Fusion protein with bifunctional activity, construction method, application and kit thereof

Abstract

The invention discloses a bifunctional active fusion protein, a construction method, application and a kit thereof, and aims to provide a fusion protein with better specificity and signal amplification effect, and a hypersensitive bioluminescent open sandwich immunoassay kit established by adopting the fusion protein and used for detecting the content of glycocholic acid in urine. Good application values and prospects are realized; the MBP-G11-VL and G11-VH-Nluc fusion protein is prepared by the following steps: taking maltose binding protein as a label of the fusion protein, fusing light chain variable regions (G11-VL, G11-RL-VL and G11-FL-VL) of an anti-glycocholic acid single-chain antibody with the maltose binding protein, cloning the fused light chain variable regions into a pMal-p2E expression vector, taking nano luciferase as a bioluminescence catalyst, and carrying out bioluminescence on the fusion protein to obtain the MBP-G11-VL and G11-VH-Nluc fusion protein. A heavy chain variable region (G11-VH) of an anti-glycocholic acid single-chain antibody and nano luciferase are fused and cloned into a pET22b expression vector, and the anti-glycocholic acid single-chain antibody is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and relates to the preparation of MBP-VL, MBP-RL-VL, MBP-FL-VL and VH-Nluc fusion proteins, and also relates to an open-heart sandwich immunoassay method constructed based on the above fusion proteins for the detection of glycocholic acid. Background Art

[0002] Glycocholic acid (GCA) is a conjugated bile acid formed by the combination of cholesterol and glycine in the liver. It is a metabolite of cholesterol in hepatocytes and one of the main components of bile acids. Glycocholic acid participates in the enterohepatic circulation of bile acids. It is synthesized and secreted by hepatocytes, discharged into the gallbladder through bile capillaries and bile ducts, then refluxed to the duodenum through bile, and finally reabsorbed at the end of the intestine and returned to the liver through the portal vein. Its main function is to help with food digestion. When the liver is damaged, the glycocholic acid reabsorbed through the portal vein cannot be effectively absorbed by hepatocytes, and the content of glycocholic acid in peripheral blood increases. Relevant studies have pointed out that the concentration of glycocholic acid is related to the degree of hepatobiliary diseases. Glycocholic acid can be used as a biomarker to indicate hepatobiliary diseases, and its concentration in serum and urine is related to the degree of hepatocyte damage. For some pediatric patients and patients who are not convenient to draw venous blood, it is also convenient to detect glycocholic acid in their urine. Therefore, detecting glycocholic acid in urine has guiding significance for the early diagnosis of liver diseases.

[0003] Traditional quantitative methods for detecting glycocholic acid mainly rely on chromatographic techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS). These methods can perform reliable and sensitive quantitative analysis of glycocholic acid in biological samples, thus promoting its application in clinical evaluation and research. High-performance liquid chromatography (HPLC) is a chromatographic technique for separating compounds in a mixture, which can achieve highly sensitive detection of glycocholic acid, but it takes a long time and requires expensive equipment and skilled personnel, so it is not very suitable for routine applications and has limitations. Mass spectrometry (MS) technology is an analytical technique for measuring the mass-to-charge ratio of ions. Among them, liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) can have high detection accuracy for glycocholic acid and other bile acids, can analyze complex mixtures, and its sensitivity and specificity both exceed traditional chromatographic techniques. Due to its specificity, accuracy, the ability to perform minimal sample preparation, and the ability to analyze low concentrations in complex biological matrices, it has also become a method for many laboratories to measure glycocholic acid. The detection limit (LOD) of the isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS / MS) method based on bracket calibration established by researchers for glycocholic acid is 0.01 ng / mL. However, due to its involvement of complex instruments, expensive equipment, and the need for specialized training and a large amount of laboratory resources, the cost is high, which limits its wide application in resource-limited environments.

[0004] Immunoassay is an analytical method for detecting antigens or antibodies in samples through immune reactions based on the specific recognition of antigens and antibodies, including radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay, lateral flow immunoassay, etc.; it has the characteristics of simplicity, rapidity, safety, high sensitivity, etc. and is the most widely used. It is divided into two types: "competitive" immunoassay and "non-competitive" immunoassay. Generally, for macromolecules such as proteins or polypeptides, since there are multiple antigenic epitopes on their surfaces, it is more suitable to use the double-antibody sandwich method for "non-competitive" detection. For small molecule compounds such as glycocholic acid, since there is usually only one antigenic epitope on its surface, the "competitive" detection method is mostly used. However, compared with the classical sandwich method, this method has deficiencies in sensitivity and is difficult to fully meet the requirements of actual detection.

[0005] Bioluminescence immunoassay is an immunoassay method that uses a luminescence reaction to detect specific analytes. Compared with traditional immunoassay techniques, it has the advantages of high sensitivity, fast detection speed, and simple workflow. Bioluminescence, as a chemical process, relies on the interaction between luciferase and substrates to emit light. Utilizing the bioluminescent enzyme luminescence characteristics of luciferase, single-chain antibodies are fused with luciferase, and the sensitivity of immunoassay is enhanced through signal amplification. In the fusion of antibodies and luciferase, different luciferases have different action mechanisms, different activities, and different effects on the detection sensitivity.

[0006] In addition, in immunoassay methods based on the specific recognition of antigens and antibodies, antibodies (immunoglobulin IgG) are one of the important factors affecting their sensitivity. Therefore, the selection, preparation, and optimization of antibodies are the keys to improving the sensitivity of immunoassay methods. Summary of the Invention

[0007] In order to overcome the disadvantages and deficiencies in sensitivity in the prior art, the first object of the present invention is to prepare MBP-VL, MBP-RL-VL, MBP-FL-VL and G11-VH-Nluc fusion proteins. By fusing single-chain antibodies with luciferase, signal amplification is achieved. The MBP-VL, MBP-RL-VL, and MBP-FL-VL fusion proteins are formed by fusing maltose-binding protein (MBP) with the light chain variable region, which improves the protein expression level and purification efficiency; the gene of nano-luciferase (Nluc) is fused with the variable region of the heavy chain of the antibody (VH), and the high-efficiency luminescence characteristics and high activity of nano-luciferase are utilized to further improve the detection sensitivity.

[0008] The second object of the present invention is to provide a construction method for the above-mentioned fusion proteins with bifunctional activities. The construction process of this construction method is simple to operate and easy to implement.

[0009] The third object of the present invention is to provide the application of the fusion protein with the above dual-functional activity as an antibody for detecting glycocholic acid in urine, which does not cross-react with other structural analogues and has high specificity.

[0010] The fourth object of the present invention is to provide a bioluminescent immunoassay kit for detecting glycocholic acid containing the fusion protein with the above dual-functional activity. The kit has the advantages of simple operation, high sensitivity and good accuracy.

[0011] Another object of the present invention is that the fusion protein with dual-functional activity is used as a detection antibody, and by using an open sandwich immunoassay method, highly sensitive and highly specific detection of glycocholic acid in urine is achieved.

[0012] To achieve the above first object, the first technical solution provided by the present invention is as follows: A fusion protein with dual-functional activity, comprising a light chain variable region of an anti-glycocholic acid single-chain antibody bound to maltose and cloned into an expression vector, and a purified light chain variable region fusion protein; and using nano-luciferase as a catalyst for bioluminescence, the heavy chain variable region G11-VH of the anti-glycocholic acid single-chain antibody is fused with nano-luciferase and cloned into an expression vector, and a purified G11-VH-Nluc fusion protein;

[0013] The light chain variable region of the anti-glycocholic acid single-chain antibody is one of G11-VL or a light chain variable region G11-RL-VL containing a rigid linker peptide or a light chain variable region G11-FL-VL containing a flexible linker peptide.

[0014] Further, for the above fusion protein with dual-functional activity, the light chain variable region is G11-VL.

[0015] Further, for the above fusion protein with dual-functional activity, the expression vector is a pET22b expression vector.

[0016] Further, for the above fusion protein with dual-functional activity, the light chain variable region fusion protein is an MBP-G11-VL or MBP-G11-RL-VL or MBP-G11-FL-VL fusion protein; the heavy chain variable region G11-VH-Nluc fusion protein.

[0017] To achieve the above second object, the second technical solution provided by the present invention is as follows: A method for constructing a fusion protein with dual-functional activity, successively comprising the following steps:

[0018] (1) Amplification of target genes

[0019] The plasmid pET22b(+)-G11-scFv-Nluc carrying the gene of nano-luciferase fusion single-chain antibody G11-scFv-Nluc was extracted using a plasmid DNA extraction kit as a template;

[0020] The variable light chain gene G11-VL of the antibody was amplified by PCR using the upstream primer MBP-VL-F and the downstream primer MBP-VL-R to obtain the PCR product fusion gene MBP-G11-VL;

[0021] The variable light chain G11-RL-VL containing a rigid linker peptide was amplified by PCR using the upstream primer RL-F and the downstream primer MBP-VL-R to obtain the PCR product fusion gene MBP-G11-RL-VL;

[0022] The variable light chain G11-FL-VL containing a flexible linker peptide was amplified by PCR using the upstream primer FL-F and the downstream primer MBP-VL-R to obtain the PCR product fusion gene MBP-G11-FL-VL;

[0023] The variable heavy chain gene G11-VH of the antibody was amplified by PCR using the upstream primer FL-F and the downstream primer MBP-VL-R to obtain the PCR product fusion gene G11-VH-Nluc;

[0024] (2) Construction of expression vectors

[0025] Using the PCR product fusion gene MBP-G11-VL prepared in step (1) as a template, the vector pMal-p2E and the variable light chain gene of the fusion gene MBP-G11-VL were double-digested with Sal I and Hind III, the product was recovered by gel cutting, and ligated through T4 DNA to obtain the ligated pMal-p2E(+)-MBP-G11-VL expression vector;

[0026] Using the PCR product fusion gene MBP-G11-RL-VL prepared in step (1) as a template, the vector pMal-p2E and the variable light chain gene of the fusion gene MBP-G11-RL-VL were double-digested with SalI and HindIII, the product was recovered by gel cutting, and ligated through T4 DNA to obtain the ligated pMal-p2E(+)-MBP-G11-RL-VL expression vector;

[0027] Using the fusion gene MBP-G11-FL-VL of the PCR product prepared in step (1) as a template, the vector pMal-p2E and the light chain variable region gene of the fusion gene MBP-G11-FL-VL were double digested with SalI and HindIIII, the product was recovered by gel cutting, and the ligated pMal-p2E(+)-MBP-G11-FL-VL expression vector was obtained through T4 DNA ligation;

[0028] Using the fusion gene G11-VH-Nluc of the PCR product prepared in step (1) as a template, the vector pET22b(+)-G11-scFv-Nluc and the heavy chain variable region of G11-VH-Nluc were digested with SfiI, the product was recovered by gel cutting, and the ligated pET22b(+)-G11-VH-Nluc expression vector was obtained through T4 DNA ligation.

