CN110540591A - anti-Glycoprotein A33 (glycoprotin A33) monoclonal antibody and immunodetection application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses an anti-Glycoprotein A33(Glycoprotein A33, GPA33) monoclonal antibody secreted and generated by a hybridoma cell strain OTI6C11 (the preservation number is CGMCC No. 18200). The invention also relates to application of the monoclonal antibody secreted by the OTI6C11 in preparing an immunodetection tool for detecting the GPA33 protein, and comprises application of an immunohistochemical detection kit and a tumor labeling kit. The monoclonal antibody can be specifically combined with the GPA33 protein, so that the specificity and the reliability of immunodetection of the GPA33 protein are obviously improved.

Description

anti-Glycoprotein A33 (glycoprotin A33) monoclonal antibody and immunodetection application thereof

Technical Field

The invention relates to the technical field of biology, and in particular relates to a method for specifically combining a monoclonal antibody secreted by a hybridoma cell strain OTI6C11 with Glycoprotein A33 (glycoprotin A33 and GPA33) for immunodetection and application thereof.

Background

Glycoprotein a33 (glycoprotin a33, GPA33) is a transmembrane protein of 319 amino acids with a molecular weight of approximately 36-43kDa, wherein glycosylation modifications are located between 1-235 amino acids, located outside the cell membrane. The GPA33 allele is located on chromosome 1q24, contains 7 exons, and has a total genomic DNA length of 37,787 bp. GPA33 exhibits a tissue-specific expression pattern, specifically expressed in normal gastrointestinal epithelial tissues, and is predominantly distributed in mucosal epithelial cells. Although the function of GPA33 is not yet fully understood, oncological studies have found that GPA33 is highly expressed in more than 95% of colon cancers. Intestinal cancer is one of the most common malignant tumors in the world, and the number of cancer diseases in the world is 1810 ten thousand and the number of deaths in 2018, wherein the morbidity of the intestinal cancer is 6.1 percent and the mortality of the intestinal cancer is 9.2 percent. Because of the high intestinal tissue specificity of the expression of GPA33, it has become a hot spot of the clinical intestinal cancer targeted therapy research. Preclinical results reported in the 2018 literature show that the anti-GPA 33/anti-CD 3 bispecific antibody has potential effect in treating colon cancer. Therefore, GPA33 not only provides an index for intestinal tumor detection, but also plays an important role in studying the occurrence and the progression of intestinal tumors.

At present, clinically, the expression condition of the GPA33 protein in tumor tissues is mainly detected through Immunohistochemistry (IHC for short), and the quality of the performance directly determines the sensitivity and specificity of the whole detection. Therefore, the development of a monoclonal antibody aiming at the GPA33 protein with higher binding specificity is of great significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a monoclonal antibody specifically binding to GPA33 protein, and its application in the preparation of an immunoassay tool for detecting GPA33 protein.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a hybridoma cell strain OTI6C11 capable of secreting monoclonal antibodies against GPA33 protein, which is preserved in China general microbiological culture Collection center (CGMCC for short), the preservation date is 7 and 11 days in 2019, and the preservation number is CGMCC No. 18200.

The invention also provides a monoclonal antibody specifically bound with GPA33, which is secreted by the hybridoma cell strain OTI6C 11.

The preparation method of the monoclonal antibody comprises the following steps:

(1) construction of recombinant expression vectors: the nucleotide sequence of the gene synthesis GPA33 is shown as SEQ ID No.1, the ORF length is 846bp, the corresponding GPA33 protein comprises 22-235 and 257-319 amino acid fragments which are connected through a linker (GGGGS) design, the amino acid sequence is shown as SEQ ID No.2, and the length is 282 aa. The ORF of synthetic GPA33 was inserted into expression vector pET23a-His (available from Origene corporation), and recombinant expression plasmid pET23a-His-rGPA33 of GPA33 was constructed.

