Polypeptide based on PDZK1IP1 protein, antibody and application thereof

Abstract

The invention relates to the technical field of antibody immunological detection, in particular to a polypeptide based on PDZK1IP1 protein, an antibody and application of the polypeptide and the antibody. On the first aspect, the invention discloses a polypeptide for preparing a tumor marker PDZK1IP1 antibody, and the polypeptide is selected from the 68-81th amino acid sequence of PDZK1IP1 protein. The second aspect discloses a polyclonal antibody based on the polypeptide, the polyclonal antibody is used for preparing or screening a pulmonary nodule detection reagent, and the expression quantity of the PDZK1IP1 protein in a subject is reflected through the polyclonal antibody. On the third aspect, the invention discloses a kit for detecting lung cancer related PDZK1IP1 protein, which comprises latex particles coated with a polyclonal antibody on the surface, and a standard substance obtained by expressing and purifying a recombinant expression vector of a PDZK1IP1 protein gene complete sequence. The invention discloses a non-invasive biomarker protein for distinguishing benign and malignant pulmonary nodules for the first time, and develops a detection method based on immunoturbidimetry and a corresponding diagnostic kit accordingly, thereby providing a novel non-invasive, rapid, sensitive and high-specificity detection approach for early lung cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody immunological detection, and in particular to a polypeptide based on PDZK1IP1 protein, an antibody and applications thereof. Background Art

[0002] Globally, lung cancer has become the leading health threat among malignant tumors due to its high morbidity and mortality, and its growth trend is intensifying year by year. According to the latest data, the five-year survival rate of lung cancer patients in my country is about 19.7%, while for those in the early stage of lung cancer (stage I), the five-year survival rate can be significantly improved to 77% to 92%. Therefore, early diagnosis and timely treatment of lung cancer are crucial to improving patient survival rate and prognosis.

[0003] At present, with the development of medical imaging technology, low-dose spiral CT is widely used to detect early lung nodules and is an effective means of early screening for lung cancer. However, the problem with low-dose spiral CT is that its test results have a high false positive rate, and multiple CT tests may also accelerate the deterioration of lung cancer. Other early lung cancer examination methods include PET, MRI, bronchial endoscopy, needle aspiration biopsy and other examinations, but they cannot be screened early, are traumatic, and are expensive. Traditional conventional tumor markers such as CEA, CYFRA21-1, and NSE have very low or even no content in the blood in the early stage of tumors. They are currently mainly used for dynamic monitoring of the condition of patients with malignant tumors and are difficult to be used as the basis for early diagnosis or confirmation of lung cancer. The lung nodule marker detection technology that relies on tissue samples or blood is currently the most accurate early diagnosis technology for lung cancer, but this method has sampling defects, such as low sampling convenience and trauma. At present, there are few reports on the discovery and application of non-invasive lung nodule tumor markers. The main reason is that the current commercialized lung cancer and lung nodule biomarkers have insufficient sensitivity and specificity in non-invasive liquids, such as sputum testing.

[0004] Regarding the above-mentioned related technologies, the inventors believe that developing a non-invasive liquid biopsy technology based on new biomarkers and applying it to the early screening of lung nodules so as to conduct regular screening and improve the detection rate of early lung cancer is a key strategy to reduce the mortality rate of lung cancer. Summary of the invention

[0005] In order to improve the current problems of low sampling convenience, traumatic nature, and insufficient sensitivity and specificity of non-invasive liquid biomarkers in early screening of lung nodules, the first purpose of the present invention is to provide a biomarker PDZK1IP1 protein and its polypeptide antigen screened from the non-invasive fluid of lung cancer subjects, which can be used to assist in the non-invasive screening of benign and malignant lung nodules.

[0006] The second object of the present invention is to provide a polyclonal antibody prepared using a polypeptide antigen based on the PDZK1IP1 protein, which has high specificity and sensitivity to the PDZK1IP1 protein and can be used for in vitro non-invasive detection of benign and malignant pulmonary nodules.

[0007] The third object of the present invention is to provide a kit for detecting lung cancer-related PDZK1IP1 protein, comprising the above-mentioned PDZK1IP1 protein standard and polyclonal antibody, and performing non-invasive in vitro lung nodule screening based on latex-enhanced immunoturbidimetry, which has the advantages of high specificity, accuracy and safety, automated analysis, simple operation and low cost.

