Nano antibody OVA-3-23 for resisting chicken ovalbumin and application of nano antibody OVA-3-23

Abstract

The invention belongs to the technical field of nano antibodies, and provides a nano antibody OVA-3-23 for resisting chicken ovalbumin and application of the nano antibody OVA-3-23. The nano antibody OVA-3-23 for resisting the chicken ovalbumin has an amino acid sequence as shown in SEQ ID NO. 1. According to the prepared nano antibody OVA-3-23 for resisting the chicken ovalbumin, in the field of directional fixation, accurate anchoring of the chicken ovalbumin is achieved with high specificity, and the defects of impurity interference and fuzzy positioning in traditional fixation are overcome; in the field of immunodetection, high specificity and strong binding force guarantee the detection accuracy, colloidal gold, enzyme and other multi-component components are matched, and the chicken ovalbumin is efficiently identified from food on-site fast screening to laboratory fine detection; in the field of enrichment and purification, a high-purity product is efficiently separated from a complex sample. Especially, reliable support is provided for application scenes such as food detection, feed quality monitoring, immunology and vaccine development, and an efficient solution is provided for detection, research and quality control related to chicken ovalbumin.

Description

Technical Field

[0001] This invention belongs to the field of nanobody technology, and in particular relates to an anti-chicken oocyte albumin nanobody OVA-3-23 and its application. Background Technology

[0002] Camelidae and cartilaginous fish possess naturally occurring heavy chain antibodies (HCAbs) lacking the light chain. Their variable domains (VHH) are single-domain heavy chain antibodies, also known as nanobodies due to their small size. Nanobodies are currently the smallest known antigen-binding fragments, with a molecular weight of only 15 kDa, approximately one-tenth that of traditional IgG antibodies. Furthermore, nanobodies possess numerous advantages over traditional antibodies, such as high stability, strong water solubility, small size, resistance to organic solvents, ease of modification, and the ability to recognize cryptic epitopes.

[0003] Ovalbumin (OVA) is a globular protein extracted from egg white. It is the most abundant protein component in egg white, accounting for approximately 54% of the total protein content. Composed of 12,269 amino acids, it has a molecular weight of approximately 172.44 kDa and an isoelectric point (pI) of 4.6. OVA exhibits good solubility in water and also possesses excellent emulsifying, foaming, and gel-forming properties. It has various important applications in food science and biomedical research, such as serving as a food additive, a blocking agent in immunoassays, and a carrier protein for conjugating small molecule haptens to prepare complete antigens for animal immunization or the construction of immunoassay methods. Furthermore, OVA is a common allergen and is frequently used as a model antigen in immunological research. In immunological and vaccine development research, the presence and concentration of OVA can be detected to assess the immune system's response. In food quality control, detecting OVA in food can prevent food allergic reactions. Therefore, the preparation and development of anti-OVA antibodies is crucial for the targeted immobilization, purification, and detection of OVA. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an anti-chicken oocyte albumin nanobody OVA-3-23 and its applications, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides an anti-chicken oocyte albumin nanobody OVA-3-23, wherein the anti-chicken oocyte albumin nanobody OVA-3-23 has an amino acid sequence as shown in SEQ ID NO.1.

[0006] Furthermore, the nucleotide sequence encoding the anti-chicken oocyte albumin nanobody OVA-3-23 is shown in SEQ ID NO.2.

[0007] Furthermore, the nanobody includes at least one of monomers, bivalent antibodies, and multivalent antibodies.

[0008] Furthermore, an anti-chicken oocyte albumin nanobody, OVA-3-23, was applied to the targeted immobilization of chicken oocyte albumin.

[0009] Furthermore, the application of an anti-chicken oocyte albumin nanobody OVA-3-23 in the immunoassay of chicken oocyte albumin.

[0010] Furthermore, the application of an anti-chicken oocyte albumin nanobody, OVA-3-23, in the enrichment and purification of chicken oocyte albumin.