(2) Expression and purification of recombinant GPA33 protein: transforming the constructed recombinant expression plasmid of GPA33 into E.coli cells, cracking and centrifuging to obtain soluble protein, and purifying by a nickel column affinity chromatography column to obtain purified recombinant GPA33 protein.

(3) Screening of monoclonal antibody hybridoma and preparation of monoclonal antibody secreted by the monoclonal antibody: immunizing a BALB/c mouse by adopting the purified recombinant GPA33 protein, fusing spleen cells of the mouse with SP2/0 cells, obtaining a monoclonal by a limiting dilution method, screening positive hybridoma cells by an ELISA method, obtaining a hybridoma cell strain capable of secreting an anti-GPA 33 protein specific antibody, and performing subtype identification; preparing the antibody through serum-free culture medium, and purifying through an affinity chromatography column to obtain the monoclonal antibody of the GPA33 protein. The sensitivity and specificity of the monoclonal antibody is verified by immunodetection methods such as Western Blot (WB), immunohistochemical experiments (IHC).

After the preparation by the method, the hybridoma capable of secreting monoclonal antibodies against GPA33 protein is screened out and named as OTI6C11, the subtype is identified as IgG1, and the hybridoma is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 7 and 11 months and is preserved with the preservation number of CGMCC No. 18200.

The invention also provides application of the monoclonal antibody secreted by the hybridoma cell strain OTI6C11 in preparation of an immunodetection tool for detecting GPA33 protein.

Preferably, the immunoassay tool is a kit, a chip or a test paper.

The invention also provides an immunohistochemical detection kit, which comprises the GPA33 protein monoclonal antibody secreted by the hybridoma cell strain OTI6C11 and can detect the expression condition of the GPA33 protein in tissue cells.

In addition, the invention also provides application of the monoclonal antibody of the anti-GPA 33 protein secreted and generated by the hybridoma cell strain OTI6C11 in preparation of a labeled tumor cell kit.

Preferably, the tumor cells comprise the intestinal cancer cell lines LoVo, COLO205, Caco-2, HT-29 and HCT 116; the tumor tissue includes intestinal cancer and gastric cancer tissue.

The monoclonal antibody which can be stably secreted by a hybridoma cell strain OTI6C11 and is specifically combined with GPA33 protein, so that the specificity, the accuracy and the reliability of immunodetection of the GPA33 protein are obviously improved, and the monoclonal antibody is widely suitable for labeling the GPA33 protein in various gastrointestinal tumors.

Biological preservation information description

The hybridoma cell strain OTI6C11 for preserving monoclonal antibody secreting GPA33 protein is classified and named as: mouse anti-human glycoprotein A33(GPA33) monoclonal hybridoma cell strain

The preservation unit is called as follows: china general microbiological culture Collection center

The preservation unit is abbreviated as: CGMCC (China general microbiological culture Collection center)

The address of the depository: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang

The preservation date is as follows: 7 month and 11 days 2019

The preservation number is: CGMCC No.18200

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the ORF cloning site design of GPA33 in example 1.

Fig. 2 is a graph showing the results of WB detection of recombinant GPA33 protein in example 2, using anti-His to detect the expression of recombinant GPA33 protein in e. Wherein, the Lane L is the detection result of E.coli cell lysate transfected with empty vector as antigen; lane R is the detection of e.coli cell lysate antigen transfected with pET-His-rGPA33 plasmid.

FIG. 3 is a graph showing the results of SDS-PAGE electrophoresis in example 2 for detecting recombinant GPA33 protein, recombinant GPA33 protein was purified using a nickel affinity column, and the purified protein was stained by SDS-PAGE and Coomassie Brilliant blue.

FIG. 4 is a graph showing the results of WB detection of monoclonal antibody secreted by OTI6C11 in example 4 on the expression of GPA33 protein in lysates of various intestinal cancer cell lines. Wherein lanes 1-5 are the detection results of lysates of human intestinal cancer cell lines LoVo, COLO205, Caco-2, HT-29 and HCT116 as antigens, respectively.

