Monoclonal antibody against surf4 protein and application thereof

Abstract

The application provides a kind of anti-SURF4 protein monoclonal antibody and its application, belong to biotechnology and oncology medical field;The application expresses SURF4 recombinant protein as immunogen with molecular biology technique, and develops a kind of high specificity, high affinity anti-SURF4 protein monoclonal antibody;The heavy chain variable region amino acid sequence of the anti-SURF4 protein monoclonal antibody is as shown in SEQ ID No:3, and the light chain variable region amino acid sequence is as shown in SEQ ID No:4;The titer of the monoclonal antibody reaches 1×10 5 The above has the characteristics of high specificity and high affinity;The monoclonal antibody can specifically bind to SURF4 protein and can be used for immunological detection of cells, and has a broad market prospect;The monoclonal antibody can be used for preparing immunological detection tools, and has important significance for early diagnosis, prognosis evaluation and targeted therapy research of hepatocellular carcinoma.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and oncology medicine, and specifically relates to a monoclonal antibody against SURF4 protein and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer. Due to its insidious onset, rapid progression, and high susceptibility to metastasis and recurrence, patients generally have a poor prognosis. Currently used serum biomarkers for liver cancer, such as alpha-fetoprotein (AFP), have certain false positive and false negative rates. Therefore, developing novel and highly effective auxiliary diagnostic biomarkers and therapeutic targets is of significant clinical importance.

[0003] SURF4 (Surfeit locus protein 4) is a transmembrane cargo receptor located in the endoplasmic reticulum (ER), primarily responsible for mediating the transport of specific proteins from the ER to the Golgi apparatus, playing a crucial role in protein secretion pathways. Recent studies have revealed that SURF4 not only functions in intracellular transport but is also widely present in exosomes secreted by tumor cells. Research shows that SURF4 protein expression is significantly upregulated in tumor tissues and serum exosomes of hepatocellular carcinoma (HCC) patients, and its abnormal expression is closely related to tumor proliferation, metastasis, and poor prognosis. Therefore, SURF4 holds promise as a novel target for the diagnosis and targeted therapy of HCC. Currently, there are no highly specific and high-affinity anti-SURF4 monoclonal antibodies available for clinical testing; this invention aims to address this issue. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides a monoclonal antibody against SURF4 protein and its application. This invention uses molecular biology techniques to express recombinant SURF4 protein as an immunogen, developing a highly specific and high-affinity monoclonal antibody against SURF4 protein. The heavy chain variable region amino acid sequence of the anti-SURF4 protein monoclonal antibody is shown in SEQ ID No:3, and the light chain variable region amino acid sequence is shown in SEQ ID No:4. The monoclonal antibody achieves a titer of 1×10⁻⁶. 5 The above features high specificity and high affinity; the monoclonal antibody can specifically bind to the SURF4 protein and can be used for cellular immunological detection, with broad market prospects; the monoclonal antibody can be used to prepare immunological detection tools, and then used for the research and development of biological diagnostic and therapeutic drugs targeting SURF4, which is of great significance for the early diagnosis, prognostic assessment and targeted therapy research of hepatocellular carcinoma.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] The present invention first provides a monoclonal antibody against SURF4 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody against SURF4 protein 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.

[0007] Preferably, the amino acid sequence of the SURF4 recombinant protein is shown in SEQ ID No:1.

[0008] Preferably, the nucleotide sequence encoding the SURF4 recombinant protein is shown in SEQ ID No:2.

[0009] The present invention also provides a polynucleotide encoding a monoclonal antibody against the above-mentioned anti-SURF4 protein.

[0010] This invention also provides a method for preparing the above-mentioned monoclonal antibody against TMED10 protein, the method comprising:

[0011] BALB / c mice were immunized with the above-mentioned recombinant SURF4 protein. Then, the spleen lymphocytes of the successfully immunized mice were fused with myeloma cells. Positive clones were screened to obtain hybridoma cell lines that specifically secrete monoclonal antibodies against SURF4 protein. Antibodies were then produced by in vivo ascites induction method to obtain the monoclonal antibodies against SURF4 protein.

[0012] The present invention also provides a hybridoma cell line capable of specifically secreting monoclonal antibodies against SURF4 protein.

[0013] The present invention also provides the use of the above-mentioned monoclonal antibody against SURF4 protein and the above-mentioned polynucleotide-encoded monoclonal antibody in the detection of SURF4 protein for non-therapeutic and diagnostic purposes.

[0014] The present invention also provides the use of the above-mentioned monoclonal antibody against SURF4 protein and the above-mentioned polynucleotide-encoded monoclonal antibody in the preparation of immunological detection tools.

[0015] Preferably, the immunological detection tool includes a biological detection reagent or kit for detecting the SURF4 protein.

