Single-chain antibody against beta-stimulant and use thereof

Abstract

The application discloses an anti-beta-stimulant single-chain antibody and application thereof, and belongs to the technical field of biology. The beta-stimulant is bromobuterol, the anti-bromobuterol single-chain antibody is composed of a heavy chain variable region, a connecting peptide and a light chain variable region, the connecting peptide is located between the heavy chain variable region and the light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2. The application couples BRO and ZIL small molecules with bovine serum albumin and chicken serum albumin, and obtains a specific and high-sensitivity mAb cell strain through screening. Then, based on the obtained monoclonal cell strain, the complete antibody gene is amplified by using a PCR technology, and finally, a high-sensitivity and high-specificity single-chain antibody is screened, thereby laying a foundation for subsequent development of a rapid detection kit for BRO and ZIL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a single-chain antibody against beta-stimulant and application thereof. BACKGROUND

[0002] Brombuterol (BRO) and Zilpaterol (ZIL) are both a kind of "lean meat powder" and belong to beta-stimulant. They can significantly improve the lean meat rate of animals and are easy to form residues in edible tissues of animals. Excessive use will cause symptoms such as rapid breathing and muscle tremor, which poses a serious threat to the safety of animal-derived food.

[0003] At present, the detection of beta-stimulant substances is mainly instrument analysis, including gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Although these methods can accurately quantify and qualitatively detect BRO and ZIL in different samples, the use of these methods is greatly limited due to the high cost of instruments, time and labor consumption, the need for a dedicated laboratory and operators, and the long and strict sample processing procedures. The antibodies used in immunological detection methods for BRO and ZIL are mainly monoclonal antibodies (mAb) and polyclonal antibodies (pAb). The conventional preparation methods of mAb and pAb have the disadvantages of complicated preparation procedures, long time consumption, non-optimization, and differences in antibody sensitivity between different batches, which can lead to poor stability of the constructed immunological detection method. Compared with traditional antibodies, single-chain antibodies (ScFv) are genetically engineered antibodies with only one chain. They can be studied at the molecular level and their affinity and stability can be improved through random mutagenesis and site-directed mutagenesis. Based on phage display technology, this antibody can be produced without the need for hybridoma cell lines, and has the advantages of convenience and easy preparation, and has gradually become an important research object for the establishment of immunological rapid detection methods. SUMMARY

[0004] To solve the above technical problems, the purpose of the present application is to provide a single-chain antibody against beta-stimulant and application thereof, in order to solve the problems of complicated procedures, high price, time and labor consumption, or poor stability in the existing detection of beta-stimulant.

[0005] The technical solution of the present application to solve the above technical problems is as follows:

[0006] The anti-beta-stimulant single-chain antibody is an anti-bromobuterol single-chain antibody, and the anti-bromobuterol single-chain antibody is composed of a heavy chain variable region, a connecting peptide and a light chain variable region, the connecting peptide is located between the heavy chain variable region and the light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

[0007] Further, the nucleotide sequence for coding the heavy chain variable region of the anti-bromobuterol single-chain antibody is shown as SEQ ID NO. 3, and the nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO. 4.

[0008] Further, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 5.

[0009] Further, the beta-stimulant is zilpaterol, the anti-zilpaterol single-chain antibody is composed of a heavy chain variable region, a connecting peptide and a light chain variable region, the connecting peptide is located between the heavy chain variable region and the light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7.

[0010] Further, the nucleotide sequence for coding the heavy chain variable region of the anti-zilpaterol single-chain antibody is shown as SEQ ID NO. 8, and the nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO. 9.

[0011] Further, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 10.

[0012] An expression vector, a bacteriophage or a recombinant bacterium, comprising all the nucleotides described above.

[0013] A monoclonal cell strain, comprising all the nucleotides described above.

[0014] A kit for detecting beta-stimulants, comprising the anti-beta-stimulant single-chain antibody described above.

[0015] The anti-beta-stimulant single-chain antibody or the kit for detecting beta-stimulants described above is applied to beta-stimulant detection.

[0016] The present application has the following beneficial effects:

[0017] The present application respectively couples BRO and modified ZIL small molecules with bovine serum albumin and chicken serum albumin by diazo method, screens the mAb cell strains capable of secreting specific and high-sensitivity antibodies after immunizing mice with the prepared complete antigens and identifying the serum titer and sensitivity, uses the obtained monoclonal cell strains as the basis, amplifies the complete antibody genes by PCR technology, and finally screens the high-sensitivity anti-bromobuterol single-chain antibody and anti-zilpaterol single-chain antibody, thereby laying a foundation for the subsequent development of the rapid detection kit of BRO and ZIL. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 BSA protein standard curve chart in example 1;

[0019] Figure 2 SDS-PAGE identification results of BRO-BSA and BRO-OVA in example 1, wherein M is rainbow maker 180 in A, 1 is the coupling product of BRO-BSA, and 2 is the BSA carrier protein, M is rainbow maker 180 in B, 1 is the OVA carrier protein, and 2 is the coupling product of BRO-OVA;

[0020] Figure 3 UV scanning chart of BRO-OVA and BRO-BSA in example 1, wherein A is BRO-OVA, and B is BRO-BSA;

[0021] Figure 4 Mouse serum titer results of BRO-BSA in example 1;

[0022] Figure 5 IC 50 determination results of the mouse polyclonal serum of BRO in example 1;

[0023] Figure 6 Microscope photograph of feeder cells in example 2;

[0024] Figure 7 Microscope photograph of the growth state of the fused cells in example 2, wherein A is the third day, B is the fourth day, C is the sixth day, D is the seventh day, E is the tenth day, and F is the thirteenth day;

[0025] Figure 8 Detection results of the inhibition rate of the positive wells of cell fusion in example 2;

[0026] Figure 9 Detection results of the inhibition rate of the positive wells of the first to third times of limited dilution in example 2, wherein A is the first time, B is the second time, and C is the third time;

[0027] Figure 10The titer and IC50 of the supernatant of 2B10 cells in Example 2 50 The determination results

[0028] Figure 11 The titer and IC50 of the mouse ascites in Example 2 50 The determination results

[0029] Figure 12 The electrophoretogram of total RNA extracted from 2B10 cells in Example 3

[0030] Figure 13 The electrophoretogram of VH gene and VL gene fragments in Example 3, wherein M is DL200 DNA Ladder, 1 and 2 are VH gene, and 3 and 4 are VL gene

[0031] Figure 14 The electrophoretogram of ScFv ligation in Example 3

[0032] Figure 15 The electrophoretogram of pCANTAB-5E phagemid vector in Example 3

[0033] Figure 16 The PCR identification results of positive bacterial solution in Example 3

[0034] Figure 17 The phage-ELISA detection results of BRO phage in Example 3

[0035] Figure 18 The Western Blotting identification results of BRO-ScFv in the periplasmic cavity in Example 3, wherein M is Rainbow Maker 180, 1 is BRO-ScFv in the periplasmic cavity, and 2 is the supernatant of induced expression bacteria

[0036] Figure 19 The titer identification results and antibody affinity identification results of BRO-ScFv single-chain antibody in Example 3, wherein the upper graph is the titer identification results, and the lower graph is the affinity identification results

[0037] Figure 20 The SDS-PAGE identification results of ZIL-OVA in Example 4, wherein M is Rainbow Maker 180, 1 is OVA carrier protein, and 2 is ZIL-OVA coupling product

[0038] Figure 21 The SDS-PAGE identification results of ZIL-BSA in Example 4, wherein M is Rainbow Maker 180, 1 is BSA carrier protein, and 2 is ZIL-BSA coupling product

[0039] Figure 22Figure 2: UV scan of ZIL-OVA and ZIL-BSA in Example 4;

[0040] Figure 23 Figure 4: Results of the indirect ELISA for the detection of mouse polyclonal serum titers in Example 4;

[0041] Figure 24 Figure 6: Results of the mouse ascites titer detection of hybridoma cell 5E7-9A in Example 6;

[0042] Figure 25 Figure 7: Sensitivity standard curve of mouse ascites in Example 6;

[0043] Figure 26 Figure 8: Indirect ELISA standard curve of ZIL single-chain antibody in Example 6. DETAILED DESCRIPTION

[0044] The principles and features of the present application are described below in conjunction with the accompanying drawings, in which the examples are used to explain the present application and are not intended to limit the scope of the present application. In the examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturers are used. The reagents or instruments used are not specified, and are the conventional products that can be purchased on the market.