FIG. 5 is a graph showing the results of measuring the GPA33 protein expression distribution in intestinal cancer tissues by the monoclonal antibody IHC secreted by OTI6C11 in example 5 (primary antibody is anti-GPA 33 monoclonal antibody secreted by OTI6C11, 0.5 ug/ml).

FIG. 6 is a graph showing the results of measuring the expression profile of GPA33 protein in gastric cancer tissues by the monoclonal antibody IHC secreted by OTI6C11 in example 5 (primary antibody is anti-GPA 33 monoclonal antibody secreted by OTI6C11, 0.5 ug/ml).

Detailed Description

The invention discloses a method for using an anti-GPA 33 protein monoclonal antibody secreted by a hybridoma cell strain OTI6C11 for immunodetection and application. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. Based on the embodiments of the present invention, those skilled in the art can appropriately modify the process parameters to implement the embodiments based on the contents of the present invention. It is particularly pointed out that all other embodiments obtained by the person skilled in the art without making any inventive step belong to the scope of protection of the present invention.

Example 1: construction of GPA33 recombinant expression plasmid

A plasmid (containing GPA33 partial nucleotide and having the length of 849bp) purchased from Nanjing Kingsler Biotech Limited is used as a template, two primers are designed and are respectively introduced into an enzyme cutting site SgfI and MluI, the partial nucleotide of GPA33 is cloned to an expression vector pET23a-His, and a recombinant expression plasmid pET23a-His-rGPA33 of a GPA33 protein fragment (22-235aa-GGGGS-257 and 319aa) is established. The ORF cloning site design for GPA33 is shown in FIG. 1.

Example 2: expression and purification of recombinant GPA33 protein

1. Experimental methods

(1) Coli cells: melting 100ul competent cells on ice, adding plasmid DNA, mixing, ice-cooling for 30min, heat-shocking at 42 deg.C for 90sec, and further ice-cooling for 1-2 min. Adding 500ul of fresh non-antibiotic LB culture medium into a super clean bench, incubating for 45min at 37 ℃ in a shaking table, taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing antibiotics, and inversely placing a culture dish in a constant temperature incubator at 37 ℃ for culturing overnight.

(2) Cell lysis: the single clone was picked up in a fresh medium, cultured at 37 ℃ and 200rpm until the OD value reached 0.4 to 0.6, and then IPTG (final concentration: 1mM) was added thereto to induce culture for 7 hrs. The cells were collected by centrifugation, then resuspended in lysis buffer, sonicated for 20min and centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant was collected. A small amount of supernatant was subjected to WB assay using anti-His antibody, as shown in FIG. 2.

(3) Purifying with a nickel column affinity chromatography column: and (3) balancing the nickel column by using a buffer solution, filtering the supernatant by using a 0.45-micrometer filter membrane, then sampling, collecting and flowing out, eluting by using the buffer solution to remove unbound protein, finally eluting by using eluents containing imidazole with different concentrations, respectively collecting, combining the eluted proteins meeting the requirements, adding 10% of glycerol, and identifying the purified recombinant GPA33 protein by SDS-PAGE electrophoresis, wherein the figure is shown in figure 3.

2. Results of the experiment

(1) As can be seen from the results in fig. 2, there was a distinct specific band (R) at about 35kD after WB detection in e.coli cell lysates transfected with pET23a-His-rGPA33 plasmid, the molecular weight was consistent with the expected molecular weight, while there was no band of the corresponding size in control lysates (L) transformed with empty vectors. Indicating that the recombinant GPA33 protein is specifically expressed in the cells.

(2) As can be seen from the results in FIG. 3, the purified protein has a distinct specific band at 35-45kD on SDS-PAGE, and the molecular weight is consistent with the expected molecular weight. The recombinant GPA33 protein with better purity is obtained.

