Polyclonal antibody targeting human CPNE7 variable splicing isomer and application thereof

Abstract

The invention discloses a polyclonal antibody targeting a human CPNE7 variable splicing isomer and application of the polyclonal antibody, and belongs to the technical field of biological medicine. The antigen epitope 'KYKQKRRSYKN' (SEQ ID NO.1) capable of being targeted by the polyclonal antibody provided by the invention is positioned in a common conserved region of all main isomers of CPNE7, and a variable splicing region is not involved. The titer of the obtained antibody serum is as high as 1: 32,000, which indicates that the antibody has extremely high immunoreactivity and sensitivity and can be used for detecting trace proteins. The antibody disclosed by the invention can be used for effectively identifying GST-CPNE7 fusion protein expressed by a prokaryotic system and CPNE7-FL and CPNE7-S protein overexpressed in an eukaryotic system (A549 cells), and the applicability of the antibody in various experimental systems is proved. Therefore, the blank in the prior art can be filled, and a core tool is provided for research of the CPNE7 in the fields of tumor biology, diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to a polyclonal antibody targeting the human CPNE7 alternative splice isoform and its application, belonging to the field of biomedical technology. Background Technology

[0002] Alternative splicing is a key molecular event in tumorigenesis and development, and the specific protein isoforms it produces are important research targets and potential diagnostic biomarkers. CPNE7, an important member of the Copine family, is abnormally highly expressed in various tumors, including colorectal cancer, and is associated with poor prognosis and chemotherapy resistance. Studies have shown that CPNE7 generates functionally differentiated isoforms (such as CPNE7-FL and CPNE7-S) through alternative splicing, but its mechanism of action in tumors remains unclear.

[0003] Currently, research on the function of CPNE7 isoforms faces a key technical bottleneck: the lack of antibodies that can specifically recognize its major isoforms. Since there are currently no reported commercial antibodies that can distinguish or simultaneously recognize different CPNE7 isoforms, researchers struggle to accurately analyze the expression patterns and functional differences among the isoforms. To address this problem, this invention has successfully developed an antibody capable of specifically recognizing the two major CPNE7 isoforms in both prokaryotic and eukaryotic cells. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention provides a polyclonal antibody targeting human CPNE7 alternative splicing isoforms and its application, aiming to solve the technical problem of the lack of high-titer, high-specificity polyclonal antibodies capable of simultaneously recognizing major CPNE7 alternative splicing isoforms in the prior art.

[0005] The first technical solution provided by the present invention is an antigenic epitope polypeptide of an anti-CPNE7 antibody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] In some embodiments, the epitope of the polypeptide is amino acid positions 344-354 of the CPNE7 protein.

[0007] The second technical solution provided by this invention is the application of the antigenic epitope polypeptide described in the first technical solution in the preparation of anti-CPNE7 antibody.

[0008] In some embodiments, the anti-CPNE7 antibody is a polyclonal antibody.

[0009] The third technical solution provided by the present invention is an anti-CPNE7 antibody, which specifically binds to the CPNE7 protein and specifically recognizes the antigenic epitope polypeptide of the first technical solution.

[0010] The fourth technical solution provided by the present invention is a polyclonal antibody containing the antigen epitope polypeptide described in the first technical solution.

[0011] In some embodiments, the polyclonal antibody is derived from mice, rabbits, or goats.

[0012] The fifth technical solution provided by this invention is the application of the antigen epitope polypeptide described in the first technical solution in the in vitro detection of CPNE7.

[0013] The sixth technical solution provided by the present invention is a product containing the antigenic epitope polypeptide of the first technical solution, or the antibody of the second technical solution, or the dolomite antibody of the third technical solution.

[0014] In some embodiments, the product includes reagents, test strips, and kits.

[0015] The seventh technical solution provided by this invention is a gene encoding the polypeptide described in the first technical solution.

[0016] The eighth technical solution provided by the present invention is a recombinant vector carrying the gene described in the seventh technical solution.

