CN113234126A - Polypeptide and WNT2 polyclonal antibody - Google Patents

Polypeptide and WNT2 polyclonal antibody Download PDF

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Publication number
CN113234126A
CN113234126A CN202110678222.9A CN202110678222A CN113234126A CN 113234126 A CN113234126 A CN 113234126A CN 202110678222 A CN202110678222 A CN 202110678222A CN 113234126 A CN113234126 A CN 113234126A
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wnt2
polypeptide
polyclonal antibody
protein
preparation
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邹畅
张彬
董少伟
黄土雄
王继刚
刘东成
周文斌
宋惠彬
张荟蓉
赵盼
许静
成志强
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

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Abstract

The invention discloses a polypeptide and a WNT2 polyclonal antibody, relates to the technical field of biology, and particularly provides a polypeptide, wherein an amino acid sequence of the polypeptide is shown as SEQ ID No.1 or SEQ ID No.2, the polypeptide sequence is used as an antigen peptide to immunize animals, so that the Wnt2 polyclonal antibody can be obtained, and a new way is provided for preparation and research of the Wnt antibody.

Description

Polypeptide and WNT2 polyclonal antibody
Technical Field
The invention relates to the technical field of biology, and particularly relates to a polypeptide and a WNT2 polyclonal antibody.
Background
Wnt is a type of secreted glycoprotein that acts through autocrine or paracrine. The Wnt signaling pathway can cause accumulation of intracellular β -catenin (β -catenin), which is a multifunctional protein (named Armadillo protein Armadillo in drosophila) that interacts with cadherin at the cell junction to participate in formation of adhesive bands, while free β -catenin can enter the nucleus of the cell and regulate gene expression. Wnt signaling plays an important role in animal development, and its aberrant expression or activation can cause tumors. The Wnt signaling pathway can be summarized as: wnt → Frz → Dsh → disintegration of the degradation complex of β -catenin → accumulation of β -catenin, entering the nucleus → TCF/LEF → gene transcription (e.g. c-myc, cyclinD 1).
Wnt2 is an important member of the Wnts protein family, and how to detect it and develop Wnt antibodies are one of the problems to be solved.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a polypeptide and a WNT2 polyclonal antibody.
The invention is realized by the following steps:
in a first aspect, the embodiments of the present invention provide a polypeptide, the amino acid sequence of which is shown in SEQ ID No.1 or SEQ ID No. 2.
In a second aspect, the embodiments provide a fusion protein comprising the polypeptide as described in the previous embodiments and a carrier protein.
In a third aspect, the embodiments provide an isolated nucleic acid encoding a polypeptide as described in the previous embodiments.
In a fourth aspect, embodiments of the present invention provide a recombinant vector comprising an isolated nucleic acid as described in the previous embodiments.
In a fifth aspect, embodiments of the present invention provide a host cell comprising a recombinant vector as described in the previous embodiments.
In a sixth aspect, the present invention provides a method for preparing Wnt2 polyclonal antibody, which includes: animals were immunized with the polypeptides as described in the previous examples.
In a seventh aspect, the present invention provides a Wnt2 polyclonal antibody, which is prepared by the preparation method of Wnt2 polyclonal antibody as described in the previous embodiments.
In an eighth aspect, the embodiments of the present invention provide an application of the Wnt2 polyclonal antibody described in the previous embodiments in the preparation of a kit for detecting Wnt2 protein.
In a ninth aspect, the embodiments of the present invention provide a kit for detecting Wnt2 protein, which includes Wnt2 polyclonal antibody as described in the previous embodiments.
The invention has the following beneficial effects:
the invention adopts a new polypeptide sequence, takes the polypeptide sequence as an antigen peptide to immunize animals, stimulates the immune system of animal organisms to generate antibodies, can obtain the polyclonal antibody of Wnt2 by obtaining the hyperimmune serum of the animals, and provides a new way for the preparation and research of the Wnt antibodies.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the results of the test of binding of Wnt2 protein to Wnt2 polyclonal antibody in test example 1;
FIG. 2 shows the result of the sensitivity test of Wnt2 polyclonal antibody in test example 2;
FIG. 3 shows the results of clinical samples tested by the commercial Wnt2 ELISA kit of Experimental example 3;
FIG. 4 shows the results of the detection of clinical specimens by the antibody provided in example 1 of Experimental example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID No.1 or SEQ ID No. 2.
Specifically, the polypeptide can be selected from any one of a sequence 1 and a sequence 2, wherein the amino acid sequence of the sequence 1 is as follows: KRFLKQECKCHG (SEQ ID No. 1);
the amino acid sequence of the sequence 2 is as follows: GRKAVKRFLKQECKC (SEQ ID No. 2).
