CN100402551C - Antigenic epitope and application of β2-microglobulin - Google Patents

Abstract

The invention relates to the study of the epitope of β2-microglobulin by matrix-assisted laser desorption time-of-flight mass spectrometry and affinity separation. The amino acid sequence of the antigenic epitope is located at positions 61-67, and the sequence is: SFYLLYY. The antigenic epitope of the present invention can be used to develop new peptide vaccines, can also be used as a therapeutic target, and can be used to provide a scientific basis for the diagnosis and prognosis of tumor-related diseases.

Description

Antigenic epitope and application of β2-microglobulin

technical field

The invention belongs to the technical field of biomedical engineering, and relates to an antigenic epitope of β2-microglobulin and its application.

Background technique

β2-microglobulin is a single-chain polypeptide low-molecular protein composed of 99 amino acids. Its amino acid sequence is:

Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly

-Lys-Ser-Asn-Phe-Leu-Asn-Cys-Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu

-Val-Asp-Leu-Leu-Lys-Asn-Gly-Glu-Arg-Ile-Glu-Lys-Val-Glu-His-Ser-Asp-Leu

-Ser-Phe-Ser-Lys-Asp-Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro

-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-Cys-Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro

-Lys-Ile-Val-Lys-Trp-Asp-Arg-Asp-Met

There is a disulfide bond between Cys 25 and Cys 80, the average molecular weight is 11729.1694, and the monoisotopic molecular weight is 11721.7718.

β2-microglobulin was first isolated from the urine of patients with renal tubular lesions by Berggard in 1968. β2-microglobulin is a low-molecular-weight globulin secreted by nucleated cells. It can freely pass through glomerular filtration and reabsorb 99.0% in the proximal tubule. It is a single-chain polypeptide composed of 99 amino acid residues with a molecular weight of 11800, it widely exists in various body fluids such as human plasma, urine and cerebrospinal fluid, and on the surface of nucleated cell membranes. Under normal conditions, β2-microglobulin on the cell surface is repeatedly combined with free β2-microglobulin in body fluids to maintain dynamic balance.

β2-microglobulin has received considerable attention in recent years, because the serum level of β2-microglobulin in patients with various tumors increases. Many scholars have conducted in-depth research on its biological properties, pathophysiology and clinical significance. Clinically, there are various tumors such as lung cancer, kidney cancer, breast cancer, and malignant tumors of the digestive system. The β2-microglobulin in blood and urine has abnormal changes, which are generally higher than normal levels. Therefore, β2-microglobulin has been used in the early serological diagnosis of various malignant tumors.

Contents of the invention

One of the objects of the present invention is to provide an antigenic epitope of β2-microglobulin.

The second object of the present invention is to provide the application of an antigenic epitope of β2-microglobulin.

The invention provides an antigen epitope of β2-microglobulin, which is located at the 61-67 positions of the antigen and is a polypeptide with the amino acid sequence: SFYLLYY.

To achieve the present invention there is provided an antigenic epitope of β2-microglobulin. We use affinity mass spectrometry (MALDI-TOF/MS) to combine the immunoaffinity extraction method with modern soft ionization mass spectrometry to systematically study the specific reaction between β2-microglobulin and its antibody, and then determine its epitope site and amino acid sequence, and conduct detailed research on the conditions that affect the antigen-antibody reaction, explore the optimal experimental conditions on the immobilized carrier of active protein, and establish a new method of micro-quantity, rapid and sensitive immunology. The experimental results show that mass spectrometry, as a rapid and effective analysis method, is a powerful means to analyze antigenic determinants.

It is first hydrolyzed with protease to generate a series of peptides, which are then immunoaffinity reacted with monoclonal antibodies immobilized on agarose beads. After the reaction, the beads were collected and washed several times with the appropriate buffer. Peptides that are not bound to the antibody are washed away, while the peptides containing the antigenic determinant bind to the antibody to form a complex and remain with the beads. Finally, mass spectrometry is directly performed to obtain the mass spectrometry data of the peptide bound to the antibody. In the experiment, we used proteases with three different cleavage sites: Endoproteinase Lys-C, Glu-C and Trypsin, and used synthetic peptides to determine the binding site between the antigen and the antibody, that is, the epitope site is 61-67: SFYLLYY .