[0017] The ninth technical solution provided by the present invention is to express the polypeptide described in the first technical solution, or to contain the gene described in the seventh technical solution, or to recombinant cells transformed with the recombinant vector described in the eighth technical solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The polyclonal antibody provided by this invention can simultaneously and specifically recognize two key alternative splicing isoforms of human CPNE7 (full-length CPNE7-FL and deletion-type CPNE7-S). The antigenic epitope “KYKQKRRSYKN” targeted by this antibody is located in a common conserved region of all major CPNE7 isoforms and does not involve alternative splicing regions. With an optimized immunization protocol, the resulting antibody serum titer is as high as 1:32000, indicating extremely high immunoreactivity and sensitivity, suitable for the detection of trace proteins. Western blot validation showed that this antibody can effectively recognize the GST-CPNE7 fusion protein expressed in prokaryotic systems, as well as the CPNE7-FL and CPNE7-S proteins overexpressed in eukaryotic systems (A549 cells), demonstrating its applicability in various experimental systems. Furthermore, this invention fills a gap in existing technologies, providing a core tool for research on CPNE7 in tumor biology, diagnosis, and treatment. Attached Figure Description

[0019] Figure 1Figure 1 shows the transcript and sequence analysis of CPNE7; A: Comparison of the structures of CPNE7-FL and CPNE7-S transcripts; B: Comparison of the secondary structures of CPNE7-FL and CPNE7-S.

[0020] Figure 2 Comparison of the secondary structures of CPNE7-FL and CPNE7-S; (green box represents the peptide of this invention, red box represents peptide 2, and blue box represents peptide 3).

[0021] Figure 3 Analysis of CPNE7 exon alternative splicing events; A: CPNE7 exon 3-4 skipping events (CPNE7-S); B: Box plot of PSI values ​​for Exon 3-4 skipping events in different cancer types, comparing differences between tumor and normal tissues. Based on the TCGA SpliceSeq database, the distribution of CPNE7 gene alternative splicing events and their PSI (Percent Spliced ​​In) values ​​in different tumor types was analyzed to assess its splicing activity in multiple cancer types.

[0022] Figure 4 The expression profile of CPNE7 protein was analyzed; based on the Human Protein Atlas (HPA) database, the protein expression level of the CPNE7 gene in different tumor types was analyzed.

[0023] Figure 5 The ELISA titer curve for anti-CPNE7 serum.

[0024] Figure 6 A: ELISA titer curve for antiserum No. 2; B: ELISA titer curve for antiserum No. 3.

[0025] Figure 7 For CPNE7 prokaryotic expression and antibody application; A: SDS-PAGE of GST-CPNE7 protein: Lane 1: after pGEX-4T-1-CPNE7-S induction; Lanes 2 and 4: before pGEX-4T-1-CPNE7 induction; Lane 3: after pGEX-4T-1-CPNE7-FL induction; Lane M: protein Mr marker; B: Mass spectrometry identification of GST-CPNE7 protein; C: Western blot detection of CPNE7 antibody recognition of CPNE7-S and CPNE7-FL prokaryotic expression proteins.

[0026] Figure 8Comparison of CPNE7 prokaryotic expression and antibody application; A: Western blot detection of the recognition of CPNE7-S and CPNE7-FL prokaryotic expression proteins by the CPNE7 antibody of this invention; B: Western blot detection of the recognition of CPNE7-S and CPNE7-FL prokaryotic expression proteins by antibody 2; C: Western blot detection of the recognition of CPNE7-S and CPNE7-FL prokaryotic expression proteins by antibody 3.

[0027] Figure 9 Applications of CPNE7 overexpressing cell lines and antibodies; A: Microscopic imaging of A549-CPNE7 overexpressing cells; B: Western blot detection of CPNE7 antibody recognizing CPNE7-S protein overexpressed in A549 cells; C: Western blot detection of CPNE7-FL protein overexpressed in A549 cells by CPNE7 antibody.

[0028] Figure 10 Application and comparison of CPNE7 overexpression cell lines and antibodies; A: Western blot detection of the recognition of CPNE7-S protein overexpressed in A549 cells by the antibody of the present invention and antibody No. 2; B: Western blot detection of the recognition of CPNE7-FL protein overexpressed in A549 cells by the antibody of the present invention and antibody No. 2. Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. Test method:

[0030] 1. ELISA to determine antibody titer Antibody titers were determined using an indirect ELISA method. A 96-well microplate was coated with 0.1 μg / well of CPNE7 peptide-BSA conjugate and incubated overnight at 4 °C. After discarding the coating solution, the plate was washed three times with TBST, and 200 μL of 5% BSA blocking buffer was added, followed by incubation at 37 °C for 2 hours. Mouse serum was then serially diluted 1:500–1:64000 as primary antibody, and the plate was incubated at 37 °C for 1 hour. After washing, HRP-labeled anti-mouse IgG (secondary antibody, 1:5000) was added, and the plate was incubated at 37 °C for 45 minutes. After washing, the plate was reacted with chromogenic reagent, and the OD value of each well was measured to calculate the antibody titer.

