A VAR2CSA recombinant protein and its preparation method and application

Abstract

The present application belongs to the field of biomedicine and relates to a VAR2CSA recombinant protein and its preparation method and application. The VAR2CSA recombinant protein and its preparation method provided in the present application have good stability and high yield, and at the same time have good detection efficiency of ofCS and its glycoprotein, and can be used for large-scale production of ofCS / ofCSPG detection protein for early screening, diagnosis, tumor load monitoring and prognosis prediction of malignant tumors.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202310054915.X, application date February 3, 2023, priority date December 9, 2022, and invention name “A VAR2CSA recombinant protein, preparation method and application thereof”. Technical Field

[0002] The present application belongs to the field of biomedicine and relates to a VAR2CSA recombinant protein and a preparation method and application thereof. Background Art

[0003] A special structure of chondroitin sulfate A (CSA) modification is commonly found in malignant tumor cells. This structure is a sugar chain composed of disaccharide units formed by alternating glucuronic acid and acetylgalactosamine, with different degrees of sulfate group modification, called oncofetal chondroitin sulfate (ofCS) or placental chondroitin sulfate (plCS). 1 Due to its similarity to the chondroitin sulfate structure on the surface of placental trophoblast cells, ofCS can be bound with high affinity by the VAR2CSA protein expressed by malarial parasites. 2 The VAR2CSA-based ofCS detection technology has great potential for application in the screening and diagnosis of malignant tumors.

[0004] Currently, VAR2CSA protein has been used to develop drugs targeting diseases with abnormal expression of chondroitin sulfate glycans and to separate circulating tumor cells from the blood of tumor patients. 3 and detection ofCS-modified proteoglycans in urine of bladder cancer patients 4 A study has constructed an ELISA detection system using VAR2CSA as the capture molecule and anti-ofCS antibody as the detection molecule by expressing a 28-amino acid VAR2CSA short peptide. 5 (Authorization Announcement No.: CN 109387627B). However, the affinity of the VAR2CSA short peptide used in this method for tumor-type chondroitin sulfate is unknown, and there are disadvantages such as low detection sensitivity (unclear effect on early tumor detection) and the need to purify ofCS antibodies. In the disclosed related invention (application publication number: CN 113740521A), rVAR2 is used to detect the level of free ofCS in the urine of patients with kidney cancer and bladder cancer for cancer diagnosis. However, the ELISA used in this method is a direct method, which also has problems such as unknown affinity between rVAR2 and CSA and low sensitivity.

[0005] VAR2CSA is a large multidomain transmembrane protein (350 kDa) expressed on the surface of red blood cells infected with Plasmodium falciparum. Its extracellular region contains an N terminal segment (NTS), six Duffy-Binding-Like (DBL) domains, and three interdomains (IDs) segments. 6 Existing studies have shown that the extracellular segment of VAR2CSA has different domains that can bind to chondroitin sulfate A (CSA), and different domains have different affinities. 7-9 The minimum region of VAR2CSA that can bind to chondroitin sulfate A is DBL2X and the flanking interdomain (ID) 10,11 The extracellular full segment of VAR2CSA ensures its specific and high affinity binding to chondroitin sulfate A 12 However, the in vitro expression of large fragments of extracellular recombinant proteins has the following technical disadvantages: 1. Low expression yield; 2. The protein is prone to misfolding and instability. 13 The large size and complex structure of the VAR2CSA protein, as well as the genetic variation between isolates, complicate current large-scale production strategies. 14 The key to developing VAR2CSA-based tumor markers is to stably produce small fragments of VAR2CSA that can efficiently detect ofCS. Summary of the invention

[0006] In order to solve the problems of unstable expression and low yield of the existing extracellular full-length VAR2CSA protein that can be used for ofCS detection, the present invention constructs VAR2CSA of different sequence lengths and different insect strain sources containing a minimum CSA binding domain (ID1-DBL2X-ID2a), which can be used for large-scale production of ofCS / ofCSPG detection proteins for early screening, diagnosis, tumor load monitoring and prognosis prediction of malignant tumors.

