Preparation Of Recombinant Protein A Affinity Adsorbent And It’s Properties And Application Research

Due to the unique immunological characteristics, Protein A from Staphylococcus aureus is not only playing an important role in medicine and biology but also occupying the irreplaceable position in the recovery of antibodies, so it has great reaearch value. In this paper, in order to realize the practical application of Protein A-based affinity adsorbent, we express two kinds of Protein A and are focused on studying the adsorption capacity and the application in the separation and purification. The main works are carried out as follows:(1) Constructing the gene engineering bacteria. Successfully constructing two strains recombinant bacterias, a piant with spa from natural Protein A gene, another plant with z from the modified Protein A. At the same time introducting the His·Tag to the N-terminal, which lays a foundation for the purified protein.(2) Expression and purification of r Protein A. Optimizing the fermentation condition and improving the protein expression level. Use the Ni affinity chromatography to purify the r Protein A and choise 400 mmol/L imidazole to elute. The effect is the best.(3) Studying the performance of homemade chromatography medium. We are focused on studying the static adsorption and adsorption kinetics of two kinds of affinity medias in the process of adsorpting Ig G. z5 affinity medium has more superior performance. On the one hand, it has the more moderate elution condition, p H 4; On the other hand it is strong resistance to alkali. After soaking by Na OH, adsorption performance does not change.(4) Application in the separation and purification. We use the z5 ligand affinity chromatography to purify the pig blood and are focused on studying sample quantity, the flow rate and stability avaluation. When sample quantity is 3 m L and the flow rate is 1.0 m L/min, it makes the balance between the purification cycle and work effifiency and the recovery rate and purity respectively are 92.8% and 65.6%, purification factor is 2.3; r Protein A affinity chromatography has the good stability, which has a huge potential to achieve business value.