As the target structure recognized by immune cells and the material basis for stimulating specific immune responses, antigenic epitopes are of great significance for the related research of immune responses.

An antigen epitope peptide SFYLLYY of the β2-microglobulin of the present invention can stimulate the human body to produce antibodies against the β2-microglobulin. β2-microglobulin can specifically bind to its corresponding antibody, so the antibody produced by an epitope peptide SFYLLYY of β2-microglobulin of the present invention can be used as a probe to recognize and interact with β2-microglobulin Immunoprecipitation reaction. Therefore, an epitope peptide SFYLLYY of the β2-microglobulin of the present invention and the corresponding antibody can be used for early serological diagnosis, treatment and prognosis of diseases.

The small molecule polypeptide of the present invention can be prepared by chemical synthesis or genetic engineering recombinant expression.

Experiments show that: SFYLLYY, an epitope peptide of β2-microglobulin of the present invention, can compete with β2-microglobulin and participate in the specific binding of multiple antibodies; it can effectively inhibit the function of antibodies, and after coupling with a suitable carrier Has similar immunogenicity to β2-microglobulin. This provides a scientific basis for the preparation of new vaccines and drugs.

Combined with some biotechnology, PCR, ELISA technology, protein chip, an epitope peptide SFYLLYY of the β2-microglobulin of the present invention can be used to develop new diagnostic kits and target proteins as diagnostic chips, both of which have clinical Application prospect.

An antigenic epitope of β2-microglobulin of the present invention has laid the foundation for various tumors, such as early serological diagnosis and treatment of lung cancer, kidney cancer, breast cancer, and digestive system malignancies; and opened up new technical approaches and new The application provides a scientific basis for the preparation of new vaccines and drugs.

Detailed ways

Example 1:

This embodiment is the determination of antigenic epitopes of β2-microglobulin:

(1) Preparation of reagents: 10 mg CHCA was weighed and dissolved in 50% ACN+0.1% TFA to prepare a 10 mg/ml matrix solution. 5 μg Endoproteinase Lys-C was dissolved in 20 μL Millpore ultrapure water to prepare a solution with a concentration of 0.25 μg/μL. 50 μg Endoproteinase Glu-C was dissolved in 200 μL Millpore ultrapure water to prepare a solution with a concentration of 0.25 μg/μL. 100 μg of Trypsin was dissolved in 200 μL Millpore ultrapure water to prepare a solution with a concentration of 0.5 μg/μL.

(2) Enzymolysis reaction of β2-microglobulin: add 60μL Endoproteinase Glu-C (enzymolysis buffer solution 50mM ammonium acetate solution, pH=4.5) into a 1.5mL centrifuge tube The ratio was added to the enzymolysis buffer solution, reacted at 37°C for 2h, and then placed at -20°C to terminate the enzymolysis reaction. The enzymatic hydrolyzate was desalted according to the instruction of ZiptipC18. Take 1 μL of the desalted enzymatic hydrolysis solution and 1 μL of CHCA matrix solution to spot the target, and then do MALDI-TOF-MS analysis after natural drying at room temperature in the air. Trypsin (50mM Tris-HCl 1mM CaCl2pH8.0) and Endoproteinase Lys-C (enzymolysis buffer solution: 50mM Tris-HCl, 0.5mM EDTA, pH=8.0) were performed according to the operation steps of Endoproteinase Glu-C enzymatic hydrolysis of β2-microglobulin .

(3) Immunoaffinity: add 100μL TSO (75mM Tris-HCl, 200mM NaCl, 0.5% N-octyl glucoside, pH=8.0) buffer solution and 10μg monoclonal antibody (Clone: GJ14, mouse IgGl) was reacted at 4°C for 4 hours. Take 4 μL of Protein G/Protein AAgarose in the solution, mix well and react at 4°C for 2 hours. After the reaction, centrifuge at 14000r/min, remove the supernatant and collect the agarose beads. The agarose beads were washed 3 times with 200 μL TSO buffer solution each time, followed by 200 μL TSMK buffer solution (10 mM Tris-HCl, 200 mM NaCl, 5 mM β-mercaptoethanol, pH=8.0). Take 4 μL of matrix solution and mix well with the washed agarose beads, and finally take 1.5 μL of the mixed solution to spot the target, and let it dry naturally in the air at room temperature, and do MALDI-TOF-MS analysis after drying.