[0007] In one aspect, the present application provides a VAR2CSA recombinant protein, wherein the VAR2CSA recombinant protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4; in some embodiments, the VAR2CSA recombinant protein comprises the amino acid sequence of SEQ ID NO: 3.

[0008] On the one hand, the present application provides a nucleic acid molecule encoding the VAR2CSA recombinant protein; in some embodiments, the sequence of the nucleic acid molecule is selected from the nucleotide sequence as described in SEQ ID NO: 5-8; in some embodiments, the sequence of the nucleic acid molecule is selected from the nucleotide sequence as described in SEQ ID NO: 7.

[0009] In one aspect, the present application provides a method for preparing the VAR2CSA recombinant protein as described, comprising the following steps:

[0010] S1. Using the amino acid sequence of Plasmodium VAR2CSA protein, the nucleic acid molecule is constructed into an expression vector by homologous recombination;

[0011] S2. Take the positive clones with correct sequencing prepared in step S1 and transform them into expression competent cells;

[0012] S3. After screening, single clones were picked and tested again by PCR;

[0013] S4. Add inducer to induce recombinant protein expression.

[0014] In some embodiments, the strain of Plasmodium is selected from FCR3 strain or 3D7 strain; preferably, the number of the FCR3 strain is GenBank No: ADG23053.1; preferably, the number of the 3D7 strain is NCBI: XP_001350415.1; preferably, the expression vector is selected from pGEX series plasmids; preferably, the pGEX series plasmid is pGEX-4T2; preferably, the pGEX-4T2 plasmid C-terminus is fused with a protease recognition site and a tag protein;

[0015] Preferably, the expression competent cells are selected from Escherichia coli cells; preferably, the screening in S3 is a dual antibody screening; preferably, the dual antibody includes ampicillin and / or streptomycin; preferably, the inducer is selected from isopropylthiogalactoside.

[0016] In some embodiments, the method further comprises purifying the recombinant protein.

[0017] In some embodiments, the purification of the recombinant protein comprises the following steps:

[0018] 1) The bacterial solution obtained by resuspending the competent cells expressing the VAR2CSA recombinant protein is broken;

[0019] 2) Centrifuging the disrupted bacterial solution;

[0020] 3) After filtering the supernatant after centrifugation, add it to a chromatography medium that can bind to the C-terminal tag protein and has been equilibrated with a resuspension buffer and incubate it sufficiently;

[0021] 4) After washing with a washing buffer, the VAR2CSA recombinant protein is eluted with an elution buffer;

[0022] 5) The eluted protein product is added to the GST affinity chromatography medium balanced with buffer for full binding;

[0023] 6) Wash thoroughly with buffer, then replace with protease cleavage buffer and digest overnight;

[0024] 7) The collected enzyme cleavage products are passed through a chromatography column that can bind to the C-terminal tag protein again, the protein eluate is collected, and the buffer is replaced with a desalting column or dialyzed against PBS, and finally the protein is concentrated.

[0025] In some embodiments, in step 1), a resuspension solution is added to resuspend the expression competent cells; preferably, the resuspension solution includes 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH=7.4, 140mM NaCl, 2.7mM KCl, 2.5mMβ-ME, 1μM DNase I, 1mM PMSF, protease inhibitor I; preferably, the resuspension is added according to the ratio of expression competent cells: resuspension of 1g:5ml; preferably, the step 1) is mechanical disruption; preferably, the conditions of the mechanical disruption are set to: pressure 800-1200bar, continuous disruption 2-5 times; preferably, in the step 2), the centrifugation conditions are: 30,000×g-50,000×g, 3-5℃ centrifugation for 0.5-2 hours; preferably, in the step 3), the supernatant after centrifugation is filtered through a 0.45μm and / or 0.22μm filter membrane; preferably, in the step 4), the washing buffer is a washing buffer with an imidazole concentration of 80-150mM; preferably, in the step 4), the elution buffer is an elution buffer with an imidazole concentration of 300-700mM; preferably, in the step 6), the enzyme digestion is performed at 3-5℃; preferably, the steps 3)-7) are performed at 2-8℃.

