Anti-human TTC22v1 monoclonal antibody, hybridoma cell and application thereof

Abstract

The invention relates to the technical field of antibodies, and particularly provides an anti-human TTC22v1 monoclonal antibody, a hybridoma cell strain prepared from the anti-human TTC22v1 monoclonal antibody and application of the anti-human TTC22v1 monoclonal antibody. The monoclonal antibody can specifically recognize the 432 -569 amino acid region of the TTC22v1 protein, and is secreted by a hybridoma cell strain TTC22v1-06 # with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C202594. The antibody has high specificity and high sensitivity, can obviously distinguish TTC22v1 and TTC22v2 proteins, is especially suitable for Western blot and cell immunofluorescence detection, can effectively detect expression of endogenous TTC22v1 proteins in colon cancer cells, provides a reliable detection tool for research of TTC22v1 in tumor research and disease diagnosis, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to an anti-human TTC22v1 monoclonal antibody, hybridoma cells, and their applications. Background Art

[0002] TTC22 has two transcripts, which translate into two proteins, one long and one short, with the same N-terminus but different C-terminus. TTC22 virant1 (amino acids 1-569) and TTC22 virant2 (amino acids 1-372) are identical only in amino acid sequences 1-340.

[0003] Based on our previous research, we found that TTC22v1 (excluding v2) is a target gene of miR663a and promotes colorectal cancer metastasis (PMID: 30664167). Therefore, the preparation of high-affinity and high-specificity antibodies against endogenous TTC22v1 is necessary to study its function and significance.

[0004] Currently, there are very few TTC22 antibodies available for Western blot analysis: Abcam's ab182537 and Santa Cruz's sc-249130 and sc-249128 cannot detect endogenous TTC22v1 in colon cancer cells in Western blot analysis, and both have been discontinued. ThrmoFisher's PA5-71278 and Wako Pure Chemical Industries' PA571278 (possibly from the same clone) are recommended for WB, but they target the N-terminus of the antigen and cannot distinguish between TTC22 virant 1 and TTC22 virant 2. There are currently no commercially available antibodies specifically targeting TTC22 virant 1. Three other commercially available antibodies target TTC22 aa359-448: Sigma's HPA035072, Novus' rabbit polyclonal antibody NBP1-93970, and ThrmoFisher's polyclonal antibody PA5-57142, but all three are recommended for IHC.

[0005] Therefore, currently available TTC22 antibodies for Western blot all recognize both TTC22 virant 1 and TTC22 virant 2, but exhibit poor specificity and limited efficacy in detecting endogenous TTC22 protein in colon cancer cells. There is an urgent need to develop an antibody that targets only TTC22 virant 1 (specifically, TTC22 virant 1 aa349-569). Summary of the Invention

[0006] Existing commercially available TTC22 antibodies generally lack specificity and cannot effectively distinguish between different TTC22 transcripts, especially failing to specifically detect the TTC22v1 protein endogenously expressed in colon cancer cells, posing challenges to research on related diseases. The purpose of this invention is to provide a highly specific, highly sensitive monoclonal antibody capable of effectively distinguishing between TTC22v1 and TTC22v2 proteins, as well as the hybridoma cell line for preparing this antibody and related applications, to overcome the aforementioned technical deficiencies.

[0007] To achieve the above objectives, the present invention specifically provides the following technical solutions: In a first aspect, the present invention provides for the first time an anti-human TTC22v1 monoclonal antibody that specifically recognizes the amino acid region 432-569 of the TTC22v1 protein. This antibody is secreted by the hybridoma cell line TTC22v1-06#. This hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202594.

[0008] In a second aspect, the present invention provides a hybridoma cell line that secretes the above-mentioned specific monoclonal antibody, namely hybridoma cell line TTC22v1-06#, whose accession number is CCTCC NO: C202594.

[0009] In a third aspect, the present invention further provides a detection kit containing the above-mentioned specific antibodies, the kit being suitable for the specific detection of endogenously or exogenously expressed TTC22v1 protein in Western blot and cell immunofluorescence experiments.

[0010] In one embodiment, the kit further includes one or more of the following: secondary antibody, chromogenic reagent, and blocking solution required for Western blot detection.

[0011] In one embodiment, the kit further comprises one or more of the following: a fluorescently labeled secondary antibody, a cell fixative, and a cell permeation solution required for cell immunofluorescence detection.

