CN113278057A - Preparation and application of retinoic acid-induced protein 16 specific polyclonal antibody - Google Patents

Preparation and application of retinoic acid-induced protein 16 specific polyclonal antibody Download PDF

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CN113278057A
CN113278057A CN202110444267.XA CN202110444267A CN113278057A CN 113278057 A CN113278057 A CN 113278057A CN 202110444267 A CN202110444267 A CN 202110444267A CN 113278057 A CN113278057 A CN 113278057A
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antibody
protein
epitope
preparing
rai16
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CN113278057B (en
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王文
丁翠玲
苗根
钱春霖
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Second Military Medical University SMMU
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Abstract

The invention provides a preparation method and application of a retinoic acid induced protein 16(RAI16) specific polyclonal antibody.

Description

Preparation and application of retinoic acid-induced protein 16 specific polyclonal antibody
Technical Field
The invention relates to the technical field of biomedicine, in particular to retinoic acid induced protein 16 immunogen protein expression and a preparation method and application of an antibody thereof.
Background
The NCBI accession number of Retinoic acid-induced protein 16(RAI16) is: NP-919326.1, Swissprot accession No. Q80YR2, IPI No. IPI 00654780.2. RAI16 is a protein molecule induced by retinoic acid, and the structure and function of the protein molecule are not clear at present. RAI16 was originally isolated when tretinoin induced cell differentiation, which has the effects of inhibiting cell proliferation, promoting apoptosis and promoting cell differentiation, and has been used for the treatment of various cancers such as acute lymphocytic leukemia, lung cancer and liver cancer. Therefore, the retinoic acid-induced protein RAI16 is likely to participate in a series of physiological processes, and plays an important role in cell proliferation, differentiation, apoptosis and the like.
The existing research shows that RAI16 is used as an A-type kinase anchoring protein in cells, regulates HSP70 phosphorylation and regulates apoptosis, and the expression of the protein is regulated by a transcription factor-androgen receptor. RAI6 promotes the development of hepatocellular carcinoma. Participate in the regulation and control of the fatty hepatitis and play an important role in colitis and related colon cancer.
Antibodies are important tools for scientific research and are also widely used in the field of biomedicine. The evaluation criteria of the antibody include specificity and sensitivity, and the strong specificity is the most basic and important requirement for the antibody to be capable of specifically recognizing the target protein. Scientific research on the RAI16 protein gradually reveals the important role in the cell physiology and pathology, however, at present, no mainstream commercial antibody aiming at the RAI16 protein can be verified by a RAI16 gene knockout mouse, and the identified protein bands are all non-specifically bound, which proves that the RAI16 protein cannot be specifically identified. Therefore, the preparation of specific RAI16 antibody by selecting proper immune antigen protein is needed to be solved.
Disclosure of Invention
The invention aims to provide preparation and application of a retinoic acid inducible protein 16(RAI16) specific polyclonal antibody.
The invention provides an epitope, which is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.
LCTLGKAEYPPGMRQQVFQFFSKVLSQVQHPLLHYLSVHRPVQKLLRLGGTVPGSLTEKEEVQFTSVLCSKIQQDPELLAYILEGKKIIGKKKTARESTAPPKDIAGYRDKDCPHSDALNRDPGLDKEHCGVPALSIHLPAETEGPENGPGESNLITSLLGLCKSKKSRLALKAQENILLLVSVASPAAATYLTQSTSCCM
The invention utilizes DNAStar software to analyze RAI16 epitope: the antigen activity of 201 amino acids at 92-292 of the RAI16 protein is determined to be better.
The invention also provides an antigen protein, which is characterized in that: after constructing the expression plasmid of the amino acid sequence, the antigen protein is obtained by purification.
The present invention also provides a composition for producing an antibody, characterized in that: comprises the above epitope;
or
The above-mentioned antigen protein.
Further, the present invention provides a composition for producing an antibody, which is characterized in that: also contains adjuvant for immunization.
In addition, the invention also provides a preparation method of the antibody, which is characterized by comprising the following steps: comprising the use of the above epitope; or the above antigenic protein; or a combination thereof.
