Specific anti-ITSN1-S-K1068 lysine lactylated antibody as well as preparation method and application thereof

Abstract

The invention discloses a specific anti-ITSN1-S-K1068 lysine lactylated antibody and a preparation method and application thereof.The preparation method comprises the following steps that 1, ITSN1-S-K1068 lactylated polypeptide is connected to an immune carrier in a sulfydryl coupling mode, a polypeptide antigen is obtained, the polypeptide antigen is mixed with a Freund's complete adjuvant and a Freund's incomplete adjuvant respectively, and an immune carrier is obtained; emulsifying to obtain water-in-oil liquid drops; and step 2, carrying out animal immunization by using the water-in-oil liquid drops, and carrying out separation from the immunized animal to obtain the specific anti-ITSN1-S-K1068 lysine lactylated antibody. The antibody provided by the invention can specifically detect the lactic acid modification condition of the human ITSN1-S protein from cell and tissue levels in practical application, is beneficial to discussing the action mechanism of post-translational modification of the ITSN1-S protein in the processes of tumor proliferation, migration and the like, and provides a potential action target for clinical diagnosis and treatment of tumor diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a specific anti-ITSN1-S-K1068 lysine lactylation antibody and a preparation method and application thereof. Background Art

[0002] Intersectin 1 (ITSN1) is a highly conserved protein in the evolutionary process. There are two splice forms in the human body, ITSN1-L and ITSN1-S. The two splice forms have multiple common protein domains. The long subtype ITSN1-L is mainly expressed in neurons, participates in the endocytosis and exocytosis of neurons and the synthesis and release of neurotransmitters, and is associated with a variety of neurodegenerative diseases; while the short subtype ITSN1-S is widely distributed in various types of tissues and cells, and can participate in physiological activities such as endocytosis, exocytosis, and cell proliferation through its various domains. Therefore, the preparation of specific bridge molecule ITSN1-S protein lysine 1068 lactylated antibodies is of great significance for in-depth research on the regulation of its lactylated state on the function of ITSN1-S itself and on various physiological activities. Summary of the invention

[0003] The purpose of the present invention is to effectively study the action mechanism of the bridge molecule ITSN1-S protein in malignant tumors, and to provide a specific anti-ITSN1-S-K1068 lysine lactylated antibody and a preparation method and application thereof.

[0004] The technical solution adopted to achieve the purpose of the present invention is:

[0005] An ITSN1-S-K1068 lactated polypeptide has an amino acid sequence SGTAGKTGSLG as described in SEQ ID No. 1, wherein the sixth lysine is lactated, and the structural formula is SGTAGK(lac)TGSLG.

[0006] In another aspect of the present invention, an ITSN1-S-K1068 lactic acid antigen is prepared by cross-linking the ITSN1-S-K1068 lactic acid polypeptide with an immune carrier.

[0007] In the above technical solution, the immune carrier is KLH-VLP.

[0008] In another aspect of the present invention, a method for preparing a specific anti-ITSN1-S-K1068 lysine lactylated antibody comprises the following steps:

[0009] Step 1, connecting the ITSN1-S-K1068 lactic acid polypeptide to an immune carrier by thiol coupling to obtain a polypeptide antigen, and mixing the polypeptide antigen with Freund's complete adjuvant and Freund's incomplete adjuvant, respectively, and emulsifying to obtain water-in-oil droplets;

[0010] Step 2, using the water-in-oil droplets to immunize animals, and isolating the specific anti-ITSN1-S-K1068 lysine lactylated antibody from the immunized animals.

[0011] In the above technical solution, in step 1, the droplets formed by mixing the polypeptide antigen with Freund's complete adjuvant are used for the first immunization of animals, and the droplets formed by mixing the polypeptide antigen with Freund's incomplete adjuvant are used for the booster immunization of animals.

[0012] In the above technical scheme, in step 2, the animal is a New Zealand white rabbit, which is immunized five times in total. The first immunization uses oil-in-water droplets obtained by mixing polypeptide antigen and Freund's complete adjuvant in a mass ratio of 1:1, and the second to fifth booster immunizations use oil-in-water droplets obtained by mixing polypeptide antigen and Freund's incomplete adjuvant in a mass ratio of 1:1.

[0013] In the above technical solution, in the preparation method, the specific anti-ITSN1-S-K1068 lysine lactylated antibody is separated from the blood of the immunized animal.

