脑心肌炎增强型绿色荧光蛋白嵌合病毒的构建

携带增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)的脑心肌炎(Encephalomyocarditis virus, EMCV)嵌合病毒是研究该病毒体内外生物学特性的有力工具。因此，本研究基于前期构建的巨细胞病毒(Cytomegalovirus, CMV)感染性克隆，在EMCV基因组2A蛋白序列之后插入EGFP基因片段，并将重组质粒转染BHK-21细胞，获得携带EGFP的嵌合病毒。通过荧光定量PCR(real-time PCR)、间接免疫荧光(Indirect immunofluorescence assay, IFA)及半数组织细胞感染量测定(Median tissue culture infective dose, TCID₅₀)等方法对拯救病毒的基因组复制、蛋白表达及病毒粒子的感染性进行测定，结果证实嵌合病毒能够在BHK-21细胞上成功表达EGFP并产生完整的病毒粒子。然而，相较于野生型亲本拯救病毒，嵌合病毒在BHK-21细胞上的复制能力降低，并且在连续传代后逐渐失去绿色荧光信号，表明嵌合病毒中EGFP...

Construction of Encephalomyocarditis Virus for Enhancement of Green Fluorescent Protein Chimeric Virus

Chimeric Encephalomyocarditis Virus (EMCV) with an enhanced green fluorescent protein (EGFP) insertion is a powerful tool for investigating biological characteristics both in vitro and in vivo. In this study,we introduced the EGFP coding gene at the downstream flank of the genome sequence that encodes EMCV 2A protein on the basis of previously constructed cytomegalo virus(CMV)-driven infectious clone. The EGFP chimeric virus was obtained by transfecting BHK-21 cells with the recombinant plasmid.We evaluated genomic replication,protein synthesis,and the infectivity of chimeric viral particles through real-time PCR,immunofluorescence assay (IFA),and TCID₅₀ assay. The collective data demonstrated that recombinant plasmid transfection resulted in viable chimeric virus progenies expressing EGFP in BHK-21 cells. However,the chimeric virus exhibited hampered replication capacity and gradually diminished green fluorescence signals through passages. This suggests that EGFP genetic insertion impaired the assembly and release of chimeric virus particles,and the accumulated depletion and mutation during continuous passaging gradually led to the malfunction of EGFP.