[0016] The present invention also provides a reagent or kit comprising the above-described monoclonal antibody against SURF4 protein.

[0017] The present invention also provides the use of the above-described reagents or kits in the detection of SURF4 protein for non-therapeutic and diagnostic purposes.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The monoclonal antibody against SURF4 protein of the present invention has the characteristics of high specificity and strong affinity, and the titer of the obtained antibody reaches 1×10⁻⁶. 5 The anti-SURF4 monoclonal antibody obtained in this invention can specifically bind to the SURF4 protein and can be used to detect the expression level of SURF4 in tissue or serum samples of hepatocellular carcinoma patients. This provides a powerful tool for the auxiliary diagnosis, efficacy monitoring, and prognosis of hepatocellular carcinoma and has broad market prospects. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis image of the recombinant SURF4 protein.

[0021] Figure 2 This is an SDS-PAGE electrophoresis image of the purified anti-SURF4 monoclonal antibody.

[0022] Figure 3 This is a graph showing the immunotiter test results of anti-SURF4 monoclonal antibodies at different dilutions.

[0023] Figure 4 This is the specificity identification result of the anti-SURF4 monoclonal antibody (verified by Western blot).

[0024] Figure 5 This is a Western blot result of detecting SURF4 expression in different hepatocellular carcinoma cell lines using an anti-SURF4 monoclonal antibody. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the methods, equipment, and materials used in the following embodiments are conventional methods, equipment, and materials in the art, and are all commercially available.

[0026] Example 1: Preparation of monoclonal antibodies against SURF4 protein

[0027] (1) Expression and purification of SURF4 recombinant protein:

[0028] The nucleotide sequence of the gene encoding the SURF4 protein was obtained from a public database (Accession: NM_033161.4). The coding region (CDS) of the SURF4 protein is located at nucleotides 92 to 901. The nucleotide sequence encoding the extracellular domain (N-terminal luminal domain) of the SURF4 protein corresponds to the 5' end of the CDS and before the first transmembrane region, i.e., positions 92 to 280, encoding 63 amino acids. This is designated as the SURF4 recombinant protein. The amino acid sequence of the SURF4 recombinant protein is shown in SEQ ID No:1, and the polynucleotide sequence encoding this SURF4 recombinant protein is shown in SEQ ID No:2.

[0029] SEQ ID No:1

[0030] MGQNDLMGTAEDFADQFLRVTKQYLPHVARLCLISTFLEDGIRMWFQWSEQRDYIDTTWNCGY

[0031] SEQ ID No:2

[0032] atgggccagaacgacctgatgggcacggccgaggacttcgccgaccagttcctccgtgtcacaaagcagtacctgccccacgtggcgcgccctctgtctgatcagcaccttcctggaggacggcatccgtatgtggttccagtggagcgagcagcgcgactacatcgacaccacctggaactgcggctac

[0033] The polynucleotide sequence (SEQ ID No:2) encoding the recombinant SURF4 protein was codon optimized, and the optimized codon was cloned into the prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-SURF4.

[0034] The recombinant plasmid pET-30a-SURF4 was transformed into *E. coli* BL21(DE3) using a 42℃ heat shock for 90 s method. The transformed bacterial culture was plated on LB agar containing kanamycin (final concentration 50 μg / mL) and incubated overnight at 37℃. Kanamycin-resistant plaques were picked and cultured in 500 mL LB agar until the OD of the culture reached 0.6–0.8. 0.5 mM IPTG was added to induce the expression of the target protein. After incubation at 25℃ for 12 h, the bacterial cells were collected, sonicated, and the supernatant was collected by centrifugation. The collected supernatant was added to a Ni-NTA affinity chromatography column (purchased from Qiagen) and incubated overnight at 4℃. The supernatant was discarded, and unbound protein was washed away with Wash Buffer, followed by elution buffer to elute the target protein. The elution peaks were collected and analyzed by SDS-PAGE. The results showed that high-purity recombinant SURF4 protein was obtained. Figure 1 ).

[0035] (2) Immunization and titer testing of BALB / c mice:

[0036] BALB / c mice were immunized with the SURF4 recombinant protein obtained in step (1) (purchased from Dongshan Kehui Trading Company, Wuzhong District). The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant. Blood was collected one week after each immunization, and serum titer was detected by indirect ELISA. Mice with the highest serum titer were selected for subsequent fusion experiments.

[0037] The method for detecting the serum titer is as follows:

[0038] (a) Dilute the obtained SURF4 recombinant protein to 1 μg / mL with carbonate buffer (pH 9.6, 0.05 mol / L) and add 100 μL to each well of a 96-well microplate. Coat overnight at 4°C. Remove the coated microplate, wash three times with TBS-T buffer, pat dry, and store at 4°C for later use.

