CN110563805A - Single-chain antibody key sequence for accurately identifying neomycin and application - Google Patents
Single-chain antibody key sequence for accurately identifying neomycin and application Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a single-chain antibody key sequence for accurately identifying neomycin and application thereof, belonging to the field of antigen detection of antibacterial drugs. The invention searches polypeptide ligands with the best binding mode and affinity with target protein in a virtual polypeptide library by means of molecular docking and virtual screening technologies on the basis of a neomycin single-chain antibody crystal structure and through the molecular docking technology, wherein the polypeptide sequences are LSEDYEDY and VLDSDGK. The ELISA binding test result shows that the neomycin single-chain antibody can have good binding capacity with a corresponding target antigen, and the polypeptide designed by the invention can be used for carrying out quantitative and qualitative rapid detection on the neomycin antigen.
Description
Technical Field
The invention relates to a neomycin single-chain antibody sequence and application thereof, belonging to the field of antibacterial drug antigen detection.
Background
The specific single-chain antibody of the aminoglycoside antibacterial drug is screened by utilizing a phage display technology, the antibody can be obtained without depending on cell fusion and animal immunity, the inhibition effect of ELISA identification after expression and purification is achieved, and the test period is short. Through connecting with NCBI DNA database on Internet, SWISS-MODEL software is used to carry out homologous modeling on scFv gene sequence, and the structure of the scFv gene sequence is analyzed.
a virtual screening technology based on molecular docking is an emerging technical means for researching the interaction between polypeptide and protein. The technology mainly uses computer fast operation to realize the butt joint of polypeptide and corresponding target protein on spatial conformation, butt joints the molecules in a virtual polypeptide database with specific active sites of a target protein crystal structure one by one, searches the optimal conformation of the polypeptide molecules and the target protein on the spatial structure through computer fast operation and continuously adjusts the position and conformation of the combination of the polypeptide and the target protein, the dihedral angle of rotatable bonds in the molecules and the amino acid residue side chain and the skeleton of the target protein, predicts the combination mode and the affinity between the two, and selects a polypeptide ligand which is close to the natural conformation and has the optimal affinity with the target protein through score evaluation.
The neomycin is a chemically synthesized aminoglycoside antibacterial agent, has wide antibacterial spectrum, has good inhibition effect on gram-negative bacteria, gram-positive bacteria, tubercle bacillus, acid-fast bacteria and the like, and is used as a common veterinary drug for inhibiting bacterial gastrointestinal tract infection of livestock and poultry. With the increasingly wide application of neomycin in the domestic and foreign breeding industry, the problem of residues of neomycin in animal-derived foods is increasingly prominent, and further enhanced monitoring is necessary.
Disclosure of Invention
The invention searches polypeptide ligands with the best binding mode and affinity with target protein in a virtual polypeptide library by means of molecular docking and virtual screening technologies on the basis of a neomycin single-chain antibody crystal structure and through the molecular docking technology, wherein the polypeptide sequences are LSEDYEDY and VLDSDGK. The ELISA binding test result shows that the neomycin single-chain antibody can have good binding capacity with the corresponding target antigen, so that the polypeptide designed by the invention can be used for carrying out quantitative and qualitative rapid detection on the neomycin antigen.
In order to achieve the purpose, the invention adopts the technical scheme that:
The key sequence of the single-chain antibody for accurately identifying the neomycin and the application thereof are LSEDYEDY and VLDSDGK.
The single-chain antibody key sequence for accurately identifying neomycin and the application thereof comprise that the polypeptide sequence is taken as a core, and any corresponding adjustment or modification is carried out on the polypeptide sequence; modifying materials include, but are not limited to, nanomaterials, fluorescent materials, enzymes, biotin, and specific proteins.
The single-chain antibody key sequence for accurately identifying neomycin and the application thereof in the identification of neomycin antigens.
The single-chain antibody key sequence is applied to the rapid detection of neomycin antigen.
Such rapid assays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) assays.
