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目的对脊髓灰质炎病毒减毒活疫苗中Ⅲ2株全基因进行测序,并与P3/Leon/37株和SabinⅢ株的全基因序列进行同源性比较。方法通过大片段克隆和小片段克隆两种方法,采用RT-PCR法扩增覆盖中Ⅲ2株基因组全长且互相重叠的4个大片段和8个小片段,基因组中间片段的PCR产物直接测序,基因组末端的片段先进行TA克隆再测序。分别将两种方法得到的c DNA片段进行拼接,并将所得全长c DNA序列与P3/Leon/37株和SabinⅢ株的全基因序列进行同源性比较。结果两种方法均能得到长度为7 432 bp的中Ⅲ2株全长c DNA序列,且碱基序列完全一致;中Ⅲ2株全基因序列与P3/Leon/37株和SabinⅢ株全基因序列的同源性均为99%;中Ⅲ2株与P3/Leon/37株的碱基序列差异共有19处(非编码区3处和编码区16处),编码区有6个基因突变导致氨基酸序列的改变;中Ⅲ2株和SabinⅢ株的碱基序列差异共有28处(非编码区13处和编码区15处),编码区有3个基因突变导致氨基酸序列的改变。结论中Ⅲ2减毒疫苗株全基因组c DNA序列为7 432 bp,与SabinⅢ株全基因序列的同源性为99%。
Objective To sequence the whole genome of Ⅲ2 strains of attenuated live vaccine of poliovirus and compare their homology with the whole gene sequences of P3 / Leon / 37 and Sabin Ⅲ strains. Methods Four large fragments and eight small fragments which overlapped with each other were amplified by RT-PCR using large fragment cloning and small fragment cloning. The PCR products of the middle fragment of the genome were directly sequenced, The fragment at the end of the genome is first TA cloned and then sequenced. The c DNA fragments obtained by the two methods were respectively spliced, and the homology comparison between the obtained full length c DNA sequences and the whole gene sequences of P3 / Leon / 37 and SabinIII strains was carried out. Results The full-length c DNA sequence of middle-length and full-length cDNA of 7 432 bp was obtained by both methods. The full-length cDNA sequence of middle- The results showed that there were 19 nucleotide differences (Ⅲ in non-coding region and 16 in coding region) between Ⅲ 2 strains and P3 / Leon / 37 strains, and 6 mutations in coding region resulted in the change of amino acid sequence There were 28 nucleotide differences (13 in the non-coding region and 15 in the coding region) in the nucleotide sequence of the Ⅲ 2 strain and the Sabin Ⅲ strain. Three gene mutations in the coding region resulted in the change of the amino acid sequence. Conclusion The complete genomic DNA sequence of Ⅲ 2 attenuated vaccine strain was 7 432 bp, which shared 99% homology with the full-length sequence of Sabin Ⅲ strain.