[0008] In order to achieve the first object of the present invention, the present invention provides a polypeptide for preparing a tumor marker PDZK1IP1 antibody, which adopts the following technical scheme:

[0009] A polypeptide for preparing a tumor marker PDZK1IP1 antibody, wherein the polypeptide is selected from the amino acid sequence at positions 68 to 81 of the PDZK1IP1 protein, and the amino acid sequence of the PDZK1IP1 protein is shown in SEQ ID NO.1.

[0010] The present invention found that the PDZK1IP1 protein has a relatively high expression level in early clinical samples of malignant pulmonary nodules, and used the PDZK1IP1 protein as a biomarker for pulmonary nodule screening, and further studied its application in in vitro non-invasive detection of benign and malignant pulmonary nodules. The full sequence of the PDZK1IP1 protein (SEQ ID NO.1) was analyzed for antigenicity, hydrophilicity and surface structure possibility, and the amino acid sequence of positions 68 to 81 with high antigenicity and strong hydrophilicity was screened as an immune peptide segment, so as to successfully prepare a highly specific polyclonal antibody.

[0011] In some embodiments, the polypeptide is connected to a cysteine ​​at the C-terminus, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.2.

[0012] In the above technical solution, a cysteine ​​is artificially added to the C-terminus of the polypeptide to provide a free thiol group for coupling with the carrier protein.

[0013] In some embodiments, the tumor is lung cancer.

[0014] In order to achieve the second object of the present invention, the present invention provides a polyclonal antibody, which adopts the following technical scheme:

[0015] A polyclonal antibody is obtained by immunizing an animal with the above polypeptide.

[0016] The present invention also provides the use of the above-mentioned polyclonal antibody in the preparation or screening of lung nodule diagnostic reagents, wherein the polyclonal antibody immunologically binds to the PDZK1IP1 protein in the subject, and the polyclonal antibody reflects the expression level of the PDZK1IP1 protein in the subject.

[0017] The experimental results show that the polyclonal antibody prepared by the present invention has a specificity of 93.33% and a sensitivity of 83.33% in distinguishing malignant lung nodules in the application of PDZK1IP1 protein detection, and can achieve ng-level identification of PDZK1IP1 protein. It has high specificity and sensitivity in the detection of benign and malignant lung nodules and can be used for early screening of lung cancer.

[0018] In some embodiments, when the polyclonal antibody immunologically binds to the PDZK1IP1 protein in the subject, the body fluid sample is selected from at least one of serum, plasma, blood, and sputum.

[0019] In some embodiments, the body fluid sample is selected from sputum, and the sputum is subjected to a separation process, wherein the separation process includes separating extracellular vesicles in the sputum, and extracting and purifying proteins from the extracellular vesicles.

[0020] In order to further reduce the difficulty of sampling and avoid trauma during early screening of lung cancer, the present invention selects sputum as the detection sample, pre-treats the detected sputum sample, and uses the extracellular vesicle protein in the sputum as the final detection sample, thereby improving the accuracy and sensitivity of the polyclonal antibody in detecting PDZK1IP1 protein in sputum.

[0021] In order to achieve the third object of the present invention, the present invention provides a kit for detecting lung cancer-related PDZK1IP1 protein, which adopts the following technical scheme:

[0022] A kit for detecting lung cancer-related PDZK1IP1 protein, comprising the above-mentioned polyclonal antibody.

[0023] In some embodiments, the kit comprises:

[0024] A first reagent composition, including a buffer and a preservation solution;

[0025] A second reagent composition, comprising a buffer, latex particles coated with the polyclonal antibody, a stabilizer and a preservation solution;

[0026] The standard product is obtained by expressing and purifying the recombinant expression vector of the full sequence of the PDZK1IP1 protein gene.

[0027] In some embodiments, the kit further comprises a sputum box, a filter, and an affinity column, and the filter membrane pore size of the filter is 0.22 μm.

[0028] Through the above technical scheme, the kit of the present invention can use sputum as a sample to perform non-invasive in vitro early screening of lung nodules based on latex-enhanced immunoturbidimetry, which has the advantages of high specificity, accuracy and safety, automated analysis, simple operation and low cost, greatly enhancing the convenience of sampling and detection.

[0029] In summary, the present invention provides a polypeptide, an antibody and an application thereof based on PDZK1IP1 protein, which have the following beneficial effects:

[0030] First, the present invention uses quantitative proteomics technology of clinical samples to clarify that the level of PDZK1IP1 in the sputum of patients with malignant pulmonary nodules is significantly higher than that in patients with benign pulmonary nodules, revealing for the first time the application of PDZK1IP1 as a biomarker for early screening of benign and malignant pulmonary nodules.

