CN120040544A

CN120040544A - Polypeptide specifically combined with endosulfan immune complex and application thereof

Info

Publication number: CN120040544A
Application number: CN202410750493.4A
Authority: CN
Inventors: 华修德; 尤天阳; 马瑾煜; 黄岳; 王鸣华
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2025-05-27

Abstract

The invention discloses a polypeptide specifically combined with a endosulfan antibody immune complex and application of the polypeptide in endosulfan immune detection. According to the invention, immune complexes formed by the endosulfan monoclonal antibodies and endosulfan are utilized, specifically bound polypeptides are elutriated from a phage display random polypeptide library, and positive clones and polypeptide sequences thereof are obtained through ELISA and Sanger sequencing. The half-saturated concentration of the non-competitive phage ELISA established based on the optimal polypeptide is 6.8 ng/mL, and can be used for sensitively and highly specifically detecting the endosulfan residue in the environment and agricultural products.

Description

Polypeptide specifically combined with endosulfan immune complex and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide specifically combined with a endosulfan immune complex and application thereof in enzyme-linked immunosorbent assay.

Background

The endosulfan (Endosulfan) is an organic chlorine pesticide, has the characteristics of high efficiency, broad spectrum, long lasting period and the like, belongs to toxic and persistent organic pollutants, and has been listed in forbidden pesticide books due to high toxicity and long half-life. The endosulfan has the characteristics of difficult degradation, high fat solubility, enrichment and amplification in food chains, and the like, is a pollutant which has extremely high toxicity and can be subjected to global migration through various transmission paths, and has stronger toxicity to almost all kinds of organisms. The maximum residue limit standard (GB 2763-2021) of pesticides in Chinese food prescribes that the maximum residue limit of the endosulfan on citrus, apples and cucumbers is 0.05 mg/kg. The accurate and rapid detection of the endosulfan residue in the environment and food is of great significance in guaranteeing the safety of the food and the environment.

The immunoassay technology is a kind of analysis technology which is based on the specific reaction of an antibody-antigen, and converts the combination of the antibody and an object to be detected into a measurable signal through tracers such as enzyme, fluorescein and the like so as to realize qualitative and quantitative detection of the object to be detected. Compared with the instrument analysis technology, the immunoassay has the advantages of rapidness, simplicity, sensitivity, economy and the like, does not need expensive detection instruments and professional operators, is suitable for high-throughput and on-site detection, is used as the supplement of the instrument analysis technology, and has wide application prospect in the field of pesticide residue detection. As a small molecule compound, the molecular weight of the pesticide is usually less than 1 KD, the pesticide cannot independently stimulate the organism to generate immune response, the pesticide can stimulate the organism to generate antibodies after being coupled with carrier protein with larger molecular weight (immune antigen), and only one epitope can be combined with one antibody. The small molecule compounds often immobilize the modified antigen (i.e., the coated antigen) on the solid phase interface, compete with the analyte for binding to the antibody site, and the amount of the analyte is characterized by a decrease in signal intensity, with the analyte concentration being inversely related to the signal intensity, known as a competitive immunoassay. After the small molecule compound binds to its antibody, most of the structure will be encapsulated by the antibody, forming an antigen-antibody immune complex, but a small portion will be exposed to the solvent. The small molecule exposed outside and adjacent part of the antibody is regarded as a new epitope, and the anti-immune complex antibody or polypeptide which can generate specific action with the small molecule is screened, so that the non-competitive immune analysis of the small molecule compound can be realized. Compared with the competition mode, the non-competition immunoassay has better sensitivity, accuracy and specificity, and is more suitable for the field rapid detection technology of test strips, biosensors and the like. In addition, the signal value of a non-competitive immunoassay is generally proportional to the analyte content, and is more intuitive when the results are interpreted. Therefore, the non-competitive detection mode has great potential in immunoassay of small molecules such as pesticides.

