Anti-trimethoprim single-chain antibody, preparation method and application

Abstract

This invention provides an anti-trimethoprim single-chain antibody for detecting trimethoprim-like drug residues, its preparation method, and its application. The amino acid sequences of the single-chain antibody are shown in SEQ ID3, SEQ ID4, and SEQ ID6. Compared with traditional monoclonal antibodies, the single-chain antibody has lower production costs, is simpler to prepare, and is easier to manipulate at the gene level. This invention utilizes trimethoprim-specific monoclonal hybridoma cells and employs phage display technology to screen for high-affinity single-chain antibodies. A single-chain antibody mutant with even higher affinity was successfully obtained through computer simulation combined with site-directed mutagenesis. Based on this mutant, an ic-ELISA was established for the detection of trimethoprim residues in animal-derived foods, meeting the needs of practical applications.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary drug residue analysis and genetically engineered antibody technology, and particularly relates to an anti-trimethoprim single-chain antibody for detecting trimethoprim drug residues, a preparation method and application thereof. BACKGROUND

[0002] Trimethoprim drugs belong to the class of synthetic antibacterial drugs of diaminopyrimidine. Currently, two commonly used trimethoprim drugs in China are trimethoprim (TMP) and diaveridine (DVD). In livestock production and veterinary clinics, trimethoprim drugs are often used in combination with sulfonamides, which can increase the activity of sulfonamides by several to dozens of times, change the bacteriostatic effect into a bactericidal effect, reduce the emergence of drug-resistant strains, and have a killing effect on gram-positive, gram-negative bacteria and toxoplasma. The combination of the two drugs also expands the antibacterial spectrum of sulfonamides, and has an effect on sulfonamide-resistant Escherichia coli, Proteus, Streptococcus pyogenes, etc. Trimethoprim drugs are often used to treat diseases such as septicemia, chicken white dysentery, chicken infectious rhinitis, fowl typhoid, cholera, and respiratory system secondary bacterial infections caused by Escherichia coli in livestock and poultry. However, trimethoprim drugs can cause bone marrow micronuclear inhibition, have sensitization, cause a decrease in white blood cells and platelets, and diaveridine even has genetic toxicity. Long-term repeated exposure to trimethoprim drugs can cause bacteria in the human body to develop drug resistance, directly affecting human disease treatment. Therefore, the residue of trimethoprim drugs has become an important monitoring object, and China has stipulated that the residue of trimethoprim in animal edible tissues is limited to 50 μg / kg.

[0003] The current analysis of trimethoprim drug residue detection technology shows that there are many methods for detecting the residue, and the methods are developing towards being newer, more precise and more accurate. In summary, instrument detection technology is not suitable for on-site monitoring and large-scale sample screening due to complex instruments and tedious pretreatment processes. Enzyme-linked immunosorbent assay (ELISA) in immunodetection technology has the advantages of high sensitivity, simple operation, low detection cost, and batch on-site detection, which better meets the requirements of actual production. Although the physical composition, construction technology and buffer of ELISA play an important role in optimizing the test, the core of these processes is the antigen and antibody itself, so developing antibodies with better performance has become the main task of current research. The antibodies currently used for trimethoprim immunodetection are monoclonal antibodies or polyclonal antibodies, which must be obtained by immunizing animals and cell culture. The whole production process is complex, time-consuming and expensive, and once prepared, the performance is fixed and not easy to genetically engineer. SUMMARY

[0004] The main purpose of the present application is to provide an anti-methicillin single-chain antibody for methicillin drug residue detection, a preparation method and application, which is suitable for rapid detection of methicillin drug residues in animal-derived food, aiming at solving the problems of complicated preparation, high cost and time-consuming and laborious of existing monoclonal antibodies.

[0005] In order to achieve the above-mentioned purpose, the present application provides an anti-methicillin single-chain antibody for methicillin drug residue detection, which comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4, and the light chain variable region and the heavy chain variable region are connected by a polypeptide from the nitrogen end to the carbon end.

[0006] Single-chain antibody light chain amino acid sequence SEQ ID NO: 3

[0007] ELDILMTQSPLSLPVSLGDQASISCRPSQNIVHFNGNTYLEWYLQKPGQSPKLLIYKVSNRR SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK;

[0008] Single-chain antibody heavy chain amino acid sequence SEQ ID NO: 4

[0009] LEVQLQESGGGLVQPGGSMKISCAASGFTFSDAWMNWVRQSPEKGLDWVAEIRSRTQNHGTY YAESVRGRFTISRDDSKSIVYLQMSSLRAEDTGIYYCTHFDYYGMDYWGQGTSVTVSSAKTTAPSV TS.