[0026] On the one hand, the present application provides an expression vector comprising the nucleic acid molecule; preferably, the expression vector is selected from one or more of a plasmid, a phage, an artificial chromosome, and a virus; preferably, the expression vector is selected from a plasmid; preferably, the plasmid is selected from a pGEX series plasmid; preferably, the plasmid is selected from pGEX-4T2.

[0027] On the one hand, the present application provides a cell comprising the expression vector; preferably, the cell is selected from prokaryotic cells and / or eukaryotic cells; preferably, the prokaryotic cells are selected from Escherichia coli; preferably, the Escherichia coli is Shuffle T7 E coli.

[0028] On the one hand, the present application provides an ELISA reagent for tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein, wherein the ELISA reagent comprises the VAR2CSA recombinant protein as a detection reagent; preferably, the ELISA reagent further comprises a capture reagent, wherein the capture reagent is an antibody against tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein; preferably, the antibody is a monoclonal antibody, a polyclonal antibody, a multispecific antibody or an antibody fragment; preferably, the capture reagent is a recombinant VAR2CSA protein; preferably, the detection The reagents also include enzyme labeling reagents; preferably, the enzyme labeling reagents are horseradish peroxidase, alkaline phosphatase (ALP), β-galactosidase or gold colloid. Preferably, when horseradish peroxidase is used, the chromogenic substrate is selected from 3,3',5,5'-tetramethylbenzidine and o-phenylenediamine; when ALP is used, the chromogenic substrate is selected from p-nitrophenyl phosphate; when β-galactosidase is used, the chromogenic substrate is selected from o-nitrophenyl-β-D-pyranogalactoside; preferably, the kit also includes blocking solution, washing solution, sample diluent, color developing solution, stop solution, and standard. Preferably, the blocking solution is 3%-5% BSA or 1%-5% gelatin; preferably, the blocking solution is 5% BSA.

[0029] On the one hand, the present application provides the use of the VAR2CSA recombinant protein or the ELISA reagent in preparing a detection reagent for tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein in a sample; preferably, the tumor-type chondroitin sulfate includes tumor-type chondroitin sulfate glycosaminoglycan; preferably, the tumor-type chondroitin sulfate-modified glycoprotein is selected from one or more of tumor-type chondroitin sulfate-modified CD44, tumor-type chondroitin sulfate-modified CSPG4, and tumor-type chondroitin sulfate-modified SDC1; preferably, the tumor-type chondroitin sulfate-modified glycoprotein is selected from tumor-type chondroitin sulfate-modified CD44.

[0030] On the one hand, the present application provides the use of the VAR2CSA recombinant protein or the ELISA reagent in the preparation of a detection reagent for detecting tumor risk.

[0031] In some embodiments, the tumor is a tumor expressing CSA; preferably, the tumor is an epithelial malignant tumor, a mesenchymal malignant tumor, a hematopoietic cancer, a malignant melanoma, a neuroepithelial malignant tumor, or a neuroendocrine cancer; preferably, the epithelial malignant tumor is: breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, basal cell skin cancer, renal clear cell carcinoma, head and neck keratinizing squamous cell carcinoma Preferably, the malignant tumor of mesenchymal tissue origin is: liposarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, lymphangiosarcoma or chondrosarcoma; preferably, the hematopoietic system cancer is: lymphoma or leukemia; preferably, the malignant tumor of neuroepithelial tissue is: glioma, diffuse astrocytoma, or neuroblastoma.

[0032] In some embodiments, the present application constructs four recombinant VAR2CSA sequences comprising the CSA minimal binding domain.

[0033] In some embodiments, the present application modifies the expression vector pGEX-4T2 plasmid to improve the solubility and expression level of the protein, successfully expresses the VAR2CSA protein, and obtains a higher yield.

[0034] Among the four VAR2CSA sequences, the rVAR2-1 protein (DBL1X-ID1-DBL2X-ID2a-ID2b) containing the most domain fragments had the lowest yield, the rVAR2-4 protein containing the domain fragments ID1-DBL2X-ID2a-ID2b had a slightly higher yield, and the rVAR2-2 and rVAR2-3 proteins containing the least domain fragments ID1-DBL2X-ID2a had relatively higher yields. Among rVAR2-2 and rVAR2-3, the yield of rVAR2-3 from the 3D7 strain was nearly twice that of rVAR2-2 from the FCR3 strain.