[0031] 2. SDS-PAGE and Western blot analysis of prokaryotic systems Protein samples were separated by 12% separating gel and 5% stacking gel SDS-PAGE. A portion of the gel was used for Coomassie Brilliant Blue staining to assess the molecular weight and expression level of the target proteins, while the remaining proteins were transferred to a 0.45 μm PVDF membrane. The membrane was blocked with 5% skim milk powder TBST for 1 hour, then incubated overnight at 4 °C with a self-made CPNE7 polyclonal antibody (1:1000). After washing with TBST, HRP-labeled anti-mouse IgG (1:5000) was added and incubated at room temperature for 1 hour. The results were then detected and recorded by ECL colorimetric analysis. For further validation, protein bands with the theoretical molecular weight were excised, destained, and digested with trypsin before identification using MALDI-TOF MS / MS.

[0032] 3. Western blot analysis of eukaryotic systems In the eukaryotic expression system, A549 empty vector control cells and cell lysate samples stably overexpressing CPNE7-FL and CPNE7-S were separated by 10% separating gel and 5% stacking gel SDS-PAGE. The proteins on the gel were transferred to a 0.45 μm PVDF membrane. After blocking the membrane with 5% skim milk powder TBST for 1 hour, self-made anti-CPNE7 polyclonal antibodies No. 1 and No. 2 (1:1000) were added and incubated overnight at 4 °C. After washing with TBST, HRP-labeled anti-mouse IgG (1:5000) was added and incubated at room temperature for 1 hour. The results were detected and recorded by ECL colorimetric analysis. Raw materials used in the examples:

[0033] 1. pGEX-4T-1 E. coli DH5α, E. coli BL21 was purchased from Shanghai Sangon Biotech Co., Ltd.

[0034] 2. A549 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0035] 3. DMEM culture medium and fetal bovine serum (FBS) were purchased from Gibco.

[0036] 4. Opti-MEMⅠ and Lipofectamine 3000 were purchased from Invitrogen.

[0037] 5. Freund's complete adjuvant, Freund's incomplete adjuvant, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sigma.

[0038] 6. The relative molecular mass (Mr) standard for proteins and the PVDF membrane were purchased from Bio-Rad.

[0039] 7. Horseradish peroxidase-labeled anti-mouse secondary antibody, RIPA lysis buffer, penicillin, streptomycin, trypsin cell digestion solution, antibody dilution buffer, and ECL chemiluminescence detection kit were purchased from Byeotime.

[0040] 8. Male BALB / c mice aged 6–8 weeks were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. Example 1: Selection of antigenic epitopes and peptide synthesis

[0041] The human CPNE7 gene was retrieved from the Ensembl database (https: / / www.ensembl.org), yielding its major alternative splicing transcripts NM_014427.5 (CPNE7-FL) and NM_153636.3 (CPNE7-S). To analyze the conservation and differences in functional domains between the major CPNE7 isoforms (CPNE7-FL and CPNE7-S), their encoded amino acid sequences were first submitted to the EBI Clustal Omega online tool (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) for multiple sequence alignment. The alignment parameters were set to default. Compared to CPNE7-FL, the CPNE7-S isoform lacks exons 3 and 4 (…). Figure 1 -A). Simultaneously, combining the CPNE7 three-dimensional structural information available in the RCSB PDB database (https: / / www.rcsb.org), and using ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) for comprehensive analysis of sequence conservation and secondary structure characteristics, this provides a basis for understanding the potential differences between the two isomers at the structural and functional levels. The comprehensive analysis based on RCSB PDB structural data and ESPript 3.0 reveals that the absence of CPNE7-S directly leads to the loss of a β-sheet. Figure 1 The -B group constitutes a key structural defect compared to its full-length isomer, which is likely to impair the conformational stability and domain function of this region. Given that the amino acid sequence “KYKQKRRSYKN” (1) at positions 344–354 is completely conserved across all isomers and does not involve alternative splicing, this peptide was selected as the basic antigen for antibody preparation.

[0042] The TCGA SpliceSeq database (http: / / bioinformatics.mdanderson.org / TCGASpliceSeq) was used to search for alternative splicing patterns of CPNE7 in different tumor types, obtaining the distribution of various splicing events and corresponding PSI values ​​(i.e., the proportion of exons included in the total transcript) to assess the splicing activity and expression characteristics of this gene in multiple cancer types. Analysis showed that the CPNE7 gene generates the CPNE7-S transcript (…) through a skipping event in exons 3-4. Figure 3 -A), and the incidence of this event (PSI value) has significant cancer type specificity ( Figure 3 -B). This tissue-specific alternative splicing pattern suggests that CPNE7 may play different roles in the development and progression of different tumors.