[0010] In order to achieve the above-mentioned purpose, the present application further provides a mutant of the anti-methicillin single-chain antibody, and the amino acid sequence of the mutant of the single-chain antibody is shown in SEQ ID NO: 6.

[0011] Single-chain antibody mutant amino acid sequence SEQ ID NO: 6

[0012] ELDILMTQSPLSLPVSLGDQASISCRPSQNIVHFNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKSSGGGGSGGGGGGSSRSSLEVQLQESGGGLVQPGGSMKISCAASGFTFSDAWMNWVRQSPEKGLDWVAEIRSRTQNHGTYYAESVRGRFTISRDDSKSIVYLQMSSLRAEDTGIYYCTHFDYYGMDYWGQGTSVTVSSAKTTAPSVTS.

[0013] To achieve the above object, the present application further provides a gene encoding the anti-methicillin single-chain antibody as described above. The nucleotide sequence of the light chain of the single-chain antibody for detecting methicillin drug residue is shown in SEQ ID: 1, and the nucleotide sequence of the heavy chain of the single-chain antibody is shown in SEQ ID: 2.

[0014] The nucleotide sequence of the light chain of the single-chain antibody is SEQ ID NO: 1

[0015] GAGCTCGATATTCTGATGACCCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGACCTAGCCAGAACATTGTACATTTTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGACGTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA;

[0016] The nucleotide sequence of the heavy chain of the single-chain antibody is SEQ ID NO: 2

[0017] CTCGAGGTACAGCTTCAGGAGTCTGGTGGAGGCTTGGTGCAACCTGGAGGATCCATGAAAATCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGACTGGGTTGCTGAAATTAGAAGCAGAACTCAAAATCATGGAACATACTATGCTGAGTCTGTGAGGGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTATTGTCTACCTGCAAATGAGCAGCTTGAGAGCTGAAGACACTGGCATTTATTACTGTACCCACTTTGATTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACAGCCCCATCTGTCACTAGT.

[0018] To achieve the above object, the present application further provides a gene encoding the mutant of the anti-methicillin single-chain antibody, and the nucleotide sequence of the mutant of the anti-methicillin single-chain antibody is shown as SEQ ID NO: 5.

[0019] The nucleotide sequence of the mutant of the anti-methicillin single-chain antibody is SEQ ID NO: 5

[0020] GAGCTCGATATTCTGATGACCCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGACCTAGCCAGAACATTGTACATTTTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAATCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGGGTGGTTCCTCTAGATCTTCCCTCGAGGTACAGCTTCAGGAGTCTGGTGGAGGCTTGGTGCAACCTGGAGGATCCATGAAAATCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGACTGGGTTGCTGAAATTAGAAGCAGAACTCAAAATCATGGAACATACTATGCTGAGTCTGTGAGGGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTATTGTCTACCTGCAAATGAGCAGCTTGAGAGCTGAAGACACTGGCATTTATTACTGTACCCACTTTGATTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACAGCCCCATCTGTCACTAGT.

[0021] To achieve the above object, the present application also provides an expression vector expressing the anti-methicillin single-chain antibody or the gene encoding the anti-methicillin single-chain antibody as described above.

[0022] To achieve the above object, the present application also provides a method for preparing the anti-methicillin single-chain antibody as described above, comprising the steps of:

[0023] (1) Extract total RNA from a methicillin-specific monoclonal hybridoma cell strain, reverse transcribe it into cDNA, and use it as a template to amplify the light chain variable region gene and the heavy chain variable region gene of the anti-methicillin single-chain antibody by PCR, and then splice the light chain variable region gene and the heavy chain variable region gene to obtain the anti-methicillin single-chain antibody gene by using SOE-PCR;

[0024] (2) Clone the anti-methicillin single-chain antibody gene fragment into a phagemid vector, and transfer it into E. coli, and obtain a phage antibody library after being attacked by a helper phage;

[0025] (3) Use solid-phase screening and gradually reduce the concentration of the coating agent to screen the methicillin-specific single-chain antibody gene through three rounds of screening;

[0026] (4) Transfer the recombinant phage containing the positive single-chain antibody gene into an E. coli expression strain, induce expression, and obtain a soluble single-chain antibody.

