A hybridoma cell line secreting anti-human Claudin 16 protein monoclonal antibody and its application

Abstract

The present invention discloses a hybridoma cell line and application that secretes an anti-human Claudin 16 protein monoclonal antibody, including the hybridoma cell line 1F8E5. The deposit number of the hybridoma cell line 1F8E5 is CCTCC NO: C202298, the deposit date is July 19, 2022, and the depository is the China Center for Type Culture Collection. The monoclonal antibody provided by the present invention can specifically bind to the human Claudin 16 protein and specifically target the cellular antigen epitope. The antigen epitope includes a segment corresponding to positions 95-150 of the amino acid sequence shown in SEQ ID NO: 1, has high titer and good affinity, and can specifically detect the human Claudin 16 protein. The antibody of the present invention can recognize the extracellular segment of the Claudin 16 protein of ovarian cancer cells and has the potential value of being developed for clinical sample detection and immunohistochemical pathological diagnosis of ovarian cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell line secreting anti-human Claudin 16 protein monoclonal antibody and its application. Background Art

[0002] Ovarian cancer, a malignant tumor that arises in the ovaries, is one of the most common malignant tumors of the female reproductive organs, second only to cervical cancer and uterine corpus cancer in incidence. Epithelial carcinomas are the most common type of ovarian cancer, followed by malignant germ cell tumors. Epithelial ovarian cancer leads the mortality rate among all gynecological cancers, posing a serious threat to women's lives. Early ovarian cancer is often asymptomatic, but in advanced stages, digestive symptoms such as lower abdominal discomfort, bloating, and decreased appetite may occur. Treatment options include surgical resection, medications, and radiotherapy, with an overall poor prognosis. In recent years, the number of women diagnosed with ovarian cancer in my country has increased significantly. Early symptoms are nonspecific and clinical screening is limited, making early diagnosis challenging. The heterogeneity of tumors, including ovarian cancer, is gaining widespread attention. Even tumors with similar clinical manifestations or traditional pathological classifications can have distinct molecular origins. This requires deeper understanding of the molecular pathology of tumors to improve the precision of diagnosis and treatment. Currently, immunological detection of key tumor-associated proteins is a crucial component of precision cancer diagnosis and treatment. Preliminary studies of this application found that the expression level of Claudin 16, a member of the human Claudin protein family, is related to the differential diagnosis of ovarian cancer. The independently developed monoclonal antibody against human Claudin 16 protein can specifically recognize the extracellular segment of human Claudin 16 protein, and thus be used for immunofluorescence detection of ovarian cancer cells.

[0003] Claudin (CLDN) is a key protein in tight junctions in normal human tissues. It possesses four transmembrane domains and is a crucial molecule in cell tight junctions. It participates in physiological processes such as the regulation of paracellular permeability and conductance, forming a paracellular barrier that controls the flow of molecules between cells. Claudin forms an integral membrane protein family and is crucial for the formation and function of tight junctions (TJs). Claudin 16, also known as Paracellin-1, belongs to the Claudin protein family, which consists of 23 integral membrane proteins. It is a renal tight junction protein required for paracellular magnesium absorption. Regulation of TJ structure and function has been shown to be associated with the development of epithelial tumors. Studies have shown that Claudin 3 and Claudin 4 are among the most highly upregulated genes in ovarian cancer. Furthermore, increased expression of Claudin 3 and Claudin 4 in ovarian cancer cells is associated with increased invasiveness, suggesting their involvement in promoting ovarian tumor development and metastasis. Research in this application demonstrates that Claudin 16 can serve as a new, specific marker for ovarian cancer. To elucidate the role of Claudin 16 in ovarian tumorigenesis, we generated a monoclonal antibody against the extracellular domain of the Claudin 16 protein and used immunofluorescence to investigate the expression pattern of Claudin 16 in normal and malignant ovarian tissues and ovarian cancer cells. The results showed that Claudin 16 is expressed at high levels in most ovarian cancer tissues, primarily distributed on the cell membrane but also in the cytoplasm. Immunofluorescence revealed that Claudin 16 is highly overexpressed in many ovarian cancer cells. Expression of Claudin 16 in Claudin-negative cells and knockdown of Claudin 16 in Claudin-positive ovarian cancer cells were associated with phenotypic changes, such as altered cell permeability and invasiveness. These results suggest that Claudin 16 may be a potential target for the differential diagnosis and treatment of ovarian cancer.

