The Expression Of Thymosin α1 In Transgenic Tomato And Escherichia Coli

Thymosin alpha-1(Tα1) is an acetylated 28-amino-acid peptide hormone from the thymus glands of mammals. Thymosin plays important roles in modulating immune response and has significant clinical applications used as therapeutic agents. Tα1 has been evaluated for its immuno-modulatory activities and therapeutic potential in several diseases including chronic hepatitis B and C, acquired immunodeficiency syndrome, and depressed response to vaccination and cancer. Nowadays, the main methods to get thymosin are via extraction from animal tissues or via chemical synthesis. However, the high price confine the application of the thymosin. In recent years there has been a growing interest of producing therapeutic proteins and vaccines in transgenic plants. Plants offer the prospect of inexpensive biopharmaceutical production without sacrificing product quality or safety. It will be interesting to see if plants can be used to produce Tα1, by which the cost and complication to produce Tα1 may be lowed down. In this study, Tα1 gene was designed and synthesized according to plant codon usage preference. The tandem Tα1 gene of 4 repeats was concatenated and identified by sequencing. A plant expression vector PG-pRD12-4×Tα1 harboring the 4×Tα1 gene was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 54 putative transgenic tomato plants were regenerated. PCR, Southern blot analyses confirmed the transgenic status of some plants. RT-PCR analysis demonstrated that the 4×Tα1 gene was expressed specifically in tomato fruits. Western blot analysis confirmed the expression of the 4×Tα1 concatemer protein in tomato fruits. ELISA analysis showed that the concentration of the 4×Tα1 concatemer protein ranged from 2.725~6.094μg/g fresh weight in mature fruit. However in the immature fruits, the concentration of the 4×Tα1 concatemer protein ranged from 2.373~2.434μg/g fresh weight. This study confirmed that the 4×Tα1 concatemer protein was successfully expressed in tomato. Biological activity of expressed 4×Tα1 concatemer protein was carried out by lymphocyte proliferation assay, and the result demonstrated that the protein had effects of immuno-modulation. This is the first report on successful expression of biologically active thymosin alpha 1 (Tα1) in plants and this research provides a basis for further development of plants as bioreactor to produce Tα1, one of the important therapeutic proteins. Although the oral immunogenic protein antigens can low down the production cost, it is difficult to control the oral therapy dose. The mainline is also widely applied in clinic. Therefore the purification of target protein from the transgenic plants was very important. In this study, some researches about protein purification were carried out. Considered the advantage of Escherichia coli (E. coli) expression system in expression time, the E. coli expression vector pQE30-4×Tα1 containing 4×Tα1 gene was constructed which was subsequently transformed into E. coli M15. Vector pQE30 was used because the target protein could be expressed as a fusion protein with a 6×His-tag for the convenience of subsequent purification. The result showed that the 4×Tα1 gene was successfully expressed in E. coli. The expressed protein was purified and found to have the normal bioactivity. This is the first report on the expression of 4×Tα1 concatemer which is synthesized according to plant codon usage preference in E. coli expression system. This study not only provides the positive protein of 4×Tα1, but also provides the reference in purification of expressed protein in plants in the future.