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• Preparation

  1. Biochemical Extraction Method
  Thymosin peptide preparations (mixtures of various peptides) are primarily obtained from animal tissues, resulting in significant differences in activity. Because they contain animal proteins, the likelihood of allergic reactions is relatively high. Thymalfasin, extracted biochemically from human embryonic thymus, has a low yield but good efficacy. Considering the limited availability of raw materials, high price, low yield, and complex extraction steps, large-scale production faces numerous difficulties, and its development prospects are not optimistic.
  2. Chemical Synthesis Method
  Commercially available Thymalfasin is mainly synthesized using the SPSS method, with synthesis and purification achieved through appropriate techniques. Using resin as a carrier, solid-phase synthesis is performed using Fmoc. 28 amino acids are sequentially coupled according to the Thymalfasin formula. The N-terminal amino acid is acetylated with acetic anhydride, and cleavage is performed using TFA as the main cleavage reagent to obtain the crude product. The crude product is then purified by HPLC to obtain pure Thymalfasin, which is further purified to obtain the Thymalfasin raw material.
  3. Genetic Engineering Methods
  There are three ways to obtain genetically engineered bacteria:
  (1) Cloning a multi-heterogeneous gene into a host to obtain the target gene bacteria, which are then expressed as inclusion bodies. It has been reported that a multi-heterogeneous gene of thymosin was constructed using random annealing and PCR techniques and cloned into *E. coli*.
  (2) Recombining the synthesized fusion protein gene into a vector and transforming the vector into a host to construct genetically engineered bacteria. For example, based on the signal peptide gene in the plasmid, an expression plasmid was constructed, transformed into *Pichia pastoris* by electroporation, and then the fusion protein was expressed by methanol induction, followed by enzyme digestion to obtain thymosin.
  (3) First, an expression vector was constructed and transformed into a host to become a genetically engineered bacterium, then fermented to obtain the target product. The synthesized thymosin alpha monomer gene is ligated to the corresponding plasmid to construct an expression vector, which is then integrated into Pichia pastoris. The target product is directly secreted into the fermentation broth, and high-purity thymosin alpha is obtained through two-step purification. This method involves fewer separation steps, lower cost, and is easy to scale up for production.;

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