[0011] Secondly, the present invention provides a chicken oocyte albumin immunoassay component having an anti-chicken oocyte albumin nanobody OVA-3-23.

[0012] Furthermore, the chicken oocyte albumin immunoassay component includes a colloidal gold immunochromatographic assay component, a fluorescence immunochromatographic assay component, an enzyme-linked immunosorbent assay (ELISA) component, or an immunoblotting assay component.

[0013] Furthermore, an ovalbumin immunoassay kit is applied in immunology and vaccine development to assess the immune system's response by detecting the presence and / or concentration of ovalbumin.

[0014] Furthermore, an ovalbumin immunoassay kit is applied in food testing and quality control to prevent allergic reactions by detecting ovalbumin residues in food.

[0015] This invention offers the following advantages: By constructing a phage display immunomodulatory nanobody library, nanobody panning, and self-induced expression and purification of nanobodies, an anti-chicken oocyte albumin nanobody, OVA-3-23, was prepared, possessing the amino acid sequence shown in SEQ ID NO.1. In the field of targeted immobilization, the anti-chicken oocyte albumin nanobody OVA-3-23 achieves high specificity for precise anchoring of chicken oocyte albumin, overcoming the drawbacks of traditional immobilization methods such as impurity interference and ambiguous positioning. In the field of immunoassay, its high specificity and strong binding force ensure accurate detection. Combined with colloidal gold, enzymes, and other multi-component components, it is suitable for everything from rapid on-site food screening to precise laboratory testing, efficiently identifying chicken oocyte albumin. In the field of enrichment and purification, it efficiently separates high-purity products from complex samples. The anti-chicken oocyte albumin nanobody OVA-3-23 prepared by this invention provides reliable support, particularly in applications such as food testing, feed quality monitoring, immunology, and vaccine development, offering an efficient solution for the detection, research, and quality control of chicken oocyte albumin. Attached Figure Description

[0016] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is a graph showing the results of the third round of screening of 1-48 monoclonal phage-ELISA in Example 1 of the present invention; Figure 2 This is a graph showing the results of the third round of screening of 49-96 monoclonal phage-ELISA in Example 1 of the present invention; Figure 3 This is a graph showing the polyacrylamide gel electrophoresis identification results of Example 2 of the present invention; Figure 4 This is a graph showing the protein immunoblotting identification results of Example 2 of the present invention; Figure 5 This is a schematic diagram of the amino acid sequence and structural domains of the anti-chicken egg albumin nanobody OVA-3-23 according to an embodiment of the present invention. Detailed Implementation

[0017] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0019] This invention provides an anti-chicken oocyte albumin nanobody OVA-3-23, which has the amino acid sequence shown in SEQ ID NO.1.

[0020] In some embodiments, the nucleotide sequence of the nanobody OVA-3-23 encoding anti-egg albumin is shown in SEQ ID NO.2.

[0021] In some embodiments, nanobodies include at least one of monomers, bivalent antibodies, and multivalent antibodies.

[0022] In some embodiments, an anti-chicken oocyte albumin nanobody OVA-3-23 is used for the targeted immobilization of chicken oocyte albumin.

[0023] In some embodiments, an anti-chicken oocyte albumin nanobody OVA-3-23 is used in the immunoassay of chicken oocyte albumin.

[0024] In some embodiments, an anti-chicken oocyte albumin nanobody OVA-3-23 is used in the enrichment and purification of chicken oocyte albumin.

[0025] In some embodiments, the present invention provides a chicken oocyte albumin immunoassay component having an anti-chicken oocyte albumin nanobody OVA-3-23.

[0026] In some embodiments, the oocyte albumin immunoassay kit includes a colloidal gold immunochromatographic assay kit, a fluorescence immunochromatographic assay kit, an enzyme-linked immunosorbent assay kit, or an immunoblotting assay kit.

[0027] In some embodiments, an ovalbumin immunoassay component is used in immunology and vaccine development to assess the immune system’s response by detecting the presence and / or concentration of ovalbumin.