The phage display technology can insert the foreign protein gene into the filiform phage coat protein gene under the condition of not affecting the normal function of phage, so that the foreign protein is expressed on the filiform phage coat protein. Because of the characteristics of simplicity, high efficiency and short experimental period, phage display technology is widely applied to the preparation of polypeptides capable of recognizing pesticides, antibody antigenic determinants and immune complexes in the field of rapid detection of pesticide residues. The pesticide small molecule non-competitive immunoassay established by the anti-immune complex polypeptide is superior to the competitive assay in sensitivity and specificity. At present, no related report of the anti-immune complex polypeptide of the endosulfan exists.

Disclosure of Invention

The invention aims to solve the technical problem of providing a polypeptide specifically combined with a endosulfan immune complex and application of the polypeptide in enzyme-linked immunosorbent assay aiming at the defects of the existing endosulfan rapid detection technology.

The aim of the invention is realized by the following technical scheme:

A polypeptide specifically combined with a endosulfan immune complex, wherein the sequence of the polypeptide is an amino acid sequence shown as SEQ ID NO. 3.

Wherein disulfide bonds are formed between cysteine residues at two ends of the polypeptide to cyclize the polypeptide.

Wherein, the preparation method of the polypeptide comprises the following steps:

(1) Fixing anti-endosulfan monoclonal antibody 4H4 on an ELISA plate, adding endosulfan to form immune complex, and adding phage display polypeptide library for panning. The eluted phage were amplified and used for the next round of panning. Three rounds of panning were performed in total. The content of a surfactant (Tween-20) in the washing liquid is sequentially increased in each round, and the coating concentration of the endosulfan antibody is reduced;

(2) After panning was completed, phage clones were selected, DNA sequences were determined, and the sensitivity of the different phage polypeptides was determined. The obtained polypeptide has the sequence shown as 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 comprises a cyclic structure formed by disulfide cysteine at two ends;

wherein, the endosulfan immune complex is a complex formed by the reaction of an anti-endosulfan monoclonal antibody and endosulfan.

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

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.

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

In a second aspect of the present invention, there is provided a gene encoding the polypeptide specifically binding to the endosulfan immune complex, wherein the gene has a nucleotide sequence shown in SEQ ID NO. 7.

In a third aspect, the present invention provides the use of a polypeptide specifically binding to an immune complex of endosulfan, or a gene thereof, as described above in the non-diagnostic detection of endosulfan.

Wherein, the application in the non-diagnostic detection of the endosulfan comprises, but is not limited to, an enzyme-linked immunosorbent assay method.

The invention has the following beneficial effects:

(1) For the first time, polypeptides capable of specifically binding to the endosulfan immune complex were reported;

(2) By utilizing the polypeptide provided by the invention, a non-competitive immunoassay method of the endosulfan can be established;

(3) The semi-saturation concentration (SC ₅₀) of the non-competitive ELISA based on the anti-immune complex polypeptide is 6.8 ng/mL, the cross reaction rate of the non-competitive ELISA based on the anti-immune complex polypeptide is less than 10.1%, and the reaction specificity is higher than that of the prior art.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 shows ELISA results for verifying the ability of phage clones obtained from the third round of panning to specifically recognize endosulfan immune complex, with the abscissa indicating the serial number of phage clones and the ordinate indicating absorbance.

FIG. 2 is a standard curve of a non-competitive ELISA based on anti-immune complex polypeptides for detecting endosulfan, with the abscissa representing endosulfan concentration (in ng/mL) and the ordinate representing absorbance at 450 nm.

Detailed Description

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. Specific materials and sources thereof used in embodiments of the present invention are provided below. It will be understood that these are merely exemplary and are not intended to limit the invention, as materials identical or similar to the type, model, quality, nature or function of the reagents and instruments described below may be used in the practice of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 panning of anti-endosulfan immune Complex Polypeptides

Step 1) the endosulfan monoclonal antibody 4H4 is preserved by the laboratory, the preparation method is shown 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;, and the constant regions of the antibody are the constant regions of the heavy chain and the constant regions of the light chain of the IgG subtype from mice. Anti-endosulfan monoclonal antibody 4H4 was diluted to 10 μg/mL with phosphate buffered saline (PBS, ph=7.4), 100 μl per well was added to a 96-well elisa plate and coated for 12 hours at 4 ℃.

