An anti-human ttc22v1 monoclonal antibody, hybridoma cells and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular, the present application relates to an anti-human TTC22v1 monoclonal antibody, hybridoma cells and application thereof. BACKGROUND

[0002] TTC22 has two transcripts, which are translated into two proteins with long and short, the N-terminal is the same, and the C-terminal is different. TTC22 virant1 (amino acid 1-569) and TTC22 virant2 (amino acid 1-372) are only the same in amino acid sequence 1-340.

[0003] According to our previous research results, it is found that TTC22v1 (not including v2) is the target gene of miR663a, which promotes colon cancer metastasis (PMID: 30664167). Therefore, it is necessary to prepare a high-affinity and high-specificity antibody for endogenous TTC22v1 to study its function and significance.

[0004] At present, there are very limited TTC22 antibodies available on the market for Western blot: ab182537 of Abeam and sc-249130, sc-249128 of Santa-cruz cannot detect endogenous TTC22v1 in colon cancer cells in Western blot, and have been discontinued. PA5-71278 of ThrmoFisher and PA571278 of Japan and light pure drug WAKO (may come from the same clone) are recommended for WB, but they are directed against the N-terminal of the antigen, and cannot distinguish TTC22 virant1 and TTC22 virant2. There is no antibody specific to TTC22 virant1 on the market at present. There are three commercial antibodies against TTC22 aa359-448: HPA035072 of sigma, rabbit polyclonal antibody NBP1-93970 of Novus, and polyclonal antibody PA5-57142 of ThrmoFisher, but these three are recommended for IHC.

[0005] Therefore, so far, all the TTC22 antibodies available on the market for Western blot can recognize TTC22 virant1 and TTC22 virant2, and have poor specificity, and the detection efficiency of endogenous TTC22 protein in colon cancer cells is not good. It is urgent to develop an antibody only for TTC22 virant1 (directed against TTC22 virant1 aa349-569). SUMMARY

[0006] The existing commercially available TTC22 antibodies generally have insufficient specificity, and cannot effectively distinguish different TTC22 transcripts, especially cannot specifically detect the endogenous expression of TTC22v1 protein in colon cancer cells, which brings difficulties to the research of related diseases. The purpose of the present application is to provide a monoclonal antibody with high specificity and high sensitivity, which can effectively distinguish TTC22v1 and TTC22v2 proteins, and a hybridoma cell strain for preparing the antibody and related applications, so as to overcome the above technical defects.

[0007] To achieve the above-mentioned purpose, the present application specifically provides the following technical solutions:

[0008] In a first aspect, the present application provides an anti-human TTC22v1 monoclonal antibody which specifically recognizes the amino acid region of 432-569 of TTC22v1 protein for the first time. The antibody is secreted by hybridoma cell strain TTC22v1-06#. The hybridoma cell strain has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C202594.

[0009] In a second aspect, the present application provides a hybridoma cell strain secreting the specific monoclonal antibody, that is, the hybridoma cell strain TTC22v1-06#, and the preservation number is CCTCC NO: C202594.

[0010] In a third aspect, the present application further provides a detection kit comprising the specific antibody, which is suitable for specific detection of endogenous or exogenous expression of TTC22v1 protein in Western blot and cell immunofluorescence experiments.

[0011] In an embodiment, the kit further comprises one or more of the secondary antibody, the color developing reagent and the blocking solution required for Western blot detection.

[0012] In an embodiment, the kit further comprises one or more of the fluorescently labeled secondary antibody, the cell fixing solution and the cell permeation solution required for cell immunofluorescence detection.

[0013] In a fourth aspect, the present application also provides the use of the above-mentioned monoclonal antibody in the preparation of a reagent for detecting endogenous TTC22v1 protein in colon cancer cells.