[0045] Example 1: Synthesis and identification of BRO complete antigen

[0046] I. Modification of BRO molecule

[0047] (1) Diazotization of BRO

[0048] Accurately weigh 3 mg of the BRO sample using a high-precision balance and dissolve it in 1.5 mL of pre-lyophilized 0.1 moL hydrochloric acid solution. Under an ice water bath environment, gradually add 680 μL of pre-cooled 1 mg / mL sodium nitrite solution at a low speed with continuous stirring. Use starch-KI test paper to detect whether the sodium nitrite is excessive, and the judgment standard is that the test paper turns to gray purple. The entire diazotization process needs to be carried out in the dark, continuously stirring in a 4°C refrigerator for 1-2 h.

[0049] (2) Coupling of BRO diazonium salt with two carrier proteins (BSA and OVA)

[0050] Precise balance was used to accurately weigh 26 mg BSA and 18.4 mg OVA, ensuring a constant molar ratio of 20:1 between BRO and carrier proteins. BSA and OVA were dissolved in 3 mL of 0.1 mol / L sodium borate solution (pH 8.6). The mixture was stirred in an ice bath for 2 h. Subsequently, the diazonium salt solution obtained in the first step was divided into two equal parts. Each part was slowly added to the solution containing BSA and OVA. During this process, dilute sodium hydroxide solution was used to adjust the pH of the mixture, ensuring that it remained stable at 8.0, and then continued to be stirred and left overnight at low temperature to promote the coupling reaction to proceed fully. The BRO molecular modification flow chart is shown below:

[0051]

[0052] (3) Purification and dialysis

[0053] After the reaction was completed the next day, the bright yellow reaction product was collected and dialyzed against phosphate buffered saline (PBS) at 4°C for 3 consecutive days, with the dialysis solution being changed during this period.

[0054] (4) Concentration determination and storage

[0055] The Bradford protein quantification kit from Bio-Rad was used to determine the protein concentration of the product after coupling. After obtaining the accurate concentration reading, the sample was appropriately diluted to achieve a concentration of 1 mg / mL. Subsequently, the sample was divided into 1.5 mL EP tubes and stored in a low-temperature refrigerator at -80°C.

[0056] II. Identification of the complete antigen of BRO

[0057] (1) Complete antigen concentration identification

[0058] The concentration of the complete antigen was determined using the Bradford method. The steps are as follows:

[0059] Take 10 μL of BSA standard with a concentration of 5 mg / mL, add 240 μL of PBS and mix well to prepare a standard solution of 0.2 mg / mL.

[0060] Dilute 5x G250 staining reagent with sterile double distilled water to prepare 1x G250 staining solution.

[0061] In a 96-well enzyme-labeled plate, different volumes (0-20 μL) of the above standard solution were added to each well, followed by dilution with PBS to a total volume of 20 μL.

[0062] The complete antigen sample was diluted by 2-256 times, and 20 μL of each dilution was added to a 96-well plate. 200 μL of 1x G250 staining solution was added to each well, and it was left for 3 min. The spectrophotometer was used to measure the absorbance value at 595 nm wavelength. The data was recorded, and the absorbance-concentration graph was plotted according to the standard curve. The protein concentration of the complete antigen sample to be tested was calculated by comparison.

[0063] The experimental results are shown in Figure 1 By measuring the absorbance value of the BSA protein standard at 595 nm wavelength, a standard curve graph of BSA protein was successfully constructed. The protein concentration of the immunogen BRO-BSA was calculated to be 1.4 mg / mL using the standard curve, and the protein concentration of the antigen BRO-OVA was detected to be 2 mg / mL.

[0064] (2) SDS-PAGE identification of complete antigen

[0065] Sample processing: Adjust the concentrations of BRO-BSA, BSA, BRO-OVA and OVA to 4 μg / mL, mix with loading buffer, denature by boiling in boiling water for 10 min, initially set the voltage to 80 V during electrophoresis, and then increase to 120 V after the sample enters the separation gel, and complete the electrophoresis when it reaches the bottom of the gel. The electrophoresed gel was stained with Coomassie brilliant blue for 45 min, and the staining solution was recovered. Decolorization was performed every 2 h, and the decolorization solution was replaced until the gel was completely transparent. Photographs were taken using an ultraviolet gel imaging system, and the results were analyzed.

[0066] The experimental results are shown in Figure 2 The prepared coupled proteins: BRO-BSA and BRO-OVA were identified by electrophoresis. The experimental results showed that the migration speed of the complete antigens BRO-BSA and BRO-OVA after coupling reaction was significantly lower than that of their respective carrier proteins BSA and OVA. The decrease in migration speed was due to the relative increase in molecular weight of BRO-BSA and BRO-OVA after coupling, which resulted in more hindrance in gel migration. This phenomenon confirmed that the BRO molecule had effectively coupled with the BSA and OVA carrier proteins to form the expected coupled protein product.

[0067] (3) UV full wavelength identification of complete antigen

[0068] The BSA, OVA and BRO standard solutions were adjusted to 1 mg / mL with PBS. The complete antigen was identified by ultraviolet full wavelength scanning with a microplate reader. After calibration with PBS as the baseline, the absorbance of each sample was measured in the range of 200 nm to 500 nm, and the spectrum was drawn using origin 2021 software. By analyzing whether the absorption peak of the sample is shifted, it can be evaluated whether the synthesis of the BRO complete antigen is successful.

[0069] The experimental results are shown in Table 2. Figure 3 There is a significant peak shift between the maximum absorption peak wavelength of the complete antigen conjugate product and the carrier protein alone. The results confirm from another angle that the BRO has successfully and effectively coupled with the carrier protein. The introduction of the new chemical group changes the spectral properties of the complete antigen, resulting in a shift in the wavelength of the maximum absorption peak in the ultraviolet spectrum. Therefore, through the analysis of the ultraviolet scanning technology, it can be confirmed that the coupling reaction between the BRO and the carrier protein has been successfully carried out, and the complete antigen with the expected characteristics has been generated.

[0070] III. Animal Immunization

[0071] The purchased 7-week-old BALB / c female mice were acclimated for one week, then divided into two groups and immunized. BRO-BSA was used as the immunogen, mixed with FCA in equal volume, prepared into an emulsifier at a dose of 200 μL per mouse, and finally formed into a stable water-in-oil emulsion in water. According to this method, FIA was used for booster immunization every two weeks, and five immunizations were performed in succession. The detailed immunization scheme is shown in Table 1.

[0072] Table 1 BRO-BSA Immunization Program

[0073]

[0074] (1) Determination of mouse antibody serum titer by indirect ELISA

[0075] Blood collection: After stunning the mice, 2 μL of blood was collected by tail clipping and dissolved in 198 μL of PBS, i.e. 1:100 dilution.

[0076] Coating: 5 μg / mL of BRO-OVA was coated in a 96-well plate at 50 μL per well, 4°C overnight, PBST was washed 5 times, and dried by tapping;

[0077] Blocking: 200 μL of 5% skim milk was added to each well for blocking, 37°C, 2h, PBST was washed 5 times, and dried by tapping;

[0078] Addition of the first antibody: 50 μL of PBS was added to each well, and the above-mentioned 1:100 diluted mouse serum was added, and then mixed by blowing, and then transferred to the next well, and mixed again by blowing, and so on, so that the mouse serum was diluted by a factor of 2 (1:200-1:25600), 37°C, 1h, 5 times of washing with PBST, and the non-immunized mouse serum was used as a negative control;

[0079] Addition of the second antibody: the HRP-goat anti-mouse IgG was diluted to 1:5000 with the blocking solution, 50 μL / well, 37°C, 40 min, 5 times of washing with PBST, and then dried by tapping;

[0080] TMB color development: 50 μL / well of TMB color developing solution was used, 37°C, 10 min;

[0081] Addition of the termination solution: 50 μL / well of H2SO4 termination solution was added to the 96-well plate to terminate the reaction;

[0082] Reading and recording the results: the OD value of the 96-well plate was detected by using an enzyme-labeled instrument 450 , and the sample well was determined as P, the negative control well was determined as N, and the maximum dilution factor of the serum when P / N≥2.1 was determined as the titer of the mouse antibody.

[0083] The experimental results are shown in Table 1. Figure 4 Six BALB / c mice were divided into two groups A and B (named as A1, A2, A3, B1, B2, B3, respectively), and the six mice immunized with BRO-BSA all successfully produced antibodies against BRO. According to the judgment standard set in the experiment, that is, the P / N value is greater than 2.1, compared with the non-immunized negative mouse serum, the titer of pAb in the serum of the immunized mouse is significantly improved, reaching 1:1.28×10 4 . This result shows that the coupled immunogen BRO-BSA can effectively induce specific antibody response in mice.

