A marker for diagnosis of ovarian cancer and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a marker for diagnosing ovarian cancer and application thereof. The serum marker is SRRM1, and the kit for detecting the ovarian cancer marker comprises an enzyme label plate coated with a capture antibody, an HRP labeled antibody, an antigen standard, a washing solution, a coloring solution and a termination solution. The present research proves that the SRRM1 protein is abnormally highly expressed in ovarian cancer patients, and can be used as a biomarker of ovarian cancer. The kit for detecting the ovarian cancer marker provided by the application has high specificity, can specifically detect the content of the SRRM1 protein in serum, can be used as an auxiliary diagnostic means for ovarian cancer, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biomarker for the diagnosis of ovarian cancer and its application. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive organs, ranking third in incidence after cervical cancer and endometrial cancer. However, it has the highest mortality rate among all gynecological tumors. The pathogenesis of ovarian cancer is not fully understood, posing a serious threat to women's lives. Because the ovaries are located in the pelvic cavity, are small, and often lack typical symptoms, early diagnosis of ovarian cancer is difficult. By the time it is clinically discovered, it has often progressed to the middle or late stages. Studies show that only 30% of ovarian cancer patients are found to have tumors confined to the ovary during surgery; most have already spread to the uterus, bilateral adnexa, greater omentum, and other pelvic organs, with a five-year survival rate hovering between 25% and 30%. If ovarian cancer is detected and treated early, the five-year survival rate exceeds 90%. Screening for early detection of ovarian cancer is a crucial way to reduce the mortality rate of this disease. Traditional diagnostic methods include abdominal X-rays, abdominal CT scans, transvaginal ultrasound (TVU), small bowel contrast radiography, and ovarian examinations, but their early diagnosis accuracy is extremely low. Therefore, there is an urgent need to find a tumor marker to solve the current international problem, improve the early diagnosis rate of ovarian cancer, and further improve the treatment effect of ovarian cancer.

[0003] Ovarian cancer biomarkers refer to abnormal substances or gene mutations produced by tumor cells or the body during the occurrence and development of ovarian cancer. These substances or mutations can be detected in biological samples such as blood, tissues, and urine, and are used for the diagnosis, prognostic assessment, and treatment monitoring of ovarian cancer. Ovarian cancer biomarkers mainly include protein biomarkers, carbohydrate antigen biomarkers, gene biomarkers, and microRNA biomarkers. With the continuous development of technologies such as high-throughput sequencing, proteomics, and metabolomics, more and more ovarian cancer biomarkers are being discovered and validated. Currently, common serum biomarkers for ovarian cancer include CA125, CA19-9, HE4, AFP, ER, estrogen, and PR; however, these conventional biomarkers have low specificity and cannot be used as diagnostic criteria for ovarian cancer.

[0004] Therefore, there is still a lack of accurate and reliable biomarkers for the early diagnosis of ovarian cancer, and there is an urgent need to develop new serum biomarkers for ovarian cancer with diagnostic value as a supplementary means of early detection in medicine. Currently, there are no products on the market that specifically target the SRRM1 protein for ovarian cancer detection. Summary of the Invention

[0005] The first objective of this invention is to provide a biomarker for the diagnosis of ovarian cancer, wherein the biomarker is SRRM1.

[0006] A second objective of this invention is to provide an application of a biomarker for the diagnosis of ovarian cancer.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A biomarker for the diagnosis of ovarian cancer, the biomarker being SRRM1.

[0009] Application of a biomarker for ovarian cancer diagnosis in the preparation of products for ovarian cancer diagnosis.

[0010] Furthermore, the product is an ELISA kit.

[0011] Furthermore, the ELISA kit includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing buffer, a chromogenic solution, and a stop solution; the capture antibody is a monoclonal antibody against SRRM1 protein, SRRM1-1, and the HRP-labeled antibody is a monoclonal antibody against SRRM1 protein, SRRM1-2.

[0012] Further, the heavy chain amino acid sequence of the monoclonal antibody SRRM1-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the monoclonal antibody SRRM1-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0013] Furthermore, the antigen standard is a recombinant SRRM1 protein antigen standard.

[0014] Furthermore, the washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

[0015] Compared with the prior art, the main advantages of the present invention are as follows:

[0016] This invention experimentally demonstrates that SRRM1 protein is abnormally highly expressed in ovarian cancer patients, and that SRRM1 protein can serve as a biomarker for ovarian cancer diagnosis. It also provides an ELISA kit for detecting ovarian cancer biomarkers. This kit detects the SRRM1 protein level in human serum samples, and can be used as an auxiliary diagnostic tool for ovarian cancer. It has advantages such as simple detection method and high accuracy, and shows promising application prospects. Attached Figure Description

[0017] Figure 1 A schematic diagram showing the results of screening differentially expressed mRNAs associated with ovarian cancer;

[0018] Figure 2A schematic diagram showing the results of Western blotting detection of SRRM1 protein levels in ovarian cancer serum and healthy serum.

