CN113278057B

CN113278057B - Preparation and application of retinoic acid induced protein 16 specific polyclonal antibody

Info

Publication number: CN113278057B
Application number: CN202110444267.XA
Authority: CN
Inventors: 王文; 丁翠玲; 苗根; 钱春霖
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2023-05-26
Anticipated expiration: 2041-04-23
Also published as: CN113278057A

Abstract

The invention provides preparation and application of a retinoic acid induced protein 16 (RAI 16) 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 for retinoic acid inducing protein 16 (Retinoic acid induced protein, rai 16) is: NP-919426.1, swissprot accession number Q80YR2, IPI accession number IPI00654780.2.RAI16 is a retinoic acid-induced protein molecule, the structure and function of which are not yet clear. RAI16 was originally isolated when retinoic acid induced cell differentiation, and retinoic acid has effects of inhibiting cell proliferation, promoting apoptosis and promoting cell differentiation, and has been used for the treatment of various cancers such as acute lymphoblastic leukemia, lung cancer and liver cancer. Therefore, retinoic acid-inducing protein RAI16 is likely to also participate in a series of physiological processes, playing an important role in cell proliferation, differentiation, apoptosis, and the like.

The prior study shows that RAI16 is used as an A-type kinase anchoring protein in cells, regulates HSP70 phosphorylation and apoptosis, and the expression of the RAI is regulated by a transcription factor-androgen receptor. RAI6 promotes the development of hepatocellular carcinoma. Is involved in regulating and controlling the steatohepatitis and plays an important role in colonitis and related colon cancer.

Antibodies are important tools for scientific research and are also widely used in the biomedical field. The criteria for antibody evaluation include specificity and sensitivity, while strong specificity, the ability to specifically recognize a target protein, is the most fundamental and important requirement for antibodies. The scientific research on RAI16 protein gradually reveals the important roles in cell physiology and pathology, however, the current commercial antibodies aiming at the RAI16 protein cannot be verified by RAI16 gene knockout mice, and the identified protein bands are all nonspecific binding, so that the identified protein bands cannot be proved to specifically identify the RAI16 protein. Thus, the selection of appropriate immune antigen proteins to prepare specific RAI16 antibodies is highly desirable.

Disclosure of Invention

The invention aims to provide preparation and application of a retinoic acid induced protein 16 (RAI 16) specific polyclonal antibody.

The invention provides an epitope, which is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

LCTLGKAEYPPGMRQQVFQFFSKVLSQVQHPLLHYLSVHRPVQKLLRLGGTVPGSLTEKEEVQFTSVLCSKIQQDPELLAYILEGKKIIGKKKTARESTAPPKDIAGYRDKDCPHSDALNRDPGLDKEHCGVPALSIHLPAETEGPENGPGESNLITSLLGLCKSKKSRLALKAQENILLLVSVASPAAATYLTQSTSCCM

The invention utilizes DNAStar software to analyze RAI16 epitope: the antigen activity of the RAI16 protein of the 201 amino acids 92-292 is determined to be better.

The invention also provides an antigen protein, which is characterized in that: after constructing the expression plasmid with the amino acid sequence, the antigen protein is obtained through purification.

The invention also provides a composition for preparing an antibody, which is characterized in that: comprising the epitope;

or (b)

The antigen protein described above.

Further, the invention provides a composition for preparing an antibody, which is further characterized in that: and adjuvants for immunization.

In addition, the invention also provides a preparation method of the antibody, which is characterized in that: comprising the use of the above epitope; or the antigen protein described above; or a step of the above composition.

Further, the preparation method of the antibody provided by the invention is further characterized by comprising the following steps of: the antibody is a polyclonal antibody.

Further, the preparation method of the antibody provided by the invention is further characterized by comprising the following steps of: the antigen epitope is treated by the method;

or the antigen protein described above;

or a combination thereof;

after immunization of the animals, polyclonal antisera are obtained.

Further, the preparation method of the antibody provided by the invention is further characterized by comprising the following steps of: comprises the following steps:

s1, emulsifying the epitope; or the antigenic proteins described above;

or the composition is subcutaneously injected on animals and the boosting is repeated at least once until the antibody titer in the serum of the animals is equal to or higher than 1:64000;

s2, collecting and separating serum containing animal antibodies, and purifying the antibodies by adopting an antigen affinity chromatography method to obtain the antibodies.