[0004] Currently, there are no patents related to monoclonal antibodies targeting the extracellular domain of human Claudin 16 protein and indirect immunofluorescence detection methods. Therefore, a hybridoma cell line secreting anti-human Claudin 16 protein monoclonal antibodies and its application are urgently needed. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention provides a hybridoma cell line that secretes monoclonal antibodies against human Claudin 16 protein and its applications. By preparing monoclonal antibodies targeting the extracellular domain of human Claudin 16 protein, the present invention establishes an indirect immunofluorescence assay for detecting human Claudin 16 protein. This assay exhibits excellent specificity and can better detect the distribution of human Claudin 16 protein in tissue cells, providing an effective means for detecting the localization and dynamic distribution of Claudin 16 protein in ovarian cancer cells.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a hybridoma cell line that secretes an anti-human Claudin 16 protein monoclonal antibody, including the hybridoma cell line 1F8E5. The hybridoma cell line 1F8E5 has a deposit number of CCTCC NO: C202298 and a deposit date of July 19, 2022. The depository is the China Center for Type Culture Collection, and the depository address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The second object of the present invention is to provide a monoclonal antibody, which is secreted by the hybridoma cell line 1F8E5 and can specifically recognize the extracellular segment of human Claudin 16 protein. The amino acid sequence of the recognition site is shown in SEQ ID NO: 2.

[0009] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0010] The third object of the present invention is to provide a detection kit, which includes a detection antibody and a fluorescent secondary antibody, wherein the detection antibody is the above-mentioned monoclonal antibody, and the fluorescent secondary antibody is FITC-labeled goat anti-mouse IgG.

[0011] Preferably, it further comprises one or more of a fixing solution, a washing solution, a blocking solution, and a diluent.

[0012] The fourth object of the present invention is to provide a use of a monoclonal antibody in the preparation of an immunofluorescence detection reagent for ovarian cancer cells.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The monoclonal antibody provided by the present invention can specifically bind to the human Claudin 16 protein. The monoclonal antibody specifically targets an antigenic epitope corresponding to the segment of amino acid sequences from positions 95 to 150 of SEQ ID NO: 1. The antibody has high titer and good affinity, and can be used to specifically detect the human Claudin 16 protein and for immunofluorescence detection of ovarian cancer cells. The antibody provided by the present invention can specifically detect the cell surface Claudin 16 protein, namely the extracellular segment of the Claudin 16 protein, and can effectively detect the expression level and location of the Claudin 16 protein in ovarian cancer tissue cells, facilitating direct interpretation of the localization and expression of the Claudin 16 protein in ovarian cancer tissue cells, and thus serving as a basis for determining the individualized heterogeneity and survival prognosis of ovarian cancer.

[0015] Cell preservation:

[0016] The present invention provides a hybridoma cell line that secretes an anti-human Claudin 16 protein monoclonal antibody, including the hybridoma cell line 1F8E5. The hybridoma cell line 1F8E5 was screened by the inventors of the present invention. The hybridoma cell line 1F8E5 has an accession number of CCTCC NO: C202298, a deposit date of July 19, 2022, and is deposited with the China Center for Type Culture Collection. The depository address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the SDS-PAGE results in Example 1 of the present invention;

[0018] Figure 2 Schematic diagram of Western Blotting analysis results in Example 1 of the present invention;

[0019] Figure 3 This is a schematic diagram of the SDS-PAGE results in Example 2 of the present invention;

[0020] Figure 4 Schematic diagram of Western Blotting results in Example 2 of the present invention;

[0021] Figure 5 This is a schematic diagram of the monoclonal antibody 1F8E5 used in Example 3 of the present invention for validating the expression of Claudin 16 in ovarian cells;

[0022] Figure 6 This is a schematic diagram of the expression of Claudin 16 on the surface of different ovarian cancer cells in Example 5 of the present invention. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0024] The GeneBank international universal sequence number of the gene Claudin 16 in the present invention is: NC_000003.12, and the GeneBank international universal sequence number of the protein encoded by the gene Claudin 16 is: NP_006571.2.