[0028] In some embodiments, a chicken ovalbumin immunoassay kit is used in food testing and quality control to detect chicken ovalbumin residues in food and prevent allergic reactions.

[0029] Example 1: Screening of anti-chicken egg albumin nanobodies 1. Chicken oocyte albumin was diluted to the required concentration using carbonate buffer (pH 8.6), as shown in Table 1. 100 μL of chicken oocyte albumin was added to a 96-well plate and incubated at 4°C for 12 h. After incubation, the 96-well plate was washed three times with PBST solution. After washing, 300 μL of 1% gelatin blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. After incubation, the 96-well plate was washed three times with PBS buffer. After washing, 100 μL of the diluted anti-chicken oocyte albumin phage display immunomodulatory nanobody library was added to each well, with a library volume of 1 × 10⁻⁶. 11 PFU / well, incubated at 37℃ for 1 h; after incubation, the 96-well plate was washed 10 times with PBST solution, followed by 10 washes with PBS buffer; after washing, 94.5 μL of pH 2.2 Gly-HCl elution buffer was added to each well, and the plate was incubated on a horizontal shaker at room temperature for 8 min; after incubation, the elution buffer was added to 5.5 μL of pH 9.0 Tris-HCl neutralization buffer to neutralize, yielding 100 μL of neutralized solution; 10 μL of the neutralized solution was serially diluted and used to infect E. coli ER2738, and plated on LB / Amp resistant plates to measure the titer of the eluted phage; the remaining 90 μL of the neutralized solution was used to infect E. coli ER2738 for phage amplification, and the resulting phage was used for the next round of screening; To obtain nanobodies with high affinity for antigens, three rounds of panning were conducted. The panning conditions and experimental parameters for each round are shown in Table 1.

[0030] Table 1. Selection conditions and experimental results of nanobodies against chicken egg white albumin.

[0031] 2. Positive clones were identified by phage-ELISA. During the third round of panning, 96 single clones were randomly selected from the plates used for phage titer determination for phage amplification. Experimental group: Chicken ovalbumin was diluted to 4 μg / mL with PBS buffer and added to each well of a 96-well plate (100 μL per well, i.e., 0.4 μg of chicken ovalbumin per well). Incubation was performed overnight at 4°C. Control group: Gelatin was diluted to 4 μg / mL with PBS buffer and added to each well (100 μL per well). Incubation was performed overnight at 4°C. After coating, the plates were washed three times with PBST solution (1 L of PBS buffer with 2.5 mL of Tween-20 added, resulting in a final PBS concentration of 10 mmol / L). After washing, 300 μL of 1% gelatin was added to each well, and the plates were blocked at 37°C. h; After blocking, wash 3 times with PBST; After washing, add 100 μL of phage amplification buffer to each well of the 96-well plates of the experimental and control groups, and incubate at 37℃ for 1 h; After incubation, wash 4 times with PBST solution; After washing, add 100 μL of diluted anti-M13-HRP secondary antibody (final concentration 0.275 μg / mL) to each well, and incubate at 37℃ for 1 h; After incubation, wash 5 times with PBST solution; After washing, add 100 μL of TMB chromogenic solution to each well, and incubate at 37℃ in the dark for 10 min; After the chromogenic reaction, add 50 μL of 2 The absorbance at 450 nm was measured using an ELISA reader. Phage clones in the experimental group with an OD450 greater than that of the control group were identified as positive clones. The positive clones were sequenced to obtain the sequence of the anti-chicken oocyte albumin nanobody. The sequence alignment showed that all the sequenced positive clones had the same sequence, and it was named nanobody OVA-3-23.

[0032] The results of the third round of selection of 1-48 monoclonal phage-ELISA samples are as follows: Figure 1 As shown, the results of the third round of screening for 49-96 monoclonal phage-ELISA are as follows: Figure 2 As shown.