[0027] The present application uses a methicillin-specific hybridoma cell strain as the mRNA source, extracts mRNA and reverses it into cDNA, and uses it as a template to amplify the heavy chain variable region (VH) and the light chain variable region (VL) gene fragments by using a mouse antibody gene full-coverage primer library, and then connects the VH and VL to obtain a single-chain antibody (scFv) gene fragment by using SOE-PCR; the scFv gene is cloned into a phagemid pcomb3xss and electroporated into E. coli XL1-Blue, a helper phage VCSM13 is added, and a phage antibody library is obtained; methicillin is used as the stationary phase, and the phage antibody library is used as the mobile phase, and through three rounds of "adsorption-elution-amplification" screening process, a methicillin-specific scFv is obtained.

[0028] To achieve the above-mentioned purpose, the present application also proposes an anti-methicillin single-chain antibody in vitro evolution method, that is, an anti-methicillin single-chain antibody recognition mechanism analysis and affinity improvement method, the anti-methicillin single-chain antibody in vitro evolution method of the present application comprises: obtaining a three-dimensional model of the single-chain antibody by using homology modeling technology, and analyzing the molecular recognition mechanism of the single-chain antibody recognizing the methicillin drug by using molecular docking technology; obtaining a single-chain antibody mutant with improved affinity by computer simulation design and site-directed mutation technology.

[0029] Specifically, the present application analyzes the key amino acids and the interaction force of the scFv recognizing TMP by using homology modeling and molecular docking technology; locks the key amino acids by using alanine scanning mutation experiment; guides the directed evolution of the scFv by using virtual mutation of multiple software, and obtains a scFv mutant with significantly improved affinity by using site-directed mutation experiment.

[0030] To achieve the above object, the application further provides application of the anti-methicillin single-chain antibody or the mutant of the anti-methicillin single-chain antibody in methicillin residue detection.

[0031] Specifically, the method for detecting methicillin drug residue by using the anti-methicillin single-chain antibody comprises the following steps: coating an enzyme-labeled plate with methicillin, taking the developed mutant of the anti-methicillin single-chain antibody as a primary antibody, taking HRP anti-HA tag antibody as a secondary antibody, and detecting the methicillin content in a sample to be detected by ic-ELISA.

[0032] More specifically, the application of the anti-methicillin single-chain antibody in methicillin residue detection takes methicillin drug coupled carrier protein as a coating agent, takes the scFv mutant as a primary antibody, configures methicillin drugs into a series of gradient concentrations, establishes an indirect competitive enzyme-linked immunoassay (ic-ELISA), determines a standard curve, a regression equation and a correlation coefficient, repeats 5 times, calculates the inter-plate variation coefficient, evaluates the precision of the standard curve, selects other drugs to replace methicillin standard products, performs ic-ELISA determination, calculates a cross-reaction rate, and takes milk, honey, eggs, pork, chicken and other tissue samples to be detected to perform drug spiking experiments and calculate a recovery rate.

[0033] The light chain nucleotide sequence of the methicillin-specific scFv gene is shown as SEQ ID:1, the heavy chain nucleotide sequence is shown as SEQ ID:2, and the scFv mutant nucleotide sequence is shown as SEQ:ID5.

[0034] Compared with the prior art, the application has the following beneficial effects:

[0035] The application first prepares a methicillin-specific single-chain antibody by phage display technology, and the variable region nucleotide and protein sequences are first reported at home and abroad. The application improves the affinity of the single-chain antibody by computer simulation combined with site-directed mutation technology, and obtains a single-chain antibody with better recognition performance, thereby providing a new method for obtaining high-quality antibody materials. The single-chain antibody prepared by the application is prepared by a prokaryotic expression system, and compared with a monoclonal antibody prepared by hybridoma technology, the method is more economical and efficient, and greatly reduces the development and production cost. The ic-ELISA method based on the single-chain antibody established by the application can be applied to methicillin residue detection in animal-derived food, is conducive to low-cost and high-efficiency monitoring of methicillin residue in production practice, and maintains food safety and human health. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is an amplified fragment of VL and VH genes of the methicillin-specific antibody;

[0037] Figure 2 VL and VH of the methicillin specific antibody are assembled into a single-chain antibody fragment;

[0038] Figure 3 monoclonal phage ELISA detection results of the phage antibody library;

[0039] Figure 4 ic-ELISA standard curve of the methicillin specific single-chain antibody;

[0040] Figure 5 ic-ELISA standard curve of the methicillin specific single-chain antibody mutant.

