Anti-enr nanobodies, encoding genes, cELISA detection methods and uses

Abstract

The application discloses an anti-ENR nanobody, a coding gene, a cELISA detection method and purposes, and belongs to the technical field of biology. The amino acid sequence of the anti-ENR nanobody is shown as SEQ ID NO. 1, and the nucleic acid sequence for coding the anti-ENR nanobody is shown as SEQ ID NO. 2. A new type of competitive ELISA (cELISA) is established by screening the specific nanobody against ENR from an immunized bactrian camel library. When detecting the enrofloxacin residue in animal-derived food, the method has high sensitivity and specificity, can detect the ENR residue as low as 6.49 ng / mL in the sample, and has no cross reaction with other antibiotics. It is shown that the cELISA established in the application is a simple, rapid, sensitive and low-cost immunodetection method, and has great application prospect in the detection of ENR residue in animal-derived food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an anti-ENR nanobody, a coding gene, a cELISA detection method and purposes. BACKGROUND

[0002] Enrofloxacin (ENR) is an oral antibiotic belonging to the third generation of fluoroquinolones. It is the first fluoroquinolone used by veterinarians to treat bacterial infections. Due to its excellent bactericidal activity against both gram-negative and gram-positive pathogens, it is widely used in the agricultural sector. However, in recent years, the misuse and residues of antibiotics have attracted widespread attention. The misuse of antibiotics can lead to the development of resistance to these drugs by pathogens. The misuse of ENR can lead to the emergence of fluoroquinolone-resistant pathogens, which can spread between humans and animals. In addition, the presence of ENR residues in the food chain can cause adverse effects and diseases in humans. In order to regulate the use of ENR and ensure that its residue levels in animal-derived food are within acceptable limits, many countries and organizations have established maximum residue limits (MRLs) for ENR in animal-derived food. These maximum residue limits vary from 100 μg / kg to 500 μg / kg depending on the specific animal product. Despite the implementation of restrictive measures, many cases of exceeding the maximum residue limit of ENR have been exposed. According to relevant data, during the period of 2015-2019, fluoroquinolone drug residues ranked first in food antibiotic residue monitoring, accounting for 41% of the total.

[0003] There are generally three conventional methods for controlling antibiotic residues in animal-derived food. Microbiological methods are the first method used to control antibiotic residues in food. These methods are based on the sensitivity of bacteria to antibiotics, simple and inexpensive. However, they are time-consuming and have low detection limits. Analytical techniques based on the physical and chemical properties of antibiotics, such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), have also been developed in parallel. These methods have low detection limits and high sensitivity, but require expensive equipment and precise experimental conditions, limiting their applicability. On the other hand, immunoassay methods based on antigen-antibody interactions are very suitable for screening large numbers of samples due to their simplicity. They have lower development costs because they do not require expensive large-scale testing instruments or rare label materials, making them the most mature and most widely accepted method. However, despite the fact that antibodies are the core of molecular recognition and detection, the widespread use of polyclonal and monoclonal antibodies has some key limitations, including large molecular weight, expensive preparation process, poor reproducibility, and loss of activity, which need to be overcome. Therefore, it is necessary to develop efficient and sensitive detection methods. SUMMARY

[0004] In view of the above problems in the prior art, the application provides an anti-ENR nanobody, a coding gene, an ENR novel competitive ELISA detection method and application, which can effectively and quickly detect the residual amount of enrofloxacin in animal-derived food.

[0005] To achieve the above object, the technical scheme adopted by the application to solve its technical problem is:

[0006] An anti-ENR nanobody, the amino acid sequence of the anti-ENR nanobody is shown in SEQ ID NO. 1.

[0007] QVQLQESGGGSVQAGGSLRLSCSASGYISRTKCLGWFRQVPGKEREGV AAIDSAGTTNYAESVKGRFTISADNAKEILYLQMNSLKPEDTAMYYCAAERR TSPATCGQLVVWGRYGGRYGWDYWGKGTQVTVSS (SEQ ID NO. 1).

[0008] Further, the anti-ENR nanobody is a fusion protein obtained by connecting a tag at the N terminal and / or C terminal shown in SEQ ID NO. 1.

[0009] Further, the tag is an HRP tag.

[0010] A nucleic acid coding the above anti-ENR nanobody, the nucleic acid sequence of which is shown in SEQ ID NO. 2;

[0011] or a DNA molecule with more than 80% homology with the nucleic acid sequence shown in SEQ ID NO. 2 and coding the nanobody in claim 1.