[0019] Figure 3 A standard curve for a kit to specifically detect serum SRRM1 protein levels;

[0020] Figure 4 ROC curve analysis was performed to determine the diagnostic value of serum SRRM1 protein in ovarian cancer. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0022] Example 1

[0023] (1) Screening for differentially expressed mRNAs associated with ovarian cancer:

[0024] Whole transcriptome sequencing was performed on serum samples from 20 ovarian cancer patients and 20 healthy individuals. The results of high-throughput sequencing were analyzed to further observe the changes in mRNA expression profiles in the serum of ovarian cancer patients and healthy individuals.

[0025] Sequencing results as follows Figure 1 As shown, the five mRNA candidate molecules with the largest differential expression folds associated with ovarian cancer were screened out: SRRM1, FOXP4, CDCA5, TOP2A, and NUPR1. Furthermore, SRRM1 was selected as the candidate molecule with the largest differential expression fold in the serum of ovarian cancer patients.

[0026] (2) Verify the relationship between SRRM1 expression and ovarian cancer:

[0027] Western blotting analysis was performed on serum samples from 5 ovarian cancer patients and 5 healthy individuals to identify the ovarian cancer. The results are as follows: Figure 2 As shown.

[0028] Figure 2 The results of Western blotting analysis of blood exosomes from ovarian cancer patients and serum SRRM1 protein levels from healthy individuals were obtained from... Figure 2 It was observed that SRRM1 protein was expressed at higher levels in ovarian cancer patients (lanes 1-5) and at lower levels in healthy individuals (lanes 6-10), suggesting that SRRM1 protein has the potential to serve as a marker for ovarian cancer.

[0029] Example 2

[0030] Preparation of ELISA kit:

[0031] Obtaining S1 monoclonal antibodies against recombinant SRRM1 protein:

[0032] (1) Immunization of experimental animals: Eight healthy female mice around 7 weeks old were selected, with SRRM1 recombinant protein as the immunogen and one unimmunized healthy female mouse as the negative control. The injection method was subcutaneous multi-point injection. For the first immunization, the immunogen was mixed with an equal amount of Freund's complete adjuvant to prepare an emulsion. For the second immunization, the immunogen was mixed with an equal amount of Freund's incomplete adjuvant to prepare an emulsion. Immunization was performed every 2 weeks for a total of 3 immunizations. After the third immunization, antibody titers were determined using a conventional indirect enzyme-linked immunosorbent assay (ELISA). Finally, the mouse with the highest antibody titer was selected for subsequent experiments.

[0033] (2) Hybridoma cell fusion: Female mice with the highest antibody titer in step (1) were euthanized by cervical dislocation, and their spleens were removed to prepare a spleen cell suspension. The ratio of spleen cells to myeloma cells SP2 / 0 was 5:1, the fusion time was 2 min, and 50% polyethylene glycol was used as the fusion reagent. After about 10 days of screening culture in HAT selection medium, the cell culture supernatant was collected 7 days later for ELISA detection, and positive hybridoma cells were screened by indirect ELISA.

[0034] (3) Subcloning and screening: positive hybridoma cells were subcloned by limiting dilution, and the single-clone stable cell line with the highest positive value was selected for expansion culture and cryopreservation.

[0035] (4) Preparation of monoclonal antibodies: 1×10 6 The hybridoma cells obtained in step (3) were inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. The state of the mouse abdomen was observed, and ascites was collected when the mouse abdomen was significantly distended. The collected ascites from different mice was purified by immunochromatography to obtain purified monoclonal antibodies. The activity of the purified monoclonal antibodies was accurately measured using a Biacore T200, and the two monoclonal antibodies with the highest activity were named SRRM1-1 and SRRM1-2. The protein concentration was determined by the BCA method, and the antibody concentration was adjusted to 5 mg / mL for later use and stored at -20°C.