[0012] In a fourth aspect, the present invention also provides the use of the above-described monoclonal antibody in the preparation of a reagent for detecting endogenous TTC22v1 protein in colon cancer cells.

[0013] In a fifth aspect, the present invention further provides a method for specifically detecting TTC22v1 protein expression in a sample using the aforementioned monoclonal antibody. This method specifically includes: The above-mentioned antibody was used as the primary antibody; The expression of TTC22v1 protein was detected using Western blot or immunofluorescence assays.

[0014] In one embodiment, the sample is selected from colon cancer cells or colon cancer tissue.

[0015] In one implementation, the specific operation of Western blot in step (2) includes the following steps: (a) Proteins were extracted from the samples after lysis and subjected to SDS-PAGE gel electrophoresis; (b) After electrophoresis, the proteins are transferred to a PVDF membrane; (c) After incubating the PVDF membrane with the aforementioned antibody, add the corresponding secondary antibody for incubation and color development.

[0016] In one implementation, the cell immunofluorescence experiment in step (2) specifically includes the following steps: (a) After fixing the cell sample to be tested with a fixative, permeabilize it; (b) After incubation with the aforementioned antibody, further incubation with a fluorescently labeled secondary antibody; (c) Use a fluorescence microscope to observe and detect fluorescence signals to determine the expression status of TTC22v1 protein.

[0017] Compared with the prior art, the present invention has the following outstanding advantages and technical effects: High specificity: The antibody of this invention specifically recognizes the amino acid region of TTC22v1 protein from position 432 to 569, and can significantly distinguish TTC22v1 from TTC22v2 protein; High sensitivity: It can effectively detect both endogenous and exogenous expression of TTC22v1 protein in cells; Highly practical: This antibody is particularly suitable for Western blot and cellular immunofluorescence experiments; Highly innovative: For the first time, a monoclonal antibody that specifically recognizes TTC22v1 and a hybridoma cell line that stably secretes the antibody were obtained, making up for the lack of commercially available antibodies. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is the result of antigenicity analysis; Figure 2 These are the sequencing results; Figure 3 The result is the antigen purification result; Figure 4 Western blot results for TTC22v1-04#, 07#, and 06# monoclonal antibodies; Figure 5Western blot results of TTC22v1-06# monoclonal antibody compared with three other commercial TTC22 antibodies; Figure 6 The left figure shows the recognition effect of TTC22v1 06# monoclonal antibody on different truncated forms of TTC22v1; Figure 6 The right figure shows the expression of the truncated and full-length variants as detected by the tagged antibody (quality control verification). Figure 7 Immunoprecipitation results for TTC22v1-04#, 07# and 06# monoclonal antibodies; Figure 8 The results of immunofluorescence assays for detecting LoVo cells in colon cancer using TTC22v1-06# monoclonal antibody (OE: overexpression; NM: normal mouse). Detailed Implementation

[0019] 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.

[0020] Example 1: Preparation of Antibody 1. Antigen analysis and selection Most commercially available TTC22 antibodies target the N-terminus aa146-195 or aa359-448 of the TTC22 protein. The former cannot specifically recognize TTC22v1, and both have very limited recognition capabilities for TTC22v1. We used TMPred software to analyze and predict that TTC22v1 lacks a hydrophobic region. For example... Figure 1 As shown, the region within the green box exhibits good antigenicity and hydrophilicity. Analysis of sequence hydrophobicity, homology, antigenicity, hydrophilicity, and the likelihood of protein expression in prokaryotic systems determined that TTC22v1 (NP_001107580.1)aa349-569 could be selected as the antigen for expression, purification, and immunization.

[0021] 2. Antigen expression and purification A prokaryotic expression vector for the TTC22v1 fragment (aa349-569) was constructed, transformed, and induced to express in small quantities. After large-scale expression and purification, sufficient protein was obtained to initiate immunization (purity greater than 80%, concentration 1 mg / ml).

