人巨细胞病毒Han株US12基因缺失株的构建及其在人胚肺成纤维细胞中的复制

Construction of Human Cytomegalovirus Han-ΔUS12-BAC Strain and Studies on Its Replication in Human Embryonic Lung Fibroblasts

目的构建人巨细胞病毒(HCMV)Han株US12基因缺失的BAC毒株,研究US12产物对HCMV Han株在人胚肺成纤维细胞(HELF)中复制的影响。方法以侧翼含有US12基因同源序列的PCR引物扩增卡那霉素抗性基因,电转至含有Han株BAC质粒的大肠杆菌DY380-Han-wt-BAC,PCR及DNA测序验证US12缺失的病毒序列。将缺失突变质粒Han-ΔUS12-BAC-P电转至HELF,获得感染性病毒颗粒Han-ΔUS12-BAC。以MOI为0.1的Han-ΔUS12-BAC毒株及Han-wt-BAC毒株感染HELF,TCID50法测定上清中病毒滴度,绘制病毒增殖曲线。结果成功构建了Han-ΔUS12-BAC克隆,转染HELF后成功获得感染性病毒。生长曲线实验结果表明,ΔUS12缺失和野生型毒株在HELF中复制增殖与扩散水平无明显差异。结论 US12为HCMV临床株Han在HELF中增殖和扩散过程中的非必需基因。

Objective To construct human cytomegalovirus （HCMV） Han-ΔUS12-BAC strain and to study the role of US12 in HCMV replication in human embryonic lung fibroblasts （HELF）. Methods Kanamycin-resistant gene was amplified with primers containing homology arms se - quence flanking US12 and then electroporated into E.coli DY380-Han-wt-BAC competent cells. Successfully recombinant Han-ΔUS12-BAC clones were identified by PCR, sequenced for confirmation Han-ΔUS12-BAC plasmids were electropomted into HELF to produce infectious virions. Han-ΔUS12-BAC and Han-wt-BAC were inoculated onto HELF at the multiplicity of infection of 0.1 pfu/cell. The viral titer in the supernatant was measured by TCID50 assay and growth kinetics of the viruses in HELF was studied. Results Han-ΔUS12-BAC clone was successfully constructed. Han-ΔUS12-BAC was reconstructed in HELF to generate infectious virions. Grouch kinetics assay indicated that Han-ΔUS12-BAC and Han-wt-BAC showed no differences in their growth and dissemination in HELF. Conclusion US12 in HCMV clinical strain Han is dispensable for HCMV growth and dissemination in HELF.