The single-chain antibody key sequence is applied to quantitative and qualitative detection of neomycin antigen.
The invention has the beneficial effects that:
1. The invention searches polypeptide ligand with best binding mode and affinity with target protein in virtual polypeptide library by molecule docking and virtual screening technology on the basis of neomycin single-chain antibody structure, and finally obtains polypeptide sequence specifically binding neomycin antigen, wherein the polypeptide sequence is LSEDYEDY (SEQ ID NO.1) and VLDSDGK (SEQ ID NO. 2). Through the identification of the affinity with neomycin antigen, the affinity is better.
2. Due to the limitation of purification technology, the antibody aiming at the neomycin is difficult to obtain, the sequence designed by the invention well avoids the problem, the rapid artificial synthesis is realized, and the detection cost is low.
3. The sequence designed by the invention has better specificity.
4. The invention provides better theoretical guidance for realizing the structural function analysis of the neomycin single-chain antibody by carrying out the molecular docking with the assistance of a computer.
5. The invention can realize qualitative and quantitative rapid detection of the neomycin antigen by marking the screening sequence. Has the advantages of simple operation, time and labor saving, low cost and the like.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of heavy (VH) and light (VL) chain genes;
In the figure, M refers to Marker, VH refers to heavy chain gene segment with the size of about 340bp, VL1, VL2, VL3 and VL4 refer to light chain gene segment with the size of about 320 bp.
FIG. 2 shows the results of the expression of neomycin NEO in E.coli;
The expression amount of the protein in the picture is higher in the sediment after the protein expression is subjected to ultrasonic disruption, the expression amount of the supernatant is less, and the protein is mainly expressed by inclusion bodies.
FIG. 3 shows the best template for homology modeling of NEO-ScFv (upper panel) and the modeling results (lower panel).
Detailed Description
Example 1: construction and panning of recombinant antibody displayed by pCANTAB5e system
First, extraction of total RNA from spleen cell
1. Mice (uninmmunized BALB/c mice) were sacrificed by cervical dislocation, soaked in 75% (V/V) alcohol for 10 minutes, removed, placed on a white board, supine to expose the abdomen, placed in a clean bench, opened with sterile scissors, and the spleen removed with forceps. The spleen was placed in a dish containing 10mL of sterilized 0.02M PBS buffer, the surrounding adipose tissues were peeled off, the outer surface was washed, and then the dish containing 10mL of sterilized 0.02M PBS buffer was rinsed 1 time to completely wash off the free adipocytes on the spleen surface.
2. The spleen was placed on a sterilized nylon mesh, cut into pieces, washed with 10mL of a sterilized 0.15M PBS buffer, and ground with scissors while washing. The spleen cells were all flowed through the mesh into a small beaker by adding 10mL of sterile 0.15M PBS buffer based on the remaining amount of tissue.
3. The mesh bag was removed, the spleen cell suspension in the dish was blown up and transferred to a 50mL centrifuge tube.
4. Centrifuge at 1000rpm for 10 minutes at 4 ℃ and discard the supernatant.
5. The pelleted cells were then washed once again by resuspension in 10mL of sterile 0.15M PBS buffer. The supernatant was discarded and a small amount of cell suspension was left in the bottom of the centrifuge tube.
6. After splenocytes were separated, RNA extraction was performed by TRIzol method, and the product was detected by UV spectrophotometer to have a concentration of 39.33 ng/. mu.L, an OD 260/280 of 1.787, and an OD 260/230 of 2.035, indicating that the extracted RNA was of higher purity.
Amplification and identification of mouse light chain (VL) and heavy chain (VH) genes
The total RNA is used as a template, the first chain of cDNA is synthesized through reverse transcription, and VL and VH primers are used for amplifying a complete set of antibody genes. The reaction conditions are as follows, pre-denaturation at 94 ℃ for 5min, pre-denaturation at 94 ℃ for 45s, pre-denaturation at 58 ℃ for 1min and pre-denaturation at 72 ℃ for 45s, and 30 cycles of pre-denaturation and extension at 72 ℃ for 10 min. And (3) identifying the reaction product by agarose gel electrophoresis, and recovering the target fragment. The results are shown in FIG. 1. The primers used are shown in Table 1.