[0035] The purified protein was further labeled with HRP and used for ELISA detection. In the case-control cohort, the level of CS-CD44 in the case group was significantly higher than that in the control group (p<0.0001), and the area under the ROC curve was above 0.8, which can be used for the detection of malignant tumors ( Figure 5 ).

[0036] In the present application, CSA is the abbreviation of chondroitin sulfate A. CSA, chondroitin sulfate A and chondroitin sulfate A can be used interchangeably.

[0037] In the present application, rVAR2 is the abbreviation of recombinant VAR2CSA; rVAR2, recombinant VAR2, recombinant VAR2, and recombinant VAR2CSA can be used interchangeably.

[0038] In the present application, ofCSPG is the abbreviation of oncofetal chondroitin sulfate proteoglycan; since ofCS glycosaminoglycan is covalently attached to a variety of proteins, there are different types of ofCSPG depending on the specific type of protein to which ofCS is bound, including but not limited to: ofCS-CD44, ofCS-CSPG4, ofCS-SDC1.

[0039] "Detection" in this application is the same as diagnosis, which includes not only early diagnosis of cancer, but also mid- and late-stage diagnosis of cancer, and also includes cancer screening, risk assessment, prognosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of SDS-PAGE Coomassie Brilliant Blue staining (A) and Western-Blot (B) of rVAR2 1-4 are shown;

[0041] Figure 2 The results are summarized as the mean fluorescence intensity of rVAR2 1-4 binding to tumor cells;

[0042] Figure 3 The flow cytometry results of rVAR2 1-4 binding to peripheral blood cells (ALL-P1: acute lymphoblastic leukemia patient leukocyte-1, ALL-P2: acute lymphoblastic leukemia patient leukocyte-2);

[0043] Figure 4 Schematic diagram of ELISA detection technology for this application;

[0044] Figure 5 This is a graph showing the effect of rVAR2 1-4 in detecting CS-CD44 in plasma in the case-control population (59 cases and 22 controls) in Example 4, and ROC analysis of the efficacy of CS-CD44 in detecting malignant tumors in the case-control population. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0046] Preparation Example

[0047] Cloning and expression of recombinant vectors in E. coli

[0048] The PreScission protease recognition site, EcoRI and HindIII restriction protease cleavage sites, V5 tag, TEV protease recognition site and His10 tag were fused to the C-terminus of the original plasmid restriction enzyme cleavage site BamHI. The amino acid sequence of the Plasmodium FCR3 strain VAR2CSA protein (GenBank No: ADG23053.1) and the Plasmodium 3D7 strain VAR2CSA protein (NCBI: XP_001350415.1) was used to optimize the codons of the peptide segment with reference to the codon preference of Escherichia coli, and the target gene was constructed into the expression vector by homologous recombination. The homologous recombination primers are shown in Table 1.

[0049] Table 1 Recombinant primers for expression of rVAR2 insert fragment in E. coli

[0050]

[0051]

[0052] The positive clones with correct sequencing were transformed into Shuffle T7 E.coli expression competent cells, and single clones were selected for PCR detection again after ampicillin and streptomycin double antibody screening. The above positive clones were added to LB liquid culture medium containing ampicillin and streptomycin, and placed in a 37°C shaker for 12 hours to recover; the activated strains were inoculated into a 2L conical flask at a ratio of 1:100 and continued to expand and culture in a 37°C shaker for 2-3 hours, and the shaker temperature was adjusted to 18°C. After the temperature dropped to 18°C, the inducer isopropylthiogalactoside (IPTG) was added to induce the expression of the recombinant protein.