[0039] (b) One week after the second immunization of mice, blood was collected from the tail vein of the mice. The serum was separated by centrifugation at 5000 g for 15 min. The serum was diluted with sample diluent (phosphate buffer containing 0.5% bovine serum albumin) at a gradient of 1:100, 1:1000, 1:10000, 1:100000, and 1:1000000. 100 μL of the diluent was added to each well of the ELISA plate to be tested. After incubation at 37°C for 1 h, the plate was washed 3 times with TBS-T buffer, dried, and then 100 μL of HRP-labeled goat anti-mouse secondary antibody (purchased from Jackson Immuno Research) diluted 1:5000 was added to each well. The plate was incubated at 37°C for 30 min.

[0040] (c) After incubation, remove the microplate, wash it 5 times with TBS-T buffer, add 100 μL of TMB substrate display solution to each well, and develop the color at 37°C in the dark for 10-15 min. Then add 50 μL of stop solution and read the absorbance at 450 nm wavelength using a microplate reader.

[0041] Select serum titers reaching 1:10 5 The mice were then immunized a third time, referring to "Modern Antibody Technology and Its Application" published by Peking University Press and "Experimental Guide to Antibody Preparation and Use" published by Science Press.

[0042] (3) Fusion and screening of hybridoma cells:

[0043] Feeder cells were prepared, and SP2 / 0 myeloma cells were prepared. Three to four days after the immunization, mouse spleen cells from the three immunizations in step (2) were fused with SP2 / 0 myeloma cells. The fused cells were plated (4 96-well plates), and hybridoma cells that produce monoclonal antibodies were screened using indirect ELISA. After two rounds of subclonal screening, the hybridoma cell line with the best antibody secretion was obtained and named SURF4-mAb.

[0044] (4) Production and purification of anti-SURF4 monoclonal antibody:

[0045] Mouse monoclonal antibody ascites was prepared using conventional methods. The ascites antibody was purified using the octanoic acid-protein G method to obtain a monoclonal antibody against SURF4 protein. The monoclonal antibody against SURF4 protein was verified by SDS-PAGE electrophoresis. Figure 2 The figure shows clear antibody bands, indicating that the purity meets the requirements. The molecular weight of the prepared antibody IgG (H+L) is approximately 160 KD, of which the IgG heavy chain is approximately 55 KD and the IgG light chain is approximately 25 KD.

[0046] Long-term preservation of monoclonal cells may lead to the loss of positive clones due to instability and contamination after multiple passages. In this embodiment, molecular biology techniques were used to amplify the heavy chain variable region (mVH) and light chain variable region (mVL) genes of positive monoclonal cell lines using the Mouse Ig-Primer Set kit (Merck Millipore), and the sequences were identified. The amino acid sequence of the heavy chain variable region of hybridoma cells 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.

[0047] SEQ ID No:3

[0048] NIQLIQSGPELKKPGETVKISCRGSAYSPTTNYLYFVKQAPGKELKWMGDLRPSQASLKFNDRPRSKLAFSLDTSASTAYLQINNLKTEDTVTYFCAKWVPFGQFGNGTSVTVSS.

[0049] SEQ ID No:4

[0050] NQTPISLPVSLGGQASISCRSSYSLVRSNAITQIEFKLQKPGQSPKLLISTLSYRFSGVPERFSGSGSGSGTDFTLKISRVEAEDLGVYFSVQASKLPWTWGAGTKLDLKR.

[0051] Example 2: Determination of anti-SURF4 monoclonal antibody titer and specificity

[0052] (1) Determination of the titer of monoclonal antibodies:

[0053] The SURF4 protein was diluted to 1 μg / mL with 0.05 mol / L carbonate buffer at pH 9.6. The diluted protein was then added to 100 μL of each well in a 96-well microplate and incubated overnight at 4°C for later use.

[0054] The monoclonal antibody against SURF4 protein obtained in Example 1 was diluted 1:10 with sample dilution buffer (phosphate buffer containing 0.5% bovine serum albumin). 3 1:10 4 1:10 5 1:10 6 Dilute 100 μL / well and incubate at 37℃ for 1 h. Remove the ELISA plate, wash 3 times with TBS-T, blot dry, and add 100 μL of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody to each well. Incubate at 37℃ for 30 min. After washing 5 times with TBS-T, add 100 μL of TMB substrate display solution to each well and incubate at 37℃ in the dark for 10-15 min. Then add 50 μL of stop solution to terminate the reaction. Read the absorbance at 450 nm using an ELISA reader. The results are as follows: Figure 3 As shown in the figure, the titer of the monoclonal antibody against SURF4 protein is 1×10⁻⁶. 5 .

