Polypeptide specifically bound with endosulfan immune complex and application thereof

Abstract

The invention discloses a polypeptide specifically bound with an endosulfan antibody immune complex, and an application of the polypeptide in endosulfan immunodetection. According to the invention, an immune complex formed by an endosulfan monoclonal antibody and endosulfan is utilized to panning out specifically bound polypeptide from a phage display random polypeptide library, and positive clone and a polypeptide sequence thereof are obtained through ELISA and Sanger sequencing. The semi-saturated concentration of the non-competitive bacteriophage ELISA established based on the optimal polypeptide is 6.8 ng / mL, and the non-competitive bacteriophage ELISA can be used for sensitively and highly specifically detecting endosulfan residues in the environment and agricultural products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a polypeptide that specifically binds to an endosulfan immune complex and its application in enzyme-linked immunosorbent assay. Background Art

[0002] Endosulfan is an organochlorine insecticide with characteristics such as high efficiency, broad spectrum, and long residual period. It belongs to toxic persistent organic pollutants. Due to its high toxicity and long half-life, it has been included in the list of banned pesticides. Endosulfan has the characteristics of being difficult to degrade, highly fat-soluble, and can be enriched and amplified in the food chain. It is a highly toxic pollutant that can migrate globally through various transmission routes and is highly toxic to almost all types of organisms. The maximum residue limit standard for pesticides in foods in China (GB 2763-2021) stipulates that the maximum residue limit of endosulfan in citrus, apples, and cucumbers is 0.05 mg / kg. Accurate and rapid detection of endosulfan residues in the environment and foods is of great significance for ensuring food and environmental safety.

[0003] Immunoassay technology is based on the specific reaction between antibody and antigen. By using tracers such as enzymes and fluorescein, the binding of antibody and analyte to be detected is converted into a measurable signal to achieve qualitative and quantitative detection of the analyte. Compared with instrumental analysis technology, immunoassay has the advantages of rapidity, simplicity, sensitivity, economy, etc. It does not require expensive detection instruments and professional operators, and is suitable for high-throughput and on-site detection. As a supplement to instrumental analysis technology, it has broad application prospects in the field of pesticide residue detection. As a small molecule compound, the molecular weight of pesticides is usually less than 1 KD, which cannot stimulate the body to produce an immune response alone. It needs to be conjugated with a carrier protein with a larger molecular weight (immunogen) to stimulate the body to produce antibodies, and there is only one antigenic determinant, which can only bind one antibody. Small molecule compounds usually immobilize the modified antigen (i.e., coated antigen) on the solid phase interface, compete with the analyte to bind to the antibody site, and characterize the amount of the analyte by the decrease in signal intensity. The analyte concentration is negatively correlated with the signal intensity, which is called competitive immunoassay. After a small molecule compound binds to its antibody, most of its structure will be wrapped by the antibody to form an antigen-antibody immune complex, but there is still a small part that will be exposed to the solvent. Regarding the exposed part of the small molecule and the adjacent part of the antibody as a new epitope, screening anti-immune complex antibodies or polypeptides that can specifically interact with it can achieve non-competitive immunoassay of small molecule compounds. Compared with the competitive mode, non-competitive immunoassay has better sensitivity, accuracy and specificity, and is more suitable for on-site rapid detection technologies such as test strips and biosensors. In addition, the signal value of non-competitive immunoassay is usually proportional to the analyte content, which is more intuitive when interpreting the results. Therefore, the non-competitive detection mode has great development potential in the immunoanalysis of small molecules such as pesticides.

[0004] Phage display technology can insert the exogenous protein gene into the filamentous phage coat protein gene without affecting the normal function of the phage, so that the exogenous protein is expressed on the filamentous phage coat protein. Due to its simplicity, high efficiency and short experimental cycle, phage display technology is widely used in the field of rapid pesticide residue detection to prepare polypeptides that can recognize pesticides, antibody antigenic determinants and immune complexes. The non-competitive immunoassay method for pesticide small molecules established by using anti-immune complex polypeptides is superior to the competitive analysis method in terms of sensitivity and specificity. Currently, there is no relevant report on anti-immune complex polypeptides of endosulfan. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a polypeptide that specifically binds to the endosulfan immune complex and its application in enzyme-linked immunosorbent assay in view of the deficiencies of existing endosulfan rapid detection technologies.