[0014] In the above technical solution, the separation method comprises the following steps:

[0015] Step 1, centrifuging the blood to obtain serum;

[0016] Step 2, first wash the empty chromatography column with glycine at pH 2.5, then rinse the column with sodium phosphate solution, sterile secondary water, and ethanol in sequence, and after the rinsing is completed, add ethanol and store at low temperature;

[0017] Step 3, adding the serum obtained in step 1 to an empty chromatography column equilibrated with a sodium phosphate solution at pH 7.0;

[0018] Step 4, eluting with glycine at pH 2.5, and then adding Tris-HCl at pH 9.0 to mix, to obtain the separated specific anti-ITSN1-S-K1068 lysine lactylated antibody.

[0019] Another aspect of the present invention also includes a specific anti-ITSN1-S-K1068 lysine lactylated antibody obtained by the preparation method.

[0020] Another aspect of the present invention also includes the use of the specific anti-ITSN1-S-K1068 lysine lactylation antibody in the preparation of anticancer drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. In practical applications, the antibody provided by the present invention can specifically detect the lactic acid modification of human ITSN1-S protein at the cellular and tissue levels.

[0023] 2. The present invention is a lactic acid polyclonal antibody prepared against the specific 1068 site of human ITSN1-S protein, which is helpful for studying the cell signal transduction pathways and interaction networks of various biological functions involved in ITSN1-S protein.

[0024] 3. The antibodies provided by the present invention are helpful to explore the mechanism of action of post-translational modification of ITSN1-S protein in tumor proliferation, migration and other processes, and provide potential targets for the diagnosis and treatment of clinical tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the Dot blot detection result of the specific anti-ITSN1-S-K1068 lysine lactylation antibody provided by the present invention.

[0026] Figure 2 The specific anti-ITSN1-S-K1068 lysine lactylated antibody provided by the present invention is used to perform Western blot to detect the expression of exogenous K1068 lactylated ITSN1-S-protein in HEK293T cells.

[0027] Figure 3 The specific anti-ITSN1-S-K1068 lysine lactylated antibody provided by the present invention is used to perform immunohistochemistry to detect the expression of K1068 lactylated ITSN1-S-protein in human breast invasive ductal carcinoma tissue.

[0028] Figure 4 It is the ELISA binding detection result of the purified specific anti-ITSN1-S-K1068 lysine lactylation antibody provided by the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] Two antigen polypeptides were designed according to the provided target sequence. Antigen number 1 was ITSN1-S-K1068 modified peptide (ITSN1-S-K1068 lactated polypeptide), and the sequence was [SGTAGK(lac)TGSLG]2K-C. Antigen number 2 was ITSN1-S-K1068 non-modified peptide (ITSN1-S-K1068 non-lactated polypeptide), and the sequence was SGTAGKTGSLG-C.

[0032] The ITSN1-S-K1068 modified peptide and the ITSN1-S-K1068 non-modified peptide were synthesized, and the quality control of the peptide synthesis was performed by HPLC-MS to ensure that the purity of the peptide was >90%.

[0033] Example 2

[0034] The immune vector was prepared by the following steps:

[0035] Step 1, KLH activated VLP (virus-like particles): 20 mg of KLH solid + 10 ml of VLP liquid, prepared into KLH liquid. Continue to add 160 ul of SMPH and 10 ml of VLP liquid to 20 mg of KLH liquid, and place in a 25 degree water bath for 2 hours. Then dialyze overnight in immunogen buffer (800 mM sodium chloride + 100 mM sodium dihydrogen phosphate + 100 mM EDTA), and after a second dialysis on the next day, the dialysis is completed to obtain KLH + VLP protein.

[0036] Step 2, KLH-VLP coupled polypeptide: dilute 100*TCEP (0.4gTCEP+10ml secondary water) to 1*TCEP with 125ul immunogen buffer to dissolve 1mg of ITSN1-S-K1068 lactic acid polypeptide; add 100ul, 0.2M disodium hydrogen phosphate to adjust the pH value to 7.0; add 250ul KLH+VLP protein to a final concentration of 2.105mg / ml, and incubate at 37°C for 4 hours or at 4°C overnight to obtain KLH-VLP coupled polypeptide.

[0037] The ITSN1-S-K1068 modified peptide was dissolved in DMSO, and the peptide was coupled to the immune carrier by sulfhydryl (SH) coupling according to the characteristics of the peptide sequence to obtain ITSN1-S-K1068 lactic acid antigen.

