CN110563804A - Polypeptide sequence for screening and identifying ofloxacin single-chain antibody based on natural immune bank and application - Google Patents

Abstract

The invention relates to a polypeptide sequence for screening and identifying ofloxacin single-chain antibody based on natural immune library and application thereof, belonging to the field of antigen detection of antibacterial drugs. The invention searches a polypeptide ligand with the best combination mode and affinity with a target protein in a virtual polypeptide library by a molecular docking and virtual screening technology on the basis of the crystal structure of the ofloxacin single-chain antibody, wherein the polypeptide sequence is FNWDFAY and YGYPH. The ELISA binding test result shows that the ofloxacin single-chain antibody can have good binding capacity with the corresponding target antigen, and the polypeptide designed by the invention can be used for carrying out quantitative and qualitative rapid detection on the ofloxacin antigen.

Description

Screening and recognition of the polypeptide sequence of ofloxacin single-chain antibody based on natural immune library application

technical field

The invention relates to a polypeptide sequence of ofloxacin single-chain antibody and application thereof, belonging to the field of antigen detection of antibacterial drugs.

Background technique

Using phage display technology to screen specific single-chain antibodies of fluoroquinolone antibacterial drugs, antibodies can be obtained without relying on cell fusion and animal immunization, and the inhibitory effect can be identified by ELISA after expression and purification, and the test period is short. By linking with the NCBI DNA database on the Internet, SWISS-MODEL software was used to model the homology of the scFv gene sequence and analyze its structure.

The virtual screening technology based on molecular docking is an emerging technical means to study the interaction between peptides and proteins. This technology mainly uses fast computer calculations to realize the docking of peptides and corresponding target proteins in spatial conformation, and docks the molecules in the virtual peptide database with the specific active sites of the target protein crystal structure one by one. The position, conformation, dihedral angle of the rotatable bond inside the molecule and the amino acid residue side chain and skeleton of the target protein are used to find the best conformation of the polypeptide molecule and the target protein in terms of spatial structure, and predict both It is a method of theoretically simulating intermolecular interactions to select the polypeptide ligand with the best affinity with the target protein that is close to the natural conformation based on the binding mode and affinity between them.

Ofloxacin is a class of chemically synthesized third-generation quinolone antibacterial drugs. It is a small molecule compound that belongs to a hapten. After coupling it with a protein carrier, it is prepared into a complete antigen, which means that the small molecule must After the carrier protein is coupled, the body can be stimulated to produce specific antibodies, and it is coated on a solid phase carrier for ELISA detection. The drug has the characteristics of broad antibacterial spectrum, strong antibacterial power, rapid action, complete absorption, low toxicity and resistance to drug resistance; with the wide application of ofloxacin, the residues in animal-derived foods will have harmful effects on the human body. It has caused great harm to health and has attracted widespread attention. It is necessary to further strengthen its monitoring.

Contents of the invention

With the help of molecular docking and virtual screening technology, on the basis of the crystal structure of ofloxacin single-chain antibody, the present invention searches for the polypeptide ligand with the best binding mode and affinity with the target protein in the virtual polypeptide library through the molecular docking technology. The polypeptide sequences are FNWDFAY and YGYPH. The results of the ELISA binding test show that the ofloxacin single-chain antibody can have a good binding ability with the corresponding target antigen, thus proving that the polypeptide designed by the present invention can be used for quantitative and qualitative analysis of the ofloxacin antigen rapid detection.

In order to achieve the above object, the technical solution adopted in the present invention is:

The polypeptide sequences that recognize the ofloxacin single-chain antibody based on natural immune library screening are SEQ ID NO.1: FNWDFAY and SEQ ID NO.2: YGYPH.

The polypeptide sequence includes the polypeptide sequence as the core, any corresponding adjustments or modifications to the polypeptide sequence; modified materials include but are not limited to nanomaterials, fluorescent materials, enzymes, biotin and specific protein.

The application of the polypeptide sequence in the identification of ofloxacin antigen.

The application of the polypeptide sequence in the rapid detection of ofloxacin antigen.

The rapid detection includes but not limited to enzyme-linked immunosorbent assay (ELISA) detection.

The application of the polypeptide sequence in quantitative and qualitative detection of ofloxacin antigen.