[0014] In a fifth aspect, the present application further provides a method for specifically detecting the expression of TTC22v1 protein in a sample using the above-mentioned monoclonal antibody. The method specifically comprises:

[0015] Using the above-mentioned antibody as a primary antibody;

[0016] The expression of TTC22v1 protein is detected by Western blot or cell immunofluorescence experiment.

[0017] In an embodiment, the sample is selected from colon cancer cells or colon cancer tissues.

[0018] In an embodiment, the specific operation of Western blot in step (2) comprises the following steps:

[0019] (a) The sample is lysed to extract protein and subjected to SDS-PAGE gel electrophoresis;

[0020] (b) After the electrophoresis is completed, the protein is transferred to a PVDF membrane;

[0021] (c) After the PVDF membrane is incubated with the aforementioned antibody, the corresponding secondary antibody is added for incubation and color development.

[0022] In an embodiment, the specific operation of cell immunofluorescence experiment in step (2) comprises the following steps:

[0023] (a) After the cell sample to be tested is fixed with a fixing solution, it is subjected to permeation treatment;

[0024] (b) After incubation with the aforementioned antibody, a fluorescently labeled secondary antibody is further incubated;

[0025] (c) The fluorescent microscope is used to observe and detect the fluorescent signal to determine the expression of TTC22v1 protein.

[0026] Compared with the prior art, the present application has the following outstanding advantages and technical effects:

[0027] High specificity: the antibody of the present application specifically recognizes the amino acid region at positions 432-569 of TTC22v1 protein, and can significantly distinguish TTC22v1 from TTC22v2 protein;

[0028] High sensitivity: it can effectively detect endogenous and exogenous expression of TTC22v1 protein;

[0029] Strong practicability: the antibody is particularly suitable for Western blot and cell immunofluorescence experiment;

[0030] Strong innovation: for the first time, a monoclonal antibody specifically recognizing TTC22v1 and a hybridoma cell strain stably secreting the antibody are obtained, which makes up for the shortcomings of commercially available antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings are used to provide a further understanding of the present application, and constitute a part of the specification, together with the embodiments of the present application, to explain the present application, and do not constitute a limitation on the present application. In the drawings:

[0032] Figure 1 Antigenic analysis results;

[0033] Figure 2 Sequencing results;

[0034] Figure 3 Antigen purification results;

[0035] Figure 4 Western blot results of TTC22v1-04#, 07#, 06# monoclonal antibodies;

[0036] Figure 5 Comparison of Western blot results of TTC22v1-06# monoclonal antibody and other three TTC22 commercial antibodies;

[0037] Figure 6 Left panel: recognition of TTC22v1 different truncations by TTC22v1-06# monoclonal antibody; Figure 6 Right panel: detection of truncations and full length expression by tag antibody (quality control verification);

[0038] Figure 7 Immunoprecipitation results of TTC22v1-04#, 07# and 06# monoclonal antibodies;

[0039] Figure 8 Immunofluorescence experiment results of TTC22v1-06# monoclonal antibody detecting colon cancer LoVo cells (OE: overexpression; NM: normal mouse). DETAILED DESCRIPTION

[0040] The preferred embodiments of the present application will be described herein below with reference to the drawings; it should be understood that the preferred embodiments described herein are intended to explain and describe the present application, and are not intended to limit the present application.

[0041] Example 1. Preparation of antibodies

[0042] 1. Antigen analysis and selection

[0043] Most of the commercialized TTC22 antibodies are directed against N-terminal aa146-195 or aa359-448 of TTC22 protein, the former cannot specifically recognize TTC22v1, and both have very limited recognition ability for TTC22v1. We used TMPred software to analyze and predict that TTC22v1 has no hydrophobic region. For example, the TTC22v1-04# monoclonal antibody was prepared by immunizing mice with the N-terminal aa146-195 of TTC22 protein, and the TTC22v1-07# monoclonal antibody was prepared by immunizing mice with the N-terminal aa359-448 of TTC22 protein. However, the TTC22v1-04# and TTC22v1-07# monoclonal antibodies could not specifically recognize TTC22v1, and the TTC22v1-04# monoclonal antibody could not recognize TTC22v2 either. Figure 1As shown, the green box region has better antigenicity and hydrophilicity. Through analysis of sequence hydrophobicity, homology, antigenicity, hydrophilicity, and the possibility of protein expression in prokaryotic system, TTC22vl (NP_001107580.1) aa349-569 was determined to be selected as an antigen for expression, purification and immunization.