Example 3: preparation and screening of OTI6C11 secretion anti-GPA 33 monoclonal antibody

BALB/c mice (Beijing Wittingle laboratory animal technology Co., Ltd.) were immunized with purified recombinant GPA33 protein (hereinafter abbreviated as GPA33 antigen) according to standard methods. The specific method comprises the following steps:

1. Animal immunization: emulsifying the purified GPA33 antigen with complete Freund's adjuvant, immunizing BALB/c mouse of 6-8 weeks old by subcutaneous or intraperitoneal injection method, with an immunization dose of 50 μ g/mouse, performing the second immunization after two weeks, emulsifying with incomplete Freund's adjuvant, with an immunization dose of 50 μ g/mouse. After twice immunization, tail blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; and determining whether to strengthen the immunity according to the result, and selecting the mouse with the highest antibody titer for cell fusion.

2. Cell fusion: the myeloma cells adopt BALB/c-derived sp2/0, and are in logarithmic growth phase when fused; taking the spleen of an immunized mouse, and preparing a lymphocyte single cell suspension; mixing mouse spleen lymphocytes and myeloma cells at a ratio of 1:5-1:10, dripping 1mL of 50% PEG (pH8.0) at 37 ℃, adding an incomplete culture medium and the rest stop solution, centrifuging, removing supernatant, adding HAT culture medium, suspending and mixing uniformly, metering the volume of MC to 50mL, subpackaging in a 3.5cm culture dish, placing in a wet box, and culturing in a constant-temperature incubator at 37 ℃ and 5% CO 2.

3. Screening and cloning: hybridoma cell clones were selected within 7-10 days of fusion and tested by ELISA using purified recombinant GPA33 protein. The cell line number was labeled. And (3) performing limited dilution on the positive well cells, measuring the ELISA value 5-6 days after each limited dilution, and selecting the monoclonal well with the higher OD280 positive value for performing limited dilution until the whole plate result of the 96-well plate is positive in ELISA measurement. And (4) selecting a monoclonal fixed strain with a high positive value. The corresponding fused plate cell line was OTI6C 11.

4. Preparing and purifying cell supernatant monoclonal antibody: the hybridoma cell line OTI6C11 was cultured in a 10cm dish in DMEM medium containing 15% serum, and when about 4X 107 cells were expanded, the cell line was centrifuged at 800rpm for 5min, the supernatant was discarded and the cells were transferred to a 2L spinner flask, and serum-free medium was added to give a cell density of about 3X 105 cells/ml. And after continuing culturing for 1-2 weeks, when the cell death rate reaches 60% -70% (at the moment, the cell density is about 1-2 × 106/ml), collecting cell suspension, centrifuging at 6000rpm for 20min, collecting supernatant, purifying the supernatant by an affinity chromatography, and selecting a corresponding column material according to the antibody subtype for purification (the subtype is IgG1, and protein G column material is adopted for purification). The purified monoclonal antibody was assayed for concentration, lyophilized and aliquoted (100 ug/tube) and finally stored at-20 ℃.

Example 4: WB detection of OTI6C11 secretion of anti-GPA 33 monoclonal antibody

WB detection was carried out using lysates of human intestinal cancer cell lines LoVo, COLO205, Caco-2, HT-29 and HCT116 as antigens and the monoclonal antibody against GPA33 protein prepared in example 3 as a primary antibody, respectively, and the results are shown in FIG. 4.

As can be seen from the results in FIG. 4, the GPA33 protein was expressed in all of LoVo, COLO205, Caco-2, HT-29 and HCT116, indicating that the monoclonal antibody secreted by OTI6C11 prepared in example 3 can specifically recognize GPA33 protein endogenous in LoVo, COLO205, Caco-2, HT-29 and HCT116 cells, indicating that the monoclonal antibody can specifically detect GPA33 protein by WB.

Example 5: IHC detection of OTI6C11 secretion anti-GPA 33 monoclonal antibody

1. The experimental method comprises the following steps:

(1) Formalin-fixed intestinal and gastric cancer tissue blocks were paraffin-embedded and sectioned using a Leica microtome, tissue thickness 4 μm.

