Engineering a Cure for Diabetes Procedures
The nucleotide sequence of the IL-13 gene was retrieved from the EMBL database (accession number X69079). Plasmid containing the human IL-13 gene was purchased from Invivogen. The IL-13 gene was in-frame fused downstream to the CTB gene. The CTB-IL-13 chimeric gene and the IL-13 gene were cloned into the modified pCaMterX vector under the control of the double CaMV 35S promoter to generate plasmids pDW6 and pDW7, respectively. The plasmids confirmed by DNA sequencing were transformed into agrobacterium strain GV3101 and the resulting strains were used to transform low-nicotine tobacco as described (Ma et al., 2005). Genomic DNA was isolated from transgenic seedlings (Ma et al., 2005). PCR was used to detect CTB-IL-13 or IL-13 in transgenic plants. RNA was purified with RNeasy Plant Mini Kit (Qiagen) following supplier's recommendation. The isolated RNA was treated to DNaseI to remove contaminated genomic DNA. RT-PCR and northern blot was used to detect IL-13. Protein purification and western blot were conducted as described (Ma et al 2005). The plant-derived recombinant protein was directly used in an in vitro bioassay to determine whether or not it is able to induce T-cell proliferation.