[0041] In order to make the objects, technical schemes and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. DETAILED DESCRIPTION

[0042] It should be understood that the specific examples described herein are intended to be illustrative only and not limiting of the present application.

[0043] Single-chain fragment variable (scFv) refers to the high-efficiency expression of the heavy chain variable region and the light chain variable region genes of an antibody after being connected by a specific method. The in-vivo expression of the antibody in Escherichia coli has the characteristics of low cost and high yield. Meanwhile, the production of scFv does not use experimental animals, and can well avoid the problem of animal welfare.

[0044] The present application takes a methicillin drug specific monoclonal hybridoma cell strain as a basic material, extracts RNA, reverses transcription into cDNA, takes this as a template, and uses a PCR method to amplify the heavy chain variable region and the light chain variable region sequences of all antibody genes. The SOE-PCR is used to further splice into a scFv gene, then the scFv gene is inserted into a phage display vector pcomb3xss, combined with phage display and in-vitro specific screening technology, an anti-methicillin single-chain antibody capable of specifically recognizing methicillin is obtained. Based on the scFv, an indirect competitive enzyme-linked immunoassay method (ic-ELISA) is established to detect the residual amount of methicillin in animal-derived food.

[0045] Example 1: Construction of antibody library

[0046] 1. Resuscitation and culture of monoclonal hybridoma cell strain: The methicillin-specific monoclonal hybridoma cell strain stored in liquid nitrogen was placed in a 37°C water bath and gently shaken to quickly melt. 10 mL of RPMI-1640 basic medium was suspended, centrifuged at 1200 r / min for 5 min, and the supernatant was discarded. 2 mL of RPMI-1640 complete medium (containing 20% fetal bovine serum) was added to resuspend, and the cells were evenly distributed by blowing and sucking. Then, the cells were inoculated into a 6-well plate and cultured in a 37°C, 5% CO2 incubator. The cell state and quantity were observed, and the cells were passaged in time according to the cell density.

[0047] 2. Extraction of total RNA from monoclonal hybridoma cells: The hybridoma cells with a quantity of more than 5 x 10 6 were collected by centrifugation, and 1 mL of Trizol reagent was added to lyse the cells, and then transferred to a 2 mL RNAase-free centrifuge tube. The total RNA was extracted using an RNA extraction kit and stored at -80°C.

[0048] 3. Amplification of antibody heavy chain (VH) and light chain (VL): The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, a high-fidelity enzyme and a mouse antibody gene full-coverage primer library (purchased from Beijing Baokewei) were used to amplify the full set of VH and VL. The PCR amplification system is shown in Table 1, and the amplification program is: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 1 min, 35 cycles; 72°C, 5 min. The PCR products were identified by 1% agarose gel electrophoresis, and the identification results are shown in Figure 1 , and the amplified VH and VL fragments were purified and recovered using a gel recovery kit.

[0049] Table 1 PCR cloning of VL and VH gene fragments

[0050]

[0051] 4. Single-chain antibody gene splicing: VH and VL were spliced into scFv by SOE-PCR technology, in which the template was the purified and recovered VH and VL fragments, and the front and rear primers were the 5' primer of VL and the 3' primer of VH, respectively. The final splicing scFv mode was VL-linker-VH. The linker used was SSGGGGSGGGGGGSSRSS containing 18 amino acids. The SOE-PCR system is shown in Table 2:

[0052] Table 2 SOE-PCR reaction system for splicing scFv

[0053]

[0054] The optimal amplification procedure of the PCR amplification setup is as follows: 95℃, 3 min; 95℃, 15 s; 60℃, 30 s; 72℃, 1 min, 35 cycles; 72℃, 5 min. The PCR product is identified by 1% agarose gel electrophoresis, and the theoretical band size of the scFv is about 800 bp. The electrophoresis identification result is shown in Fig. 1, and the scFv gene fragment is purified and recovered by using a gel recovery kit for constructing a recombinant phagemid. Figure 2

[0055] 5. Construction of recombinant phagemid: the scFv gene fragment and the phagemid pcomb3xss are simultaneously subjected to enzyme digestion by using a restriction endonuclease sfi I, and the enzyme digestion system is shown in Table 3.