[0006] The object of the present invention is achieved by the following technical solutions: A polypeptide that specifically binds to an endosulfan immune complex, wherein the sequence of the polypeptide is the amino acid sequence shown in SEQ ID NO. 3.

[0007] Wherein, a disulfide bond is formed between the cysteine residues at both ends of the polypeptide to be cyclized.

[0008] Wherein, the preparation method of the polypeptide comprises the following steps: (1) Fix the anti-endosulfan monoclonal antibody 4H4 on the enzyme-linked immunosorbent assay (ELISA) plate, add endosulfan to form an immune complex, and add a phage display polypeptide library for panning. The eluted phages are amplified and used for the next round of panning. A total of three rounds of panning are carried out. In each round, the content of the surfactant (Tween-20) in the washing solution is increased in turn, and the coating concentration of the endosulfan antibody is decreased; (2) After the panning is completed, select phage clones, determine the DNA sequence, and determine the sensitivity of different phage polypeptides. The obtained polypeptide sequences are the amino acid sequences shown in SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5 or SEQ ID NO. 6. Preferably, the polypeptide amino acid sequence SEQ ID NO. 3 is CPSYLSPEFC. The polypeptide consists of 10 amino acids and contains a cyclic structure formed by disulfide formation of cysteines at both ends; Wherein, the endosulfan immune complex is a complex formed by the reaction of an anti-endosulfan monoclonal antibody and endosulfan.

[0009] Wherein, the anti-endosulfan monoclonal antibody comprises a heavy chain variable region and a light chain variable region.

[0010] Wherein, the heavy chain variable region has the amino acid sequence of SEQ ID NO. 1, and the light chain variable region has the amino acid sequence of SEQ ID NO. 2.

[0011] Wherein, the anti-endosulfan monoclonal antibody comprises a heavy chain constant region and a light chain constant region of a murine IgG subtype.

[0012] In the second aspect of the present invention, there is provided a gene encoding the polypeptide that specifically binds to the endosulfan immune complex as described above, wherein the gene has the nucleotide sequence shown in SEQ ID NO. 7.

[0013] In the third aspect of the present invention, there is provided the use of the polypeptide that specifically binds to the endosulfan immune complex as described above, or its gene in the non-diagnostic detection of endosulfan.

[0014] Wherein, the use in the non-diagnostic detection of endosulfan includes but is not limited to the enzyme-linked immunosorbent assay method.

[0015] The present invention has the following beneficial effects: (1) A polypeptide capable of specifically binding to an endosulfan immune complex was reported for the first time; (2) Using the polypeptide provided by the present invention, a non-competitive immunoassay method for endosulfan can be established; (3) The semi-saturation concentration (SC 50 ) of the non-competitive enzyme-linked immunosorbent assay for endosulfan established based on the polypeptide against the immune complex is 6.8 ng / mL; the cross-reactivity rate of the non-competitive enzyme-linked immunosorbent assay for endosulfan established based on the polypeptide against the immune complex is less than 10.1%, and the reaction specificity is higher than that of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further specifically described below in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0017] Figure 1 For the ELISA results verifying the ability of the phage clones obtained by the third round of panning to specifically recognize the endosulfan immune complex, the abscissa is the serial number of the phage clones, and the ordinate is the absorbance value.

[0018] Figure 2 For the standard curve of the non-competitive ELISA for endosulfan established based on the polypeptide against the immune complex, the abscissa is the endosulfan concentration (in ng / mL), and the ordinate is the absorbance value at 450 nm. SPECIFIC EMBODIMENTS