Step 2) the coated wells were washed five times with PBS (PBST) containing 1%Tween-20, then blocked with 300. Mu.L of 5% nonfat dry milk per well for 1.5 hours, and washed 5 times with 1%PBST.

Step 3) 100. Mu.L of 5. Mu.g/mL endosulfan standard was added to each well and incubated at 25℃for 1 hour at 300 rpm times with 1%.

Step 4) phage display random circular seven, eight, nine, decapeptide libraries (1×10 ¹¹ pfu each) were added per well and incubated at 25 ℃ for 300: 300 rpm for 1 hour.

Step 5) 100 μl of elution solution (0.2M glycine-hydrochloric acid solution containing 10% BSA, ph=2.2) was added per well, and incubated at 25 ℃ for 15 min at 300: 300 rpm.

Step 6) 15 μl of neutralization solution (1M Tris-HCl, ph=9.1) was added per well, the supernatant was collected after mixing, 1 μl of the phage titer was measured and the rest was stored to 4 ℃.

Phage titer assay method was as follows:

Phage-containing supernatants were diluted to 106, 108 and 1010, 10. Mu.L each was added to 100. Mu.L of ER2738 E.coli in the logarithmic growth phase, and infected at 37℃for 30 minutes. The bacterial solution was spread on LB solid medium containing 20. Mu.g/mL tetracycline and cultured at 37℃for 12 hours. Phage titer (pfu/mL) =100×dilution×colony count was calculated from the colony count on the plate.

Step 7) the supernatant collected in step 6) was added to ER2738 E.coli in the logarithmic growth phase at 3 mL, infected for 1 hour at 37℃and grown in an expanded culture of 50 mL in LB liquid medium at 37℃and 250 rpm to the logarithmic growth phase, and helper phage M13KO7 at a complex number of infection (helper phage number/E.coli number) >20 was added and infected for 1 hour at 37 ℃.

Step 8) the bacterial liquid was centrifuged, and the pellet was collected and added to 100mL SB medium containing 25. Mu.g/mL IPTG and cultured at 37℃for 12 hours at 250℃ 250 rpm.

Step 9) the supernatant was collected after centrifugation and added to 25 mL of 20% PEG2000, 2.5M NaCl solution and allowed to stand on ice for 4 hours.

Step 10) 14000 g, centrifugation at 4℃for 30min, discarding the supernatant, resuspension of the pellet with sterile PBS, addition of an equal volume of glycerol, determination of titer, storage at-20 ℃.

Step 11) steps 1) to 10) are performed in the first round of panning, the second round and the third round of panning are performed in the same round of panning, but the coating concentration of the endosulfan monoclonal antibody is 5 and 2.5 mug/mL respectively, and the content of Tween-20 in PBST is 3 permillage and 5 permillage, so that anti-immune complex polypeptide with stronger affinity is obtained.

Step 12) 30 clones were picked from the third titer plate and incubated in 1mL 2 XYT medium containing 50. Mu.g/mL ampicillin and 20. Mu.g/mL tetracycline at 37℃for 4 hours at 300 rpm. 500 mu L of the culture medium is stored, and the residual bacterial liquid is added with helper phage M13KO7 with complex infection number (helper phage number/escherichia coli number) >20 and is infected for 1 hour at 37 ℃. To the infected bacterial liquid, 2 XYT medium containing 50. Mu.g/mL of ampicillin, 200. Mu.g/mL of kanamycin and 0.5. Mu.g/mL of IPTG was added, and the mixture was cultured at 37℃for 300 rpm for 12 hours, followed by centrifugation to obtain a supernatant.