[0084] (2) icELISA determination of the sensitivity of mouse serum

[0085] The serum was collected, coated and blocked by the method in (1).

[0086] Addition of the standard and serum: different concentrations of BRO standard solution (500 ng-0.5 ng) were added to the 96-well plate, and 50 μL of PBS was added to the last well as a blank control, and the titer with an OD 450 value close to 1 was used as the working concentration for dilution, 25 μL of mouse polyclonal serum was added to each well, 5 times of washing with PBST, and then dried by tapping;

[0087] Add secondary antibody: 1:5000 HRP-goat anti-mouse IgG, 50 μL / well, 37°C, 40 min, wash 5 times with PBST, dry;

[0088] TMB color development: add 50 μL TMB color developing solution per well, 37°C, 10 min;

[0089] Add stop solution: add 50 μL / well of H2SO4 to the 96-well plate to stop the reaction;

[0090] Read and analyze results: use the microplate reader to measure the OD value of each well 450 . Process and analyze the data using Origin 2021 software, take the concentration of the BRO standard as the abscissa, take B / B0 (B: absorbance value of the BRO standard; B0: absorbance value without the addition of the BRO standard) as the ordinate, and use the Logistics5 function equation to fit the curve. From the resulting curve, the IC 50 value can be obtained.

[0091] The experimental results are shown in Figure 5 . The IC 50 value of the B1 mouse serum was calculated to be the lowest at 25.28 ng / mL.

[0092] Example 2: Screening and identification of hybridoma cell lines against BRO

[0093] I. Preparation of anti-BRO mAb

[0094] (1) Culture of SP2 / 0 cells

[0095] Culture the SP2 / 0 cells in a cell culture flask in a constant temperature incubator. When the cell density reaches about 80%, perform subculture.

[0096] (2) Mouse booster immunization

[0097] The B1 mouse with the optimal IC 50 value of the immune response in Example 1 was subjected to booster immunization. Three days before cell fusion, 100 μg of the immunogen was mixed with 100 μL of PBS, and then the mouse was directly injected intraperitoneally. No adjuvant was used during the booster immunization process.

[0098] (3) Preparation of feeder layer

[0099] Select BALB / c mice that have not been immunized, perform decapitation using forceps, and then immediately immerse them in 75% (mass fraction) alcohol for disinfection, and then fix them on a sterile wax plate. Thoroughly disinfect the entire body of the mouse, especially the neck, abdomen, and other areas, to achieve a sterile state.

[0100] After the sterilization and drying, under aseptic operation, using high-pressure sterilized surgical scissors and forceps, the skin of the mouse is cut along the midline of the abdomen to the right, exposing the peritoneal membrane. Special attention should be paid to not touching or piercing the intestines during the operation to prevent contamination of the intestinal contents. Then, the HAT medium pre-cooled to the appropriate temperature is slowly injected into the mouse abdominal cavity through a 5 mL syringe, while ensuring that the needle tip is away from the intestinal tissue. After injecting the medium, the mouse abdomen is gently patted with an alcohol cotton ball to encourage the macrophages in the abdominal cavity to detach from the peritoneum and mix into the medium as much as possible. Then, through the same needle insertion path, the abdominal fluid mixed with macrophages is slowly extracted and combined with the pre-prepared HAT medium, and the cells are gently mixed using a pipette to ensure uniform distribution.

[0101] Next, the macrophages are distributed into 96-well cell culture plates, with each well containing 100 μL of the cells. The cell-filled culture plates are placed in a constant temperature and humidity incubator at 37°C and 5% CO2(volumetric fraction) for overnight culture.

[0102] The next day, the cells in each well are carefully observed under a microscope, and the medium is checked for signs of microbial contamination, and the growth state of the feeder layer cells is evaluated, including cell adhesion, morphology, and activity, to ensure that the quality of the feeder layer cells meets the needs of subsequent experiments. The state of the feeder layer cells is shown in FIG. 1. Figure 6

[0103] (3) Cell fusion

[0104] Eight cell culture bottles of SP2 / 0 cells with round and transparent growth are selected. The cells are rinsed once with preheated GNK solution to remove cell debris and a small number of cells in poor condition, improving the fusion efficiency.

[0105] The B1 mouse is subjected to orbital blood collection and placed at -80°C for subsequent use as positive blood. After the mouse is decapitated, it is immersed in 75% (mass fraction) alcohol and then moved to a clean bench. The mouse is fixed on a fully sterilized wax plate and the mouse is disinfected. Using sterile scissors and forceps, the mouse's spleen is removed and placed on a high-pressure beaker with a nylon mesh. The spleen is rinsed with GNK solution, then cut into small pieces using scissors and forceps, and finally rinsed with 40 mL of GNK solution and collected in a centrifuge tube. Centrifuge at 1000 r / min for 10 min, discard the supernatant.

[0106] The SP2 / 0 cells prepared in the above step are mixed with the spleen cells in the centrifuge tube, and the cell pellet is retained after centrifugation at 1000 r / min for 5 min. Gently shake the cell mass to disperse it.

[0107] ​In a super-clean bench, 1 mL of PEG 1500 was added dropwise to the above mixed cell mass within 60 s. Then, within 80 s, 15 mL of preheated GNK solution was added dropwise into the centrifuge tube, and finally the centrifuge tube was supplemented with GNK solution to 40 mL, and then incubated in a 42°C water bath for 5 min. Centrifugation was performed at 1000 r / min for 5 min, and the cell pellet was gently dispersed.

[0108] Slowly add 40 mL of HAT medium to the cell pellet, mix gently, and then transfer to a flat dish. Add 100 μL per well of 40 mL of HAT medium to a total of 8 96-well plates, and then incubate in a 37°C incubator.

[0109] (4) Screening of positive hybridoma cells

[0110] To ensure that the fused cells maintain a relatively stable growth state in the early stage, and to avoid the decline in cell viability caused by excessive disturbance, the cells in the 96-well plate were not observed for the first 3 days after inoculation. By the 3rd day, the cells had aggregated to form small cell masses of 2 to 5 cells under microscopic observation. By the 5th and 10th days, as the cell masses continued to proliferate and expand, if the color of the culture medium was observed to be yellow, indicating the accumulation of metabolic products, semi-volume replacement was required. By the 13th day, it was expected that the hybridoma cell masses would occupy 40% to 70% of the culture well area (as shown in Figure 7 At this time, in addition to semi-volume replacement, indirect ELISA and icELISA detection were simultaneously performed to evaluate the titer and sensitivity of the antibodies in the supernatant of the hybridoma cells, and positive cell clones with high titer and high sensitivity were selected accordingly. With repeated semi-volume replacement and subsequent limiting dilution, the use of 1640 medium to replace the original HT medium and HAT medium was gradually transitioned.

[0111] icELISA was used to determine the OD 450 value of the supernatant, and the experimental results are shown in Figure 8 , obtaining 13 positive clone wells. The 5C3 well had an inhibition rate of 57.0% at a concentration of 50 ng / mL BRO, and was therefore selected as the positive well for subcloning.

[0112] (5) Limiting dilution of positive cell wells

[0113] Viable cell count: The viability of the cells was identified by trypan blue staining, and the number of viable cells was counted under a microscope to determine the cell concentration according to the calculation formula.

[0114] Feeder layer preparation: The feeder layer cells were prepared one day before the limiting dilution was performed to ensure that their density was moderate enough to support the proliferation of the hybridoma cells.

[0115] Cell dilution procedure: Take 1000 cells from dilution I and add them to 5 mL of culture medium, mixing thoroughly by pipetting. Take 1.1 mL of cell suspension from dilution I and add it to 9.9 mL of fresh culture medium, mixing again by pipetting; this is recorded as dilution II. Further dilution is performed in two cases, A and B. For each case, take 3 mL of cell suspension from dilution II and add it to 7 mL of fresh culture medium, mixing thoroughly; this is recorded as dilution III.

[0116] Cell seeding layout: In dilution I, seed 100 μL of cell suspension into each well, covering half of a 96-well plate. Approximately 20 cells are expected per well. In dilution II, seed 100 μL into each well, covering half the plate. At this dilution, approximately 2 cells are expected per well. Similarly, in dilution Ш, seed 100 μL into each well. Due to the higher dilution, two plates are needed, theoretically containing approximately 0.6 cells per well.

[0117] Limiting dilution screening and amplification: Typically, three rounds of subclonal screening are performed. For monoclonal cells with stable titers and excellent sensitivity, they are progressively transferred to 24-well and 6-well plates for culture, and finally expanded on a large scale in T25 cell culture flasks. The screened cell lines are cryopreserved in liquid nitrogen for subsequent experiments.

