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Method for purifying egg yolk antibody by using affinity immune medium

A technology of egg yolk antibody and medium, which is applied in the preparation method of peptides, chemical instruments and methods, immunoglobulin from eggs, etc., can solve the problems of inactivation, high price, high purity of ligands and other problems in the preparation of target substances, and achieve Effect of fixed process safety, increased production efficiency, improved extraction efficiency and extraction purity

Active Publication Date: 2020-11-06
YOULIKANG (JIANGSU) BIOPHARMACEUTICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are also some problems in affinity immunochromatography, such as the lack of carrier development leading to high prices (agarose particles, etc.), the high purity requirements of ligands, the difficulty of preparation, and the inactivation of target substances during the elution process.

Method used

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  • Method for purifying egg yolk antibody by using affinity immune medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the construction of fusion protein expression vector and host bacterium

[0039] Preparation of fusion protein expression vector:

[0040] chemical synthesis of the gene encoding ribosomal L2 protein (nucleotide sequence as shown in SEQ ID NO.6) and the gene encoding urease UerB from Helicobacter pylori (nucleotide sequence as shown in SEQ ID NO.8) , connect the gene encoding L2 protein with the gene UerB encoding urease from Helicobacter pylori by fusion PCR to obtain the target gene fusion protein; connect the target gene and pET28a plasmid after restriction endonuclease BamHI / XhoI digestion , to obtain the ligation product; transform the ligation product into Escherichia coli E coli BL21(DE3) to obtain the transformation product; insert the transformation product into ZYM-5052 medium for self-induction culture, first cultivate at 37°C for 4 hours, and then at 20 Cultivate at -28°C for 12-16 hours; after cultivation, extract the plasmid for enzyme diges...

Embodiment 2

[0041] Example 2: Preparation of Diatomaceous Earth Affinity Immunity Medium

[0042] (1) The recombinant Escherichia coli E coli BL21 (DE3) / fusion protein that embodiment 1 obtains is inoculated on LB medium, cultivates overnight at 37 ℃, obtains seed liquid; Above-mentioned seed liquid is inoculated with 2% inoculum ZYM-5052 medium, cultured at 37°C, 200r / min for 4 hours, then cultured at 28°C for 12 hours to make the OD of the bacterial liquid in the medium reach 1.8, collect the bacterial liquid, and collect the bacterial cells by centrifugation;

[0043] (2) After resuspending the bacterial cells obtained in step (1) with 10mM / L phosphate buffer solution of pH 7.0, the cells were broken by ultrasonic wave or high-pressure homogenization, and then clarified after being filtered through filter cloth. The cell disruption solution containing the fusion protein;

[0044] (3) Stir and mix the clarified cell disruption liquid with the selected diatomite for 1 hour, then filter ...

Embodiment 3

[0046] Example 3: Purification of specific urease egg yolk antibody

[0047] (1) Separation of egg yolks from eggs produced by laying hens immunized with urease from Helicobacter pylori: after the eggs are washed and dried, egg yolks are obtained by separating them with an egg separator;

[0048] (2) Rough extraction of egg yolk immunoglobulin: mix the separated egg yolk with water at a ratio of 1:4 to liquid, adjust the pH of the liquid to 7.0 with citric acid, let stand at 4°C for 16 hours, and extract the supernatant , obtain a clarified crude extract after ceramic microfiltration;

[0049] (3) Purification of specific egg yolk antibody: Mix the clarified crude extract in step (2) with the diatomite affinity medium immobilized with the specific antigen protein obtained in Example 2, stir and mix for 1 hour, and then remove the water Phase solution, first clean the diatomite with deionized water at a material-to-water ratio of 1:10, and then use 0.1mol / L NaCl solution to cl...

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Abstract

The invention discloses a method for purifying an egg yolk antibody by utilizing an affinity immune medium and belongs to the technical field of foods. According to the method of the invention, a novel antibody affinity immunopurification method is developed by using a diatomite affinity protein label. According to the affinity purification technology, a cheap diatomite filter aid is adopted as acarrier, an antigen protein with a ribosomal protein L2 label is adopted as a coupling ligand, the specific adsorption effect of the ribosomal protein L2 on diatomite is utilized to construct the affinity immune purification method which is low in cost and high in efficiency. The development of the efficient and low-cost specific yolk antibody affinity purification technology can effectively improve the separation and extraction efficiency of specific yolk immunoglobulin, improve the additional value of the egg yolk antibody, and expand the application field of the egg yolk antibody, so that the efficient and low-cost specific yolk antibody affinity purification technology has important significance on improving the competitiveness of the specific egg yolk immunoglobulin in the high-end biological antibody market.

Description

technical field [0001] The invention relates to a method for purifying egg yolk antibody by using an affinity immune medium, which belongs to the technical field of food. Background technique [0002] At present, for the purification of antibodies, ion-exchange chromatography using electrostatic interaction, hydrophobic chromatography using hydrophobic interaction, and protein A chromatography using affinity interaction for antibodies are mainly used, but now the general Antibody purification methods all have disadvantages such as complicated operation and high price. [0003] Egg yolk immunoglobulin is an important biologically active antibody. It has shown good substitution effect in the immune prevention and control of animal diseases, and the reduction or withdrawal of sub-therapeutic antibiotics in animal diets. Some domestic and foreign manufacturers have successfully realized the application of egg yolk immunoglobulin in the fields of biomedicine and daily food, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/26C07K16/02C07K1/22
CPCB01D15/26C07K16/02C07K1/22
Inventor 杨严俊张秀芳李俊华常翠华苏宇杰顾璐萍
Owner YOULIKANG (JIANGSU) BIOPHARMACEUTICAL CO LTD
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