[0012] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTCAGCCTCTGGATACATCTCTCGTACGAAGT GCCTGGGTTGGTTCCGCCAGGTTCCAGGGAAAGAGCGCGAGGGGGTCGCCGCTATTGATAGTGCTGGAACCACAAATTACGCAGAGTCCGTGAAGGGCCGATTCACCATCTCCGCAGACAACGCCAAGGAGATTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGCGCGGCAGAACGGAGGACAAGTCCTGCGACATGTGGACAATTAGTCGTATGGGGGAGATACGGCGGGAGATACGGCTGGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA (SEQ ID NO.2).

[0013] A recombinant vector comprising the nucleic acid.

[0014] A transgenic cell line comprising the nucleic acid, or the recombinant vector.

[0015] An engineered bacterium comprising the nucleic acid, or the recombinant vector.

[0016] Use of the anti-ENR nanobody in the preparation of a preparation for detecting enrofloxacin residues.

[0017] A novel competitive ELISA method for detecting enrofloxacin residues in food, using the anti-ENR nanobody.

[0018] Advantages of the present application:

[0019] The present application utilizes the specific nanobody against ENR screened from the immunized Bactrian camel library, and after fusion with horseradish peroxidase (HRP), it is expressed in HEK293T cells. A novel competitive ELISA (cELISA) based on ENR-OVA and nanobody-horseradish peroxidase fusion is established, which has high sensitivity and specificity when used for rapid detection of enrofloxacin residues in animal-derived food, and can detect as low as 6.49 ng / mL of ENR residues in the sample, and has no cross-reaction with other antibiotics such as CIP.

[0020] In addition, the method has good repeatability, and the batch and batch variation coefficients are less than 10%. It is shown that the cELISA established in the application is a simple, rapid, sensitive and low-cost immunodetection method, and has great application prospect in the detection of ENR residues in animal-derived food. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is full wavelength scanning identification chart, PAGE identification chart for ENR-BSA and ENR-OVA; wherein, a is ultraviolet scanning spectrum chart of BSA, ENR and BSA-ENR; b is ultraviolet scanning spectrum chart of OVA, Gent and OVA-ENR; c is SDS-PAGE detection chart of BSA-ENR and OVA-ENR;

[0022] Figure 2 It is immune double-humped camel post antibody titer determination chart, two rounds of PCR chart and screening chart; wherein, a is titer detection of anti-ENR antibody in immune camel serum; b is first round of PCR detection chart; c is second round of PCR detection chart; d is 10-fold gradient dilution;

[0023] Figure 3 It is anti-ENR nanobody expression and identification chart; wherein, a is nanobody positive identification chart; b is nanobody sequence comparison chart;

[0024] Figure 4 It is anti-ENR nanobody and vHRP protein fusion expression and identification chart;

[0025] Figure 5 It is cELISA standard curve and ENR-Nb66-vHRP cross-reactivity determination result;

[0026] Figure 6 It is nanobody structure and its combination with ligand and ENR schematic diagram;

[0027] Figure 7 It is ENR-Nb66-vHRP fusion protein expression identification and immunofluorescence result chart;

[0028] Figure 8 It is technical flow chart of the application. DETAILED DESCRIPTION

[0029] The specific embodiments of the application are described below to facilitate the understanding of the application by those skilled in the art, but it should be clear that the application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the application defined and determined by the appended claims, and all the application and creation utilizing the concept of the application are within the scope of protection.

[0030] Construction and screening of nanobody phage display library

[0031] 1. Synthesis of ENR-BSA immunizing antigen and ENR-OVA coating antigen

[0032] ENR-BSA and ENR-OVA were synthesized using glutaraldehyde method. Briefly, 10.94 mg ENR and 15 mg BSA were dissolved in 3 mL PBS, then 50 μL 25% glutaraldehyde was added to the mixture, which was incubated at 25 °C for 16 h, and then centrifuged at 5000 g for 10 min. The supernatant was then dialyzed in PBS at 4 °C for 72 h, and the dialysis solution was changed every 8 h, finally obtaining ENR-BSA. The synthesis of ENR-OVA was the same as the above steps, except that 15 mg BSA was replaced by 9.8 mg OVA. The prepared ENR-BSA immunizing antigen and ENR-OVA coating antigen were scanned at full wavelength, and the results are shown in Figure 1 .

[0033] 2. Immunization of Bactrian camel and construction of VHH library

[0034] To obtain a highly specific VHH library, ENR-BSA was mixed with adjuvant 1:1 and immunized a 4-year-old Bactrian camel after complete emulsification. After the first immunization, the camel was given subsequent immunization every two weeks using an emulsified mixture of ENR-BSA and incomplete Freund's adjuvant, a total of four times. Four days after the last immunization, indirect ELISA (iELISA) was performed using HRP-conjugated rabbit anti-camel IgG, with 5 μg / mL of ENR-BSA as the coating antigen, to evaluate the antibody titer of the Bactrian camel serum (see Figure 2 a of FIG.).