[0031] Second, the present invention obtains highly specific immune peptides through sequence analysis verification, and uses the immune peptides to prepare polyclonal antibodies, realizing for the first time the non-invasive screening technology for benign and malignant lung nodules, avoiding the risk of traumatic sampling for patients, and providing a new non-invasive identification biological resource for early screening of lung nodules.

[0032] Third, based on the PDZK1IP1 protein, the present invention constructs a recombinant expression vector of the full sequence of the PDZK1IP1 protein gene, prepares a recombinant protein standard, and prepares a kit for detecting the lung cancer-related marker PDZK1IP1 protein. The latex-enhanced immunoturbidimetry method is used to quantitatively determine the PDZK1IP1 protein in sputum, which can quickly, non-invasively, low-cost, highly specific, accurately and safely identify benign and malignant lung nodules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It shows the result of antigen polypeptide analysis of the sequence of PDZK1IP1 protein using the antigen design tool in Example 1. The horizontal axis 1-5 represents 5 different antigen fragments, and the vertical axis shows the immunogenicity score of the antigen polypeptide fragment.

[0034] Figure 2 It shows the ROC curve obtained by processing in Example 5, with the horizontal axis representing specificity and the vertical axis representing sensitivity.

[0035] Figure 3 It is a scatter plot of the expression level of PDZK1IP1 protein in sputum of patients with malignant pulmonary nodules and benign pulmonary nodules in Example 5, the horizontal axis shows the two groups of specimens of malignant pulmonary nodules and benign pulmonary nodules, and the vertical axis shows the PDZK1IP1 protein level (ng / ML) in sputum detected by this kit. DETAILED DESCRIPTION

[0036] The first aspect of the present invention is to disclose a biomarker for preparing or screening a lung cancer detection reagent, wherein the biomarker is PDZK1IP1 protein, and the protein expression level of PDZK1IP1 in a subject is positively correlated with the presence or severity of malignant pulmonary nodules. Although PDZK1IP1 protein is used as a marker for lung cancer in this embodiment, in other embodiments, PDZK1IP1 protein can be used as a marker for other types of tumors.

[0037] The second aspect of the present invention is based on the human PDZK1IP1 protein, and discloses a polypeptide for preparing a tumor marker PDZK1IP1 antibody. The polypeptide is used as an immune peptide, and is not limited to preparing polyclonal antibodies, but can also prepare monoclonal antibodies. The prepared polyclonal antibodies are not limited to immunodetection of PDZK1IP1 protein in lung cancer samples, but can also immunodetect PDZK1IP1 protein in other tumor samples.

[0038] The polypeptide is an analysis of the antigenicity, hydrophilicity, surface accessibility and flexibility of the human PDZK1IP1 protein sequence (SEQ ID NO.1). Among the candidate fragments, the polypeptide is analyzed using an antigen design tool to minimize cross-reactions and ensure specificity, and finally the immune peptide with the highest score is screened as the polypeptide antigen.

[0039] The third aspect of the present invention is to provide a method for preparing polyclonal antibodies by immunizing animals with the above-screened polypeptide antigens. The animals used for the immunization can be rabbits, goats, mice and other animals commonly used for antibody preparation.

[0040] The fourth aspect of the present invention is to provide the use of the polyclonal antibody in the preparation or screening of a lung nodule diagnostic reagent, wherein the polyclonal antibody is immunologically bound to the PDZK1IP1 protein in the subject, and the polyclonal antibody is used to reflect the expression of the PDZK1IP1 protein in the subject. The immunological binding can be an enzyme-linked immunosorbent assay, an immunofluorescence assay, an immunochemiluminescence assay, an immunoturbidimetry assay, or an immunoblotting assay to detect the expression of the PDZK1IP1 protein. When detecting the expression level of the PDZK1IP1 protein in the subject, the test sample is taken from the subject's body fluid, which can be at least one of serum, plasma, blood, and sputum.