[0118] Experimental results are as follows Figure 9 As shown, the selected positive well 5C3 underwent the first round of subcloning, as follows: Figure 9 As shown in Figure A, 41 positive wells were tested. The figure shows the inhibition rate of 15 strongly positive wells. It can be seen that well 3C2 has the highest inhibition rate, reaching 65.3% at 50 ng of BRO. A second round of subcloning was performed on the screened positive well 3C2. Figure 9 As shown in Figure B, for 10 6 The 13 positive wells were tested, and the figure shows the inhibition rate of the 13 strongly positive wells. It can be seen that well 3H1 had the highest inhibition rate, reaching 81.3% at 25 ng of BRO. The selected positive wells (3H1) underwent the first round of subcloning, as shown in the figure. Figure 9 As shown in Figure C, 48 positive wells were tested. The figure shows the inhibition rate of 13 strong positive wells. It can be seen that the 2B10 well has the highest inhibition rate, which reaches 81.1% under 5ng BRO small molecules.

[0119] (6) Determination of mAb titer and sensitivity

[0120] The mAb titer and sensitivity were determined using the ELISA and icELISA methods described in Example 2.

[0121] Experimental results are as follows Figure 10 As shown in the figure, the titer of the 2B10 cell supernatant was 1:1.28×10⁻⁶. 4Its IC 50 was 2.94 ng / mL, showing that it has high affinity and high sensitivity.

[0122] (7) Identification of mAb subtypes

[0123] The murine mAb was identified by subtypes using an antibody subtype identification kit.

[0124] The experimental results are shown in Table 2.

[0125] Table 2 Subtype identification of BRO mAb

[0126]

[0127] As shown in Table 2, the antibody heavy chain subtype of anti-BRO mAb cell 2B10 is IgG1, and the light chain subtype is Kappa.

[0128] (8) Preparation of anti-BRO mAb mouse ascites and determination of titer and sensitivity

[0129] Six healthy and stable female Balb / c mice were selected for monoclonal cell injection, and 0.5 mL of paraffin oil was injected into the abdominal cavity of each mouse one week before cell injection to promote ascites production. Then, the frozen cell tube was taken out from liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were centrifuged and washed to remove the cryoprotectant, then resuspended and inoculated into cell culture bottles, and expanded after 80% growth. The mice were injected intraperitoneally one week later, and the hybridoma cells with good growth were collected. According to the cell number of 2 x 10 6 cells per mouse, a sterile syringe was used to inject the mouse abdomen. Three days after injection, the state of the mouse was observed, and the ascites from the mouse abdomen was collected 7-14 days later. If the ascites produced is small, the ascites collection can be performed in 2-3 times. Finally, the titer and sensitivity of the ascites were detected.

[0130] The titer and sensitivity of mouse ascites were determined by the ELISA and icELISA methods in Example 2.

[0131] The experimental results are shown in Table 2. Figure 11 The titer of mouse ascites can reach 1:1.024 x 10 5 The IC 50 of mouse ascites was 2.13 ng / mL.

[0132] Example 3: Preparation of anti-BRO single-chain antibody

[0133] I. Amplification of BRO single-chain antibody gene

[0134] (1) Extraction of 2B10 cell RNA

[0135] The total RNA extraction kit was used to extract the RNA of the 2B10 cells obtained in Example 2, and the concentration of the extracted RNA was determined using a micro nucleic acid protein analyzer. 100 μg of the RNA was used for agarose gel electrophoresis experiment to determine its integrity. The extracted total RNA was converted into cDNA by reverse transcription, and the reverse transcription system was 20 μL: total RNA 10 μL, 5x PrimeScript RT Master Mix 4 μL, ddH2O 6 μL. The reverse transcription process was as follows: 37°C, 15 min, 85°C, 5 s. The electrophoretogram of the extracted RNA of 2B10 is shown in FIG. 2. Figure 12

[0136] (2) Amplification of VH and VL fragments

[0137] According to the structure characteristics of mouse IgG, specific primers were designed for amplifying the variable region VH and VL fragments of the antibody.

[0138] The primer sequences were as follows: VH for: 5'-TGAGGAGACGGTGACCGTGGTGCC-3'(SEQ ID NO. 11);

[0139] VH back: 5'-CAGGTSMARCTGCAGSAGTCWGG-3'(SEQ ID NO. 12);

[0140] VL for 1: 5'-CCGTTTTATTTCCAACTTTGTCCC-3'(SEQ ID NO. 13);

[0141] VL for 2: 5'-CCGTTTGATTTCCAGCTTGGTGCC-3'(SEQ ID NO. 14);

[0142] VL for 3: 5'-CCGTTTTATTTCCAGCTTGGTCCC-3'(SEQ ID NO. 15);

[0143] VL for 4: 5'-CCGTTTCAGCTCCAGCTTGGTCCC-3'(SEQ ID NO. 16);

[0144] VL back: 5'-GACATCGAGCTCACTCAGTCTCCA-3'(SEQ ID NO. 17).

[0145] ​The amplification system was 50 μL system: PrimeSTAR Max Premix 25 μL, VH for / VL for mix 1.0 μL, VH back / VL back 1.0 μL, cDNA 1.0 μL, and ddH2O to 50 μL.

[0146] The amplification conditions were: using cDNA as a template, amplifying VL: 96°C, 10 min; 96°C, 30 s, 56°C, 45 s, 72°C, 1 min, 30 cycles, 72°C, 7 min; amplifying VH: 96°C, 10 min; 96°C, 30 s, 58°C, 45 s, 72°C, 1 min, 30 cycles, 72°C, 7 min.

[0147] The electrophoretogram of the amplification product is shown in Figure 13 The size of the obtained VH gene fragment is about 350 bp, and the size of the VL gene fragment is about 320 bp, which is consistent with the expected size, indicating that the VH and VL gene fragments are successfully amplified, providing a basis for the subsequent construction of ScFv.

[0148] (3) Splicing ScFv sequence by SOE-PCR

[0149] The purified VH and VL gene fragments by gel recovery were spliced by using Linker, and specific enzyme cutting sites VH Back Sfi I and VL For Not I were introduced at both ends. The primer sequences of Linker and two enzyme cutting sites are as follows:

[0150] Linker1: 5'-GGCTCTGGCGGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCA-3'(SEQ ID NO. 18);

[0151] Linker2: 5'-GACATCGAGCTCACTCAGTCTCCAGAGGCGGTTCAGGCGGAGGT-3'(SEQ ID NO. 19);

[0152] VL-Not I: 5'-ATACGGCACCGGCGCACCTgcggccgcCCGTTTTATTTCCAGCCTGGTCCC-3'(SEQ ID NO. 20);

[0153] VH-Sfi I: 5'-GTTGTTCCTTTCTATggcccagccggccSCAGGTSMARCTGCAGSAGTCWGG-3'(SEQ ID NO. 21).

[0154] The ScFv needs to be connected in two steps. The first step connection PCR reaction system is:

[0155] PrimeSTAR Max Premix 25 μL, VH 10 μL, VL 10 μL, Linker mix 2 μL, and ddH2O to 50 μL. The reaction conditions are: 96℃, 5 min, 96℃, 30 s, 56℃, 45 s, 72℃, 1 min; 10 cycles; 72℃, 7 min.

[0156] The second step connection PCR reaction system is: PrimeSTAR Max Premix 25 μL, the first step connection PCR reaction final solution 10 μL, VH-Sfi I 1 μL, VL-Not I mix 1 μL, and ddH2O to 50 μL. The reaction conditions are: 96℃, 1 min; 96℃, 30 s, 56℃, 45 s, 72℃, 1 min, 35 cycles; 72℃, 7 min, 4℃ storage. The electrophoresis map of the constructed ScFv fragment is shown in Figure 14 An obvious band appears at about 750 bp, which is consistent with the expected size.

[0157] II. Construction and identification of ScFv library

[0158] (1) Construction of ScFv-pCANTAB5E recombinant plasmid

[0159] Two restriction endonucleases Not I and Sfi I are used to cut the pCANTAB5E vector specifically, so that it can be effectively spliced with the ScFv fragment. The specific components of the enzyme cutting reaction are: 10×QuickCut Buffer 5 μL, Quick Cut Sfi I 1 μL, Quick Cut Not I 1 μL, pCANTAB5E 20 μL, and ddH2O to 50 μL. The enzyme cutting PCR program is: 37℃, 30 min, 50℃, 30 min.

