Ovalbumin shark single-domain antibody and application thereof

Abstract

The application discloses ovalbumin shark single-domain antibody and application thereof, and the amino acid sequence is shown as SEQ ID NO. 01 or SEQ ID NO. 02. The application is derived from striped spiny dogfish, has the characteristics of small molecular weight, easy modification and expression, low immunogenicity, high antigen binding, strong tissue penetration and the like, and is suitable for application in multiple fields such as immune analysis detection of ovalbumin (OVA) and protein structure research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of single-domain antibodies, and particularly relates to a shark-derived single-domain antibody of ovalbumin and application thereof. BACKGROUND

[0002] Ovalbumin (OVA) is the most abundant protein in egg white, which is widely used in the food industry due to its good gelation, emulsification and water retention. However, OVA is also one of the main allergens in egg white, which is stable in nature and difficult to be destroyed. When ingested by the human body, OVA can trigger an IgE-mediated immediate allergic reaction, leading to allergic reactions in the respiratory tract, digestive tract and skin, and even can endanger life. Therefore, the detection of OVA in food has important practical significance.

[0003] The detection methods of OVA mainly include mass spectrometry and immunoassay. Immunoassay includes Western Blot, ELISA and LFIS. The detection principle of these methods relies on the ability of anti-OVA antibodies to specifically recognize and bind to OVA antigens in food matrix, and produce color reaction to realize detection. Although traditional anti-OVA monoclonal antibodies or polyclonal antibodies play an important role in these immunoassay techniques, due to their difficulty in expression, poor stability, difficulty in modification, and inability to recognize hidden epitopes, the detection sensitivity is limited, which limits their further application.

[0004] In the late 1980s, the successful separation of single heavy chain variable region domain opened a new chapter of single domain antibody research. In 1995, Greenberg et al. found heavy chain antibodies naturally lacking light chains in nurse sharks, which are called immunoglobulin new antigen receptors (IgNAR). Antibodies composed entirely of antibody heavy chain or light chain variable domains are called single-domain antibodies (sdAb). Shark-derived sdAb is called variable domain of immunoglobulin new antigen receptor (VNAR). The molecular weight of VNAR is only one-tenth of that of traditional antibodies, about 12-17 kDa, close to the nanometer level, so it is also called nanobody. Compared with traditional antibodies, the complementarity determining region 1 (CDR1) and CDR3 of VNAR are longer than those of ordinary antibodies, and the structural composition of the antigen binding loop is larger. The CDR3 of part of VNAR can form a disulfide bond with the framework region 2 (FR2), so VNAR has high stability, high affinity, high specificity, low preparation cost, and the advantage of resisting complex external environment, which is obviously superior to traditional antibodies. SUMMARY

[0005] The present application aims to provide an ovalbumin shark single-domain antibody.

[0006] Another object of the present application is to provide the application of the above-mentioned ovalbumin shark single-domain antibody.

[0007] The technical solutions of the present application are as follows:

[0008] An ovalbumin shark single-domain antibody, the amino acid sequence of which is shown in SEQ ID NO. 01 or SEQ ID NO. 02.

[0009] In a preferred embodiment of the present application, the nucleotide sequence is shown in SEQ ID NO. 03 or SEQ ID NO. 04.

[0010] The use of the above-mentioned ovalbumin shark single-domain antibody in the preparation of an ovalbumin in vitro detection component.

[0011] In a preferred embodiment of the present application, the ovalbumin in vitro detection component includes a colloidal gold immunochromatographic detection component, a fluorescent immunochromatographic detection component, an enzyme-linked immunosorbent assay detection component, a lateral flow immunochromatographic detection component, and an immunoblotting detection component.

[0012] An ovalbumin in vitro detection component has the above-mentioned ovalbumin shark single-domain antibody.

[0013] In a preferred embodiment of the present application, it is a colloidal gold immunochromatographic detection component, a fluorescent immunochromatographic detection component, an enzyme-linked immunosorbent assay detection component, a lateral flow immunochromatographic detection component or an immunoblotting detection component.

[0014] An ovalbumin in vitro detection method is applied to the above ovalbumin in vitro detection component.

[0015] The present application has the following advantages:

[0016] 1. The present application is derived from the striped catshark and has the characteristics of small molecular weight, easy modification and expression, low immunogenicity, high antigen binding property and strong tissue penetration, which makes it suitable for the immunological analysis and detection of ovalbumin (OVA) and the study of protein structure in multiple fields.

[0017] 2. The present application is equivalent to the original heavy chain antibody in terms of structural stability and antigen binding activity, and is the smallest unit known to bind to target antigens, which has strong specificity, good affinity and high stability, making it suitable for a wide range of applications.

