JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES) ›› 2017, Vol. 55 ›› Issue (8): 7-12.doi: 10.6040/j.issn.1671-7554.0.2016.1383

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Rescue of 3D recombinant enterovirus 71

BAI Yongjuan1, ZHUANG Zhichao1, HAO Shubin2, WANG Lihong1, LI Chun1, LU Yan1, ZHAO Li1, WANG Zhiyu1, WEN Hongling1   

  1. 1. Department of Virology, School of Public Health, Shandong University, Jinan 250012, Shandong, China;
    2. Department of Microbiology, Shandong Medical Equipment Quality Supervision and Inspection Center, Jinan 250101, Shandong, China
  • Received:2016-10-26 Online:2017-08-10 Published:2017-08-10

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Abstract: Objective To replace 3D region of enterovirus 71(EV71)high virulent strain SDLY107 with low virulent strain SDLY1 by reverse genetics technology, and to rescue recombinant virus, providing a platform for investigation on EV71 3D protein. Methods The recombine fragment of 3D protein was obtained by overlap PCR, and then inserted into pMD19-T vector. 3D region of pMD19-T-107 containing the full-length gene of SDLY107 was replaced with recombine fragment through double digestion and ligase T4 to construct pMD19-T-107(1-3D). The infectious RNA was obtained by in vitro transcription, then transfected into Vero cells and gained recombinant virus particles after three passages. PCR and qRT-PCR were performed for recombinant virus identification. Results ApaI-3D anterior region(600 bp), 3D region(1 400 bp)and 3'UTR-XbaI region(200 bp)were obtained by PCR, and 1-3D(2 100 bp)was obtained by overlap PCR. The recombinant plasmid was identified by double enzyme digestion(7 400 and 2 700 bp), and sequencing results further confirmed the successful replacement of 3D region. Cell shrinkage, roundness and refractive index enhancement were observed after transfecting the infectious clone into Vero cells and cultivating to the third generation. Identification of recombinant virus by PCR gained fragment of 226 bp; qRT-PCR detected the amount of 山 东 大 学 学 报 (医 学 版)55卷8期 -白永娟,等.肠道病毒71型3D重组病毒的拯救 \=-virus increased slowly from 24 h to 48 h and increased rapidly from 48 h to 72 h, and the amount of virus no longer growed after 72 h. Conclusion The recombinant virus SDLY107(1-3D)is rescued successfully. The study provids the basis for further research on the virulence of 3D protein and its role in the pathogenesis of EV71.

Key words: Reverse genetics, Recombinant virus, 3D protein, Enterovirus 71

CLC Number: 

  • R373.2
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