Step 13) Sudan standard was diluted to 2.5. Mu.g/mL with PBS containing 5% nonfat milk powder, 50. Mu.L of the diluted solution and 50. Mu.L of the supernatant collected in step 12) were added to the wells of the enzyme-labeled plate coated with 2.5. Mu.g/mL Sudan monoclonal antibody, while 50. Mu.L of PBS containing 5% nonfat milk powder and 50. Mu.L of the supernatant were set for each clone as a control, and incubated at 37℃for 1 hour.

Step 14) 0.5%PBST washing 5 times, each hole added with 100 u L HRP labeled anti M13 antibody, 37 degrees C1 hours incubation.

Step 15) 0.5%PBST washing 15 times, each hole added with 100 u L TMB color development liquid, reaction for 15 minutes.

Step 16) the reaction was terminated by adding 50. Mu.L of H2SO4 of M to each well, and the absorbance at 450 nm was measured using a microplate reader, as shown in FIG. 1.

Step 17) the bacterial liquid stored in step 12) corresponding to the positive clone is sequenced, and the primer is 5'-TAGTCCTCAAAGCCTCTGTA-3'. Wherein the nucleotide sequence of the polypeptide sequence of the SEQ ID NO. 3 is shown as SEQ ID NO. 7.

Sequencing results were as follows:

TABLE 1 anti-endosulfan immune complex polypeptide sequence

Numbering device	Sequence(s)
		SEQ ID NO. 3	CPSYLSPEFC
SEQ ID NO. 4	CLDWHPPEFC
		SEQ ID NO. 5	CMSVMGVMEC
SEQ ID NO. 6	CLATGGQTWC

EXAMPLE 2 non-competitive ELISA determination of endosulfan

Step 1) determining the optimal working concentration, namely adding 1 mug/mL endosulfan standard (positive) or 5% skimmed milk powder (negative) under different antibody coating concentration and phage dosage combinations, comparing signal-to-noise ratio (positive signal/negative signal), and selecting the combination with the maximum signal-to-noise ratio for subsequent experiments.

Step 2) coating, namely diluting the endosulfan monoclonal antibody to the optimal coating concentration, adding 100 mu L of the endosulfan monoclonal antibody into the enzyme-labeled wells, and incubating at 4 ℃ for 12 hours.

Step 3) blocking, namely adding 300 mu L of 5% skimmed milk powder into each well after 1 millPBST washing, and incubating for 2 hours at 37 ℃.

Step 4) adding phage polypeptide and endosulfan standard substance, namely adding 50 mu L of endosulfan standard substance with a certain concentration and 50 mu L of phage polypeptide with the optimal dosage into each hole after washing by 1 per mill of PBST, and incubating for 1 hour at 37 ℃.

Step 5) steps 14) to 16) are the same as in example 1).

Step 6) fitting a nonlinear equation with the thiodan standard concentration on the abscissa and the absorbance on the ordinate using a logistic function in Origin 2021 software to obtain the half-saturated concentrations of the different polypeptides (the analyte concentration required to reach half of the maximum signal, SC ₅₀), the results are shown in table 2:

TABLE 2 optimal antibody and phage concentration combinations for different anti-immune Complex polypeptides and SC ₅₀

Numbering device	Antibody concentration (μg/mL)	Phage concentration (pfu/mL)	SC₅₀ (ng/mL)
				SEQ ID NO. 3	5.0	5.0×10¹⁰	6.8
SEQ ID NO. 4	5.0	2.5×10¹⁰	35.37
				SEQ ID NO. 5	5.0	2.5×10¹⁰	33.12
SEQ ID NO. 6	2.5	5.0×10¹⁰	51.50

The results showed that SC ₅₀ based on the endosulfan non-competitive ELISA of polypeptide CPSYLSPEFC (SEQ ID NO. 3) was 6.8 ng/mL, with a standard curve as shown in FIG. 2. Therefore, the prepared anti-endosulfan immune complex polypeptide has great application value in endosulfan immune detection, and can be used for developing a endosulfan detection method with high sensitivity.