[0056] Table 3 Sfi I enzyme digestion system of the pcomb3xss vector and the scFv

[0057]

[0058] After the enzyme digestion system is subjected to enzyme digestion at 37℃ for 2 h, the enzyme digestion result is detected by using 1% agarose gel electrophoresis, the scFv gene fragment after enzyme digestion is recovered by using a gel recovery kit, and the scFv gene fragment and the phagemid pcomb3xss are connected by using a T4 DNA ligase at 16℃ overnight to construct a recombinant phagemid.

[0059] 6. Construction of phage antibody library: the recombinant phagemid and the XL1-Blue electrocompetent cells are mixed uniformly on ice, and are allowed to stand for 90 s. After being subjected to electric shock transformation by using an electrotransformation instrument, 37℃ preheated SOC liquid medium is added to resuspend and culture, which is a primary antibody library. The primary antibody library is gradiently diluted and coated on a solid plate, and the antibody library capacity is calculated to be 3.3×10 7 The remaining antibody library is stored at -80℃ after being added with 15% sterile glycerol.

[0060] Example 2: Phage screening and identification

[0061] 1. Preparation of phage display library: 1 mL of the antibody library stored at -80℃ is added to 250 mL of 2YT-G medium, and is cultured at 37℃ and 250 r / min with shaking until OD600 is about 0.4-0.6; 100 μg / mL of ampicillin, 50 μg / mL of tetracycline and 2% glucose are added to a final concentration, and 2×10 11 ​PFU (MOI = 20:1), incubate at 37°C for 30 min, then shake at 220 rpm for 30 min; centrifuge at 1000 rpm for 10 min, carefully discard the supernatant. Resuspend the bacterial pellet in 500 mL of 2×YT-ATK medium, incubate overnight at 37°C with shaking at 220 rpm; centrifuge at 10,000 rpm for 20 min, collect the supernatant, add 20% PEG / NaCl, incubate on ice at 4°C for 1 h; centrifuge at 12,000 rpm for 20 min at 4°C, and resuspend the phage pellet in 2 mL of PBS to obtain the phage display library.

[0062] 2. Phage display library screening: Coat the original coated immunotubes with 2 mL of 100 μg / mL trimethoprim and incubate overnight at 4°C; block the immunotubes with 2% skim milk (MPBS) at 37°C for 2 h; discard the blocking solution and wash the tubes 3 times with PBST. During washing, mix the phage antibody supernatant obtained in the previous section with OVA, MPBS and phage supernatant in a ratio of 1% OVA: 2% MPBS: supernatant = 1:1:3, and perform deinterference treatment at room temperature for 20 min to remove interference from non-specific binding; add 2 mL of the treated phage supernatant mixture to the blocked immunotubes, gently shake and incubate for 30 min, then incubate for 1.5 h; discard the phage supernatant in the immunotubes, wash 3 times with PBST, then wash 3 times with PBS, and pat dry; add 2 mL of glycine buffer (0.1 mol / L, pH 1.5) to each tube. 2.5) Gently elute at room temperature for 6 min, then immediately add an equal volume of Tris-HCl (pH 7.4) buffer to neutralize the elution buffer. Add the eluted phage to a sterile 50 mL centrifuge tube containing 10 mL of host bacteria XL1-Blue (OD600 = 0.5), and incubate at 37°C with shaking at 150 rpm for 1 h. This completes the first round of screening, obtaining the primary antibody library. Repeat the above steps for the second and third rounds of screening using the primary antibody library. The original coating concentration used in the three rounds of screening is progressively reduced to 100 mg / L, 50 mg / L, and 25 mg / L, respectively. Take the bacterial culture from the third round of shaking culture and perform serial dilutions with 2×YT medium. Spread 100 μL of the diluted solution onto SOB-TKG plates and incubate overnight at 37°C.