[0055] (2) Monoclonal antibody specific recognition and identification:

[0056] The recombinant SURF4 protein, vascular endothelial growth factor A (VEGFA), T-cell immune receptor with Ig and ITIM domains (TIGIT), heparin-binding epidermal growth factor (HB-EGF), and heat shock protein 90 (HSP90) were diluted to 1 μg / mL using carbonate buffer, coated onto an ELISA plate, and a blank control was set up. The blank control group consisted of 1 μg / mL BSA protein. The immunotiter of the monoclonal antibody against SURF4 protein was detected by indirect ELISA using the same method as step (2) in Example 1, in order to identify the specificity of the monoclonal antibody. The detection results are as follows: Figure 4 As shown.

[0057] As can be seen from the figure, the monoclonal antibody against SURF4 protein showed no cross-reactivity with VEGFA, TIGIT, HB-EGF, and HSP90 proteins, indicating that the monoclonal antibody has good specificity.

[0058] Example 3: Western blot experiment of monoclonal antibody against SURF4 protein

[0059] Human hepatocellular carcinoma cell line HepG2 (Chinese Academy of Sciences Cell Bank, catalog number: SCSP-510) was collected, cells were lysed with RIPA lysis buffer, centrifuged at 5000 g at 4℃ for 20 min, and the supernatant was collected. Protein concentration was detected using a BCA kit (purchased from Beyotime Biotechnology Co., Ltd.).

[0060] The collected supernatant was separated by 12% SDS-PAGE electrophoresis. The PAGE gel (purchased from Beyotime Biotechnology Co., Ltd.) was then placed on a PVDF membrane and transferred at 120 mA for 240 min. The PVDF membrane was then blocked in 5 wt% skim milk at room temperature for 1 h, and incubated overnight at 4°C with a 1:5000 dilution of the anti-SURF4 protein monoclonal antibody obtained in Example 1 as the primary antibody. The PVDF membrane was removed, washed three times with PBS-T for 5 min each time, and incubated for 1 h at room temperature with a 1:5000 dilution of HRP-labeled goat anti-mouse secondary antibody. The secondary antibody was discarded, and the membrane was washed five times with PBS-T for 5 min each time. The membrane was then added to a chromogenic solution for light-protected development, and the experimental results were photographed and recorded. The results are as follows: Figure 5 As shown.

[0061] from Figure 5 As can be seen from the above, the anti-SURF4 monoclonal antibody prepared in Example 1 of the present invention can specifically recognize the SURF4 protein. A clear SURF4-specific band was detected in HepG2 cells, indicating that the anti-SURF4 monoclonal antibody produced in the present invention has high specificity and can be used to detect the expression of SURF4 protein in hepatocellular carcinoma cells.

[0062] In summary, this invention utilizes molecular biology techniques to express recombinant SURF4 protein as an immunogen, developing a highly specific and high-affinity monoclonal antibody against SURF4 protein. The amino acid sequence of the heavy chain variable region of the anti-SURF4 protein monoclonal antibody 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. The monoclonal antibody achieves a titer of 1×10⁻⁶. 5 The above features high specificity and high affinity; the monoclonal antibody can specifically bind to the SURF4 protein and can be used for cellular immunological detection, with broad market prospects; the monoclonal antibody can be used to prepare immunological detection tools, and then used for the research and development of biological diagnostic and therapeutic drugs targeting SURF4, which is of great significance for the early diagnosis, prognostic assessment and targeted therapy research of hepatocellular carcinoma.

[0063] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against SURF4 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody against SURF4 protein 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.

2. The monoclonal antibody against SURF4 protein according to claim 1, characterized in that, The amino acid sequence of the SURF4 recombinant protein is shown in SEQ ID No:

1.

3. The monoclonal antibody against SURF4 protein according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant SURF4 protein is shown in SEQ ID No:

2.

4. A polynucleotide encoding a monoclonal antibody against the anti-SURF4 protein as described in any one of claims 1-3.

5. A hybridoma cell line capable of specifically secreting a monoclonal antibody against the anti-SURF4 protein as described in any one of claims 1-3.

6. Use of the monoclonal antibody against SURF4 protein according to any one of claims 1-3, and the polynucleotide-encoded monoclonal antibody according to claim 4, in the detection of SURF4 protein for non-therapeutic and diagnostic purposes.

7. Use of the monoclonal antibody against SURF4 protein as described in any one of claims 1-3, and the polynucleotide-encoded monoclonal antibody as described in claim 4, in the preparation of immunological detection tools.

8. The use according to claim 7, characterized in that, The immunological detection tools include biological detection reagents or kits for detecting SURF4 protein.

9. A reagent or kit, characterized in that, The reagent or kit contains a monoclonal antibody against the SURF4 protein as described in any one of claims 1-3.

10. Use of the reagent or kit of claim 9 in the detection of SURF4 protein for non-therapeutic and non-diagnostic purposes.

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