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Preparation and Application of Cellulose Membrane Immobilized Fc Fragment of Antibody

Author: ZhangZhengHua
Tutor: JiaLingYun
School: Dalian University of Technology
Course: Biomedical Engineering
Keywords: Fc fragment Affinity membranes rProteinA Purification Functional groups
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 41
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Abstract

Protein A (Protien A) are some of Staphylococcus aureus cell wall proteins, and human and mammalian serum immunoglobulin Fc fragments, which may be specific binding characteristics, has been used in the purification of antibodies and autoimmune treatment of disease. To obtain large amounts of high purity and high biological activity of Protein A or class function of Protein A small molecule is one of the one of the key issues, in order to achieve this purpose, this thesis prepared coupled Fc fragment of the cellulose film, and studied the feasibility of recombinant Protein A purification and screening molecular cluster structure Bionic small molecule affinity ligand affinity membrane. The main results are as follows: 1. Optimized conditions through the papaya enzyme Enzymolysis IgG Fc fragment optimal conditions of 37 ° C, pH 7.5, for enzymolysis 2 h, enzyme substrate ratio of 480 U / mg. Protein A affinity chromatography and Fc fragments isolated and purified from the enzymatic hydrolysis mixture. 2 coupling the Fc fragment of the preparation process of the cellulose film. Determine the better the the NaIO4 oxidation conditions for pH 5, 0.1 mol / L citric acid buffer NaIO4 concentration of 0.2 mol / L, oxidation time control 15 min. Sequentially coupling spacer arm hexamethylenediamine and glutaraldehyde, and finally to the Fc fragment fixed to the nitrocellulose membrane after activation, the fixed amount of the Fc fragment of the affinity membrane is 11.75 mg / g. 3 Coupling of the Fc fragment of cellulose film static saturated adsorption amount of Protein A is 8.22 mg / g, a dynamic adsorption capacity was 6.84 mg / g; the film has good selectivity of E. coli cell lysate of recombinant Protein A , the adsorption capacity of 5.38 mg / g, affinity membrane reused eight times, has not changed its basic dynamic adsorption; can be used for the separation and purification of recombinant Protein A. Immobilized antibody Fc fragment cellulose film screening platform using chitosan as the structural basis of molecular clusters, tyramine mode molecular evaluation of the structure of molecular clusters that can be played in a small molecule affinity ligand screening to the role. The results indicate that the chitosan - tyramine compared with separate chitosan, tyramine, the adsorption and antibody Fc fragment cellulose film stronger, each fixed FC average per molecule can be adsorbed 31.1 functional groups is Fc 11.6 times the combined solution of free tyramine molecules, to verify such screening model for screening small molecule affinity ligands immobilized protein feasibility. In summary, the above results indicate antibody Fc fragment cellulose film rProtein A Purification and biomimetic molecular clusters structure can be applied to small molecule affinity ligand screening.

Preparation and Application of Cellulose Membrane Immobilized Fc Fragment of Antibody

Abstract

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