[0035] Mononuclear cells were isolated from 200 mL peripheral blood of the immunized camel. After RNA extraction, cDNA synthesis was performed, CALL001 and CALL002 were used as primer pairs for the first round of PCR amplification, and VHH-FOR and VHH-REV were used as primer pairs for the second round of PCR amplification of VHH genes. The pMECS plasmid and VHH genes were digested with restriction enzymes Pst I and Not I, and then T4 ligase ligation, TG1 electrocompetent preparation, plasmid electroporation, and library collection were performed. Forty-eight library colonies were randomly selected, PCR amplification was performed using primer pairs MP57 and GIII to determine the positive rate of the library, and positive clones were sequenced to identify diversity. Finally, the VHH library was stored in LB medium supplemented with 20% glycerol and 100 μg / mL ampicillin at -80 °C for use (see Figure 2 b, c, d of FIG.).

[0036] 3. Screening and identification of enrofloxacin-specific nanobodies

[0037] Specific ENR Nanobodies were selected by three rounds of biopanning. 200 pL of VHH library was grown in 2xTY medium to log phase and infected with M13KO7 helper phage to obtain rescue phage. ENR-OVA was coated on 96-well plates (20 pg / well, 10 pg / well and 5 pg / well for the first to third round of panning, respectively) at 4°C overnight. Coated wells were washed three times with PBS containing 2% Tween-20 (PBST, v / v) and then blocked with 3% skim milk (w / v) for 1 h.

[0038] Then 5 x 10 11 PFU of rescue phage were added and incubated at 37°C for 1 h. Each well was then washed 15 times with PBST, 100 pL of TAE solution (100 mM triethylamine, pH = 11.0) was added and incubated at RT for 10 min to elute specific phage particles and immediately neutralized with 100 pL of 1.0 M Tris-HCl (pH = 7.4).

[0039] Subsequently, for the next round of selection, the eluted phage particles were transferred to infect TG1 cells for titering assessment and amplification. Infected TG1 cells were counted to quantify the input and output of phage and enriched phage particles were detected using iELISA with anti-M13 antibody.

[0040] After three rounds of selection, 48 clones were randomly selected from the third round eluted phage. After incubation in TB medium to log phase, soluble Nanobodies were expressed in E. coli periplasm in 96-well plates using 1 mmol / L IPTG induction, respectively. Multiple freeze-thaw cycles generated periplasmic extracts, which consisted of Nanobodies with HA and His tags. In addition, iELISA using mouse anti-HA monoclonal antibody was used to determine the presence of anti-ENR Nanobodies. Finally, positive colonies (P / N > 3.0) can be identified and classified by sequencing of the amino acid sequence based on complementarity determining regions (CDRs) (see Figure 3 ).

[0041] Example 2 Expression and characterization of Nanobody-HRP fusion against ENR

[0042] The VHH-encoding gene was amplified by PCR using the primers Nb-vHRP-F and Nb-vHRP-R. The amplified gene was then ligated into the pCMV-N1-vHRP vector, thus constructing the pCMV-Nb-vHRP plasmid. Positive plasmids were transfected into HEK293T cells, and 72 hours after transfection, the culture supernatant was collected by centrifugation at 1000 x g for 5 min to remove cell debris. The secreted nanobody HRP fusion was supplemented with 0.02% NaN3(w / v) and stored at 4°C for direct use. Next, indirect immunofluorescence assay (IFA), SDS-PAGE and Western blotting were used to evaluate the expression of the nanobody HRP fusion protein in HEK293T cells and cell culture supernatant (see Figure 4 and Figure 7 ).

[0043] Example 3 Construction of a competitive ELISA (cELISA) detection method

[0044] 1. After determining the optimal dilution of the ENR-Nb66-vHRP fusion in the supernatant of HEK393T cells and the concentration of ENR-OVA in the cELISA, the cELISA was performed by chessboard titration, the specific process was as follows:

[0045] The optimal concentration of ENR-OVA was coated on the microtiter plate by incubation at 4°C overnight. Then the plate was washed with PBST three times. Unbound sites were blocked by incubation with 3% skim milk at 37°C for 1 hour. After washing three times with PBST, 100 μL / well of ENR-Nb-vHRP was pre-diluted with antibody dilution solution and a series of ENR standard concentrations (200, 100, 50, 20, 10, 5, 2, 1 and 0.1 ng / mL). The mixture was incubated for 1 hour, then the plate was washed with PBST (PBS with 0.05% Tween 20) 5 times, then 100 μL TMB was added to each well, then washed with PBST three times, and the reaction was stopped by adding 50 μL 2M H2SO4 to each well. Finally, the absorbance was measured at 450 nm. The logarithm of the ENR concentration was used as the x-axis, and B / B0(where B is the average absorbance at a given ENR concentration, and B0is the average absorbance when the ENR concentration is zero) as the y-axis, and a four-parameter logistic equation was used to fit the immunoassay data.