[0063] 3. Monoclonal phage ELISA identification: Take a 96-well plate and add 2×YT-ATG medium, 400 μL / well; using a sterile pipette tip, randomly pick 48 monoclonal colonies from the third-round output library and inoculate them into each well. Label this plate as the Master Plate. Place the Master Plate on a shaker and incubate overnight at 37℃ and 250 rpm; the next day, take another 96-well plate and add 600 μL of medium containing 2.5×10⁻⁶ phages. 10Pfu / mL VCSM13 of 2xYT-TKG to each well of the plate, which is labeled as P1 Plate; 60 μL of culture solution from each well of the Master Plate of overnight culture was taken to the P1 Plate. The P1 Plate was placed in a shaker at 37°C, 150 r / min, and shaken for 2 h. Centrifugation was performed at 1500 r / min for 20 min, and the supernatant was carefully removed; 600 μL of 2xY-ATK medium was added to each well of the P1 Plate, which was shaken at 37°C, 250 r / min, and incubated overnight. Centrifugation was performed at 1500 r / min for 20 min, and the supernatant was used for ELISA detection; the enzyme-labeled plate was coated with ampicillin, and 1% OVA was set as a negative control; 250 μL of 2% MPBS was added to the enzyme-labeled plate, which was blocked at 37°C for 2 h; 100 μL of phage antibody was added to PBST, which was incubated in a wet box at 37°C for 2 h; the phage antibody was discarded, and the enzyme-labeled plate was washed with PBST for 3 times, washed with PBS for 3 times, and dried; the HRP-labeled M13 antibody was diluted with PBS at 1:5000, 100 μL / well, which was incubated in a wet box at 37°C for 1 h; the enzyme-labeled antibody solution was discarded, and the enzyme-labeled plate was washed with PBST for 3 times, washed with PBS for 3 times, and dried; 100 μL / well of TMB color developing solution was added, which was incubated at 37°C for 15 min or so; 50 μL / well of 2 mol / L H2SO4 was added to terminate the color development, which was immediately placed in an enzyme-labeled instrument to measure the absorbance value at a wavelength of 450 nm; the OD450nm value of the sample to be tested was set as P, and the OD450nm value of the negative control was set as N; when P / N≥2.1, it was determined as a positive clone, and when P / N<1.5, it was determined as a negative clone. The results of ELISA of the monoclonal phage (as shown in Figure 3 Table 1) showed that most of the bacteria in the phage antibody library could display ampicillin specificity after 3 rounds of specific screening.

[0064] Example 4: Soluble expression of single-chain antibody and ELISA identification

[0065] 1. Soluble expression of single-chain antibody: According to the results of monoclonal phage ELISA, the most active clone was selected and added to 15 mL of 2xYT-AT medium for expansion culture. After 16 h of culture at 37°C with 220 r / min shaking, the positive recombinant phagemid was extracted using a plasmid extraction kit. The positive phagemid was heat-shocked and transformed into a soluble expression strain TOP10F'. After correct identification by sequencing, it was added to 20 mL of 2xYT-A medium and cultured at 37°C with 220 r / min shaking until OD600≈0.8. Then, 1 mM of IPTG was added, and expression was induced at 18°C with 180 rpm for 20 h. The bacterial solution was centrifuged at 13,000 r / min for 5 min at 4°C to collect the bacterial precipitate. After resuspending the precipitate with 2 mL of pre-cooled PBS, it was transferred to a 10 mL centrifuge tube. The centrifuge tube was placed in an ice water bath for ultrasonic crushing, with the following instrument settings: ultrasonic for 10 s, pause for 15 s, power 45%, total ultrasonic for 10 min, and the liquid was clarified. The soluble expression of single-chain antibody was present in the supernatant after centrifugation at 12,000 r / min for 10 min at 4°C.

[0066] 2. ELISA identification of single-chain antibody: The activity of single-chain antibody was identified according to the operation process of ELISA method. 100 μL of 50 mg / L methicillin was used to coat a 96-well enzyme-labeled plate, and the coating was performed at 4°C overnight. 250 μL of blocking solution (2% MPBS) was added per well, and the blocking was performed at 37°C for 2 h. After PBST washing, different dilutions of single-chain antibody and an equal volume of methicillin standard were added, and incubation was performed at 37°C for 2 h. After PBST washing, 100 μL of enzyme-labeled secondary antibody HRP-anti HA-tag antibody (1:5000) was added to the 96-well enzyme-labeled plate per well, and incubation was performed at 37°C for 1 h. After PBST washing, 100 μL of substrate (A:B=1:100) was added per well, and incubation was performed at 37°C for 15 min. The color development was terminated with 50 μL of 2 mol / L H2SO4, and the OD value was read at 450 nm. The standard curve was drawn with the logarithmic value of the competitor concentration as the abscissa and the corresponding inhibition rate B / B0 as the ordinate, as shown in Figure 4 The regression equation was y=(A-D) / [1+(1000x / C)^B]+D, where A=0.91008, B=14.43192, C=3.25493, D=0.04297, R2=0.99991, and the half-inhibition rate IC 50 value calculated according to the regression equation was 14.4 μg / L.

