CN113121669B

CN113121669B - Antigen mimic epitope of human adiponectin and preparation method thereof

Info

Publication number: CN113121669B
Application number: CN202110390702.5A
Authority: CN
Inventors: 万蒙; 何庆华
Original assignee: Jiangxi Lecheng Biological Medical Co ltd
Current assignee: Jiangxi Lecheng Biological Medical Co ltd
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2023-01-24
Anticipated expiration: 2041-04-12
Also published as: CN113121669A

Abstract

The invention provides an antigen mimic epitope of human Adiponectin (Adiponectin), and relates to the technical field of biology. The invention adopts an optimized liquid phase affinity panning mode to rapidly pan and obtain a polypeptide molecule (antigen mimic epitope) which can be specifically combined with the anti-human adiponectin monoclonal antibody, the antigen mimic epitope has immunoreactivity with the anti-human adiponectin monoclonal antibody, can be used as a substitute of the human adiponectin and recombinant adiponectin protein prepared by a genetic engineering technology to be applied to an immunoreaction analysis system, has the advantages of simple preparation, rapidness, high efficiency and the like, provides a new path for replacing the human adiponectin antigen, and has better application value.

Description

Antigen mimic epitope of human adiponectin and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to an antigen mimic epitope of human Adiponectin (Adiponectin) and a preparation method thereof.

Background

Adiponectin (Adiponectin) is a biologically active protein secreted by adipocytes and highly expressed in adipocytes. Human adiponectin is also known as adipose tissue most abundant gene transcript-1 and gelatin binding protein 28. It has been found that the adiponectin level in humans is predictive of type II diabetes and coronary heart disease.

The prior art has not found reports about the antigenic mimotope of human adiponectin.

Disclosure of Invention

The invention takes anti-human adiponectin monoclonal antibody as a target molecule, combines the target molecule to an enzyme label plate carrier, puts phage into randomly displaying dodecapeptide library, and obtains a polypeptide molecule (antigen simulation epitope) capable of specifically combining the anti-human adiponectin monoclonal antibody through optimized affinity panning conditions, wherein the amino acid sequence of the polypeptide molecule is as follows: E-P-N-S-G-G-Y-H-D-L-M-N.

In the molecular structure of the polypeptide, capital English letters respectively represent known natural L-type amino acid residues or one of D-type isomers thereof, namely E represents glutamic acid residues, P represents proline residues, N represents asparagine residues, S represents serine residues, G represents glycine residues, Y represents tyrosine residues, H represents histidine residues, D represents aspartic acid residues, L represents leucine residues, and M represents methionine (methionine) residues.

The invention also relates to the nucleotide for coding the amino acid sequence (E-P-N-S-G-G-Y-H-D-L-M-N) of the polypeptide molecule, and the sequence is GAG CCG AAT TCT GGT GGT TAT CAT GAT TTG ATG AAT.

The polypeptide molecule can be prepared in a large scale by means of phage amplification and chemical synthesis. Phage amplification refers to the mass propagation of phage displaying polypeptide molecules in a biological amplification manner to produce phage particles displaying polypeptide molecules. Chemical synthesis refers to polypeptide synthesis by means of chemical synthesis of polypeptides according to published polypeptide amino acid sequences.

The beneficial effects of the invention are: the polypeptide molecule (antigen mimic epitope) can be used as a substitute of natural human adiponectin and recombinant human adiponectin protein prepared by a genetic engineering technology and applied to an immune reaction analysis system. The complex extraction and purification process faced by the traditional human adiponectin is avoided; the complex preparation processes of gene sequence optimization, expression vector construction, prokaryotic/eukaryotic expression condition optimization and the like in the traditional preparation process of the recombinant adiponectin protein are avoided, and the method has the advantages of simple preparation, rapidness, high efficiency and the like.

In addition, the invention rapidly screens polypeptide molecules (antigen mimic epitope) which can be specifically combined with the anti-human adiponectin monoclonal antibody from a phage display polypeptide library by a phage display polypeptide library technology and an affinity panning mode. The antigen mimic epitope has immunoreactivity with an anti-human adiponectin monoclonal antibody, and can be used as a substitute of human adiponectin and recombinant adiponectin protein prepared by a genetic engineering technology to be applied to an immunoreaction analysis system.

Drawings

FIG. 1 is a graph showing the binding characteristics of ELISA for identifying human adiponectin antigenic mimotope.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings. Several embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 affinity panning and identification of human adiponectin antigenic mimotopes

1) The specific method for affinity panning and anti-human adiponectin monoclonal antibody specific binding polypeptide molecules comprises the following steps: 50 μ g of anti-human adiponectin monoclonal antibody was coated on a well of an enzyme-labeled plate (200 μ L/well), and incubated at 37 ℃ for 4 hours. The microplate was removed, washed 15 times with PBST (1.0% Tween-20 (v/v) in 1.0 mM PBS pH 7.4), incubated at 37 ℃ for 2 hours with 300. Mu.l of blocking solution (3% skim milk), and washed 10 times with PBST.

