摘要
目的研究人巨细胞病毒(human cytomegalovirus,HCMV)感染对人血管平滑肌细胞同源盒基因(homeobox gene,HOX)表达的影响,并克隆受HCMV诱导表达的同源框基因,为其功能研究奠定基础。方法以1MOI的HCMV感染人血管平滑肌细胞,分别于感染后24h,48h及72h用基因表达谱芯片检测血管平滑肌细胞基因表达水平的改变,筛选差异表达的同源框基因并进行全长cDNA的克隆和原核表达。结果人血管平滑肌细胞感染HCMV后24、48、72h有9个HOX基因的表达发生变化。其中上调基因3个(HHEX、ZEB2、HOXD8),下调基因6个(EMX2OS、SIX5、PKNOX1、IRX3、MEIS1、ESX1)。在HCMV感染后48、72h,TLX2基因表达上调。成功构建pGEX-4T-1/TLX2重组原核表达质粒,转化BL21后经IPTG诱导表达58×103的重组蛋白,该蛋白能被相应抗体识别。结论 HCMV感染可诱导人血管平滑肌细胞多个HOX基因表达的改变,构建的pGEX-4T-1/TLX2重组质粒获得体外表达,为进一步研究TLX2的功能及HCMV致血管平滑肌增生性疾病的分子机制奠定了基础。
Objectives To study the influence of human cytomegalovirus（HCMV）infection on the expression of homeobox genes in human vascular smooth muscle cells and to clone the homeobox genes induced by HCMV in order to lay the foundation for the further study of the function of those genes. Methods Twenty-four,48,and 72 hafter vascular smooth muscle cells were infected with 1 MOI HCMV,changes in the level of expression of homeobox genes were identified with a gene expression array.In addition,homeobox genes that were differentially expressed in vascular smooth muscle cells were cloned and expressed in a prokaryotic expression system. Results（1）The level of expression of 9 homeobox genes changed significantly 24,48,and 72 hafter infection with HCMV.Expression of 3 of those genes（HHEX,ZEB2,and HOXD8）was up-regulated while expression of 6（EMX2 OS,SIX5,PKNOX1,IRX3,MEIS1,and ESX1）was down-regulated.（2）Expression of the TLX2 gene increased markedly 48 and 72 hafter infection with HCMV.（3）The recombinant pGEX-4 T-1/TLX2 plasmid was successfully constructed.After the plasmid was transformed into E.coli BL21 cells,expression of a recombinant protein with a molecular mass of 58×10^3 Da was induced with IPTG,and the protein was recognized by the corresponding antibody. Conclusion HCMV infection can change the expression of homeobox genes in human vascular smooth muscle cells.The recombinant pGEX-4 T-1/TLX2 plasmid was successfully expressed in vitro.This work has provided additional information for the further study of TLX2 and for understanding the mechanism of vascular smooth muscle hyperplasia due to HCMV infection.
作者
杨长江
苏颖慧
赵婵玥
曹家敏
马琼山
谭宇蓉
邬国军
YANG Chang-jiang;SU Ying-hui;ZHAO Chan-yue;CAO Jia-min;MA Qiong-shan;TAN yu-rong;WU Guo-jun(Department of Clinical Medicine,Xiangya School of Medicine,Central South University,Changsha 410078,China;Department of Medical Microbiology,School of Basic Medical Science,Central South University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2018年第9期953-957,共5页
Journal of Pathogen Biology
基金
中南大学大学生创新类项目(No.YC20170673
YC20170979)
