阅读说明：本技术 一种利用亲和免疫介质纯化蛋黄抗体的方法 (Method for purifying yolk antibody by using affinity immune medium ) 是由 杨严俊 张秀芳 李俊华 常翠华 苏宇杰 顾璐萍 于 2020-07-10 设计创作，主要内容包括：本发明公开了一种利用亲和免疫介质纯化蛋黄抗体的方法,属于食品技术领域。本发明利用一种硅藻土亲和蛋白标签开发了一种新型的抗体亲和免疫纯化方法。该亲和纯化技术采用廉价的硅藻土助滤剂作为载体,以带有核糖体蛋白L2标签抗原蛋白为偶联配基,利用了核糖体蛋白L2对硅藻土的特异性吸附作用,构建了一种成本低、效率高的亲和免疫纯化方法。高效低成本的特异性蛋黄抗体亲和纯化技术的开发不仅能有效提高特异性蛋黄免疫球蛋白分离提取效率,也能进一步提高蛋黄抗体的附加值和扩大应用领域,这对于提高特异性蛋黄免疫球蛋白在高端生物抗体市场中竞争力具有重要意义。(The invention discloses a method for purifying yolk antibodies by using an affinity immune medium, belonging to the technical field of food. The invention develops a novel antibody affinity immune purification method by utilizing a diatomite affinity protein label. The affinity purification technology adopts a cheap diatomite filter aid as a carrier, takes an antigen protein with a ribosomal protein L2 label as a coupling ligand, and utilizes the specific adsorption effect of the ribosomal protein L2 on diatomite to construct an affinity immune purification method with low cost and high efficiency. The development of the high-efficiency low-cost affinity purification technology for the specific egg yolk immunoglobulin can not only effectively improve the separation and extraction efficiency of the specific egg yolk immunoglobulin, but also further improve the additional value of the egg yolk antibody and expand the application field, which has important significance for improving the competitiveness of the specific egg yolk immunoglobulin in the high-end biological antibody market.)

1. An affinity immune medium for purifying an antibody is diatomite adsorbing a fusion protein, and the fusion protein is obtained by fusing an antigen protein derived from pathogenic bacteria or viruses and a ribosome L2 protein.

2. The affinity immune media of claim 1, wherein the ribosomal L2 protein comprises a polypeptide sequence as set forth in SEQ id No. 2.

3. The affinity immune media of claim 1 or 2, wherein the amino acid sequence of ribosomal L2 protein is shown in SEQ ID No. 1.

4. A method of preparing the affinity immune media of claim 1, comprising the steps of: (1) synthesizing a gene sequence for encoding ribosome L2 protein; (2) the gene of the antigen protein which is coded and derived from pathogenic bacteria or viruses is fused with the gene which is coded and used for ribosome L2 protein, and then the fused gene is cloned into a secretory expression vector with a signal peptide to construct a recombinant expression vector; (3) transforming the recombinant expression vector into an expression host, and culturing and expressing the expression host; (4) centrifuging the fermentation liquor obtained after the culture is finished, and taking supernatant; (5) mixing the supernatant with diatomite to enable the fusion protein in the supernatant to be adsorbed on the diatomite to obtain an affinity immune medium;

or, the method comprises the following steps: (1) synthesizing a gene sequence for encoding ribosome L2 protein; (2) the gene of the antigen protein which is coded and derived from pathogenic bacteria or viruses is fused with the gene which is coded and generated by ribosome L2 protein, and then the fused gene is cloned into an intracellular expression vector which does not contain signal peptide, so as to construct a recombinant expression vector; (3) transforming the recombinant expression vector into an expression host, and culturing and expressing the expression host; (4) breaking host cells after the culture is finished, centrifuging cell breaking liquid and taking supernatant; (5) and mixing the supernatant with diatomite to enable the fusion protein in the supernatant to be adsorbed on the diatomite to obtain the affinity immune medium.

5. A method for purifying egg yolk antibodies is characterized by comprising the following steps: the egg yolk antibody is purified by using the affinity immune medium immobilized with the corresponding antigen protein as claimed in any one of claims 1 to 3 as an adsorbent.