[0053] Purification of VAR2CSA expressed in E. coli recombinant vector

[0054] Collect the cells and add 5 mL of resuspension buffer (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4, pH = 7.4, 140mM NaCl, 2.7mM KCl, 2.5mMβ-ME, 1μM DNase I, 1mM PMSF, protease inhibitor I) ratio to fully resuspend the bacteria. The resuspended bacterial solution was mechanically disrupted in an ice bath, and the disruption conditions were set to: pressure 1000bar, continuous disruption 3 times. After disruption, the bacterial solution was centrifuged at 40,000×g, 4℃ for 1 hour. To prevent the degradation of recombinant protein, the following operations were all performed at 2-8℃. The supernatant after centrifugation was double-filtered through 0.45μm and 0.22μm filter membranes, and added to the Ni-NTA column equilibrated with the resuspension buffer for metal chelation. Wash with a washing buffer with an imidazole concentration of 100mM. After sufficient washing, rVAR2 was eluted with an elution buffer with an imidazole concentration of 500mM. The eluted protein product was added to the GST affinity chromatography medium equilibrated with PBS, and placed on a vertical rotating mixer for 1 hour to ensure that the recombinant protein was fully bound to the medium. The protein and chromatography medium were transferred to the gravity chromatography column, and after the liquid was drained, they were fully washed with PBS solution, and then replaced with PreScission Protease cleavage buffer. An appropriate amount of Prescission Protease was added and cleaved overnight at 4°C. The next day, the collected cleavage product was passed through the Ni-NTA column again, and the protein eluate with an imidazole concentration of 500mM was collected, and the buffer was replaced with a desalting column, and the protein was concentrated with an ultrafiltration tube. BCA protein quantification, SDS-PAGE and Western-Blot identification ( Figure 1 ).

[0055] Table 24 Recombinant VAR2CSA protein information

[0056] serial number Contains domain Host cells parasite strains Sequence number rVAR2-1 DBL1X-ID1-DBL2X-ID2a-ID2b E. coli FCR3 SEQ ID NO: 1 rVAR2-2 ID1-DBL2X-ID2a E. coli FCR3 SEQ ID NO: 2 rVAR2-3 ID1-DBL2X-ID2a E. coli 3D7 SEQ ID NO: 3 rVAR2-4 ID1-DBL2X-ID2a-ID2b E. coli FCR3 SEQ ID NO: 4

[0057] Example 1 Production of VAR2CSA

[0058] After the VAR2CSA was expressed by the E. coli recombinant vector and purified, the yields of the four recombinant VAR2CSA prepared in the preparation example are shown in Table 3.

[0059] Table 3 Yields of 4 recombinant VAR2CSA proteins

[0060]

[0061] Different VAR2CSAs have different expression yields. Some VAR2CSA proteins with specific sequence structures are more likely to be stably expressed and show higher yields. The yield of rVAR2-2 reached 58.1ug / L; and rVAR2-3, which has a comparable sequence length, had a yield of up to 99.6ug / L. Such a high yield is of great significance for solving the problem of unstable expression and low yield of existing VAR2CSA proteins that can be used for ofCS detection.

[0062] Example 2 Flow cytometry detection of VAR2CSA binding to tumor cells

[0063] After the cells were blocked, they were incubated with rVAR2 for 1 hour. After washing, they were incubated again with FITC-labeled anti-V5 tag monoclonal antibody and incubated at room temperature in the dark for 1 hour. After washing again, the cells were resuspended in pre-cooled PBS solution containing 5% BSA, and the cells were immediately analyzed using a flow cytometer. The fluorescence intensity of each group was recorded, and the ratio of the average fluorescence intensity of each group to the blank control was used as an evaluation index for the ability of cells to bind to rVAR2. The flow cytometry results showed that the four recombinant proteins could bind to lung adenocarcinoma cells (A549), colorectal cancer cells (SW480, HCT116, LoVo, HT29, CaCo2, SW620), and esophageal squamous cell carcinoma cells (KYSE180, KYSE30), and the average fluorescence intensity increased with the increase of the incubated protein concentration ( Figure 2 ).

[0064] In addition, rVAR2 can also bind to peripheral blood leukocytes of patients with acute lymphoblastic leukemia, but not to peripheral blood leukocytes of healthy controls ( Figure 3 ).

[0065] This application example is from the cell level (attached Figure 2 , 3 ) verified that the prepared VAR2CSA protein can specifically bind to ofCS and ofCSPG.