[0044] 2. Antigen expression and purification

[0045] The prokaryotic expression vector of TTC22vl fragment (aa349-569) was constructed, transformed and small-scale induced expression, large-scale expression and purification were performed to obtain sufficient protein for immunization (purity greater than 80%, concentration 1 mg / ml).

[0046] a. The overexpression plasmid of TTC22vl (Origene, RC225966) was used as a template to design and synthesize primers for PCR amplification, which was inserted into pET28a vector through BamHI and HindIII, and the enzyme digestion was consistent with the expected result, and the sequencing result was correct (e.g. Figure 2 );

[0047] b. The constructed expression vector was transformed into BL21 (DE3) competent cells, and 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 centrifugal collection, the bacteria were broken by ultrasonic (300W, ultrasonic for 10 seconds, interval for 10 seconds, 30 times). The precipitate and supernatant were collected after centrifugation, and the results showed that TTC22vl (aa349-569) protein was mainly expressed in the form of inclusion bodies. The precipitate collected after centrifugation was dissolved with urea and loaded onto a pre-equilibrated NTA purification column. Different components were collected and detected by electrophoresis, and finally the components containing the target protein were mixed together. The gel running results of purification are as follows Figure 3 .

[0048] 3. Animal immunization and detection

[0049] Seven BALB / c mice (purchased from Shanghai Xipu-Bike Experimental Animal Co., Ltd.) were immunized with the antigen. After the third immunization, the mouse serum titer was detected, and after 6 times of immunization, the mouse serum titer was more than 1:10000, which was used for cell fusion.

[0050] 4. Cell fusion and screening

[0051] The spleens of mice 04#, 05#, 06#, and 07# with high serum titers were selected for fusion, and 3-4 rounds of screening and subcloning were performed to obtain one hybridoma cell strain capable of stably secreting antibodies against TTC22v1 (aa349-569) from the spleen of each mouse. The four obtained hybridoma cell strains were expanded and cryopreserved, and the next day the cell viability was >80% under microscopic examination, indicating that the cryopreservation was qualified.

[0052] 5. Monoclonal antibody production

[0053] The above four hybridoma cells were recovered. Paraffin oil was injected intraperitoneally into new BALB / c mice (0.5 ml per mouse) in advance. At least 7 days later, 1-2 x 10 6 The mice were observed daily, and were sacrificed by cervical dislocation before death. The ascites was aspirated with a dropper, centrifuged, and the supernatant was taken. A small amount of ascites was detected by Elisa, and the antibodies obtained from each hybridoma were purified using a protein G column. The antibodies were named TTC22v1-04# monoclonal antibody, TTC22v1-05# monoclonal antibody, TTC22v1-06# monoclonal antibody, and TTC22v1-07# monoclonal antibody, respectively, according to their mouse sources. Glycine solution was used for elution, PBS was used for dialysis overnight, and the titers were detected by Elisa. The results are shown in the following table: The titers of TTC22v1 04# / 05# / 06# / 07# monoclonal antibodies meet the standard, and the Elisa detection results are qualified: the binding force of TTC22v1 04# / 05# / 06# / 07# monoclonal antibodies to the antigen his-TTC22v1 (aa349-569) protein is 29 times, 23 times, 27 times, and 23 times that of blank mouse IgG, respectively, under the same dilution ratio. Among them, 05# has low overall yield and is less used. In subsequent Western blot detection, it is shown that the specificity of 06# is better than that of 04# and 07# monoclonal antibodies. The TTC22v1-06# hybridoma cell strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation on March 27, 2025, with the preservation number CCTCC NO: C202594 and the preservation name Hybridoma cell line TTC22v1-06# Hybridoma cell line TTC22v1-06#.

