Study On The Technique Of Purifying Thymosinβ₄ From Calf Thymus

Thymosin is a series of bioactive peptides secreted from the thymus, which is a kind of immunostimulants. Thymosin P₄(Tβ₄) is a kind of thymosin widely presented in various animal organs. Research had showed that thymosinβ₄ had the effect of anti-inflammatory and wound healing, which can promote angiogenesis, hair follicle development and corneal wound healing. So thymosinβ₄ has high medical value. The main research work we have done was the purification of thymosinβ₄, the main work include: the filtration of the proper ion exchange media, establishment of ion-exchange separation process of thymosinβ₄, the optimization of the separation process.10 kinds of ion exchange media were filtrated by absorption in static state.CM-Sepharose FF weak cation gel and DEAE-Sepharose FF weak anion gel were selected for the purification process.Thymosinβ₄ purification process include 1) thymosin fraction5 extraction: ammonium sulfate precipitation was used, and 25%-50% ammonium sulfate precipitation from calf thymus was thymosin fraction 5; 2) ion exchange separation: Firstly, the thymosin fraction 5 solution was passed though CM-Sepharose FF cation exchange column and washed by successive elution with NaCl of increasing concentrations (1%(w/v), 4%(w/v) and 5%(w/v)) in a stepwise manner. The fraction washed by 4%(w/v)NaCl was collected and adjusted to pH6.8. Then, this fraction was adsorbed in DEAE-Sepharose FF anion exchange column and washed by de-ionized water, then followed by successive elution of NaCl with step increased concentrations(1%(w/v) ,4%(w/v) and 5%(w/v)). After the fraction de-absorbed by 4%(w/v) NaCl was collected and desalted, thymosinβ₄ of 85.2% was obtained and characterized.Optimization of separation process has three parts: By comparison, more thymosin fraction5 was gotten using perchloric acid precipitation; Optimized the operating conditions of anion and cation column, CM-Sepharose FF optimized condition was column length 30cm, elution flow rate 40cm/h, DEAE-Sepharose FF column optimized condition was sample concentration 4g/L, velocity 60cm/h, column length 40cm; desalination process optimization, Desalination using gel filtration was more efficient than dialysis. After the optimization, product of 90.8% was obtained.Product of thymosin β₄ was detected. Using nondenaturing electrophoresis, activity of thymosinβ₄ was tested. Result showed product was active. Using mass spectrometry to detect amino acid sequence of the product, result was consistent with the literature. These proved the product was thymosinβ₄.