T-PA and PAI-1 pseudo compound based on bispecific functional antibody skeleton, construction method and determination kit

Abstract

The invention discloses a t-PA and PAI-1 pseudo compound based on a bispecific functional antibody skeleton, a construction method and a determination kit, the pseudo compound takes a bifunctional antibody as the skeleton, a tissue-type plasminogen activator t-PA and a tissue-type plasminogen activator inhibitor PAI-1 are used for replacing two variable region arms in a natural antibody structure, and the t-PA and PAI-1 pseudo compound is prepared by using the bifunctional antibody as the skeleton. The protein takes an antibody constant region as a support skeleton, and a tissue type plasminogen activator t-PA and a tissue type plasminogen activator inhibitor PAI-1 are respectively arranged at the Y-type top of the antibody. The tissue-type plasminogen activator t-PA and the tissue-type plasminogen activator inhibitor PAI-1 are constructed into the pseudo-complex in a ratio of 1: 1, and a kit using the pseudo-complex as a freeze-drying calibrator or reference has the characteristics of high sensitivity, good stability and difficulty in interference.

Description

Technical Field

[0001] The present application relates to the technical field of t-PAI-C determination, and specifically to a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, a construction method, and a determination kit. Background Art

[0002] When coagulation is activated and fibrin is formed, it can activate the tissue plasminogen activator t-PA synthesized in vascular endothelial cells. The activated t-PA further activates plasminogen to convert into plasmin, which degrades fibrin to produce fibrin degradation products. Tissue plasminogen activator inhibitor PAI-1 is a physiological inhibitor of t-PA. After the two form a 1:1 complex, namely t-PAI-C, the activity of t-PA in activating plasminogen is inhibited, thereby inhibiting fibrin degradation. The concentration of PAI-1 in plasma is 5 times that of t-PA, which is much higher than t-PA. Therefore, it can be considered that almost all t-PA released into the blood forms a complex with PAI-1. The level of t-PAI-C is positively correlated with the concentration of t-PA and the degree of vascular endothelial damage, and is a direct marker for evaluating both. Elevated t-PAI-C is mainly seen in DIC, vascular endothelial damage and various arterial and venous thrombosis, such as deep vein thrombosis and acute myocardial infarction. Studies have shown that t-PAI-C can be used as a risk predictor for myocardial infarction.

[0003] However, during the development of clinical test kits, t-PAI-C was very unstable in plasma and easily degraded. It was also found that the stability of the purified eukaryotic expression products of tissue plasminogen activator t-PA and tissue plasminogen activator inhibitor PAI-1 was also relatively poor. Furthermore, the difficulty and stability of the two proteins forming a complex in vitro were also poor, which brought great difficulties to the stable storage of reference or calibrators used in clinical diagnostic reagents.

[0004] Application Contents

[0005] The purpose of the present application is to provide a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, a construction method and an assay kit, which realizes the quantitative detection of t-PAI-C and has the advantages of high detection sensitivity, high accuracy, good stability, simple operation, high throughput, etc., and can meet the needs of clinical t-PAI-C quantitative detection.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, wherein the pseudo-complex uses a bifunctional antibody as a skeleton, and replaces the two variable region arms in the natural antibody structure with tissue plasminogen activator t-PA and tissue plasminogen activator inhibitor PAI-1, thereby forming a protein with an antibody constant region as a supporting skeleton, and the top of the antibody Y-shape is tissue plasminogen activator t-PA and tissue plasminogen activator inhibitor PAI-1, respectively. The present application constructs a 1:1 pseudo-complex of tissue plasminogen activator t-PA and tissue plasminogen activator inhibitor PAI-1 by combining the technical method of bispecific functional antibodies. The kit using the pseudo-complex as a freeze-dried calibrator or reference product has the characteristics of high sensitivity, good stability, and is not easily interfered with.

[0007] The present application also provides a method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody framework, comprising the following steps:

[0008] Step 1: Molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002 are both artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis method;

[0009] Step 2: Select appropriate host cells according to the characteristics of the proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002 in step 1, and transiently express the proteins of SEQ ID NO: 001 and SEQ ID NO: 002, respectively;

[0010] Step 3: Purify the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002 transiently expressed in step 2;

[0011] Step 4: Use the purified SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins in step 3 to prepare a bispecific functional antibody backbone, and finally form a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1.

