Marker for ovarian cancer diagnosis and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a marker for ovarian cancer diagnosis and application thereof. The serum marker is SRRM1, and the kit for detecting the ovarian cancer marker comprises an elisa plate coated with a captured antibody, an HRP labeled antibody, an antigen standard substance, a cleaning solution, a developing solution and a stop solution. Experiments prove 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 is high in specificity, can specifically detect the SRRM1 protein content in serum, serves as an auxiliary diagnosis means of ovarian cancer, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a marker for ovarian cancer diagnosis and application thereof. Background Art

[0002] Ovarian cancer is one of the common malignant tumors of female reproductive organs. Its incidence rate ranks third after cervical cancer and uterine body cancer, but its mortality rate ranks first among all kinds of gynecological tumors. The pathogenesis of ovarian cancer has not been fully clarified, which poses a serious threat to women's lives. Because the ovaries are located in the pelvic cavity, are small in size, and lack typical symptoms when they occur, it is difficult to diagnose ovarian cancer in the early stage. When clinically discovered, it has often developed to the middle and late stages. Studies have shown that only 30% of ovarian cancer patients have tumors confined to the ovaries during surgery, and most have spread to the uterus, bilateral adnexa, greater omentum and pelvic organs. Their five-year survival rate hovers between 25% and 30%. If the patient's ovarian cancer is discovered and treated in the early stage, their five-year survival period is about more than 90%. Screening for early ovarian cancer is an important way to reduce the mortality rate of the disease. Traditional diagnostic methods include imaging examinations such as abdominal plain film, abdominal CT, transvaginal ultrasound (TVU), small intestine angiography, and ovarian examination, but their early diagnosis rate is extremely low. Therefore, it is urgent to find a tumor marker to solve the current international problem, increase 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, prognosis assessment, and treatment monitoring of ovarian cancer. Ovarian cancer biomarkers mainly include protein markers, carbohydrate antigen markers, gene markers, and microRNA markers. With the continuous development of technologies such as high-throughput sequencing, proteomics, and metabolomics, more and more ovarian cancer biomarkers have been discovered and verified. At present, common serum markers for ovarian cancer include CA125, CA19-9, HE4, AFP, ER, estrogen, and PR, but these conventional biomarkers are not specific 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 it is urgent to develop some new serum markers of ovarian cancer with diagnostic value as a supplementary means of early detection of ovarian cancer in medicine. Currently, there is no product on the market that detects ovarian cancer for SRRM1 protein. Summary of the invention

[0005] The first object of the present invention is to provide a marker for diagnosing ovarian cancer, wherein the marker is SRRM1.

[0006] The second object of the present invention is to provide an application of a marker for ovarian cancer diagnosis.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: A marker for diagnosing ovarian cancer, wherein the marker is SRRM1.

[0008] A marker for ovarian cancer diagnosis is used in the preparation of a product for diagnosing ovarian cancer.

[0009] Furthermore, the product is an ELISA kit.

[0010] Furthermore, the ELISA kit includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a color developing solution, and a stop solution; the capture antibody is a monoclonal antibody SRRM1-1 against the SRRM1 protein, and the HRP-labeled antibody is a monoclonal antibody SRRM1-2 against the SRRM1 protein.

[0011] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody SRRM1-1 is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO.2; the heavy chain amino acid sequence of the monoclonal antibody SRRM1-2 is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO.4.

[0012] Furthermore, the antigen standard is a recombinant SRRM1 protein antigen standard.

[0013] Furthermore, the washing solution is PBST; the color developing solution is TMB color developing solution; and the stop solution is sulfuric acid.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly: The present invention proves through experiments that SRRM1 protein is abnormally highly expressed in ovarian cancer patients, and SRRM1 protein can be used as a biomarker for ovarian cancer diagnosis. At the same time, an ELISA kit for detecting ovarian cancer markers is provided. The kit can be used as an auxiliary diagnostic method for ovarian cancer by detecting the content of SRRM1 protein in human serum samples, has the advantages of simple detection method, high accuracy, etc., and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the results of screening for differentially expressed mRNAs associated with ovarian cancer; Figure 2 Schematic diagram of Western blotting test results of SRRM1 protein levels in ovarian cancer serum and healthy human serum; Figure 3 The standard curve of the kit for specific detection of serum SRRM1 protein content; Figure 4 ROC curve was used to analyze the diagnostic value of serum SRRM1 protein in ovarian cancer. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0017] Example 1 (1) Screening of differentially expressed mRNAs related to ovarian cancer: Whole transcriptome sequencing was performed on serum samples from 20 ovarian cancer patients and 20 healthy controls. 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 controls.

[0018] The sequencing results are as follows Figure 1 As shown, the five mRNA candidate molecules with the largest differential expression folds associated with ovarian cancer, SRRM1, FOXP4, CDCA5, TOP2A, and NUPR1, were screened out, and then the candidate molecule SRRM1 with the largest differential expression folds in the serum of ovarian cancer patients was screened out.

[0019] (2) Verify the relationship between SRRM1 expression and ovarian cancer: Western blotting analysis was performed on serum samples from 5 ovarian cancer patients and 5 healthy controls. Figure 2 shown.