[0029] (3) Preparation of fusion protein

[0030] The expression vectors Mal-p2E(+)-MBP-G11-VL of the antibody light chain, the expression vector

[0031] pMal-p2E(+)-MBP-G11-RL-VL containing a rigid linker peptide, the expression vector pMal-p2E(+)-MBP-G11-FL-VL containing a flexible linker peptide, and the expression vector pET22b(+)-G11-scFv-Nluc containing nanoluciferase and the antibody heavy chain were respectively transformed into competent cells BL21 by heat shock transformation, and the positive monoclonal colonies were sent for sequencing verification. The sequencing primers for the expression vectors of the proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL containing the light chain were malefor, and the sequencing primer for the expression vector containing the antibody heavy chain was T7; For the positive monoclonal bacteria of the proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL containing the light chain and the protein G11-VH-Nluc containing the heavy chain with correct sequencing, the positive monoclonal bacteria were respectively expanded in culture, and then IPTG inducer was added for induction expression and purification to obtain the MBP-G11-VL, MBP-G11-RL-VL, MBP-G11-FL-VL, and G11-VH-Nluc fusion proteins.

[0032] Further, for the construction method of the above-mentioned bifunctional active fusion protein, the purification methods for the MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL proteins after induced expression are as follows: First, use the MBP-tagged bacterial lysate to separately induce and extract the MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL proteins after expression, and then purify the target proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL through an amylose-agarose medium affinity chromatography column. Finally, use SDS-PAGE electrophoresis to determine the molecular weight and purity of the proteins.

[0033] For the purification method of the G11-VH-Nluc protein after induced expression, use B-per bacterial lysate to extract the G11-VH-Nluc protein after induced expression, and then purify the target protein G11-VH-Nluc fusion protein through a nickel affinity chromatography column. Finally, use SDS-PAGE electrophoresis to determine the molecular weight and purity of the protein.

[0034] Further, for the construction method of the above-mentioned bifunctional active fusion protein, the plasmid pET22b(+)-G11-scFv-Nluc of the nano-luciferase fusion single-chain antibody G11-scFv-Nluc gene is prepared through the following steps:

[0035] 4) Amplify the single-chain antibody G11-scFv gene against glycocholic acid using SNF1 and SNR1 as primers;

[0036] Amplify the nano-luciferase Nluc gene using SNF2 and SNR2 as primers;

[0037] 5) Using the PCR products G11-scFv gene and Nluc gene in step 1) as templates respectively, and using SNF1 and SNR2 as primers, use the overlap extension PCR technique to amplify the G11-scFv-Nluc recombinant gene of the single-chain antibody against glycocholic acid fused with nano-luciferase;

[0038] 6) Double-digest the G11-scFv-Nluc recombinant gene and the vector pET-22b(+) with XhoI and EcoRI, and use T4 ligase for ligation to obtain the pET22b(+)-G11-scFv-Nluc expression vector.

[0039] To achieve the above third object, the third technical solution provided by the present invention is to provide the application of the above-mentioned bifunctional active fusion protein as an antibody for detecting glycocholic acid in urine.

[0040] The fourth technical solution provided by the present invention is to provide a bioluminescent immunoassay kit for detecting glycocholic acid, including the fusion protein with dual-functional activity described in the first technical solution.

[0041] The fifth technical solution provided by the present invention is to provide a method for detecting glycocholic acid in urine. The fusion protein with dual-functional activity described in the first technical solution is used as the antibody for detecting glycocholic acid in urine, and an open sandwich immunoassay method is utilized.

[0042] The beneficial effects of the present invention compared with the prior art are as follows:

[0043] 1) In the technical solution provided by the present invention, maltose-binding protein is used as the tag of the fusion protein. The light-chain variable regions (G11-VL, G11-RL-VL, G11-FL-VL) of the single-chain antibody against glycocholic acid are fused with maltose (MPB)-binding protein and cloned into the pMal-p2E expression vector. Nano luciferase is used as the catalyst for bioluminescence. The heavy-chain variable region (G11-VH) of the single-chain antibody against glycocholic acid is fused with nano luciferase and cloned into the pET22b expression vector to obtain a fusion protein with dual-functional activities of maltose-binding protein-antibody light chain (MBP-G11-VL) and antibody heavy chain-enzyme (G11-VH-Nluc). Using MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL as the light-chain coating antibodies respectively, the fusion protein (G11-VH-Nluc) as the detection heavy-chain antibody, and glycocholic acid as the standard product, an open sandwich bioluminescent enzyme-linked immunoassay kit is established. This kit has high specificity, good sensitivity, and the lowest detection limit is 52.076 ng / mL. The sensitivity is increased by 98 times compared with the ordinary enzyme-linked immunoassay method of 5.08 μg / mL, providing an accurate and highly sensitive detection method for the rapid detection of glycocholic acid in urine;

[0044] 2) The MBP-G11-VL fusion protein and G11-VH-Nluc fusion protein provided by the present invention have good specificity and signal amplification effects. A hypersensitive bioluminescent immunoassay kit is established using the MBP-G11-VL fusion protein and G11-VH-Nluc fusion protein to detect the content of glycocholic acid in urine, which has good application value and prospects. Description of the Drawings

[0045] Figure 1 It is the DNA sequence of the PCR amplification primer and the restriction enzyme cutting site map described in the present invention;

[0046] Figure 2 The schematic diagram of the open sandwich immunoassay principle of the antibody light-chain fusion protein and the antibody heavy-chain fusion protein;

[0047] Figure 3 PCR amplification electrophoresis map of antibody light chain variable region and heavy chain variable region genes;

[0048] Figure 4 Restriction enzyme analysis electrophoresis map of antibody light chain variable region and heavy chain variable region;

[0049] Figure 5 SDS-PAGE electrophoresis maps of MBP-G11-VL, MBP-G11-RL-VL, MBP-G11-FL-VL fusion proteins and G11-VH-Nluc fusion protein described in the present invention.

[0050] Figure 6 Optimization curves of different coating types in Example 4;

[0051] Figure 7 Optimization curves of different coating concentrations in Example 4;

[0052] Figure 8 Effect curves of different methanol concentrations on immunoassay sensitivity in Example 4;

[0053] Figure 9 Effect curves of different ionic strengths on immunoassay sensitivity in Example 4;

[0054] Figure 10 Effect curves of different solution pH values on immunoassay sensitivity in Example 4;

[0055] Figure 11 "Non-competitive" OS-ELISA standard curve for detecting glycocholic acid based on open sandwich method in Example 4;

[0056] Figure 12 "Competitive" BLEIA standard curve for detecting glycocholic acid based on G11-scFv-Nluc fusion antibody in Example 4. Specific implementation method

[0058] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.

[0059] Experimental materials

[0060] 1) Experimental reagents are shown in Table 1:

[0061] Table 1 Experimental reagent information

[0062]

[0063] 2) Experimental instruments are shown in Table 2:

[0064] Table 2 Experimental instrument information

[0065]

[0066] 3) The strains and vectors are shown in Table 3:

[0067] Table 3 Information on strains and vectors

[0068]

[0069] 4) The primers are shown in Table 4:

[0070] Table 4 Primer sequence information

[0071]

[0072]

[0073] The underlined parts are the restriction sites of SfiI, SalI and HindIII in sequence.

[0074] 5) Solution preparation

[0075] (1) 50×TAE Buffer electrophoresis buffer: In a 1L beaker, add 242 g of tris(hydroxymethyl)aminomethane (Tris base) and 37.2 g of disodium ethylenediaminetetraacetate dihydrate (Na2EDTA·2H2O) in sequence, inject 800 mL of deionized pure water for dissolution. After the solute is completely dissolved, slowly mix and add 57.1 mL of glacial acetic acid. After thoroughly mixing with a magnetic stirrer for 30 minutes, adjust the pH to 8.3 with NaOH, and make up the volume to the 1L scale line with deionized pure water. The prepared 50×TAE solution needs to be sealed and stored at room temperature.

[0076] (2) 1×TAE Buffer electrophoresis buffer: Accurately measure 10.0 mL of 50×TAE Buffer electrophoresis buffer, transfer it to a 500 mL graduated cylinder, and supplement 490 mL of deionized pure water. Mix the solution evenly with a vortex oscillator for 30 seconds.

[0077] (3) 0.5 M IPTG: Weigh 1.2 g of IPTG, dissolve it in 8 mL of sterile water, make up the volume to 10 mL, filter it with a 0.22 μm syringe filter, dispense it into 1 mL aliquots, and store at -20°C.

[0078] (4) 50 mg / mL Carbenicillin sodium (CA): Weigh 500 mg of carbenicillin sodium, dissolve it in 8 mL of sterile water, make up the volume to 10 mL, filter it with a 0.22 μm syringe filter, dispense it into 1 mL aliquots, and store at -20°C.

[0079] (5) LB medium: Weigh 10 g of sodium chloride, 5 g of yeast extract, and 10 g of tryptone, add 800 mL of deionized water, stir well until completely dissolved, then add deionized water to make up to 1 L. After autoclaving at high temperature and high pressure, store at room temperature.

[0080] (6) LB plate: In 100 mL of LB medium, add 1.5 g of agar powder, autoclave at high temperature and high pressure. When it cools down to a temperature that is not too hot to touch, pour it into plates in a laminar flow hood. After solidification, invert the plates and store at 4 °C.

[0081] (7) 10× Electrophoresis buffer: Weigh 30 g of Tris-base, 144 g of glycine, and 10 g of SDS, add 800 mL of deionized water, stir well until completely dissolved, then add deionized water to make up to 1 L.

[0082] (8) Staining solution: Weigh 1 g of Coomassie Brilliant Blue, add 500 mL of decolorizing solution, mix well, and store at room temperature in the dark.

[0083] (9) Decolorizing solution: Measure 400 mL of methanol and 100 mL of glacial acetic acid, mix well, add deionized water to make up to 1 L, and store at room temperature in the dark.

[0084] (10) Transfer buffer: Weigh 14.42 g of glycine and 3.04 g of Tris-base, add 700 mL of deionized water to dissolve, add 200 mL of methanol, and add deionized water to make up to 1 L.

[0085] (11) 10× TBS buffer: Weigh 60.57 g of Tris-base and 87.64 g of sodium chloride, add 800 mL of deionized water to dissolve, adjust the pH to 7.5 with concentrated hydrochloric acid, and add deionized water to make up to 1 L. Store at room temperature.

[0086] (12) TBST buffer: Measure 100 mL of 10× TBS buffer, add 900 mL of deionized water, and add 0.5 mL of Tween 20 and mix well.

[0087] (15) 0.1 L cell washing solution: Take 1 mL of Tris-HCL (pH = 7.4, 10 mM), 1.5 mL of 2 M sodium chloride solution (30 mM), and make up to 100 mL with deionized water.

[0088] (16) 0.1 L cell lysis solution: Take 3 mL of Tris-HCL (pH = 7.4, 30 mM), 0.1 mL of 0.1 M EDTA solution (0.1 mM), 20 g of sucrose (20% m / V), and make up to 100 mL with deionized water.