[0038] The ITSN1-S-K1068 lactic acid antigen was packaged according to the immunization dose, and Freund's complete adjuvant (for the first immunization) or Freund's incomplete adjuvant (for booster immunization) was added in a 1:1 ratio, and emulsified using an emulsifier to form oil-in-water droplets.

[0039] Animal Immunization:

[0040] According to the immunization dose of 500ug per rabbit, New Zealand white rabbits were immunized at multiple points on the back, with a total of 6-8 injections. Four healthy rabbits were immunized with each antigen. A total of five immunizations were performed, with an interval of 14 days between immunizations, and blood sampling was performed 7 days after the third immunization.

[0041] First immunization: 500ug immunogen (ITSN1-S-K1068 lactic acid antigen) was mixed evenly with the corresponding volume of Freund's complete adjuvant and injected subcutaneously at multiple points; 2nd to 5th booster immunization: 150ug immunogen (ITSN1-S-K1068 lactic acid antigen) was mixed evenly with the corresponding volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points. Titer detection after three immunizations, according to the titer after five immunizations, the rabbits were killed and whole blood was collected for antibody purification.

[0042] Example 3

[0043] Purification of specific anti-ITSN1-S-K1068 lysine lactylation antibody:

[0044] Step 1, centrifuge the serum obtained by the above technical solution at 10000 rpm and 4°C for 5 minutes (twice), take out the supernatant and put it into a clean 50ml centrifuge tube, and discard the precipitate.

[0045] Step 2, first wash the empty chromatography column with 100mM glycine (pH2.5), then rinse the column with 20mM sodium phosphate solution, sterile secondary water, and 20% ethanol in sequence. After rinsing, add 20% ethanol, cover the bottom cap tightly, and store at 4 degrees.

[0046] In step 3, the serum is added to an empty chromatography column equilibrated with 20 mM sodium phosphate solution (pH 7.0).

[0047] Step 4: Elution was performed with 8 ml of 100 mM glycine (pH 2.5), and then 1 M Tris-HCl (pH 9.0, neutralizing solution) was added to obtain the purified specific anti-ITSN1-S-K1068 lysine lactylated antibody.

[0048] Example 4

[0049] Antibody detection by ELISA assay:

[0050] The detection peptide was coupled to the BSA carrier and then coated on the ELISA plate. The first well was coated with 10 ng, and the coating amount was reduced by one-fold dilution. Blocked with 1% BSA / PBS-T, the antibody / serum was diluted 1:1000, 100 ul was added to each well for primary antibody incubation for 1 hour, and the secondary antibody was added after washing for 45 minutes, and then the plate was read (OD450).

[0051] The purified antibody (specific anti-ITSN1-S-K1068 lysine lactate antibody obtained in Example 3) was tested by ELISA binding. The results are shown in Figure 4 . In the primary antibody information, antibodies B1-B4 are all specific anti-ITSN1-S-K1068 lysine lactylated antibodies, which come from four different New Zealand white rabbits. The results show that the specific anti-ITSN1-S-K1068 lysine lactylated antibodies bind to lactylated peptides, and the cross-reaction rate with non-lactated peptides is less than 10%, and the antibody specificity is high. The ELISA titers of all specific anti-ITSN1-S-K1068 lysine lactylated antibodies against ITSN1-S-K1068 lactylated peptides are ≤1ng; the ratio of B1 antibody against ITSN1-S-K1068 lactylated peptide and ITSN1-S-K1068 non-lactated peptide is 128 times; the ratio of B2 antibody is 256 times; the ratio of B3 antibody is 256 times; the ratio of B4 antibody is 256 times.

[0052] Example 5

[0053] Molecular biology experimental detection:

[0054] Dot blot test results are as attached Figure 1 As shown, 200ng, 100ng, 10ng, and 1ng of ITSN1-S-K1068 non-lactated peptide and ITSN1-S-K1068 lactated peptide were spotted on the nitrocellulose membrane in sequence, and then a specific anti-ITSN1-S-K1068 lysine lactylation antibody was added at a ratio of 1:2000 for hybridization detection. The results showed that the specific anti-ITSN1-S-K1068 lysine lactylation antibody could specifically recognize ITSN1-S-K1068 lactic acid-modified peptide, while ITSN1-S-K1068 non-lactated peptide could not be recognized by the antibody.