[0020] Figure 2 Western blotting results of SRRM1 protein levels in exosomes from blood of patients with ovarian cancer and in serum of healthy controls. Figure 2 It can be observed that the expression level of SRRM1 protein is higher in ovarian cancer patients (lanes 1-5) and lower in healthy people (lanes 6-10), which indicates that SRRM1 protein has the potential to serve as a marker for ovarian cancer.

[0021] Example 2 Preparation of ELISA kit: S1. Acquisition of monoclonal antibodies against SRRM1 recombinant protein: (1) Immunization of experimental animals: 8 healthy female mice of about 7 weeks old were selected, and SRRM1 recombinant protein was used as the immunogen, and 1 unimmunized female healthy mouse was used as the negative control. The injection method was subcutaneous multiple injections. For the first immunization, the immunogen was mixed with an equal amount of Freund's complete adjuvant to make an emulsion. For the second immunization, the immunogen was mixed with an equal amount of Freund's incomplete adjuvant to make an emulsion. Immunization was performed once every 2 weeks for 3 consecutive times. After the third immunization, the antibody titer was determined by conventional indirect enzyme-linked immunosorbent assay, and finally the mouse with the highest antibody titer was selected for subsequent experiments.

[0022] (2) Fusion of hybridoma cells: Take the female mouse with the highest antibody titer in step (1), kill it by cervical dislocation, and then remove the spleen to prepare a spleen cell suspension. The cell number ratio of spleen cells to myeloma cells SP2 / 0 is 5:1, the fusion time is 2 minutes, and 50% polyethylene glycol is used as the fusion reagent. After about 10 days of screening and culture in HAT screening medium, collect the cell culture medium supernatant 7 days later for ELISA detection, and screen positive hybridoma cells by indirect ELISA.

[0023] (3) Subcloning and screening: Subclone positive hybridoma cells by limiting dilution method, select the monoclonal stable cell line with the highest positive value, expand the culture and freeze it.

[0024] (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. When the mouse abdomen was obviously bulging, the mouse ascites was collected. The collected ascites of different mice were purified by immunochromatography to obtain purified monoclonal antibodies. The activity of the purified monoclonal antibodies was accurately measured using 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 standby use and stored at -20°C.

[0025] 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.

[0026] Table 1 Sequence Listing SEQ ID NO.1 QIQLEGSGAGLVQPGASVKASCKASGFTFTLYVSDWVKQPGKKALEWIGPILPGSGIYTTNEKTGGKATGHYSRDSSTAYMQLSRSELRASAVYYCARTGGGDYDLAWGTTQGTSVTVSA SEQ ID NO.2 DVVMTQTPLSLPGSPGEKVAISCRASQSLVSGMADWYLYTGQQKPGQAPNLLIYKVSNRPARFSGSSGRDFTGTDITSMEDVEAEYFLGVYNNYPREFPGGTFGGKLEIKR SEQ ID NO.3 EVKFLESGAELAKPGGSLGLSCKASSYTFTDYYSWVVKQRPGQGLEWLDINLPGDISTKYEYSKDRKATLTADKSNSQYQMQLSSLASEDEDSCIAARTNQRNYGGLHAMPYWGQGLSVTS SEQ ID NO.4 DAVMTQAGDLMALMAGDHVSVTCKASQNVGTNVSWYQQKTGQSPKISPGQSYSALIYLSGGVPSGVGSGSGRTSYSLTFTLQSEVEAATFCHQYHNYPGTFPGGHTKLELK S2. Preparation of ELISA plate coated with SRRM1-1 antibody: Use SRRM1-1 as the capture antibody and an enzyme-labeled 96-well plate as a solid phase carrier. Dilute the capture antibody to 3 μg / mL with a coating buffer (50 mM carbonate buffer) to obtain a coating solution. Add 200 μL of the coating solution to the ELISA plate, seal the plate, and coat overnight at 4°C. Discard the coating solution, wash three times with a washing solution, add 200 μL / well of the blocking solution, block at room temperature for 4 hours, discard the liquid in the plate, dry it, and store it at 4°C.

[0027] S3. Preparation of HRP-labeled SRRM1-2 antibody working solution: SRRM1-2 was used as the HRP labeled antibody. The monoclonal antibody SRRM1-2 was dialyzed in 50 mM carbonate buffer for 20 h, during which the solution was changed 3 times to adjust the concentration to 2 mg / mL. 5 mg of HRP dry 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 sugar group of HRP to an aldehyde group. 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 dialyzed solution overnight 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 hours, and then 0.1 mL of 4 mg / L NaBH4 was added. After mixing, the mixture was placed at 4°C for 2 hours to obtain a stable enzyme labeled antibody. The above samples were placed in 0.15 M PBS, pH 7.6, and dialyzed overnight at 4°C to remove unbound antibodies and other impurities. The dialyzate was centrifuged to remove the precipitate, and the supernatant was the HRP-labeled SRRM1-2 antibody working solution.

[0028] S4. Preparation of ELISA kit: The prepared enzyme labeling plate coated with the capture antibody, the HRP labeled antibody working solution, the antigen standard SRRM1, the washing solution PBST, the color developing solution TMB, and the stop solution sulfuric acid are packaged separately to form the ELISA kit of the present invention.