[0025] Example 1: Expression, purification and identification of recombinant human Claudin 16 protein.

[0026] The claudin 16 gene was cloned from ovarian cancer cells (OVCA R3) using RT-PCR using primers Claudin 16-F and Claudin 16-R (the gene sequences of Claudin 16-F are shown in SEQ ID NO: 5, the gene sequence of Claudin 16-R is shown in SEQ ID NO: 6, and the gene sequence of Claudin 16 is shown in SEQ ID NO: 7). The cloned claudin 16 gene fragment was then ligated into the His-tag pcDNA3.1 vector to generate the pcDNA3.1-Claudin 16 recombinant fragment. The pcDNA3.1-Claudin 16 fragment was then transformed into DH5α cells, and positive clones were selected and expanded. The recombinant plasmid pcDNA3.1-Claudin 16 was then extracted and digested with Hind III and Bam HI, indicating that the expected size fragment was obtained. The recombinant plasmid pc DNA3.1-Claudin 16 with the correct enzyme digestion band was sequenced and confirmed to be correct. The gene was then transfected into 293 suspension cells using PEI and cultured in 293 serum-free medium (37°C, 8% CO2) for 6 days. Supplement was added midway, and the culture supernatant was collected and purified with a nickel column. The cell expression product was analyzed by SDS-PAGE. The results are as follows. Figure 1 The results showed that a specific expression band was found at around 25KD, which was consistent with the size of the recombinant protein of the extracellular segment of Claudin 16 predicted by the gene sequence. Western Blotting analysis was performed using Anti-6x His antibody, and the results were as follows Figure 2 As shown, there is only one specific band, indicating that the protein is the target protein.

[0027] The cells were expanded using the transfection method described above and the protein was purified. Specifically, the cell culture medium was collected, the cells were centrifuged at 1200 rpm, the supernatant was collected, and the supernatant was filtered through a 0.45 μm filter. The filtrate was loaded onto a 5 ml Ni-NTA column, washed with 5 column volumes of 10 mM imidazole solution, and then eluted with 300 mM imidazole. The eluted protein solution was collected from each tube. Protein purity was assessed by SDS-PAGE and quantified. The obtained protein purity reached 95%, and the concentration after concentration was 5.86 mg / ml.

[0028] Example 2: Preparation, purification and identification of Claudin 16 monoclonal antibody.

[0029] The purified Claudin 16 recombinant protein in Example 1 was used to immunize BALB / c mice by intraperitoneal injection. Each mouse was immunized with 50 μg of antigen in an immunization volume of 100 μl. For the first immunization, Claudin 16 protein was mixed with Freund's complete adjuvant in a 1:1 ratio, and immunization was performed every two weeks. Claudin 16 protein was mixed with Freund's incomplete adjuvant in a 1:1 ratio, and immunization was performed three times in total. Finally, Claudin 16 protein (without adjuvant) was used for intraperitoneal booster immunization (30ug). Three days later, spleen cells of the immunized mice were collected, and then PEG-4000 was used to fuse the spleen cells of the immunized mice with mouse myeloma cells SP2 / 0. Hybridoma cells were selected on a 384-well cell culture plate using HAT selection medium, and cells producing anti-Claudin 16 protein extracellular segment were identified by ELISA. They were hybridoma cell lines 1F8E5, hybridoma cell line 3H6F11, hybridoma cell line 4F7D7, hybridoma cell line 4F11G6, and hybridoma cell line 3E8G12. Finally, the hybridoma cell lines obtained by roller bottle culture were collected, the cell culture supernatant was collected, the solution was filtered using a 0.45um filter, and the cell supernatant containing monoclonal antibodies was purified by AKTA On the purification system, the sample was loaded onto a Protein A-Sepharose affinity chromatography column at a flow rate of 1 ml / min, washed with 5 column volumes of binding buffer (50 mM PBS pH 7.0), and then eluted with 0.1 M glycine-hydrochloric acid solution pH 2.7 (1 M pH 9.0 Tris buffer was pre-neutralized in each collection tube) to obtain the target antibody. The isolated and purified monoclonal antibodies were identified by SDS-PAGE and Western Blotting and named 1F8E5 monoclonal antibody, 3H6F11 monoclonal antibody, 4F7D7 monoclonal antibody, 4F11G6 monoclonal antibody, and 3E8G12 monoclonal antibody, respectively. The SDS-PAGE results are shown in the figure. Figure 3As shown in Figure 2, the purified monoclonal antibody contains only a heavy chain at 50 kD and a light chain at 25 kD. Grayscale analysis shows that the antibody purity reaches 90%. 5 μg of Claudin 16 recombinant protein was loaded into each well. The above monoclonal antibody was diluted 1:10,000. The Western Blotting results are shown in Figure 2. Figure 4 As shown, the antibodies were shown to react specifically with the recombinant protein.

