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通过构建乙型脑炎病毒减毒株SA14-14-2的全长cDNA克隆,来初步探讨将其作为表达载体的可行性,为下一步利用乙型脑炎病毒作为载体构建嵌合病毒打下基础。利用长片段RT-PCR的方法分两段扩增出乙型脑炎病毒cDNA,通过片段两端的酶切位点,将cDNA依次连接到pACYC184载体。进一步利用分子克隆的手段,在乙型脑炎病毒基因组cDNA的3′非编码区插入增强型绿色荧光蛋白(Ehanced green fluorescent portein,EGFP)基因作为报告基因,通过体外转录和转染,拯救乙型脑炎病毒和表达绿色荧光蛋白的乙型脑炎嵌合病毒。采用RT-PCR、蚀斑实验和荧光显微镜观察等方法对恢复病毒进行鉴定。对恢复病毒进行连续6次细胞传代,从病毒生长特性和结构基因稳定性的角度对恢复病毒传代稳定性进行研究。结果表明成功地扩增并构建得到乙型脑炎病毒的全长cDNA,在此基础上进一步构建得到了乙型脑炎嵌合病毒rJEV-EGFP全长cDNA,经过体外转录和转染获得了活的rJEV病毒及嵌合病毒rJEV-EGFP,并采用了多种方法对其进行了鉴定,拯救出的恢复病毒在已传代的代次内稳定性良好,嵌合病毒rJEV-EGFP可稳定地表达绿色荧光蛋白。本研究应用反向遗传学技术构建并在BHK-21细胞中成功地拯救出了rJEV和rJEV-EGFP活病毒,同时也表明乙型脑炎病毒SA14-14-2株可以作为载体来表达外源基因。
Abstract：This study aimed to construct full-length cDNA clones of the Japanese encephalitis virus(JEV)SA14-14-2strain and discuss the feasibility of constructing chimeric viruses for exogenous gene expression based on the JEV genetic skeleton.Long-fragment RT-PCR techniques were applied to amplify JEV cDNAs,and two amplified fragments with corresponding restriction endonuclease sites at both ends were cloned into the pACYC184 vector sequentially.Using standard molecular techniques,the enhanced green fluorescent protein(EGFP)gene was inserted into the 3′non-coding region of JEV as a reporter gene.After in vitro transcription and transfection procedures,wild-type JEV and chimeric JEV that expressed the EGFP as the reporter gene were successfully rescued.The recovered viruses were characterized by RTPCR,plaque assays,and direct fluorescence microscopy.After six serial passage generations,the stability of the recovered viruses were studied in terms of virus growth characteristics and structural gene expression.The results showed that cDNA clones of rJEV and rJEV-EGFP were successfully constructed and rescued in BHK-21 cells after in vitro transcription and transfection.Each generation of the recovered viruses was stable and the chimeric virus rJEV-EGFP could stably express EGFP.The findings of this study indicate that both rJEV and rJEV-EGFP could be constructed and rescued in BHK-21 cells,and the JEV SA14-14-2strain could be obtained as a viral vector to express foreign genes.
[1]赵荣乐,郑光宇.乙型脑炎病毒研究进展[J].生物学通报,2007,42(7):6-8.
[2]Yun S I,Lee Y M.Japanese Encephalitis:The virus and vaccines[J].Hum Vaccine Immunother,2013,10(2):263-279.
[3]Boyer J C,Haenni A L.Infectious transcripts and cDNA clones of RNA viruses[J].Virology,1994,198(2):415-426.
[4]黄莺,贾丽丽,孙志伟,等.乙型脑炎病毒(JEV)全长感染性克隆的制备及恢复病毒的获得[J].病毒学报,2003,19(4):313-319.
[5]李静,俞永新,董关木,等.乙型脑炎减毒株SA14-14-2全长cDNA克隆感染性的研究[J].中国生物制品学杂志,2010,23(1):13-16.
[6]Zhao Z J,Date T,Li Y H,et al.Characterization of the E-138(Glu/Lys)mutation in Japanese encephalitis virus by using a stable,full-length,infectious cDNA clone[J].J Gen Virol,2005,86(8):2209-2220.
[7]Monath T P,Arroyo J,Levenbook I,et al.Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys:relevance to development and safety testing of live,attenuated vaccines[J].J Virol,2002,76(4):1932-1943.
[8]Wei Y,Li C,Lu P,et al.Identification and construction of replicon vectors of Japanese encephalitis virus?(strain SA14-14-2)[J].Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi,2009,23(6):418-420.
[9]邓淑珍,张海林,李金梅.流行性乙型脑炎病毒研究进展[J].中国热带医学,2008,8(2):306-310.
[10]Ni H,Chang G J,Xie H,et al.Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14[J].J Gen Virol,1995,76(pt2):409-413.
[11]郭雄明,吴东.黄病毒属病毒感染性克隆的构建及应用[J].军医进修学院学报,2007,228(1):75-77.
[12]Yun S I,Kim S Y,Rice C M,et al.Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus[J].J Virol,2003,77(11):6450-6465.
[13]Rasmussen T B,Risager P C,Fahnoe U,et al.Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA[J].BMC Genomics,2013,14(1):819.
[14]Shi P Y,Tilgner M,Lo M K,et al.Infectious cDNA clone of the epidemic West Nile virus from New York City[J].J Virol,2002,76(12):5847-5856.
[15]Yamshehikov V,Mishin V,Comineni F.A new strategy in design of RNA virus infectious clones enabling their stable propagation in E.coli[J].Virology,2001,281(2):272-280.
[16]Pu S Y,Wu R H,Yang C C,et al.Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes[J].J Virol,2011,85(6):2927-2941.
[17]Fan Y H,Nadar M,Chen C C,et al.Small noncoding RNA modulates Japanese encephalitis virus replication and translation in trans[J].Virol J,2011,8:492.
[18]Chien H L,Liao C L,Lin Y L.FUSE binding protein1interacts with untranslated regions of Japanese encephalitis virus RNA and negatively regulates viral replication[J].J Virol,2011,85(10):4698-4706.
[19]Bonaldo M C,Garratt R C,Caufour P S,et al.Surface Expression of an Immunodominant Malaria Protein B Cell Epitope by Yellow Fever Virus[J].J Mol Biol,2002,315(4):873-885.
[20]Li X F,Deng Y Q,Yang H Q,et al,A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates[J].J Virol,2013,87(24):13694-13705.
[21]Yun S I,Choi Y J,Yu X F,et al.Engineering the Japanese encephalitis virus RNA genome for the expression of foreign genes of various size:Implications for packaging capacity and RNA replication efficiency[J].J Neuro Virol,2007,13(6):522-535.
[22]Bonaldo M C,Mello S M,Trindade G F,et al.Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1genes[J].Virol J,2007,4:115.
[23]Irwin C R,Farmer A,Willer D O,et al.In-fusionR○cloning with vaccinia virus DNA polymerase[J].Methods Mol Biol,2012,890:23-35.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002579
中图分类号:R392
引用信息:
[1]胡兵,杨爽,方志正.乙型脑炎减毒活疫苗株全长感染性克隆的构建及表达外源基因的初步研究[J].病毒学报,2014,30(06):652-660.DOI:10.13242/j.cnki.bingduxuebao.002579.
2014-12-02
2014-12-02
2014-12-02