[0012] Preferably, the step 1: molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the codons encoding SEQ ID NO: 001 and SEQ ID NO: 002 are artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis method, specifically comprising: molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the codons encoding SEQ ID NO: 001 and SEQ ID NO: 002 are artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis method, a restriction site is respectively inserted at the 5' and 3' ends of the synthesized fragment, and the protein genes verified by XbaI and EcoRV after sequencing are respectively inserted into the corresponding restriction sites of plasmid PCDNA3.4 with the corresponding restriction sites, and a large amount of transfection plasmids are obtained by using Escherichia coli cloning bacteria, and filtered and sterilized.

[0013] Preferably, HEK293 is selected as the host cell, and the chemical transfection reagent PEI is used to transiently express the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002, respectively.

[0014] Preferably, the transient expression of sequence protein specifically includes:

[0015] A1, add KPM and sterile plasmid DNA into sterile centrifuge tube No. 1, pipette and mix well;

[0016] A2, add KPM and TA-293 transfection reagent to the second sterile centrifuge tube and mix well by pipetting;

[0017] A3, transfer all the liquid in sterile centrifuge tube No. 2 to sterile centrifuge tube No. 1, and mix well by pipetting;

[0018] A4, leaving the sterile centrifuge tube No. 2 obtained in 2.3 at room temperature to prepare the plasmid-vector complex;

[0019] A5, remove the cells and add the plasmid-vector complex prepared in 2.4, and culture with shaking until the recombinant protein is expressed to obtain a transiently expressed sequence protein.

[0020] Preferably, the purification of the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002 includes the following steps: starting the protein detector in advance and preheating it for 15-25 minutes, then selecting a column and performing pre-treatment of the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002; connecting, cleaning and balancing the column; loading the sample; eluting; rebalancing; cleaning and storing; in-situ cleaning and regeneration of the column; collecting the sequence proteins and determining their concentration to obtain the purified sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002.

[0021] Preferably, the in-place cleaning and regeneration of the column starts when the column pressure increases significantly or the purity of the purified sample decreases.

[0022] Preferably, step 4: using the SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins purified in step 3 to prepare a bispecific functional antibody backbone, and finally forming a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1 specifically includes step 4: using DTT to break the disulfide bonds of the SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins purified in step 3, respectively, mixing the SEQ ID NO: 001 and SEQ ID NO: 002 proteins after the disulfide bonds are broken, placing the mixed protein in a dialysis bag for dialyzing, and finally forming a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1.

[0023] The present application also provides a kit for determining a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, wherein the kit uses any of the pseudo-complexes described above as a lyophilized calibrator or reference.

[0024] Compared with the prior art, the beneficial effects of this application are:

[0025] (1) The present application uses the technology of combining bispecific functional antibodies to construct a 1:1 pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1, which has the advantages of simple production and low cost, making clinical diagnosis more accurate and reliable;

[0026] (2) The pseudo-complex of the present application uses the antibody constant region as the supporting skeleton, and the top of the antibody Y-shaped protein is respectively the tissue-type plasminogen activator t-PA and the tissue-type plasminogen activator inhibitor PAI-1. The kit using the pseudo-complex as a freeze-dried calibrator or reference product has the characteristics of high sensitivity, good stability, and is not easily interfered. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the transient expression operation of sequence proteins for a method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton proposed in this application;

[0028] Figure 2 This is a diagram showing the detection results of different process complexes for a pseudo-complex determination kit of t-PA and PAI-1 based on a bispecific functional antibody skeleton proposed in the present application;

[0029] Figure 3 This is a comparison chart of the assay results of the same sample using a pseudo-complex assay kit of t-PA and PAI-1 based on a bispecific functional antibody skeleton proposed in this application and a kit of Guangzhou Wondfo Biotech Co., Ltd. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0031] Embodiment 1

[0032] A method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton comprises the following steps:

[0033] Step 1: Molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the coding sequences of SEQ ID NO: 001 and SEQ ID NO: 002 are both artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis. In order to facilitate the insertion of the obtained gene target fragment into a specific site of the expression vector, a restriction site is inserted at the 5' and 3' ends of the synthesized fragment, respectively, and the protein genes verified by sequencing by XbaI and EcoRV are inserted into the corresponding restriction sites of plasmid PCDNA3.4 with the corresponding restriction sites, and a large amount of transfection plasmids are obtained by using Escherichia coli cloning bacteria, and filtered for sterilization;