2) After incubating the plate for 2 hours at 37 ℃ in 300. Mu.l of blocking solution (3% skim milk), it was washed 15 times with PBST (1.0% Tween-20 (v/v) in 1mM PBS pH 7.4), and 100. Mu.l of phage peptide library (phage display random dodecapeptide library from NEB, which was obtained by diluting phage stock solution 10-fold with PBS, about 2.0X 10 ¹¹ pfu), incubated at 37 ℃ for 3 minutes with shaking.

3) Gently sucking out the supernatant of the phage peptide library incubated in the plate hole in the step 2), and then putting the supernatant into the plate hole coated with the anti-human adiponectin monoclonal antibody in the step 1), and carrying out oscillation reaction for 6 min at 37 ℃. After 15 PBST washes to remove unbound phage, 100. Mu.L of eluent (Gly-HCl, pH 2.2) was added and shaken gently at 37 ℃ for 10min to elute phage bound to monoclonal antibody. The eluate was aspirated, and 15. Mu.L of 1M Tris-HCl (pH 9.1) was added to neutralize the buffer. 10 μ l of the neutralization solution was taken for phage titer determination and the rest was used to infect 20mL of E.coli ER2738 strain grown to early log. The next day, phages were purified by PEG/NaCl precipitation and the titer of the amplified phages was determined.

4) Then, the panning of 2 nd round and 3 rd round was performed in sequence, the panning step was substantially the same as that of the first round, and the amount of phage added was 2X 10 each time ¹¹ pfu, except for: incubation times of the phage display polypeptides screened in the 2 nd and 3 rd rounds and the laths coated with the anti-human adiponectin monoclonal antibodies are respectively 30min and 15min; the coating concentration of the antihuman adiponectin monoclonal antibody is 30 and 15 mu g respectively, the eluent of the 2 nd round is serum solution containing enriched human adiponectin, the eluent of the 3 rd round is (Gly-HCL, pH2.2), and the elution time of the competitive eluent is 5min and 3min respectively.

5) Identification of positive phage clones: randomly picking 100 phage plaques from a plate for determining the phage titer after the 3 rd round of panning, amplifying the phage, and identifying positive phage clones by adopting an enzyme-linked immunosorbent assay method, wherein the specific method comprises the following steps: first, an anti-human adiponectin monoclonal antibody was diluted with 10mM PBS (pH 7.4), coated with 1. Mu.g/mL of a 96-well plate, and incubated overnight at 4 ℃. The following day, washing with PBST (10mM PBS,0.05% Tween-20 (v/v)) 3 times, blocking with PBS containing 3% skimmed milk powder, and incubating at 37 ℃ for 1 hour; mu.l of phage plaque amplification solution (1.0X 10) was put in ¹² pfu) with the original phage peptide library as negative control, incubated for 1 hour at 37 ℃; adding 1: 100 μ l of a 5000-fold dilution of HRP-labeled anti-M13 phage secondary antibody, incubated at 37 ℃ for 1 hour; adding 100 μ l TMB substrate solution, developing for 5min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument. Selection of OD ₄₅₀ Phage clones 2 times larger than the negative control were positive clones.

6) And (3) polypeptide molecule identification of the specific binding anti-human adiponectin monoclonal antibody: the method for identifying the binding capacity and specificity of the polypeptide molecules and the anti-human adiponectin monoclonal antibodies by adopting an ELISA method comprises the following steps: respectively coating an anti-human adiponectin monoclonal antibody, bovine serum albumin (100 mu L/hole and 1 mu g/ml), normal human serum (100 mu L/hole) and mouse anti-human IgG (100 mu L/hole and 1 mu g/ml) on an enzyme label plate by using 10mM PBS (pH 7.4), and incubating overnight at 4 ℃; the following day, washing with PBST (10mM PBS,0.05% Tween-20 (v/v)) 3 times, blocking with PBS containing 3% skimmed milk powder, and incubating at 37 ℃ for 1 hour; after washing the plate, 100. Mu.l of the solution was addedPhage clones identified as positive by ELISA (1.0X 10) ¹¹ pfu), incubated at 37 ℃ for 1 hour; adding 1: 100. Mu.l of HRP-labeled secondary anti-M13 phage antibody was diluted at 5000 ℃ and incubated at 37 ℃ for 1 hour; adding 100 μ l TMB substrate solution, developing in dark for 5min, stopping reaction, and reading OD ₄₅₀ . The experimental results show that: after adding positive clone phage, coated with anti-human adiponectin monoclonal antibody, bovine serum albumin (100. Mu.L/well, 1. Mu.g/ml), healthy human serum (100. Mu.L/well), mouse anti-human IgG (100. Mu.L/well, 1. Mu.g/ml) ELISA plate hole OD ₄₅₀ The values were 2.0, 0.08, 0.1, 0.06, 0.05, respectively, indicating that the obtained polypeptide molecules had good binding properties to the anti-human adiponectin monoclonal antibody (fig. 1).