(2) dewaxing and hydrating: analytically pure xylene 10min × 3 times, anhydrous ethanol 10min × 3 times, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, and deionized water soaking for 3min × 3 times

(3) Adding antigen repairing solution [1mM EDTA,10mM Tris buffer (pH8.0) ] into the autoclave, performing high-pressure heat repairing for 3min, opening the autoclave when the temperature of the autoclave is reduced to about 90 ℃, taking out the sample, and naturally cooling to room temperature. Soaking in deionized water for 3min × 3 times.

(4) Tissue endogenous peroxidase was inactivated with 3% hydrogen peroxide and allowed to stand at room temperature for 10 min. Soaking in deionized water for 5min × 3 times.

(5) Blocking solution (PBS + 5% skim milk powder + 5% normal goat serum) was added and incubated at 37 ℃ for 60 min.

(6) The blocking solution was removed, no rinsing was performed, and monoclonal antibody secreted by OTI6C11 (0.5. mu.g/ml) was added, and the mixture was incubated in a wet box at 37 ℃ for 60 min. Wash 5min × 2 times with PBST (0.1% Tween-20). PBST (0.02% Tween-20) was washed for 5 min.

(7) Polink-kit 2(Catalog No. D37-15) reagent 1 was added dropwise and incubated at 37 ℃ for 10-20 minutes. Wash 5min × 3 times with PBS. Polink-2 kit reagent 2 was added dropwise, incubated at 37 ℃ for 10-20 min, and washed 5min × 3 times with PBS.

(8) Applying DAB solution (China fir Jinqiao ZLI-9019) for color development, and developing for 3-10 min. Washing with distilled water.

(9) Hematoxylin counterstain nuclei for 2min, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinsing with distilled water for 3 times, and standing at room temperature for 1 min.

(10) Dehydration and transparency: 75% ethanol for 5min, 100% ethanol for 5min × 3 times, 85% ethanol for 5min, 95% ethanol for 5min, and 100% ethanol for 5min × 3 times; xylene 5min × 3 times, and sealing with neutral gum.

(11) Microscopic examination, see fig. 5 and 6.

2. The experimental results are as follows:

(1) From the results of fig. 5, it can be seen that monoclonal antibody IHC secreted by hybridoma OTI6C11 detected that GPA33 protein was expressed in specific cytoplasm in intestinal cancer tissues.

(2) As can be seen from the results of fig. 6, monoclonal antibody IHC secreted by hybridoma OTI6C11 detected a GPA33 protein expressed in specific cytoplasm in gastric cancer tissues.

The result shows that the anti-GPA 33 monoclonal antibody secreted by the hybridoma cell strain OTI6C11 can specifically recognize GPA33 protein in intestinal cancer and gastric cancer tissues.

sequence listing

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Claims (7)

1. A monoclonal antibody characterized by: specifically binds to Glycoprotein A33 (glycoprotin A33, GPA33), and the antibody is secreted by hybridoma cell strain OTI6C11 with the preservation number of CGMCC No. 18200.

2. A hybridoma cell strain OTI6C11, which is characterized in that: secretes monoclonal antibody which is combined with GPA33 protein specificity, the preservation number of the hybridoma cell strain is CGMCC No. 18200.

3. Use of the monoclonal antibody of claim 1 for the preparation of an immunoassay tool for the detection of GPA33 protein.

4. The use according to claim 3, wherein the immunoassay means is a kit, chip or strip.

5. An immunohistochemical detection kit comprising the monoclonal antibody of claim 1.

6. Use of the monoclonal antibody of claim 1 for the preparation of a kit for labeling tumor cells and tumor tissue.

7. The use of claim 6, wherein the tumor cells comprise the intestinal cancer cell lines LoVo, COLO205, Caco-2, HT-29 and HCT 116; the tumor tissue includes intestinal cancer and gastric cancer tissue.