[0034] Step 2: According to the characteristics of the proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002 in step 1, HEK293 is selected as the host cell, and the chemical transfection reagent PEI is used to transiently express the proteins of SEQ ID NO: 001 and SEQ ID NO: 002, respectively;

[0035] like Figure 1 As shown, the sequence protein transient expression specifically includes:

[0036] A1, add 5 ml KPM and 100 μg sterile plasmid DNA into a 15 ml No. 1 sterile centrifuge tube, and gently pipette to mix;

[0037] A2, add 5 ml KPM and 500 μl TA-293 transfection reagent to a 15 ml No. 2 sterile centrifuge tube, and gently pipette to mix;

[0038] A3, transfer all the liquid in sterile centrifuge tube No. 2 to sterile centrifuge tube No. 1, and mix by gently pipetting;

[0039] A4, place the sterile centrifuge tube No. 2 obtained in 2.3 at room temperature for 10 minutes to prepare the plasmid-vector complex;

[0040] A5. Remove the cells from the constant temperature shaker, add the plasmid-vector complex prepared in 2.4 while shaking, and then put them back into the CO2 constant temperature shaker for shaking culture until the recombinant protein is expressed to obtain a transiently expressed sequence protein.

[0041] It should be noted that after 3 hours of shaking culture, an appropriate amount of antibiotics can be added as needed. The expression level of the recombinant protein can reach the highest value around the 6th day after transfection and can be used for subsequent protein purification.

[0042] Step 3: Purify the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002 transiently expressed in step 2;

[0043] Specifically include:

[0044] First, prepare the reagents: binding buffer: 1×PBS; elution buffer: 0.1M Glycine-HCl, 0.15M NaCl, pH 3.0-3.5, different elution pH can be selected according to different antibodies; dialysate: 1×PBS; cleaning and storage buffer: 0.1M NaOH, 20% ethanol; in-situ cleaning buffer: 0.5M NaOH.

[0045] Then turn on the protein detector and preheat it for 20 minutes.

[0046] Select the column and pre-treat the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002: select the column with 50% of the total amount of the primary purified antibody as the binding amount and 25mg monoclonal antibody / ml filler as the loading capacity, and the monoclonal antibody binding amount is preferably 50%-80% of the column loading capacity. If the primary purified sequence protein is frozen at -20℃, it should be taken out the night before and dissolved at 4℃ or put in water to accelerate thawing on the same day, and after thawing, centrifuge at 12000rpm and 4℃ for 30min before loading.

[0047] Then connect, clean and balance the column: first rinse the column with purified water for at least 2 column volumes (CV) to remove ethanol. If the antibody purified last time is not the same as the antibody purified this time, rinse with 0.1M NaOH solution for 3-5CV, then balance the column with 3-5CV binding buffer, and balance the column with binding buffer for at least 5 column volumes.

[0048] Then load the sample: measure the concentration of the sequence protein after centrifugation again to determine the total amount of sample loading. Keep 1-5 mg of the original solution before loading. If the concentration of the antibody solution is too high, dilute the antibody solution to about 5 mg / mL before loading. Adjust the loading flow rate to half of the working flow rate. After the loading is completed, A280 drops to the baseline and then returns to the working flow rate. Rinse with loading buffer for at least 3CV.

[0049] Then elution: elution with elution buffer. To protect the activity of the antibody, when about 5 mL is collected in the collection container each time, slowly add about 50 μL 1M Tris-HCl pH 10 to mix, and stir and collect with a stirrer. Finally, adjust the pH of the antibody solution to 7.0-7.5 with pH test paper, put it into a dialysis bag for dialysis, and dialyze it with 1× PBS buffer at 4°C with 30-50 times the sample volume. Change the solution twice in the middle. After stopping collection, continue to rinse the column with elution buffer for at least 3CV.

[0050] Re-balance: After elution, rinse the column with at least 3CV 1× PBS to restore it to neutrality and keep records of its use.

[0051] Then wash and store: put the sample port into purified water to rinse 2 column volumes, then put the sample port into 20% ethanol and rinse for at least 2CV to fully immerse the gel in ethanol. After rinsing, remove the column from the instrument in the order of first down and then up, keep a record of use, and store at 4℃.