[0067] Example 5: Analysis of methicillin single-chain antibody recognition mechanism and directed evolution

[0068] 1. Homology Modeling and Molecular Docking: E. coli strains validated for soluble expression were sent to a biotechnology company for sequencing. The light chain nucleotide sequence of the single-chain antibody was obtained (SEQ ID:1), and the heavy chain nucleotide sequence (SEQ ID:2). Sequence translation revealed the light and heavy chain amino acid sequences of the single-chain antibody (SEQ ID:3 and SEQ ID:4, respectively). The amino acid sequences of the single-chain antibody were uploaded to the SWISS-MODEL website for homology modeling. Molecular docking of the single-chain antibody with trimethoprim was performed using Autodock Vina software. The docking results showed that Arg-62, Ser-129, Gln-136, Trp-242, and Gln-244 were contact amino acids. Virtual saturation mutagenesis of the contact amino acids was performed using Discovery Studio (DS) software, demonstrating that mutating Arg-62 of the single-chain antibody to phenylalanine (Phe) enhances the activity of the single-chain antibody. The correctness of this mutation scheme was also verified using Autodock Vina.

[0069] 2. Site-directed mutagenesis and bioactivity verification of single-chain antibodies: Using recombinant phage particles of single-chain antibodies as templates, the upstream primer for site-directed mutagenesis was designed as ATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTC, and the downstream primer was designed as GAACCTGTCTGGGACCCCAGAAAATCGGTTGGAAACTTTGTAGAT. Site-directed mutagenesis PCR amplification was performed to obtain single-chain antibody mutant phage particles. These particles were then transformed into soluble expression strains, and after correct sequencing, soluble expression was performed to obtain single-chain antibody mutants. The nucleotide and amino acid sequences of the single-chain antibody mutants are shown in SEQ ID: 5 and SEQ ID: 6, respectively. Using the above-mentioned single-chain antibody ELISA identification scheme, an indirect competitive ELISA method based on single-chain antibody mutants was established. The standard curve equation is y = 0.06681 + 0.9091 / [1 + (x / 0.5761)^ 4 . 1721 ](R 2 =0.9998)(e.g. Figure 5 (as shown), half-inhibitory concentration (IC50) 50 The concentration was 3.88 μg / L, and the detection range was 1.25–40 μg / L. Compared with the parent scFv, the affinity of the scFv-24 mutant was increased by 3.7 times.

[0070] Example 6: Application of single-chain antibodies in the detection of trimethoprim residues in animal-derived foods

[0071] 1. Precision: After establishing the standard curve, TMP was prepared into 1.25 μg / L, 2.5 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L series of gradient concentrations, 5 parallel holes were set for each concentration, the logarithmic value of the concentration of the drug standard was taken as the abscissa, and the inhibition rate (B / B0) corresponding to each concentration was taken as the ordinate to draw the standard curve, the regression equation and the correlation coefficient were calculated, the IC50 was calculated according to the regression equation. The same method was repeated for 5 times, the average value of IC50 was calculated, and the inter-plate and intra-plate variation coefficients were calculated to evaluate the precision of the standard curve. As shown in Table 4, after 5 times of intra-plate repetition and 5 times of inter-plate repetition, the intra-plate variation coefficient was less than 13.4%, and the inter-plate variation coefficient was less than 12.6%, indicating that the precision of the ic-ELISA method was good.

[0072] Table 4 Intra-plate and inter-plate variation coefficients of the standard curve

[0073]

[0074]

[0075] 2. Specificity: Other drugs of the methicillin class were selected as standard substances of TMP, and the ic-ELISA method was used to determine the IC 50 values of each drug, and the cross-reactivity was calculated. As shown in Table 5, the IC 50 of scFv-24 mutant recognizing DVD was 6.17 μg / L, and the cross-reactivity was 62.88%. In addition, the scFv-24 mutant had no recognition activity to non-methicillin drugs.