[0089] (17) 0.1L Binding Buffer: Take 2 mL of 1M Tris-HCL (pH = 7.4, 20 mM), 1 mL of 0.1M EDTA solution (1 mM), 10 mL of 2M sodium chloride solution (200 mM), and make up to 100 mL with deionized water.

[0090] (18) 0.1L Maltose Elution Buffer: 2 mL of 1M Tris-HCL (pH = 7.4, 20 mM), 1 mL of 0.1M EDTA solution (1 mM), 10 mL of 0.1M maltose solution (10 mM), and make up to 100 mL with deionized water.

[0091] Example 1

[0092] This example provides a bifunctional active fusion protein, which consists of a light chain variable region fusion protein MBP-G11-VL and a heavy chain variable region G11-VH-Nluc fusion protein.

[0093] It is prepared by the following method:

[0094] I) Amplification of G11-scFv-Nluc gene

[0095] 1) Amplification of G11-scFv gene and Nluc gene

[0096] (1) Take out the pComb3x-G11-scFv in E.coli DH5α glycerol bacteria stored in the laboratory -80°C ultra-low temperature storage refrigerator. Take an LB solid plate medium containing 50 μg / mL sodium carbenicillin, streak-culture it on a clean bench, and invert the LB solid plate in a constant temperature bacterial incubator at 37°C for 16 h overnight for activation.

[0097] The next day, in the clean bench, use a 10 μL sterile pipette tip to pick a G11-scFv monoclonal colony into 5 mL of LB liquid medium containing 50 μg / mL sodium carbenicillin, culture it overnight at 37°C and 225 rpm in an oscillating incubator, and then use a plasmid DNA extraction kit to extract the positive plasmid pComb3x-G11-scFv.

[0098] The operation of extracting the positive plasmid pComb3x-G11-scFv is as follows:

[0099] 1) Take the overnight cultured monoclonal bacterial solution, centrifuge at 10000g and 37°C for 2 min;

[0100] 2) Pour off the supernatant and leave the precipitate. Invert the centrifuge tube and place it on absorbent paper to remove excess liquid;

[0101] 3) Add 250 μL of Solution I to the centrifuged tube pellet, and use a pipette tip to repeatedly pipette up and down to dissolve the pellet and completely resuspend the bacterial cell pellet;

[0102] 4) Transfer the resuspended pellet to a sterilized EP tube;

[0103] 5) Add 250 μL of Solution II, cover the sterilized EP tube, and gently rotate it up and down 6 times to fully lyse the bacterial solution until it becomes transparent. Then wait for 2 - 3 minutes and complete it within no more than 5 minutes;

[0104] 6) Add 350 μL of Solution III, immediately cover the sterilized EP tube, and gently invert it several times to fully mix until a flocculent white precipitate appears in the solution;

[0105] 7) Immediately centrifuge the sterilized EP tube at a maximum speed of 13000 g at 4°C for 10 minutes. After centrifuging to form a dense white pellet, proceed to the next step immediately;

[0106] 8) Insert the HiBind DNA adsorption column into a 2 mL filter tube. Use a pipette to carefully aspirate 700 μL of the supernatant of the centrifuged dense white pellet into the adsorption column at one time, taking care not to aspirate the white pellet and cell debris;

[0107] 9) Centrifuge the adsorption column and the filter tube at a maximum speed of 13000 g at 4°C for 1 minute;

[0108] 10) Discard the filtrate in the filter tube after centrifugation. Put the adsorption column back into the filter tube, add 500 μL of HBC solution diluted with isopropanol to the adsorption column, and centrifuge at a maximum speed of 13000 g at 4°C for 1 minute;

[0109] 11) Discard the filtrate in the filter tube after centrifugation. Put the adsorption column back into the filter tube, add 700 μL of DNA Wash Buffer solution diluted with 100% ethanol to the adsorption column, and centrifuge at a maximum speed of 13000 g at 4°C for 1 minute. Discard the filtrate and repeat this step once;

[0110] 12) Centrifuge the empty adsorption column at a maximum speed of 13000 g at 4°C for 2 minutes, discard the filter tube, air-dry the adsorption column, and remove the ethanol;

[0111] 13) Place the adsorption column in a clean, sterile, and enzyme-free centrifuge tube. Slowly add 30 - 100 μL of Elution Buffer solution to the center of the adsorption membrane of the adsorption column. Let it stand at room temperature for 1 minute, then centrifuge at a maximum speed of 13000 g at 4°C for 1 minute. Repeat the process of letting it stand at room temperature for 1 minute and then centrifuge at a maximum speed of 13000 g at 4°C once to increase the concentration;

[0112] 14) The solution collected in the centrifuge tube is the extracted plasmid pComb3x-G11-scFv. Measure the concentration and purity of the collected plasmid DNA using a Nanodrop micro ultraviolet spectrophotometer, and store the plasmid DNA in a -20 °C refrigerator.

[0113] (2) Using the plasmid pComb3x-G11-scFv of the anti-glycocholic acid single-chain antibody as a template in a PCR tube, add 5×PrimeSTAR buffer, dNTPs, and Taq enzyme to amplify the G11-scFv gene (nucleotide sequence shown in SEQ ID NO.1). At the same time, use the plasmid pNL1.1-Nluc of the nano luciferase Nluc gene (nucleotide sequence shown in SEQ ID NO.2) as a template to amplify the Nluc gene (nucleotide sequence shown in SEQ ID NO.3). The reaction system is shown in Table 5:

[0114] Table 5

[0115]

[0116] (3) Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand still to be evenly distributed. Set the conditions in the PCR instrument as shown in Table 6:

[0117] Table 6

[0118]

[0119] (4) After the G11-scFv gene and Nluc gene amplification reactions are completed in the PCR instrument, perform 1.2% agarose gel electrophoresis on the PCR product (nucleotide sequence shown in SEQ ID NO.4).

[0120] The procedure for agarose gel electrophoresis of nucleic acid products is as follows:

[0121] 1) Pre-treat the gel preparation mold. Rinse the bottom plate, card slot, and comb of the gel preparation mold with deionized water to remove residual impurities. After wiping, place it on the workbench to dry. After ensuring that the bottom plate fits perfectly with the mold card slot, place the bottom plate on the mold card slot and place the comb vertically on the black stripe port of the bottom plate;

[0122] 2) Prepare a 1.2% agarose gel. Weigh the agarose dry powder quantitatively using a precision electronic balance. Transfer the powder to a clean and high-temperature-resistant conical flask, and then inject the newly prepared 1×TAE Buffer electrophoresis solution;

[0123] 3) Seal the clean and high-temperature-resistant conical flask with aluminum foil throughout the process to prevent evaporation. Use the gradient heating method to initially heat it in a microwave oven at medium power for 30 seconds after shaking. Take it out and vortex for 10 seconds to disperse the undissolved particles. Then repeat the heating for 30 seconds each time until the solution is clear and free of suspended matter. Shake to maintain the fluidity of the solution and cool the temperature until it reaches 45°C;

[0124] 4) When the solution cools down to the operable temperature of 45°C, quickly mix GoldView nucleic acid dye in the conical flask to avoid dye degradation caused by high temperature. After adding, shake well. Do not shake too hard to avoid generating bubbles in the solution;

[0125] 5) Immediately pour the solution onto the bottom plate and comb in the mold slot. Pull the comb on the bottom plate up and down to avoid generating bubbles in the solution. Wait for 30 minutes at room temperature until it solidifies;

[0126] 6) Pull out the bottom plate and comb from the mold slot. Remove the excess solidified gel on the bottom plate. Slowly remove the comb on the bottom plate to avoid damaging the solidified gel. Fix the gel with the black band end facing the negative electrode and place it in the horizontal electrophoresis tank. The nucleic acid product sample of PCR will move from the negative electrode to the positive electrode in the horizontal electrophoresis tank;

[0127] 7) Pour the newly prepared 1×TAE Buffer electrophoresis solution into the horizontal electrophoresis tank just to cover the gel. Pour it slowly to avoid generating bubbles in the loading wells at the black band end of the gel;

[0128] 8) Load the sample into the loading wells at the black band end of the gel. Add DNA marker to the loading wells of the gel. Mix the 10×Loading buffer with the PCR nucleic acid product DNA sample at a volume ratio of 1:10 and then add it to the loading wells of the gel;

[0129] 9) Confirm the electrode correspondence. That is, cover the lid of the horizontal electrophoresis tank with the black band end facing the negative electrode and the red band end facing the positive electrode. At the same time, connect the electrophoresis instrument and set the electrode of the electrophoresis instrument to the constant voltage mode of 100V;

[0130] 10) Immediately turn off the power of the electrophoresis instrument after 30 minutes of electrophoresis to prevent molecular diffusion. Wear ethylene gloves when taking the gel to avoid nucleic acid contamination. Take out the agarose gel electrophoresis on the bottom plate and place it in the dark box ultraviolet analyzer. After turning on the ultraviolet lamp, observe the PCR nucleic acid product bands in the dark through the dark box ultraviolet analyzer;

[0131] (5) After agarose gel electrophoresis, use the gel extraction kit OmegaBIO-TEKE.Z.N.A. Gel ExtractionKit to perform gel extraction of the PCR nucleic acid product.

[0132] The operation of cutting and recovering PCR nucleic acid products is as follows:

[0133] 1) After the agarose gel electrophoresis is completed, quickly cut the target band of the PCR nucleic acid product with a clean blade under ultraviolet light using a dark box ultraviolet analyzer. Remove the excess agarose gel outside the target band. Do not expose the PCR nucleic acid product to ultraviolet light for a long time to avoid DNA mutation;

[0134] 2) Place the cut gel block of the target band of the PCR nucleic acid product in a sterile and enzyme-free centrifuge tube, and weigh the volume of the gel block of the target band using a precision electronic balance;

[0135] 3) Add XP2 Binding Buffer solubilization buffer with a volume of 1 mL for every 1 g of the volume of the gel block of the target band. Add an equal volume of XP2 Binding Buffer solubilization buffer to the centrifuge tube as the volume of the weighed gel block of the target band;

[0136] 4) Place the centrifuge tube containing an equal volume of XP2 Binding Buffer solubilization buffer in a water bath at 50 - 60 °C and shake it once every 3 min with a micro vortex mixer, then put it back into the water bath for constant incubation for a total of 7 min until the gel block completely melts and no solid gel block can be seen;

[0137] 5) Set the HiBind DNA adsorption column onto the filter tube, and transfer the DNA sol liquid in the centrifuge tube to the HiBind DNA adsorption column at no more than 700 μL each time;

[0138] 6) Centrifuge at 10000 g and 37 °C for 1 min, discard the filtrate in the filter tube, and put the adsorption column back onto the filter tube. If the DNA sol liquid in the centrifuge tube exceeds 700 μL, repeat this step until all the DNA sol liquid passes through the column;

[0139] 7) Add 300 μL of XP2 Binding Buffer solubilization buffer to the adsorption column, centrifuge at a maximum speed of 13000 g and 4 °C for 1 min, discard the filtrate in the filter tube, and put the adsorption column back onto the filter tube;

[0140] 8) Add 700 μL of SPW Buffer rinsing buffer diluted with absolute ethanol to the adsorption column, centrifuge at 10000 g and 4 °C for 1 min, discard the filtrate in the filter tube, and put the adsorption column back onto the filter tube. This step can be repeated once to reduce the sensitivity to the content of salt ions;

[0141] 9) Centrifuge at a maximum speed of 13000 g and 4 °C for 2 min to discard the residual liquid in the matrix of the adsorption binding column;

[0142] 11) Place the adsorption column on a clean, sterile, and enzyme-free centrifuge tube. Slowly add 15 - 30 μL of Elution Buffer to the center of the adsorption membrane in the adsorption column using a pipette. After standing at room temperature for 2 min, centrifuge at a maximum speed of 13,000 g at 4°C for 1 min. Repeat the process of standing at room temperature for 2 min and then centrifuging at a maximum speed of 13,000 g at 4°C for 1 min for elution to increase the concentration.