[0018] 3. The present application has high solubility and strong renaturation ability, and can be obtained in large quantities through a recombinant expression system.

[0019] 4. The present application not only solves the problem of insufficient research on shark-derived single-domain antibodies, but also optimizes the shortcomings of traditional human monoclonal antibodies, such as large molecular weight, weak affinity and complex operation. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the reduced SDS-PAGE pattern of OVA isolated and purified in Example 1 of the present application; wherein M, standard protein; 1, OVA.

[0021] Figure 2 is the mass spectrometry identification result of OVA in Example 1 of the present application;

[0022] Figure 3 is the ELISA result graph of the monoclonal phage in Example 1 of the present application;

[0023] Figure 4 is the amino acid sequence alignment result graph of the single-domain antibody in Example 1 of the present application;

[0024] Figure 5 is the SDS-PAGE result graph of the single-domain antibody Fc fusion protein in Example 1 of the present application; wherein M, standard protein.

[0025] Figure 6Figure showing the affinity between the single-domain antibody and OVA in Example 1 of the present application using BLIz for characterization; wherein the solid line is the kinetic curve of real-time monitoring, the dotted line is the curve fitted by software, and the kinetic curves of different OVA concentration gradients from top to bottom correspond to the concentrations from top to bottom on the right side identification in turn.

[0026] Figure 7 Figure showing the determination results of the single-domain antibody Tm of the present application in Example 1 of the present application using circular dichroism.

[0027] Figure 8 Figure showing the results of verifying the specificity of the single-domain antibody of the present application in Example 1 of the present application using Western Blot. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described and explained in detail below by specific embodiments in conjunction with the accompanying drawings.

[0029] Example 1

[0030] (1) Purification of antigen ovalbumin: based on the prior art (Geng, Xie, Wang, et al. Large-scale purification of ovalbumin using polyethylene glycol precipitation and isoelectric precipitation [J]. Poultry Science, 2019, 98(3): 1545-1550. DOI: 10.3382 / ps / pey402. and Zhao Yongjuan, Weng Ling, Yan Longjie, et al. Competitive ELISA detection of egg white content in surimi products [J]. Food Science, 2017, 38(14): 284-289. DOI: 10.7506 / spkx1002-6630-201714044.), the egg white was diluted with 50 mmol / L NaCl solution at a ratio of 1:3 (v:v), and stirred at room temperature for 2 h. The pH value of the solution was adjusted to pH 5.0, and after standing for 0.5 h, the supernatant was taken by centrifugation. Polyethylene glycol 8000 (PEG 8000) was added to the egg white solution in three times, and the final PEG 8000 addition amount was 15% (v:w) of the solution volume, and after standing for 0.5 h, the supernatant was taken by centrifugation. The supernatant was dialyzed in 20 mmol / L Tris-HCl buffer to equilibrium, and then loaded on a DEAE-ion exchange chromatography column, and then separated and purified with 20 mmol / L Tris-HCl buffer containing 0-0.2 mol / L NaCl, and the third protein elution peak was collected as OVA.

[0031] The third protein elution peak was collected and analyzed by reducing SDS-PAGE, and the final results are shown in Figure 1 The results show that there is only a single band at the position of about 45 kDa. In order to verify whether the protein band is OVA, mass spectrometry was performed after cutting the gel, and the detection results are shown in Figure 2 The results show that the protein band at the position of about 45 kDa is OVA, and the purity is high.

[0032] (2) Immunize the striped bonnet shark with ovalbumin: The OVA purified in step (1) is mixed with Freund's complete adjuvant at a volume ratio of 1:1 as an antigen, and after complete emulsification, it is injected subcutaneously into the lateral fin of the striped bonnet shark, and this subcutaneous injection is the first immunization; the single immunization dose is 100 μg per striped bonnet shark; the striped bonnet shark is immunized a total of four times, each time using subcutaneous injection, and the interval between subcutaneous injections is 20 days.

[0033] (3) Constructing a phage synthetic library: In order to construct a VNAR synthetic library with randomized CDR3 region, the natural framework of VNAR is amplified by three-step PCR:

[0034] A. The first step PCR uses cDNA extracted from peripheral blood lymphocytes of striped bonnet shark as template, and amplifies the CDR1-FR3 region of shark-derived single-domain antibody VNAR using specific primers and high-fidelity enzyme Prime STAR Max DNA Polymerase, and the amplification program is: 94℃, 5min, 98℃, 10s, 50℃, 15s, 72℃, 30s, 72℃, 5min, a total of 35 cycles;

[0035] B. The second step PCR uses the product of the first step PCR as template, and amplifies the FR1 to CDR3-FR4 region of shark-derived single-domain antibody VNAR using specific primers, and the amplification program is: 95℃, 3min, 98℃, 10s, 68℃, 5s, 72℃, 25s, 72℃, 1min, a total of 30 cycles;

[0036] C. The third step PCR: using the product of the second step PCR as template, the specific primers are used to amplify the pR2 vector homologous sequence to both ends, and the amplification program is: 95℃, 3min, 98℃, 10s, 68℃, 5s, 72℃, 25s, 72℃, 1min, a total of 30 cycles, and the amplified VNAR fragment is recovered using a kit.