Further, the present invention provides a method for producing an antibody, which is characterized in that: the antibody is a polyclonal antibody.
Further, the present invention provides a method for producing an antibody, which is characterized in that: the epitope is added;
or the above antigenic protein;
or the above composition;
after immunization of the animals, multiple antisera were obtained.
Further, the present invention provides a method for producing an antibody, which is characterized in that: comprises the following steps:
s1, emulsifying the epitope; or the above-mentioned antigenic protein;
or the composition is injected subcutaneously in animals, and at least one boosting is repeated until the antibody titer in the serum of the animals is equal to or higher than 1: 64000;
s2, serum containing animal antibodies is obtained through collection and separation, and the antibodies are purified through an antigen affinity chromatography method, so that the antibodies are obtained.
Further, the present invention provides a method for producing an antibody, which is characterized in that: in S1, the injection for the first immunization injection contains Freund' S complete adjuvant;
the injection for boosting immunity injection contains Freund incomplete adjuvant;
and/or
In S2, the specific method for purifying the antibody by antigen affinity chromatography is as follows: adding the antigen dissolved in the coupling buffer solution into agarose gel, gently mixing and shaking at room temperature, and standing overnight at a temperature below 0 ℃; adding BSA solution and incubating for 1-3 hours at room temperature; washing the medium at least once with a buffer; diluting antiserum with buffer solution in equal volume, centrifuging, collecting supernatant, washing antigen affinity column with 5-50 times of buffer solution to balance column, and adding diluted antiserum into the balanced column; gently mixing and shaking at room temperature for 2-4 hours; washing the antigen column with 5-50 times of buffer solution to wash away the hetero protein bound on the column; the column is washed with 1-10 times the volume of the antibody eluent to obtain specific antibodies.
In addition, the invention also provides an application of the active factor, which is characterized in that:
the active factor is selected from:
A. an epitope as described above;
B. an antigenic protein as described above;
C. the antibody as described above;
the above applications include at least one of the following applications:
1. for preparing a medicament for preventing and treating acute lymphocytic leukemia;
2. a vaccine for preparing acute lymphocytic leukemia;
3. the kit is used for preparing a diagnostic kit for diagnosing acute lymphocytic leukemia;
4. used for preparing medicaments for preventing and treating lung cancer;
5. for preparing a vaccine for lung cancer;
6. preparing a diagnostic kit for diagnosing lung cancer;
7. used for preparing medicines for preventing and treating liver cancer;
8. the vaccine is used for preparing the liver cancer;
9. is used for preparing a diagnostic kit for diagnosing liver cancer.
The invention has the following functions and effects:
the main technical scheme of the invention is to specifically detect the expression and natural existence of RAI16 through an immune experiment, establish the corresponding basic biological products required by in vitro immunoassay, and then provide an antibody specifically aiming at RAI16 and a preparation method and application thereof.
The invention also solves the problems that the currently used RAI16 is expensive in price, the cost of the prepared antibody is too high, and the commercialized RAI16 antibody is non-specifically bound.
In particular, the amount of the solvent to be used,
in one aspect, the invention provides a retinoic acid-induced protein 16(RAI16) immunogenic protein.
Secondly, the invention provides a polyclonal antibody of retinoic acid-induced protein 16(RAI16), which is obtained by immunizing an animal with an amino acid sequence shown as SEQ ID NO. 1.
Thirdly, the invention provides a preparation method of the antibody of the retinoic acid induced protein 16(RAI 16).
The invention provides an application of retinoic acid induced protein 16(RAI16) immunogen protein in preparation of immunogen. Namely, the immunogen protein and the application of the antibody thereof in the preparation of vaccines or diagnostic kits for preventing and treating acute lymphocytic leukemia, lung cancer, liver cancer and the like.
In addition, the research of the invention shows that the RAI16 immunogen protein has excellent hydrophilic structure, flexible region, antigen index and surface probability structure.