[0036] Analysis of monoclonal antibody sequence structure: The heavy chain and light chain variable region sequences of the obtained monoclonal antibodies SRRM1-1 and SRRM1-2 were further determined. The heavy chain amino acid sequence of monoclonal antibody SRRM1-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of monoclonal antibody SRRM1-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0037] Table 1 Sequence List

[0038] SEQ ID NO.1 QIQLEGSGAGLVQPGASVKASCKASGFTFTLYVSDWVKQPGKKALEWIGPILPGSGIYTTNEKTGGKATGHYSRDSSTAYMQLSRSELRASAVYYCARTGGGDYDLAWGTTQGTSVTVSA SEQ ID NO.2 DVVMTQTPLSLPGSPGEKVAISCRASQSLVSGMADWYLYTGQQKPGQAPNLLIYKVSNRPARFSGSSGRDFTGTDITSMEDVEAEYFLGVYNNYPREFPGGTFGGKLEIKR SEQ ID NO.3 EVKFLESGAELAKPGGSLGLSCKASSYTFTDYYSWVVKQRPGQGLEWLDINLPGDISTKYEYSKDRKATLTADKSNSQYQMQLSSLASEDEDSCIAARTNQRNYGGLHAMPYWGQGLSVTS SEQ ID NO.4 DAVMTQAGDLMALMAGDHVSVTCKASQNVGTNVSWYQQKTGQSPKISPGQSYSALIYLSGGVPSGVGSGSGRTSYSLTFTLQSEVEAATFCHQYHNYPGTFPGGHTKLELK

[0039] S2. Preparation of ELISA plates coated with SRRM1-1 antibody:

[0040] SRRM1-1 was used as the capture antibody, and a 96-well ELISA plate was used as the solid-phase carrier. The capture antibody was diluted to 3 μg / mL with coating buffer (50 mM carbonate buffer) to obtain the coating solution. 200 μL of the coating solution was added to the ELISA plate, and the plate was sealed and coated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with washing buffer. 200 μL of blocking buffer was added per well, and the plate was blocked at room temperature for 4 h. The liquid in the plate was discarded, the plate was dried, and stored at 4°C to obtain the final product.

[0041] Preparation of S3 and HRP-labeled SRRM1-2 antibody working solution:

[0042] SRRM1-2 was used as the HRP-labeled antibody. The monoclonal antibody SRRM1-2 was dialyzed in 50 mM carbonate buffer for 20 h, with three buffer changes during this period, adjusting the concentration to 2 mg / mL. 5 mg of HRP powder was weighed and dissolved in 1 mL of ddH2O, and 200 μL of 0.1 M NaIO4 solution was added. The mixture was stirred at room temperature in the dark for 60 min to oxidize the glycosyl groups of HRP to aldehyde groups. The aldehyde-modified HRP solution was placed in a dialysis bag and dialyzed overnight in 10 mM sodium acetate buffer at 4 °C. Carbonate buffer was added to the overnight dialyzed solution to adjust the pH to 9.2, and then an equal volume of monoclonal antibody SRRM1-2 was immediately added. The mixture was gently stirred at room temperature in the dark for 4 h, followed by the addition of 0.1 mL of 4 mg / L NaBH4. After mixing, the mixture was incubated at 4 °C for 2 h to obtain a stable enzyme-labeled antibody. The above sample was placed in 0.15 M PBS (pH 7.6) and dialyzed overnight at 4°C to remove unbound antibodies and other impurities. The dialysate was centrifuged to remove the precipitate, and the supernatant was the HRP-labeled SRRM1-2 antibody working solution.

[0043] S4. Preparation of ELISA kit:

[0044] The ELISA kit of this invention is constructed by packaging the prepared enzyme-labeled plate coated with capture antibody, HRP-labeled antibody working solution, antigen standard SRRM1, washing solution PBST, colorimetric solution TMB, and stop solution sulfuric acid separately.

[0045] S5. Plotting the standard curve for the reagent kit:

[0046] Remove the ELISA plate coated with SRRM1-1 antibody and allow it to return to room temperature. Wash the plate three times with PBST buffer. Add 100 μL of serum SRRM1 protein standards at different dilutions (12.5 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL), incubate at 37°C for 1 h, and wash three times with PBST buffer. Add 100 μL of HRP-labeled SRRM1-2 antibody working solution to the reaction wells, incubate at 37°C for 1 h, wash three times with PBST buffer, then add 100 μL of TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 60 min, and terminate the reaction with 100 μL of sulfuric acid. Perform dual-wavelength detection using an ELISA reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. Plot the standard concentration (ng / mL) on the x-axis. 450 - OD 630 Plot a standard curve for the ordinate, and the result is as follows: Figure 3 As shown. The standard curve for the kit used in this application for the detection of serum SRRM1 protein content is y = 0.0223x + 0.0359, R... 2 = 0.9967.

