Separation and application of BCG specific protection antibody

Abstract

The invention discloses separation and application of a BCG specific protective antibody, the BCG specific antibody is separated by adopting a biotin-streptavidin chromatography method, and the antibody connected sugar chain is analyzed through high performance liquid chromatography, so that the sugar chain mixture has increased glycoform percentage of digalactose mono / disialic acid, increased glycoform percentage of digalactose mono / disialic acid, increased glycoform percentage of digalactose mono / disialic acid and increased glycoform percentage of digalactose mono / disialic acid. The antibody is beneficial to killing mycobacterium tuberculosis, and a new thought is provided for research and development of tuberculosis immune markers and vaccines.

Description

Technical Field

[0001] The present invention specifically relates to the separation and application of BCG-specific protective antibodies. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Newborns in my country need to be vaccinated with Bacillus Calmette-Guerin (BCG) to prevent tuberculosis. BCG is an attenuated strain of bovine tuberculosis Mycobacterium tuberculosis. In areas with a high incidence of tuberculosis, it is the only vaccine recommended by the World Health Organization for the prevention of tuberculosis.

[0004] The human body can produce both cellular immunity and humoral immunity against tuberculosis infection. Because Mycobacterium tuberculosis is an intracellular parasite, cellular immunity is considered to be an immune response that plays a protective role for the subject, but some vaccines designed for cellular immunity are not very effective. In recent years, the protective effect of humoral immunity, namely antibody IgG, has attracted attention. For example, Mycobacterium tuberculosis capsular polysaccharide-specific IgG has shown a good anti-tuberculosis effect in human experiments, and transporter and lipoprotein-specific IgG can resist infection with Mycobacterium tuberculosis. Capsular polysaccharide-specific IgG can also promote neutrophils to kill Mycobacterium tuberculosis. The sugar chain connected to IgG also affects the protective effect of the antibody. The 297th amino acid asparagine of the IgG Fc segment is connected to the sugar chain. There are many sugar forms, and the basic structure is acetylglucosamine-acetylglucosamine-mannose-mannose-acetylglucosamine. In the sugar chain complex, if the percentage of sugar chains containing digalactosyl sialic acid at the end is high, it can enhance the killing effect of macrophages on Mycobacterium tuberculosis. Figure 1 The glycoform with the largest molecular weight is shown, i.e., the digalactose-disialoglycoform.

[0005] As mentioned above, since BCG is widely administered to newborns in my country, studying the protective effect of specific antibodies after BCG vaccination has important practical significance and can be used to establish new immune markers and prepare biological products in vaccine development. However, the separation of BCG-specific antibodies and the identification of the percentage of antibody-linked glycoforms are difficult problems and have not been reported at home or abroad. There have also been no patent applications or reports on the protective effect of such specific antibodies at home or abroad. Summary of the invention

[0006] Immunity research on tuberculosis mainly focuses on cellular immunity, with less research on antibodies. European and American countries do not vaccinate with BCG, so BCG-specific antibodies cannot be separated. The content of pathogen or antigen-specific antibodies in the blood is low, and the separation procedure is relatively cumbersome. Analysis of the percentage of antibody-linked sugar forms is also a technical challenge, and no reports have been seen in China yet.

[0007] In view of the research on the protective role of antibodies in tuberculosis and BCG as the only currently used vaccine to prevent tuberculosis, the separation of BCG-specific antibodies and the analysis of the percentage of glycoforms attached to BCG antibodies are of great significance.

[0008] The technical solution adopted by the present invention is as follows: In a first aspect of the present invention, a method for isolating BCG-specific protective antibodies is provided, the method comprising the following steps: (1) Whole blood density gradient centrifugation was used to obtain serum, and total IgG was separated by affinity chromatography; (2) Separating BCG-specific IgG from the total IgG using a biotin-streptavidin method.

[0009] In one or some embodiments of the present invention, in step (1), the affinity chromatography method is specifically as follows: a filler is added to the chromatography column, and then a purification buffer is added, and serum is added. When the serum flows through the filler, all proteins in the serum except IgG are adsorbed to the filler, and IgG is collected.

[0010] Preferably, the filler is a gel resin.

[0011] In one or some embodiments of the present invention, in step (2), BCG purified protein derivative (PPD) is labeled with biotin-streptavidin magnetic beads, and BCG-specific antibodies are separated by magnetic force.