6. The method of claim 5, wherein the antibody-containing clear solution is mixed with an affinity immune medium immobilized with the corresponding antigenic protein, the mixture is left to stand and the clear solution is extracted, the affinity immune medium is washed with deionized water and a low ionic strength salt solution to remove non-specifically adsorbed foreign proteins, the affinity immune medium is eluted with a higher ionic strength salt solution for specific antibody, and finally, the desalted high-purity specific antibody is obtained by ultrafiltration desalting.

7. The method of purifying egg yolk antibodies according to claim 5 or 6, wherein the egg yolk antibodies comprise urease Uerb egg yolk antibodies, flagellin HpaA egg yolk antibodies, vacuolar cytotoxin VacA egg yolk antibodies and/or cytotoxin CagA egg yolk antibodies.

8. The method of purifying egg yolk antibodies as claimed in claim 7, wherein the antibody-containing serum is prepared by: separating egg yolk from eggs laid by laying hens immunized with antigen protein, mixing the egg yolk with deionized water or buffer solution, standing, collecting supernatant, and filtering.

9. A method for purifying an egg yolk antibody as claimed in any one of claims 5 to 8, wherein said antigen protein comprises urease Uerb derived from helicobacter pylori, flagellin HpaA, vacuolar cytotoxin VacA and/or cytotoxin CagA.

10. Use of the immunoaffinity medium according to any one of claims 1 to 3 or the process for the preparation according to claim 4 or the process for the purification of egg yolk antibodies according to claim 5 or 6 for the purification of egg yolk antibodies.

Technical Field

The invention relates to a method for purifying yolk antibody by using an affinity immune medium, belonging to the technical field of food.

Background

Currently, although purification of antibodies is mainly performed by ion exchange chromatography using electrostatic interaction, hydrophobic chromatography using hydrophobic interaction, and protein a chromatography using affinity interaction for antibodies, all of the conventional methods for purifying antibodies have disadvantages such as complicated operation and high cost.

Yolk immunoglobulin is an important bioactive antibody, and has shown a better substitution effect in animal disease immune control, reduction or discontinuation of sub-therapeutic amounts of antibiotics in animal diets. Some domestic and foreign manufacturers have successfully realized the application of yolk immunoglobulin in the fields of biological medicines and daily foods, developed immunoglobulin oral spray which can have auxiliary treatment effect on oral ulcer, chewing gum with anti-dental caries effect, beverage with anti-helicobacter pylori (preventing gastritis and gastric ulcer) property, candida albicans specific antibody care solution with bacteriostatic and itching relieving effect, anti-influenza virus toothpaste and other various products.

Chromatographic or non-chromatographic purification methods in biological proteins have historically had to trade off separation efficiency, cost and ease of use. Because the traditional aqueous phase dilution-membrane column separation method has low content of specific antibodies in antibody extraction, particularly in extracted egg yolk antibodies, the application of the traditional aqueous phase dilution-membrane column separation method in the aspects of industrial and commercial, scientific research, analytical detection, theoretical research, disease diagnosis, prevention and treatment and the like has certain limitations. In recent years, the affinity immunochromatography technique has been attracting attention as a technique for separating antibodies, and it has been reported that a desired specific yolk immunoglobulin with high purity can be separated from a crude clarified solution of a yolk sample by simple column chromatography. However, affinity immunochromatography also has some problems, such as high price (agarose particles and the like) caused by insufficient development of the carrier, high requirement on the purity of the ligand, difficulty in preparation, easy inactivation of target substances in the elution process and the like.

Research and development of new carriers and high-purity preparation of coupling ligands are bottlenecks which restrict the development of high-purity antibody high-efficiency separation technology.

Disclosure of Invention

In order to solve the problems, the invention develops a novel antibody affinity immune purification method by utilizing a diatomite affinity protein label. The affinity purification technology adopts a cheap diatomite filter aid as a carrier, takes an antigen protein with a ribosomal protein L2 label as a coupling ligand, and utilizes the specific adsorption effect of the ribosomal protein L2 on diatomite to construct an affinity immune purification method with low cost and high efficiency. The development of the high-efficiency low-cost affinity purification technology for the specific egg yolk immunoglobulin can not only effectively improve the separation and extraction efficiency of the specific egg yolk immunoglobulin, but also further improve the additional value of the egg yolk antibody and expand the application field, which has important significance for improving the competitiveness of the specific egg yolk immunoglobulin in the high-end biological antibody market.

