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目的研究EV71VP1上的变异位点(P639S/G),构建突变体拯救病毒,分析病毒突变前后在细胞上的复制差异性。方法采用反向遗传学方法,在EV71感染性全长cDNA的基础上构建突变体病毒的全长感染性cDNA,转染获得拯救病毒并传代验证;通过全基因测序,RT-PCR、Western blot、免疫荧光法检测拯救病毒,并绘制拯救病毒的生长曲线。结果 RT-PCR检测到EV71VP1特异性核酸片段,大小为227bp,与理论值相符;Western blot、免疫荧光检测到EV71特异蛋白;突变前后拯救病毒的生长曲线无明显改变。结论突变体病毒拯救成功且能稳定传代;EV71-P639变异不影响病毒的复制。
Objective To study the mutation site of EV71VP1 (P639S / G) and construct a mutant-rescue virus to analyze the replication differences on the cells before and after the mutation. Methods The reverse transcriptase-PCR method was used to construct the full-length infectious cDNA of the mutant virus based on the full-length EV71 infectious cDNA. The full-length infectious cDNA of the mutant virus was transfected and verified by passage. Immunofluorescence assay to rescue the virus, and draw the growth curve to save the virus. Results The specific fragment of EV71VP1 was detected by RT-PCR and its size was 227bp, which was in good agreement with the theoretical value. The specific protein of EV71 was detected by Western blot and the virus-free growth curve did not change significantly. Conclusion The mutant virus was successfully rescued and passaged steadily. The mutation of EV71-P639 did not affect the replication of virus.