[0143] 12) The solution collected in the centrifuge tube is the PCR nucleic acid product recovered by gel cutting. Measure the concentration and purity of the collected PCR nucleic acid product using a Nanodrop micro ultraviolet spectrophotometer, and store the PCR nucleic acid product in a -20°C refrigerator.

[0144] II) Amplification of the G11-scFv-Nluc gene by overlap extension PCR

[0145] (1) Using the G11-scFv recovery product and the Nluc recovery product as templates in a PCR tube, add 5×PrimeSTAR buffer, dNTPs, and Taq enzyme, and amplify the G11-scFv-Nluc recombinant gene by overlap extension PCR. The reaction system is shown in Table 7:

[0146] Table 7

[0147]

[0148] (2) Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand evenly. Set the conditions in the PCR instrument as shown in Table 8:

[0149] Table 8

[0150]

[0151] (3) After the G11-scFv-Nluc recombinant gene amplification reaction is completed in the PCR instrument, perform 1.2% agarose gel electrophoresis on the PCR nucleic acid product, place it in a dark box ultraviolet analyzer, turn on the ultraviolet lamp, and observe the DL2000 DNA Marker band and the PCR nucleic acid product band under darkness through the dark box ultraviolet analyzer.

[0152] (4) After agarose gel electrophoresis, use a gel recovery kit to perform gel cutting and recovery of the PCR nucleic acid product. The collected gel-cut and recovered PCR nucleic acid product is the G11-scFv-Nluc recombinant gene fragment of the anti-glycocholic acid single-chain antibody fused with nano-luciferase (the nucleotide sequence is shown in SEQ ID NO.4). Measure the concentration and purity of the collected PCR nucleic acid product using a Nanodrop micro ultraviolet spectrophotometer, and store the PCR nucleic acid product in a -20°C refrigerator.

[0153] (iii) Construction of pET-22b(+)-G11-scFv-Nluc expression vector

[0154] Using the recombinant gene G11-scFv-Nluc of the second-round PCR product as a template, since restriction enzyme sites EcoR I and Xho I were inserted at both ends of the G11-scFv-Nluc gene, the vector pET-22b(+) and the recombinant gene G11-scFv-Nluc were double digested with EcoRI and Xho I. After gel extraction and recovery, they were ligated together with T4 DNA ligase to obtain the ligated pET22b(+)-G11-scFv-Nluc positive plasmid (the nucleotide sequence is shown in SEQ ID NO.5).

[0155] (iv) Amplification of G11-VL gene and G11-VH gene

[0156] (1) Extract the positive plasmid pET22b(+)-G11-scFv-Nluc of the nanoluciferase fusion antibody.

[0157] The operation of extracting the positive plasmid pET22b(+)-G11-scFv-Nluc is as follows:

[0158] 1) Take out the pET22b(+)-G11-scFv-Nluc in DH5α glycerol bacteria stored in the -80°C ultra-low temperature storage refrigerator in the laboratory. Take an LB solid plate medium containing 50 μg / mL sodium carbenicillin, streak it for culture on a clean bench, and invert the LB solid plate in a constant temperature bacterial incubator at 37°C for 16 h for overnight activation. The next day, use a 10 μL sterile pipette tip to pick the G11-scFv monoclonal colony into 5 mL of LB liquid medium containing 50 μg / mL sodium carbenicillin, and culture it overnight at 37°C and 225 rpm in an oscillating incubator. The overnight cultured pET22b(+)-G11-scFv-Nluc in DH5α glycerol bacteria was centrifuged at 10000g and 37°C for 2 min;

[0159] 2) Pour off the supernatant and leave the precipitate. Invert the centrifuge tube and place it on absorbent paper to remove the excess liquid;

[0160] 3) Add 250 μL of Solution I to the precipitate in the centrifuge tube, and use a pipette tip to pipette up and down repeatedly to dissolve the precipitate and completely resuspend the bacterial cell precipitate;

[0161] 4) Transfer the resuspended precipitate to a sterilized EP tube;

[0162] 5) Add 250 μL of Solution II, cover the sterilized EP tube, and gently invert it up and down 6 times in a mild manner to fully lyse the bacterial solution until it becomes transparent. Then wait for another 2 - 3 minutes and complete the process within no more than 5 minutes.

[0163] 6) Add 350 μL of Solution III, immediately cover the sterilized EP tube, and gently invert it several times to mix well until a flocculent white precipitate appears in the solution.

[0164] 7) Immediately centrifuge the sterilized EP tube at a maximum speed of 13000 g at 4 °C for 10 minutes. After centrifuging to form dense white particles, proceed to the next step immediately.

[0165] 8) Fit the HiBind DNA adsorption column onto the 2 mL filter tube. Use a pipette to carefully aspirate 700 μL of the supernatant of the centrifuged dense white particles into the adsorption column at one time, taking care not to aspirate the white particles and cell debris.

[0166] 9) Centrifuge the adsorption column and the filter tube at a maximum speed of 13000 g at 4 °C for 1 minute.

[0167] 10) Discard the filtrate in the filter tube after centrifugation. Put the adsorption column back onto the filter tube. Add 500 μL of the HBC solution diluted with isopropanol to the adsorption column and centrifuge at a maximum speed of 13000 g at 4 °C for 1 minute.

[0168] 11) Discard the filtrate in the filter tube after centrifugation. Put the adsorption column back onto the filter tube. Add 700 μL of the DNA Wash Buffer solution diluted with 100% ethanol to the adsorption column and centrifuge at a maximum speed of 13000 g at 4 °C for 1 minute. Discard the filtrate and repeat this step once.

[0169] 12) Centrifuge the empty adsorption column at a maximum speed of 13000 g at 4 °C for 2 minutes. Discard the filter tube, air-dry the adsorption column, and remove the ethanol.

[0170] 13) Place the adsorption column in a clean, sterile, and enzyme-free centrifuge tube. Slowly add 30 - 100 μL of Elution Buffer solution to the center of the adsorption membrane of the adsorption column. Let it stand at room temperature for 1 minute and then centrifuge at a maximum speed of 13000 g at 4 °C for 1 minute. Repeat the process of letting it stand at room temperature for 1 minute and then centrifuge at a maximum speed of 13000 g at 4 °C for 1 minute once to increase the concentration.

[0171] 14) The solution collected in the centrifuge tube is the extracted plasmid pET22b(+)-G11-scFv-Nluc. Measure the concentration and purity of the collected plasmid DNA using a Nanodrop micro ultraviolet spectrophotometer and store the plasmid DNA in a -20 °C refrigerator.

[0172] (2) Using the plasmid pET22b(+)-G11-scFv-Nluc of the nano-luciferase fusion antibody as a template in a PCR tube, add 5×PrimeSTAR buffer, dNTPs and Taq enzyme. At the same time, use the MBP-VL-F upstream primer (nucleotide sequence as shown in SEQ ID NO.6) and the MBP-VL-R downstream primer (nucleotide sequence as shown in SEQ ID NO.7), and the VH-F upstream primer (nucleotide sequence as shown in SEQ ID NO.8) and the VH-R downstream primer (nucleotide sequence as shown in SEQ ID NO.9) to amplify the antibody light chain G11-VL (nucleotide sequence as shown in SEQ ID NO.10) and the antibody heavy chain G11-VH gene (nucleotide sequence as shown in SEQ ID NO.11). The reaction system is shown in Table 9:

[0173] Table 9

[0174]

[0175] (3) Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand still to be uniform. Set the conditions in the PCR instrument as shown in Table 10:

[0176] Table 10

[0177]

[0178]

[0179] After the PCR instrument completes the amplification reaction of the antibody light chain G11-VL and the antibody heavy chain G11-VH gene, perform 1.2% agarose gel electrophoresis; turn on the ultraviolet lamp on the dark box ultraviolet analyzer to observe the Marker band and the PCR amplification product band. Finally, use the gel extraction kit Omega BIO-TEK E.Z.N.A. Gel Extraction Kit to cut and recover the gel, measure the concentration and purity of the collected PCR product with Nanodrop, and store the PCR product in a -20°C refrigerator.

[0180] The process of agarose gel electrophoresis for nucleic acid products is as follows:

[0181] 1) Pre-treat the gel preparation mold. Rinse the bottom plate, card slot and comb of the gel preparation mold with deionized water to remove residual impurities. After wiping, place it on the workbench to dry. After ensuring that the bottom plate fits perfectly with the mold card slot, place the bottom plate on the mold card slot and place the comb vertically on the black stripe port of the bottom plate;

[0182] 2) Prepare a 1.2% agarose gel. Weigh the dry agarose powder quantitatively using a precision electronic balance. After transferring the powder to a clean and heat-resistant conical flask, pour in the freshly prepared 1×TAE Buffer electrophoresis solution.

[0183] 3) Seal the clean and heat-resistant conical flask with aluminum foil throughout the process to prevent evaporation. Use the gradient heating method. First, heat it in the microwave at medium power for 30 seconds after shaking, take it out and vortex for 10 seconds to disperse the undissolved particles, then repeat the heating for 30 seconds each time until the solution is clear and free of suspended matter. Shake to maintain the fluidity of the solution and cool it down to 45°C.

[0184] 4) When the solution cools down to the operable temperature of 45°C, quickly mix in the GoldView nucleic acid dye in the conical flask to avoid dye degradation caused by high temperature. After adding, shake well, but don't use too much force when shaking to avoid generating bubbles in the solution.

[0185] 5) Immediately pour the solution into the bottom plate and comb in the mold slot. Pull the comb on the bottom plate up and down to avoid generating bubbles in the solution. Wait for 30 minutes at room temperature until it solidifies.

[0186] 6) Pull out the bottom plate and comb from the mold slot, remove the excess solidified gel on the bottom plate, and slowly remove the comb on the bottom plate to avoid damaging the solidified gel. Fix the gel with the black band end facing the negative electrode and place it in the horizontal electrophoresis tank. The nucleic acid product samples of the antibody light chain G11-VL and the antibody heavy chain G11-VH gene PCR will move from the negative electrode to the positive electrode in the horizontal electrophoresis tank.

[0187] 7) Pour the freshly prepared 1×TAE Buffer electrophoresis solution into the horizontal electrophoresis tank until it just covers the gel. Pour it slowly to avoid generating bubbles in the sample wells at the black band end of the gel.

