Disclosure of Invention
A first object of the present invention is to provide a kit for detecting breast cancer.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a kit for detecting breast cancer, the kit comprises an ELISA plate coated with a capture antibody L4-CAP, a biotin-labeled detection antibody L4-SIG, a protein standard, HRP, a sample diluent, a washing solution, a chromogenic solution and a stop solution;
the capture antibody L4-CAP is a monoclonal antibody L4-CAP of anti-URCL 4 protein, the heavy chain amino acid sequence is shown as SEQ ID NO.1, the light chain amino acid sequence is shown as SEQ ID NO.2, the detection antibody L4-SIG is a monoclonal antibody L4-SIG of anti-URCL 4 protein, the heavy chain amino acid sequence is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO. 4.
Further, the amino acid sequence of the URCL4 protein is shown as SEQ ID NO. 5.
Further, the kit is an enzyme-linked immunosorbent assay kit.
Further, the protein standard is recombinant URCL4 protein antigen, the sample diluent is PBS, the washing solution is PBST, the color developing solution is TMB color developing solution, and the termination solution is sulfuric acid.
Further, the concentration of the developing solution is 2mg/L-4mg/L, and the concentration of the stopping solution is 2M-4M.
A second object of the present invention is to provide a detection method of a kit for detecting breast cancer.
The detection method of the kit for detecting breast cancer comprises the following steps:
(1) Taking out the ELISA plate coated with the capture antibody L4-CAP, washing the plate by using a washing liquid, adding a sample to be detected and a protein standard substance diluted in a gradient manner, incubating for 1-3h at room temperature, and washing and spin-drying the washing liquid;
(2) Adding the biotin-labeled detection antibody L4-SIG into the system in the step (1), incubating for 1-3h at room temperature, and washing and spin-drying the washing solution;
(3) Adding HRP into the system after the reaction in the step (2), and after incubating for 1-3 hours at room temperature, washing the washing solution and spin-drying;
(4) Adding a color development liquid into the system after the reaction in the step (3), developing color for 5-10min at room temperature in a dark place, and adding a stop solution to stop the reaction;
(5) The two-wavelength detection is carried out by using an enzyme-labeled instrument, the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm is measured, and the result is calculated through a standard curve.
Further, the sample to be detected is serum.
Compared with the prior art, the invention has the following main beneficial effects:
experiments prove that the URCL4 protein is abnormally high in breast cancer patients, and the inhibition of the expression level of the URCL4 can inhibit proliferation of breast cancer cells, so that the URCL4 protein can be used as a diagnosis biomarker of breast cancer. Meanwhile, the ELISA kit for detecting the breast cancer marker is provided, can specifically detect the content of URCL4 protein in a human serum sample, can be used as an auxiliary diagnosis means for high-expression URCL4 protein breast cancer, and has good application prospect.
Detailed Description
The technical scheme of the invention is further described below with reference to the specific embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention and are not to be construed as limiting the invention. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used, unless otherwise specified, are all conventional products obtained from commercial sources.
Example 1
(1) Screening for differentially expressed mRNA associated with breast cancer:
high-throughput sequencing is carried out on the whole transcriptome of the serum of 30 breast cancer patients and 30 healthy people, the variation of mRNA expression profiles in the serum of the breast cancer patients and the serum of the healthy people is analyzed, and mRNA with larger differential expression multiples in the serum of the breast cancer patients and the serum of the healthy people is screened out as a candidate molecule.
As shown in FIG. 1, the results are shown in the figure, through GEO2R and R language screening differential expression genes, KEGG and GO function enrichment analysis, 5 mRNA candidate molecules URCL4, COL9A3, SOX10, MMP28 and MMP19 with the largest differential expression multiple relative to breast cancer are finally screened out, and then candidate molecules URCL4 with the largest differential expression multiple in serum of breast cancer patients are screened out.
(2) Verifying the relationship of URCL4 expression to breast cancer:
western blotting analysis and identification are carried out on serum samples of 5 breast cancer patients and 5 healthy people, and the results are shown in FIG. 2.
