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

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.