[0022] a. Using the TTC22v1 overexpression plasmid (Origene, RC225966) as a template, primers were designed and synthesized for PCR amplification. The plasmid was inserted into the pET28a vector using BamHI and HindIII. Enzyme digestion confirmed the results were consistent with expectations, and sequencing results were correct (e.g., ...). Figure 2 ); b. The constructed expression vector was transformed into BL21(DE3) competent cells. Single colonies were picked and gradually expanded to 1000 mL of bacteria. 0.5 mM IPTG was added and induced at 20°C for 12 hours. After centrifugation, the cells were sonicated (300W, 10 seconds sonication, 10-second intervals, 30 times) in 100 mL of lysis buffer. After centrifugation, the precipitate and supernatant were collected separately. The results showed that TTC22v1 (aa349-569) protein was mainly expressed in the form of inclusion bodies. The precipitate collected after centrifugation was dissolved in urea and loaded onto a pre-equilibrated NTA purification column. Different fractions were collected stepwise and detected by electrophoresis. Finally, the fractions containing the target protein were mixed together. The purification results were obtained by gel electrophoresis. Figure 3 .

[0023] 3. Animal immunization and testing Seven BALB / c mice (purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were immunized with antigen. The serum titer of the mice was tested after the third immunization. After six immunizations, the serum titer of the mice exceeded 1:10000, and the mice were used for cell fusion.

[0024] 4. Cell fusion and screening Spleens from mice (04#, 05#, 06#, and 07#) with high serum titers were selected for fusion. After 3-4 rounds of selection and subcloning, one hybridoma cell line stably secreting antibodies against TTC22v1 (aa349-569) was obtained from each mouse spleen. The four obtained hybridoma cell lines were expanded and cryopreserved. Microscopic examination the next day showed a cell viability >80%, indicating successful cryopreservation.

[0025] 5. Monoclonal antibody production The four hybridoma cell lines were revived. New BALB / c mice were pre-injected with paraffin oil via intraperitoneal injection (0.5 ml / mouse). At least 7 days later, each mouse was injected intraperitoneally with 1-2 × 10⁻⁶ cells. 6Cells. Mice were observed daily and euthanized by cervical dislocation shortly before death. Ascites fluid was aspirated with a dropper, centrifuged, and the supernatant was collected. A small amount of ELISA was used to detect the ascites fluid titer. Antibodies obtained from each hybridoma were purified using a Protein G column and named according to their mouse origin: TTC22v1-04# monoclonal antibody, TTC22v1-05# monoclonal antibody, TTC22v1-06# monoclonal antibody, and TTC22v1-07# monoclonal antibody. Elution with glycine solution, dialyzed overnight with PBS, and titer was detected by ELISA. The results are shown in the table below: The titers of TTC22v1 monoclonal antibodies 04# / 05# / 06# / 07# met the standards, and the ELISA results were satisfactory. At the same dilution ratio, the binding efficiencies of TTC22v1 monoclonal antibodies 04# / 05# / 06# / 07# to the antigen his-TTC22v1 (aa349-569) protein were 29, 23, 27, and 23 times that of blank mouse IgG, respectively. Among them, 05# had a low overall yield and was used less frequently. Subsequent Western blot analysis showed that 06# had better specificity than 04# and 07# monoclonal antibodies. The hybridoma cell line TTC22v1-06# was deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2025, with accession number CCTCCNO:C202594 and accession name: Hybridoma cell line TTC22v1-06#.

[0026] Judgment criterion: S / N > 2.1 (S: antibody detection OD value, N: blank control detection OD value) Example 2: Specificity Identification and Application of TTC22v1-06# Monoclonal Antibody 1. The TTC22v1-06# monoclonal antibody exhibits superior specificity for recognizing the TTC22v1 protein compared to TTC22v1-04# and 07#. Whole-cell lysates of colon cancer cell lines SW480 (a gift from Professor Chen Yuanjia, Peking Union Medical College) and LoVo (a gift from Professor Shou Chengchao, Peking University Cancer Hospital) were cultured and collected. Simultaneously, the full-length plasmid expressing TTC22v1 and the control empty vector were overexpressed in colon cancer cell line HCT116 (a gift from Professor Chen Yuanjia, Peking Union Medical College). 10 μL of whole-cell lysate was loaded onto a 12% SDS-PAGE gel, and wet-transferred at 200 mA onto a PVDF membrane. Western blot analysis was performed using the aforementioned TTC22v1 04#, TTC22v1 07#, and TTC22v1 06# monoclonal antibodies (05# had low overall yield and was used less frequently later) as primary antibodies (primary antibody concentration 1 μg / ml, diluted with 5% milk, reaction at room temperature for 1.5 h).

