A CDC42 acetylation-specific antibody, preparation method, and application

Abstract

The present invention discloses a specific antibody for acetylation at the CDC42-K153 site, a preparation method and an application thereof, and relates to the field of biotechnology. The amino acid sequence of the antibody is shown in SEQ ID NO: 1; the preparation method thereof is as follows: a CDC42-K153 peptide segment DLKAVKYVECSALTQKGL is prepared by a synthetic method, and the nucleotide sequence thereof is shown in SEQ ID NO: 2; and an acetylated modified peptide segment is obtained, and a peptide segment without an acetylated group is used as a control peptide segment. The acetylated modified peptide segment is immunized with New Zealand white rabbits to produce a site-specific acetylated polyclonal antibody and obtain an antiserum; the application of the specific antibody for acetylation at the K153 site in determining the expression level of acetylation at K153 of CDC42. The present invention develops a specific antibody for acetylation at the K153 site, which is applied to the determination of acetylated antigens at the K153 site of the CDC42 protein, and has good specificity and sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a CDC4 K153 site acetylated specific antibody, preparation method and application. Background Art

[0002] There are no highly specific serum tumor markers for colon cancer. Currently, the primary serum tumor marker used clinically for auxiliary diagnosis and prognosis assessment of colon cancer is carcinoembryonic antigen (CEA). In addition, cancer antigens CA19-9, CA24-2, CA50, and CA74-2 are also commonly used in clinical practice to assess prognosis and treatment efficacy. However, these markers are far less specific and sensitive than CEA and are often tested in combination with CEA. Furthermore, HE and Ki67 staining are often performed during pathological tissue biopsies, but these staining methods are limited.

[0003] Existing colorectal cancer tumor markers and immunohistochemical staining indicators are relatively limited, lacking specificity and sensitivity. Acetylation at the K153 site of the CDC42 protein is highly expressed in normal tissues, but its expression is significantly reduced in cancer tissues.

[0004] Therefore, those skilled in the art are committed to developing an antibody prepared from an acetylated peptide fragment of CDC42-K153 with good specificity and sensitivity, which is applied to the determination of acetylated antigen at the K153 site of CDC42 protein. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a specific antibody for K153 acetylation with good specificity and sensitivity, which is applied to the determination of acetylated antigen at K153 site of CDC42 protein.

[0006] To achieve the above objectives, the present invention provides a CDC42-K153 acetylated modified peptide segment CDC42-K153 acetylated modified peptide segment, the amino acid sequence of the K153 acetylated portion is shown in SEQ ID NO: 1, and the acetylation site is the sixth amino acid K in the amino acid sequence SEQ ID NO: 1, namely DLKAVK (acetylated) YVECSALTQKGL.

[0007] The present invention also provides an application of a CDC42-K153 acetylated modified peptide segment in the preparation of a K153 acetylated antibody, which comprises the following steps:

[0008] Step 1. Prepare the CDC42-K153 peptide by a synthetic method. The amino acid sequence of the CDC42-K153 peptide is shown in SEQ ID NO: 3, which is DLKAVKYVECSALTQKGL. The nucleotide sequence encoding the CDC42-K153 peptide is shown in SEQ ID NO: 2. An acetylated group is added to the amino acid K at the sixth position of the amino acid sequence to obtain an acetylated peptide. The CDC42-K153 peptide without the acetylated group is used as a control peptide.

[0009] Step 2: using the acetylated peptide obtained in step 1 as an antigen to immunize New Zealand white rabbits with site-directed acetylated polyclonal antibodies to obtain antiserum;

[0010] Step 3: The control peptide obtained in step 1 is used to remove nonspecific antibodies in the antiserum obtained in step 2 to obtain specific antibodies.

[0011] Furthermore, the amino acid sequence of the acetylated peptide obtained in step 1 is DLKAVK(acetylated)YVECSALTQKGL;

[0012] Furthermore, step 2 also includes: immunizing rabbits four times within two months and collecting antiserum.

[0013] Furthermore, the sensitivity and specificity of specific antibodies were evaluated by ELISA and western blot.

[0014] The present invention also provides an application of a specific antibody for acetylation at K153 site in determining the expression level of acetylation at K153 of CDC42 protein.

[0015] Furthermore, the application method includes the following steps:

[0016] Step a, coating the K153 acetylation-specific antibody on a solid phase carrier to obtain a coated antibody;

[0017] Step b: using the coated antibody obtained in step a to detect the antigen using a double antibody sandwich method.

[0018] Furthermore, the solid phase carrier is a 96-well plate.

[0019] Furthermore, the double antibody sandwich method in step b is as follows: the coated antibody obtained in step a is washed and then added to the CDC42 protein to be tested containing the antigen, and combined with the specific antibody coated on the solid phase carrier, after incubation and washing, an enzyme-labeled specific antibody is added, and after further incubation and washing, a substrate is added for color development and measurement.

[0020] Furthermore, the labeling enzyme is horseradish peroxidase (HRP).

[0021] Furthermore, the parameter measured is the OD value.

[0022] In a preferred embodiment of the present invention, it is described in detail how to prepare a specific antibody against acetylation at K153 and how to measure its specificity and sensitivity.