[0054]

[0055] Judgment standard: S / N > 2.1 (S: antibody detection OD value, N: blank control detection OD value)

[0056] Example 2 Identification of TTC22v1-06# Monoclonal Antibody Specificity and Application

[0057] 1. The specificity of TTC22v1-06# monoclonal antibody in recognizing TTC22v1 protein is superior to TTC22v1-04# and 07#

[0058] The whole cell lysate of colon cancer cell lines SW480 (Professor Chen Yuanjia of Beijing Union Medical College Hospital) and LoVo (Professor Shoucheng Chou of Peking University Cancer Hospital) was cultured and collected. Meanwhile, the full-length plasmid of TTC22v1 and the control empty vector were overexpressed in colon cancer cells HCT116 (Professor Chen Yuanjia of Beijing Union Medical College Hospital), 10ul whole cell lysate was loaded on 12% SDS-PAGE gel, wet transferred to PVDF membrane at 200mA, and Western blot detection was performed using the aforementioned TTC22v1 04#, TTC22v1 07# and TTC22v106# monoclonal antibodies (05# total yield is low and less used later) as primary antibodies (primary antibody concentration 1μg / ml, 5% milk dilution, room temperature reaction 1.5h).

[0059] The results are shown in Figure 4 : The recognition efficiency of 04# and 06# monoclonal antibodies to overexpressed TTC22v1 is superior to 07#, and the recognition of endogenous TTC22v1 in SW480 and LoVo cells is superior to 04# monoclonal antibody.

[0060] 2. The recognition ability of TTC22v1-06# monoclonal antibody to TTC22v1 is superior to three commercial TTC22 monoclonal antibodies purchased by the laboratory

[0061] According to the same method as described above, the laboratory's TTC22v1-06# monoclonal antibody and three commercial antibodies against TTC22 (Abcam's ab-182537 / Lot:GR3177364-I, Sigma:HPA035072 / Lot:R32445, Santacruz:sc-249128 / Lot:#12112) were used as primary antibodies for Western blot detection (primary antibody concentration 0.2μg / ml for HPA035072 and 1μg / ml for the rest, 5% milk dilution, room temperature reaction 1.5h).

[0062] The results are shown in Figure 5 : The recognition efficiency of TTC22v1-06# monoclonal antibody to overexpressed TTC22v1 is significantly superior to Abcam's ab-182537 and Santa cruz:sc-249128, and the recognition of endogenous TTC22v1 in SW480 and LoVo cells is superior to Sigma:HPA035072 and the above two commercial antibodies.

[0063] 3. TTC22v1-06# monoclonal antibody recognizes the 432-569 amino acids of TTC22v1

[0064] Western blot analysis of the overexpression of TTC22v1 full-length plasmid (RC225966, purchased from OriGene Tech Inc. USA) and different truncated bodies (obtained by PCR and molecular cloning using the full-length plasmid as a template, see PMID: 35798874) in colon cancer cells HCT116. 10ul whole cell lysate was loaded onto a 12% SDS-PAGE gel, wet transferred to a PVDF membrane at 200mA, and Western blot detection was performed using TTC22v1-06# monoclonal antibody. At the same time, the rabbit polyclonal antibody of TTC22 full-length immunized in the laboratory (obtained by immunizing a New Zealand white rabbit with TTC22v1 full-length protein, see PMID: 35798874) and commercial anti-Flag antibody (66008-4-Ig, purchased from Proteintech Group, USA) and GAPDH antibody (60004-1-Ig, purchased from Proteintech Group, USA) were used as quality control to detect the overexpression effect of each truncated body (TTC22 rabbit polyclonal antibody and mouse monoclonal antibody at a concentration of 1ug / ml, diluted with 5% milk, and reacted at room temperature for 1.5h).