And (3) specificity measurement of non-competitive ELISA, namely preparing a series of standard solutions of the endosulfan structural similar compounds (chlordane, diels, endosulfan sulfate, heptachlor and driping nasal discharge) with different concentrations under the optimal working buffer, respectively fitting corresponding standard curves through PHAGE ELISA, and calculating the cross reaction rate according to a formula. The cross-reactivity of the five endosulfan structural analogs is shown in table 3.

TABLE 3 Cross-reactivity of non-competitive immunochromatographic test strips to five structural analogues of endosulfan

Compounds of formula (I)	SC₅₀(ng/mL)	CR(%)
			Sudan (pill) for treating cancer	6.80	100.0
Dirichter reagent	101.49	6.7
			Chlordane	117.24	5.8
Sudan sulfate	67.33	10.1
			Drop nasal discharge	>10000.0	<0.1

Example 3 detection of added samples by non-competitive ELISA

And (3) preparing and pre-treating an added sample, namely crushing a soil sample, sieving the crushed soil sample with a 20-mesh sieve, and crushing citrus, apples and cucumbers by using a broken wall cooking machine. The substrate is respectively taken 10g to 50 mL centrifuge tubes, 20mL optimal PBS buffer containing 60% methanol is added, after vortexing for 10min, ultrasound is carried out for 10min, 2500 rpm is oscillated for 5min, 4000 rpm is centrifuged for 5min, the supernatant is taken out and transferred to a new 50 mL centrifuge tube, then the volume is fixed to 30 mL, and after proper multiple dilution, the substrate is used for non-competitive ELISA detection. The results are shown in Table 4.

TABLE 4 results of detection of the added samples by the non-competitive immunochromatographic test strip

Sample of	Concentration (ng/g)	Concentration (ng/g)	Recovery (%)	Relative standard deviation (%)
					Citrus fruit	50	35.55±0.92	71.1	2.6
Citrus fruit	100	76.85±2.69	76.9	3.5
					Citrus fruit	200	160.92±7.24	80.5	4.5
Citrus fruit	400	326.83±8.29	81.7	3.5
					Apple tree	50	36.25±0.94	72.5	2.6
Apple tree	100	76.55±3.14	76.6	4.1
					Apple tree	200	158.86±9.85	79.5	6.2
Apple tree	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 and method for producing soil	50	38.15±0.88	76.3	2.3
Soil and method for producing soil	100	81.45±3.10	81.5	3.8
					Soil and method for producing soil	200	169.43±8.64	84.7	5.1
Soil and method for producing soil	400	353.61±16.97	88.4	4.8

The results show that the non-competitive ELISA has better accuracy in detecting the endosulfan in soil, citrus, apple and cucumber samples.

The invention provides a polypeptide specifically combined with a endosulfan immune complex, an application idea and a method thereof, and a method and a way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to a person skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A polypeptide specifically combined with a endosulfan immune complex, which is characterized in that the sequence of the polypeptide is an amino acid sequence shown as SEQ ID NO. 3.

2. The polypeptide of claim 1, wherein disulfide bonds are formed between cysteine residues at both ends of the polypeptide and are cyclized.

3. The polypeptide of claim 1, wherein the endosulfan immune complex is a complex formed by reacting an anti-endosulfan monoclonal antibody with endosulfan.

4. The polypeptide of claim 3, wherein the anti-endosulfan monoclonal antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 1 and a light chain variable region having the amino acid sequence of SEQ ID No. 2.

5. The polypeptide of claim 4, wherein the anti-endosulfan monoclonal antibody comprises a heavy chain constant region and a light chain constant region of murine IgG subtype.

6. A gene encoding the polypeptide of claim 1.

7. The gene according to claim 6, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 7.

8. Use of the polypeptide of claim 1 or 2, or the gene of claim 6 or 7, in a non-diagnostic assay for endosulfan.