[0047] Experimental Example 1

[0048] Serum sample testing:

[0049] Fifteen serum samples from healthy individuals (numbered 1-15) and fifteen serum samples from ovarian cancer patients (numbered 16-30) were collected. The purified serum samples (numbered 1-20) were added to ELISA plates coated with SRRM1-1 antibody and incubated for 1 hour, followed by washing three times with PBST buffer. 100 μL of HRP-labeled SRRM1-2 antibody working solution was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing three times with PBST buffer. 100 μL of TMB chromogenic buffer was added to each well, and the plates were incubated at room temperature in the dark for 60 minutes. The reaction was terminated by adding 100 μL of sulfuric acid. Dual-wavelength detection was performed using an ELISA reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The SRRM1 protein content in the serum was calculated based on the standard curve. The results were obtained by repeating the assay three times and taking the average. The results are shown in Table 1.

[0050] Table 1 Clinical Sample Test Results

[0051] Sample OD450 - OD630 Detected Concentration (ng / mL) Sample OD450 - OD630 Detected Concentration (ng / mL) 1 0.5028 20.9372 16 1.9265 84.7803 2 0.4594 18.9910 17 1.9916 87.6996 3 0.3934 16.0314 18 2.0400 89.8700 4 0.4389 18.0717 19 2.1053 92.7982 5 0.3900 15.8789 20 2.1731 95.8386 6 0.5055 21.0583 21 1.8607 81.8296 7 0.5365 22.4484 22 2.1711 95.7489 8 0.4422 18.2197 23 2.3085 101.9103 9 0.4828 20.0404 24 2.3745 104.8700 10 0.4264 17.5112 25 2.1945 96.7982 11 0.4853 20.1525 26 2.4213 106.9686 12 0.5412 22.6592 27 2.3975 105.9013 13 0.5102 21.2691 28 2.4165 106.7534 14 0.4795 19.8924 29 2.2263 98.2242 15 0.4874 20.2466 30 2.3142 102.1659

[0052] Table 1 shows that the kit of the present invention can accurately detect the serum SRRM1 protein content. The concentration of SRRM1 protein in the serum of healthy individuals is 15-30 ng / mL, while the concentration in the serum of ovarian cancer patients is 80-110 ng / mL. These results further demonstrate that serum SRRM1 protein can be used as a biomarker for differential diagnosis between healthy individuals and ovarian cancer patients. This indicates that the kit of the present invention can use serum as a sample to complete the detection of ovarian cancer, meeting the needs of basic research and clinical diagnosis.

[0053] Experimental Example 2

[0054] ROC curve assessment of the diagnostic value of SRRM1 protein in ovarian cancer:

[0055] Sixty samples were included, comprising 30 serum samples from healthy individuals as the control group and 30 serum samples from ovarian cancer patients as the observation group. SRRM1 protein levels were measured according to the procedures outlined in Experiment Example 1. ROC curves were plotted based on the results, and AUC values ​​were calculated to evaluate the diagnostic value of SRRM1 protein in ovarian cancer. The results are as follows: Figure 4 As shown.

[0056] An AUC between 0.7 and 0.9 indicates that the model has good classification ability. Figure 4 The AUC value of SRRM1 protein in diagnosing ovarian cancer is 0.803, indicating that SRRM1 protein can specifically distinguish between ovarian cancer patients and healthy individuals. By detecting the level of SRRM1 protein in serum, the purpose of screening for ovarian cancer can be achieved.

[0057] In summary, this study experimentally demonstrated that SRRM1 protein is abnormally highly expressed in ovarian cancer patients and can serve as a biomarker for ovarian cancer. The kit provided by this invention for detecting ovarian cancer biomarkers has high specificity and can specifically detect the level of SRRM1 protein in serum. It can be used as an auxiliary diagnostic tool for ovarian cancer with high expression of SRRM1 protein and has good application prospects.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. The application of a reagent for detecting the serum biomarker SRRM1 protein in the preparation of an ELISA kit for diagnosing ovarian cancer.

2. The application according to claim 1, characterized in that, The ELISA kit is used to detect the SRRM1 protein content in human serum.

3. The application according to claim 1, characterized in that, The ELISA kit includes an enzyme-labeled plate coated with capture antibody, HRP-labeled antibody, antigen standard, washing buffer, chromogenic solution, and stop solution; the capture antibody is a monoclonal antibody against SRRM1 protein SRRM1-1, and the HRP-labeled antibody is a monoclonal antibody against SRRM1 protein SRRM1-2.

4. The application according to claim 3, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody SRRM1-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the monoclonal antibody SRRM1-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

5. The application according to claim 3, characterized in that, The antigen standard is a recombinant SRRM1 protein antigen standard.

6. The application according to claim 3, characterized in that, The washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

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