[0055] Western blot test results of cell samples are shown in the attached Figure 2 As shown, a group of cells overexpressing ITSN1-S and another group of cells overexpressing ITSN1-S-K1068R (amino acid position 1068 was mutated to arginine) were used for protein enrichment and protein electrophoresis blotting experiments.

[0056] The results showed that the overexpressed ITSN1-S protein could be recognized by the specific anti-ITSN1-S-K1068 lysine lactylated antibody, while the ITSN1-S-K1068R protein could not be recognized by the antibody due to the change in amino acid sequence. The results showed that the specific anti-ITSN1-S-K1068 lysine lactylated antibody could recognize the ITSN1-S protein lactylated at 1068 lysine in cells.

[0057] The results of immunohistochemistry are shown in the attached Figure 3 As shown, paraffin specimens of 4 breast cancer patients with invasive ductal carcinoma of the breast were collected, and immunohistochemistry experiments were performed using specific anti-ITSN1-S-K1068 lysine lactylated antibody. The results showed that the ITSN1-S protein lactylated at position 1068 was located in the cytoplasm, without nonspecific staining, which fully demonstrated that the antibody can recognize the ITSN1-S protein lactylated at position 1068 lysine in a certain degree of spatial structure, and can specifically and accurately detect the expression of the ITSN1-S protein lactylated at position 1068 lysine in tissues.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ITSN1-S-K1068 lactic acid polypeptide, characterized in that The amino acid sequence is SGTAGKTGSLG as shown in SEQ ID No. 1, wherein the sixth lysine is lactylated.

2. An ITSN1-S-K1068 lactic acid antigen, characterized in that The ITSN1-S-K1068 lactic acid polypeptide according to claim 1 is cross-linked with an immune carrier.

3. The ITSN1-S-K1068 lactic acid antigen according to claim 2, characterized in that The immune carrier is KLH-VLP.

4. A method for preparing a specific anti-ITSN1-S-K1068 lysine lactylated antibody, characterized in that: The following steps are involved: Step 1, connecting the ITSN1-S-K1068 lactic acid polypeptide as claimed in claim 1 to an immune carrier by thiol coupling to obtain a polypeptide antigen, and mixing the polypeptide antigen with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, and emulsifying to obtain water-in-oil droplets; Step 2, using the water-in-oil droplets to immunize animals, and isolating the specific anti-ITSN1-S-K1068 lysine lactylated antibody from the immunized animals.

5. The preparation method according to claim 4, characterized in that: In the step 1, the droplets formed by mixing the polypeptide antigen with Freund's complete adjuvant are used for the first immunization of the animal, and the droplets formed by mixing the polypeptide antigen with Freund's incomplete adjuvant are used for the booster immunization of the animal.

6. The preparation method according to claim 5, characterized in that: In step 2, the animal is a New Zealand white rabbit, which is immunized five times in total. The first immunization uses oil-in-water droplets obtained by mixing polypeptide antigen and Freund's complete adjuvant in a mass ratio of 1:1, and the second to fifth booster immunizations use oil-in-water droplets obtained by mixing polypeptide antigen and Freund's incomplete adjuvant in a mass ratio of 1:

1.

7. The preparation method according to claim 4, characterized in that: In the preparation method, the specific anti-ITSN1-S-K1068 lysine lactylated antibody is isolated from the blood of the immunized animal.

8. The preparation method according to claim 7, characterized in that: The separation method comprises the following steps: Step 1, centrifuging the blood to obtain serum; Step 2, first wash the empty chromatography column with glycine at pH 2.5, then rinse the column with sodium phosphate solution, sterile secondary water, and ethanol in sequence, and after the rinsing is completed, add ethanol and store at low temperature; Step 3, adding the serum obtained in step 1 to an empty chromatography column equilibrated with a sodium phosphate solution at pH 7.0; Step 4, eluting with glycine at pH 2.5, and then adding Tris-HCl at pH 9.0 to mix, to obtain the separated specific anti-ITSN1-S-K1068 lysine lactylated antibody.

9. A specific anti-ITSN1-S-K1068 lysine lactylated antibody obtained by the preparation method according to claim 4.

10. Use of the specific anti-ITSN1-S-K1068 lysine lactylation antibody according to claim 9 in the preparation of anticancer drugs.

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