with the original vaccine prepared from the seed of the main seed. Results The genome of RV PV-2061 strain was 11 932 bp in length with 5 structural genes of N, P, M, G and L arranged in turn from the 3 ’end to the 5’ end. Each gene consisted of 3 ’end to 5’ end Non-coding area and the middle of the coding area. Compared with the genomic sequences known in GenBank, there was no length change in the non-coding region of the 3 ’leader and N genes in the genome. The length of the gene non-coding region began to vary from the P gene, Strain length varies. The antigen value of RV PV-2061 strain was 7.46 IU / ml; the protection index of the original vaccine prepared by the main seed batch virus was 1 585. Conclusion The complete genome sequence of RV PV-2061 strain was identified as genotype 1 with RV-specific antigenicity and good immunogenicity, which provided an experimental basis for the design of rabies vaccine and screening of strains.