[0188] 8) Load the sample into the sample wells at the black band end of the gel. Add the DNA marker to the sample wells of the gel. Mix the 10×Loading buffer with the PCR nucleic acid product DNA sample at a volume ratio of 1:10 and then add it to the sample wells of the gel.

[0189] 9) Confirm the electrode correspondence. That is, cover the lid of the horizontal electrophoresis tank by making the black band end face the negative electrode and the red band end face the positive electrode. At the same time, connect the electrophoresis instrument and set the electrophoresis instrument electrode to the constant voltage mode of 100V.

[0190] 10) After 30 minutes of electrophoresis, immediately turn off the power of the electrophoresis apparatus to prevent molecular diffusion. Wear ethylene gloves when taking the gel to avoid nucleic acid contamination. Take out the agarose gel electrophoresis on the bottom plate and place it in the dark box UV analyzer. After turning on the UV lamp, observe the nucleic acid product bands of the PCR of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes under the dark through the dark box UV analyzer;

[0191] The operation of cutting and recovering the PCR nucleic acid products is as follows:

[0192] 1) After the agarose gel electrophoresis is completed, quickly cut the target bands of the PCR nucleic acid products of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes with a clean blade under the UV lamp using the dark box UV analyzer. Remove the excess agarose gel outside the target bands. Do not expose the PCR nucleic acid products under the UV lamp for a long time to avoid DNA mutations;

[0193] 2) Place the cut gel blocks of the target bands of the PCR nucleic acid products of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes in a sterile and enzyme-free centrifuge tube, and weigh the volume of the gel blocks of the target bands with a precision electronic balance;

[0194] 3) Add 1 mL of XP2 Binding Buffer sol buffer for every 1 g of the volume of the gel blocks of the target bands. Add the XP2 Binding Buffer sol buffer with the same volume as the weighed volume of the gel blocks of the target bands into the centrifuge tube;

[0195] 4) Place the centrifuge tube with the same volume of XP2 Binding Buffer sol buffer added in a water bath at 50-60 °C and oscillate it with a micro vortex mixer every 3 minutes, then put it back into the water bath for constant incubation for a total of 7 minutes until the gel blocks completely melt and no solid gel blocks can be seen;

[0196] 5) Put the HiBind DNA adsorption column on the filter tube, and transfer the DNA sol liquid in the centrifuge tube to the HiBind DNA adsorption column at no more than 700 μL each time;

[0197] 6) Centrifuge at 10000 g and 37 °C for 1 minute, discard the filtrate in the filter tube, and put the adsorption column back on the filter tube. If the DNA sol liquid in the centrifuge tube exceeds 700 μL, repeat this step until all the DNA sol liquid passes through the column;

[0198] 7) Add 300 μL of XP2 Binding Buffer sol buffer to the adsorption column, centrifuge at the maximum speed of 13000 g and 4 °C for 1 minute, discard the filtrate in the filter tube, and put the adsorption column back on the filter tube;

[0199] 8) Add 700 μL of SPW Buffer rinsing buffer diluted with absolute ethanol to the adsorption column, centrifuge at 10,000 g and 4 °C for 1 min, discard the filtrate in the filter tube, and put the adsorption column into the filter tube. This step can be repeated once to reduce the sensitivity to the content of salt ions;

[0200] 9) Centrifuge at the maximum speed of 13,000 g and 4 °C for 2 min to discard the residual liquid in the matrix of the adsorption binding column;

[0201] 11) Place the adsorption column on a clean, sterile and enzyme-free centrifuge tube. Slowly add 15 - 30 μL of Elution Buffer to the center of the adsorption membrane of the adsorption column with a pipette gun. After standing at room temperature for 2 min, centrifuge at the maximum speed of 13,000 g and 4 °C for 1 min. Repeat the step of standing at room temperature for 2 min and then centrifuge at the maximum speed of 13,000 g and 4 °C for 1 min for elution to increase the concentration;

[0202] 12) The solution collected in the centrifuge tube is the PCR nucleic acid product of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes recovered by gel cutting. Measure the concentration and purity of the PCR nucleic acid product of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes collected with a Nanodrop micro ultraviolet spectrophotometer, and store the PCR nucleic acid product of the antibody light chain G11-VL and the antibody heavy chain G11-VH genes (nucleotide sequence as SEQ ID NO.12) in a -20 °C refrigerator.

[0203] V) Construction of expression vector

[0204] Using the antibody light chain PCR product G11-VL (nucleotide sequence as SEQ ID NO.13) and the heavy chain and G11-VH gene (nucleotide sequence as SEQ ID NO.14) as templates, the vector pMal-p2E and the G11-VL light chain variable region gene can be double digested with Sal I and HindⅢ. After gel cutting and recovery, they are ligated together with T4 DNA ligase to obtain the ligated pMal-p2E-MBP-G11-VL (nucleotide sequence as SEQ ID NO.15). At the same time, the vector pET22b(+)-G11-scFv-Nluc and the G11-VHL heavy chain variable region can be digested with Sfi I. After gel cutting and recovery, they are ligated with T4 to obtain the ligated pET22b(+)-G11-VH-Nluc expression vector (nucleotide sequence as SEQ ID NO.16).

[0205] (1) Double digestion of plasmid vector pMal-p2E and antibody light chain variable region gene

[0206] 1) Using the plasmid vector pMal-p2E as a template in a PCR tube, add 10×M buffer, 10×H buffer, restriction endonuclease Sal I and restriction endonuclease Hind III, and double-digest the plasmid vector pMal-p2E. The reaction system is shown in Table 11:

[0207] Table 11

[0208]

[0209] 2) Using the antibody light chain recovery product G11-VL as a template in a PCR tube, add 10×M buffer, 10×H buffer, restriction endonuclease Sal I and restriction endonuclease Hind III, and double-digest the PCR product of the antibody light chain variable region. The reaction system is shown in Table 12:

[0210] Table 12

[0211]

[0212] 3) Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand still to be uniform. The double-digestion temperature of restriction endonuclease Sal I and restriction endonuclease Hind III is 37°C, and use the program set in the PCR instrument as shown in Table 13 for double-digestion:

[0213] Table 13

[0214]

[0215] 4) After the double-digestion reaction of the plasmid vector pMal-p2E and the antibody light chain PCR product is completed in the PCR instrument, the digested products are subjected to 1.2% agarose gel electrophoresis, and then the target fragments of the plasmid and the antibody light chain PCR digested products are observed through a dark box ultraviolet analyzer. Use a gel recovery kit to collect the gel-cut recovery. The recovered products are the target fragment of the digested plasmid vector pMal-p2E (nucleotide sequence as SEQ ID NO.17) and the digested antibody light chain gene fragment (nucleotide sequence as SEQ ID NO.18). Measure the concentration and purity of the PCR digested products with a Nanodrop micro ultraviolet spectrophotometer and store them in a -20°C refrigerator.

[0216] (2) Digestion of plasmid vector pET22b(+)-G11-scFv-Nluc and G11-VH gene

[0217] 1) Using the plasmid vector pET22b(+)-G11-scFv-Nluc as a template in a PCR tube, add 10×M buffer and the restriction endonuclease Sfi I to digest the plasmid vector pET22b(+)-G11-scFv-Nluc. The reaction system is shown in Table 14:

[0218] Table 14

[0219]

[0220] 2) Using the recovered product of the antibody heavy chain G11-VH gene as a template in a PCR tube, add 10×M buffer and the restriction endonuclease Sfi I to digest the variable region of the antibody heavy chain G11-VH gene. The reaction system is shown in Table 15:

[0221] Table 15

[0222]

[0223] 3) Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand still to be uniform. The digestion temperature of the restriction endonuclease Sfi I is 50°C. Set the conditions in the PCR instrument as shown in 16 for digestion:

[0224] Table 16

[0225]

[0226] 4) After the digestion reaction of the plasmid vector pET22b(+)-G11-scFv-Nluc and the antibody heavy chain G11-VH gene is completed in the PCR instrument, the digestion products are subjected to 1.2% agarose gel electrophoresis. Then, observe the target fragments of the plasmid and the variable region gene of the antibody heavy chain through a dark box ultraviolet analyzer. Use a gel recovery kit to collect the gel cutting and recovery. The recovered product is the target fragment of the digested plasmid vector pET22b(+)-G11-scFv-Nluc (the nucleotide sequence is shown in SEQ ID NO.19), and the digested antibody heavy chain gene fragment (the nucleotide sequence is shown in SEQ ID NO.20). Measure the concentration and purity of the PCR digestion products with Nanodrop and store them in a -20°C refrigerator.

[0227] (2) Construction of the expression vector by T4 DNA ligation

[0228] 1) Using the double-digested pMal-p2E vector and the antibody light chain variable region gene fragment as templates in a PCR tube, add T4 DNA Buffer and T4 DNA ligase to construct the expression vector pMal-p2E-MBP-G11-VL (the nucleotide sequence is shown in SEQ ID NO.21) through T4 DNA ligation. The reaction system is shown in 17:

[0229] Table 17

[0230]

[0231] (4) Using the digested pET22b(+)-G11-scFv-Nluc and the digested G11-VH gene fragment as templates in a PCR tube, construct the expression vector pET22b(+)-G11-VH-Nluc (the nucleotide sequence is shown in SEQ ID NO.22) through T4 ligation. The reaction system is as follows in Table 18:

[0232] Table 18

[0233]

[0234] Gently shake the bottom of the PCR tube by hand on an ice box and then let it stand still to be evenly distributed. The T4 DNA ligation temperature is 16°C, and the inactivating temperature is 65°C. Set the following conditions in a PCR instrument for T4 ligation. The PCR amplification program is shown in Table 19:

[0235] Table 19

[0236]

[0237] (4) After the T4 ligation is completed in the PCR instrument, the obtained expression vectors pMal-p2E-MBP-G11-VL and pET22b(+)-G11-VH-Nluc are in the PCR tube and stored in a -20°C refrigerator.

[0238] VI) Prokaryotic expression of MBP-G11-VL and G11-VH-Nluc proteins

[0239] (1) Respectively transfer the expression vector pMal-p2E-MBP-G11-VL containing maltose (MBP) binding protein and antibody light chain and the expression vector pET22b(+)-G11-VH-Nluc containing nanoluciferase and antibody heavy chain into the competent cell BL21(DE3)pLysS, and send the positive monoclonal colonies for sequencing verification. The sequencing primer for the expression vector containing antibody light chain is malefor (the nucleotide sequence is shown in SEQ ID NO.23), and the sequencing primer for the expression vector containing antibody heavy chain is T7 (the nucleotide sequence is shown in SEQ ID NO.24).

