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[目的]对重组人巨细胞病毒进行体外包装培养并鉴定,为该病毒基因组功能研究提供稳定的实验材料。[方法]用试剂盒抽提、纯化重组人巨细胞病毒Towne基因组(Towne-BAC),构建pcDNA3.1(+)-UL82表达质粒;将Towne-BAC与pcDNA3.1(+)-UL82共转人胚肺成纤维细胞;培养病毒、观察细胞病变,抽提病毒RNA;设计人巨细胞病毒IE1、UL138、UL145基因PCR引物并扩增序列,电泳鉴定。[结果]成功构建pcDNA3.1(+)-UL82,Towne-BAC与pcDNA3.1(+)-UL82共转后发生细胞病变;重组病毒的细胞培养物中分别检测到IE1、UL145基因表达,无UL138基因表达。[结论]成功包装出重组人巨细胞病毒,表型与Towne株吻合。
[Objective] The recombinant human cytomegalovirus (HCMV) was cultured in vitro and identified, which provided a stable experimental material for the study of the genomic function of the virus. [Method] The Towne-BAC of Tow cells was extracted and purified by using the kit to construct the pcDNA3.1 (+) - UL82 expression plasmid. Towne-BAC was co-transfected with pcDNA3.1 (+) - UL82 Human embryo lung fibroblasts were cultured, the virus was observed, the cytopathic effect was observed and the viral RNA was extracted. The PCR primers of human cytomegalovirus IE1, UL138 and UL145 were designed and amplified by PCR. [Results] The cytopathic effect of co-transfected with pcDNA3.1 (+) - UL82, Towne-BAC and pcDNA3.1 (+) - UL82 resulted in the expression of IE1 and UL145 gene in the cell culture of recombinant virus UL138 gene expression. [Conclusion] The recombinant human cytomegalovirus was successfully packaged and its phenotype was consistent with Towne strain.