Ovalbumin shark source single-domain antibody and application thereof

Abstract

The invention discloses an ovalbumin shark-source single-domain antibody and application thereof. The amino acid sequence of the ovalbumin shark-source single-domain antibody is shown as SEQ ID NO.01 or SEQ ID NO.02. The invention further discloses a preparation method of the The antibody is derived from mottled bamboo shark, has the characteristics of small molecular weight, easiness in transformation and expression, low immunogenicity, high antigen binding property, strong tissue penetrability and the like, and is suitable for a plurality of fields such as ovalbumin (OVA) immunoassay detection, protein structure research and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of single domain antibodies, and specifically relates to an ovalbumin shark-derived single domain antibody and an application thereof. Background Art

[0002] Ovalbumin (OVA) is the most abundant protein in egg white. It is widely used in the food industry due to its good gelling, emulsifying and water-retaining properties. However, OVA is also one of the main allergens in egg white. It is stable and its allergenicity is not easily destroyed. When ingested by the human body, OVA may trigger an immediate allergic reaction mediated by IgE, leading to allergic reactions in the respiratory tract, digestive tract and skin, and may even be life-threatening. Therefore, the detection of OVA in food has important practical significance.

[0003] The detection methods of OVA mainly include mass spectrometry and immunoassay. Immunoassay covers Western Blot, enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatography (LFIS). The detection principles of these methods rely on the ability of anti-OVA antibodies to specifically recognize and bind to OVA antigens in food matrices to produce a color reaction for detection. Although traditional anti-OVA monoclonal antibodies or polyclonal antibodies have played an important role in these immunoassay techniques, they are difficult to express, have poor stability, are difficult to modify, and cannot recognize hidden epitopes, resulting in limited detection sensitivity, which limits their further application.

[0004] In the late 1980s, the successful separation of a single heavy chain variable region domain opened a new chapter in the study of single-domain antibodies. In 1995, Greenberg et al. discovered a heavy chain antibody with a natural lack of light chain in nurse sharks, called the new antigen receptor (Immunoglobulin new antigen receptor, IgNAR). Antibodies composed entirely of antibody heavy chain or light chain variable domains are called single-domain antibodies (sdAb). Shark-derived sdAbs are called variable domains of immunoglobulin new antigen receptors (VNAR). The molecular mass 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 a nanobody. Compared with traditional antibodies, the complementary 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 some VNARs can form a disulfide bond with the framework region 2 (FR2), so VNAR has the advantages of high stability, high affinity, high specificity, low preparation cost, and resistance to complex external environments, which is significantly superior to traditional antibodies. Summary of the invention

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

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

[0007] The technical solution of the present invention is as follows:

[0008] An ovalbumin shark-derived 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 invention, the nucleotide sequence is shown as SEQ ID NO.03 or SEQ ID NO.04.

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

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

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

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

[0014] An in vitro detection method for ovalbumin is carried out using the above-mentioned in vitro detection component for ovalbumin.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention is derived from the striped bamboo shark and has the characteristics of small molecular weight, easy transformation and expression, low immunogenicity, high antigen binding and strong tissue penetration, making it suitable for immunoassay detection of ovalbumin (OVA) and protein structure research and other fields.

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

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

[0019] 4. The present invention 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 THE DRAWINGS

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

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

[0022] Figure 3 The results of monoclonal phage ELISA in Example 1 of the present invention are shown in FIG.

[0023] Figure 4 This is a diagram showing the result of comparing the amino acid sequences of the single domain antibodies in Example 1 of the present invention;

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

[0025] Figure 6Schematic diagram of using BLIz to characterize the affinity between the single-domain antibody and OVA in Example 1 of the present invention; wherein the solid line is the kinetic curve monitored in real time, the dotted line is the curve fitted by the software, and the kinetic curves of different OVA concentration gradients correspond from top to bottom to the concentrations marked on the right from top to bottom.

[0026] Figure 7 The results of measuring the Tm of the single domain antibody of the present invention using circular dichroism spectroscopy in Example 1 of the present invention are shown.