FIG. 2 shows Western blottig of serum and healthy human serum URCL4 protein levels from breast cancer patients, and it can be seen from FIG. 2 that URCL4 protein is expressed at a higher level in breast cancer patients (lanes 1-5) and at a lower level in healthy humans (lanes 6-10), indicating the potential of URCL4 protein as a marker for breast cancer.
Example 2
The preparation of the URCL4 protein monoclonal antibody specifically comprises the following steps:
(1) Immunization of experimental animals healthy female mice about 8 weeks were selected, and the recombinant URCL4 protein (C1 orf 74) was used as an immunogen to immunize the mice by subcutaneous multipoint injection immunization. The first immunization was performed by mixing the recombinant URCL4 protein with an equal volume of complete Freund's adjuvant, and the subsequent immunization was performed by mixing the recombinant URCL4 protein with Freund's incomplete adjuvant. The immunization was performed again in the third and fifth weeks after the primary immunization, and the whole immunization was performed three times, 200. Mu.g each time. After the third immunization, the mouse serum was collected and analyzed for antibody titers using ELISA, and the mice with the highest titers were selected for subsequent experiments.
(2) Cell fusion and screening, namely taking the female mice with the highest antibody titer in the step (1), killing the immunized mice by adopting a cervical dislocation method, and placing the mice in a beaker filled with 75% alcohol for soaking for 2min. Spleens of mice were removed in a sterile super clean bench and placed in a 200 mesh sterile screen for milling to obtain spleen cell suspensions. SP2/0 cells in good growth state and in logarithmic growth phase were collected, and spleen cells and myeloma cells SP2/0 were fused in a water bath at 37 ℃ for 2min at a cell number ratio of 5:1, using 50% polyethylene glycol as a fusion reagent. Culturing the fused cells in a HAT selective medium, collecting cell culture supernatant after one week, screening positive hybridoma cells by ELISA method, subcloning the screened positive hybridoma cells for 3 times by limiting dilution method, and selecting the monoclonal cells with strongest positive reaction for expansion culture.
(3) Preparation of URCL4 protein monoclonal antibody 2X 106 hybridoma cells obtained in step (2) are inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. And observing the state of the abdomen of the mouse, and collecting ascites of the mouse when the abdomen of the mouse is obviously raised. Purifying the collected different mouse ascites by adopting an immunochromatography method to obtain the purified monoclonal antibody.
(4) And analyzing the sequence of the monoclonal antibody, namely accurately measuring the activity of the purified monoclonal antibody, and determining the variable region sequence of the monoclonal antibodies with the highest activity by named L4-CAP and L4-SIG. The monoclonal antibody L4-CAP and the monoclonal antibody L4-SIG respectively comprise a heavy chain and a light chain, wherein the heavy chain amino acid sequence of the monoclonal antibody L4-CAP is shown as SEQ ID NO.1, the light chain amino acid sequence is shown as SEQ ID NO.2, the heavy chain amino acid sequence of the monoclonal antibody L4-SIG is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO. 4.
Table 1 sequence listing
Example 3
Preparation of ELISA kit:
the ELISA kit comprises an ELISA plate coated with a capture antibody L4-CAP, a biotin-labeled L4-SIG detection antibody, a recombinant URCL4 protein standard, horseradish peroxidase (HRP), a sample diluent (PBS), a washing solution (PBST), a chromogenic solution (TMB) and a stop solution (sulfuric acid).
The protein standard is recombinant URCL4 protein antigen, and the amino acid sequence of the URCL4 protein is shown as SEQ ID NO. 5.
SEQ ID NO.5:
MLLLDLMSSPSPQLLVAAAQQTLGMGKRRSPPQAICLHLAGEVLAVARGLKPAVLYDCNCAGASELQSYLEELKGLGFLTFGLHILEIGENSLIVSPEHVCQHLEQVLLGTIAFVDVSSCQRHPSVCSLDQLQDLKALVAEIITHLQGLQRDLSLAVSYSRLHSSDWNLCTVFGILLGYPVPYTFHLNQGDDNCLALTPLRVFTARISWLLGQPPILLYSFSVPESLFPGLRDILNTWEKDLRTRFRTQNDFADLSISSEIVTLPAVAL;
The preparation of the ELISA plate for coating the capture antibody L4-CAP comprises the steps of taking L4-CAP as the capture antibody, taking an ELISA 96-well plate as a solid phase carrier, and diluting the capture antibody to 2 mug/mL by using a coating buffer (50 mM carbonate buffer) to obtain a coating solution. 200. Mu.L of coating solution was added to the ELISA plate, and the plate was sealed and coated overnight at 4 ℃. Discarding the coating liquid, washing for 6 times with a washing liquid, adding 200 mu L/hole of a sealing liquid, sealing for 3 hours at room temperature, discarding the liquid in the plate, drying, and preserving at 4 ℃ to obtain the coating.