The sequence 1 and the sequence 2 are peptide fragments capable of mutually combining with Wnt2 protein and can be obtained by artificial synthesis.
The peptide fragments capable of being combined with the Wnt2 protein are many, but not all peptide fragments can be used as antigen peptides to prepare the Wnt2 polyclonal antibody, and the inventor finds that the sequence 1 or the sequence 2 can be used as the antigen peptides to immunize animals to obtain the Wnt2 polyclonal antibody through a series of creative works, thereby providing a new way for the preparation and research of the Wnt antibody.
Preferably, the sequence of the polypeptide is shown as SEQ ID No. 1. Compared with other polypeptides capable of being combined by Wnt2, the sequence provided by the embodiment of the invention has stronger immunogenicity as an antigen peptide, and can be used for preparing Wnt2 polyclonal antibodies more effectively.
The embodiment of the invention also provides a fusion protein which contains the polypeptide as described in the previous embodiment and a carrier protein.
The fusion protein is obtained by coupling the polypeptide provided by the invention with a carrier protein, and the coupling mode of the antigen peptide and the carrier protein can be obtained by the prior art, and is not described again.
Alternatively, the carrier protein may be selected from any of the known carrier proteins, including but not limited to keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, and the like.
The immunogenicity of the antigenic peptide can be improved by coupling to a carrier protein.
The invention also provides an isolated nucleic acid encoding a polypeptide as described in any of the preceding embodiments.
The embodiments also provide a recombinant vector comprising an isolated nucleic acid as described in any of the preceding embodiments.
The vector in the embodiment of the present invention may be selected from any existing vectors, such as expression vectors and cloning vectors. The vectors may contain various elements that facilitate replication and/or expression of the polypeptides provided herein, such as replication elements, promoters, enhancers, terminators, Internal Ribosome Entry Sites (IRES), and the like. These elements are well known to those skilled in the art.
The present invention also provides a host cell comprising a recombinant vector as described in the preceding examples.
Alternatively, the host cell may be selected from the group consisting of prokaryotic organisms such as E.coli, eukaryotic organisms such as yeast-like unicellular eukaryotic organisms, insect, animal, etc. multicellular eukaryotic cells, and the like.
The recombinant vector can be replicated in a host cell and expression of a target protein can be performed, and the target protein can be obtained by introducing the recombinant vector such as an expression plasmid into an appropriate host cell to obtain a transformant and culturing the transformant in an appropriate medium.
Examples of methods for introducing a target gene into a host cell include a calcium phosphate method, a DEAE-dextran method, and an electric pulse method.
After the target protein is obtained by culturing, the obtained target protein or peptide fragment can be separated and purified by a biochemical method.
The embodiment of the invention also provides a preparation method of the Wnt2 polyclonal antibody, which comprises the following steps: immunizing an animal with a polypeptide as described in any of the preceding examples.
The "animal" herein may be selected from any existing immunized animal for polyclonal antibody production.
Preferably, the animal is selected from: any one of mouse, rat, guinea pig, goat, rabbit, horse and mule.
The invention provides a Wnt2 polyclonal antibody, which is prepared by the preparation method of the Wnt2 polyclonal antibody according to any embodiment.
The invention also provides application of the Wnt2 polyclonal antibody in preparation of a kit for detecting the Wnt2 protein.
In addition, the embodiment of the invention also provides a kit for detecting the Wnt2 protein, which comprises the Wnt2 polyclonal antibody as described in the previous embodiment.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present invention provides a method for preparing Wnt2 polyclonal antibody, which comprises the following steps:
(1) obtaining antigenic peptides
Artificially synthesizing polypeptide with amino acid sequence shown as SEQ ID No. 1.
(2) Preparation of antibodies
Immunizing rabbits with the polypeptide provided in step (1) according to the steps shown in Table 1, stimulating the immune system of the immune animal body to produce antibodies, and finally obtaining Wnt2 polyclonal antibody by obtaining animal immune serum.
TABLE 1 immunization procedure
Figure BDA0003121689200000071
Figure BDA0003121689200000081
Example 2
The present invention provides a method for preparing Wnt2 polyclonal antibody, which comprises the following steps:
(1) obtaining antigenic peptides
Artificially synthesizing to obtain the polypeptide with the amino acid sequence shown as SEQ ID No. 2.
(2) Preparation of antibodies
And (2) immunizing rabbits by using the polypeptide provided in the step (1), and obtaining immune serum of animals to obtain Wnt2 polyclonal antibody.
Comparative example 1