[0240] The operation of chemically transforming the T4 ligation product into Escherichia coli competent cell DH5α is as follows:

[0241] 1) Quickly transfer the E. coli competent cell DH5α cryopreserved in an -80°C ultra-low temperature storage refrigerator in the laboratory to an ice box in an ice bath environment and wait for the competent cell DH5α to thaw and melt;

[0242] 2) Add the ligation product pMal-p2E-MBP-G11-VL to an EP tube containing 100 μL of competent cells DH5α, gently flick the bottom of the EP tube of competent cells DH5α to promote mixing, insert it into an ice box in an ice bath environment and incubate statically for 30 min;

[0243] 3) Place the EP tube in a water bath for heat shock transformation. After heat shock treatment at 42 °C in a water bath for 90 s, immediately transfer it to an ice box in an ice bath environment and cool in the ice bath for 2 min;

[0244] 4) Add 900 μL of preheated sterile and antibiotic-free LB liquid medium at 42 °C, place it in an orbital shaker and incubate at 180 rpm and 37 °C for 45 min to resuscitate the bacteria in the EP tube;

[0245] 5) Centrifuge at 5000 rpm and 4 °C for 1 min, retain 300 μL of the supernatant to resuspend the bacterial pellet, pipette to mix well, and take 200 μL of the mixed bacterial solution and spread it on an LB solid plate resistant to CA;

[0246] 6) Incubate upright at 37 °C in a constant temperature bacterial incubator for 1 h to allow the liquid to be absorbed, then invert it and incubate at 37 °C in the constant temperature bacterial incubator overnight for 16 h;

[0247] (2) For the positive monoclonal bacteria containing the light chain protein MBP-G11-VL and the heavy chain protein G11-VH-Nluc with correct sequencing, expand the culture respectively and induce expression with an IPTG inducer at a final concentration of 1 mM.

[0248] Expansion culture and induction expression of pMal-p2E-MBP-G11-VL fusion protein

[0249] (1) Take 100 μL of positive monoclonal bacteria with accurate sequencing verification and already transferred into competent cells BL21(DE3)pLysS for prokaryotic expression, inoculate them into 10 mL of LB liquid medium containing 50 μg / mL carbenicillin, and incubate at 37 °C and 225 rpm in an orbital shaker overnight for 16 h;

[0250] (2) The next day, perform an expansion culture. Add antibiotics to the LB medium at a ratio of 1000:1 and add the bacterial solution at a ratio of 100:1, that is, transfer 1 mL of 50 μg / mL carbenicillin and 10 mL of the bacterial solution to 1 L of LB liquid culture in a biosafety cabinet. Tighten the lid of the medium and place it in an orbital shaker and shake at 225 rpm and 37 °C for 5 - 6 h. After the medium becomes turbid visibly, detect OD600 ≈ 0.6 with a multifunctional microplate reader;

[0251] (3)Induce expression. Wait until the LB medium is ice-bathed to about 20 °C. In a laminar flow hood, add 0.5 mol / L isopropyl-β-D-thiogalactoside (IPTG) inducer to the LB medium at a ratio of 1000:1. Then put it back into the shaking incubator and shake at 200 rpm and 20 °C for 16 hours overnight.

[0252] (4)The next day, use a sterilized centrifuge tube to collect the bacterial liquid that has been induced to express overnight. Centrifuge it at 10000 rpm and 4 °C for 10 min in a refrigerated centrifuge. After centrifugation, the precipitate is retained in the centrifuge tube, and the supernatant yellow bacterial liquid is removed and placed in an empty medium to await the destruction of waste bacteria. Continue centrifugation until 1 L of bacterial liquid is completely centrifuged.

[0253] (5)Add PBS to the precipitate retained in the centrifuge tube for reconstitution. Use a disposable pipette to blow and suck the precipitate. Finally, resuspend it at 10000 rpm and 4 °C for 10 min. Retain the precipitate and weigh the weight of the bacterial precipitate on an analytical balance. The net weight of the bacteria can be placed in a -80 °C ultra-low temperature refrigerator. After 1 h, the bacteria can be restored to activity and the bacterial membrane can be lysed with warm water at 37 °C. This can be done multiple times for subsequent protein extraction and purification.

[0254] VII) Purification of MBP-G11-VL and G11-VH-Nluc proteins

[0255] (1)Purification of MBP-G11-VL protein containing MBP tag

[0256] The MBP tag containing maltose-binding protein can increase the solubility of the fusion protein, and the MBP-G11-VL protein can be purified by cross-linked starch affinity chromatography.

[0257] The specific operation using the MBP tag protein purification kit is as follows:

[0258] 1) Centrifuge the bacterial liquid at 10000 rpm and 4 °C for 10 min to collect the induced bacterial precipitate. Wash it in 20 mL of freshly prepared and pre-cooled cell wash buffer per 1 g of bacterial precipitate, centrifuge at 10000 rpm and 4 °C for 15 min, and then resuspend it again in the pre-cooled cell wash buffer.

[0259] 2) Centrifuge at 10000 rpm and 4 °C for 15 min, then collect the bacterial precipitate and resuspend it in freshly prepared and pre-cooled cell lysis buffer containing 20% sucrose for lysis.

[0260] 3) Add 250 μL of 10 mg / mL PMSF, stir at 37 °C for 15 min, then centrifuge at 15000 g and 4 °C for 10 min to collect the bacteria. Spin the bacterial precipitate, add pre-cooled 0.1 mM magnesium chloride solution, and stir at 4 °C for 10 min.

[0261] 4) Centrifuge the shocked cells at 15,000 g for 10 min at 4 °C, collect the supernatant, and filter the supernatant through a 0.22-μm filter membrane;

[0262] 5) After thoroughly mixing the amylose-agarose medium with a pipette tip, add it to an affinity chromatography column pre-placed with a sieve plate. Wash the column 3 times with 5 column volumes of deionized water, and then wash the column once with 5 column volumes of freshly prepared binding buffer for equilibration;

[0263] 6) Add the sample to the equilibrated amylose-agarose medium and incubate with shaking overnight at 4 °C;

[0264] 7) Add the binding buffer for washing to remove non-specifically adsorbed miscellaneous proteins until the absorbance value of the liquid is less than 0.02;

[0265] 8) Add freshly prepared 0.1 M maltose elution solution for elution to elute the bound fusion protein. Collect the eluate, which is the purified protein, and store it at -20 °C.

[0266] (2) Purification of His-tagged G11-VH-Nluc protein

[0267] The His-tagged G11-VH-Nluc protein was purified by nickel column affinity chromatography after being lysed with B-per bacteria and stored at -20 °C.

[0268] The purification procedure of G11-VH-Nluc fusion protein is as follows:

[0269] 1) Take an appropriate amount of evenly mixed 50% His-tag reducible chelating Ni-NTA purification packing material and 50% PBS. Add one column volume of PBS to it, mix well, centrifuge at 1000 g for 10 s at 4 °C, discard the liquid for equilibration, and repeat the equilibration 1 - 2 times;

[0270] 2) Use a 10-mL syringe and a 0.45-μm filter membrane to filter the collected soluble bacterial protein supernatant through the filter membrane, and collect the bacterial lysate CL;

[0271] 3) Add the soluble bacterial protein lysate to the purification packing material and mix, and slowly shake overnight in a shaker at 4 °C;

[0272] 4) Vertically fix the empty affinity chromatography column, load the mixture shaken overnight into the empty affinity chromatography column, and let the liquid in the column flow out under gravity. Repeat the loading 3 - 5 times, and collect the loading flow-through FT;

[0273] 5) Add 2 column volumes of non-denaturing washing solution to wash the column. The washing solution is PBS containing 2 mM imidazole. Wash the miscellaneous proteins with the washing solution until the absorbance value of the liquid is less than 0.02;

[0274] 6) Add 1 column volume of non-denaturing eluent for elution, the eluent is PBS containing 50 mM imidazole, and all the measurable liquid absorbance values ​​of the eluent are collected. The eluent is the purified G11-VH-Nluc fusion protein (nucleotide sequence is shown in SEQ ID NO.25);

[0275] 7) The purified eluate was concentrated by ultrafiltration at 6000 rpm and 4°C for 15 min to concentrate the fusion protein and stored at -20°C.

[0276] (3) Protein identification

[0277] The MBP-G11-VL and G11-VH-Nluc fusion proteins were purified, and the protein concentration was first determined using a BCA protein quantification kit, and then the relative molecular weight was determined by gel electrophoresis and Coomassie brilliant blue staining using an SDS-PAGE kit.

[0278] The BCA protein quantification method for the concentration determination of MBP-G11-VL and G11-VH-Nluc proteins is as follows:

[0279] 1) Prepare BCA working solution A and BCA working solution B, with the ratio of A to B being 50:1;

[0280] 2) Using 2 mg / mL bovine serum albumin standard BSA Standard Solution, 100 μL of the standard was added to 100 μL of PBS for dilution, and then diluted in sequence to make the final concentration of the standard 2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL;

[0281] 3) Add 25 μL of standard and purified fusion protein samples (MBP-G11-VL or G11-VH-Nluc protein) and 200 μL of BCA working solution to each well of the ELISA plate, and incubate in a water bath at 37°C in the dark for 30 min;

[0282] 4) Immediately after incubation, measure the absorbance OD at 562 nm using a multifunctional microplate reader. 562 The detection was completed within 3-5 minutes, and the protein concentration of the purified fusion protein sample was calculated by comparing with the standard.

[0283] (2) Use SDS-PAGE kit for gel electrophoresis to determine the molecular weight and purity of G11-scFv-Nluc fusion protein.

[0284] The SDS-PAGE gel electrophoresis of GMBP-G11-VL and G11-VH-Nluc fusion proteins was performed as follows:

[0285] 1) Sample preparation: Select purified sample bacterial lysate CL, loading flow-through FT, washing solution W1, and elution solution E1. Add the sample, dilute it with 1× Loading Buffer, mix well, and incubate in a 95°C metal bath for 10 min. Then place it on ice for later use.

[0286] 2) Prepare acrylamide PAGE gel: Prepare two 1.0 mm glass plates for gel electrophoresis, clamp them with an electrophoresis clamp, place them on a gel-making stand and fix them. Add pure water to fill the glass plates for 10 min to check for leaks. After the glass plates are successfully leak-free, pour out the pure water and blot dry with filter paper.

[0287] 3) Prepare 10 mL of 10% separating gel: Prepare the separating gel mixture according to the ratio. Add 4160 μL of pure water, 2500 μL of Separating Buffer, 3340 μL of 30% acrylamide - bisacrylamide (29:1), and 100 μL of 10% ammonium persulfate APS solution, mix well. Then, in a fume hood, add 4 μL of N,N,N',N'-tetramethylethylenediamine TEMED, mix well, and gently add it to the comb line of the glass plate with a pipette tip. At the same time, fill the glass plate with methanol MeOH to form a horizontal liquid seal.

[0288] 4) Let it stand at room temperature for 40 min until the gel is completely stratified. Pour out the methanol for the horizontal liquid seal and blot dry with filter paper.

[0289] 5) Prepare 4 mL of 5% stacking gel: Prepare the stacking gel mixture according to the ratio. Add 2280 μL of pure water, 1000 μL of Stacking Buffer, 680 μL of 30% acrylamide - bisacrylamide (29:1), and 40 μL of 10% ammonium persulfate APS solution, mix well. Then, in a fume hood, add 4 μL of N,N,N',N'-tetramethylethylenediamine TEMED, mix well, and gently add it to the glass plate with a pipette tip. At the same time, press a 1.0 mm comb on the glass plate, taking care not to generate bubbles under the comb.

