【摘要】 利用RT-PCR方法扩增获得了乙型脑炎病毒PrM-E基因,并将之克隆至慢病毒载体系统中的转移质粒pHR-IRES-EGFP中,得到重组质粒pHR-PrM-E。将该质粒与包装质粒pCMV△8.2、囊膜质粒pVSV/G共转染293FT细胞,培养48h后收集细胞上清,并将之转导293FT细胞。通过报告基因EGFP表达、间接免疫荧光和Western-blot检测,结果表明,上述三质粒共转染293FT细胞后,获得了假病毒,并且该假病毒可以高效转导正常293FT细胞。同时,PrM-E基因可以在被转导细胞中正确表达,从而为研制基于慢病毒载体的乙型脑炎病毒新型疫苗奠定了基础。更多还原

【Abstract】 PrM-E gene was amplified from Japanese encephalitis virus(JEV) by RT-PCR and cloned into the transfer plasmid pHR-IRES-EGFP.The recombinant plasmid pHR-PrM-E was then co-transfected into 293FT cells with packaging vector pCMV△8.2 and envelope vector pVSV/G.At 48 hour post-transfection,the supernatant was harvested and transduced 293FT cells.Subsequently,the expression of target protein was examined by indirect immunofluorescence assay and Western-blot.The results demonstrated that the lentiviral particles were successfully packaged by co-transfection of these three plasmids and could transduce 293FT cells efficiently.Also PrM-E gene was expressed correctly in the transduced cells,which provided the experimental basis for the development of novel JEV vaccines in future.更多还原