不同毒力表型EV71 VP1氨基酸变异的研究  

Study of amino acid mutations in the VP1 capsid protein of enterovirus 71 strains with different levels of virulence

作　　者：张亚杰[1,2] 李鹏[1] 岳盈盈[1] 宋楠楠[1] 李冰清[1] 李志会[1] 秦立增[1] 孟红[1] 

机构地区：[1]山东省医学科学院基础医学研究所,山东济南250062 [2]济南大学,山东省医学科学院医学与生命科学学院,山东济南250200

出　　处：《中国病原生物学杂志》2015年第11期989-993,共5页Journal of Pathogen Biology

基　　金：国家科技重大专项(No.2014ZX09509-001001008);山东省高校中医药抗病毒协同创新中心项目(No.XTCX2014);免疫牛奶在控制手足口病疫情中的应用(No.201303041)

摘　　要：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 an amino acid mutation （P639 S/G） in the VP1 capsid protein of enterovirus 71 （EV71） strains with different levels of virulence. A mutant rescue virus was generated to determine if the amino acid mutation af- fects viral production or virulence in cells. Methods A reverse genetics approaeh was used to construct a mutant virus based on the full-length cDNA of infectious EV71. A rescue virus was obtained by transfecting that cDNA into cells. The rescue virus was stably passaged and characterized using RT-PCR, Western blotting, and an indirect fluorescent antibody assay （IFA）, and the resulting rescue virus was compared to the native virus. A growth curve was created for the rescue virus. Results RT-PCR, Western blotting, and an indirect immunofluorescence assay （IFA） revealed an EV71-specific nucleotide （227 bp） and protein from the rescued virus by, but the virus growth curve changed little when the mutation was present. Conclusion A mutant rescue virus （EV71） was successfully constructed and stably passaged. However, an amino acid mutation in the VP1 protein did not affect viral replication.

关 键 词：肠道病毒71型 VP1 变异 

分 类 号：R373.2[医药卫生—病原生物学]