[0029] S5. Drawing of the standard curve of the kit: Remove the ELISA plate coated with SRRM1-1 antibody, return to room temperature, and wash the plate three times with PBST buffer. Add 100 μL of serum SRRM1 protein standards of 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 washing solution. Add 100 μL of HRP-labeled SRRM1-2 antibody working solution to the reaction well, incubate at 37°C for 1 h, wash three times with PBST washing solution, and then add 100 μL of TMB color development solution to the reaction well, color at room temperature in the dark for 60 min, and add 100 μL of sulfuric acid to terminate the reaction. Use an ELISA reader for dual-wavelength detection to measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. With the concentration of the standard as the horizontal axis (ng / mL), OD 450 -OD 630 Draw a standard curve for the ordinate, and the result is as follows Figure 3 The standard curve of the kit used in the present application for detecting the serum SRRM1 protein content is y=0.0223x+0.0359, R 2 = 0.9967.

[0030] Test Example 1 Serum sample testing: Fifteen healthy human serum samples (numbered 1-15) and 15 ovarian cancer patient serum samples (numbered 16-30) were collected. After separation and purification, serum samples No. 1-20 were added to the ELISA plate coated with SRRM1-1 antibody and incubated for 1 hour, and washed 3 times with PBST washing solution. 100 μL of HRP-labeled SRRM1-2 antibody working solution was added to the reaction well, incubated at 37°C for 1 hour, washed 3 times with PBST washing solution, and 100 μL of TMB color development solution was added to the reaction well. The reaction was developed at room temperature in the dark for 60 min, and 100 μL of sulfuric acid was added to terminate the reaction. The OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm was measured using an ELISA reader for dual wavelength detection. The SRRM1 protein content in the serum to be tested was calculated based on the standard curve, and the average was taken after three replicates. The results are shown in Table 1.

[0031] Table 1 Clinical sample test results 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 It can be seen from Table 1 that the kit of the present invention can accurately detect the serum SRRM1 protein content, wherein the concentration of SRRM1 protein in the serum of the healthy group is 15-30 ng / mL, and the content of SRRM1 protein in the serum of the ovarian cancer patient group is 80-110 ng / mL. The above results once again show that the SRRM1 protein in serum can be used as a biomarker for distinguishing and diagnosing healthy people and ovarian cancer patients. It shows that the kit of the present invention can use serum as a sample to complete the detection of ovarian cancer, which can meet the needs of basic research and clinical diagnosis.

[0032] Test Example 2 ROC curve evaluates the diagnostic value of SRRM1 protein in ovarian cancer: For 60 samples, including 30 healthy human serum samples as a control group and 30 ovarian cancer patient serum samples as an observation group, the SRRM1 protein content was detected according to the operating steps of Experimental Example 1, and the detection results were plotted into an ROC curve, and the AUC value was calculated to evaluate the diagnostic value of SRRM1 protein in ovarian cancer. The results are as follows Figure 4 shown.

[0033] When the AUC is between 0.7 and 0.9, it means that the model has good classification ability. Figure 4 It can be seen that the AUC value of SRRM1 protein in diagnosing ovarian cancer is 0.803, which indicates that SRRM1 protein can specifically distinguish ovarian cancer patients from healthy people. By detecting the level of SRRM1 protein in serum, the purpose of screening ovarian cancer can be achieved.

[0034] In summary, this study experimentally proved that SRRM1 protein is abnormally highly expressed in ovarian cancer patients and can be used as a biomarker for ovarian cancer. The kit for detecting ovarian cancer markers provided by the present invention has strong specificity and can specifically detect the SRRM1 protein content in serum. It can be used as an auxiliary diagnostic method for ovarian cancer with high expression of SRRM1 protein and has good application prospects.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A marker for ovarian cancer diagnosis, characterized in that: The marker is SRRM1.

2. Use of the marker for ovarian cancer diagnosis according to claim 1 in the preparation of a product for diagnosing ovarian cancer.

3. The use of the marker for ovarian cancer diagnosis according to claim 2 in the preparation of a product for diagnosing ovarian cancer, characterized in that: The product is an ELISA kit.

4. Use of the marker for ovarian cancer diagnosis according to claim 3 in the preparation of a product for diagnosing ovarian cancer, characterized in that: The ELISA kit comprises an enzyme labeling plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a color developing solution and a stop solution; the capture antibody is a monoclonal antibody SRRM1-1 against the SRRM1 protein, and the HRP-labeled antibody is a monoclonal antibody SRRM1-2 against the SRRM1 protein.

5. Use of the marker for ovarian cancer diagnosis according to claim 4 in the preparation of a product for diagnosing ovarian cancer, 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.

6. Use of the marker for ovarian cancer diagnosis according to claim 4 in the preparation of a product for diagnosing ovarian cancer, characterized in that: The antigen standard is a recombinant SRRM1 protein antigen standard.

7. Use of the marker for ovarian cancer diagnosis according to claim 4 in the preparation of a product for diagnosing ovarian cancer, 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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