Abstract: Thymulin (facteur thymique serique, FTS) is a bioactive thymic peptide involved in several aspects of intra- and extrathymic T-cell differentiation. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5’-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3’, was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 pg/ml in experimental supernatants versus <0.1 pg/ml in control supernatants). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single injection (107 pfu/mouse; i.m.) of RAd-metFTS in thymectomized mice and rats (nondetectable serum thymulin) restored serum thymulin levels for several weeks post-injection. We conclude that this synthetic gene for met-FTS constitutes a suitable biotechnological tool for basic research and for the implementation of thymulin gene therapy in animal models of thymodeficiency and other pathologies like brain inflammatory processes.