As mentioned above, the anti-RAI 1692-292 AA antibody prepared by the invention has high titer and good specificity, and can generate specific binding reaction with natural human RAI16 molecules; the antibody disclosed by the invention is low in preparation cost, can specifically recognize RAI16 protein, can be verified by a RAI16 knockout mouse, and can be used for establishing in vitro immunoassay. The anti-RAI 1692-292 AA immunogen antibody prepared by the invention provides a powerful tool for the research of RAI16 molecules and the research of related diseases (such as the formulation of diagnosis strategies, the analysis of disease correlation, epidemiological investigation, prevention and control), and has important significance and value in the aspects of determination of the characteristics and content of target proteins, analysis of distribution conditions, confirmation of proteins and the like in biomedical research.
Drawings
FIG. 1 shows the result of analyzing the amino acid sequence of the RAI16 protein using DNAstar software.
FIG. 2 shows the electrophoresis results of the expression and purification of the immunogenic protein of RAI 16.
FIG. 3 shows the results of ELISA assay of anti-RAI 16 antibody titers.
FIG. 4 is the immunoblot results of the non-specific detection of RAI16 protein by a commercial anti-RAI 16 antibody.
FIG. 5 is the immunoblotting result of the invention involving the specific detection of RAI16 protein by anti-RAI 16 antibody.
Detailed Description
The invention will now be further described with reference to examples, but the practice of the invention is not limited thereto.
Example 1: preparation of anti-RAI 1692-292 AA antibody
The anti-RAI 1692-292 AA antibody is prepared by the following steps:
(1) analysis of the RAI16 epitope: the amino acid sequence of the RAI16 protein is analyzed by using DNAStar software, and the analysis result is shown in figure 1, and the amino acids 92-292 of the RAI16 protein have excellent hydrophilic structure, flexible region, antigen index and surface probability structure. Determining 201 amino acids from 92 to 292 of RAI16 protein as an expression immunogen protein sequence:
the amino acid sequence is shown as follows:
lctlgkaeyppgmrqqvfqffskvlsqvqhpllhylsvhrpvqkllrlggtvpgsltekeevqftsvlcskiqqdpellayilegkkiigkkktarestappkdiagyrdkdcphsdalnrdpgldkehcgvpalsihlpaetegpengpgesnlitsllglckskksrlalkaqenilllvsvaspaaatyltqstsccm
(2) RAI 1692-292 AA protein expression: constructing 92-292AA expression plasmid, expressing by using escherichia coli, purifying by using an affinity chromatography column and an HPLC column, wherein the purity after purification is 95 percent, the plasmid can be used as an antigen to immunize animals, and the result of purification electrophoresis is shown in figure 2.
(3) Immunizing animals: RAI16 protein was mixed with an equal volume of complete Freund's adjuvant [ Freund's complete adjuvant, Sigma, cat # F5881, 1mg heat-inactivated dry Mycobacterium tuberculosis (H37Ra, ATCC 25177), 0.85mL paraffin oil and 0.15mL mannide-monooleic acid) ] and emulsified thoroughly and injected subcutaneously into the back of rabbits at 5 injection points, 100pg per injection point, first booster injections were given 2 weeks after the first booster injection, and booster injections were given every two weeks thereafter, and the dose of each booster injection was the same as that of the first booster injection: booster immunizations were given of RAI16 antigen protein with incomplete freund's adjuvant [ freund's incomplete adjuvant, sigma, cat No. F5506. (containing 0.85mL of paraffin oil and 0.15mL of mannide monooleate per mL) ] to emulsify. Booster immunizations were repeated until the antibody titer was equal to or higher than 1: 64000;
antibody purification: collecting rabbit blood containing rabbit anti-RAI 16 antibody by using carotid artery blood sampling to obtain serum containing rabbit anti-RAI 16 antibody, and purifying RAI16 antibody by using antigen affinity chromatography.