[0027] The results are as follows Figure 4 The 04# and 06# monoclonal antibodies showed better recognition efficacy against overexpressed TTC22v1 than the 07# monoclonal antibody. However, the 06# monoclonal antibody showed better recognition efficacy against endogenous TTC22v1 in SW480 and LoVo cells than the 04# monoclonal antibody.

[0028] 2. The TTC22v1-06# monoclonal antibody demonstrates superior recognition ability for TTC22v1 compared to the three commercially available TTC22 monoclonal antibodies purchased by our laboratory. Following the same method described above, our laboratory's TTC22v1-06# monoclonal antibody and three commercially available antibodies against TTC22 (Abcam's ab-182537 / Lot:GR3177364-I, Sigma's HPA035072 / Lot:R32445, and Santacruz's sc-249128 / Lot:#12112) were used as primary antibodies for Western blot analysis (primary antibody concentrations were 1 μg / ml except for HPA035072, which was 0.2 μg / ml; the others were diluted with 5% milk and reacted at room temperature for 1.5 h).

[0029] The results are as follows Figure 5 As shown, the TTC22v1-06# monoclonal antibody has significantly better recognition efficacy against overexpressed TTC22v1 than Abcam's ab-182537 and Santa Cruz's sc-249128; and its recognition efficacy against endogenous TTC22v1 in SW480 and LoVo cells is better than Sigma's HPA035072 and the two commercial antibodies mentioned above.

[0030] 3. The TTC22v1-06# monoclonal antibody recognizes amino acids 432-569 of TTC22v1. The full-length plasmid of TTC22v1 (RC225966, purchased from OriGene Tech Inc. USA) and different truncated forms (obtained by PCR and molecular cloning using the full-length plasmid as a template, see reference PMID:35798874) were overexpressed in HCT116 colon cancer cells. The cells were loaded with 10 μL of whole-cell lysis buffer and run on a 12% SDS-PAGE gel. The cells were then wet-transferred to a PVDF membrane at 200 mA and detected by Western blot using the TTC22v1-06# monoclonal antibody. Simultaneously, our laboratory's full-length TTC22 immunization rabbit polyclonal antibody (obtained by immunizing New Zealand white rabbits with full-length TTC22v1 protein, see literature PMID: 35798874) and commercial antibody Flag (66008-4-Ig, purchased from Proteintech Group, USA) and GAPDH antibody (60004-1-Ig, purchased from Proteintech Group, USA) were used for quality control to detect the overexpression effect of each truncated variant (TTC22 rabbit polyclonal antibody and mouse monoclonal antibody primary antibody concentration were both 1 μg / ml, diluted with 5% milk, and reacted at room temperature for 1.5 h).

[0031] The results showed that the TTC22v1-06# monoclonal antibody only bound to the full-length plasmid containing TTC22v1 aa432-569, and did not bind to any of the truncated variants of TTC22v1 aa1-431. Figure 6 (Left) The results of detection of TTC22 rabbit polyclonal antibody, Flag antibody, and GAPDH antibody using tag antibodies demonstrated that the full-length and various truncated forms of TTC22 rabbit polyclonal antibody showed good protein expression. Figure 6 (Right). TTC22v1-06# monoclonal antibody binds to a specific region of TTC22v1, effectively distinguishing TTC22v1 from TTC22v2. TTC22v1-06# monoclonal antibody binds significantly to TTC22v1, and its detection efficacy is superior to the aforementioned TTC22 rabbit polyclonal antibody and commercial antibody Flag.

[0032] The TTC22v1-06# monoclonal antibody recognizes the antigenic epitope of the TTC22v1 protein, located at amino acids 432-569 of TTC22v1, as shown in the following sequence: PELQLLRGKCLRIKGEDANAACFKRAVELDDAGSSHTDGFGCLLEALLAQWSQAQLSDGELGR EVDAWLRRAQDKYPAARLRQELQRVWRGHTDEVLGLARALVAQGRPALVRLLFETMEREGEGAS APRDRRAVSF (SEQ ID NO. 1).