[0076] Table 5 Determination of cross-reactivity of scFv-24 mutant

[0077]

[0078] 3. Sample pretreatment: milk: 10 mL milk was taken in a 50 mL centrifuge tube, 20 μL diluted drug was added, and after centrifugation at 6000 r / min for 10 min at 4°C, the middle layer of milk emulsion was diluted 10 times with PBST and used for ELISA determination. Honey: 2.0 g of honey was weighed, 20 μL of diluted drug was added, and after vortexing and oscillation, it was diluted 10 times with PBST and used for ELISA determination. Egg: 1 g of egg sample was weighed in a 50 mL centrifuge tube, 10 mL of PBST and 20 μL of diluted drug were added, and after vortexing and oscillation, it was centrifuged at 6000 r / min for 15 min. The upper liquid was filtered through filter paper and used for ELISA determination. Pork and chicken: fresh pork and chicken were purchased from the market, cut into small pieces and put into a meat grinder; 2.0 g of homogenized sample was weighed, 20 μL of diluted drug was added, and after vortexing and oscillation, 8 mL of acetonitrile was added, vortexed thoroughly, and centrifuged at 8000 r / min for 15 min. The supernatant was blown dry with N2, and 2 mL of PBST containing 10% methanol was used to redissolve it for ELISA determination.

[0079] 4. Recovery rate: 20 blank samples of different sources were taken, ELISA detection was performed according to the above sample pretreatment method, OD value was determined, average value of blank sample OD value was calculated, average value was brought into standard curve to calculate corresponding drug concentration (C), and standard deviation (SD) was calculated. As shown in Table 6, the detection limit (LOD) and the quantification limit (LOQ) of 20 different blank samples were 2.04-2.75 μg / L and 2.43-4.64 μg / L, respectively. The actual sample was added according to 0.5 times, 1 times and 2 times of the highest residue limit, 5 parallel samples were set for each sample concentration, and the sample pretreatment steps were the same as above. The drug concentration in the sample was determined by ELISA method, and 3 batches were repeated to calculate the recovery rate. As shown in Table 6, the recovery rate of the added sample of the established ic-ELISA method was 81.05%-101.91%, and the coefficient of variation was <13.6% (Tables 3-6 and Tables B3-B7). It is proved by the addition of recovery test that the scFv-24 mutant can be used for the detection of trimethoprim residues in pork, chicken, milk, honey and egg samples.

[0080] Table 6 LOD, LOQ recovery rate and coefficient of variation analysis of ic-ELISA method in actual samples

[0081]

[0082] Compared with the prior art, the beneficial effects of the present application are:

[0083] The present application first prepares the trimethoprim specific single-chain antibody by phage display technology, and the variable region nucleotide and protein sequence are first reported at home and abroad. The present application improves the affinity of the single-chain antibody by computer simulation and site-directed mutation technology, and obtains the single-chain antibody with better recognition performance, thereby providing a new method for obtaining high-quality antibody material. The single-chain antibody prepared by the present application is prepared by a prokaryotic expression system, and compared with the monoclonal antibody prepared by hybridoma technology, the method is more economical and efficient, and greatly reduces the development and production cost. The ic-ELISA method based on the single-chain antibody established by the present application can be applied to the detection of trimethoprim residues in animal-derived food, is conducive to the low-cost and high-efficiency monitoring of trimethoprim residues in production practice, and maintains food safety and human health.

[0084] The above only describes the preferred embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structure or process transformation made by using the content of the specification and drawings, or directly or indirectly applied to other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. An anti-methicillin single chain antibody, characterized in that, The light chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 3 and the heavy chain variable region with the amino acid sequence as shown in SEQ ID NO: 4 are connected by a polypeptide from the nitrogen end to the carbon end.

2. A mutant of an anti-methicillin single chain antibody, characterized in that, The amino acid sequence of the mutant of the anti-methicillin single-chain antibody is shown as SEQ ID NO:

6.

3. A gene encoding the anti-methicillin single chain antibody according to claim 1, characterized by, The nucleotide sequence encoding the light chain variable region of the anti-methicillin single-chain antibody is shown as SEQ ID NO: 1, and the nucleotide sequence encoding the heavy chain variable region of the anti-methicillin single-chain antibody is shown as SEQ ID NO:

2.

4. A gene encoding the mutant of the anti-methicillin single-chain antibody according to claim 2, characterized by, The nucleotide sequence encoding the mutant of the anti-methicillin single-chain antibody is shown as SEQ ID NO:

5.

5. An expression vector, characterized by, The expression vector expresses the anti-methicillin single-chain antibody as claimed in claim 1 or comprises the gene as claimed in claim 3.

6. Use of the anti-methicillin single-chain antibody as claimed in claim 1 or the mutant of the anti-methicillin single-chain antibody as claimed in claim 2 in methicillin drug residue detection.

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