Example 2: determination of titer of anti-RAI 1692-292 AA protein antibody
In the embodiment, the titer of the anti-RAI 1692-292 AA protein antibody is detected by ELISA: coating an ELISA detection plate with RAI16-BSA with the concentration of 1mg/L, incubating for 8-12h at 4 ℃ with 100ul of each well, adding 200ul of bovine serum with the concentration of 100ul into each well, sealing for 2h at 37 ℃, adding anti-RAI 1692-292 AA protein antibody diluted in multiple times (normal rabbit serum is added into a control group) after washing, incubating for 60min at 37 ℃, and adding 1:100 ul of HRP-labeled goat anti-rabbit IgG diluted by 5000, incubation for 30min at 37 ℃, washing for 3 times, TMB color development, measurement of the light absorption value of a sample at A450 by using an enzyme-labeling instrument, and calculation of the antibody titer, wherein the antibody titer of the embodiment is 1: 64000 (as shown in fig. 3).
Example 3: application of anti-RAI 1692-292 AA protein antibody in detection of RAI16 protein expression
The RAI16 protein expression was detected by immunoblotting (Western blotting).
1×107Cells or 100mg mouse colon tissue are added with lysis solution (50mM Tris-HCl,150mM NaCl,0.5mM EDTA, protease inhibitor cocktail) to be lysed at 4 ℃ for 30min, centrifuged at 4 ℃ for 20min under 10000g, and supernatant protein is taken to be quantified to obtain a protein sample. Suspending the prepared cell or colon tissue protein sample by using a 5xSDS loading buffer solution, heating for 10min at 95 ℃, placing on ice, cooling for 10min, and then loading; after being separated by SDS-PAGE gel, the solution is electrically transferred to a PVDF membrane by a wet transfer method; sealing with skimmed milk powder (room temperature for 2h), adding purified RAI16 antibody diluted at a ratio of 1:1000, and standing at 4 deg.C overnight; washing membrane with PBST for 3 times, adding 1:2000 HRP-labeled goat anti-rabbit IgG, incubating at room temperature for 1h, washing membrane with PBST for 3 times, treating with chemiluminescence agent kit for 1min, and exposing in dark room.
The immunoblotting results of the antibody of this embodiment are shown in FIG. 4, and the results of the detection of colon tissue samples of three commercial antibodies (Abcam, cat No. ab 102566; Atlas, cat No. HPA 025040; Abclonal, cat No. A392) against wild-type (WT) mice and RAI16 knockout (RAI16-/-) mice, respectively. It can be seen that the antibodies of three companies still detected signal bands in the colon tissue sample of RAI 16-/-mice, which are identical to those of WT mice, indicating that the signal bands (70kD and/or 55kD) are non-specific.
FIG. 5 shows the immunoblotting results of the antibody of the present embodiment, and the results of the detection of colon tissue samples of the antibody of the present invention in wild-type (WT) mice and RAI16 knockout (RAI16-/-) mice. As can be seen, the antibody of the present invention specifically recognized the 70kD band in the WT mouse colon tissue sample, while the above band was not detected in the RAI 16-/-mouse colon tissue sample, indicating that the above signal band (70kD) is specifically recognized by RAI16 protein.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Figure BDA0003036159360000091
Figure BDA0003036159360000101
Sequence listing
<110> China people liberation army navy military medical university
<120> preparation and application of retinoic acid-induced protein 16 specific polyclonal antibody
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> PRT
<213> RAI16
<400> 1
Leu Cys Thr Leu Gly Lys Ala Glu Tyr Pro Pro Gly Met Arg Gln Gln
1 5 10 15
Val Phe Gln Phe Phe Ser Lys Val Leu Ser Gln Val Gln His Pro Leu
20 25 30
Leu His Tyr Leu Ser Val His Arg Pro Val Gln Lys Leu Leu Arg Leu
35 40 45
Gly Gly Thr Val Pro Gly Ser Leu Thr Glu Lys Glu Glu Val Gln Phe
50 55 60
Thr Ser Val Leu Cys Ser Lys Ile Gln Gln Asp Pro Glu Leu Leu Ala
65 70 75 80
Tyr Ile Leu Glu Gly Lys Lys Ile Ile Gly Lys Lys Lys Thr Ala Arg
85 90 95
Glu Ser Thr Ala Pro Pro Lys Asp Ile Ala Gly Tyr Arg Asp Lys Asp
100 105 110
Cys Pro His Ser Asp Ala Leu Asn Arg Asp Pro Gly Leu Asp Lys Glu
115 120 125
His Cys Gly Val Pro Ala Leu Ser Ile His Leu Pro Ala Glu Thr Glu
130 135 140
Gly Pro Glu Asn Gly Pro Gly Glu Ser Asn Leu Ile Thr Ser Leu Leu
145 150 155 160
Gly Leu Cys Lys Ser Lys Lys Ser Arg Leu Ala Leu Lys Ala Gln Glu
165 170 175
Asn Ile Leu Leu Leu Val Ser Val Ala Ser Pro Ala Ala Ala Thr Tyr
180 185 190
Leu Thr Gln Ser Thr Ser Cys Cys Met
195 200

Claims (10)

