人脂联素基因慢病毒载体在293 T细胞中的表达及意义  

Expression of human apM1 gene lentiviral vector in 293 T cells and its significance

作　　者：黄杰[1] 谢良地[1] 

机构地区：[1]福建医科大学附属第一医院福建省高血压研究所,福建福州350005

出　　处：《中国药理学通报》2008年第7期928-932,共5页Chinese Pharmacological Bulletin

基　　金：福建医科大学教授基金资助课题(NoJS06042)

摘　　要：目的构建携带人脂联素(human apM1,hapM1)基因的慢病毒载体,并观察其在293 T细胞中的表达及意义。方法将hapM1的编码区(CDS区)从克隆载体上亚克隆到慢病毒载体上,构建重组慢病毒载体质粒。在脂质体介导下与包装质粒HELPER、包膜质粒VSVG共转染293 T细胞包装生产慢病毒。慢病毒感染293 T细胞后,RT-PCR和Westernblot法检测在293 T细胞中hapM1基因的表达。采用抑制血管平滑肌细胞增殖实验,检测表达产物的生物学活性。结果hapM1基因经测序后与GenBank报道序列完全一致。重组慢病毒载体质粒PNL-hapM1-IRES2-EGFP经鉴定正确。三质粒共转染293 T细胞成功,收集、浓缩病毒后测定其滴度为2.0×105TU·L-1。感染293 T细胞后RT-PCR、Western blot检测hapM1组细胞有hapM1蛋白表达,其余两组(Mock-293 T组、293 T组)均无表达。表达产物hapM1蛋白能够抑制血管平滑肌细胞的增殖。结论成功构建带有hapM1基因慢病毒载体,并且能够在293 T细胞中表达具有生物学活性的hapM1蛋白。Aim To investigate the expression of the lentiviral vector carrying a human apM1 （hapM1） gene in 293 T cells and its bioactivity. Methods The CDS region of hapM1 gene was subcloned into the lentiviral vector from a cloning vector, and the transfer plasmid （PNL-hapMI-IRES2-EGFP）of the lentiviral vector was constructed. The three plasmids of the lentiviral vector： PNL-hapM1-IRES2-EGFP, HELPER, and VSVG were cotransfected to 293 T cells to produce the recombinant lentivirus; 293 T cells were infected by the recombinant lentivirus and were identified with fluorescent microscope, RT-PCR and western blot. Inhibitory effect of the recombinant hapM1 on proliferation of vascular smooth muscle cells （VSMCs） was identified.Results The sequencing results showed that the sequence of the subcloned hapM1 gene was completely identical to that reported in GeneBank. The virus titre of the recombinant hapM1 lentivirus was 2.0×105 TU·L^-1. After the recombinant lentivirus infected 293 T cells, RT-PCR and Western blot results showed that the expression of hapM1 in the hapM1-293 T group could be measured. The recombinant hapM1 inhibited the proliferation of VSMCs. Conclusions The recombinant lentivirus carrying a human apM1 gene was constructed successfully. The recombinant hapM1 could be expressed in 293 T cells and had its bioactivity.

关 键 词：脂联素 慢病毒载体 293 T细胞 血管平滑肌细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R373.9[医药卫生—基础医学]