The continuous epitope range of β2-microglobulin obtained by Endoproteinase Glu-C is a peptide segment (51-69), and the amino acid sequence is: HSDLSFSKDWSFYLLYYTE; the continuous epitope range of β2-microglobulin obtained by Endoproteinase Lys-C is a peptide Segment (59-75), the amino acid sequence is: DWSFYLLYYTEFTPTEK; the continuous epitope range of β2-microglobulin obtained by Trypsin is peptide segment (59-75), and the amino acid sequence is: DWSFYLLYYTEFTPTEK. It is known that the epitope range of β2-microglobulin is within the peptide segment (59-69), namely: DWSFYLLYYTE.

(4) Affinity mass spectrometry analysis of synthetic peptides

01--SFYLLYYTE, 02---DWSFYLLYY, 03---SFYLLY, 04---FYLLYY; these four peptides were co-immunoprecipitated with monoclonal antibodies, and the results of mass spectrometry showed that 01 and 02 samples could be compared with Antibody binding, while 03 and 04 did not co-immunoprecipitate with the antibody. 01 and 02 samples can be combined with the antibody, then the peptide of the same sequence of the two - SFYLLYY can be combined with the antibody, so the conclusion obtained from the affinity mass spectrometry analysis of the 01 and 02 samples is: SFYLLYY contains β2-microglobulin antigen determinant cluster. The peptide sequence synthesized in 03 is SFYLLYY with one amino acid residue Y missing at the C-terminus, namely: SFYLLY. The peptide sequence synthesized by 04 is that SFYLLYY lacks an amino acid residue S at its N-terminus. The affinity mass spectrometry analysis of 03 and 04 shows that: 03 and 04 do not undergo co-immunoprecipitation with antibodies, and do not interact with antibodies. Combine.

Therefore, the result obtained through the affinity spectrum analysis of samples 01, 02, 03 and 04 is: the amino acid sequence of the β2-microglobulin epitope is: SFYLLYY

Example 2:

Utilize antigenic epitope peptide of the present invention to screen out positive serum from patient's serum, prepare corresponding antibody, and adopt enzyme-linked immunosorbent assay (ELISA) to detect the corresponding antibody of antigenic epitope SFYLLYY in patient's serum and healthy person The titer levels between the serums of the 50 samples confirmed that the serum titer levels of the patients were significantly higher than those of the healthy ones, and the statistical difference was p<0.01.

The β2-microglobulin epitope peptide was implanted into the mice to immunize the mice. After immunization twice, ELISA and Westen Blot were used to detect the production of specific antibodies. The experiment proves that the antibody serum produced can specifically combine with β2-microglobulin. The experimental results show that SFYLYY, an antigenic epitope peptide of β2-microglobulin, has similar immunogenicity to β2-microglobulin after being coupled with a suitable carrier, and further can be used to prepare new vaccines and medicines.

sequence listing

<110>Changchun Institute of Applied Chemistry, Chinese Academy of Sciences

<120> Antigen epitope and application of β2-microglobulin

<160>1

<210>1

<211>7

<212>PRT

<400>1

Ser Phe Tyr Leu Leu Tyr Tyr

1 5

Claims (3)

1. the epitope of B2M, it is characterized in that having the aminoacid sequence shown in the following formula: be positioned at antigenic 61-67 site, aminoacid sequence is: SFYLLYY.

2. the application of the epitope of B2M as claimed in claim 1 is characterized in that being used to prepare anti-lung cancer, kidney, mammary cancer, alimentary system malignant tumour vaccine or medicine.

3. the application of the epitope of the described B2M of claim 1 is characterized in that being used to develop medicine relevant with tumour and diagnostic kit as target protein.