Further, the preparation method of the antibody provided by the invention is further characterized by comprising the following steps of: in S1, the injection for the first time contains Freund' S complete adjuvant;

the injection for enhancing immunity comprises Freund's incomplete adjuvant;

and/or

In S2, the specific method for purifying the antibody by adopting the antigen affinity chromatography method is as follows: adding the antigen dissolved in the coupling buffer solution into agarose gel, and gently mixing and shaking at room temperature, wherein the temperature is lower than 0 ℃ overnight; 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 balanced column; gently mixing and shaking for 2-4 hours at room temperature; washing the antigen column with 5-50 times of buffer solution to wash off the foreign proteins bound to the column; the column was washed with 1-10 volumes of antibody eluent to give specific antibodies.

In addition, the invention also provides an application of the active factor, which is characterized in that:

the active factors are selected from:

A. an epitope as described above;

B. an antigenic protein as described above;

C. antibodies as described above;

the above-mentioned applications include at least one of the following applications:

1. is used for preparing medicines for preventing and treating acute lymphoblastic leukemia;

2. a vaccine for the preparation of acute lymphoblastic leukemia;

3. a diagnostic kit for preparing a diagnostic of acute lymphoblastic leukemia;

4. is used for preparing medicines for preventing and treating lung cancer;

5. vaccine for preparing lung cancer;

6. the diagnosis kit is used for preparing the diagnosis kit for diagnosing lung cancer;

7. is used for preparing medicines for preventing and treating liver cancer;

8. vaccine for preparing liver cancer;

9. the kit is used for preparing a diagnosis kit for diagnosing liver cancer.

The invention has the following functions and effects:

the invention adopts the main technical scheme that the expression and natural existence of RAI16 are specifically detected through an immune experiment, and the problem of basic biological products required by corresponding in-vitro immune analysis is established, so that an antibody specific to the RAI16 is provided, and a preparation method and application thereof.

The invention also solves the problems of high price of RAI16 used at present, high cost of prepared antibodies and nonspecific binding of commercial RAI16 antibodies.

In particular, the method comprises the steps of,

one aspect of the invention provides a retinoic acid-induced protein 16 (RAI 16) immunogenic protein.

Secondly, the invention provides a polyclonal antibody of retinoic acid induced protein 16 (RAI 16), which is obtained by immunizing animals with an amino acid sequence shown as SEQ ID NO. 1.

Thirdly, the invention provides a preparation method of an antibody of retinoic acid induced protein 16 (RAI 16).

Fourth, the invention provides an application of retinoic acid induced protein 16 (RAI 16) immunogen protein in the preparation of immunogen. Namely, the application of the immunogenic protein and the antibody thereof in preparing vaccines or diagnostic kits for preventing and treating acute lymphoblastic 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.

The anti-RAI 16 92-292AA antibody prepared by the invention has high titer and good specificity, and can generate specific binding reaction with natural human RAI16 molecules; the antibody provided by the invention has low preparation cost, can specifically identify RAI16 protein, can be verified by a RAI16 gene knockout mouse, and can be used for establishing in vitro immunoassay. The anti-RAI 16 92-292AA 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 establishment of diagnosis strategies, analysis of disease correlation, epidemiological investigation, prevention and control), and has important significance and value in the aspects of the characteristics, the content measurement, the analysis of distribution situation, the confirmation of proteins and the like of target proteins in biomedical research.

Drawings

FIG. 1 shows the results of analysis of the amino acid sequence of the RAI16 protein using DNAstar software.

FIG. 2 shows the results of electrophoresis of RAI16 immunogen protein expression and purification.

FIG. 3 shows the results of ELISA for anti-RAI 16 antibody titer determination.

FIG. 4 is the result of immunoblotting of a commercial anti-RAI 16 antibody to nonspecifically detect RAI16 protein.

FIG. 5 shows the immunoblotting results of the specific detection of RAI16 protein by the anti-RAI 16 antibody of the present invention.

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 16 92-292AA antibodies

The anti-RAI 16 92-292AA antibody was prepared as follows:

(1) Analysis of RAI16 epitope: the amino acid sequence of the RAI16 protein is analyzed by DNAStar software, and the analysis result is shown in figure 1, wherein the amino acids at 92-292 of the RAI16 protein have excellent hydrophilic structure, flexible region, antigen index and surface probability structure. Determining the 201 amino acids 92-292 of RAI16 protein as the expressed immunogen protein sequence:

the amino acid sequence is shown as follows:

(2) RAI16 92-292AA protein expression: the 92-292AA expression plasmid is constructed, the expression is carried out by using escherichia coli, the purification is carried out by adopting an affinity chromatography column and an HPLC column, the purity after the purification is 95 percent, the purification can be used as antigen for immunizing animals, and the purification electrophoresis result is shown in figure 2.