Beneficial effects of the present invention:

1. The present invention uses molecular docking and virtual screening technology, on the basis of the structure of ofloxacin single-chain antibody, to search for the polypeptide ligand with the best binding mode and affinity with the target protein in the virtual polypeptide library through molecular docking technology, Finally, the polypeptide sequences specifically binding to the ofloxacin antigen were obtained, and the polypeptide sequences were FNWDFAY (SEQ ID NO.1) and YGYPH (SEQ ID NO.2). After affinity identification with ofloxacin antigen, the affinity is good.

2. Due to the limitations of purification technology, it is difficult to obtain antibodies against ofloxacin. The sequence designed by the present invention avoids this problem well, and can realize rapid artificial synthesis and low detection cost.

3. The sequence specificity designed by the present invention is better.

4. The present invention can provide better theoretical guidance for the analysis of the structure and function of the ofloxacin single-chain antibody through computer-assisted molecular docking.

5. The present invention can realize rapid qualitative and quantitative detection of ofloxacin antigen by marking the screening sequence; it has the advantages of simple operation, saving time and effort, and low cost.

Description of drawings

Fig. 1 is the amplification agarose gel electrophoresis result of heavy chain (VH), light chain (VL) gene;

In the figure, M refers to Marker, VH refers to the heavy chain gene fragment, the size is about 340bp, and VL1, VL2, VL3, VL4 refers to the light chain gene fragment, the size is about 320bp.

Fig. 2 is the expression result of ofloxacin OFX in escherichia coli;

The figure shows that after sonication for protein expression, there is a certain amount of expression in both the supernatant and the precipitate, and the expression level in the precipitate is slightly higher than that in the supernatant.

Figure 3 shows the best template (upper panel) and modeling results (lower panel) for OFX-scFv homology modeling.

Detailed ways

Example 1: Using the pCANTAB5e system to display recombinant antibody library construction and panning

1. Extraction of total RNA from splenocytes

1. The mice (unimmunized BALB/c mice) were killed by cervical dislocation, and the mice were soaked in 75% (V/V) alcohol for 10 minutes. Put it into the ultra-clean workbench, open the abdomen with sterilized scissors, and take out the spleen with tweezers. Put the spleen into a plate containing 10 mL of sterilized 0.02M PBS buffer, peel off the surrounding fat tissue, wash the outer surface, and then put it into a plate containing 10 mL of sterilized 0.02M PBS buffer for rinsing once. Thoroughly wash away the free fat cells on the surface of the spleen.

2. Place the spleen on a sterilized nylon mesh, cut it into pieces, wash it with 10mL of sterilized 0.15M PBS buffer solution, and grind it with scissors while washing. Add 10mL of sterilized 0.15M PBS buffer according to the remaining amount of the tissue, so that all the splenocytes flow into the small beaker through the mesh.

3. Remove the mesh bag, pipette the splenocyte suspension evenly in the plate, and transfer it to a 50mL centrifuge tube.

4. Centrifuge at 1000 rpm at 4°C for 10 minutes, discard the supernatant.

5. Resuspend the pelleted cells with 10 mL of sterilized 0.15M PBS buffer and wash once by centrifugation. Discard the supernatant and leave a small amount of cell suspension at the bottom of the centrifuge tube.

6. After the splenocytes were separated, RNA was extracted by TRIzol method, and the product was detected by ultraviolet spectrophotometer. It shows that the purity of the extracted RNA is high.

2. Amplification and identification of mouse light chain (VL) and heavy chain (VH) genes

Using the obtained total RNA as a template, the first strand of cDNA was synthesized by reverse transcription, and the complete set of antibody genes was amplified using VL and VH primers. The reaction conditions were as follows: pre-denaturation at 94°C for 5min, 45s at 94°C, 1min at 58°C, 45s at 72°C for a total of 30 cycles, and extension at 72°C for 10min. After the reaction product was identified by agarose gel electrophoresis, the target fragment was recovered. The results are shown in Figure 1. The primers used are listed in Table 1.

Table 1. Primer sequences and their significance

Note: The underlined part in VH for in the table is the Sfi I restriction site, the underlined part in VL back is the Not I restriction site; the underlined part in VH back and VL for is the (Gly4Ser) ₃ sequence; the degenerate base in the sequence The basic symbols are W=A/T; S=G/C; M=A/C; R=A/G.