[0019] The present invention will be further described below in conjunction with the drawings by way of examples, but it is not intended to limit the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials of the same or similar type, model, quality, nature or function as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0020] Example 1: Panning of the polypeptide against the endosulfan immune complex Step 1) The endosulfan monoclonal antibody 4H4 is stored in our laboratory. The preparation method can be found in Chinese Patent ZL2021112980049. The variable region sequence of the heavy chain of the antibody is QVQLQESGPELVKPGASVKMSCKASGYTFTNYVMYWVKQKPGQGLEWIGYINPFNDGTKYNEKFKGKATLTSDKSSDTAYMELSSLTSEDSAVYYCVRGSDDEDYWGQGTTLTVSS, SEQ ID NO. 1; the variable region sequence of the light chain is: DVVMTQTPLTLSVTIGQPASISCKSSQSLLSVNGKTYLNWLLQRPGQSPKRLIYLVSKLVSGVPDRITGSGSGTDFTLKFSRVEAEDLGIYYCVQGTHFPFTFGSGTKLEIK, SEQ ID NO.2; the constant region of the antibody is the heavy chain constant region and the light chain constant region derived from the IgG subtype of mouse. Dilute the endosulfan monoclonal antibody 4H4 with phosphate buffer solution (PBS, pH = 7.4) to 10 μg / mL, add 100 μL per well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and coat at 4 °C for 12 hours.

[0021] Step 2) Wash the coated wells five times with PBS containing 1‰ Tween-20 (PBST), then add 300 μL of 5% skim milk powder to each well and incubate for 1.5 hours, and wash 5 times with 1‰ PBST.

[0022] Step 3) Add 100 μL of 5 μg / mL endosulfan standard to each well, incubate at 25 °C and 300 rpm for 1 hour, and wash 5 times with 1‰ PBST.

[0023] Step 4) Add the phage display random cyclic heptapeptide, octapeptide, nonapeptide, decapeptide library (each 1×10 11 pfu) to each well, and incubate at 25 °C and 300 rpm for 1 hour.

[0024] Step 5) Add 100 μL of elution solution (0.2 M glycine-hydrochloric acid solution containing 10% BSA, pH = 2.2) to each well, and incubate at 25 °C and 300 rpm for 15 minutes.

[0025] Step 6) Add 15 μL of neutralization solution (1 M Tris-HCl, pH = 9.1) to each well, mix well and collect the supernatant. Take 1 μL to measure the phage titer, and store the rest at 4 °C.

[0026] The method for measuring the phage titer is as follows: Dilute the supernatant containing phage to 10^6, 10^8, and 10^10. Take 10 μL of each dilution and add it to 100 μL of ER2738 Escherichia coli in the logarithmic growth phase. Infect at 37 °C for 30 minutes. Spread the bacterial solution on an LB solid medium containing 20 μg / mL tetracycline and culture at 37 °C for 12 hours. Calculate the phage titer based on the number of colonies on the plate: Phage titer (pfu / mL) = 100 × dilution factor × number of colonies.

[0027] Step 7) Add the supernatant collected in step 6) to 3 mL of ER2738 Escherichia coli in the logarithmic growth phase. Infect at 37 °C for 1 hour, then expand the culture to 50 mL with LB liquid medium. Culture at 37 °C and 250 rpm until the logarithmic growth phase, and then add helper phage M13KO7 with a multiplicity of infection (number of helper phages / number of Escherichia coli) > 20. Infect at 37 °C for 1 hour.

[0028] Step 8) Centrifuge the bacterial solution, collect the precipitate, and add it to 100 mL of SB medium containing 25 μg / mL IPTG. Culture at 37 °C and 250 rpm for 12 hours.

[0029] Step 9) After centrifugation, collect the supernatant and add it to 25 mL of a solution containing 20% PEG2000 and 2.5 M NaCl. Let it stand on ice for 4 hours.

[0030] Step 10) Centrifuge at 14000 g and 4 °C for 30 minutes. Discard the supernatant, resuspend the precipitate with sterile PBS, add an equal volume of glycerol, measure the titer, and store at -20 °C.

[0031] Step 11) Steps 1)-10) are the first round of panning. The second and third rounds of panning are the same as the first round, but the coating concentrations of endosulfan monoclonal antibody are 5 and 2.5 μg / mL respectively, and the content of Tween-20 in PBST is 3‰ and 5‰. The purpose is to obtain anti-immune complex polypeptides with stronger affinity.

[0032] Step 12) Pick 30 clones from the plate of the third round of titer measurement and transfer them to 1 mL of 2×YT medium containing 50 μg / mL ampicillin and 20 μg / mL tetracycline. Culture at 37 °C and 300 rpm for 4 hours. Save 500 μL, and add helper phage M13KO7 with a multiplicity of infection (number of helper phages / number of Escherichia coli) > 20 to the remaining bacterial solution. Infect at 37 °C for 1 hour. Add 2 mL of 2×YT medium containing 50 μg / mL ampicillin, 200 μg / mL kanamycin, and 0.5 μg / mL IPTG to the infected bacterial solution. Culture at 37 °C and 300 rpm for 12 hours, and then centrifuge to collect the supernatant.