[0027] Figure 8 The results of verifying the specificity of the single domain antibody of the present invention by Western Blot in Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0029] Example 1

[0030] (1) Purification of antigen ovalbumin: Based on the existing technology (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.), 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 the supernatant was obtained by centrifugation after standing for 0.5 h. Polyethylene glycol 8000 (PEG 8000) was added to the egg white solution three times, and the final amount of PEG 8000 added was 15% (v:w) of the solution volume. The solution was allowed to stand for 0.5 h and then centrifuged to obtain the supernatant. The supernatant was dialyzed in 20mmol / L Tris-HCl buffer to equilibrium, loaded on a DEAE-ion exchange chromatography column, and separated and purified using 20mmol / L Tris-HCl buffer containing 0-0.2mol / L NaCl. The third protein elution peak was collected, which was OVA.

[0031] The third protein elution peak was collected and analyzed by reducing SDS-PAGE. The final result was as follows Figure 1 As shown, the results show that there is only a single band at around 45 kDa. In order to verify whether the protein band is OVA, mass spectrometry identification was performed after gel cutting. The test results are shown in Figure 2 As shown, the comprehensive results show that the protein band with a molecular weight of about 45 kDa is OVA, and its purity is relatively high.

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

[0033] (3) Construction of phage synthetic library: To construct a VNAR synthetic library with randomized CDR3 region, the natural framework of VNAR was amplified by three consecutive PCR steps:

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

[0035] B. In the second step, the product recovered from the first step of PCR was used as a template to amplify the FR1 to CDR3-FR4 region of the shark-derived single-domain antibody VNAR using specific primers. The amplification program was: 95°C, 3 min, 98°C, 10 s, 68°C, 5 s, 72°C, 25 s, 72°C, 1 min, for a total of 30 cycles;

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

[0037] Then, pR2 phagemid was used as a template and specific primers were used to amplify pR2 phagemid. The DNA polymerase used for amplification was the high-fidelity enzyme Prime STARGXL DNA Polymerase. The amplification program was: 98°C denaturation for 10s, 57°C annealing for 15s, 72°C extension for 25s, and a total of 35 cycles. The product was recovered using a kit and the pR2 amplification recovery product was digested and placed in a 37°C water bath for 1h. The digestion site was DnpⅠ. The VNAR target gene recovery product and the pR2 digestion recovery product were seamlessly cloned, and the connection product was recovered using a kit. The connection product and TG1 cells were mixed at a volume ratio of 1:20 and then electroporated. The electroporator was set to a voltage of 2.5kv and 5ms, and then cultured at 37°C for 1h. The cultured bacterial solution was diluted in a gradient manner (0.2 μL, 0.02 μL, and 0.002 μL of the stock solution were taken respectively) and then spread on a 9 cm LB / Amp / 2% glucose plate and cultured overnight at 37°C. 2×YT liquid culture medium containing 20% ​​glycerol was added to the plate cultured overnight, and the bacterial lawn was scraped with a spreading rod and collected in a centrifuge tube. After vortex mixing, it was packaged, quickly frozen in liquid nitrogen, and stored at -80°C, which is the phage synthetic library.

[0038] (4) Screening for specific single domain antibodies: Activate the bacteria of the frozen phage synthesis library and add KM13 helper phage. Take the bacterial culture supernatant and measure the phage titer. This is the amplified phage. Coat blood with OVA at a final concentration of 0.1 mg / mL in a 96-well immunoplate and add 1×10 11 pfu and incubate the amplified phages at room temperature for 1 hour. Use a trypsin solution with a final concentration of 2 mg / mL to elute the phages that specifically bind to OVA, and use it to infect TG1 bacteria. Take 5 μL and 1 μL of the infected bacterial solution to coat solid plates, respectively, and record the total number of colonies. Randomly pick 95 single colonies from the above plates and activate them overnight. Add KM13 helper phage, centrifuge and collect the supernatant after lysis, which is the monoclonal phage. Coat ovalbumin at a concentration of 1 μg / mL to a 96-well immunoplate, add the monoclonal phage solution prepared above to each well, and incubate at room temperature for 1 hour. Use HRP-anti M13 antibody to capture the phages bound to ovalbumin, and use TMB substrate to develop color for 10 minutes, and terminate the reaction with a final concentration of 1 mol / L H2SO4 solution. Use an enzyme reader to record OD 450 Value, the selection result is as follows Figure 3 shown.

