Construction of a bicistronic recombinant adenoviral vector for human interleukin-10 and enhanced green fluorescent protein expression in bone marrow mesenchymal stem cells

Jian-Qing Lin, Cai-Zhu Lin, Xian-Zhong Lin, Kai Zeng, You-Guang Gao
Chinese Medical Journal 2012, 125 (1): 102-8

BACKGROUND: Human interleukin-10 (hIL-10) is a cytokine synthesis inhibitory factor, which is involved in various immune responses. The purpose of this study was to construct an adenoviral vector carrying the hIL-10 gene for expression of biologically active hIL-10 in rat bone marrow mesenchymal stem cells (rMSCs).

METHODS: A pSNAV2.0-hIL10 plasmid was used as a template to obtain a hIL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene. The pDC316-hIL-10-IRES-EGFP plasmid was linearized by PmeI digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax. Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange. After the number of virus particles and titer was determined, rMSCs were infected with the adenoviral vector. The infection rate was determined by fluorescence microscopy and flow cytometry, and hIL-10 protein expression in rMSCs was measured by Western blotting.

RESULTS: The virus particle concentration, OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2 × 10(11) VP/ml, approximately 2.0, and 1.1 × 10(10) TCID50/ml, respectively. Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs. GFP expression was considered the multiplicity of infection (MOI) and was time-dependent. The infection rate was 92.9% at 100 MOI.

CONCLUSIONS: A bicistronic recombinant adenoviral vector for hIL-10 and EGFP gene expression were successfully constructed. The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hIL-10 was highly expressed in cells. The present study provides an experimental basis for further research of immunosuppressive therapy using hIL-10. The expression level of hIL-10 protein as detected by Western blotting was also MOI- and time-dependent.

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