[0030] Example 3: Claudin 16 immunofluorescence antibody screening.

[0031] Ovarian cells (OVCA R3) were cultured to 90% confluency. Fixative solution (formaldehyde: acetone in a 1:1 ratio) was added to the cells and incubated at room temperature for 10 minutes. The cells were washed three times with PBS, each wash lasting 5 minutes. Blocking solution (PBST containing 1% BSA) was added. The cells were incubated at 37°C for 1 hour. The cells were washed three times with PBS, each wash lasting 5 minutes. Claudin 16 monoclonal antibody (antibody) diluted in PBST containing 1% BSA was added. The cells were incubated at 37°C for 1 hour. The cells were washed four times with PBST, each wash lasting 5 minutes. FITC-conjugated goat anti-mouse IgG was diluted 1:1000 in diluent and incubated at 37°C for 1 hour. The cells were washed four times with PBST, each wash lasting 5 minutes. Nuclei were stained with DAPI for 10 minutes at room temperature. The cells were washed three times with PBST, each wash lasting 5 minutes. Finally, buffered glycerol (50% glycerol in water) was added, and the cells were photographed under a fluorescence microscope to observe the antibody interaction. The results showed that the 1F8E5 monoclonal antibody had high affinity and specificity and had no specific cross-reaction with enzyme-labeled antibodies (e.g. Figure 5 shown).

[0032] Example 4: Claudin 16 immunofluorescence detection kit.

[0033] Kit components: Reagent 1: Fixative; Reagent 2: Blocking solution; Reagent 3: Mouse anti-human Claudin 16 monoclonal antibody (1F8E5); Reagent 4: FITC-labeled goat anti-mouse IgG; Reagent 5: Washing solution.

[0034] Example 5: Claudin 16 immunohistochemistry kit was used to detect ovarian cancer cell samples.

[0035] Different ovarian cancer cells were cultured in a conventional manner and detected using the Claudin 16 immunofluorescence detection kit described in Example 4 ( Figure 6 ).

[0036] Results under a fluorescence microscope: The expression levels of Claudin 16 protein vary in different ovarian cancer cells. The immunofluorescence detection method using the antibody provided by the present invention can effectively detect the expression level and expression location of Claudin 16 protein in ovarian cancer cells. This facilitates direct interpretation of the localization and expression of Claudin 16 protein in ovarian cancer tissue cells from the immunofluorescence image, and can be used as a basis for determining the individual heterogeneity of ovarian cancer and providing medication guidance.

[0037] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A hybridoma cell line secreting anti-human Claudin 16 protein monoclonal antibody, characterized in that: Including hybridoma cell line 1F8E5, the preservation number of the hybridoma cell line 1F8E5 is CCTCC NO: C202298, the preservation date is July 19, 2022, the preservation unit is China Center for Type Culture Collection, and the preservation unit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University.

2. A monoclonal antibody, characterized in that The monoclonal antibody is secreted by the hybridoma cell line 1F8E5 according to claim 1, and can specifically recognize the extracellular segment of human Claudin 16 protein. The amino acid sequence of the recognition site is shown in SEQ ID NO:

2.

3. The monoclonal antibody according to claim 2, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

4. A detection kit, characterized in that The detection kit comprises a detection antibody and a fluorescent secondary antibody, wherein the detection antibody is the monoclonal antibody according to claim 2 or 3, and the fluorescent secondary antibody is a FITC-labeled goat anti-mouse IgG.

5. The detection kit according to claim 4, characterized in that It also includes one or more of a fixing solution, a washing solution, a blocking solution, and a diluent.

6. Use of the monoclonal antibody according to claim 2 or 3 in the preparation of a reagent for immunofluorescence detection of ovarian cancer cells.

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