[0290] 6) Let it stand at room temperature for 20 min until the gel is formed. Place the glass plate in the inner liquid of the electrophoresis tank. Add the freshly prepared 1× electrophoresis buffer to both the inner and outer liquids of the electrophoresis tank, making the amount of the inner liquid higher than that of the outer liquid. Remove the comb from the glass plate.

[0291] 7) Loading samples: Slowly add the prepared samples and the 10 - 180 kDa colored prestained protein marker to the loading wells on the glass plate one by one, align the positive and negative electrodes, and cover the lid of the electrophoresis tank.

[0292] 8) After connecting the electrophoresis tank to the electrophoresis apparatus, first run the stacking gel at 60 mV for 35 min, and then run the separating gel at 120 mV for 45 min. After the electrophoresis is completed, take out the glass plate, and under the action of water flow, separate the gel from the glass plate and place it on a glassware;

[0293] 9) Pour Coomassie Brilliant Blue staining solution onto the sodium dodecyl sulfate-polyacrylamide SDS-PAGE gel, place it on a shaker at room temperature and stain for 25 min at 120 rpm, then discard the staining solution;

[0294] 10) Prepare a decolorizing solution with a ratio of pure water: methanol: acetic acid of 5:4:1. Pour the decolorizing solution onto the gel, wait for 30 min and then discard the decolorizing solution. Repeat several times by adding a new decolorizing solution until the background of the SDS-PAGE gel is reduced.

[0295] Example 2

[0296] This example provides a fusion protein with dual functional activities, which is composed of a light chain variable region fusion protein MBP-G11-RL-VL containing a rigid linker peptide and a heavy chain variable region G11-VH-Nluc fusion protein.

[0297] Its preparation method and parameters are basically the same as those in Example 1, and the differences are as follows:

[0298] In step (2) of the PRC amplification process in the preparation process of amplifying the G11-VL gene and the G11-VH gene in step iv): Replace the MBP-VL-F upstream primer (nucleotide sequence as shown in SEQ ID NO.6) with the MBP-RL-F upstream primer (nucleotide sequence as shown in SEQ ID NO.26); Use the MBP-VL-R downstream primer (nucleotide sequence as shown in SEQ ID NO.7) to amplify the light chain variable region G11-RL-VL gene containing a rigid linker peptide to obtain the amplification product of the light chain variable region MBP-G11-RL-VL gene.

[0299] In step v), using the PCR product MBP-G11-RL-VL of the light chain variable region with a rigid linker peptide and the heavy chain G11-VH gene (nucleotide sequence as shown in SEQ ID NO.14) as templates, the vector pMal-p2E and the light chain variable region gene of MBP-G11-RL-VL can be double digested with Sal I and HindⅢ, and after gel cutting and recovery, they are ligated together with T4 DNA ligase to obtain the ligated pMal-p2E-MBP-G11-RL-VL (nucleotide sequence as shown in SEQ ID NO.27).

[0300] In step (vi), the prokaryotic expression of MBP-G11-RL-VL and G11-VH-Nluc proteins involves transferring the expression vector pMal-p2E-MBP-G11-RL-VL containing the maltose (MBP) binding protein and the light chain of the antibody, and the expression vector pET22b(+)-G11-VH-Nluc containing NanoLuc luciferase and the heavy chain of the antibody into the competent cell BL21(DE3)pLysS respectively. The positive monoclonal colonies are sent for sequencing verification. The sequencing primer for the expression vector containing the light chain of the antibody is malefor (nucleotide sequence as shown in SEQ ID NO.28), and the sequencing primer for the expression vector containing the heavy chain of the antibody is T7 (nucleotide sequence as shown in SEQ ID NO.24).

[0301] Example 3

[0302] This example provides a bifunctional active fusion protein, which consists of a light chain variable region fusion protein MBP-G11-FL-VL containing a flexible linker peptide and a heavy chain variable region G11-VH-Nluc fusion protein.

[0303] Its preparation method and parameters are basically the same as those in Example 1, except that:

[0304] In step (iv), in the step (2) PCR amplification process of the preparation process of amplifying G11-VL gene and G11-VH gene:

[0305] Replace the MBP-VL-F upstream primer (nucleotide sequence as shown in SEQ ID NO.6) with the MBP-FL-F upstream primer (nucleotide sequence as shown in SEQ ID NO.29); use the MBP-VL-R downstream primer (nucleotide sequence as shown in SEQ ID NO.7) to amplify the light chain variable region G11-FL-VL gene containing the flexible linker peptide to obtain the amplification product of the light chain variable region MBP-G11-FL-VL gene.

[0306] In step (v), using the PCR product MBP-G11-FL-VL of the light chain variable region containing the flexible linker peptide and the heavy chain and G11-VH gene in step (iv) as templates, the vector pMal-p2E and the light chain variable region gene G11-FL-VL containing the flexible linker peptide can be double digested with Sal I and HindⅢ, and after gel extraction and recovery, they are ligated together with T4 DNA ligase to obtain the ligated pMal-p2E-MBP-G11-FL-VL (nucleotide sequence as shown in SEQ ID NO.30).

[0307] In step (vi), the prokaryotic expression of MBP-G11-FL-VL and G11-VH-Nluc proteins involves the expression vector pMal-p2E-MBP-G11-FL-VL containing the maltose (MBP) binding protein and the antibody light chain, and the expression vector

[0308] pET22b(+)-G11-VH-Nluc containing the nanoluciferase and the antibody heavy chain were respectively transformed into the competent cell BL21(DE3)pLysS, and the positive monoclonal colonies were sent for sequencing verification. The sequencing primer for the expression vector containing the antibody light chain was malefor (nucleotide sequence as SEQ ID NO.31), and the sequencing primer for the expression vector containing the antibody heavy chain was T7 (nucleotide sequence as SEQ ID NO.24).

[0309] Experimental Results and Discussion

[0310] (i) Construction of Expression Vectors

[0311] 1) Amplification of Antibody Light Chain Variable Region and Heavy Chain Variable Region Genes

[0312] Figure 1 This is the DNA sequence of the PCR amplification primers and the restriction enzyme site map described in the present invention. Using PCR amplification, the results are as Figure 3 shown. Using the plasmid pET22b(+)-G11-scFv-Nluc as a template, the antibody light chain variable region G11-VL, G11-RL-VL, G11-FL-VL gene fragments of approximately 350 bp were amplified with MBP-VL-F and MBP-VL-R as primers, and the G11-VH gene fragment of approximately 400 bp was amplified with VH-F and VH-R as primers, which was consistent with the expected results.

[0313] (ii) Construction of Antibody Light Chain Variable Region and Heavy Chain Variable Region Expression Vectors

[0314] The plasmid vector pMal-p2E and the antibody light chain recovery products G11-VL, G11-RL-VL, G11-FL-VL genes were double-digested with Sal I enzyme and HindIII enzyme using PCR, and the plasmid vector and the antibody heavy chain recovery product G11-VH gene were double-digested with SfiI enzyme. The double-digestion results are as Figure 4 shown. After gel extraction and recovery, T4 ligation was performed to obtain the ligated expression vectors pMal-p2E-MBP-G11-VL and pET22b(+)-G11-VH-Nluc.

[0315] (iii) Expression and Purification of MBP-G11-VL and G11-VH-Nluc Proteins

[0316] In the SDS-PAGE electrophoresis of the MBP-G11-VL fusion protein, MBP-G11-RL-VL fusion protein, and MBP-G11-FL-VL fusion protein prepared in Examples 1, 2, and 3 and the G11-VH-Nluc fusion protein, a 10% separating gel was used. After electrophoresis at 60 V for 30 min, electrophoresis was carried out at 120 V for 1 hour. Refer to Figure 5 , which includes MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL. Band 1 is the marker, and bands 2 and 3 (E1, E2) are the eluates. The molecular weight of the fusion protein can be obtained from the electrophoresis diagram to be approximately 69 kDa.

[0317] Construction of the "non-competitive" open sandwich immunoassay method in Example 4

[0318] I) Optimization of the coating type and concentration of MBP-G11-VL

[0319] Antibody light chain variable region fusion proteins (MBP-G11-VL, MBP-G11-RL-VL, MBP-G11-FL-VL) with different linker peptides formulated with phosphate buffer PBS and different coating concentrations of 10, 4, 2, and 1 μg / mL were coated, and optimization was carried out through the OS-ELISA experiment.

[0320] II) Establishment of the "non-competitive" OS-ELISA standard curve

[0321] Using the coating type and concentration optimized in step I), the experimental operation for establishing the open sandwich OS-ELISA standard curve is as follows:

[0322] (1) Prepare a 4 μg / mL MBP-G11-FL-VL light chain variable region fusion protein with phosphate coating buffer PBS as the coating solution, and add 100 μL per well to a 96-well white chemiluminescent enzyme-labeled plate strip, and coat overnight at 4 °C for 16 h;

[0323] (2) The next day, discard the coating solution, wash the plate four times with PBST, and tap the white enzyme-labeled plate until there is no obvious liquid in the wells. Add 300 μL per well of 2% skim milk powder formulated with phosphate buffer PBS as the blocking solution, and block at 37 °C in a water bath for 1 h;

[0324] (3) Discard the blocking solution, wash the plate four times with PBST, and tap the white enzyme-labeled plate until there is no obvious liquid in the wells. Add 50 μL of 5 μg / mL G11-VH-Nluc heavy chain variable region fusion protein, and at the same time add 50 μL of a series of gradient-diluted glycocholic acid standards GCA of 1000, 200, 40, 8, 1.6, 0.32, and 0.064 ng / mL, mix well, and incubate at 37 °C in a water bath for 1 h;

[0325] (4) Pour out the solution in the wells, wash the plate four times with PBST, and tap the white ELISA plate until there is no obvious liquid in the wells;

[0326] (5) Dilute the 0.5 mg / mL coelenterazine CTZ-h stock solution 100-fold with phosphate buffer PBS, and add 1×coelenterazine substrate luminescence solution at 100 μL per well, and immediately place it in a multi-functional microplate reader to detect the chemiluminescence value, denoted as RLU.

[0327] (6) Using the concentration of glycocholic acid as the abscissa and the chemiluminescence value as the ordinate, plot the standard curve of the "non-competitive" open sandwich OS-ELISA.

[0328] III) Influence of methanol content on the reaction

[0329] Prepare phosphate buffer PBS containing 5%, 10%, 20% and 40% methanol as the dilution solution of glycocholic acid standard GCA, and use it to determine the influence of methanol content on the sensitivity of the detection method of the established "non-competitive" OS-ELISA standard curve at different methanol concentrations.

[0330] IV) Influence of ionic strength in the reaction system on the reaction

[0331] Using the optimized methanol content in step III), prepare phosphate buffer PBS containing 10% methanol of 0.5×PBS, 1×PBS, 2×PBS and 4×PBS as the dilution solution of glycocholic acid standard GCA, and use it to determine the influence of ionic strength in the reaction system on the sensitivity of the detection method of the established "non-competitive" OS-ELISA standard curve at different ionic strengths.