7) Double antibody sandwich determination of human adiponectin antigenic mimotope: the specific method comprises the following steps: coating an anti-human adiponectin monoclonal antibody (5 mu g/ml, capture antibody 1) on an enzyme-labeled plate hole, and incubating overnight at 4 ℃; the following day, washing 3 times with PBST (10mM PBS,0.05% Tween-20 (v/v)), blocking with PBS containing 3% skimmed milk powder, and incubating at 37 ℃ for 1 hour; washing the plate, adding different dilutions of human adiponectin antigen mimic epitope (50-1000 ng/mL), and incubating at 37 ℃ for 30min; washing the plate, adding HRP enzyme-labeled anti-human adiponectin monoclonal antibody (5. Mu.g/ml, detection antibody 2), incubating at 37 ℃ for 30min, washing the plate, adding 100. Mu.l TMB substrate solution, developing in dark for 5min, stopping reaction, and reading OD ₄₅₀ . The results showed that the OD values increased with increasing concentrations of the human adiponectin mimotope administered, showing good correspondence (table 1).

TABLE 1 double antibody sandwich ELISA assay for different concentrations of human adiponectin antigenic mimotope

Example 2 sequencing of genes encoding polypeptide molecules and determination of amino acid sequences thereof

Amplifying the phage which is identified and displayed with the antigen mimic epitope by ELISA, and extracting a DNA sequencing template of the phage. The brief procedure is as follows: phage amplification was performed and after the first centrifugation step 800. Mu.l were addedl phage-containing supernatants were transferred to a new centrifuge tube. Add 200 u l PEG/NaCl precipitation phage. After centrifugation, the pellet was resuspended in 100. Mu.l of iodide buffer (10 mM Tris-HCl (pH 8.0), 1mM EDTA,4M NaI), 250. Mu.l of absolute ethanol was added to precipitate the DNA, and after centrifugation, the pellet was washed with 70% ethanol (DNA sequencing template). The precipitate was finally resuspended in 20. Mu.l of sterile water, 2. Mu.l were taken for agarose gel electrophoresis analysis; taking 5 mu L of phage template for DNA sequencing, wherein-96 gIII sequencing primers are as follows: 5' - ^HO CCC TCA TAG TTA GCG TAA CG-3'. The amino acid sequence of the polypeptide molecule can be obtained according to the DNA sequencing result and the codon table: E-P-N-S-G-G-Y-H-D-L-M-N.

EXAMPLE 3 Mass preparation of human adiponectin antigenic mimotopes

1) By means of phage amplification

Phage particles displaying human adiponectin antigenic mimotope were added to 20ml of E.coli ER2738 inoculated culture and cultured with shaking at 37 degrees and 220rpm for 4.5h. Transferring the culture into another centrifuge tube, centrifuging at 4 deg.C 10000rpm for 10min, transferring the upper 80% of the supernatant into a fresh tube, adding 1/6 volume of PEG/NaCl, and standing at 4 deg.C for 120min. The PEG/NaCL solution was centrifuged at 10000rpm at 4 ℃ for 15min. The supernatant was discarded, centrifuged briefly and the residual supernatant was aspirated. Adding 1mL TBS for resuspension to obtain phage amplification solution.

2) By chemical synthesis

According to the analyzed polypeptide amino acid sequence of the antigen mimic epitope, the polypeptide is chemically synthesized in a large amount based on a polypeptide solid phase synthesis method.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. An epitope peptide of human adiponectin having an amino acid sequence of: E-P-N-S-G-G-Y-H-D-L-M-N.

2. A nucleotide encoding the amino acid sequence of claim 1.

3. The nucleotide sequence according to claim 2 corresponding to:

GAG CCG AAT TCT GGT GGT TAT CAT GAT TTG ATG AAT。

4. the method for producing an epitope peptide of human adiponectin according to claim 1, wherein the preparation is carried out in a large amount by means of phage amplification or chemical synthesis; the phage amplification refers to the mass propagation production of phage particles displaying the adiponectin antigen mimic epitope peptide in a biological amplification mode by using phage displaying the antigen mimic epitope peptide; the chemical synthesis refers to polypeptide synthesis by means of chemical synthesis of polypeptides according to the amino acid sequence of the antigen mimic epitope peptide.