[0039] Pick OD 450 Wells with values ​​greater than 1.0 were sequenced and analyzed using the sequencing primer: 5'-caggaaacagctatgac-3' (SEQ ID NO.05). After comparing the antibody sequences obtained and excluding duplicate clones, two different single-domain antibody sequences were obtained. The comparison of the two sequences is shown in Figure 2. Figure 4 The specific information of the above single domain antibody is as follows:

[0040] Name: 14-A2

[0041] Source: Striped Bamboo Shark (Chiloscyllium plagiosum)

[0042] Amino Acid Sequence:

[0043]

[0044] Nucleotide sequence:

[0045]

[0046] Name: 14-A8

[0047] Source: Striped Bamboo Shark (Chiloscyllium plagiosum)

[0048] Amino Acid Sequence:

[0049]

[0050] Nucleotide sequence:

[0051]

[0052] (5) Recombinant expression and purification of single-domain antibodies: The single-domain antibodies 14-A2 and 14-A8 obtained in step (4) were constructed into the mammalian expression vector pTT5 with a signal peptide, and fused with human IgG1Fc. The Fc fragment was expressed at the C-terminus and the single-domain antibody could be obtained by enzymatic digestion with TEV enzyme. 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. Figure 5 As shown, the molecular weight of the Fc fusion proteins of 14-A2 and 14-A8 are both about 42 kDa.

[0053] (6) Characterizing the properties of the single domain antibody:

[0054] BLIz was used to characterize the affinity of 14-A2 and 14-A8 to OVA: OVA was first labeled with biotin to obtain biotinylated OVA (Bio-OVA). After that, it was solidified on the SA biosensor, and different single-domain antibody concentration gradients were set to detect the affinity of different single-domain antibody Fc fusion protein concentration gradients to ovalbumin. The results are shown in Figure 2. Figure 6 As shown in Figure 2, the binding strength of the two single-domain antibodies to OVA is as follows: 14-A2>14-A8, and their affinity constants K DThe values ​​are 9.35×10- 6 M and 5.14×10- 5 M, reaching the micromolar level. The affinity of the original heavy chain antibody is generally in the micromolar to high micromolar level, so the affinity of 14-A2 and 14-A8 has reached the affinity level of the original heavy chain antibody.

[0055] The Tm of 14-A2 and 14-A8 was measured by circular dichroism to characterize their stability. Figure 7 As shown, the Tm of 14-A2 and 14-A8 reached about 86°C and 87°C, respectively, with high thermal stability.

[0056] Western Blot was used to verify whether 14-A2 and 14-A8 had cross-reactivity with other proteins in hen and duck egg whites except OVA and proteins in surimi. Figure 8 As shown, both 14-A2 and 14-A8 reacted only to OVA and had no cross-reaction to other proteins in egg white and surimi.

[0057] The physicochemical properties of 14-A2 and 14-A8 were predicted using the ExPASy website (http: / / web.expasy.org / protparam). The prediction results are shown in the following table. The average hydrophobicity values ​​(GRAVYA) of 14-A2 and 14-A8 are -0.548 and -0.521, respectively, indicating that 14-A2 and 14-A8 have a high content of hydrophilic amino acids and a high solubility.

[0058] Prediction of physicochemical properties of 14A2 and 14A8

[0059]

[0060] Note: GRAVY (average hydrophobicity), positive GRAVY value: protein tends to be hydrophobic; positive GRAVY value: protein tends to be hydrophobic;

[0061] Negative value: the protein tends to be hydrophilic.

[0062] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

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

02.

2. The ovalbumin shark-derived single domain antibody according to claim 1, characterized in that: The nucleotide sequence thereof is shown as SEQ ID NO.03 or SEQ ID NO.

04.

3. Use of the ovalbumin shark-derived single-domain antibody according to claim 1 or 2 in the preparation of an in vitro ovalbumin detection component.

4. The use according to claim 3, characterized in that: The ovalbumin in vitro detection component comprises a colloidal gold immunochromatography detection component, a fluorescent immunochromatography detection component, an enzyme-linked immunosorbent assay detection component, a lateral flow immunochromatography detection component and an immunoblotting detection component.

5. An in vitro detection component for ovalbumin, characterized in that: A shark-derived single domain antibody containing ovalbumin as claimed in claim 1 or 2.

6. An ovalbumin in vitro detection assembly as claimed in claim 5, characterized in that: It is a colloidal gold immunochromatography detection component, a fluorescent immunochromatography detection component, an enzyme-linked immunosorbent assay component, a lateral flow immunochromatography detection component or an immunoblotting detection component.

7. An in vitro detection method for ovalbumin, characterized in that: The method is carried out using the ovalbumin in vitro detection component according to claim 5 or 6.

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