The invention provides an affinity immune medium for antibody purification, which is diatomite adsorbing fusion protein obtained by fusing pathogen or virus antigen protein and ribosome L2 protein.

In one embodiment of the invention, the ribosomal L2 protein contains a polypeptide sequence as shown in SEQ ID No. 2.

In one embodiment of the invention, the ribosomal L2 protein is encoded by a nucleotide sequence shown in SEQ ID No. 7.

In one embodiment of the invention, the amino acid sequence of the ribosomal L2 protein is shown in SEQ ID No. 1.

In one embodiment of the invention, the nucleotide sequence encoding the ribosomal L2 protein is shown in SEQ ID NO. 6.

In one embodiment of the present invention, the gene encoding the L2 protein can be obtained by designing PCR primers, and amplifying the genes from L2-containing strains such as Escherichia coli strain HB101, BL21(DE3), TOP10, etc.; the corresponding gene sequence can also be synthesized according to the amino acid sequence and the gene codon preference of the expression cells.

The invention provides a preparation method of an affinity immune medium for antibody purification, which comprises the following steps: (1) synthesizing a gene sequence for encoding ribosome L2 protein; (2) the gene of the antigen protein which is coded and derived from pathogenic bacteria or viruses is fused with the gene which is coded and used for ribosome L2 protein, and then the fused gene is cloned into a secretory expression vector with a signal peptide to construct a recombinant expression vector; (3) transforming the recombinant expression vector into an expression host, and culturing and expressing the expression host; (4) centrifuging the fermentation liquor obtained after the culture is finished, and taking supernatant; (5) mixing the supernatant with diatomite to enable the fusion protein in the supernatant to be adsorbed on the diatomite to obtain an affinity immune medium;

or, the method comprises the following steps: (1) synthesizing a gene sequence for encoding ribosome L2 protein; (2) the gene of the antigen protein which is coded and derived from pathogenic bacteria or viruses is fused with the gene which is coded and generated by ribosome L2 protein, and then the fused gene is cloned into an intracellular expression vector which does not contain signal peptide, so as to construct a recombinant expression vector; (3) transforming the recombinant expression vector into an expression host, and culturing and expressing the expression host; (4) breaking host cells after the culture is finished, centrifuging cell breaking liquid and taking supernatant; (5) and mixing the supernatant with diatomite to enable the fusion protein in the supernatant to be adsorbed on the diatomite to obtain the affinity immune medium.

In one embodiment of the invention, the expression host is a bacterium or a fungus.

In one embodiment of the invention, the expression host is E.coli.

The invention also provides a method for purifying the yolk antibody, which comprises the following steps: and (3) purifying the egg yolk antibody by using the affinity immune medium fixed with the corresponding antigen protein as an adsorbent.

The invention also provides a yolk antibody purification method, which comprises the steps of mixing the clear liquid containing the antibody with the affinity immune medium fixed with corresponding antigen protein, standing and extracting the clear liquid, washing the affinity immune medium by deionized water and low ionic strength salt solution, washing off non-specific adsorbed foreign protein, eluting the affinity immune medium by salt solution with higher ionic strength, and finally obtaining the desalted high-purity specific antibody by ultrafiltration and desalination.

In one embodiment of the invention, the egg yolk antibody is urease UerB egg yolk antibody, flagellin protein HpaA egg yolk antibody, vacuolar cytotoxin VacA egg yolk antibody and/or cytotoxin CagA egg yolk antibody.

In one embodiment of the present invention, the method for preparing the antibody-containing clear solution comprises: separating egg yolk from eggs laid by laying hens immunized with antigen protein, mixing the egg yolk with deionized water or buffer solution, standing, collecting supernatant, and filtering.

