Method for purifying lysozyme dimer and preparing standard substance of lysozyme dimer

Abstract

The invention discloses a method for purifying a lysozyme dimer and preparing a standard substance of the lysozyme dimer. The method comprises the following steps: carrying out volume exclusion chromatography separation and purification on a lysozyme monomer / polymer mixture obtained after crosslinking by using a high performance liquid chromatography system. The chromatographic column is a hydrophilic silica gel column; the mobile phase comprises 50mMPB, 0.5M urea, 0.3M mmonium sulfate and 10% acetonitrile; a flow rate of 0.35ml / min is adopted for driving, and the sample analysis time is 40min; and a UV 280nm ultraviolet detector is used for detection. And dimer fraction in the mixture is collected, after freeze drying, and an ultrafiltration tube is employed for desalination treatment. And concentration determination is conducted on an obtained lysozyme dimer aqueous solution by adopting a BCA quantitative kit, and freeze-drying is carried out after split charging to obtain a lysozymedimer standard substance. The process of the method can be completed at room temperature within 5h, the process steps are simple, the operation is simple and feasible, the yield of the lysozyme dimeris high, the cost is low, and the effect is good.

Description

technical field [0001] The invention relates to a method for purifying and preparing dimers, in particular to a method for separating and purifying dimers of lysozyme, which is applied to the technical fields of separation and purification of chemical substances and preparation of test standard products. Background technique [0002] Lysozyme is a common globular basic protein in nature, its molecular weight MW is about 14.4kDa, and it has antibacterial, anti-inflammatory, antiviral and other functions. Lysozyme dimer uses egg white lysozyme as raw material to modify monomeric lysozyme into lysozyme dimer, which retains the enzymatic activity of monomeric lysozyme, and also has quite new properties and antibacterial Activity [Ibrahim et al.1991, et al. 2009]. In experimental animals, dimers have been shown to increase nonspecific antigen responses and immunoglobulin production, stimulate enzymatic activity in lung macrophages, prevent antibiotic immunosuppression, elimin...

AI Technical Summary

Problems solved by technology

The separation effect of size exclusion chromatography (SEC) is mainly affected by the selection of stationary phase and mobile phase. There are great differences in the condition parameters used for different purification target substances. Determination of lysozyme by size exclusion chromatography The molecular weight of the dimer, thereby effectively controlling the purity of the lysozyme dimer, has the advantages of being accurate, intuitive, fast, and less sampling, but so far, there has been no direct monitoring of the lysozyme dimer by size exclusion chromatography. Related reports on the separation and purification process

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