(3) Immunization of animals: RAI16 protein was fully emulsified with an equal volume of complete Freund's adjuvant [ Freund's complete adjuvant, sigma, cat# F5881, 1mg heat inactivated dried Mycobacterium tuberculosis (H37 Ra, ATCC 25177), 0.85mL paraffin oil and 0.15mL mannitol monooleate) ] and injected subcutaneously into the back of rabbits at 5 injection points, 100pg each, with a first booster injection at week 2 after the first booster injection, and a second booster injection at two weeks later, with the same dose as the first booster injection: boost injection of RAI16 antigen protein with incomplete freund's adjuvant, sigma, cat No. F5506. (containing 0.85mL paraffin oil and 0.15mL mannitol monooleate per milliliter). The immunization was repeated until the antibody titer was equal to or higher than 1:64000;

antibody purification: collecting rabbit blood containing the rabbit anti-RAI 16 antibody by carotid artery blood taking, obtaining serum containing the rabbit anti-RAI 16 antibody, and purifying the RAI16 antibody by antigen affinity chromatography.

Example 2: determination of anti-RAI 16 92-292AA protein antibody titers

In this embodiment, ELISA was used to detect the titers of anti-RAI 16 92-292AA protein antibodies: ELISA assay plates were coated with 1mg/L of RAI16-BSA, incubated at 4℃for 8-12h per well, then 200ul of 100ul bovine serum was added per well, blocked at 37℃for 2h, washed, and then a double-diluted anti-RAI 16 92-292AA protein antibody (control added normal rabbit serum) was added, incubated at 37℃for 60min,3 washes were followed by 1:5000 dilution of HRP marked goat anti-rabbit IgG 100ul, incubation for 30min at 37 ℃, washing for 3 times, TMB color development, measuring the absorbance value of the sample at A450 by using an enzyme-labeling instrument, and calculating the antibody titer, wherein the antibody titer is 1:64000 (as shown in figure 3).

Example 3: detection of RAI16 protein expression by anti-RAI 16 92-292AA protein antibody

RAI16 protein expression was detected by immunoblotting (Western blotting).

1×10 ⁷ Cells or 100mg of colon tissue of mice are added with lysate (50 mM Tris-HCl,150mM NaCl,0.5mM EDTA, protease inhibitor cocktail) and lysed at 4 ℃ for 30min, centrifuged at 4 ℃ for 20min under 10000g, and the supernatant protein is quantified to obtain a protein sample. Suspending the prepared cell or colon tissue protein sample with 5xSDS loading buffer solution, heating at 95deg.C for 10min, cooling on ice for 10min, and loadingSampling; separating by SDS-PAGE gel, and electrically transferring to PVDF membrane by wet transfer method; adding 1:1000 diluted purified RAI16 antibody after being blocked by skimmed milk powder (room temperature for 2 h) at 4 ℃ overnight; PBST was washed 3 times, then incubated with goat anti-rabbit IgG at room temperature for 1h with 1:2000HRP, washed 3 times with PBST, then treated with chemiluminescent kit for 1min, and then exposed to dark room.

The results of immunoblotting of the antibodies of this embodiment are shown in FIG. 4, and the results of detection of colon tissue samples of wild-type (WT) mice and RAI16 gene knockout (RAI 16-/-) mice with three commercial antibodies (Abcam, cat. No. ab102566; atlas, cat. No. HPA025040; abclonal, cat. No. A392, respectively). It can be seen that antibodies from three companies still detected signal bands in RAI 16-/-mouse colon tissue samples that were completely identical to WT mouse colon tissue samples, suggesting that the signal bands (70 kD and or 55 kD) were non-specifically recognized.

The results of immunoblotting of the antibody of the present embodiment are shown in FIG. 5, and the results of detection of colon tissue samples of wild-type (WT) mice and RAI16 gene knockout (RAI 16-/-) mice with the antibody of the present invention. It can be seen that the antibodies of the invention specifically recognize the 70kD band in WT mouse colon tissue samples, whereas the absence of detection of the above band in RAI 16-/-mouse colon tissue samples suggests that the above signal band (70 kD) is specific for the RAI16 protein.