[0041] The fifth aspect of the present invention is to provide a kit for detecting lung cancer-related PDZK1IP1 protein and a preparation method thereof, wherein the kit comprises:

[0042] The first reagent composition includes a buffer solution and a preservation solution, which may be a mixed liquid prepared by the buffer solution and the preservation solution according to a certain final concentration, or may be a buffer solution and a preservation solution that are packaged and used separately;

[0043] The second reagent composition includes a buffer, latex particles coated with the polyclonal antibody, a stabilizer and a preservation solution, which can be a mixed liquid prepared by the buffer, latex particles coated with the polyclonal antibody, the stabilizer and the preservation solution according to a certain final concentration, or a buffer, a suspension of latex particles coated with the polyclonal antibody, a stabilizer and a preservation solution that are packaged and used separately;

[0044] The standard product is obtained by expressing and purifying the recombinant expression vector of the full sequence of the PDZK1IP1 protein gene.

[0045] The buffer in the first reagent composition and the second reagent composition may be, but not limited to, a phosphate buffer, and the preservation solution in the first reagent composition and the second reagent composition may be, but not limited to, a liquid with a preservative function, such as, but not limited to, sodium azide.

[0046] The particle size of the latex particles is 100nm to 500nm.

[0047] The kit quantitatively determines the expression amount of PDZK1IP1 protein in the subject's body fluid by latex enhanced immunoturbidimetry. The subject's body fluid can be at least one of serum, plasma, blood, and sputum. As a preferred embodiment, the subject's body fluid is sputum, and the sputum is subjected to separation treatment, and the separation treatment includes the steps of separating extracellular vesicles in the sputum, and extracting and purifying proteins from the extracellular vesicles.

[0048] In some embodiments, the kit further comprises a sputum box, a filter, and a CD63 affinity column, and the filter membrane pore size of the filter is 0.22 μm.

[0049] The following will further illustrate the embodiments of the present invention in conjunction with the accompanying drawings and specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. It should be noted that many specific details are set forth in the following description to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] This embodiment discloses a PDZK1IP1 protein polypeptide antigen and a screening method thereof, comprising the following steps:

[0052] Combined with the Uniprot database, the NOVOFOCUS antigen design tool was used to analyze the peptides, and the antigenicity, hydrophilicity and surface structure possibility of the PDZK1IP1 protein sequence (SEQ ID NO.1) were analyzed. The sequence with the highest score was selected as the immune peptide segment. The sequence of the immune peptide segment corresponds to the amino acids 68 to 81 of the PDZK1IP1 protein. Cysteine ​​was artificially added to the end (C-terminus) of the immune peptide segment to finally obtain a polypeptide antigen for the preparation of the tumor marker PDZK1IP1 antibody. The sequence of the polypeptide antigen is VGNKADGVLVGTDGC (SEQ ID NO.2), named IPM-1FJJ.

[0053] Figure 1 This is an antigenic peptide analysis diagram of the PDZK1IP1 protein sequence using NOVOFOCUS, wherein the horizontal axis 5 represents the VGNKADGVLVGTDGC (SEQ ID NO. 2) fragment, which has the highest polypeptide score, indicating the highest antigenicity.

[0054] Example 2

[0055] This embodiment discloses a polyclonal antibody and a method for preparing the same, comprising the following steps:

[0056] S1: Synthesize the polypeptide IPM-1FJJ described in Example 1.

[0057] S2: The polypeptide synthesized in S1 is coupled to hemocyanin (KLH).

[0058] S3: Animal immunization: the initial immune antigen polypeptide-coupled KLH protein solution obtained in S2 is mixed with an equal volume of Freund's complete adjuvant to fully emulsify the antigen and adjuvant;

[0059] Rabbits were injected subcutaneously at multiple points in the peritoneum;

[0060] On the 14th day, the booster immunization was performed with 50% of the initial immunization dose, which was fully emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points in the abdominal cavity of the rabbit.

[0061] On the 21st day, venous blood was collected and the antibody titer was tested by indirect ELISA;

[0062] Reinforce immunization on the 28th day until a satisfactory immune effect is achieved;

[0063] For shock immunization, rabbit blood was obtained after an immunization three days before fusion.

[0064] S4: Purification and preparation of polyclonal antibodies

[0065] S41: Preparation of antiserum

[0066] The rabbit blood obtained by S3 was placed at room temperature for 1 hour to coagulate and precipitate serum. The serum was collected and centrifuged at 4°C and 15000g for 10 minutes to obtain the supernatant and antiserum.