[0037] Then, the pR2 phagemid was used as a template for specific primer amplification. The DNA polymerase used for amplification was high-fidelity enzyme Prime STAR® GXL DNA Polymerase. The amplification program was as follows: denaturation at 98°C for 10 s, annealing at 57°C for 15 s, extension at 72°C for 25 s, and a total of 35 cycles. The product was recovered using a kit, and the pR2 amplification product was digested. The digestion was performed in a 37°C water bath for 1 h, and the enzyme digestion site was Dnp I. The VNAR target gene product was recovered and subjected to seamless cloning with the pR2 digestion product. The ligation product was recovered using a kit. The ligation product was mixed with TG1 cells at a volume ratio of 1:20, and then electroporation was performed at a voltage of 2.5 kv and a time of 5 ms. The mixture was then incubated at 37°C for 1 h. The bacterial liquid was gradiently diluted (0.2 μL, 0.02 μL, and 0.002 μL of the original liquid were taken, respectively), and then spread on a 9 cm LB / Amp / 2% glucose plate. The plate was incubated at 37°C overnight. Then, 2xYT liquid medium containing 20% glycerol was added to the plate, and the bacterial lawn was scraped with a spreader and collected in a centrifuge tube. After vortex mixing, the mixture was aliquoted, frozen in liquid nitrogen, and stored at -80°C. The phage synthesis library was obtained.

[0038] (4) Screening of specific single-domain antibodies: The bacteria of the frozen phage synthesis library were activated, and KM13 helper phage was added. The bacterial culture supernatant was taken, and the phage titer was measured. This was the amplified phage. The 96-well immunoplate was coated with OVA at a final concentration of 0.1 mg / mL. The amplified phage was added at a concentration of 1x10 11 pfu. The plate was incubated at room temperature for 1 h. The phage specifically bound to OVA was eluted with a trypsin solution at a final concentration of 2 mg / mL, and then the phage was infected into TG1 bacteria. 5 μL and 1 μL of the infected bacterial liquid were spread on solid plates, respectively, and the total number of colonies was recorded. Ninety-five single colonies were randomly picked from the plates, and then activated overnight. KM13 helper phage was added, and the supernatant after lysis was collected by centrifugation. This was the monoclonal phage. The 96-well immunoplate was coated with ovalbumin at a concentration of 1 μg / mL. The monoclonal phage solution prepared above was added to each well, and the plate was incubated at room temperature for 1 h. The phage bound to ovalbumin was captured using HRP-anti M13 antibody, and then the reaction was developed with TMB substrate for 10 min. The reaction was terminated with a 1 mol / L H2SO4 solution. The OD 450 value was recorded by an enzyme-labeled instrument. The results of the panning are shown in Figure 3

[0039] The wells with an OD 450 value greater than 1.0 were picked for sequencing analysis. The sequencing primer was as follows: 5'-caggaaacagctatgac-3'

[0040] ​(SEQ ID NO. 05). Two different single-domain antibody sequences were obtained after alignment analysis of the measured antibody sequences, excluding repeated clones, and the alignment of the two is shown in Figure 4 The specific information of the single-domain antibody is as follows:

[0041] Name: 14-A2

[0042] Source: Chiloscyllium plagiosum

[0043] Amino acid sequence:

[0044] AQRVEQTPTTTTKEAGESLTINCVLRDSSYALGSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLR ISDLRVEDSGTYHCKAYRGYQLFWLIRPSPAEGGGTILTVKA (SEQ ID NO. 01)

[0045] Nucleotide sequence:

[0046] gcacaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagcta

[0047] tgcattgggtagcacgtactggtatttcacaaaaaagggcgctacaaagaaggagagtttatcaaatggcggacgatacgcggaaacagt

[0048] gaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgtatcgggggtaccagctgttctggttgatcaggccgagtcccgcggaaggaggcggcaccattctgactgtaaaagca (SEQ ID NO. 03);

[0049] Name: 14-A8

[0050] Source: Chiloscyllium plagiosum

[0051] Amino acid sequence:

