[Construction of a recombinant adenovirus vector harboring human transforming growth factor-beta type II receptor-IgG1Fc fusion gene]

Li Jia, Jian-xin Xue, You Lu
Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University 2008, 28 (12): 2136-8

OBJECTIVE: To construct a recombinant adenoviral vector harboring human transforming growth factor-beta type II receptor-IgG1Fc (TbetaRII-IgG1Fc) fusion gene.

METHODS: The cDNA fragments of human TbetaRII and IgG1Fc genes were amplified by RT-PCR and fused with overlap PCR to obtain the fusion gene TbetaRKK-IgG1Fc. The TbetaRII-IgG1Fc gene was cloned into the shuttle plasmid pAdTrack-CMV, which was linearized and transfected into E.coli BJ 5183 strain containing the adenoviral backbone vector. The recombinant adenovirus vector was constructed by homologous recombination. The recombinant adenoviral plasmid was linearized and transfected into 293 cells, followed by amplification and purification of the virus and detection of TbetaRII-IgG1Fc mRNA expression by RT-PCR. The functional activity of the recombinant adenoviral plasmid was assessed using enzyme-linked immunosorbent assay (ELISA).

RESULTS: The results of restriction endonuclease digestion and DNA sequencing indicated correct sequence of the target TbetaRII-IgG1Fc fusion gene. The recombinant adenoviral plasmid expressed hTbetaRII-IgG1Fc and neutralized TGF-beta1 in vitro after infection of the human lung fibroblasts (HLF), as confirmed by RT-PCR and ELISA.

CONCLUSIONS: The recombinant adenoviral plasmid capable of neutralizing TGF-beta1 in vitro is constructed successfully.

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