3. Assembly of full-length single-chain antibody (scFv) gene

Using the gene sequence encoding the flexible peptide (Gly4Ser) ₃ as a linker, the VL and VH genes were assembled into a full-length scFv gene containing enzyme cleavage sites by overlapping extension PCR technology. The PCR program is as follows: first add equimolar VL and VH genes to the conventional PCR reaction system (50 μL), pre-denature at 94°C for 5 min, 94°C for 45 s, 68°C for 1 min, and 72°C for 45 s for a total of 10 cycles, and extend at 72°C for 10 min ; Using this product as a template, a second PCR was performed with primers scFv for and scFv back, pre-denaturation at 94°C for 5 min, 94°C for 45 s, 60°C for 1 min, and 72°C for 45 s for a total of 30 cycles, and finally 72°C for 10 min. After the reaction product was identified by agarose gel electrophoresis, the target fragment was recovered.

4. Construction and identification of phage single-chain antibody surface display library

The scFv gene and the pCANTAB5E phagemid vector were digested with SfiI and NotI and ligated, transformed into E.coli TG1 competent cells and plated, and counted colonies after overnight culture at 37°C. The colony count was 5.2×10 ⁵ (ie is the primary library capacity). Randomly pick a single colony for PCR identification, BstNI enzyme digestion identification, and extract phagemids for EcoRI and HindIII double enzyme digestion identification; expand the remaining transformed bacterial liquid, add M13KO7 helper phage to infect for 30 minutes at 37°C, and centrifuge to precipitate Bacterial cells were resuspended in 2×YT-AK medium, shaken and cultured overnight at 37°C, centrifuged to take the supernatant, added to PEG/Nacl ice bath for 1 hour, centrifuged and resuspended, and filtered through a 0.45 μm filter membrane to obtain Primary phage antibody library.

5. Affinity enrichment and immune screening of phage single-chain antibody library

The obtained primary phage antibody library was added to an OFX-BSA-coated 96-well plate, incubated at 37°C for 1 h, washed, and the phage adsorbed to the antigen was eluted with 100 mmol/L triethylamine, and then 1 mol/L Tris ( pH8.5) for neutralization, infect E.coli TG1 in the logarithmic growth phase, collect the bacterial cells after culture, add M13KO7 helper phage again for infection, repeat the above enrichment procedure 3 to 4 times, and pick a single colony Expand the culture and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.

Example 2. ELISA identification of the titer and inhibition of sequence and ofloxacin

(1) ELISA identification of the potency of ofloxacin

1. The ofloxacin antigen was coated with 1 μg/mL (protein amount) on the microplate; the PBS buffer was coated on the microplate as a control. Among them, the coated antigen was diluted with carbonate (CBS) buffer, 50 μL per well was added to a 96-well microtiter plate, placed at 4°C overnight, washed 5 times with phosphate Tween PBST buffer, and then Block with 1% bovine serum albumin (BSA) solution.

2. Dilute the artificially expressed and purified ofloxacin single-chain antibody with PBS buffer (pH 7.4), add it to the above microtiter plate at a volume of 50 μL per well, mix well, and place at 37°C Under conditions, incubate for 30 min in the dark.

3. Wash 5 times with PBST buffer, and dry the liquid in the wells of the microtiter plate; use PBST buffer to dilute the avidin coupled with horseradish peroxidase 1000 times, and add the spin-off at a volume of 50 μL per well. In a dry microtiter plate, after mixing, place it at 37°C and incubate for 30min in the dark.

4. According to the amount required for the test, add the TMB chromogenic solution to the above microtiter plate at a volume of 100 μL per well, mix well for 30 seconds, and then develop the color for 10 minutes at room temperature.

5. Add 2M sulfuric acid solution stop solution to the above microplate at a volume of 50 μL per well, mix well for 30 seconds, and read the absorbance value of each well at 450 nm on the microplate instrument, when the P/N value When (Positive/Negative) is greater than 2.1, the judgment result is positive. The measurement results are shown in Table 2 below.

Dilution factor 1 2 4 8 16 32 64 128 256 512 1024 Negative+PBS Antibody OD value 2.149 1.848 1.523 1.421 1.205 0.873 0.579 0.397 0.125 0.076 0.038 0.039 OD value of cell supernatant 2.158 1.976 1.639 1.487 1.317 0.979 0.604 0.384 0.173 0.084 0.053 0.045

The results showed that the OD value measured by the combination of the ofloxacin single-chain antibody and the antigen was equivalent to the affinity of the cell supernatant, indicating that the ofloxacin single-chain antibody had a high titer and a good affinity.