[0052] AQRVEQTPTTTTKEAGESLTINCVLKGSSYALGSTYWYFTKKGATKKESLSNGGRYAET VNKASKSFSLRISDLRVEDSGTYHCEAYHTNPWLIAFWPRYAEGGGTILTVKA (SEQ ID NO. 02)

[0053] Nucleotide sequence:

[0054] gcacaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaaaaggttccagcta

[0055] tgcattgggtagcacgtactggtatttcacaaaaaagggcgctacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagt

[0056] gaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtgaagcgtatcatactaatccgtggctgattgccttttggcctcgctatgccgaaggaggcggcaccattctgactgtaaaagca (SEQ ID NO. 04);

[0057] (5) Recombinant expression and purification of single domain antibody: The single domain antibodies 14-A2 and 14-A8 obtained in step (4) were respectively constructed into a mammalian expression vector pTT5 with a signal peptide, so as to be expressed in fusion with human IgG1 Fc, the Fc fragment being expressed at the C terminal end and the single domain antibody being obtained by TEV enzyme digestion. The recombinant plasmid was transfected into HEK 293F cells, the culture supernatant was collected, and the single domain antibody Fc fusion protein was purified by rProtein A affinity chromatography column. As shown in FIG. 2, the molecular weight of the Fc fusion protein of 14-A2 and 14-A8 is about 42 kDa. Figure 5

[0058] (6) Property characterization of the single domain antibody:

[0059] ​The BLIz was used to characterize the affinity of 14-A2 and 14-A8 to OVA. First, OVA was labeled with biotin to obtain biotinylated OVA (Bio-OVA). Then, the Bio-OVA was immobilized on a SA biosensor, and different single-domain antibody concentrations were set to detect the affinity of different single-domain antibody Fc fusion protein concentrations to OVA. The results are shown in Figure 6 The binding strength of the two single-domain antibodies to OVA was in the order of 14-A2 > 14-A8, and the affinity constants K D were 9.35 x 10 -6 M and 5.14 x 10 -5 M, respectively, reaching the micromolar level. The affinity of the original heavy chain antibody was generally in the micromolar to high micromolar range, and thus the affinities of 14-A2 and 14-A8 both reached the level of the original heavy chain antibody.

[0060] Circular dichroism was used to determine the Tm of 14-A2 and 14-A8 to characterize their stability. The results are shown in Figure 7 The Tm of 14-A2 and 14-A8 reached about 86°C and 87°C, respectively, and they had high thermal stability.

[0061] Western Blot was used to verify whether 14-A2 and 14-A8 had cross-reactivity with other proteins in chicken egg white and duck egg white in addition to OVA and proteins in surimi. The results of antibody specificity are shown in Figure 8 Both 14-A2 and 14-A8 only reacted with OVA and had no cross-reactivity with other proteins in egg white and surimi.

[0062] The physicochemical properties of 14-A2 and 14-A8 were predicted by the ExPASy website (http: / / web.expasy.org / protparam). The prediction results are shown in the table below. The average GRAVY of 14-A2 and 14-A8 was -0.548 and -0.521, respectively, indicating that 14-A2 and 14-A8 had a high content of hydrophilic amino acids and high solubility.

[0063] Prediction of physicochemical properties of 14A2 and 14A8

[0064]

[0065] Note: GRAVY (average GRAVY), positive value: protein tends to be hydrophobic; negative value: protein tends to be hydrophilic.

[0066] The above merely describes preferred embodiments of the present application, and therefore cannot limit the scope of the present application, i.e. equivalent changes and modifications made according to the patent scope and content of the present application should still be within the scope of the present application.

Claims

1. An ovalbumin shark single-domain antibody, characterized in that: The amino acid sequence is shown as SEQ ID NO. 01 or SEQ ID NO.

02.

2. Use of the ovalbumin shark single-domain antibody of claim 1 in the preparation of an ovalbumin in-vitro detection component.

3. Use according to claim 2, characterized in that: The ovalbumin in-vitro detection component includes a colloidal gold immunochromatographic detection component, a fluorescent immunochromatographic detection component, an enzyme-linked immunosorbent assay detection component, and an immunoblotting detection component.

4. An ovalbumin in vitro test assembly characterized by: The ovalbumin shark single-domain antibody of claim 1.

5. An ovalbumin in vitro test kit as claimed in claim 4, characterised in that: It is a colloidal gold immunochromatographic detection component, a fluorescent immunochromatographic detection component, an enzyme-linked immunosorbent assay detection component, or an immunoblotting detection component.

6. A method for the in vitro detection of ovalbumin for non-diagnostic purposes, characterized in that: The ovalbumin in-vitro detection component of claim 4 or 5 is applied.

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