The foregoing has shown and described the basic principles, principal 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, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

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Sequence listing

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<120> preparation and use of retinoic acid-induced protein 16-specific polyclonal antibody

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Val Phe Gln Phe Phe Ser Lys Val Leu Ser Gln Val Gln His Pro Leu

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Gly Gly Thr Val Pro Gly Ser Leu Thr Glu Lys Glu Glu Val Gln Phe

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Tyr Ile Leu Glu Gly Lys Lys Ile Ile Gly Lys Lys Lys Thr Ala Arg

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Glu Ser Thr Ala Pro Pro Lys Asp Ile Ala Gly Tyr Arg Asp Lys Asp

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Cys Pro His Ser Asp Ala Leu Asn Arg Asp Pro Gly Leu Asp Lys Glu

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His Cys Gly Val Pro Ala Leu Ser Ile His Leu Pro Ala Glu Thr Glu

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Gly Pro Glu Asn Gly Pro Gly Glu Ser Asn Leu Ile Thr Ser Leu Leu

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Gly Leu Cys Lys Ser Lys Lys Ser Arg Leu Ala Leu Lys Ala Gln Glu

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Claims

1. An antigenic protein, characterized in that: constructing an expression plasmid with an amino acid sequence shown as SEQ ID NO.1, and purifying to obtain the antigen protein.

2. A composition for the preparation of a retinoic acid-induced protein 16-specific polyclonal antibody, characterized in that: comprising the antigenic protein of claim 1.

3. A composition for the preparation of retinoic acid-induced protein 16-specific polyclonal antibodies as defined in claim 2, wherein: and adjuvants for immunization.

4. A preparation method of retinoic acid induced protein 16 specific polyclonal antibody is characterized in that: comprising using the antigenic protein of claim 1;

or a composition as claimed in claim 2 or 3; is carried out by a method comprising the steps of.

5. The method for preparing the retinoic acid-induced protein 16-specific polyclonal antibody according to claim 4, wherein:

the antigenic protein of claim 1;

or a composition as claimed in claim 2 or 3;

after immunization of the animals, polyclonal antisera are obtained.

6. The method for preparing retinoic acid-induced protein 16-specific polyclonal antibody as defined in claim 4, comprising the steps of:

s1, emulsifying the antigen protein as claimed in claim 1;

or the composition of claim 2 or 3 is subcutaneously injected on an animal and the booster immunization is repeated at least once until the retinoic acid-inducing protein 16-specific polyclonal antibody titer in the serum of the animal is equal to or higher than 1:64000;

s2, collecting and separating serum containing the animal retinoic acid induced protein 16 specific polyclonal antibody, and purifying the retinoic acid induced protein 16 specific polyclonal antibody by adopting an antigen affinity chromatography method to obtain the retinoic acid induced protein 16 specific polyclonal antibody.

7. The method for preparing retinoic acid-induced protein 16-specific polyclonal antibody according to claim 6, wherein:

in S1, the injection for the first time contains Freund' S complete adjuvant;

the injection for enhancing immunity comprises Freund's incomplete adjuvant;

and/or

In S2, the specific method for purifying the retinoic acid induced protein 16-specific polyclonal antibody by adopting an antigen affinity chromatography method comprises the following steps: adding the antigen dissolved in the coupling buffer solution into agarose gel, and gently mixing and shaking at room temperature, wherein the temperature is lower than 0 ℃ overnight; 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 balanced column; gently mixing and shaking for 2-4 hours at room temperature; washing the antigen column with 5-50 times of buffer solution to wash off the foreign proteins bound to the column; washing the column with 1-10 times volume of retinoic acid-induced protein 16-specific polyclonal antibody eluent to obtain specific retinoic acid-induced protein 16-specific polyclonal antibody.

8. An active factor application, characterized in that:

the active factor is selected from:

A. the antigenic protein of claim 1;

B. a retinoic acid-inducing protein 16-specific polyclonal antibody as defined in any one of claims 2-7;

the application comprises at least one of the following applications:

I. is used for preparing medicines for preventing and treating acute lymphoblastic leukemia;

II, vaccine for preparing acute lymphoblastic leukemia;

III, preparing a diagnosis kit for diagnosing acute lymphoblastic leukemia;

IV, preparing a medicine for preventing and treating lung cancer;

v. vaccine for preparing lung cancer;

VI, preparing a diagnosis kit for diagnosing lung cancer;

VII, preparing a medicament for preventing and treating liver cancer;

VIII, preparing vaccine for liver cancer;

IX. is used for preparing diagnostic kit for diagnosing liver cancer.