Preparation of biotin-labeled L4-SIG detection antibody L4-SIG was used as detection antibody, NHS-biotin derivative was selected and dissolved in PBS. The L4-SIG detection antibody was mixed with biotin at a molar ratio of 1:10 in a carbonate buffer at pH9.0 and reacted at 4℃overnight. The mixture after the reaction was filled into a dialysis bag and dialyzed against PBS buffer to ensure complete removal of unreacted biotin. After the dialysis was completed, the dialysis bag was removed and replaced with PBS buffer. Adding glycerol into the solution, packaging, and storing in a refrigerator at-20deg.C.
Drawing a standard curve of the kit, namely taking out an ELISA plate coated with the capture antibody L4-CAP, and washing 3 times by using PBST washing liquid. 100. Mu.L of recombinant URCL4 protein standard （50pg/mL、100pg/mL、200pg/mL、300pg/mL、400pg/mL、500pg/mL、600pg/mL、700pg/mL、800pg/mL、900pg/mL、1000pg/mL）, at different dilutions was added and incubated for 1h at room temperature, and the PBST washes were washed 3 times and spin-dried. Adding 100 mu L of biotin-labeled detection antibody L4-SIG into a reaction hole, incubating for 1h at room temperature, washing with PBST washing liquid for 3 times and spin-drying, adding horseradish peroxidase (HRP), incubating for 1h at room temperature, washing with PBST washing liquid for 3 times and spin-drying, adding 100 mu L of TMB developing solution of 2mg/L into the reaction hole, developing for 10 min at room temperature in dark, and adding 100 mu L of 2M sulfuric acid to terminate the reaction. Dual wavelength detection was performed using an enzyme-labeled instrument, and the OD values at 450 nm absorption wavelengths and 630 nm reference wavelengths were determined. The standard curve is plotted with the standard concentration (pg/mL) on the abscissa and the OD 450-OD 630 on the ordinate, and the results are shown in FIG. 3. The standard curve of the kit for detecting the serum URCL4 protein content is y=0.0049x+0.0997, R2 = 0.9972, and the detection range of the kit is 100-900 pg/mL.
Test example 1
Transwell attack migration experiments:
(1) Human breast cancer MCF-7 cells were placed in DMEM medium containing 10% FBS and penicillin (100U/mL) and streptomycin (100 μg/mL) and incubated in an incubator at 37℃with 5% CO2.
(2) Human breast cancer MCF-7 cells in the growth log phase are inoculated into a 6-well plate according to 6X 105 cells/well, and are collected for standby when the cell fusion degree in the 6-well plate reaches 70 percent.
(3) Mixing 5 μl of transfection reagent si-URCL4 and si-NC with 120 μl LOpti-MEN serum-free medium, standing at room temperature for 5min to obtain diluted si-URCL4 and si-NC reagents, wherein the sequences of si-URCL4 and si-NC are shown in Table 2.
TABLE 2 si-URCL4, si-NC sequence
(4) The transfection reagent Lipo-2000 was diluted to 1. Mu.g/. Mu.L. Mixing the si-URCL4 diluted in the step (3), the si-NC reagent and the diluted transfection reagent, and incubating for 30min at room temperature to form a transfection mixture.