[0046] The limit of detection (LOD), IC 50 and linear range (IC 20 -IC 80 ) of the ENR standard curve were calculated using Origin 8.5 software. The LOD, IC 20 , IC 50 and IC 80Indicating the presence of 10%, 20%, 50% and 80% competitive inhibition to assess the ENR concentration of the cELISA (see Figure 5 a).

[0047] 2. Cross-reactivity detection

[0048] To assess the specificity of ENR-Nb-vHRP under optimized conditions, the cross-reactivity (CR) with a set of structurally related FQS compounds was measured. The CR values were calculated according to the following equation and then the IC 50 values of these compounds were compared to the IC 50 value of ENR, calculated as follows:

[0049] CR (%) = [IC 50 (ENR) / IC 50 (analyte)] x 100%

[0050] As Figure 5 b, the cross-reactivity (%) of ENR-Nb66-vHRP with CIP, PEF, OFL and LOM was 4.67, 4.58, 25.28 and 34.28, respectively.

[0051] Example 4 Application of the cELISA

[0052] Recovery studies were performed with milk, chicken, pork and beef samples obtained from local markets. ENR (1000 pg / mL prepared in PBS) was added to each sample to produce spiked concentrations of 0, 50, 100 and 200 pg / kg for analysis, following the procedure described below:

[0053] An equal amount of tissue sample (1 g wet weight) was homogenized and transferred to a 50 mL polypropylene centrifuge tube. A mixture of 5 mL of 5% trichloroacetic acid and 10 mL of 0.2M PBS was added to the tissue sample. The mixture was incubated at 60°C for 30 minutes. Then, the suspension was centrifuged at 4000 g for 15 minutes at room temperature and the supernatant was separated. 50 pL of supernatant was transferred to a microtiter plate for analysis. Different concentrations (0, 50, 100, 200 pg / L) of ENR were added to the milk sample, which was then centrifuged at 4500 g for 20 minutes at 4°C to remove the fat. Next, 60 pL of sodium nitrite (0.36 mol / L) and 60 pL of zinc sulfate (1.04 mol / L) were added to 2 mL of defatted milk sample. The mixture was vortexed for 1 minute and then centrifuged at 4000 g for 15 minutes at 4°C. The supernatant was taken for analysis. The recovery rate was calculated according to the cELISA standard curve constructed in Example 3, and the results are shown in Table 1.

[0054] Table 1 Recovery rate

[0055]

[0056]

[0057] wherein the ENR addition concentration is in μg / kg or μL / kg and the detection concentration is in μg / kg or μL / kg.

[0058] Finally, it should be noted that the above detailed description is only used to explain the technical solutions of the present application and not to limit the present application. Although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. An anti-enrofloxacin (ENR) nanobody, characterized in that, The amino acid sequence of the anti-enrofloxacin (ENR) nanobody is shown in SEQ ID NO.

1.

2. A fusion protein, characterized in that, The fusion protein is obtained by attaching a tag to the N-terminus and / or C-terminus shown in SEQ ID NO.1; the tag is an HRP tag.

3. A nucleic acid encoding the anti-enrofloxacin (ENR) nanobody of claim 1, characterized in that, The nucleic acid sequence is shown in SEQ ID NO.2; Or a DNA molecule that has more than 80% homology with the nucleic acid sequence shown in SEQ ID NO.2 and encodes the anti-enrofloxacin (ENR) nanobody described in claim 1.

4. A recombinant vector, characterized in that, Includes the nucleic acid described in claim 3.

5. A transgenic cell line, characterized in that, It includes the nucleic acid described in claim 3, or the recombinant vector described in claim 4.

6. An engineered bacterium, characterized in that, It includes the nucleic acid described in claim 3, or the recombinant vector described in claim 4.

7. Use of the anti-enrofloxacin (ENR) nanobody of claim 1 or the fusion protein of claim 2 in the preparation of formulations for detecting enrofloxacin residues.

8. A competitive ELISA method for detecting enrofloxacin residues in food, characterized in that, The detection was performed using the anti-enrofloxacin (ENR) nanobody as described in claim 1 or the fusion protein as described in claim 2.

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