TABLE 1 primer sequences and meanings
Note: in the table, the underlined position in VH for is Sfi I restriction enzyme site, and the underlined position in VL back is Not I restriction enzyme site; the underlined parts in VHback and VL for are (Gly4Ser)3A sequence; the symbols of the degenerate basic groups in the sequence are W ═ A/T; G/C; m is A/C; r is A/G.
Assembly of three, full-length single chain antibody (scFv) genes
To code for flexible peptides ((Gly4Ser)3) The gene sequence of (a) is a joint, and VL and VH genes are assembled into a gene containing enzyme digestion by an overlap extension PCR technologyFull-length scFv gene of the site.
The PCR procedure was as follows: adding VL and VH genes with equal molar weight into a conventional PCR reaction system (50 mu L), pre-denaturing at 94 ℃ for 5min, 94 ℃ for 45s, 68 ℃ for 1min and 72 ℃ for 45s, carrying out 10 cycles, and extending at 72 ℃ for 10 min; the product is used as a template, secondary PCR is carried out by primers scFv for and scFv back, pre-denaturation is carried out at 94 ℃ for 5min, at 94 ℃ for 45s, at 60 ℃ for 1min and at 72 ℃ for 45s for 30 cycles, and finally extension is carried out at 72 ℃ for 10 min. And (3) identifying the reaction product by agarose gel electrophoresis, and recovering the target fragment.
Construction and identification of phage single-chain antibody surface display library
Carrying out Sfi I and Not I double enzyme digestion and connection on the scFv gene and the pCANTAB5E phagemid vector respectively, transforming E.coliTG1 competent cells and plating, carrying out colony counting after overnight culture at 37 ℃, wherein the colony counting is 5.2 multiplied by 105(i.e., primary library capacity). Randomly selecting a single colony for PCR identification and BstNI enzyme digestion identification, and extracting phagemids for EcoRI and HindIII double enzyme digestion identification; and performing amplification culture on the residual transformed bacterium liquid, adding M13KO7 to assist the bacteriophage to stand at 37 ℃ for 30min for infection, performing centrifugal precipitation on the somatic cells, performing heavy suspension on the somatic cells by using a 2 XYT-AK culture medium, performing shake culture at 37 ℃ overnight, centrifuging to obtain a supernatant, adding PEG/NaCl into an ice bath for 1h, centrifuging, performing heavy suspension precipitation, and filtering by using a 0.45-micrometer filter membrane to obtain the primary bacteriophage antibody library. Affinity enrichment and immune screening of phage single-chain antibody library
Adding the obtained primary phage antibody library into a 96-well plate coated by NEO-BSA, standing and incubating for 1h at 37 ℃, washing, eluting phage adsorbed by antigen by 100mmol/L triethylamine, immediately adding 1mol/L Tris (pH8.5) for neutralization so as to infect E.coli TG1 in logarithmic growth phase, collecting bacterial cells after culture, adding M13KO7 helper phage again for infection, repeating the enrichment procedure for 3-4 times, picking up a single colony for amplification culture, and sending the single colony to a company Limited in the engineering bioengineering (Shanghai) for sequencing.
Example 2 ELISA identification of the Titers and inhibition of sequences and neomycin
(I) ELISA for identifying the potency of neomycin
1. Coating the neomycin antigen by an ELISA plate at 1 mu g/mL (protein amount); PBS buffer was used as a control for microplate coating. Wherein, the coating antigens are diluted by Carbonate (CBS) buffer solution, 50 mu L of each well is added into a 96-well enzyme label plate, the 96-well enzyme label plate is placed at 4 ℃ for overnight, and is washed for 5 times by phosphate Tween PBST buffer solution and then is blocked by bovine serum albumin BSA solution with the mass fraction of 1%.
