Reggiani P C, Hereñú C B, Rimoldi O J, Brown O A, Pléau J-M, Dardenne M, Goya R G
Institute for Biochemical Research-Histology B, Faculty of Medicine, National University of La Plata, La Plata, Argentina.
Gene Ther. 2006 Aug;13(16):1214-21. doi: 10.1038/sj.gt.3302775. Epub 2006 Apr 13.
Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. 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'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-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 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.
胸腺素是一种胸腺肽，在大脑中具有促垂体活性和抗炎作用。我们构建了一个编码met-FTS（胸腺素的生物活性类似物）的合成DNA序列，随后将其克隆到不同的表达载体中。一个针对在啮齿动物中表达met-FTS优化的序列5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3'，被克隆到哺乳动物表达载体pCDNA3.1(+)和phMGFP（表达怪物绿色荧光蛋白）中，从而获得分别表达met-FTS和荧光融合蛋白metFTS-hMGFP的pcDNA3.1-metFTS和p-metFTS-hMGFP。该合成序列还被用于构建表达met-FTS的腺病毒载体RAd-metFTS。用pcDNA3.1-metFTS（实验组）或pcDNA3.1（对照组）转染HEK293和BHK细胞，导致胸腺素生物活性水平较高（实验组和对照组上清液中分别>600与<0.1 pg/ml）。用pmetFTS-hMGFP转染HEK293和BHK细胞显示荧光融合蛋白在细胞质和细胞核中分布。在胸腺切除的动物（血清胸腺素不可检测）中单次肌肉注射（10^7空斑形成单位（PFU）/小鼠或10^8 PFU/大鼠）RAd-metFTS，分别在小鼠和大鼠注射后至少110天和130天恢复血清胸腺素水平。我们得出结论，RAd-metFTS构成了在慢性脑炎症动物模型中实施胸腺素基因治疗的合适生物技术工具。