[0160] The recombinant enzyme is used to connect the linearized pCANTAB5E vector with the ScFv fragment. The splicing PCR program is: 37℃, 30 min. The connection system is: pCANTAB5E 5 μL, ScFv 5 μL, Exnase II 2 μL, 5×CE II Buffer 4 μL, and ddH2O to 20 μL. The gel electrophoresis result map of double enzyme digestion pCANTAB-5E is shown in Figure 15

[0161] (2) Preparation of competent bacteria E. coli TG1

[0162] ​The E. coli TG1 strain sample was removed from the -80 °C freezer and inoculated onto a 2xYT solid medium plate using a streak plate method on a clean bench, and the plate was incubated at 37 °C for 12 to 16 h.

[0163] After observing the clear TG1 E. coli colonies on the plate, a single colony was selected and inoculated into a 20 mL 2xYT liquid medium in a shake flask. Subsequently, the shake flask was placed in a constant temperature shaker, set at 37 °C and 180 rpm, and incubated for 12 to 16 h to allow the strain to grow and multiply sufficiently.

[0164] After the overnight culture was completed, 500 μL of the TG1 bacterial solution was inoculated into a new 50 mL 2xYT liquid medium, maintained at 37 °C and 180 rpm, and the OD 600 value was monitored every 30 min to determine the bacterial density.

[0165] When the OD 600 of the E. coli TG1 reached 0.6, the conical flask containing the bacterial solution was immediately transferred to an ice bath for cooling for 30 min, and the conical flask was gently shaken during this period to ensure uniform cooling.

[0166] After the cooling process, the bacterial solution was carefully transferred to a sterile 50 mL centrifuge tube. The tube was centrifuged at 4 °C and 1000 g for 15 min. Then, the supernatant was carefully poured out. 50 mL of pre-frozen ddH2O was added, and the tube was gently shaken to thoroughly resuspend the precipitate.

[0167] The same centrifugation conditions (4 °C, 1000 g for 20 min) were repeated, the supernatant was discarded, and the bacterial precipitate was resuspended with 25 mL of sterile 10% glycerol solution (mass fraction) pre-cooled to 4 °C.

[0168] The above centrifugation and resuspension steps were repeated, i.e., centrifugation at 4 °C and 1000 g for 20 min, and after discarding the supernatant, the precipitate was resuspended with only 5 mL of pre-cooled 10% glycerol solution.

[0169] The same centrifugation procedure was continued, and then the supernatant was carefully removed, 20 μL of the remaining trace amount of supernatant was aspirated using a pipette, and 100 μL of pre-chilled 2xYT medium was added, and the bacteria were gently mixed to resuspend them thoroughly.

[0170] The prepared competent bacteria were diluted 100-fold, and a 1 μL sample was taken to determine the OD 600 value, and the concentration was further adjusted to 2.5x10 10 / mL using ice-cold 2xYT medium.

[0171] Finally, the treated competent E. coli TG1 bacteria were aliquoted at a volume of 50 μL per tube and stored in a -80 °C refrigerator for long-term preservation.

[0172] (3) Transformation and identification of the ligation product

[0173] The TG1 competent cells prepared in step (2) were placed on ice to maintain a low temperature state, and an electrotransformation operation was performed in accordance with the product manual of the electrotransformation instrument, with the transformation parameters set to a voltage of 2000 volts and a pulse time of 5 ms. 5 μL of the ligation product was gently mixed with 5 μL of the competent bacteria to ensure uniform mixing, and the mixture was immediately injected into a pre-cooled (-80 °C) and sterile shock cup, and the outer wall of the shock cup was ensured to be dry and free of water stains.

[0174] After the end of the electrotransformation process, 1 mL of 2xYT liquid medium pre-heated at 37 °C was quickly added to the shock cup. Then, the bacteria were incubated at 37 °C at a speed of 200 rpm for 1 h to promote recovery. After the incubation, the bacteria were precipitated at 5000 rpm for 10 min. The supernatant was carefully removed, and the bacterial precipitate was resuspended with 100 μL of 2xYT liquid medium. Subsequently, the bacterial suspension was uniformly coated onto a 2xYT solid culture plate containing antibiotics using a spreader. At the same time, untransformed TG1 competent bacteria were used as a control. The coated plate was transferred to an incubator at 37 °C for overnight culture. After 12-16 h of culture, the colonies formed after transformation were observed and counted.

[0175] On the second day, a single colony for expansion culture was selected for overnight culture. The following sequences were used for PCR. Primer S1: 5'-CAACGTGAAAAAATTATTATTCGC-3' (SEQ ID NO. 22); S2: 5'-GTAAATGAATTTTCTGTATGAGG-3' (SEQ ID NO. 23). PCR system: 2xTaq Master Mix 6.25 μL, bacterial solution 1.0 μL, S1 0.5 μL, S6 0.5 μL, and ddH2O to 1.25 μL.

[0176] The bacterial solution sample of the selected positive monoclonal strain was sent to Shanghai Sangon Laboratory for DNA sequence analysis. The well-grown transformed colonies on the plate were washed and collected using 2xYT liquid medium, and then the bacterial solution was cultured at 37 °C and 180 rpm for 1 h. After the culture, 50% glycerol (mass fraction) was added to the bacterial solution for long-term preservation treatment, and the treated bacterial solution was transferred to a frozen environment at -80 °C for storage.

[0177] (4) Preparation of M13K07 phage

[0178] Bacterial culture and phage infection:

[0179] A single TG1 strain colony was picked from the solid culture medium and inoculated into 10 mL of 2xYT liquid medium. The culture was incubated at 37°C with 160 rpm overnight.

[0180] The next day, 2 mL of the overnight culture was taken and incubated in 200 mL of 2xYT liquid medium at 37°C with 180 rpm until the OD 600 approached 0.6.

[0181] Subsequently, 10 μL of the M13K07 helper phage stock stored at -80°C was added to the bacterial culture and incubated at 37°C for 30 min to promote phage adsorption. Then, the culture was shaken at 180 rpm for 30 min to enhance the infection efficiency of the phage.

[0182] Finally, 200 μL of kanamycin was added to the culture for screening of successfully transformed bacteria. The culture was incubated at 37°C with 200 rpm overnight.

[0183] (5) Phage precipitation and purification

[0184] The prepared phage culture was centrifuged at 8000 rpm for 10 min to collect the supernatant. Then, PEG / NaCl solution was added to the collected supernatant and mixed well. The mixed solution was then placed in an ice bath for 4 h. Next, the solution was centrifuged at 12000 rpm for 20 min to collect the precipitate. The supernatant was discarded and the precipitate was the phage. The precipitate was resuspended with 1 mL of PBS buffer and filtered through a 0.45 μm filter to remove impurities, then aliquoted and stored at 4°C.

[0185] (6) Titer determination of helper phage M13K07

[0186] The TG1 bacterial culture was pre-incubated to an OD 600 of 0.6. 1 μL of the M13K07 helper phage was taken to determine the titer. It was diluted by PBS to 1 x 10 -12The concentration of the M13K07 helper phage was determined by measuring the OD280 of the phage stock solution. 10 μL of each dilution was added to 100 μL of the TG1 bacterial solution described above. The mixture was allowed to stand at 37°C for 30 min to give the phage enough time to infect the bacteria. After that, the mixture was placed on a shaker and shaken at 180 rpm for 30 min to speed up the infection. After the infection was completed, the bacterial solution of each dilution was evenly spread on a 2 x YT-K medium plate using a spreader. The plate was incubated at 37°C overnight. The next day, the colonies on all the plates were counted. According to the number of colonies formed on each plate, the titer of the M13K07 helper phage was calculated.

[0187] (7) Construction of Anti-BRO Phage ScFv Library

[0188] The anti-BRO ScFv library solution was inoculated into 20 mL of 2 x YT-A liquid medium at a ratio of 1:100. The mixture was cultured in a 37°C incubator at 200 rpm until the OD 600 of the solution reached about 0.6. Then, the solution was cooled on ice for 15 min to improve the infection efficiency. Then, M13K07 (titer: 10 12 pfu / mL) was added to ensure that the ratio of bacteria to helper phage was 1:20.

[0189] The mixture was cultured at 37°C and 200 rpm. After the culture was completed, the bacterial solution was centrifuged at 4°C and 8000 rpm for 10 min to precipitate the bacteria, and the supernatant was discarded. The precipitate was resuspended in 10 mL of 2 x YT-AK medium. The resuspended bacterial solution was further cultured overnight at 37°C and 200 rpm, and the next day, the bacterial solution was centrifuged at 4°C and 10000 rpm for 20 min, and the supernatant was collected. One-fifth volume of PEG / NaCl solution was added to the supernatant to help the phage precipitate. The final precipitate was resuspended in 1 mL of PBS, filtered through a 0.45 nm filter, and stored at 4°C for subsequent screening and enrichment.