[0066] Example 3 “Chessboard method” optimizes the experimental conditions of sandwich ELISA

[0067] Determination of the optimal antibody / rVAR2 coating concentration: The coating concentrations of the antibody and rVAR2 were set to 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, and 0.125μg / mL, respectively. Determination of the optimal coating buffer: The candidate coating buffers are the common 0.05M bicarbonate buffer (pH=9.6), 0.01M Tris buffer (pH=8.0), and 0.01M PBS buffer (pH=7.2). Determination of the optimal blocking buffer: 1% gelatin, 3% gelatin, 5% BSA solution, and 5% milk were selected as candidate blocking buffers. Determination of the optimal plasma dilution to be tested: The plasma dilution ratio was tested in 8 gradients from 1:25 to 1:3200. Determination of the optimal action time of the sample to be tested: After adding the plasma sample to be tested, the reaction was allowed to stand for 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively. Determination of the optimal HRP-labeled rVAR2 dilution: HRP-labeled rVAR2 was set to 3.2μg / mL, 1.6μg / mL, 0.8μg / mL, 0.4μg / mL, 0.2μg / mL, 0.1μg / mL, 0.05μg / mL, 0.025μg / mL respectively. Determination of the optimal HRP-labeled rVAR2 action time: It was set to 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, and 90 minutes respectively. Determination of the optimal TMB action time: The TMB action time was set to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes respectively.

[0068] Each round of experiments was carried out according to the operating procedures, using positive plasma and negative plasma, and each plasma sample was repeated 2 wells, and the average value was taken. The experimental conditions corresponding to the maximum P / N value were determined as the optimal conditions and used in subsequent steps. Finally, it was determined that the best reaction P / N value could be obtained by coating rVAR2-5, anti-CD44 monoclonal antibody, anti-SDC1 monoclonal antibody and anti-CSPG4 polyclonal antibody at concentrations of 1-8μg / mL, 1-5μg / mL, 1-5μg / mL, and 0.5-5μg / mL, respectively.

[0069] The plasma to be tested was diluted at 1:5-1:100, and the concentration of HRP-labeled rVAR2-3 was 0.1 μg / mL: 1:10-1:5000 dilution can obtain a better reaction P / N value. Through the gradient reaction time exploration, the reaction time of the plasma sample to be tested was 60-120 minutes, the reaction time of HRP-labeled rVAR2-3 was 60-120 minutes, and the color development time of TMB was 5-30 minutes. In addition, using 0.05M bicarbonate buffer with pH = 9.6 as the coating solution and 5% BSA solution as the blocking buffer, a better reaction P / N value can be obtained.

[0070] Example 4 Effect of CSPG Detection in Case-Control Population of VAR2CSA

[0071] Detection principle: Anti-CD44 antibodies were coated on 96-well ELISA plates, incubated at 4°C overnight, and the excess antibody molecules not bound to the plate were washed away. The plates were blocked with 5% BSA, and the diluted plasma samples to be tested were added and incubated at room temperature. HRP-labeled rVAR2 was added and TMB was used for color development after incubation at room temperature ( Figure 4 ).

[0072] The reaction parameters are: the concentration of anti-CD44 antibody is 1-8 μg / mL; the plasma to be tested is diluted at 1:5-1:100, the concentration of HRP-labeled rVAR2 is 0.1 μg / mL: 1:10-1:5000 dilution; the reaction time of the plasma sample to be tested is 60-120 minutes, the reaction time of HRP-labeled rVAR2 is 60-120 minutes, and the color development time of TMB is 5-30 minutes. A 0.05M bicarbonate buffer with a pH of 9.6 is used as the coating solution, and 5% BSA is used as the blocking buffer.