2. The neomycin single-chain antibody expressed and purified artificially is diluted in PBS buffer (pH 7.4) in a multiple ratio, added to the above enzyme label plate in a volume of 50. mu.L per well, mixed uniformly, placed at 37 ℃, and incubated for 30min in the absence of light.
3. Washing with PBST buffer solution for 5 times, and spin-drying the liquid in the holes of the enzyme-labeled plate; the avidin coupled with the horseradish peroxidase is diluted by 1000 times by using PBST buffer solution, added into a spin-dried enzyme label plate in the volume of 50 mu L per hole, mixed evenly, placed at 37 ℃ and incubated for 30min in a dark place.
4. According to the amount required for the test, TMB developing solution is added to the above enzyme label plate in a volume of 100. mu.L per well, and after thoroughly mixing for 30s, development is performed for 10min at room temperature.
5. Adding 2M sulfuric acid solution stop solution into the enzyme label plate in a volume of 50 mu L per hole, fully mixing for 30s, reading the light absorption value of each hole at 450nm on an enzyme label instrument, and judging the result to be Positive when the P/N value (Positive/Negative) is more than 2.1. The results of the measurements are shown in Table 2 below.
| Dilution factor | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | 512 | 1024 | negative + PBS |
| OD value of antibody | *** | *** | 2.817 | 2.587 | 2.393 | 2.005 | 1.641 | 1.572 | 1.337 | 0.736 | 0.479 | 0.069 |
| OD value of cell supernatant | 1.643 | 1.262 | 1.128 | 1.096 | 0.897 | 0.792 | 0.707 | 0.684 | 0.458 | 0.204 | 0.063 | 0.052 |
The results show that: the OD value measured by combining the neomycin single-chain antibody with the antigen is equivalent to the affinity of cell supernatant, which shows that the neomycin single-chain antibody has high titer and better affinity.
(II) identification of neomycin inhibition by ELISA
1. Coating the neomycin antigen by an ELISA plate at 1 mu g/mL (protein amount); PBS buffer was used as a control for microplate coating. Wherein, the coating antigens are diluted by Carbonate (CBS) buffer solution, 50 mu L of each hole is added into a 96-hole enzyme label plate, the 96-hole enzyme label plate is placed at 4 ℃ for overnight, and the 96-hole enzyme label plate is washed by PBST buffer solution for 5 times and then is blocked by BSA solution with the mass fraction of 1 percent.
2. The standard neomycin drug is diluted to the concentration of 500 ng/. mu.L by PBS buffer solution (pH 7.4), added into the ELISA plate in the volume of 50. mu.L per well, diluted to 15.625 ng/. mu.L in a multiple ratio, the artificially expressed and purified neomycin single-chain antibody is diluted to the concentration of 500 ng/. mu.L by PBS buffer solution (pH 7.4), and added into the ELISA plate in the volume of 50. mu.L per well to be mixed with the standard substance. A positive control well is provided with 100 mu L of neomycin single-chain antibody without adding a standard substance. The microplate was incubated at 37 ℃ for 30min in the absence of light.
3. Washing with PBST buffer solution for 5 times, and spin-drying the liquid in the holes of the enzyme-labeled plate; the avidin coupled with the horseradish peroxidase is diluted by 1000 times by using PBST buffer solution, added into a spin-dried enzyme label plate in the volume of 50 mu L per hole, mixed evenly, placed at 37 ℃ and incubated for 30min in a dark place.
4. According to the amount required for the test, TMB developing solution is added to the above enzyme label plate in a volume of 100. mu.L per well, and after thoroughly mixing for 30s, development is performed for 10min at room temperature.