A preparation method of Wnt2 polyclonal antibody, which comprises the following steps:
(1) obtaining antigenic peptides
Artificially synthesizing the polypeptide with the amino acid sequence shown as SEQ ID No.3(5 '-KAVKRFLKQECKCHG-3').
(2) Preparation of antibodies
Immunizing a rabbit with the polypeptide provided in step (1); obtaining animal immune serum to obtain Wnt2 polyclonal antibody.
Comparative example 2
A preparation method of Wnt2 polyclonal antibody, which comprises the following steps:
(1) obtaining antigenic peptides
Artificially synthesizing the polypeptide with the amino acid sequence shown as SEQ ID No.4(5 '-KAVKRFLKQECKC-3').
(2) Preparation of antibodies
And (2) immunizing rabbits by using the polypeptide provided in the step (1), and obtaining immune serum of animals to obtain Wnt2 polyclonal antibody.
Comparative example 3
A preparation method of Wnt2 polyclonal antibody, which comprises the following steps:
(1) obtaining antigenic peptides
Artificially synthesizing the polypeptide with the amino acid sequence shown as SEQ ID No.5(5 '-AVKRFLKQECKC-3').
(2) Preparation of antibodies
And (2) immunizing rabbits by using the polypeptide provided in the step (1), and obtaining immune serum of animals to obtain Wnt2 polyclonal antibody.
It should be noted that the polypeptide sequences provided in references 1 to 3 are all peptide fragments capable of binding to Wnt2 protein.
Test example 1
The polyclonal antibody prepared in example 1-2 was prepared, and Western blot (Western blot) was performed to confirm whether the antibody can bind to Wnt2 protein.
Results referring to fig. 1, in fig. 1, C228KFF300 is the result of example 1, and C1897FF300 is the result of example 2.
Test example 2
The sensitivity of the antibody provided by the invention is verified.
The antibody was prepared based on the method for preparing Wnt2 polyclonal antibody provided in example 1, and the sensitivity of the prepared antibody in ELISA detection was determined using a standard. The results are shown in FIG. 2.
As can be seen from FIG. 2, the sensitivity of Wnt2 polyclonal antibody prepared from the polypeptide provided by the present invention can reach 0.001.
Test example 3
The detection effectiveness of the antibody provided by the invention is verified.
The antibody is prepared based on the preparation method of the Wnt2 polyclonal antibody provided in example 1, the prepared antibody is used for detecting a serum sample of a clinical patient, meanwhile, a group of control groups is arranged, the control groups adopt Wnt2 ELISA detection kits on the existing market to detect the same sample, the detection result of the control groups is shown in figure 3, and the detection result of example 1 is shown in figure 4.
As can be seen from the results of the tests, the test results of the antibody provided in example 1 of the present invention were consistent with those of the products on the market.
In conclusion, the invention adopts a new polypeptide sequence, and the polypeptide sequence is used as an antigen peptide to immunize animals, so that the polyclonal antibody of Wnt2 can be obtained, and a new way is provided for the preparation and research of the Wnt antibody.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> wrinkle free
Zhang Bin
Dong Shaowei
<120> a polypeptide and WNT2 polyclonal antibody
<160> 5
<170> SIPOSequenceListing 1.0
<210> 3
<211> 12
<212> PRT
<213> Artificial sequence
<400> 3
Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys His Gly
1 5 10
<210> 2
<211> 15
<212> PRT
<213> Artificial sequence
<400> 2
Gly Arg Lys Ala Val Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys
1 5 10 15
<210> 3
<211> 15
<212> PRT
<213> Artificial sequence
<400> 3
Lys Ala Val Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys His Gly
1 5 10 15
<210> 4
<211> 13
<212> PRT
<213> Artificial sequence
<400> 4
Lys Ala Val Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys
1 5 10
<210> 5
<211> 12
<212> PRT
<213> Artificial sequence
<400> 5
Ala Val Lys Arg Phe Leu Lys Gln Glu Cys Lys Cys
1 5 10

Claims (10)

1. A polypeptide, characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No.1 or SEQ ID No. 2.
2. A fusion protein comprising the polypeptide of claim 1 and a carrier protein.
3. An isolated nucleic acid encoding the polypeptide of claim 1.
4. A recombinant vector comprising the isolated nucleic acid of claim 3.
5. A host cell comprising the recombinant vector of claim 4.
6. A preparation method of Wnt2 polyclonal antibody, which is characterized by comprising the following steps: immunizing an animal with the polypeptide of claim 1.
7. The method for producing a Wnt2 polyclonal antibody according to claim 6, wherein the animal is selected from the group consisting of: any one of mouse, rat, guinea pig, goat, rabbit, horse and mule.
8. The Wnt2 polyclonal antibody, which is prepared by the method for preparing the Wnt2 polyclonal antibody according to claim 6 or 7.
9. Use of the Wnt2 polyclonal antibody of claim 8 in the preparation of a kit for detecting Wnt2 protein.
10. A kit for detecting Wnt2 protein, comprising the Wnt2 polyclonal antibody of claim 8.
CN202110678222.9A 2021-06-18 2021-06-18 Polypeptide and WNT2 polyclonal antibody Withdrawn CN113234126A (en)

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