去甲基化酶腺病毒载体构建及其在人颌下腺细胞中的表达

Cloning and expression of methyl-CpG binding domain 2 gene in human submandibular gland cell line by adenovirus vector transfection

目的构建去甲基化酶(methyl-CpG binding domain 2,MBD2)基因腺病毒载体,并观测其在人颌下腺(human submandibular gland,HSG)细胞中的表达。方法以HSG细胞总RNA为模板,RT-PCR法扩增MBD2基因全长编码序列,克隆入载体pMD18-T后,亚克隆入pAdTrack-CMV腺病毒穿梭载体,构建pAdTrack-MBD2重组体。该重组体与腺病毒骨架质粒pAdEasy-1于BJ5183菌中同源重组,产生重组腺病毒质粒Ad-MBD2,该质粒经293细胞包装,获得具有感染力的Ad/MBD2重组腺病毒颗粒,将该病毒颗粒感染HSG细胞,倒置显微镜检测转染后HSG细胞生长变化及MBD2在其中的表达等情况。图像分析法计算感染效率。RT-PCR法检测MBD2基因在该细胞中的表达变化。结果成功克隆到909 bp大小的MBD2基因,构建了其腺病毒载体Ad/MBD2,该腺病毒载体经293细胞成功包装,并获具感染力的Ad/MBD2重组腺病毒颗粒,其感染效率约70%。感染的HSG高表达MBD2。结论成功克隆到MBD2基因,构建了其重组腺病毒载体Ad/MBD2,并证实了其感染HSG细胞的有效表达。

Objective To construct the adenovirus vector carrying methyl-CpG binding domain 2（MBD2） gene and express this gene in human submandibular gland（HSG）cell line.Methods cDNA fragment encoding human MBD2 gene was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR） with the total mRNA isolated from HSG cells as template.The PCR amplified product was first cloned into pMD18-T vector,and then subcloned into the shuttle vector pAdTrack-CMV in order to construct the recombinant plasmids pAdTrack-MBD2,which homologously recombinated with the adenoviral backbone vectors Adeasy-1 in BJ5183 bacterial cells,so as to generate recombinant adenoviral plasmids Ad-MBD2.The recombinant adenoviruses Ad/MBD2,which were employed to infect HSG cells,were generated by transfecting the recombinant adenoviral DNA into 293 cells.The growth of transfected HSG cells and the expression of enhanced green fluorescent protein（EGFP） were observed by invert microscopy.Image analysis was employed to calculate the transfection efficiency.Subsequently the expression of MBD2 in transfected HSG cells was tested by RT-PCR.Results Human MBD2 gene fragment,with a length of 909 bp,was successfully cloned,and recombinant adenoviral vector of Ad/MBD2 was constructed.The recombinant adenoviruses Ad/MBD2 were ge-nerated by transfecting the recombinant adenoviral DNA into 293 cells.Adenoviral virus particle which possessed infectious competent was made from Ad/MBD2.Its transfection efficiency was about 70%.HSG cells expressed MBD2 gene obviously after infection.Conclusion Human MBD2 gene is successfully cloned,and recombinant adenoviral vector of Ad/MBD2 is constructed.The valid expression of MBD2 gene in HSG cells has been confirmed.