5. Adding 2M sulfuric acid solution stop solution into the enzyme label plate in a volume of 50 mu L per hole, fully and uniformly mixing for 30s, reading the light absorption value of each hole at 450nm on an enzyme label instrument, and judging the result. The results of the measurements are shown in Table 3 below.
| Concentration of standard substance | 500 | 250 | 125 | 62.5 | 31.25 | 15.625 | 0 |
| OD value | 0.193 | 0.361 | 0.429 | 0.443 | 0.520 | 0.596 | 1.113 |
Wherein the concentration unit of the standard substance is ng/mu L, and the half inhibition concentration IC50 is 31.25 ng/mu L, which indicates that the neomycin single-chain antibody has better specificity.
Example 3 use of neomycin Single chain antibodies
The internationally common detection method for neomycin drug residues mainly comprises a high performance liquid chromatography-mass spectrometry (HPLC-MS) method, a gas chromatography-mass spectrometry (GC-MS) method and the like. Although the methods are sensitive and accurate, the detection time is long, the cost is high, the requirement on the quality of personnel is high, the methods are not suitable for measuring a large number of samples and are not suitable for the rapid and simple market monitoring requirement. Enzyme-linked immunosorbent assay (ELISA) has the characteristics of sensitivity, specificity, simplicity, rapidness, stability, easiness in automatic operation and the like, and if the ELISA can be applied to detection of neomycin residues, the detection cost can be greatly reduced, and the detection time can be shortened. In the prior experiment, complete antigen of neomycin is prepared by an EDC method, a mouse is immunized by the prepared antigen, and a hybridoma cell strain which stably secretes the anti-neomycin monoclonal antibody is successfully obtained after cell fusion and cloning, has higher antibody titer, has an antibody subtype of IgG1 and high antibody affinity, and can be used for detecting residue of aminoglycoside drugs.
the invention can obtain the antibody with high affinity without cell fusion or animal immunization, and has low detection cost, more convenience and high efficiency.
Sequence listing
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<120> single-chain antibody key sequence for accurately identifying neomycin and application thereof
<130> molecular docking and virtual screening techniques
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Claims (6)
1. The single-chain antibody key sequence for accurately identifying neomycin is characterized in that the polypeptide sequence is LSEDYEDY and VLDSDGK.
2. The key sequence of the single-chain antibody for accurately identifying neomycin according to claim 1, which comprises the polypeptide sequence as core, any corresponding modifications or modifications of said polypeptide sequence; modifying materials include, but are not limited to, nanomaterials, fluorescent materials, enzymes, biotin, and specific proteins.
3. The use of the key sequence of a single chain antibody for accurately recognizing neomycin according to claim 1 in the identification of neomycin antigen.
4. Use of the key sequence of the single chain antibody of claim 1 or 2 for the rapid detection of neomycin antigen.
5. The use of claim 4, wherein the rapid assay comprises, but is not limited to, an enzyme-linked immunosorbent assay (ELISA) assay.
6. Use of the key sequence of the single chain antibody of claim 1 or 2 for quantitative and qualitative detection of neomycin antigen.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040031072A1 (en) * | 1999-05-06 | 2004-02-12 | La Rosa Thomas J. | Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement |
| CN103454423A (en) * | 2013-08-03 | 2013-12-18 | 河南百奥生物工程有限公司 | Up-conversion fluorescence immune chromatography test paper for quantitative detection of neomycin and preparation method thereof |
-
2019
- 2019-09-19 CN CN201910886323.8A patent/CN110563805A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040031072A1 (en) * | 1999-05-06 | 2004-02-12 | La Rosa Thomas J. | Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement |
| CN103454423A (en) * | 2013-08-03 | 2013-12-18 | 河南百奥生物工程有限公司 | Up-conversion fluorescence immune chromatography test paper for quantitative detection of neomycin and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| QIAOQIAO SHI等: ""Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for ultrasensitive detection of antibiotics in milk"", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 * |
| TOHRU SHIMIZU等: ""Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater"", 《PNAS》 * |
| 刘宣兵等: ""新霉素单克隆抗体的制备及其免疫学特性鉴定"", 《华北农学报》 * |
| 王方雨等: ""基于分子对接技术的抗新霉素单链抗体制备和进化"", 《安徽农业科学》 * |
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Application publication date: 20191213 |