In one embodiment of the present invention, the method for preparing the antibody-containing clear solution comprises: separating egg yolk from eggs laid by laying hens immunized by antigen protein to obtain egg yolk, mixing the egg yolk with 4-10 times of deionized water or buffer solution with the pH value of 5.5-7.0, mechanically cooling to 0-5 ℃, standing for 3-16 hours, then extracting supernatant, performing microfiltration by a ceramic membrane to obtain clear liquid, mixing the clear liquid with corresponding antigen affinity immune media for 0.5-1 hour, after extracting the clear liquid, washing by deionized water and low ionic strength salt solution, washing off non-specific adsorbed foreign proteins, then performing elution of specific antibodies by using higher ionic strength salt solution, and finally performing ultrafiltration desalination to obtain the desalted high-purity specific egg yolk immunoglobulin.

In one embodiment of the invention, the antigenic protein is urease Uerb from helicobacter pylori, flagellin protein HpaA, vacuolar cytotoxin VacA and/or cytotoxin CagA.

In one embodiment of the invention, the amino acid sequence of urease Uerb is shown as SEQ ID No.3, the amino acid sequence of flagellin HpaA is shown as SEQ ID No.4, the amino acid sequence of vacuolar cytotoxin VacA is shown as SEQ ID No.5, and the amino acid sequence of cytotoxin CagA is shown as SEQ ID No. 11.

In one embodiment of the invention, the nucleotide sequence encoding the urease Uerb gene is shown as SEQ ID NO.8, the nucleotide sequence encoding the flagellin protein HpaA gene is shown as SEQ ID NO.9, the nucleotide sequence encoding the vacuolar cytotoxin VacA gene is shown as SEQ ID NO.10, and the nucleotide sequence encoding the cytotoxin CagA gene is shown as SEQ ID NO. 12.

The invention also provides the application of the immunoaffinity medium or the preparation method or the method for purifying the egg yolk antibody in the purification of the egg yolk antibody.

The invention has the beneficial effects that:

(1) the invention provides a brand-new method for purifying yolk antibody, which adopts cheap diatomite material to prepare an affinity immune medium, thus utilizing the affinity immune medium provided by the invention to purify the antibody and solving the problem of high price of the affinity immune matrix; the method of the invention not only improves the extraction efficiency and the extraction purity of the antibody, but also reduces the production cost, and is a feasible industrialized large-scale production method.

(2) The preparation method of the affinity immune medium provided by the invention utilizes the special affinity adsorption capacity of ribosome L2 protein to cheap diatomite materials, target protein or polypeptide can be recombined and expressed by a conventional method after being fused with ribosome L2 protein, specific antigen fusion protein can be immobilized on a diatomite matrix through one-step adsorption, no toxic reagent is used in the immobilization process, the immobilization process is safe, green and rapid, and the production efficiency of the affinity immune medium can be effectively improved.

(3) The ribosomal protein L2 can be used as a connecting arm for immobilizing a recombinase on diatomite, and provides more choices for immobilizing active proteins.

Drawings

FIG. 1: specific urease egg yolk antibody electrophoresis elution maps under different sodium chloride concentrations; wherein, the 1-4 bands are respectively 0.25, 0.50, 0.75 and 1mol/L sodium chloride concentration gradient elution to flow out specific urease egg yolk antibody components in the diatomite affinity medium.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

Diatomaceous earth referred to in the following examples was purchased from kieselguhr ltd, yokuro-leishi.

The media involved in the following examples are as follows:

ZYM-5052 Medium: the culture medium contains 10g of peptone, 5g of yeast extract, 4.46 of disodium hydrogen phosphate, 1.7 of potassium dihydrogen phosphate, 1.33 of ammonium chloride, 0.354 of sodium sulfate, 2.5 of glycerol, 0.25 of glucose, 1 of lactose, 24.65 of magnesium sulfate and trace element mixture per liter.

LB culture medium: each liter of culture medium contains 10g of protein, 5g of yeast extract and 5g of NaCl, the pH value is adjusted to 7.4 by using 1mol/L NaOH, and the volume is fixed to 1 liter by using deionized water.

The detection methods referred to in the following examples are as follows:

and (3) detecting the purity of the yolk immunoglobulin: by adopting a conventional polyacrylamide gel electrophoresis method, 4% of concentrated gel, 8% of separation gel, 80V of concentrated voltage and 120V of separation voltage are adopted. And (3) dyeing the separation gel by using Coomassie brilliant blue, decoloring and photographing, and then performing strip optical density analysis by using Quality One software.