人巨细胞病毒IE1真核表达载体的构建与表达

目的构建人巨细胞病毒（HCMV）IEl真核表达载体，观察其在真核细胞内的表达，为HC—MVIEl核酸疫苗的相关研究奠定基础。方法将质粒pNEBl93-IEl用Sal I、BamH I双酶切与载体pEGFP—C1连接构建重组载体pEGFP—C1-IEl；双酶切鉴定后，取重组载体pEGFP—C1-IEl用EcoR I 、Hind Ⅲ／双酶切与载体pcDNA3．1（-）连接构建重组质粒pcDNA3．I（-）-IEl，双酶切和测序鉴定；将重组质粒pcDNA3．1（-）-IEl转染HeLa细胞，RT—PCR检测IElmRNA的表达。结果重组载体pEGFP—C1-IEl和pcDNA3．1（-）-IEl双酶切后均可切出约1476bp目的片段；重组载体pcDNA3．1（-）-IEI测序结果登录C,enBank，作BLAST分析，含有1476bp目的基因片段，无碱基错配和移码突变，与读码框完全-致；转染重组载体pcDNA3．1（-）-IEl的细胞中可扩增出约1476bp的目的条带。结论成功地构建了真核表达载体pcDNA3．1（-）-IEl，且其能在真核细胞内袁达。

Construction and Expression of Eukaryotic Expression Vector of Human Cytomegalovirus IE1

Objective To establish the substructure of Human Cytomegaloviru （HCMV） IE1 DNA vaccine, this study was designed to construct a recolnbinant eukaryotic expression vector of IE1 and analyze its expression in eukaryotic cells. Methods HCMV IE1 gene fragment obtained from pNEB193 - IE1 by enz）ane digestion was inserted into expression vector pEGFP - C1 and was confiixned by Sal I/BamH I. Then the recombinant vectorpEGFP - C1 - IE1 cut by enzyne digestion was inseded into eukaryotic expression vector pcDNA3.1 （ - ） and confirmned by EcoR I/Hind Ⅲ and sequencing. The recombinant eukaryotic expression vector pcDNA3.1 （ - ） - IE1 was transfected into HeLa cells. The expressed product of IE1 mRNA was amplified by RT- PCR. Results Restricted enzyme analysis and DNA sequencing showed that recombinant vectors were correct. The expressed product of HC- MV IE1 mRNA could be be detected in HeLa cells. Conclusion The recombinant eukaryotie expression vector of HCMV IE1 was successfully constructed, HCMV IE1 mRNA could be expressed in HeLa ceils.