Construction Of An Adenoviral Vector Encoding Human Interleukin-12 And Its Expression On Human Mesenchymal Stem Cells

Aim:To construct an adenoviral vector encoding human interleukin-12 (hIL-12) gene, infect human bone-marrow mesenchymal stem cells (hBMSCs) in vitro, and detect the expression of hIL-12 for providing a potential approach to the clinical application of IL-12.Methods:The experiments were conducted at the Laboratory of Biotherapy Center of Beijing General Hospital of PLA from May 2009 to December 2009. The total RNA of human dendritic cells (DCs) was obtained by RNA Extraction Kit after the treatment of DCs with LPS for 24 h. hIL-12 p35 and p40 cDNA were amplified from the total RNA by RT-PCR, cloned into the eukaryotic expression vectors pcDNA3.1(-) and pmRNA IRES, respectively, and then subcloned into an adenoviral shuttle vector pDC515, in which p35 and p40 fragments were linked by internal ribosomal entry sites (IRES), to construct the shuttle vector pDC515 p35/IRES/p40. Using Ad MaxTM adenovirus vector system, pDC515 p35/IRES/p40 and pBHGfrtΔE1,3FLP were cotransfected into 293 cells, and Ad hIL-12 was generated by FLP recombinase-mediated site-specific recombination. hBMSCs was isolated from a health volunteer and cultured with a-MEM medium, the hBMSCs of the third passage was inoculated into a 12-well plate at the density of 5×105 cells per well, and Ad hIL-12 was added into hBMSCs at the multiple of infection (MOIs) of 100 per cell. After 2-hour incubation, hBMSCs was irradiated with 10 Gy y-ray to lose proliferative activity and the medium was refreshed every 24 h. The cell supernatants were consecutively collected for 5 days and the amounts of interleukin-12 (IL-12) were detected by hIL12p70 ELISA.Results:The sequences of p35 and p40 fragments were identical with those provided by GenBank NM₀00882(762 bp) and NM₀02187(987 bp) by the sequencing, respectively. The formation of plagues began 10 days after the cotransfection of pDC515 hIL-12 p40/IRES/p35 and pBHGfrtAEl,3 FLP into 293 cells, a few obvious plagues were chosen and propagated in another fresh 293 cells. Through the amplification by two-three rounds, the 293 cells were collected and lysed by freezing and thawing of three cycles. PCR verified there existed the hIL-12 p40 and p35 fragments in the lysates. After the infection of hBMSCs with Ad IL-12 at the MOIs of 100 pfu per cell, the analysis of IL-12 by ELISA showed that the production of hIL-12 began at 12 h, reached to the peak between 24～48 h, and kept at least for 5 days post-infection,. The amounts of IL-12 at 12 h,24,48 h,72 h,96 h, and 120 h were (4.91±1.09), (59.40±1.17), (106.88±4.64), (144.53±6.10), (182.72±12.14), and (205.83±10.26) ng/106 cells/24 h, respectively.Conclusion:Ad MaxTM is an efficiently and quickly packaging system of adenoviral vectors. IL-12 gene in which the two subunits p35 and p40 were linked by IRES can efficiently express the protein of IL-12p70. The high expression of IL-12 is consecutively expressed after the infection of hBMSCs with Ad hIL-12, suggesting that a potential application to the gene therapy of hBMSCs as a carrier.