[0052] Then, the column was regenerated by in-situ cleaning: 3CV was used for washing with 0.5M NaOH solution, and the column was immediately equilibrated with 3-5CV of binding buffer, and then 2CV was used for washing with purified water and finally stored in 20% ethanol.

[0053] It should be noted that if the column pressure increases significantly or the purity of the purified sample decreases, the column should be cleaned and regenerated.

[0054] Finally, samples were collected and concentrations were measured: the antibody solution was dialyzed and filtered with a 0.22 μm filter membrane, the concentration was measured after dilution, and then Proclin 300 was added to a final concentration of 0.05%, the proteins were packaged and stored in a -20°C refrigerator to obtain purified SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins.

[0055] Step 4, using 5mM DTT to break the disulfide bonds of the SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins purified in step 3, respectively, and mixing the SEQ ID NO: 001 and SEQ ID NO: 002 proteins after the disulfide bonds are broken, and the mixed protein is dialyzed in a 30KDa dialysis bag to finally form a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1 for subsequent reference or calibration use.

[0056] Antigen assignment, antigen / calibrator diluent (1000ml)

[0057]

[0058]

[0059] The eukaryotically expressed tandem protein was diluted with antigen / calibrator diluent and detected and assigned using the tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI-C) detection kit (chemiluminescence method) produced by Sysmex Medical Electronics (Shanghai) Co., Ltd.

[0060] The stability of the pseudo-complex of t-PA and PAI-1 based on the bispecific functional antibody skeleton was studied. The high and low concentration reference proteins were placed in a 4-degree refrigerator, and the signal value was detected on the 1st, 3rd, 5th, 7th and 14th day (the average value was repeated 5 times), and the CV change of the detection signal was measured. The results are as follows:

[0061] Incubation time at 2-8°C Low sample signal value (4ng / mL) Low sample signal value (32ng / mL) Day 0 32560 501136 1 day 32202 499902 3 days 32004 489356 7 days 31869 480345 14 days 28964 450386 Protein degradation rate in 1 day 1.10％ 0.25％ Protein degradation rate in 3 days 1.71％ 2.35％ 7-day protein degradation rate 2.12％ 4.15％ Protein degradation rate at 14 days 11.04％ 10.13％

[0062] Embodiment 2

[0063] A kit for determining a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, wherein the pseudo-complex prepared by the above method is used as a freeze-dried calibrator or reference.

[0064] It should be noted that the performance evaluation of the t-PA and PAI-1 pseudo-complex assay kit based on the bispecific functional antibody skeleton of Example 2 was performed as follows:

[0065] P1: Standard curve

[0066] The pseudo-complex-1 complex t-PAI-C of tissue plasminogen activator t-PA and plasminogen activator inhibitor PAI-1 prepared in Example 1 was diluted with standard diluent to prepare calibration solution SO-S6 of different concentrations, with concentrations of 4ng / mL, 8ng / mL, 16ng / mL, 32ng / mL, and 64ng / mL, respectively, and stored at -20°C for standby use, and then the calibration product was detected using the kit provided in Example 2 of the present invention, and the luminescence intensity values ​​corresponding to each calibration product were read respectively. The standard curve was obtained by fitting with concentration as the horizontal axis and luminescence intensity as the vertical axis.

[0067] P2: Linear

[0068] The basic bovine plasma sample with added antigen (concentration ≤ blank limit) or the calibration diluent (try to use real high-value serum samples) is used as the high-concentration sample, and the bovine plasma sample or the calibration diluent is used as the low-concentration sample. The high-value samples close to the upper limit of the linear range are diluted to several concentrations according to a certain ratio, as shown in the following table, where the low-value concentration samples must be close to the lower limit of the linear range. The sample of each concentration is tested 3-5 times, and the average value is calculated. The concentration value of the analyte (as the horizontal axis) and the average value of the test result (as the vertical axis) are fitted with a straight line using the least squares method. And calculate the correlation coefficient r within the linear range. The acceptance standard is correlation coefficient r>0.99, such as Figure 2 shown.

