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

摘要:

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1.5×109 pfu/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。

Abstract:

Objective：To construct recombinant adenoviral vector encoding human LIM mineralization protein-1（LMP-1） and to in－vestigate 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 remark－ably increased in U2OS infected with Ad-LMP-1. Conclusion：The recombinant adenovirus Ad-LMP-1 has been successfully con－structed and can be helpful for the further research involved in LMP-1.