论文部分内容阅读
目的体外快速繁殖人巨细胞病毒(HCMV)。方法用试剂盒抽提、纯化重组HCMV细菌人工染色体(BAC),以重组HCMV细菌人工染色体(BAC-HCMV)为模板扩增UL82基因,酶切后连接至pcDNA载体,构建pcDNA-pp71重组质粒,转化感受态宿主菌,抽提及鉴定重组质粒。通过电穿孔技术将BAC-HCMV与鉴定成功的pcDNA-pp71重组质粒共转染人包皮成纤维细胞,通过多重PCR法及观察细胞病变效应、绿色荧光蛋白等方法鉴定培养后获得的HCMV。结果 (1)获得pcDNA-pp71重组质粒,经PCR及双酶切后,片段大小与预期相符,进一步测序鉴定成功。(2)BAC-HCMV与pcDNA-pp71共转染人包皮成纤维细胞,出现细胞病变效应,经荧光显微镜观察可见绿色荧光。(3)以培养出的病毒DNA为模板进行多重PCR扩增,获得两条与预期大小相符的条带,鉴定HCMV体外繁殖成功。结论应用BAC技术成功在体外快速繁殖HCMV,为HCMV研究提供实验材料,也为进一步研究HCMV分子机制奠定基础。
Objective To rapidly propagate human cytomegalovirus (HCMV) in vitro. Methods Recombinant HCMV bacterial artificial chromosome (BAC) was extracted and purified by kit. UL82 gene was amplified by recombinant human chromosomal HCMV (BAC-HCMV) as template. After digestion, the gene was ligated into pcDNA vector to construct pcDNA-pp71 recombinant plasmid. Transformed competent host bacteria, extraction and identification of recombinant plasmids. The human foreskin fibroblasts were co-transfected with BAC-HCMV and the successfully identified pcDNA-pp71 recombinant plasmid by electroporation, and the HCMV obtained after culturing was identified by multiplex PCR and the observation of cytopathic effect, green fluorescent protein and other methods. Results (1) The pcDNA-pp71 recombinant plasmid was obtained by PCR and double enzyme digestion, the fragment size was in line with the expected, and further sequencing was identified. (2) BAC-HCMV and pcDNA-pp71 cotransfection of human foreskin fibroblasts, cytopathic effect, the fluorescence observed by fluorescence microscopy. (3) Multiplex PCR amplification was carried out with the cultured virus DNA as a template to obtain two bands consistent with the expected size, and the success of in vitro propagation of HCMV was identified. Conclusion The rapid propagation of HCMV in vitro using BAC technology provides experimental materials for HCMV research and lays the foundation for further study on the molecular mechanism of HCMV.