[0069]

[0070]

[0071] P3: Minimum detection limit

[0072] The zero-concentration calibration dilution was used as the sample for detection, and the measurement was repeated 20 times to obtain the light signal value of the 20 measurement results. The mean value (M) and standard deviation (SD) were calculated to obtain M+2SD. A two-point regression fitting was performed based on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibration products to obtain a linear equation. The light signal value of M+2SD was substituted into the equation to obtain the corresponding concentration value, which is the minimum detection limit.

[0073] P4: Correctness

[0074] Prepare samples with two concentration levels, high and low, using a pseudo-complex of tissue plasminogen activator t-PA and plasminogen activator inhibitor PAI-1. Repeat the measurement three times, and calculate the relative deviation between the average value (denoted as M) and the labeled value. Calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples with two concentrations, high and low, that have been determined by the reference method. Repeat the measurement three times for each concentration sample, and take the average of the test results to calculate the relative deviation from the labeled value. Acceptance criteria: Relative deviation within ±10%.

[0075] B=(MT) / TX100%

[0076] Wherein, B: relative deviation; M: average value of measured concentration; T: indicated value.

[0077] P5: Precision

[0078] 5.1 Repeatability evaluation

[0079] Use samples with 2-3 concentration levels (high level, low level) to repeat the test 10 times each, calculate the mean value M and standard deviation SD of the 10 measurement results, and obtain the coefficient of variation CV according to formula (2).

[0080] CV=SD / M×100%

[0081] Wherein, CV: coefficient of variation; SD: standard deviation of 10 measurement results; M: mean value of 10 measurement results.

[0082] 5.2: Batch-to-batch precision

[0083] The precision of the measurement results of the same test sample (commonly used quality control product) measured by the same operator (group) on the same instrument, using the same method and the same type and batch of reagents over a period of time (usually one month or 20 working days).

[0084]

[0085] P6: Interfering substances

[0086] Weigh the interfering substances to be studied. Prepare a mother solution of a certain concentration of the interfering substances. Add bilirubin, triglycerides, hemoglobin and total protein mother solutions to the high and low value plasmas used for interference according to the calibrated concentrations. Prepare high concentration interfering substance plasma samples containing bilirubin, triglycerides, hemoglobin and total protein at the concentrations required by the specifications. The control plasma sample is the one without added interfering substances. The added volume is controlled within 1 / 20 to avoid the matrix effect of the solvent. Test the high concentration interfering substance sample and the control plasma sample 2-3 times respectively. The mean result of the test of the high concentration interfering substance sample is recorded as M. The mean result of the test of the control plasma sample is recorded as T, and the relative deviation B of the measured concentration is calculated according to formula (⑷).

[0087] B=(MT) / TX100%⑷

[0088] Where: Add the interfering substance to the high and low value plasma

[0089] Wherein, B: relative deviation; M: mean of the concentration of the analyte in samples with high and low concentrations of interfering substances; T: mean of the concentration of the analyte in control plasma samples.

[0090] The performance evaluation results of the above-mentioned assay kit are shown in the following table:

[0091]

[0092]

[0093] It should also be noted that, regarding the test of the clinical performance of the kit of the present invention, 151 clinical plasma samples were tested simultaneously using the kit of Example 2 and an international brand kit (Sysmex kit).

[0094] The concentration of the above-mentioned samples was also tested using the test kit of Guangzhou Wondfo Biotech Co., Ltd., and the test was performed according to the instructions of the kit.

[0095] like Figure 3 As shown, the detection concentration obtained by the kit prepared in Example 2 was analyzed and compared with the detection result concentration of the kit of Guangzhou Wondfo Biotech Co., Ltd., and the clinical correlation results are shown in the figure below. The clinical correlation is R2=0.902, and the result meets the acceptance standard (acceptance standard: R2>0.9), indicating that the kit of the present invention has a good correlation with the kit of Guangzhou Wondfo Biotech Co., Ltd.

[0096] Further, the detection concentration obtained by the kit prepared in Example 2 of the present invention and the detection result concentration of the kit of Guangzhou Wanfu Biotechnology Co., Ltd. were statistically analyzed for the positive and negative coincidence rate, according to the reference interval (or clinical judgment value). The positive and negative coincidence rate calculation table is as follows:

[0097]

[0098]

[0099] The final test results are as follows:

[0100]

[0101] The above experiments show that the test kit for the pseudo-complex of tissue plasminogen activator t-PA and tissue plasminogen activator inhibitor PAI-1 of the present invention realizes the quantitative detection of t-PAI-C in the sample, has the advantages of high detection sensitivity, high accuracy, good stability, simple operation, high throughput, etc., and can meet the needs of clinical t-PAI-C quantitative detection. Compared with the limited detection kits at home and abroad, it has higher detection stability and lower detection cost.