[0023] The positive technical effects of the present invention are as follows:

[0024] The present invention first coats a K153 acetylation-specific antibody onto a solid-phase carrier. After washing, the antigen-containing CDC42 protein to be tested is added, allowing it to bind to the specific antibody coated on the solid-phase carrier. After incubation and washing, an enzyme-labeled specific antibody is added. After further incubation and washing, a substrate is added for color development and measurement. By comparing the OD values ​​of adjacent tissues with those of cancerous tissue, the presence of colon cancer can be determined. The K153 acetylation antibody used has excellent specificity. Animal experiments comparing the expression levels of K153 acetylation of CDC42 in the intestines of mice with colorectal cancer and those of normal mice revealed significantly lower K153 acetylation in mice with colorectal cancer than in normal mice. Furthermore, testing of samples from clinical patients also revealed significantly lower K153 acetylation in tumor tissue than in adjacent tissue.

[0025] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic electrophoresis diagram of a specific antibody acetylated at the CDC42-K153 site according to a preferred embodiment of the present invention;

[0027] Figure 2 This is a statistical graph of immunohistochemical staining results of K153 acetylation in 69 cases of colon cancer according to a preferred embodiment of the present invention;

[0028] Figure 3 This is a representative immunohistochemical assay result showing the expression of CDC42-K153 acetylation in stage I, II, III, and IV human colorectal cancer tissues according to a preferred embodiment of the present invention;

[0029] Figure 4 This is a statistical graph of CDC42-K153 acetylation levels in patients with stage I, II, III, and IV colorectal cancer in a preferred embodiment of the present invention;

[0030] Figure 5 This is a diagram showing the effect of CDC42-K153 acetylation level on patient survival rate in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0031] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0032] Example 1 Preparation of a K153 acetylated antibody and determination of its specificity and sensitivity

[0033] A synthetically prepared acetylated peptide at CDC42-K153 was used as an antigen to immunize New Zealand white rabbits with a site-specific acetylated polyclonal antibody. Rabbits were immunized four times over two months, and antisera were collected. A control peptide, an unacetylated acetylated peptide at CDC42-K153, was used to eliminate nonspecific antibodies. The sensitivity and specificity of the antibody were evaluated by ELISA and western blot. The K153 acetylated antibody demonstrated excellent specificity.

[0034] K153 acetylation-specific antibodies Figure 1 As shown, different amounts of acetylated or non-acetylated K153 peptides were spotted on the membrane as antigens, and detected using CDC42-K153 site-directed acetylation antibodies with and without non-specific modifications removed. It was found that after removing the non-specific modifications, the CDC42-K153 site-directed acetylation antibody had very good specificity.

[0035] Colorectal cancer tumor tissues and adjacent normal tissues from 69 patients were collected from the hospital and detected by immunohistochemistry using CDC42-K153 acetylation antibody. The results of the CDC42-K153 acetylation level test were as follows: Figure 2 ; The immunohistochemical results of CDC42-K153 acetylation expression in human colorectal cancer tissues, among which the representative Figure 3 As shown in the figure, the upper part represents adjacent normal tissue and the lower part represents colon cancer tissue; it was found that K153 acetylation in adjacent normal tissue was significantly higher than that in tumor tissue, and this phenomenon was more obvious in patients with stage II, III, and IV colorectal cancer than in stage I. The statistical results of CDC42-K153 acetylation levels in patients with stage I, II, III, and IV colorectal cancer are shown in the figure. Figure 4 As shown in the table, immunohistochemistry was performed on patients in the four stages, and it was found that K153 acetylation in adjacent normal tissues was significantly higher than that in tumor tissues. This phenomenon was more obvious in patients with stage II, III, and IV colorectal cancer than in stage I. In addition, the effect of CDC42-K153 acetylation level on patient survival rate is shown in the table. Figure 5As shown in the results, patients with high CDC42-K153 acetylation showed worse survival than those with low acetylation. These results indicate that K153 acetylation level can be used as one of the indicators for determining colorectal cancer.

[0036] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A CDC42-K153 acetylated peptide segment, characterized in that: The peptide segment is obtained by acetylation on the peptide segment of the amino acid sequence in CDC42 protein as shown in SEQ ID NO: 1, and the acetylation site is the sixth amino acid K of the amino acid sequence SEQ ID NO:

1.

2. Use of the CDC42-K153 acetylated modified peptide according to claim 1 in the preparation of K153 acetylated antibodies, characterized in that: The application comprises the following steps: Step 1: preparing a CDC42-K153 peptide segment by a synthetic method, wherein the amino acid sequence of the CDC42-K153 peptide segment is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the CDC42-K153 peptide segment is shown in SEQ ID NO: 2; adding an acetylation group to the amino acid K at the sixth position of the amino acid sequence to obtain an acetylated peptide segment; and using the CDC42-K153 peptide segment without the addition of the acetylation group as a control peptide segment; Step 2: Using the acetylated peptide obtained in step 1 as an antigen to immunize New Zealand white rabbits with site-directed acetylated polyclonal antibodies to obtain antiserum; Step 3: The control peptide obtained in step 1 is used to remove nonspecific antibodies in the antiserum obtained in step 2 to obtain the specific antibody.

3. The preparation method according to claim 2, wherein The step 2 further comprises: immunizing the rabbit four times within two months and collecting the antiserum.

4. The preparation method according to claim 2, wherein The method further comprises evaluating the sensitivity and specificity of the specific antibody by ELISA and western blot.

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