[0033] Example 2: Self-induced expression and purification of OVA-3-23, a nanobody against chicken oocyte albumin. (1) The phage vector pComb3XSS-OVA-3-23 was transformed into chemically competent cells E. coli Rosetta by heat shock at 42℃ for 90s. After being plated on 2×YT / Amp resistance plates, single colonies were picked one day later. (2) Add the above single colony and 5 μL of 100 mg / mL ampicillin (Amp) to 5 mL of 2×YT liquid medium. The final concentration of Amp is 100 μg / mL. Incubate at 37℃ and 180 rpm for 12 h. After the culture is completed, bacterial solution 1 is obtained. (3) Inoculate bacterial culture 1 into 100 mL of self-induction medium at an inoculation rate of 1% (v / v), and add 50 μL of 100 mg / mL Amp, so that the final concentration of Amp is 50 μg / mL; culture at 37℃ and 180 rpm for 3 h until the logarithmic phase (OD600 reaches 0.6-0.8) to obtain culture 1; (4) Culture 1 was placed in a shaker at 23°C and 130 rpm for 20 h to induce protein expression and obtain culture 2; (5) Centrifuge the culture at 8000 rpm for 10 min; resuspend the precipitate obtained by centrifugation in 60 mL of purification equilibration buffer to obtain a bacterial resuspension. (6) Add 30 mg of lysozyme to the bacterial suspension, the final concentration of lysozyme is 0.5 mg / mL, and hydrolyze on a horizontal shaker at 4℃ for 1 h to obtain the hydrolysate; (7) Place the enzymatic hydrolysate in a beaker containing an ice-water mixture and use a cell sonicator to disrupt the cells for 1 hour. The cell disruption conditions are 30W power, 3 seconds working and 12 seconds rest. Stop disruption when the enzymatic hydrolysate changes from turbid to clear and transparent to obtain the disrupted product. (8) Centrifuge the broken product at 13000g, 4℃ for 30min and collect the protein supernatant; use a 0.22μm aqueous filter membrane to filter the protein supernatant to remove impurities; (9) Add 2 mL of Ni-NTA column material to the purification column. After the Ni-NTA settles naturally, wash with at least 5 column volumes of purified water to remove impurities and equilibrate the purification column with 25 mL of equilibration buffer. Add the protein supernatant to the purification column in batches, allowing the protein supernatant to bind with Ni-NTA for 10 min, and then let it flow out slowly, collecting each flow-through. (10) Use equilibration buffer (containing imidazole) to equilibrate the purification column to achieve the purpose of eluting proteins. Elute stepwise from low imidazole concentration to high imidazole concentration. Low imidazole concentration is used to elute the impurities bound in the purification column. Each imidazole concentration requires at least 20 mL of rinsing. (11) The first 10 mL of elution buffer eluted with 250 mmol / L imidazole was used to elute the target protein bound in the purification column, and the target protein solution was collected and stored in a centrifuge tube. (12) The collected target protein solution was transferred to a 3kDa dialysis bag for desalting. At 4°C, it was placed in 10mmol / L PBS dialysis solution (pH=7.4) for dialysis. The dialysis solution was changed every 4 hours, and the dialysis time was 48 hours. (13) After dialysis, the protein solution was concentrated using a 3kDa ultrafiltration tube, centrifuged at 3000g for 10 min at 4℃, and centrifuged multiple times until the final protein solution was retained at about 1 mL. The protein concentration was measured using a NanoDrop 2000 micro spectrophotometer. After the measurement, glycerol was added to make the final concentration 50%. The purified anti-chicken egg white protein nanobody OVA-3-23 was dispensed into 600μL centrifuge tubes and stored in a freezer at -80℃.

[0034] The anti-chicken egg albumin nanobody OVA-3-23 was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The results of the polyacrylamide gel electrophoresis are as follows: Figure 3 As shown, lane M is Maker, and lanes 1-5 are OVA-3-23 nanobodies against chicken oocyte albumin; the results of Western blotting identification are as follows. Figure 4 As shown, lane M is Maker, and lanes 1-5 contain the anti-chicken oocyte albumin nanobody OVA-3-23. The results show that the anti-chicken oocyte albumin nanobody OVA-3-23 was successfully expressed and purified, with high purity. Furthermore, the molecular weight of this nanobody (approximately 18 kDa) is consistent with the theoretical molecular weight of nanobodies.