1. An epitope, characterized by: the amino acid sequence is shown in SEQ ID NO. 1.
2. An antigenic protein, characterized by: the antigenic protein is obtained by purification after constructing the expression plasmid of the amino acid sequence of claim 1.
3. A composition for producing an antibody, characterized in that: comprising the epitope of claim 1;
or
The antigenic protein of claim 2.
4. A composition for the production of antibodies according to claim 3, characterized in that: also contains adjuvant for immunization.
5. A method for producing an antibody, comprising: comprising the use of an epitope according to claim 1;
or the antigenic protein of claim 2;
or the composition of claim 3 or 4; the step (2).
6. The method for producing an antibody according to claim 5, wherein: the antibody is a polyclonal antibody.
7. The method for producing an antibody according to claim 5, wherein:
combining the epitope of claim 1;
or the antigenic protein of claim 2;
or the composition of claim 3 or 4;
after immunization of the animals, multiple antisera were obtained.
8. The method for producing an antibody according to claim 5, comprising the steps of:
s1, emulsifying the epitope as claimed in claim 1; or the antigenic protein of claim 2;
or the composition of claim 3 or 4, is injected subcutaneously in an animal and the booster is repeated at least once until the antibody titer in the animal serum is equal to or higher than 1: 64000;
s2, serum containing animal antibodies is obtained through collection and separation, and the antibodies are purified through an antigen affinity chromatography method, so that the antibodies are obtained.
9. The method for producing an antibody according to claim 8, wherein:
in S1, the injection for the first immunization injection contains Freund' S complete adjuvant;
the injection for boosting immunity injection contains Freund incomplete adjuvant;
and/or
In S2, the specific method for purifying the antibody by antigen affinity chromatography is as follows: adding the antigen dissolved in the coupling buffer solution into agarose gel, gently mixing and shaking at room temperature, and standing overnight at a temperature below 0 ℃; adding BSA solution and incubating for 1-3 hours at room temperature; washing the medium at least once with a buffer; diluting antiserum with buffer solution in equal volume, centrifuging, collecting supernatant, washing antigen affinity column with 5-50 times of buffer solution to balance column, and adding diluted antiserum into the balanced column; gently mixing and shaking at room temperature for 2-4 hours; washing the antigen column with 5-50 times of buffer solution to wash away the hetero protein bound on the column; the column is washed with 1-10 times the volume of the antibody eluent to obtain specific antibodies.
10. Use of an active factor, characterized by:
the active factor is selected from:
A. the epitope of claim 1;
B. the antigenic protein of claim 2;
C. the antibody of any one of claims 5-9;
the application comprises at least one of the following applications:
I. for preparing a medicament for preventing and treating acute lymphocytic leukemia;
a vaccine for the preparation of acute lymphocytic leukemia;
preparing a diagnostic kit for diagnosing acute lymphocytic leukemia;
IV, preparing a medicament for preventing and treating lung cancer;
v. vaccines for the preparation of lung cancer;
preparing a diagnostic kit for diagnosing lung cancer;
VII, preparing a medicament for preventing and treating liver cancer;
viii, vaccines for the preparation of liver cancer;
IX. can be used for preparing diagnostic kit for diagnosing hepatocarcinoma.
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CN103102392A (en) * 2013-01-28 2013-05-15 中国人民解放军第二军医大学 N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof
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