[0033] 4. Poor efficacy against endogenous TTC22v1 protein IP (immunoprecipitation). 1 μg of TTC22v1-04# / 07# / 06# monoclonal antibody was bound to 20 μl of protein A+G Agrose (REF11134515001 and REF11243233001, purchased from Roche, Germany), and incubated overnight at 4°C with 1 mg of HCT116 cell lysis buffer (a gift from Professor Chen Yuanjia, Peking Union Medical College). After gel electrophoresis and transfer of the IP product, Western blots were performed using TTC22v1-06# monoclonal antibody and TTC22 rabbit polyclonal antibody, respectively. The results showed no obvious enrichment signal at the corresponding position of TTC22v1. Figure 7 This indicates that none of the three monoclonal antibodies, TTC22v1-04#, 06#, and 07#, significantly enriched TTC22v1 in immunoprecipitation. A possible reason is that the cell lysis buffer used in the above IP experiments was an undenatured protein, which may have resulted in amino acids 432-569 of TTC22v1 being encapsulated by secondary structures and not fully exposed. Current results do not support the use of these antibodies for immunoprecipitation enrichment of TTC22v1 protein, but TTC22v1 can be specifically detected in Western blot and immunofluorescence assays.

[0034] 5. Used for IF (Immunofluorescence) detection of TTC22v1 protein LoVo cells were fixed with ice-cold methanol (a gift from Professor Shou Chengchao of Peking University Cancer Hospital). TTC22v1-06# monoclonal antibody was diluted 1:200 as the primary antibody, and normal mouse IgG (CS200621, Millipore, USA) was used as a control. It was observed that TTC22v1-06# monoclonal antibody could detect specific signals in LoVo cells (e.g., ...). Figure 8 ): Regardless of whether it is in LoVo cells overexpressing the full length of TTC22v1 ( Figure 8 (Above) or in wild-type LoVo cells ( Figure 8 In both groups (Chinese and Western medicine), TTC22v1 was detected by the TTC22v1-06# monoclonal antibody. The signal was stronger in the TTC22v1 overexpression group, and there was no difference in the cellular sublocalization of the fluorescence signal between the two groups. Control normal mouse IgG showed no fluorescence signal in LoVo cell assays. Figure 8 (See below). The results above demonstrate that at a working concentration of 5 μg / mL, the TTC22v1-06# monoclonal antibody can recognize not only overexpressed TTC22v1 but also endogenous TTC22v1. The TTC22v1-06# monoclonal antibody can sensitively, specifically, and accurately recognize TTC22v1 in cellular immunofluorescence experiments.

[0035] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A monoclonal antibody against human TTC22v1, characterized in that: The antibody specifically recognizes amino acid region 432-569 of the TTC22v1 protein. The antibody is obtained by secretion from hybridoma cell line TTC22v1-06#, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC N0: C202594.

2. A hybridoma cell line TTC22v1-06#, characterized in that: It is deposited at the China Center for Type Culture Collection, with accession number CCTCC N0: C202594.

3. A kit comprising the antibody of claim 1, characterized in that: The kit is used for the specific detection of TTC22v1 protein in Western blot and cellular immunofluorescence assays.

4. The reagent kit according to claim 3, characterized in that: The kit further includes one or more of the following: secondary antibody, chromogenic reagent, and blocking solution required for Western blot detection.

5. The reagent kit according to claim 3, characterized in that: The kit further includes one or more of the following: a fluorescently labeled secondary antibody, a cell fixative, and a cell permeation solution required for cell immunofluorescence detection.

6. Use of the antibody of claim 1 in the preparation of a reagent for detecting endogenous TTC22v1 protein in colon cancer cells.

7. A method for in vitro detection of TTC22v1 protein in a sample, comprising the following steps: (1) The antibody described in claim 1 is used as the primary antibody; (2) The expression of TTC22v1 protein was detected by Western blot or cell immunofluorescence assay.

8. The method according to claim 7, characterized in that: The samples were selected from colon cancer cells or colon cancer tissue.

9. The method according to claim 7, characterized in that: The specific operation of Western blot in step (2) includes the following steps: (a) the sample is lysed to extract protein and then subjected to SDS-PAGE gel electrophoresis; (b) after electrophoresis, the protein is transferred to a PVDF membrane; (c) after incubating the PVDF membrane with the antibody described in claim 1, the corresponding secondary antibody is added for incubation and color development.

10. The method according to claim 7, characterized in that: The specific operation of the cell immunofluorescence experiment in step (2) includes the following steps: (a) after fixing the cell sample to be tested with fixative, perform permeabilization treatment; (b) after adding the antibody described in claim 1 for incubation, further incubate with fluorescently labeled secondary antibody; (c) observe and detect the fluorescence signal using a fluorescence microscope to determine the expression status of TTC22v1 protein.

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