(2) ELISA identification of inhibition of ofloxacin

1. The ofloxacin antigen was coated with 1 μg/mL (protein amount) on the microplate; the PBS buffer was coated on the microplate as a control. Among them, the coated antigen was diluted with carbonate (CBS) buffer, 50 μL per well was added to a 96-well microtiter plate, placed at 4°C overnight, washed 5 times with PBST buffer, and then used with a mass fraction of 1 % BSA solution for blocking.

2. Dilute the standard substance of ofloxacin to a concentration of 500 ng/μL with PBS buffer (pH 7.4), add it to the microtiter plate at a volume of 50 μL per well, and then serially dilute to 31.25 ng/μL, The artificially expressed and purified ofloxacin single-chain antibody was diluted to a concentration of 500 ng/μL with PBS buffer (pH 7.4), added to the microtiter plate at a volume of 50 μL per well and mixed with the standard. Set the positive control well without adding the standard and only adding 100 μL ofloxacin single-chain antibody. Place the microtiter plate at 37°C and incubate for 30 min in the dark.

3. Wash 5 times with PBST buffer, and dry the liquid in the wells of the microtiter plate; use PBST buffer to dilute the avidin coupled with horseradish peroxidase 1000 times, and add the spin-off at a volume of 50 μL per well. In a dry microtiter plate, after mixing, place it at 37°C and incubate for 30min in the dark.

4. According to the amount required for the test, add the TMB chromogenic solution to the above microtiter plate at a volume of 100 μL per well, mix well for 30 seconds, and then develop the color for 10 minutes at room temperature.

5. Add 2M sulfuric acid stop solution to the above microplate at a volume of 50 μL per well, mix well for 30 seconds, and read the absorbance value of each well at 450 nm on the microplate instrument to determine the result. The measurement results are shown in Table 3 below.

Standard Concentration 500ng/μL 250ng/μL 125ng/μL 62.5ng/μL 31.25ng/μL 0 OD value 0.053 0.057 0.226 0.497 0.506 1.119

The half-inhibitory concentration IC50 was 31.25ng/μL, indicating that the ofloxacin single-chain antibody had better specificity.

Example 3. Ofloxacin single chain antibody is applied to the detection of ofloxacin drug residues

The commonly used detection methods of ofloxacin residues in the world mainly include high performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Although these methods are sensitive and accurate, they are time-consuming, costly, and require high personnel quality. They are not suitable for the determination of large quantities of samples, and are not suitable for fast and simple market monitoring requirements. Enzyme-linked immunosorbent assay (ELISA) has the characteristics of sensitivity, specificity, simplicity, speed, stability and ease of automatic operation. If it can be applied to the detection of ofloxacin residues, it will greatly reduce the cost of detection and shorten the detection time. There have been experiments to prepare the complete antigen of ofloxacin by EDC method, immunize mice with the prepared antigen, and successfully obtain a hybridoma cell line stably secreting anti-ofloxacin monoclonal antibody after cell fusion and cloning, with high The antibody titer is high, the antibody subtype is IgG1, and the antibody has high affinity, which can be used for the detection of fluoroquinolone drug residues.

The invention can obtain antibodies with high affinity without relying on cell fusion or animal immunization, and the detection cost is low, more convenient and efficient.

sequence listing

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Claims (6)

1. Based on the innate immune library to screen the polypeptide sequence that recognizes the ofloxacin single-chain antibody, it is characterized in that the polypeptide sequence is SEQ ID NO.1: FNWDFAY and SEQ ID NO.2: YGYPH. 2. The polypeptide sequence according to claim 1, characterized in that it includes any corresponding adjustments or modifications to the polypeptide sequence with the polypeptide sequence as the core; modified materials include but are not limited to nanomaterials, fluorescent Materials, enzymes, biotin and specific proteins. 3. The application of the polypeptide sequence according to claim 1 in the identification of ofloxacin antigen. 4. The application of the polypeptide sequence according to claim 1 or 2 in the rapid detection of ofloxacin antigen. 5. The application according to claim 4, wherein the rapid detection includes but not limited to enzyme-linked immunosorbent assay (ELISA) detection. 6. The application of the polypeptide sequence according to claim 1 or 2 in the quantitative and qualitative detection of ofloxacin antigen.