[0067] S42: Purification of polyclonal antibodies

[0068] The antiserum obtained from S41 was diluted with TBS buffer solution at a ratio of 1:3 and filtered, and then loaded onto a protein A column at a rate of 0.5 ml per minute;

[0069] The column was washed with TBS buffer solution, and the polyclonal antibody bound to the column was eluted with pH 2.7 elution buffer at a rate of 2 ml / min;

[0070] The eluate was neutralized with a neutralization buffer and its pH was adjusted to about pH 7.2 to prevent polyclonal antibody denaturation, thereby obtaining rabbit anti-human PDZK1IP1 protein polyclonal antibody. The purified polyclonal antibody was determined to have a titer of approximately 1:1.28×10 6 above.

[0071] Example 3

[0072] This embodiment discloses a kit for detecting lung cancer-related PDZK1IP1 protein and a preparation method thereof.

[0073] 1. Preparation of latex particles coated with rabbit anti-human PDZK1IP1 protein polyclonal antibody.

[0074] Rabbit anti-human PDZK1IP1 protein polyclonal antibody is coupled to the surface of latex particles by chemical cross-linking, which specifically includes the following steps:

[0075] S1: Dissolve 100 mg of latex particles in 5 mL MES buffer (50 mM, pH 6.5), add cross-linking surfactant SDS (final mass concentration 0.01%) to obtain a latex particle solution, wherein the particle size of the latex particles is 100-500 nm.

[0076] S2: The rabbit anti-human PDZK1IP1 protein polyclonal antibody prepared in Example 2 was dissolved in 5 mL MES cross-linking buffer solution (50 mM, pH 6.5) to a concentration of 1 μg / mL to obtain a rabbit anti-human PDZK1IP1 protein polyclonal antibody solution.

[0077] S3: The latex particle solution is mixed with the rabbit anti-human PDZK1IP1 protein polyclonal antibody solution, 100 mg of a chemical cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is added, and the mixture is reacted at room temperature for 2 h to obtain latex particles coated with the rabbit anti-human PDZK1IP1 protein polyclonal antibody.

[0078] 2. Preparation of recombinant protein standards.

[0079] S1, strain construction:

[0080] According to the common methods of genetic engineering, the prokaryotic expression plasmid pET-28a(+) of the complete sequence of PDZK1IP1 protein gene (SEQ ID NO.1), i.e., the recombinant plasmid, was constructed and named pET-IPM-1FJJ.

[0081] S2, expression and purification of recombinant antigen protein:

[0082] Expression and purification of recombinant plasmid pET-IPM-1-FJJ in E.coli BL21 (DE3): Inoculate a single colony of E.coli BL21 (DE3) containing recombinant plasmid pET-IPM-1-FJJ in 5 mL of LB medium containing 50 μg / mL kanamycin, shake and culture at 200 r / min at 37°C for 7-8 hours, add 500 mL of LB medium containing 50 μg / mL kanamycin to 5 mL of bacterial solution, and further expand and culture for 2 hours at 1:100 until OD600 is about 0.6. Add IPTG with a final concentration of 0.6mmoL / L to the bacterial solution, shake and culture at 16°C overnight. Collect the overnight induced bacteria by centrifugation, resuspend the bacterial precipitate with Lysis Buffer, ice bath for 30 minutes, ultrasonically disrupt for 15 minutes, and centrifuge to obtain the supernatant. The recombinant protein was purified by Ni-NTA, and impurities were washed away with 20 mmoL / L imidazole eluent, and then eluted with 200 mmoL / L imidazole eluent to obtain the PDZK1IP1 recombinant protein mother solution.

[0083] S3, preparation of recombinant protein standards:

[0084] The recombinant protein standard includes stabilizer bovine serum albumin and recombinant protein PDZK1IP1. The PDZK1IP1 recombinant protein mother solution obtained by gradient dilution of S2 with bovine serum albumin is used to prepare the recombinant protein standard, hereinafter referred to as the standard. The concentrations of the calibrators are 0 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, and 300 ng / mL, respectively.

[0085] 3. Preparation of the first reagent composition and the second reagent composition.

[0086] The first reagent composition is a mixed solution, including a phosphate buffer and sodium azide. The final concentration of the phosphate buffer in the first reagent composition is 100mmoL / L, the final concentration of sodium azide is 0.1%, and the pH of the first reagent composition is 8.

[0087] The second reagent composition is a mixed solution, including phosphate buffer, bovine serum albumin, sodium azide and the rabbit anti-human PDZK1IP1 antibody latex particles prepared as above. The final concentration of phosphate buffer in the second reagent composition is 100mmoL / L, the final concentration of rabbit anti-human PDZK1IP1 antibody latex particles is 5mg / mL, the final concentration of bovine serum albumin is 20g / L, the final concentration of sodium azide is 0.1%, and the pH of the second reagent composition is 8.