[0190] III. Screening of Phage Library and Obtaining of Positive Single Chains

[0191] (1) The screening of the phage antibody library is mainly to enrich phage clones that can specifically bind to the target antigen through multiple rounds of affinity screening. The screening process includes the following steps:

[0192] Coating stage: BRO-OVA, OVA, BRO-BSA, and BSA were coated in 96-well plates at 50 μL / well at 4°C overnight. PBST was washed 5 times. Four rounds were coated at concentrations of 50, 25, 12, and 6 μg / mL, respectively.

[0193] Blocking step: 200 μL of blocking solution was added to the 96-well plate and incubated at 37°C for 2 h. The plate was washed three times with PBST solution.

[0194] Antibody addition: 50 μL of PBS buffer was added to each well, followed by the addition of 50 μL of the phage ScFv library and incubation at 37°C for 1 h. The plate was washed five times with PBST solution.

[0195] Elution of positive phage: 200 μL of elution solution (pH 2.2) was added to each well, and the plate was shaken on a constant-temperature shaker for 10 min. Immediately after, 50 μL of neutralization solution (pH 9.2) was added, and the resulting solution was the first round of elution solution.

[0196] Titer determination: 1 μL of the elution solution was used for titer determination, and the same determination method as for M13K07 was used. The next day, the colonies formed on the plate were counted accurately, and the actual number of recovered phage was calculated.

[0197] Phage amplification: 500 μL of the elution solution from the previous step was added to 20 mL of TG1 bacterial solution that had been cultured to the logarithmic growth phase. Then, 20 μL of ampicillin was added to the mixture. The bacterial solution was left to stand at room temperature for 30 min, after which 2 x 10 10 pfu of helper phage M13K07 was added, and the solution was left to stand again for 30 min. After the standing period was complete, the bacterial solution was shaken at 37°C at a speed of 180 rpm for 30 min. Next, the solution was centrifuged at 8000 rpm for 10 min to precipitate the phage. After the centrifugation was complete, the supernatant was carefully discarded, and the precipitate was resuspended with 30 mL of 2 x YT-AK. The resuspended bacterial solution was cultured overnight at 37°C at a speed of 180 rpm.

[0198] Phage precipitation: After the amplification of the phage was complete, the phage was concentrated using the PEG / NaCl precipitation method. This marked the completion of the first round of screening enrichment. Then, to further purify and screen the phage, a total of four rounds of screening were performed. The pressure of the screening was increased with each round to improve the efficiency of the screening and ensure that the screened phage had higher specificity and affinity.

[0199] The results of the four rounds of screening enrichment of the BRO phage antibody library are shown in Table 3.

[0200] Table 3 Four rounds of screening enrichment of the BRO phage antibody library

[0201]

[0202] According to Table 3, after the fourth round of screening, the recovery rate of phage was increased by 190 times compared with the first round. This result shows that through the continuous screening process, the specific phage is effectively enriched, and the screening strategy has achieved remarkable success. After such a screening process, the final phage library obtained contains a large number of ScFv with high specificity and high affinity to BRO, providing a material basis for subsequent research.

[0203] (2) Bacterial liquid PCR identification of phage ScFv positive bacteria

[0204] The eluate obtained after the fourth round of screening was gradient diluted, and then it was used to infect the TG1 bacterial liquid in the logarithmic growth phase. After standing at 37°C for 30 min, it was continued to be cultured in a shaking incubator at 180 rpm for 30 min. Then, the mixture was spread on 2xYT-A solid plates and incubated at 37°C overnight.

[0205] The next day, 25 representative colonies were selected from the formed single colonies, and each colony was inoculated into 1 mL of 2xYT-A liquid medium for overnight culture for further amplification.

[0206] 5 μL of the above-mentioned bacterial liquid after overnight culture was taken as a PCR reaction template for bacterial liquid PCR operation. For the positive bacterial liquid sample, it was sent to Shanghai Biosciences for sequencing analysis.

[0207] The experimental results are shown in Table 3. Figure 16 As shown in Table 3, the results show that 18 of the 20 single colonies can amplify bands, with a positive rate of 90%. Sequencing of the positive colonies confirmed the approximately 750 bp insert, and through Blast alignment analysis, it was found that there were 8 different ScFv gene sequences in the library. The library capacity diversity was 44.4%, indicating that the antibody library had good diversity.

[0208] (3) Amplification and titer determination of positive phage ScFv

[0209] The amplification and titer determination of phage M13KO7.

[0210] (4) phage-ELISA of BRO positive phage ScFv

[0211] First, 50 μL of phage sample which had been sequenced and confirmed positive was selected as the primary antibody, and was added to a 96-well enzyme-labeled plate which was previously coated with 50 μg / mL BRO-OVA, and was incubated at 37°C for 2 h. After incubation, 5 times of PBST solution was used for cleaning to remove the unbound primary antibody. Then, 50 μL of HRP-labeled anti-M13 antibody with a concentration of 0.4 μg / mL was added to each well as the secondary antibody, and was continued to be incubated at 37°C for 1 h. After the incubation was completed, 5 times of PBST solution was also used for cleaning. Subsequently, 50 μL of TMB color developing solution was added to each well, and color development was performed at 37°C for 10 min. After the color development was completed, 50 μL of stop solution was quickly added to the well. Finally, the OD 450 value of each well was determined by using an enzyme-labeled instrument.

[0212] The experimental results are shown in Table 1. Figure 17 After the OD 450 value was determined, the P / N value was calculated to measure the binding force of the phage surface ScFv to the antigen, and P / N > 2.1 indicated that it had a higher binding capacity. The results showed that the P / N values of 4 strains of monoclonal phage were all more than 2.1, indicating that the ScFv carried by them had a higher binding strength to the target antigen, and the P / N value of No. 12 was 7.46.

[0213] The phagemids with P / N value ≥ 2.1 were selected, and a total of 4 strains were sent to Shengong for sequencing. The sequence results of No. 12 phagemid were analyzed to be consistent with the characteristics of ScFv, and the sequencing results were as follows:

[0214] The sequence of the heavy chain variable region of the single-chain antibody is as follows:

[0215] MAQVQLQESGPELKKPGETVKISCKTSGNTFTDYGMDWVKQAPGKGLKWMGRINTKTGVPT YAEEFKGRFAFSLETSASTAYLQINNLRNEDMASYFCASPIYFYSGWYFDVWGQGTTVTV(SEQ ID NO.1);

[0216] The sequence of the light chain variable region is as follows:

[0217] DIELTQSPALMAASPGEKVTITCSVSSSISSSYLHWYQQKSGISPKPWIYGTSNLASGVPTRFSGS GSGTSYSLTIRSMEAEDAATYYCQQWSRSPPRSEGGPSWS(SEQ ID NO.2);

[0218] The amino acid sequence of the connecting peptide is as follows: SSGGGGSGGGGSGGGGS (SEQ ID NO. 5).

[0219] IV. Expression and identification of anti-BRO single-chain antibody

[0220] (1) Preparation of anti-BRO phage ScFv

[0221] Culture of positive bacteria: The positive bacteria were inoculated into 2xYT-A liquid medium at a ratio of 1:100, and cultured at 37°C with 180 rpm shaking until the logarithmic growth phase was reached, usually 2h 20min.

[0222] Phage infection: The helper phage M13K07 (2x10 10 pfu / mL) was added to the bacterial culture and incubated at 37°C for 30 min. Then it was placed in a shaker at 37°C with a speed of 180 rpm for 30 min.

[0223] Phage amplification: The bacterial culture was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2xYT-AK medium, then incubated at 37°C overnight with a speed of 180 rpm.

[0224] Phage harvesting and purification: The bacterial culture was centrifuged at 10,000 rpm for 15 min at 4°C to precipitate the bacteria. The supernatant was collected and mixed with 1 / 5 volume of PEG / NaCl solution for phage precipitation. After thorough mixing, the mixture was cooled on ice for 4 h, then centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was discarded, and the phage precipitate was resuspended in 1 mL PBS. Then the suspension was filtered through a 0.45 μm filter to remove bacterial debris, obtaining a phage ScFv solution which was stored at 4°C for further use.

[0225] (2) Soluble expression of anti-BRO phage ScFv

[0226] Phage inoculation: 2 μL of the selected BRO positive phage was added to 100 μL of E. coli HB2151 culture in the logarithmic growth phase for phage inoculation.