[0073] A total of 22 healthy controls from the Guangdong natural population cohort (ChiCTR1800015736) and 59 cancer patients from the Cancer Center of Sun Yat-sen University were included. The results of sandwich ELISA test showed that the expression level of ofCS-modified CD44 in the plasma of the tumor patients was significantly higher than that in the healthy control group (OD value at 450 nm). The ofCS-modified CD44 in plasma was used as an independent variable for logistic regression to predict the probability of tumor occurrence. The area under the curve (AUC) after age and gender correction was 0.864 (95% CI = 0.7669 to 0.9604), Se = 0.845, Sp = 0.864; 0.8258 (95% CI = 0.7324 to 0.9192), Se = 0.793, Sp = 0.727; 0.8429 (95% CI = 0.748 to0.9378), Se = 0.879, Sp = 0.682; 0.8716 (95% CI = 0.7822 to 0.9611), Se=0.810, Sp=0.864.

[0074] Among them, Se = Sensitivity, Sp = Specificity, CI = Confidence Internal.

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Claims

1. An ELISA reagent for detecting tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein, It is characterized in that The ELISA reagent comprises a detection reagent and a capture reagent; the detection reagent is a VAR2CSA recombinant protein with a sequence as shown in SEQ ID NO: 4; and the capture reagent is an antibody against tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein.

2. The ELISA reagent according to claim 1, It is characterized in that The antibody is a monoclonal antibody, a polyclonal antibody, a multispecific antibody or an antibody fragment.

3. The ELISA reagent according to claim 1, It is characterized in that The detection reagent also includes an enzyme-labeled reagent.

4. The ELISA reagent according to claim 3, It is characterized in that The enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase (ALP), beta-galactosidase or gold colloid.

5. The ELISA reagent according to claim 4, It is characterized in that When horseradish peroxidase is used, the chromogenic substrate is selected from 3,3',5,5'-tetramethylbenzidine and o-phenylenediamine; when ALP is used, the chromogenic substrate is selected from p-nitrophenyl phosphate; when β-galactosidase is used, the chromogenic substrate is selected from o-nitrophenyl-β-D-pyranogalactoside.

6. The ELISA reagent according to claim 1, It is characterized in that The ELISA reagent also includes a blocking solution, a washing solution, a sample diluent, a color developing solution, a stop solution, and a standard substance.

7. The ELISA reagent according to claim 6, It is characterized in that The blocking solution is 3%-5% BSA or 1%-5% gelatin.

8. The ELISA reagent according to claim 6, It is characterized in that The blocking solution is 5% BSA.

9. The method for preparing the ELISA reagent according to claim 1, It is characterized in that The method for preparing the VAR2CSA recombinant protein comprises the following steps: S1. Using the amino acid sequence of Plasmodium VAR2CSA protein, a nucleic acid molecule having a sequence as shown in SEQ ID NO: 8 is constructed into an expression vector by homologous recombination; S2. Take the positive clones with correct sequencing prepared in step S1 and transform them into expression competent cells; S3. After screening, single clones were picked and tested again by PCR; S4. Add inducer to induce recombinant protein expression.

10. The method according to claim 9, It is characterized in that The strain of Plasmodium is selected from the FCR3 strain.

11. The method according to claim 9, It is characterized in that The expression vector is selected from pGEX series plasmids.

12. The method according to claim 11, It is characterized in that The pGEX series plasmid is pGEX-4T2 plasmid.

13. The method according to claim 12, It is characterized in that The C-terminus of the pGEX-4T2 plasmid is fused with a protease recognition site and a tag protein.

14. The method according to claim 9, It is characterized in that The expression competent cells are selected from Escherichia coli cells.

15. The method of claim 9, It is characterized in that The screening in S3 is double antibody screening.

16. The method of claim 15, It is characterized in that The dual antibody comprises ampicillin and / or streptomycin.

17. The method of claim 9, It is characterized in that The inducing agent is selected from isopropylthiogalactoside.

18. The method of claim 9, It is characterized in that The method for preparing the VAR2CSA recombinant protein also includes purifying the recombinant protein.