[0088] 4. A kit for detecting lung cancer-related PDZK1IP1 protein.

[0089] The composition of the kit is shown in Table 1, including the standard prepared above, the first reagent composition (hereinafter referred to as reagent 1), the second reagent composition (hereinafter referred to as reagent 2), as well as a sputum box, a 0.22 μm filter, and an affinity column.

[0090] Table 1 Kits for detecting lung cancer-related PDZK1IP1 protein

[0091]

[0092] The test kit specifications given in Table 1 are that each box can test 20 people, but this is not the only specification and production adjustments can be made based on actual conditions.

[0093] Example 4

[0094] This embodiment discloses a method for using a kit for detecting lung cancer-related PDZK1IP1 protein according to embodiment 3.

[0095] 1. Sample collection and processing.

[0096] Sample collection: 5 ml of sputum from patients with pulmonary nodules was collected into a 20 ml plastic container, 15 ml of phosphate buffered saline (PBS) was added, the mixture was rotated for 30 s, centrifuged at 800 g, 4 °C for 10 min, and the supernatant was collected;

[0097] Sample separation: The supernatant was passed through a 0.22 μm filter and extracellular vesicles were isolated using the extracellular vesicle CD63 affinity method;

[0098] Sample processing: RIPA lysis buffer (RadioImmunoprecipitation Assay Lysis buffer) containing protease and phosphatase inhibitors was used to extract and purify the extracellular vesicles. After lysis on ice for 1 hour, centrifuge at 4°C and 12,000 g for 30 minutes, collect the supernatant into a pre-cooled EP tube to obtain the sample.

[0099] 2. Prepare the kit standard curve and ROC curve.

[0100] (1) Prepare the standard curve of the kit.

[0101] Prepare a PDZK1IP1 recombinant protein standard diluted with bovine serum albumin, with concentrations including S5: 160 ng / mL, S4: 80 ng / mL, S3: 40 ng / mL, S2: 20 ng / mL, S1: 10 ng / mL, S0: 0 ng / mL.

[0102] Use an automatic biochemical analyzer (model: HITACHI7170S automatic biochemical analyzer) to detect the standard products in sequence, make a standard curve, and test at 340nm. According to the above S0~S5 standard product concentrations, refer to Table 1, prepare test tubes, take 180μL of reagent 1 in each tube, add 3μL of standard products to each tube according to the S0~S5 standard product concentrations, react at 37℃ for 5 minutes, and read the absorbance A1 标 Then add 50 μL of reagent 2 to each tube and keep it at 37°C. After 5 minutes, measure the absorbance A2 again. 标 , calculate the absorbance difference ΔA 标 =A2 标 -A1 标 . Using a series of standard protein ΔA 标 The standard curve of the kit was fitted using the value and concentration as variables.

[0103] (2) Create a ROC curve for the test kit.

[0104] The test kit of the present invention was used to measure the sputum of the subjects in the experimental group and the control group using a fully automatic biochemical analyzer (model: HITACHI7170S fully automatic biochemical analyzer). The ROC curve was obtained using MedCalc software, and the relevant standard of the Youden index was the cut-off value. Samples exceeding this value were defined as protein positive, and samples less than or equal to this value were considered protein antigen negative.

[0105] 3. Sample testing.

[0106] Use a fully automatic biochemical analyzer (model: HITACHI7170S fully automatic biochemical analyzer) to test the samples. Prepare the corresponding number of test groups according to the number of samples. Take 180 μL of reagent 1 in each group, add 3 μL of sample, react at 37°C for 5 minutes, and read the absorbance A1 测 Then, 50 μL of reagent 2 was added to each test group and kept at 37°C. After 5 minutes, the absorbance A2 was measured again. 测 , calculate the absorbance difference ΔA 测 =A2 测 -A1 测 , and then the concentration value of the sample to be tested is obtained through the standard curve, and the cut off value is used as the judgment standard to judge the test result of the sample to be tested.

[0107] Example 5

[0108] This embodiment discloses Example 3, a kit for detecting lung cancer-related PDZK1IP1 protein, and its use in distinguishing benign and malignant pulmonary nodules.

