Construction of Human Cytomegalovirus Han?ΔUS12?BAC Strain and Studies on Its Replication in Human Embryonic Lung Fibroblasts
10.12007/j.issn.0258?4646.2017.06.003
- VernacularTitle: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
- Author:
Ying LU
;
Yanping MA
;
Zhongyang LIU
;
Hongyan WANG
;
Ying QI
;
Liying HAN
;
Shuang GAO
;
Bo ZHENG
;
Chang LIU
;
Yujing HUANG
;
Qiang RUAN
- Keywords:
cytomegalovirus;
US12;
bacterial artificial chromosome;
growth kinetics
- From:
Journal of China Medical University
2017;46(6):489-494,500
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct human cytomegalovirus(HCMV)Han?ΔUS12?BAC strain and to study the role of US12 in HCMV replica?tion 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 electroporated into HELF to produce infectious viri?ons. 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 con?structed. Han?ΔUS12?BAC was reconstructed in HELF to generate infectious virions. Growth 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.
