脊髓灰质炎病毒减毒活疫苗中Ⅲ_2株全基因测序及序列分析

Complete genome sequencing of Zhong Ⅲ_2 strain of live attenuated poliovirus vaccine

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 complete genome of Zhong Ⅲ2strain of live atteanuted poliovirus vaccine,and analyze for homologies to those of P3 / Leon / 37 and Sabin Ⅲ strains. Methods The four large and eight small fragments which were overlapping and covered full-length genome of Zhong Ⅲ2strain were amplified by RT-PCR. The PCR products of intermediate fragments were sequenced directly,while the fragments at the end of genome were subjected to TA cloning and sequencing. The c DNA fragments obtained by two methods were spliced respectively,and the obtained fulllength c DNA sequence were analyzed for homologies to those of P3 / Leon / 37 and Sabin Ⅲ strains. Results The fulllength c DNAs of Zhong Ⅲ2strain,at a length of 7 432 bp,were obtained by both the methods,of which the base sequences were completely in agreement. Both the homologies of complete genome sequence of Zhong Ⅲ2strain to those of P3 / Leon / 37 and Sabin Ⅲ strains were 99%. There were 19 variations of base sequences in Zhong Ⅲ2and P3 / Leon /37 strains,3 in non-encoding region and 16 in encoding region. The six gene mutations in encoding region resulted in the variation of amino acid sequence. However,there were 28 variations of base sequences in Zhong Ⅲ2and Sabin Ⅲstrains,13 in non-encoding region and 15 in encoding region,and the three gene mutations in encoding region resulted in the variation of amino acid sequence. Conclusion The full-length of Zhong Ⅲ2strain of live attenuated poliovirus vaccine was 7 432 bp,while the homology to SabinⅢ strain was 99%.