[0035] The amino acid sequence and domain diagram of the anti-chicken egg albumin nanobody OVA-3-23 are shown below. Figure 5 As shown, the anti-chicken oocyte albumin nanobody OVA-3-23 has the amino acid sequence shown in SEQ ID NO.1, the nucleotide sequence encoding the anti-chicken oocyte albumin nanobody OVA-3-23 is shown in SEQ ID NO.2, the amino acid sequence of CDR1 of the anti-chicken oocyte albumin nanobody OVA-3-23 is shown in SEQ ID NO.3, the amino acid sequence of CDR2 is shown in SEQ ID NO.4, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5.

[0036] SEQ ID NO:1: QLQLVESGGGLVQAGDSLRLRCAASGRTFGRMAWFRQAPGKEREFVAAIRWRDGTTHYSDSVKGRFTISRDIGQRTVYLQMNSLKFEDTARYYCNVPFYPHIWGQGTQVTVSS

[0037] SEQ ID NO.2: CAGTTGCAGCTCGTGGAGTCGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCCGCTGTGCAGCCTCTGGACGCACCTTCGGTCGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAACGTGAGTTTGTAGCGGCTATTAGGTGGAGGGATGGTACGACGC ATTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATTGGACAGCGCACGGTGTATCTGCAAATGAACAGCCTGAAATTTGAGGACACGGCCCGCTATTACTGTAATGTCCCCTTCTATCCGCACATCTGGGGCCAGGGGACCCAGGTAACCGTCTCCTCA

[0038] SEQ ID NO:3: GRTFGR

[0039] SEQ ID NO:4: IRWRDGTT

[0040] SEQ ID NO:5: NVPFYPHI

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody OVA-3-23 against chicken oocyte albumin, characterized in that: The anti-egg oocyte albumin nanobody OVA-3-23 has the amino acid sequence shown in SEQ ID NO.

1.

2. The nanobody OVA-3-23 against chicken oocyte albumin as described in claim 1, characterized in that: The nucleotide sequence of the nanobody OVA-3-23 encoding the anti-chicken oocyte albumin is shown in SEQ ID NO.

2.

3. The nanobody OVA-3-23 against chicken oocyte albumin as described in claim 1, characterized in that: The nanobody includes at least one of monomers, bivalent antibodies, and multivalent antibodies.

4. The application of the anti-chicken oocyte albumin nanobody OVA-3-23 as described in claim 1 or 2 in the directional immobilization of chicken oocyte albumin.

5. The application of the anti-chicken oocyte albumin nanobody OVA-3-23 as described in claim 1 or 2 in the immunoassay of chicken oocyte albumin.

6. The application of the anti-chicken oocyte albumin nanobody OVA-3-23 as described in claim 1 or 2 in the enrichment and purification of chicken oocyte albumin.

7. A chicken oocyte albumin immunoassay kit, characterized in that: OVA-3-23, a nanobody containing anti-chicken oocyte albumin as described in claim 1 or 2.

8. The chicken oocyte albumin immunoassay kit as described in claim 7, characterized in that: The chicken oocyte albumin immunoassay kit includes a colloidal gold immunochromatographic assay kit, a fluorescence immunochromatographic assay kit, an enzyme-linked immunosorbent assay kit, or an immunoblotting assay kit.

9. The application of the chicken oocyte albumin immunoassay component as described in claim 7 in immunology and vaccine development, characterized in that: The immune system's response is assessed by detecting the presence and / or concentration of oocyte albumin.

10. The application of the chicken oocyte albumin immunoassay kit as described in claim 7 in food testing and quality control, characterized in that: Allergic reactions can be prevented by detecting residual oocyte albumin in food.

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