[0227] Colony isolation and culture: The mixture was incubated in an incubator for 30 min, then transferred to a constant temperature shaker and cultured at a speed of 180 rpm / min for 30 min. The treated mixed bacterial solution was evenly spread on 2xYT-A culture plates and incubated at 37°C overnight to achieve colony isolation and growth.

[0228] Colony propagation and culture: The next day, single colonies were selected from the plate and inoculated into 10 mL of 2xYT-A liquid medium, maintained at 37°C, 180 rpm / min, and cultured overnight. On the third day, the overnight culture was inoculated into 200 mL of 2xYT-A medium at a ratio of 1:100, and continued to be cultured at 30°C, 180 rpm / min, until the logarithmic growth phase.

[0229] Induction of expression and sample collection: When the bacterial solution entered the logarithmic growth phase, IPTG was added for induction, with a final concentration of 1 mmol / L (equivalent to 476 μL). The culture temperature was set to 30°C, and the induction was continued for 10 h. After the induction was completed, the bacterial solution sample was collected, and E. coli HB2151 bacterial solution and HB2151 strain carrying pCANTAB5E vector were used as controls.

[0230] (3) Acquisition of BRO-ScFv in the periplasmic cavity

[0231] After the induction of expression, the bacterial culture was centrifuged at 5 000 rpm for 20 min to separate the supernatant and the precipitate. The supernatant was filtered through a 0.45 μm filter and stored at 4°C for the next step of identification analysis. For the precipitate part, it was resuspended in 1 mL of ice-cold 1xTES solution. After vortex mixing, the suspension was incubated on ice for 30 min. Subsequently, the suspension was centrifuged at 11 000 rpm for 15 min at 4°C. After centrifugation, the supernatant of the soluble antibody secreted into the periplasmic cavity was collected. Finally, the protein expression in the obtained supernatant was analyzed using SDS-PAGE and Western Blotting methods.

[0232] The results of Western Blotting identification are shown in Figure 18 As shown, the protein bands on the transferred NC membrane were clearly visible, proving the presence of the target protein.

[0233] (4) Detection of the biological activity of BRO-ScFv

[0234] ① Indirect ELISA method for detecting soluble BRO-ScFv recombinant antibody, the operation steps are as follows:

[0235] Antigen coating: 50 μL of a solution of BRO-OVA antigen with a concentration of 3 μg / mL was added to each well of the enzyme-labeled plate. Subsequently, the enzyme-labeled plate was placed at 4°C overnight. The next day, the PBST solution was washed 5 times, each time for 5 min.

[0236] Blocking: 200 μL of PBST solution containing 5% skimmed milk was added to each well of the enzyme-labeled plate coated with antigen. The blocking process was carried out at 37°C for 2 h. Washing was performed 5 times with PBST solution, each time for 5 min.

[0237] Sample incubation: 50 μL of the soluble BRO-ScFv recombinant antibody solution to be tested was added to each well of the enzyme-labeled plate, with an initial dilution of 1:5. At the same time, the E. coli HB2151 empty strain was set as a negative control. The enzyme-labeled plate was incubated at 37°C for 1 h to allow the antibody to fully react with the antigen. After incubation, washing was performed 5 times with PBST solution, each time for 5 min.

[0238] Secondary antibody incubation: 50 μL of HRP-labeled anti-E-tag antibody (dilution ratio 1:5000) was added to each well, and incubation was continued at 37°C for 30 min. After incubation, washing was performed 5 times with PBST solution, each time for 5 min.

[0239] Color development reaction: Subsequently, 50 μL of TMB color developing solution was added to each well, and reaction was carried out at room temperature for 7 min. After reaction, 50 μL of 2M H2SO4 termination solution was added to stop the color development reaction.

[0240] Absorbance determination and result analysis: The absorbance of each well was determined at a wavelength of 450 nm using an enzyme-labeled instrument. The reading of the blank control well was taken as zero point, and the OD 450 values of the sample well (P) and the negative control well (N) were recorded. By comparing the OD values of the sample well and the negative control well, the P / N ratio was calculated. If P / N≥2.1, it was determined as a positive reaction, indicating that the soluble BRO-ScFv recombinant antibody had specific binding with the BRO-OVA antigen. If the P / N ratio was lower than the threshold value, it was determined as a negative reaction.

[0241] ② Sensitivity detection of soluble BRO-ScFv

[0242] The affinity of soluble BRO-ScFv was analyzed, following the steps below:

[0243] Antigen coating: 50 μL of BRO-OVA at a concentration of 3 μg / mL was added to each well of the enzyme-labeled plate. The enzyme-labeled plate was placed at 4°C overnight. Then, washing was performed 5 times with PBST solution, each time for 5 min.

[0244] Blocking: 200 μL of PBST solution containing 5% skimmed milk was added to each well. It was placed in a 37°C incubator for 2 h. After blocking, washing was performed 5 times with PBST solution, each time for 5 min.

[0245] Competitive reaction: 50 μL of periplasmic cavity extract was added to each well, followed by the immediate addition of 50 μL of different concentrations (1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL) of the BRO standard. Among them, the well without the addition of the BRO standard was used as a positive control. It was placed in a constant temperature incubator at 37°C for 1 h. After incubation, it was washed with PBST solution for 5 times, each time for 5 min.

[0246] Incubation of secondary antibody: 50 μL of HRP-labeled anti-E-tag antibody solution diluted 1:5000 was added to each well. The enzyme-labeled plate was returned to the 37°C constant temperature incubator for continued incubation for 30 min. After incubation, it was washed with PBST solution for 5 times, each time for 5 min.

[0247] Color development: 50 μL of TMB single-component color developing solution was added to each well, and the reaction was carried out at room temperature for 10 min to allow the color development reaction to proceed fully. Subsequently, a stop solution (2M H2SO4) was added, with an amount of 50 μL per well.

[0248] Reading data: The absorbance value of each well was detected at OD 450 of 450 nm using an enzyme-labeled instrument, and the absorbance value of the blank control well was zeroed to eliminate background interference.

[0249] Calculation: The obtained data was fitted using the Logistics5 function equation by using Origin 2021 software. The standard curve was drawn with the logarithmic value of the standard concentration as the abscissa and the absorbance value B / B0 (the ratio of the absorbance value of the sample-added well to the absorbance value of the blank well) as the ordinate. Through the fitted curve, the IC 50 value was calculated, which reflects the sensitivity level of the soluble BRO-ScFv to the BRO-OVA binding.

[0250] The experimental results are shown in Figure 19 Table 2, the P / N of the BRO-ScFv is ≥2.1, which is judged to be positive, the titer is about 1:64, and the calculated IC 50 value is 13.16 ng / mL.

[0251] (5) Specificity analysis of soluble BRO-ScFv

[0252] CL, CIB, SAL, RAC, TBL, ZIL and CIM were selected as competitive small molecules for specificity analysis experiments to determine whether cross-reactions would occur, calculate the IC 50 value, and use the formula CR% = IC 50 (standard) / IC 50(structural analogues) x 100%, the respective cross-reactivity rates were calculated.

[0253] The experimental results are shown in Table 4.

[0254] Table 4 Cross-reactivity rates of BRO-ScFv with structural analogues

[0255]

[0256] The results show that the cross-reactivity rates of the BRO-ScFv antibody with CL and CIB are 31.1% and 26.25%, respectively, and the cross-reactivity rates with other small molecules are less than 0.1%.

[0257] Example 4: Synthesis and identification of ZIL complete antigen

[0258] I. ZIL molecular modification

[0259] (1) Before synthesizing the ZIL complete antigen, the ZIL molecule is modified to introduce a side chain -COOH.

[0260] The 100 mg ZIL-HCL was weighed on a millionth scale and added to 10 mL of 1 mol / L NaOH solution, and 5 mL of ethyl acetate was repeatedly extracted 5 times to obtain ZIL free base. The ethyl acetate layer was dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated with a rotary evaporator.

[0261] After adding 5 mL of acetone, 4-bromobutyric acid ethyl ester and potassium carbonate to the product, it was refluxed, the potassium carbonate was removed by filtration, and the acetone was dried by rotary evaporation.

[0262] It was refluxed with 10 mL of 66% (v / v) ethanol and 34% KOH (v / v) aqueous solution at 80°C for 3h, the heater was turned off and the reaction was allowed to proceed overnight at room temperature. The ethanol was dried by rotary evaporation, the pH was adjusted to about 3.0 with 2mL HCL, and the ethyl acetate and water phases were extracted separately.