[0043] Based on the Human Protein Atlas (HPA) database, statistical graphs of the positive expression ratio of CPNE7 protein in different cancer types were retrieved and obtained in the "Pathology" module. The results showed that the expression of CPNE7 protein in malignant tumors exhibited significant histological differences. Figure 3 -A). It is most enriched in lymphoma, thymic carcinoma, and testicular carcinoma; moderately expressed in colorectal cancer and skin cancer; and significantly less positive in liver cancer, cervical cancer, and endometrial cancer. Figure 4 ).

[0044] After systematically aligning the amino acid sequences of the CPNE7-FL and CPNE7-S protein isoforms, amino acids 344–354, “KYKQKRRSYKN” (SEQ ID NO. 1), which are highly conserved across all major transcripts and unaffected by alternative splicing, were selected as epitope 1, and “GEASPGCTP” (SEQ ID NO. 2) as epitope 2. Figure 2 “CRGGHTQ” (SEQ ID NO.3) is the 3rd antigenic epitope. Figure 2 The institute commissioned Suzhou Qiangyao Biotechnology Co., Ltd. to synthesize and conjugate the relevant peptides. The screened amino acid sequences were prepared into peptides conjugated with Keyhole Limpet Hemocyanin (KLH) to enhance immunogenicity. At the same time, homologous peptides conjugated with Bovine Serum Albumin (BSA) were prepared as coating antigens for subsequent ELISA detection and antibody screening processes. Example 2: Preparation of murine polyclonal antibodies

[0045] This study used 6–8-week-old female BALB / c mice weighing 18–22 g, with 8 mice in each group. Based on a dose of 25 μg antigen per mouse, the synthesized CPNE7 peptides No. 1 and No. 2 were emulsified with aluminum adjuvant at a volume ratio of 9:1 to prepare 200 μL immunoemulsions containing 25 μg antigen. Mice were initially immunized via subcutaneous injection at multiple sites on the back, and booster immunizations were performed on days 14 and 28 with the same dose and schedule. 7–10 days after the final immunization, blood was collected from the heart of the mice under anesthesia. After coagulation at room temperature, the blood was centrifuged at 4°C and 12000 rpm for 10 minutes to separate the supernatant serum, which was then aliquoted and stored at -80°C to obtain antibody 1 serum and antibody 2 serum. Example 3: Validation of antibody titer and specificity

[0046] 1. Valence testing: The 96-well plates were coated with BSA-conjugated antigenic epitope peptides 1 and 2 from Example 1 (0.1 μg / well), and after overnight coating at 4°C, the non-specific binding sites were blocked with 5% skim milk powder solution.

[0047] The two antisera obtained in Example 2 were serially diluted (1:500 to 1:64000) and added to the wells for incubation.

[0048] HRP-labeled goat anti-mouse IgG (1:5,000 dilution) was used as a secondary antibody, and OD450nm was measured after color development.

[0049] The results are as follows Figures 5-6 As shown, serum containing antibodies 1 and 3 is considered positive when the dilution ratio reaches 1:32,000 and the OD450 is ≥0.2. Serum containing antibody 2 is considered positive when the dilution ratio reaches 1:16,000 and the OD450 is ≥0.2. This indicates that serum containing antibody 1 has a higher titer, thus showing that antibody 1 is more sensitive than antibody 2 in detection.

[0050] 2. Specificity verification (Western Blot): The coding sequences of CPNE7-FL and CPNE7-S (shown in SEQ ID NO. 4~5) obtained from the NCBI database were cloned into the pGEX-4T-1 vector to construct recombinant plasmids. After sequencing verification, the correct recombinant plasmids were transformed into expression strains. E. coli In BL21. Cultured in LB medium containing 100 μg / mL Ampicillin at 37°C until OD. 600When the concentration was 0.6–0.8, 0.5 mmol / L IPTG was added, and expression was induced overnight at 20°C. After induction, the bacterial cells were collected by centrifugation, and after sonication, the lysate was centrifuged at 4°C and 12000×g for 20 minutes. The supernatant containing soluble recombinant protein was collected, and the results were analyzed using antibody serum No. 1 and antibody serum No. 2 (1:1000 dilution) prepared in Example 2.

