The Study On Extraction With Ultrasonic-assisted Salting-out Method And Extraction And Purification With Aqueous Two-phasesystem From Yak Blood ImmunoglobulinG

In this study, fresh yak blood as the research object, The extracting process of Ig G were optimized by using single factor and response surface analysis method. Next, Ig G was purified by using DEAE-52 ion chromatography and sephadex-G200 gel filtration layer analysis. The results were as follows:(1) The optimum extracted parameters of the yak blood Ig G with ultrasonic-assisted saturated ammonium sulphate method were obtained as ultrasonic time 4 min, the standing time after ultrasound 24 min, p H 4.3 and temperature 49℃. The Ig G content was 25.22mg/ml under these conditions, and the relative error with the theoretical value 25.79mg/ml was 2.20%. And the study compared with Saturated ammonium sulphate method can reduce 66 min standing time.(2) The optimum extracted parameters of the yak blood Ig G with PEG/K2HPO4 aqueous two-phase system were obtained as 13%(w/w)PEG, 17%(w/w)K2HPO4 and 11%(w/w)Na Cl at p H 6.0. The Ig G content was 13.90mg/ml under these conditions, and the relative error with the theoretical value 14.04mg/ml was 0.96%, and the purity of Ig G was 70.50%.(3) The optimum extracted parameters of the yak blood Ig G with PEG/HPS aqueous twophase system were obtained as 6.5%(w/w)PEG, 17.5%(w/w)HPS, 14%(w/w)Na Cl and at p H 6.1. The Ig G content was 12.48mg/ml under these conditions, and the relative error with the theoretical value 12.67mg/ml was 1.49%, and the purity of Ig G was 63.30%.(4) By comparing the result of the two extraction methods of aqueous two-phase system, PEG/K2HPO4 aqueous two-phase system is better. Therefore, Ig G extracted with it was used at subsequent experiment.(5) The optimum purified parameters of the yak blood Ig G with DEAE-52 chromatography were obtained as sample sizes 0.9m L and flow rate 0.8ml/min at p H of PBS solution 6.1. The purity and recovery rate of Ig G were 88.79% and 44.89% under these conditions. Sephadex-G200 gel filtration chromatography was used to futher purify Ig G, and the purity and recovery rate of Ig G were 95.80% and 38.80%.(6) The results identifying the purity of Ig G showed that electrophoretic bands showed a single band and has reached electrophoretic pure. The molecular weight of Ig G heavy and light chain were about 60 KD and 23 KD.In summary, The PEG/K2HPO4 aqueous two-phase system is superior to PEG/HPS aqueous two-phase system. Using DEAE-52 ion chromatography and sephadex-G200 gel filtration chromatography, electrophoresis pure Ig G can be obtained. The molecular weight of Ig G was about 166 KD.