[0012] The specific method includes: labeling PPD with biotin, incubating with streptavidin magnetic beads, and then incubating with total IgG. According to the principle of specific binding between antigen and antibody, BCG-specific IgG will bind to PPD and be separated by a magnetic rack. BCG-specific IgG will be obtained after elution and concentration.

[0013] In the second aspect of the present invention, a BCG-specific protective antibody prepared by the separation method is provided.

[0014] In the third aspect of the present invention, there is provided the use of the BCG-specific protective antibody in the preparation of an anti-tuberculosis product.

[0015] Preferably, the anti-tuberculosis product may be an immune marker or an anti-tuberculosis biological product in the development of a vaccine to prevent tuberculosis.

[0016] In a fourth aspect of the present invention, a method for identifying a digalactosylsialic acid-modified BCG-specific IgG glycoform is provided, the method comprising the following steps: BCG-specific IgG was hydrolyzed into Fab and Fc segments using IdeZ enzyme, and the two fragments were separated by electrophoresis and stained with Coomassie Brilliant Blue. The Fc segment gel strip was removed and dehydrated with acetonitrile. The sugar chains were removed by glycosidase PNGase F, labeled with 2-aminobenzamide, and separated and analyzed by high performance liquid chromatography for sugar chain percentage composition.

[0017] In one or some embodiments of the present invention, the sugar chains containing digalactosyl mono / disialic acid in the HPLC chromatogram are determined according to Ludger 2AB labelled IgG N-Glycan library.

[0018] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects: The present invention uses a biotin-streptavidin affinity chromatography method to separate BCG-specific antibodies, and uses high performance liquid chromatography to analyze antibody-linked sugar chains, and finds that in the sugar chain mixture, the percentage of digalactose mono / disialyl sugar forms increases. This antibody helps to kill Mycobacterium tuberculosis, providing new ideas for the development of tuberculosis immune markers and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 : Sugar chains attached to the IgG Fc segment.

[0021] Figure 2 :M, marker; 1-2, BCG-specific IgG.

[0022] Figure 3 : A is the sugar chain analysis of total IgG; B is the sugar chain analysis of BCG-specific IgG.

[0023] Figure 4 : Macrophage bactericidal assay. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0026] The present invention separates BCG-specific IgG through biotin-streptavidin; it is identified that among the glycoforms connected to the antibody, the proportion of digalactose mono / disialyl glycoforms increases; the BCG-specific antibody enhances the bactericidal ability of macrophages, showing an anti-tuberculosis protective effect.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0028] Sources: Whole blood: whole blood from healthy people.

[0029] Embodiment 1: 1. Separation of serum IgG and BCG-specific IgG Serum was obtained by density gradient centrifugation of whole blood, and total IgG was separated by affinity chromatography. The specific chromatography method was as follows: an appropriate amount of filler-gel resin (Melon gel resin, Thermo) was added to the chromatography column, followed by a purification buffer (purchased from Dalian Haichen Trading Co., Ltd., batch number RE234257) with a volume 10 times that of the filler, and serum was added. When the serum flowed through the filler, all proteins in the serum except IgG were adsorbed to the filler, and IgG was collected.

[0030] Isolation of BCG-specific IgG: The BCG purified protein derivative (PPD) was labeled with PPD biotin and then incubated with streptavidin magnetic beads (Dynabeads M-270 streptavidin, invitrogen), and then incubated with total serum IgG. Based on the principle of antigen-antibody specific binding, BCG-specific IgG will bind to PPD and be separated by a magnetic rack. BCG-specific IgG was eluted with 100 mM citric acid and concentrated to obtain BCG-specific IgG.

[0031] SDS-PAGE and Western blot identification: After transfer, the membrane was first incubated with biotin-labeled PPD, then incubated with horseradish peroxidase-labeled streptavidin, and chemiluminescence was used to identify whether BCG-specific IgG was separated.

[0032] 2. Identification of glycoforms of total serum IgG and BCG-specific IgG The two IgGs were hydrolyzed into Fab and Fc segments using IdeZ enzyme (NEB), and the two fragments were separated by electrophoresis and stained with Coomassie Brilliant Blue. The Fc segment gel strip was removed and dehydrated with acetonitrile. The sugar chains were removed by glycosidase PNGaseF, labeled with 2-aminobenzamide (2-AB), and separated and analyzed by high-performance liquid chromatography (HPLC).