[0051] MALDI-TOFMS / MS identification showed that the obtained peptide was highly consistent with the CPNE7 protein sequence. Figure 7 -B), confirming that the band is indeed the CPNE7 recombinant protein. Western blot analysis showed that ( Figure 8 Anti-CPNE7 polyclonal antibody #1 clearly and definitively identified the target protein band corresponding to the theoretical molecular weight, with a clean background and no obvious non-specific signals, demonstrating the best overall specificity and signal-to-noise ratio. Antibody #2 also identified the target protein, but its background was relatively dirty, with non-specific bands visible in the empty pGEX-4T-1 control channel and some non-target lanes, indicating that its specificity was inferior to antibody #1. In contrast, antibody #3 failed to identify the target protein band, and its background was more complex, with obvious non-specific bands appearing in the empty lane, indicating that its non-specific signal was strong and lacked practical detection value.

[0052] In summary, antibody 1 showed the best performance in terms of recognition ability and specificity; antibody 2 could recognize the target protein, but had significant background interference; antibody 3 was not suitable for subsequent experiments because it could not recognize the target and was accompanied by obvious non-specific bands.

[0053] Lentiviral constructs carrying CPNE7-FL and CPNE7-S were synthesized by Gemma Gene Technology Co., Ltd., with a FLAG tag fused to their N-terminus and an EGFP fluorescent reporter gene (expressed via an independent promoter) for real-time observation of infection efficiency and transduction rate. The obtained virus was used to infect A549 cells. Approximately 24 hours before infection, A549 cells in logarithmic growth phase were plated at a predetermined density to ensure 50%–60% cell confluence at infection. A working medium containing 5–8 μg / mL polybrene was used to prepare the virus infection solution. The old medium was discarded, the virus working solution was added, and the cells were cultured at 37°C and 5% CO2 for 12–16 hours. After infection, the medium was replaced with fresh complete medium to remove viral residues and allow cell recovery. 48–72 hours later, the medium was replaced with a specific concentration of puromycin for pressure selection. The medium was changed periodically. The selected positive cells were digested, passaged, and expanded to obtain stable transfected cell lines for subsequent experiments. When the cell confluence reached 80%-90%, the culture medium was replaced with fresh medium and the cells were washed with PBS. Lysis buffer was then added and incubated for 20 minutes to obtain cell lysates. The lysates were centrifuged at 4 °C and 12000 × g for 20 minutes. The supernatant was collected and the protein concentration was determined. The samples were then stored at -80 °C and tested using antibody serum No. 1 and antibody serum No. 2 (1:1000 dilution) prepared in Example 2.

[0054] The results showed that antibody 1 produced clear, specific bands at positions corresponding to the theoretical molecular weight in both A549-CPNE7-FL and A549-CPNE7-S cell lysates, while no corresponding bands were observed in the empty A549 cell lanes; antibody 2, however, did not produce any bands in the overexpressing cell lanes. Figure 9 , 10 ).

[0055] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An antigenic epitope polypeptide against CPNE7 antibody, characterized in that, The amino acid sequence of the antigenic epitope polypeptide is shown in SEQ ID NO.

1.

2. The polypeptide according to claim 1, characterized in that, The epitope of the polypeptide is amino acids 344-354 of the CPNE7 protein.

3. The use of the antigenic epitope polypeptide according to claim 1 or 2 in the preparation of anti-CPNE7 antibody, characterized in that, The anti-CPNE7 antibody is a polyclonal antibody.

4. An anti-CPNE7 antibody, characterized in that, The anti-CPNE7 antibody specifically binds to the CPNE7 protein and specifically recognizes the antigenic epitope polypeptide of claim 1 or 2.

5. A polyclonal antibody containing the antigenic epitope polypeptide of claim 1 or 2, characterized in that, The polyclonal antibodies were derived from mice, rabbits, and goats.

6. The use of the antigenic epitope polypeptide according to claim 1 or 2 in the in vitro detection of CPNE7.

7. A product characterized in that, The product contains the antigenic epitope polypeptide of claim 1 or 2, or the antibody of claim 4, or the dolomite antibody of claim 5, and the product includes reagents, test strips, and kits.

8. A gene encoding the polypeptide of claim 1 or 2.

9. A recombinant vector carrying the gene of claim 8.

10. A recombinant cell expressing the polypeptide of claim 1 or 2, or containing the gene of claim 8, or transformed with the recombinant vector of claim 9.

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