[0065] Results: TTC22v1-06# monoclonal antibody only binds to the full-length plasmid containing TTC22v1 aa432-569, and does not bind to each truncated body of TTC22v1 aa1-431 Figure 6 Left, the results of detecting TTC22 rabbit polyclonal antibody, commercial anti-Flag antibody and GAPDH antibody by tag antibody prove that the protein expression of TTC22 rabbit polyclonal antibody full-length and each truncated body is good Figure 6 Right). TTC22v1-06# monoclonal antibody binds to the specific region of TTC22v1, which can effectively distinguish TTC22v1 and TTC22v2. TTC22v1-06# monoclonal antibody binds to TTC22v1 significantly, and the detection efficiency is better than the above TTC22 rabbit polyclonal antibody and commercial anti-Flag antibody.

[0066] The antigen epitope recognized by TTC22v1-06# monoclonal antibody is located at the 432-569th amino acid of TTC22v1, and the sequence is as follows:

[0067] PELQLLRGKCLRIKGEDANAAACFKRAVELDDAGSSHTDGFGCLLEALLAQWSQAQLSDGELGR EVDAWLRRAQDKYPAARLRQELQRVWRGHTDEVLGLARALVAQGRPALVRLLFETMEREGEGAS APRDRRAVSF (SEQ ID NO. 1).

[0068] 4. Poor IP (Immunoprecipitation) efficacy of endogenous TTC22v1 protein

[0069] Using 1 μg of TTC22v1-04# / 07# / 06# mAb to bind to 20 μl of protein A+G Agrose (REF 11134515001 and REF 11243233001, purchased from Roche, Germany) and incubate with 1 mg of HCT116 (Professor Chen Yuanjia, Beijing Union Hospital) cell lysate at 4°C overnight, after running the IP product on gel and transferring to membrane, using TTC22v1-06# mAb and TTC22 rabbit polyclonal antibody for Western blot, it was found that there was no obvious enrichment signal corresponding to TTC22v1, as shown in Figure 7 , indicating that TTC22v1-04# / 06# / 07# mAb did not significantly enrich TTC22v1 in immunoprecipitation. Possible reasons are: the cell lysate used in the above IP experiment is undenatured protein, which may cause the 432-569 amino acids of TTC22v1 to be wrapped by secondary structure and not fully exposed. The current results do not support the use of this antibody to immunoprecipitate and enrich TTC22v1 protein, but it can specifically detect TTC22v1 in western blot and cell immunofluorescence experiments.

[0070] 5. TTC22v1 protein for IF (Immunofluorescence) detection

[0071] Using ice methanol to fix LoVo cells (Professor Shoucheng Chou, Peking University Cancer Hospital), diluting TTC22v1-06# mAb 1:200 as primary antibody, and using normal mouse IgG (CS200621, Millipore, USA) as control, it can be seen that TTC22v1-06# mAb can detect specific signals in LoVo cells (as shown in Figure 8 ): whether in LoVo cells overexpressing TTC22v1 full-length (Fig. Figure 8 ) or in wild-type LoVo cells (Fig. Figure 8 ), TTC22v1-06# mAb can detect TTC22v1, and the signal is stronger in the TTC22v1 overexpression group, and there is no difference in the subcellular localization of the fluorescence signal between the two groups. The control normal mouse IgG has no fluorescence signal in LoVo cell detection (Fig. Figure 8The above results show that at the working concentration of 5 μg / mL, the TTC22vl-06# monoclonal antibody can not only recognize the overexpressed TTC22vl, but also recognize the endogenous TTC22vl, and the TTC22vl-06# monoclonal antibody can sensitively, specifically and accurately recognize TTC22vl in the cell immunofluorescence experiment.

[0072] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

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.

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