人LMP-1基因腺病毒重组体的构建及其在骨肉瘤细胞U2OS中的表达

Construction of recombinant adenoviral vector encoding human LMP-1 and its expression in osteosarcoma cell U2OS

目的:构建表达人LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)基因的腺病毒重组体,体外感染骨肉瘤(Osteosarcoma,OS)细胞系U2OS,并鉴定LMP-1基因在U2OS中的表达。方法:以K562细胞的cDNA为文库,采用PCR方法对LMP-1进行扩增,通过TA克隆与pGEM-T载体连接并DNA测序。双酶切后并将目的基因插入至腺病毒穿梭质粒pAdtrack-CMV,对腺病毒穿梭质粒pAdtrack-CMV-LMP-1行双酶切和DNA测序鉴定,并通过荧光显微镜、RT-qPCR和Western blot检测pAdtrack-CMV-LMP-1在HEK-293T细胞中的表达。线性化pAdtrack-CMV-LMP-1,在BJ5183菌内完成与骨架质粒pAdeasy-1的同源重组,构建重组腺病毒质粒Ad-LMP-1。通过脂质体介导,在HEK-293A细胞内包装出复制缺陷的重组腺病毒Ad-LMP-1,大量扩增、纯化并测定滴度。用Ad-LMP-1感染OS细胞系U2OS,通过荧光显微镜、RT-qPCR和Western blot检测LMP-1在U2OS细胞中的表达。结果:双酶切和DNA测序鉴定穿梭质粒pAdtrack-CMV-LMP-1构建成功,荧光显微镜证实转染了pAdtrack-CMV-LMP-1质粒的HEK-293T细胞内有绿色荧光蛋白表达,RT-qPCR和Western blot验证了LMP-1基因的表达量明显高于对照组。通过扩增、纯化,Ad-LMP-1滴度达到1.5×109pfu/ml。荧光显微镜下观察重组腺病毒感染的U2OS内有绿色荧光蛋白表达,RT-qPCR和Western blot检测发现重组腺病毒感染U2OS后,LMP-1的mRNA和蛋白表达量明显高于对照组。结论:成功构建了人LMP-1基因腺病毒重组体,为进一步的实验研究奠定了基础。

Objective：To construct recombinant adenoviral vector encoding human LIM mineralization protein-1（LMP-1） and to investigate its expression in osteosarcoma cell line U2OS.Methods：The cDNA of LMP-1 was amplified by PCR from K562 cell line and cloned into pGEM-T,then subcloned into shuttle plasmid pAdtrack-CMV.After restrictive enzyme digestion and DNA sequencing,the recombinant shuttle plasmid pAdtrack-LMP-1 was transferred into HEK-293T cells,which were detected by fluorescent microscopy,RT-qPCR and Western blot to confirm the expression of LMP-1.Then linearized pAdtrack-LMP-1 was homologously recombined with pAdEasy-1 in BJ5183 competent cell to transform into Ad-LMP-1 which subsequently transfected HEK-293A cells under the mediation of lipofectamine.Ad-LMP-1 was packaged and amplified in HEK-293A cells.After Ad-LMP-1 infection,the gene and protein expressions of LMP-1 in U2OS were identified by fluorescent microscopy,RT-qPCR and Western blot.Results：Restrictive enzyme digestion and DNA sequencing analysis revealed that the shuttle plasmid pAdtrack-LMP-1 was constructed successfully.Fluorescence microscopy found that expressed green fluorescent protein was distributed in cytoplasm of transfected HEK-293T.RT-qPCR and Western blot verified LMP-1 to be highly expressed in HEK-293T.After purification,the virus titer reached 1.5×109 pfu/ml.The result of fluorescent microscopy,RT-qPCR and Western blot indicated that LMP-1 mRNA and protein expression were remarkably increased in U2OS infected with Ad-LMP-1.Conclusion：The recombinant adenovirus Ad-LMP-1 has been successfully constructed and can be helpful for the further research involved in LMP-1.