[0263] (2) Coupling of ZIL with carrier protein

[0264] According to the EDC / NHS method, ZIL was coupled with carrier proteins BSA / OVA, and the specific process was as follows:

[0265] The obtained product was dissolved in MES (pH 5.0) buffer solution, 1 mL of DMF was added, mixed, 10 mg of EDC and 6 mg of NHS were added, stirred in ice water bath for 2 h, recorded as A liquid, and the solution was divided into two parts; 12 mg of BSA was weighed and dissolved in 1 mL of sodium borate buffer, 40°C water bath for 30 min, dropwise added to A liquid under stirring at room temperature, 4°C. Stir overnight at room temperature; 10 mg of OVA was weighed and dissolved in 1 mL of sodium borate buffer, 30°C water bath for 30 min, dropwise added to the activated A liquid under stirring at room temperature, stirred overnight at room temperature; the reaction product was collected, and 0.01 mol / L PBS was used as the dialysis buffer to dialyze the reaction product. Dialysis for 3 days, changing the dialysate 3 times a day. Centrifuged at 5000 r / min for 5 min. The product was collected, labeled as ZIL-BSA and ZIL-OVA, and stored in a -20°C refrigerator.

[0266] (3) Complete antigen concentration identification, SDS-PAGE identification and ultraviolet full wavelength identification

[0267] The identification method was the same as in Example 1, and the experimental results are shown in Figures 20-22 .

[0268] The linear regression equation was y = 2.506x + 0.5623, R 2 = 0.99, which met the calculation requirements. The OD values of the corresponding dilution concentrations of the complete antigens ZIL-BSA and ZIL-OVA were substituted into the standard curve, and it was calculated that the protein concentration of the immunogen ZIL-BSA was 4.8 mg / mL, and the protein concentration of the coating ZIL-OVA was 0.71 mg / mL.

[0269] As shown in Figure 20 and Figure 21 , it can be observed that the migration rate of the coupled complete antigens ZIL-BSA and ZIL-OVA is smaller than that of the carrier proteins BSA and OVA, indicating that the molecular weight of ZIL-BSA and ZIL-OVA obtained after coupling increases. It is shown that ZIL is successfully coupled with the carrier proteins BSA / OV.

[0270] As shown in Figure 22 , the maximum absorption peak wavelength of the coupling product has shifted from that of the carrier protein, which can prove that ZIL is successfully coupled with the carrier protein.

[0271] II. Animal immunization

[0272] The experimental method was the same as in Example 1, and the experimental results are shown in Figure 23 . The 3 BALB / c mice immunized with the immunogen ZIL-BSA all produced anti-ZIL antibodies, with OD 450 nmThe judgment standard is that P / N > 0.3 and P / N > 2.1, and the polyantibody titer of the mice after immunization is 1:1.128x10 4 It is shown that the prepared complete antigen ZIL-BSA\OVA can be used for subsequent experiments.

[0273] Example 5: Screening and identification of hybridoma cell lines

[0274] (1) Cell fusion and positive hybridoma cell screening were performed according to the method in Example 2

[0275] The experimental results are shown in Table 5.

[0276] Table 5: Inhibition rate results of initial screening of cell fusion

[0277]

[0278] As shown in Table 4, OD 450nm P / N > 1.0 was determined as a positive well, and 26 positive wells were obtained after the initial screening after fusion. The inhibition rate of 100 ng / mL ZIL was 57.0%, and the positive well 5E7 with the highest inhibition rate was subcloned to prepare a single-chain antibody.

[0279] (2) Limited dilution of positive cell wells was performed according to the method in Example 2

[0280] After three subcloning dilutions, the positive well 5E7-A9 was obtained, and its IC 50 value was 1.75 ng / mL. The hybridoma cell 5E7-A9 was finally screened, and it was preserved in the Guangzhou City Martyrs Road 100 Courtyard No. 59 Building 5th Floor, Guangdong Microbial Culture Collection Center on August 9, 2023, with the preservation number: GDMCC No: 63432.

[0281] (3) Stability determination of hybridoma cells

[0282] The frozen hybridoma cell 5E7-A9 was repeatedly frozen and thawed 4 times, and the cell supernatant was collected for titer detection when the cell growth state was best. The results are shown in Table 6, and the titer of the cell supernatant was stable at 1:3.2x10 3 , indicating that the hybridoma cell 5E7-A9 has a good growth state in vitro and can stably secrete ZIL mAb.

[0283] Table 6: Determination of 5E7-A9 cell supernatant titer

[0284]

[0285] Example 6: Preparation of single-chain antibody

[0286] I. Preparation of anti-ZIL single-chain antibody

[0287] The hybridoma cell 5E7-A9 was injected into the abdominal cavity of a mouse to prepare an anti-ZIL single-chain antibody, and the specific process was as follows:

[0288] (1) 10 BALB / c mice were injected with 500 μL of sterilized liquid paraffin in the abdominal cavity 1W in advance. The liquid paraffin was sterilized by high pressure sterilization and filtered by a 0.22 μm filter membrane in a clean bench to remove impurities;

[0289] (2) The monoclonal hybridoma cells were collected, washed twice with PBS, and adjusted to a cell density of 1×10 6 cells were injected into the abdominal cavity of a mouse.

[0290] (3) About 7-10 days later, the abdominal cavity of the mouse was swollen and enlarged, and the ascites was collected on the 10th day, centrifuged at 3500 r / min for 5 min to remove impurities, and stored at -80°C for standby. Then, the titer of the mouse ascites was detected by ELISA, and the detection results are shown in Figure 24 , and the IC 50 value of the mouse ascites was detected by icELISA, and the detection results are shown in Figure 25 .

[0291] As shown in Figure 24 and Figure 25 , the titer of the mouse ascites can reach 1:4.096×10 5 , and the IC 50 of the mouse ascites is 1.50 ng / mL. It is shown that the hybridoma cell 5E7-A9 can stably secrete ZIL mAb, and the secreted single-chain antibody has high titer and sensitivity, which can be used for subsequent experiments.

[0292] II. Construction and identification of ZIL single-chain antibody

[0293] The construction and screening method of the ZIL single-chain antibody is the same as that in Example 3. The ZIL-phage-ScFv-5 positive phage was obtained after screening, and the obtained anti-ZIL single-chain antibody was sent to Shanghai Generay Biotech for sequencing. The sequence of the heavy chain variable region of the single-chain antibody is as follows:

[0294] EVKLQQSGGGLVKPGGSIKISCAASGFAFSSYGVSWVRQTPEKRLEWVAYISGGGGTTYYADTV RGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCSRHEARRGYALDYWGQGTTVTVSS (SEQ ID NO. 6);

[0295] The sequence of the light chain variable region is as follows:

[0296] DIELTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATSLADGVPSRFSGS GSGQDYSLTISSLESDDTATYYCLQHGESPLTFGAGTKLELKR (SEQ ID NO. 7).

[0297] The connecting peptide sequence is as follows: GGGGSGGGGSGGGGS (SEQ ID NO. 10).

[0298] The ZIL-phage-ScFv-5 single-chain antibody was expressed and identified according to the method in Embodiment 3, a recombinant expression vector was constructed, and the titer, sensitivity and specificity of the obtained ZIL single-chain antibody were identified.

[0299] The experimental results are shown in Tables 7, 8 and Figure 26

[0300] Table 7 Determination of titer of ZIL single-chain antibody protein

[0301]

[0302] Table 8 Specificity identification of ZIL single-chain antibody

[0303]

[0304] As shown in Table 7, the titer of the ZIL single-chain antibody protein is 1:16, indicating that the ZIL single-chain antibody protein can recognize ZIL-BSA. The dilution of 1:6 is used as the optimal dilution of ScFv in indirect ELISA, and the standard curve is shown in Figure 26 , and the IC 50 of the ZIL single-chain antibody is calculated to be 9.63 ng / mL. According to the specificity identification results, the ZIL single-chain antibody basically does not recognize other structural analogs, and has excellent specificity.

[0305] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A single-chain antibody against a β-agonist, characterized in that, The β-agonist is brombuterol, wherein the anti-brobuterol single-chain antibody consists of a heavy chain variable region, a linker peptide, and a light chain variable region. The linker peptide is located between the heavy chain variable region and the light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The single-chain antibody against β-agonists according to claim 1, characterized in that, The amino acid sequence of the linker peptide of the anti-brobuterol single-chain antibody is shown in SEQ ID NO.

5.

3. A kit for detecting β-agonists, characterized in that, The single-chain antibody comprising the anti-β-agonist as described in claim 1 or 2.

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