19. The method of claim 18, It is characterized in that The purification of the recombinant protein comprises the following steps: 1) The bacterial solution obtained by resuspending the competent cells expressing the VAR2CSA recombinant protein is broken; 2) Centrifuging the disrupted bacterial solution; 3) After filtering the supernatant after centrifugation, add it to a chromatography column that has been equilibrated with a resuspension buffer and is capable of binding to the C-terminal tag protein and incubate it sufficiently; 4) After washing with a washing buffer, the VAR2CSA recombinant protein is eluted with an elution buffer; 5) The eluted protein product is added to the GST affinity chromatography medium balanced with buffer for full binding; 6) Wash thoroughly with buffer, then replace with protease cleavage buffer and digest overnight; 7) The collected enzyme cleavage products are passed through a chromatography column that can bind to the C-terminal tag protein again, the protein eluate is collected, and the buffer is replaced with a desalting column or dialyzed against PBS, and finally the protein is concentrated.

20. The method of claim 19, It is characterized in that In step 1), resuspension solution is added to resuspend the expression competent cells.

21. The method of claim 20, It is characterized in that The resuspension solution includes 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH=7.4, 140 mM NaCl, 2.7 mM KCl, 2.5 mM β-ME, 1 μM DNase I, 1 mM PMSF, protease inhibitor I.

22. The method of claim 20, It is characterized in that Add resuspension according to the ratio of expression competent cells: resuspension at 1g:5ml.

23. The method of claim 19, It is characterized in that The step 1) is mechanical crushing.

24. The method of claim 23, It is characterized in that The mechanical crushing conditions are set as: pressure 800-1200 bar, continuous crushing 2-5 times.

25. The method of claim 19, It is characterized in that In the step 2), the centrifugation conditions are: 30,000×g-50,000×g, 3-5°C for 0.5-2 hours.

26. The method of claim 19, It is characterized in that In the step 3), the supernatant after centrifugation is filtered through a 0.45 μm and / or 0.22 μm filter membrane.

27. The method of claim 19, It is characterized in that In the step 4), the washing buffer is a washing buffer having an imidazole concentration of 80-150 mM.

28. The method of claim 19, It is characterized in that In the step 4), the elution buffer is an elution buffer with an imidazole concentration of 300-700 mM.

29. The method of claim 19, It is characterized in that In the step 6), the enzyme digestion is performed at 3-5°C.

30. The method of claim 19, It is characterized in that The steps 3) to 7) are carried out at 2-8°C.

31. Use of the ELISA reagent according to any one of claims 1 to 8 in preparing a detection reagent for detecting tumor-type chondroitin sulfate and / or tumor-type chondroitin sulfate-modified glycoprotein in a detection sample.

32. The use according to claim 31, It is characterized in that The tumor-type chondroitin sulfate includes tumor-type chondroitin sulfate glycosaminoglycan.

33. The use according to claim 31, It is characterized in that The tumor-type chondroitin sulfate-modified glycoprotein is selected from one or more of tumor-type chondroitin sulfate-modified CD44, tumor-type chondroitin sulfate-modified CSPG4, and tumor-type chondroitin sulfate-modified SDC1.

34. The use according to claim 33, It is characterized in that The tumor-type chondroitin sulfate-modified glycoprotein is selected from tumor-type chondroitin sulfate-modified CD44.

35. Use of the ELISA reagent according to any one of claims 1 to 8 in the preparation of a detection reagent for detecting the risk of a tumor expressing CSA.

36. The use according to claim 35, It is characterized in that The tumor is a malignant tumor of epithelial origin, a malignant tumor of mesenchymal tissue origin, a hematopoietic system cancer, a malignant melanoma, a malignant tumor of neuroepithelial tissue, or a neuroendocrine cancer.

37. The use according to claim 36, It is characterized in that The epithelial malignant tumor is: breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, basal cell skin cancer, renal clear cell carcinoma, head and neck keratinizing squamous cell carcinoma, skin squamous cell carcinoma, vulvar keratinizing squamous cell carcinoma, vulvar basal cell carcinoma, gastric cancer, thyroid cancer, intrahepatic bile duct cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, or bladder cancer.

38. The use according to claim 36, It is characterized in that The malignant tumor derived from mesenchymal tissue is: liposarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, lymphangiosarcoma or chondrosarcoma.

39. The use according to claim 36, It is characterized in that The hematopoietic cancer is lymphoma or leukemia.

40. The use according to claim 36, It is characterized in that The neuroepithelial tissue malignant tumor is: glioma, diffuse astrocytoma, or neuroblastoma.

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