[0332] V) Influence of pH value in the reaction system on the reaction

[0333] Prepare phosphate buffer PBS with pH values of 6.2, 6.8, 7.4 and 8.0 containing 10% methanol as the dilution solution of glycocholic acid standard GCA, and use it to determine the influence of pH value in the reaction system on the sensitivity of the detection method of the established "non-competitive" OS-ELISA standard curve at different pH values.

[0334] VI) Specificity

[0335] To detect the specificity of the "non-competitive" OS-ELISA method, the specificity of the assay method was analyzed by detecting the cross-reactivity rate. A series of standard solutions with gradient concentrations of 5 cholic acid structural analogs, namely TCA, TUDCA, GUDCA, UDCA, and HDCA, were prepared respectively to replace the glycocholic acid standard GCA to detect the standard curve of the "non-competitive" OS-ELISA, which was used to evaluate the established "non-competitive" OS-ELISA method. The cross-reactivity rate CR(%) = [IC 50 (GCA) / IC 50 (TCA, TUDCA, GUDCA, UDCA, HDCA)]

[0336] 7) Spiked recovery detection of artificial urine samples

[0337] Using artificial urine samples, glycocholic acid standard GCA with final concentrations of 50, 100, and 200 ng / mL was added to the samples respectively. The spiked recovery rate was detected by the established "non-competitive" OS-ELISA addition recovery analysis method, which was used to evaluate the established "non-competitive" open sandwich immunoassay method OS-ELISA.

[0338] Experimental results

[0339] 1. Plot the standard curve of bioluminescence immunoassay: Using origin8.5 software, with RLU as the ordinate and the concentration of glycocholic acid standard solution as the abscissa, a four-parameter curve was fitted to obtain the standard curve of the non-competitive open sandwich immunoassay for glycocholic acid. Refer to Figure 11 . The effects of MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL at their optimal detection concentrations on the sensitivity of the glycocholic acid open sandwich immunoassay method were detected. The best was 4 μg / mL MBP-G11-FL-VL. Refer to Figures 6 - 7 . The effect of methanol organic solvent on the sensitivity of the glycocholic acid open sandwich immunoassay method was detected. The best was 10% MeOH. Refer to Figure 8 . The effect of PBS ionic strength on the sensitivity of the glycocholic acid open sandwich immunoassay method was detected. The best was 1×PBS. Refer to Figure 9 . The effect of the pH value in the reaction system on the sensitivity of the glycocholic acid open sandwich immunoassay method was detected. The best was pH 7.4. Refer to Figure 10 . The effect of the cross-reactivity rate with other cholic acids on the sensitivity of the open sandwich immunoassay method was detected. The cross-reactivity rate of other cholic acids except glycocholic acid was low. Refer to Figure 8 . The application of spiked recovery detection for the content of 50, 100, and 200 ng / mL glycocholic acid standards in urine was detected. The recovery rate was 99.77 - 104.87%. Refer to Figure 9 .

[0340] Example 5

[0341] A bioluminescence immunoassay kit for detecting glycocholic acid, comprising the fusion protein with dual functional activities of MBP-G11-VL and G11-VH-Nluc provided in Examples 1-3, and glycocholic acid. Specifically, the MBP-G11-VL fusion protein and the G11-VH-Nluc fusion protein are used as coating and detection antibodies, and an open sandwich immunoassay method for bioluminescence is established with glycocholic acid as the standard. For the schematic diagram of the open sandwich immunoassay of the antibody light chain fusion protein and the antibody heavy chain fusion protein, refer to Figure 2 , this method has good sensitivity, the lowest detection limit is 52.076 ng / mL, and the sensitivity is 98 times higher than that of the ordinary enzyme-linked immunosorbent assay method of 5.08 μg / mL. For the "competitive" BLEIA standard curve of the G11-scFv-Nluc fusion antibody for detecting glycocholic acid, refer to Figure 12 , which provides an accurate and highly sensitive detection method for the rapid detection of glycocholic acid in urine.

[0342] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A fusion protein with dual functional activities, characterized in that, Including binding the light chain variable region of the anti-glycocholic acid single-chain antibody to maltose and cloning it into an expression vector, and purifying the obtained light chain variable region fusion protein; and using nano-luciferase as a catalyst for bioluminescence, fusing the heavy chain variable region G11-VH of the anti-glycocholic acid single-chain antibody with nano-luciferase and cloning it into an expression vector, and purifying the obtained G11-VH-Nluc fusion protein; The light chain variable region of the anti-glycocholic acid single-chain antibody is one of G11-VL or the light chain variable region G11-RL-VL containing a rigid linker peptide or the light chain variable region G11-FL-VL containing a flexible linker peptide.

2. The fusion protein with dual functional activity according to claim 1, wherein, The light chain variable region is G11-VL.

3. The bifunctional active fusion protein according to claim 1, characterized in that, The expression vector is the pET22b expression vector.

4. The bifunctional active fusion protein according to claim 1, characterized in that, The light chain variable region fusion protein is the MBP-G11-VL or MBP-G11-RL-VL or MBP-G11-FL-VL fusion protein; the heavy chain variable region G11-VH-Nluc fusion protein.

5. A method for constructing a fusion protein with dual functional activities, characterized in that, Successively includes the following steps: (1) Amplification of the target gene Extracting the plasmid pET22b(+)-G11-scFv-Nluc of the nano-luciferase fusion single-chain antibody G11-scFv-Nluc gene by a plasmid DNA extraction kit as a template; Performing PCR amplification on the antibody light chain variable region G11-VL gene with the MBP-VL-F upstream primer and the MBP-VL-R downstream primer to obtain the PCR product fusion gene MBP-G11-VL; Performing PCR amplification on the light chain variable region G11-RL-VL containing a rigid linker peptide with the RL-F upstream primer and the MBP-VL-R downstream primer to obtain the PCR product fusion gene MBP-G11-RL-VL; Performing PCR amplification on the light chain variable region G11-FL-VL containing a flexible linker peptide with the FL-F upstream primer and the MBP-VL-R downstream primer to obtain the PCR product fusion gene MBP-G11-FL-VL; Performing PCR amplification on the heavy chain variable region G11-VH gene of the antibody with the FL-F upstream primer and the MBP-VL-R downstream primer to obtain the PCR product fusion gene G11-VH-Nluc; (2) Construction of the expression vector Using the PCR product fusion gene MBP-G11-VL prepared in step (1) as a template, double-digesting the vector pMal-p2E and the light chain variable region gene of the fusion gene MBP-G11-VL with Sal I and Hind III, cutting and recovering the product, and performing T4 DNA ligation to obtain the ligated pMal-p2E(+)-MBP-G11-VL expression vector; Using the PCR product fusion gene MBP-G11-RL-VL prepared in step (1) as a template, double-digesting the vector pMal-p2E and the light chain variable region gene of the fusion gene MBP-G11-RL-VL with SalI and HindIII, cutting and recovering the product, and performing T4 DNA ligation to obtain the ligated pMal-p2E(+)-MBP-G11-RL-VL expression vector; Using the fusion gene MBP-G11-FL-VL of the PCR product prepared in step (1) as a template, the vector pMal-p2E and the light chain variable region gene of the fusion gene MBP-G11-FL-VL were double digested with SalI and HindIIII, the product was recovered by gel cutting, and the ligated pMal-p2E(+)-MBP-G11-FL-VL expression vector was obtained through T4 DNA ligation; Using the fusion gene G11-VH-Nluc of the PCR product prepared in step (1) as a template, the vector pET22b(+)-G11-scFv-Nluc and the heavy chain variable region of G11-VH-Nluc were digested with SfiI, the product was recovered by gel cutting, and the ligated pET22b(+)-G11-VH-Nluc expression vector was obtained through T4 DNA ligation. (3) Preparation of fusion protein The expression vectors of the antibody light chain, Mal-p2E(+)-MBP-G11-VL, the expression vector containing the rigid linker peptide pMal-p2E(+)-MBP-G11-RL-VL, the expression vector containing the flexible linker peptide pMal-p2E(+)-MBP-G11-FL-VL, and the expression vector containing the nano luciferase and the antibody heavy chain pET22b(+)-G11-scFv-Nluc were respectively transformed into the competent cell BL21 by heat shock transformation, and the positive monoclonal colonies were sent for sequencing verification. The sequencing primers for the expression vectors containing the light chain proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL were malefor, and the sequencing primer for the expression vector containing the antibody heavy chain was T7; For the positive monoclonal bacteria of the light chain-containing proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL and the heavy chain-containing protein G11-VH-Nluc with correct sequencing, the positive monoclonal bacteria were respectively expanded in culture, and then the IPTG inducer was added for induction expression and purification to obtain the MBP-G11-VL, MBP-G11-RL-VL, MBP-G11-FL-VL, and G11-VH-Nluc fusion proteins.

6. The method for constructing the fusion protein with dual functional activity according to claim 5, wherein For the purification method of the MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL proteins after induction expression: First, the MBP-tagged bacterial lysate was used to induce and extract the expressed MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL proteins respectively, and then the target proteins MBP-G11-VL, MBP-G11-RL-VL, and MBP-G11-FL-VL were purified by an amylose-agarose medium affinity chromatography column. Finally, SDS-PAGE electrophoresis was used to determine the molecular weight and purity of the proteins; The purification method of the G11-VH-Nluc protein after induced expression is to extract the G11-VH-Nluc protein after induced expression using B-per bacterial lysis solution, then purify the target protein G11-VH-Nluc fusion protein through a nickel affinity chromatography column, and finally use SDS-PAGE electrophoresis to determine the molecular weight and purity of the protein.

7. The method for constructing a fusion protein with dual functional activities according to claim 5, characterized in that, The plasmid pET22b(+)-G11-scFv-Nluc of the nanoluciferase fusion single-chain antibody G11-scFv-Nluc gene is prepared by the following steps: 1) Amplify the single-chain antibody G11-scFv gene against glycocholic acid using SNF1 and SNR1 as primers; Amplify the nanoluciferase Nluc gene using SNF2 and SNR2 as primers; 2) Using the PCR products G11-scFv gene and Nluc gene in step 1) as templates respectively, and SNF1 and SNR2 as primers, amplify the G11-scFv-Nluc recombinant gene of the single-chain antibody against glycocholic acid fused with nanoluciferase by overlapping extension PCR technology; 3) Double-digest the G11-scFv-Nluc recombinant gene and the vector pET-22b(+) with XhoI and EcoRI, and ligate them using T4 ligase to obtain the pET22b(+)-G11-scFv-Nluc expression vector.

8. Application of the bifunctional active fusion protein described in claim 1 as an antibody for detecting glycocholic acid in urine.

9. A bioluminescence immunoassay kit for detecting glycocholic acid, characterized in that, Including the bifunctional active fusion protein of claim 1.

10. A method for detecting glycocholic acid in urine, characterized in that, Using the bifunctional active fusion protein described in claim 1 as an antibody for detecting glycocholic acid in urine, and using an open sandwich immunoassay method.

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