[0033] 3. In vitro bactericidal test, i.e. analysis of the protective effect of BCG-specific IgG THP-1 differentiated macrophages were infected with Mycobacterium tuberculosis, and then serum total IgG and BCG-specific IgG were added to count colony forming units (CFU) to evaluate the effects of BCG-specific antibodies and glycosylation modifications on the bactericidal effect of macrophages.

[0034] Test results: 1. Isolation of BCG-specific (PPD-specific) IgG BCG-specific IgG was separated by PPD-biotin-streptavidin and identified by SDS-PAGE and Western blot. Figure 2 .

[0035] 2. Identification of BCG-specific IgG glycoform percentage According to the Ludger 2AB labelled IgG N-Glycan library, the five peaks 8-13 are sugar chains containing digalactosyl mono / di-sialic acid. The area normalization method was used to analyze the percentage of sugar chains separated by HPLC: in the total IgG linked sugar chain complex, the percentage of digalactosyl sialic acid glycoforms was 22.54±2.04, and in the BCG-specific IgG linked sugar chain, the percentage of digalactosyl sialic acid was 34.68±3.87, which was higher than that of the total IgG (p<0.05). Figure 3 .

[0036] 3. Protective effect of BCG-specific IgG BCG-specific IgG can enhance the bactericidal ability of macrophages and show an anti-tuberculosis protective effect.

[0037] The colony counts in the experimental group with BCG-specific IgG were less than those in the control group, indicating that macrophages had enhanced bactericidal ability. Figure 4 .

[0038] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for separating BCG-specific protective antibodies, characterized in that: The method comprises the following steps: (1) Whole blood density gradient centrifugation was used to obtain serum, and total IgG was separated by affinity chromatography; (2) Separating BCG-specific IgG from the total IgG using a biotin-streptavidin method: labeling BCG purified protein derivative (PPD) using biotin-streptavidin magnetic beads, and separating BCG-specific antibodies using magnetic force.

2. The method for separating BCG-specific protective antibodies according to claim 1, characterized in that: In step (1), the affinity chromatography method is specifically as follows: add filler to the chromatography column, then add purification buffer, add serum, and when the serum flows through the filler, all proteins in the serum except IgG are adsorbed to the filler, and IgG is collected.

3. The method for separating BCG-specific protective antibodies according to claim 2, characterized in that: The filler is gel resin.

4. The method for isolating BCG-specific protective antibodies according to claim 1, characterized in that: In step (2), the specific method includes: labeling the BCG purified protein derivative (PPD) with biotin, incubating it with streptavidin magnetic beads, and then incubating it with the total IgG. According to the principle of specific binding between antigen and antibody, the BCG-specific IgG will bind to the PPD and be separated by a magnetic rack, and the BCG-specific IgG will be obtained after elution and concentration.

5. The BCG-specific protective antibody prepared by the separation method according to any one of claims 1 to 4.

6. Use of the BCG-specific protective antibody according to claim 5 in the preparation of anti-tuberculosis products.

7. The use according to claim 6, characterized in that: The anti-tuberculosis product is an immune marker or an anti-tuberculosis biological product developed for the vaccine to prevent tuberculosis.

8. A method for identifying the glycoform of BCG-specific IgG modified with digalactosyl sialic acid, characterized in that: The method comprises the following steps: The BCG-specific IgG described in claim 1 is hydrolyzed into Fab and Fc segments using IdeZ enzyme, the two fragments are separated by electrophoresis, stained with Coomassie Brilliant Blue, the Fc segment strip is removed, and after dehydration with acetonitrile, the sugar chain is removed by glycosidase PNGase F, labeled with 2-aminobenzamide, and the sugar chain percentage composition is separated and analyzed by high performance liquid chromatography.

9. The method for identifying the glycoform of BCG-specific IgG modified with digalactosyl sialic acid as claimed in claim 8, characterized in that: The percentage of glycans containing digalactosyl mono / disialic acid in the HPLC chromatogram was determined based on the Ludger 2AB labelled IgG N-Glycan library.

10. The method for identifying the glycoform of BCG-specific IgG modified with digalactosyl sialic acid according to claim 8, characterized in that: The percentage of sugar chains separated by HPLC was analyzed using the area normalization method.

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