Expression of Bioactive Thymosin Alpha 1 (Tα1) in Marker-free Transgenic Lettuce (Lactuca sativa)

Thymosin alpha 1 (T alpha 1) was widely used for the treatment of hepatitis (B and C) and several cancers. However, current production of T alpha 1 is difficultly meeting clinical needs. To develop a novel and safety approach for T alpha 1 production, we synthesized a T alpha 1 gene (124 bp) based on the plant codon usage bias and constructed a four-copy T alpha 1 gene concatemer (408 bp) by using isocaudamer technique. This 4 x T alpha 1 structure was cloned into plant binary expression vector pCAMBIA2300 with twin transfer deoxyribonucleic acids (T-DNAs) and integrated into lettuce genome via Agrobacterium-mediated transformation. Thirteen positive plants were identified by polymerase chain reaction and confirmed by Southern blot analysis, and 11 marker-free lettuce plants were obtained in T2 generation. The content of recombined T alpha 1 (rT alpha 1) protein reached 1798.317 +/- 87.312 ng/g in fresh leaves of transgenic lettuce. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay demonstrated that rT alpha 1 protein stimulated mouse splenic lymphocyte proliferation in vitro. These data suggest that biologically active rT alpha 1 was successfully expressed in marker-free transgenic lettuce, and this method could provide an alternative choice for large-scale production of T alpha 1 in the future.