[0033] Step 13) Dilute the endosulfan standard to 2.5 μg / mL with PBS containing 5% skim milk powder. Add 50 μL of the dilution and 50 μL of the supernatant collected in Step 12) into the wells of an ELISA plate coated with 2.5 μg / mL endosulfan monoclonal antibody. At the same time, set 50 μL of PBS with 5% skim milk powder and 50 μL of the supernatant as a control for each clone, and incubate at 37 °C for 1 hour.

[0034] Step 14) Wash 5 times with 0.5‰ PBST. Add 100 μL of HRP-labeled anti-M13 antibody to each well and incubate at 37 °C for 1 hour.

[0035] Step 15) Wash 15 times with 0.5‰ PBST. Add 100 μL of TMB chromogenic solution to each well and react for 15 minutes.

[0036] Step 16) Add 50 μL of 2 M H2SO4 to each well to terminate the reaction. Measure the absorbance at 450 nm with an ELISA reader. The results are shown in the appendix Figure 1 。

[0037] Step 17) For the bacterial solution saved in Step 12) corresponding to the positive clone, perform sequencing. The primer is 5′-TAGTCCTCAAAGCCTCTGTA-3′. Among them, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO. 3 is as shown in SEQ ID NO.7.

[0038] The sequencing results are as follows: Table 1. Polypeptide sequence of endosulfan immune complex Number Sequence SEQ ID NO. 3 CPSYLSPEFC SEQ ID NO. 4 CLDWHPPEFC SEQ ID NO. 5 CMSVMGVMEC SEQ ID NO. 6 CLATGGQTWC Example 2: Determination of endosulfan by non-competitive ELISA Step 1) Determination of the optimal working concentration: Under different combinations of antibody coating concentrations and phage dosages, add 1 μg / mL endosulfan standard (positive) or 5% skim milk powder (negative), compare the signal-to-noise ratio (positive signal / negative signal), and select the combination with the largest signal-to-noise ratio for subsequent experiments.

[0039] Step 2) Coating: Dilute the endosulfan monoclonal antibody to the optimal coating concentration and add 100 μL per well to the ELISA wells, and incubate at 4 °C for 12 hours.

[0040] Step 3) Blocking: After washing with 1‰ PBST, add 300 μL of 5% skim milk powder to each well and incubate at 37 °C for 2 hours.

[0041] Step 4) Add phage polypeptide and endosulfan standard: After washing with 1‰ PBST, add 50 μL of endosulfan standard at a certain concentration and 50 μL of the optimal dosage of phage polypeptide to each well, and incubate at 37 °C for 1 hour.

[0042] Step 5) is the same as steps 14)-16) in Example 1.

[0043] Step 6): Using the endosulfan standard concentration as the abscissa and the absorbance value as the ordinate, fit the non-linear equation with the logistic function in Origin 2021 software to obtain the half-saturation concentration (the analyte concentration required to reach half of the maximum signal, SC 50 ) of different polypeptides. The results are shown in Table 2: Table 2. Optimal antibody and phage concentration combinations and SC of different anti-immunocomplex polypeptides 50 Number Antibody concentration (μg / mL) Phage concentration (pfu / mL) <![CDATA[SC 50 (ng / mL)]]> SEQ ID NO. 3 5.0 <![CDATA[5.0×10 10 > 6.8 SEQ ID NO. 4 5.0 <![CDATA[2.5×10 10 > 35.37 SEQ ID NO. 5 5.0 <![CDATA[2.5×10 10 > 33.12 SEQ ID NO. 6 2.5 <![CDATA[5.0×10 10 > 51.50 The results show that the SC of the endosulfan non-competitive ELISA based on polypeptide CPSYLSPEFC (SEQ ID NO. 3) 50 is 6.8 ng / mL, and the standard curve is as attached Figure 2 . Therefore, the prepared anti-endosulfan immunocomplex polypeptide has great application value in endosulfan immunoassay and can be used to develop a highly sensitive endosulfan detection method.

