Synthetic Thymosin α1 Gene And Lactoferrin Gene Expression In Tomato

Thymosin alpha-1 (Tα1) is an acetylated 28 amino acid peptide hormone from the thymus glands of mammals. Tal exerts effects of antiviralinfection. anti-tumor and immunomodulation. Thymosin alpha 1 has been evaluated for its immunomodulatory activities and therapeutic potential in several diseases, including chronic hepatitis B and C, acquired immunodeficiency syndrome (AIDS), depressed response to vaccination, and cancer. But Tal concentration decrease in serum related to a gradual decline in thymus function and thymic hormone production with aging process. So we transformed Tal gene into tomato for the development of oral vaccines. At the same time, we transformed human lactoferrin (LF) gene. LF coordinates the body's cellular immune defensive response that protects us from most infections, tumors and cancers. It is our body's first defense.In the experiment, we resynthesized Tal gene with a plant codon bias, in order to increased Tal concentration in tomato, then constructed a tandem four gene arrays, named Tal④. comparted by three enterokinase sites. Tal gene. Tal④ gene and lf gene were cloned into an expression vector pBI121 to obtain pBI121Tal. pBI121Tal④and pBI121LF. We also constructed expression vector pBIE8LF, in which lf gene under the control of tomato late-ripening-specific E8 promoter cloned by the PCR method, and PBITL contained Tal gene and lf gene. They were mobilized to Agrobacterium tumefaciens strain LBA4404 through the freeze-thaw method.Cotyledon leaf explants of tomato were transformed by co-cultivation with Agrobacterium contained target gene. Kanamycin-resistant transformants were regenerated and established in glasshouse. T0-generation putative transgenic plants obtained were screened by PCR. Southern hybridization tests, which confirmed the incorporation and expression of the target gene. Western blot assay of the transgenic plants confirmed the expression of proteins in tomato fruits except transformed with pBIE8LF, because of immature fruits. The results of ELISA showed the concentration of LF ranged from 7.9 to 15.7μg/g tomato fruits transformed with pBI121LF and PBITL, and the concentration of Tal ranged from 6.0 to 20.2μg/g fruit transformed with pBI121Tal, pBI121Tal④ and PBITL. We could get conclusion that not only Tal④ gene can express in tomato, but also concentration is higher. Bioassay of Tal and LF were done by lymphocyte proliferation assay and bacteriostatic qualities test. Results demonstrated that the transgenic plants had effects of immunomodulation.The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. Our research had theoretically and potential...