人巨细胞病毒IE1真核表达载体的构建与表达

Construction and Expression of Eukaryotic Expression Vector of Human Cytomegalovirus IE1

目的构建人巨细胞病毒(HCMV)IE1真核表达载体,观察其在真核细胞内的表达,为HC-MVIE1核酸疫苗的相关研究奠定基础。方法将质粒pNEB193-IE1用SalⅠ、BamHⅠ双酶切与载体pEGFP-C1连接构建重组载体pEGFP-C1-IE1;双酶切鉴定后,取重组载体pEGFP-C1-IE1用EcoRⅠ、HindⅢ双酶切与载体pcDNA3.1(-)连接构建重组质粒pcDNA3.1(-)-IE1,双酶切和测序鉴定;将重组质粒pcDNA3.1(-)-IE1转染HeLa细胞,RT-PCR检测IE1 mRNA的表达。结果重组载体pEGFP-C1-IE1和pcDNA3.1(-)-IE1双酶切后均可切出约1 476 bp目的片段;重组载体pcDNA3.1(-)-IE1测序结果登录GenBank,作BLAST分析,含有1 476 bp目的基因片段,无碱基错配和移码突变,与读码框完全一致;转染重组载体pcDNA3.1(-)-IE1的细胞中可扩增出约1 476 bp的目的条带。结论成功地构建了真核表达载体pcDNA3.1(-)-IE1,且其能在真核细胞内表达。

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.