(5) Washing the breast cancer MCF-7 cells obtained in the step (2) by using preheated PBS, adding 1mL of serum-free culture medium into each 6-well plate, adding the transfection mixture obtained in the step (4), slowly and uniformly mixing, placing the mixture into a 37 ℃ incubator with 5% CO2 for continuous culture of 6h, discarding the original culture medium, changing the culture medium into DMEM culture medium with 10% FBS, penicillin (100U/mL) and streptomycin (100 mug/mL), culturing, and observing the transfection efficiency under an inverted fluorescence microscope after culturing to obtain the transfected successful human breast cancer cells si-URCL4 and si-Nc which are respectively marked as si-URCL4 groups and si-NC groups.
(6) The matrix is constructed in advance in a chamber by Matrigel gel and RPMI-1640 medium, after the matrix is solidified overnight, the si-URCL4 group and the si-NC group cells are resuspended and counted, 200 mu L of cell suspension is added in the chamber, 500 mu L of complete medium is added in the lower chamber, the chamber with the inoculated cells is placed in a 37 ℃ cell incubator with 5% CO2, and the cells are cultured at constant temperature for 24 h. After that, the cells were washed with PBS and fixed in paraformaldehyde solution. 1% crystal violet staining solution is added and stained 15 min at room temperature. After the dyeing is finished, the floating color is washed off, the indoor cells are wiped off by using a cotton swab, and the indoor cells are dried at room temperature. The film in the chamber was removed, mounted on a slide glass, and covered with a cover glass. Observation under a microscope and photographing ensures that cells migrating to the bottom of the cell can be clearly observed. The experimental results were analyzed using Image J quantification, and the number of migrated cells was counted, and the results are shown in fig. 4.
The results are shown in FIG. 4, which shows that the number of cells passing through the bottom of the cell is significantly reduced in the si-URCL4 group compared to the si-NC group, indicating that the invasion and migration ability of breast cancer MCF-7 cells is significantly reduced after silencing of the URCL4 gene. The result shows that the URCL4 gene silencing has obvious inhibiting effect on invasion and migration of breast cancer MCF-7 cells, and the URCL4 gene can be used as a potential treatment target of breast cancer.
Test example 2
Blood sample detection:
collecting 15 healthy human serum samples (No. 1-15) and 15 serum samples (No. 16-30) of breast cancer patients, adding the separated and purified serum samples into an ELISA plate coated with a capture antibody L4-CAP, incubating for 1h at room temperature, washing the plate 3 times by using PBST washing liquid, and spin-drying. Adding 100 mu L of biotin-labeled detection antibody L4-SIG into a reaction hole, incubating for 1h at room temperature, washing with PBST washing liquid for 3 times and spin-drying, adding horseradish peroxidase (HRP), incubating for 1h at room temperature, washing with PBST washing liquid for 3 times and spin-drying, adding 100 mu L of TMB developing solution of 2mg/L into the reaction hole, developing for 10min at room temperature in dark, and adding 100 mu L of 2M sulfuric acid to terminate the reaction. Dual wavelength detection was performed using an enzyme-labeled instrument, and the OD values at 450 nm absorption wavelengths and 630 nm reference wavelengths were determined. The URCL4 protein content in the serum to be detected is calculated according to a standard curve, and the average is calculated three times, and the result is shown in Table 3.
TABLE 3 clinical sample test results
As can be seen from Table 3, the kit of the invention can accurately detect the content of URCL4 protein in serum of healthy volunteers, and the content of URCL4 protein in serum of breast cancer patient groups is 600-900 pg/mL. The results again show that the serum URCL4 protein can be used as a biomarker for distinguishing diagnosis of healthy people and breast cancer patients. The kit provided by the invention can be used for completing detection of breast cancer by taking serum as a sample, and can meet the requirements of basic research and clinical diagnosis.
In conclusion, the experiment proves that the URCL4 protein is abnormally high expressed in breast cancer patients and can be used as a biomarker of breast cancer. The kit for detecting the breast cancer marker provided by the invention has strong specificity, can specifically detect the content of URCL4 protein in serum, can be used as an auxiliary diagnosis means for breast cancer with high expression of URCL4 protein, and has good application prospect.
Finally, the above embodiments are only for illustrating the technical solution of the present invention, and are not limited thereto. While the basic principles and main features of the present invention have been described above with specific embodiments, modifications or substitutions may be made thereto without departing from the spirit of the invention as claimed.