| Thymosins is a group polypeptide hormone, and was firstly isolated from calf thymus by Allan Goldstein in 1966, which including over ten components, and among thymosin fraction 5(TF5) has strongest bioactive. The thymosinα1(Tα1) is a kind of the TF5, which was composed of 28 amino acid residues, and isoelectric point(pI) is 4.2, and molecular weight of the 3,108 Da. As an immune booster, play critical roles in the maturation, differentiation and function of T-cells. Tα1 mainly was used to cure various diseases of immunodeficiency and virus infection in clinic, such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), cancers and so on. Currently, several reasons confine application of the Tα1 in clinic, which include the available thymus of calf or porcine for extraction of Tα1, higher cost to yield Tα1 by chemical synthesis method, and existing safety problems by traditional expression system such as Escherichia coli and transgenic animal using genetic engineering way, because of contamination of E.coil-derived endotoxin and some zoonotic pathogens.Recently research results demonstrated that plant expression systems have advantages at cost, scale and safety to produce therapeutic proteins compare with traditional expression systems such as Escherich coli, yeast and transgenic animals.Consider above-mentioned reasons, in order to explore new ways to produce Tα1, and safety of the transgenic plants was also considered, the plant binary expression vector of the p35s::4×Tα1 which with twin T-DNAs was constructed, and the 4×Tα1 gene was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation. We obtained a series research results as follows:(1) The Tα1 gene (124 bp) was designed and synthesized according to the plant codon usage bias and created a novel 4×Tα1 concatemer (four copies of the Tα1 gene arranged end-to-end in tandem, designated 4×Tα1). Subsequently, a plant binary expression vector, p35s::4×Tα1 with twin T-DNAs was constructed.(2) One hundred sixty-five plants of the kanamycin resistant generated tobacco were obtained, and seventeen positive plants were confirmed by PCR, and positive percentage was 10.3%.(3) Fourteen independence transgenic tobacco plants were confirmed by southern blot, and copy numbers of the integrated 4×Tα1 gene in transgenic tobacco genome range from 1 (line 40) to 7 (line 12) copies. However, we found that selective nptII gene was integrated into tobacco genome at different copies or sites in T0 generation. In order to analyze the separation of 4×tα1 and nptII, 177 transgenic tobacco plants was assayed with PCR in T1 generation, we obtained 2 transgenic tobacco plats which only carried 4×tα1, but without selective gene nptII.(4) Western blot result suggested that tobacco-derived 4×Tα1 has immunocompetence. ELISA assay showed that content of the Tα1 protein ranged from 180.46 (line 81) ~756.87 ng/g·fw (line 86) in tobacco fresh leaves.(5) MTT result revealed the 4×Tα1 protein derived from transgenic tobacco plants exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes (Balb/c) in vitro.This study can provide important reference to produce therapeutic proteins using plant or improve plant genetic traits in safety way.
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