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重组肠道病毒71型反向遗传系统构建 被引量:2

Construction of reverse genetics system for recombinant entervirus 71
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摘要 目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3'UTR(107)和3CD(1)-3'UTR(107),并克隆入p M D19-T。利用双酶切、T4连接酶将两种重组3CD-3'UTR分别置换入本室构建并保存的EV71的SDLY107株的感染性c DNA克隆p M D19-T-107,得到重组p M D19-T-107后,将其线性化并转染入横纹肌肉瘤(RD)细胞,盲传得到重组病毒107(1-3C)和107(1-3CD)。对收获病毒进行鉴定。结果构建了重组EV71全长c DNA克隆;RNA转染并盲传至第3代,36 h后RD细胞出现细胞病变(CPE),48 h出现明显的CPE,成功拯救重组病毒SDLY107(1-3C)和SDLY107(1-3CD);重组病毒产生的CPE更接近SDLY107。结论成功构建了重组EV71反向遗传系统,拯救了重组病毒SDLY107(1-3C)和SDLY107(1-3CD),为进一步研究3C与3D蛋白在EV71致病机制中的作用提供基础。 Objective To recombine 3C and 3CD region of virulent strain SDLY107 and low virulent strain SDLY1 of entervirus 71 (EV71)with reverse genetics, and to rescue recombinant viruses. Methods Recombinant fragments,3C ( 1 ) -3 D -3' UTR ( 107 ) and 3 CD ( 1 ) -3' UTR ( 107 ), were obtained with PCR, and then cloned to pMD 19 -T. 3 CD -3' UTR of pMD19-T-107 constructed previously was replaced by recombinant 3CD-3' UTR through double digestion and ligase T4 to construct recombinant pMD19-T-107. Then the in vitro synthesized RNA transcripts were transfected into rhabdomyosarcoma (RD) cells to produce the rescued recombinant virus, SDLY 107 (1-3C) and SDLY 107 (1-3CD). DNA sequences of the recombinant viruses were analyzed. Results The full length cDNA clone was constructed successfully. After 36 hours of third passages, cytopathic effect(CPE) was observed, and the CPE was observed obviously after 48 hours, suggesting the recombinant viruses were rescued successfully. There were a few differences between the CPE induced by recombinant viruses and SDLY107. Conclusion Reverse genetics system for recombinant EV71 was constructed successfully,and recombinant viruses were rescued successfully. The study lays a foundation for further research on pathogenesis of EV71.
出处 《中国公共卫生》 CAS CSCD 北大核心 2016年第10期1437-1440,共4页 Chinese Journal of Public Health
基金 国家自然科学基金(81371833) 山东省医药卫生科技发展计划(2013WS0211)
关键词 肠道病毒71型 反向遗传技术 3CD蛋白 entervirus 71 reverse genetics 3 CD protein
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  • 1Sun LL,Wang JK,Cui XQ, et al. Association of vital replication capacity with the pathogenicity of enterovirus 71 [ J]. Virus Res, 2014,189:1 -7.
  • 2McMinn PC. An overview of the evolution of euterovirus 71 and its clinical and public health significance [ J ]. FEMS Microbiol Rev ,2002,26( 1 ) :91 - 107.
  • 3温红玲,郝树彬,高峰,赵丽,司鲁莹,袁晓晶,王东旭,王志玉.肠道病毒71型山东临沂分离株全基因组序列分析[J].中华微生物学和免疫学杂志,2011,31(7):603-608. 被引量:11
  • 4Ho M,Chen ER,Hsu KH,et al. An epidemic of enterovirus 71 in- fection in Taiwan[ J ]. N Engl J Med, 1999,341 (13) :929 - 935.
  • 5Mao LX, Wu B, Bao WX, et al. Epidemiology of hand, foot, and mouth disease and genotype characterization of enterovirus 71 in Jiangsu, China[ J ]. J Clin Viro1,2010,49 (2) : 100 - 104.
  • 6AbuBakar S, Chee HY, A1-Kobaisi MF, et al. Identification of enterovirus 71 isolates from an outbreak of hand,foot and mouth disease(HFMD) with fatal cases of encephalomyelitis in Malay- sia[ J]. Virus Res, 1999,61 ( 1 ) : 1 - 9.
  • 7McMinn P, Lindsay K, Perera D, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malay- sia, Singapore, and Western Australia [ J ]. J Virol, 2001,75 (16) :7732 -7738.
  • 8Kehle J, Roth B, Metzger C, et al. Molecular characterization of an enterovirus 71 causing neurological disease in Gennany[ J]. J Neuroviro1,2003,9 ( 1 ) : 126 - 128.
  • 9吴家兵,方益荣,王建军,张进,胡岱霖,曹明华.安徽省2008年手足口病流行病学分析[J].安徽预防医学杂志,2010,16(2):96-98. 被引量:45
  • 10刘威龙,韦清,杨桂林,喻爽,张明霞,陈心春,刘映霞,李晶晶,王威,高杨,周伯平.深圳5例肠道病毒71型手足口病病毒株全基因组测序分析[J].中华微生物学和免疫学杂志,2010,30(5):409-409. 被引量:4

二级参考文献31

  • 1田波,段海生,荣一兵,周世力.肠道病毒71型分子流行病学研究进展[J].中国病毒学,2004,19(4):426-429. 被引量:90
  • 2周世力,李琳琳,何雅青.深圳市肠道病毒71型血清流行病学初步调查[J].热带医学杂志,2007,7(1):66-67. 被引量:185
  • 3中华人民共和国卫生部.肠道病毒(EV71)感染诊疗指南[s].2008.
  • 4Yoke-Fun C, AbuBakar S. Phylogenetic evidence for inter-typicrecombination in the emergence of human enterovirus 71 subgenotypes. BMC Microbiol, 2006, 6: 74.
  • 5Shih SR, Ho MS, Lin KH, et al. Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res, 2000, 68(2) : 127-136.
  • 6McMinn P, Stratov I, Nagarajan L, et al. Neurological manifesta- tions of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis, 2001, 32(2): 236-242.
  • 7Singh S, Poh CL, Chow VT. Complete sequence analyses of en- terovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000). Microbiol Im- munol, 2002, 46( 11 ) : 801-808.
  • 8Chang GH, Lin L, Luo YJ, et al. Sequence analysis of six entero- virus 71 strains with different virulences in humans. Virus Res, 2010, 151(1) : 66-73.
  • 9Kung YH, Huang SW, Kuo PH, et al. Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase. Virology, 2010, 396( 1 ) : 1- 9.
  • 10Arita M, Ami Y, Wakita T, et al. Cooperative effect of the atten- uation determinants derived from poliovirus sabin 1 strain is essen- tial for attenuation of enterovirus 71 in the NOD/SCID mouse in- feetion mlxtel. J Virol, 2008, 82(4) : 1787-1797.

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