[0109] According to clinical procedures, sputum samples of 30 patients with benign pulmonary nodules and 30 patients with malignant pulmonary nodules were collected using sterile sputum boxes, 5 ml per person. The collected samples were processed according to the method of use of the kit disclosed in Example 4.

[0110] According to the method of Example 4, the 60 sputum specimens collected and processed were measured using the kit of the present invention using a fully automatic biochemical analyzer (HITACHI7170S fully automatic biochemical analyzer), and the sputum specimens of 30 patients with benign pulmonary nodules and 30 patients with malignant pulmonary nodules were divided into a control group and an experimental group. The test values ​​were processed using MedCalc software to obtain the ROC curve ( Figure 2 ), the cut-off value was set by data statistics, and sputum specimens exceeding this value were defined as protein positive (>19.97 ng / mL was considered malignant pulmonary nodules), and samples less than or equal to this value were considered protein antigen negative.

[0111] The clinical tests of 60 samples are shown in Table 2. Among the 30 patients with malignant pulmonary nodules, 25 samples were tested positive; while among the 30 patients with benign pulmonary nodules, only 2 samples were tested positive. There was a significant difference between the two groups. The specificity of PDZK1IP1 protein detection for distinguishing malignant pulmonary nodules was 93.33% (28 / 30), and the sensitivity was 83.33% (25 / 30). Figure 3 This is a scatter plot of PDZK1IP1 protein immunoturbidimetric detection in sputum of patients with malignant pulmonary nodules and benign pulmonary nodules in this embodiment. It can be seen that compared with benign pulmonary nodules, the expression of PDZK1IP1 protein in sputum of malignant pulmonary nodules is increased, and there is a significant difference. Therefore, the composition designed according to the PDZK1IP1 protein marker in this kit is closely related to the early occurrence and development of lung cancer, and can provide a non-invasive screening method for benign and malignant pulmonary nodules in the clinic, and provide an effective means for individualized treatment of patients.

[0112] Table 2 Analysis of clinical results of pulmonary nodule detection kit based on marker combination Number of cases: 60

[0113]

[0114] Of course, the above embodiments are the best embodiments of the present invention, which are only used to illustrate the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any modifications made according to the spirit of the main technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide for preparing a tumor marker PDZK1IP1 antibody, characterized in that: The polypeptide is selected from the amino acid sequence at positions 68 to 81 of the PDZK1IP1 protein, and the amino acid sequence of the PDZK1IP1 protein is shown in SEQ ID NO.

1.

2. A polypeptide for preparing a tumor marker PDZK1IP1 antibody according to claim 1, characterized in that: The polypeptide is connected to a cysteine ​​at the C-terminus, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

3. A polypeptide for preparing a tumor marker PDZK1IP1 antibody according to claim 2, characterized in that: The tumor is lung cancer.

4. A polyclonal antibody, characterized in that The method is obtained by immunizing an animal with the polypeptide according to any one of claims 1 to 3.

5. Use of the polyclonal antibody according to any one of claims 3 or 4 in the preparation or screening of diagnostic reagents for pulmonary nodules, characterized in that: The polyclonal antibody immunologically binds to the PDZK1IP1 protein in the subject, and the polyclonal antibody reflects the expression level of the PDZK1IP1 protein in the subject.

6. The use according to claim 4, characterized in that When the polyclonal antibody immunologically binds to the PDZK1IP1 protein in the subject, the body fluid sample is selected from at least one of serum, plasma, blood, and sputum.

7. The use according to claim 6, characterized in that The body fluid sample is selected from sputum, and the sputum is subjected to a separation process, wherein the separation process includes the steps of separating extracellular vesicles in the sputum, and extracting and purifying proteins from the extracellular vesicles.

8. A kit for detecting lung cancer-related PDZK1IP1 protein, characterized in that: The invention comprises the polyclonal antibody according to any one of claims 3 to 4.

9. A kit for detecting lung cancer-related PDZK1IP1 protein according to claim 8, characterized in that: include: A first reagent composition, including a buffer and a preservation solution; A second reagent composition, comprising a buffer, latex particles coated with the polyclonal antibody, a stabilizer and a preservation solution; The standard product is obtained by expressing and purifying the recombinant expression vector of the full sequence of the PDZK1IP1 protein gene.

10. A kit for detecting lung cancer-related PDZK1IP1 protein according to claim 8, characterized in that: It also includes a sputum box, a filter, and an affinity column, wherein the filter membrane pore size of the filter is 0.22 μm.

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