Claims

1. A pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody framework, characterized in that: The pseudo-complex uses a bifunctional antibody as a skeleton, and replaces the two variable region arms in the natural antibody structure with tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1, thereby forming a protein with the antibody constant region as a supporting skeleton and the Y-shaped top of the antibody being tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1 respectively.

2. A method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, characterized in that: The following steps are involved: Step 1: Molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002 are both artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis method; Step 2: Select appropriate host cells according to the characteristics of the proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002 in step 1, and transiently express the proteins of SEQ ID NO: 001 and SEQ ID NO: 002, respectively; Step 3: Purify the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002 transiently expressed in step 2; Step 4: Use the purified SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins in step 3 to prepare a bispecific functional antibody backbone, and finally form a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1.

3. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 2, characterized in that: The step 1: molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the codons for encoding SEQ ID NO: 001 and SEQ ID NO: 002 are artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis methods, specifically comprising: molecular construction, gene construction of sequence proteins encoding SEQ ID NO: 001 and SEQ ID NO: 002, wherein the codons for encoding SEQ ID NO: 001 and SEQ ID NO: 002 are artificially optimized CHO or HEK293 cell preferred codons, and are obtained by chemical synthesis methods, a restriction site is respectively inserted into the 5' and 3' ends of the synthesized fragment, and the protein genes verified by XbaI and EcoRV after sequencing are respectively inserted into the corresponding restriction sites of plasmid PCDNA3.4 with the corresponding restriction sites, and a large amount of transfection plasmids are obtained by using Escherichia coli cloning bacteria, and filtered and sterilized.

4. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 3, characterized in that: HEK293 was selected as the host cell, and the chemical transfection reagent PEI was used to transiently express the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002, respectively.

5. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 4, characterized in that: Sequence protein transient expression specifically includes: A1, add KPM and sterile plasmid DNA into sterile centrifuge tube No. 1, pipette and mix well; A2, add KPM and TA-293 transfection reagent to the second sterile centrifuge tube and mix well by pipetting; A3, transfer all the liquid in sterile centrifuge tube No. 2 to sterile centrifuge tube No. 1, and mix well by pipetting; A4, leaving the sterile centrifuge tube No. 2 obtained in 2.3 at room temperature to prepare the plasmid-vector complex; A5, remove the cells and add the plasmid-vector complex prepared in 2.4, and culture with shaking until the recombinant protein is expressed to obtain a transiently expressed sequence protein.

6. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 5, characterized in that: The purification of sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002 includes the following steps: starting the protein detector in advance and preheating for 15-25 minutes, then selecting a column and performing pre-treatment of the sequence proteins of SEQ ID NO: 001 and SEQ ID NO: 002; connecting, cleaning and balancing the column; loading the sample; eluting; rebalancing; cleaning and storing; in-situ cleaning and regeneration of the column; collecting the sequence proteins and determining the concentration to obtain the purified sequence proteins of SEQ ID NO: 001 and SEQ ID NO:

002.

7. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 6, characterized in that: The column in-place cleaning regeneration starts when the column pressure increases significantly or the purity of the purified sample decreases.

8. The method for constructing a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton according to claim 6, characterized in that: Step 4: using the SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins purified in step 3 to prepare a bispecific functional antibody backbone, and finally forming a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1, specifically comprising: step 4: using DTT to break the disulfide bonds of the SEQ ID NO: 001 and SEQ ID NO: 002 sequence proteins purified in step 3, respectively, mixing the SEQ ID NO: 001 and SEQ ID NO: 002 proteins after the disulfide bonds are broken, placing the mixed protein in a dialysis bag for dialyzing, and finally forming a pseudo-complex of tissue-type plasminogen activator t-PA and tissue-type plasminogen activator inhibitor PAI-1.

9. A kit for measuring a pseudo-complex of t-PA and PAI-1 based on a bispecific functional antibody skeleton, characterized in that: The assay kit uses the pseudo complex as described in any one of claims 1 to 7 as a lyophilized calibrator or reference.

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