[0044] Specificity determination of non-competitive ELISA: Under the optimal working buffer, prepare a series of standard solutions of endosulfan structural analogs (chlordane, dieldrin, endosulfan sulfate, heptachlor, DDT) with different concentrations, fit the corresponding standard curves by phage ELISA respectively, and calculate the cross-reactivity rate according to the formula. The cross-reactivity rates of the five endosulfan structural analogs are shown in Table 3.

[0045] Table 3. Cross-reactivity rates of non-competitive immunochromatographic test strips to five endosulfan structural analogs Compound <![CDATA[SC 50 (ng / mL)]]> CR (%) Endosulfan 6.80 100.0 Dieldrin 101.49 6.7 Chlordane 117.24 5.8 Endosulfan sulfate 67.33 10.1 DDT >10000.0 <0.1 Example 3: Detection of spiked samples by non-competitive ELISA Preparation and pretreatment of spiked samples: Crush the soil samples and pass them through a 20-mesh sieve, and crush citrus, apples, and cucumbers with a wall-breaking blender. Take 10 g of the above matrices into 50 mL centrifuge tubes, add 20 mL of the optimal PBS buffer containing 60% methanol, vortex for 10 min, then sonicate for 10 min, then shake at 2500 rpm for 5 min, and then centrifuge at 4000 rpm for 5 min. Take out the supernatant and transfer it to a new 50 mL centrifuge tube, then make up the volume to 30 mL, dilute by an appropriate multiple, and use it for non-competitive ELISA detection. The results are shown in Table 4.

[0046] Table 4. Results of detecting spiked samples by non-competitive immunochromatographic test strips Sample Added concentration (ng / g) Detected concentration (ng / g) Recovery rate (%) Relative standard deviation (%) Citrus 50 35.55±0.92 71.1 2.6 Citrus 100 76.85±2.69 76.9 3.5 Citrus 200 160.92±7.24 80.5 4.5 Citrus 400 326.83±8.29 81.7 3.5 Apple 50 36.25±0.94 72.5 2.6 Apple 100 76.55±3.14 76.6 4.1 Apple 200 158.86±9.85 79.5 6.2 Apple 400 353.72±15.21 88.6 4.3 Cucumber 50 42.55±2.34 85.1 5.5 Cucumber 100 83.72±6.03 83.7 7.2 Cucumber 200 183.81±11.21 91.9 6.1 Cucumber 400 414.84±17.84 103.8 4.3 Soil 50 38.15±0.88 76.3 2.3 Soil 100 81.45±3.10 81.5 3.8 Soil 200 169.43±8.64 84.7 5.1 Soil 400 353.61±16.97 88.4 4.8 The results showed that non-competitive ELISA had good accuracy in detecting endosulfan in soil, citrus, apple and cucumber samples.

[0047] The present invention provides a polypeptide that specifically binds to an endosulfan immune complex, as well as ideas and methods for its application. There are many ways to specifically implement this technical solution. The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented using existing technologies.

Claims

1. A polypeptide that specifically binds to an endosulfan immune complex, characterized in that: The sequence of the polypeptide is the amino acid sequence shown in SEQ ID NO.

3.

2. The polypeptide according to claim 1, characterized in that The polypeptide is cyclized by forming a disulfide bond between the cysteine ​​residues at both ends of the polypeptide.

3. The polypeptide according to claim 1, characterized in that The endosulfan immune complex is a complex formed by the reaction of anti-endosulfan monoclonal antibody and endosulfan.

4. The polypeptide according to claim 3, characterized in that The anti-endosulfan monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO. 1, and the light chain variable region has an amino acid sequence of SEQ ID NO.

2.

5. The polypeptide according to claim 4, characterized in that The anti-endosulfan monoclonal antibody comprises a heavy chain constant region and a light chain constant region of mouse IgG subtype.

6. A gene encoding the polypeptide according to claim 1.

7. The gene according to claim 6, characterized in that The gene has a nucleotide sequence as shown in SEQ ID NO.

7.

8. Use of the polypeptide according to claim 1 or 2, or the gene according to claim 6 or 7 in non-diagnostic detection of endosulfan.

Images