Nanobodies specifically binding mouse igg, methods of making and uses thereof

Abstract

The application discloses a nanobody specifically binding to mouse IgG, a preparation method and application thereof, and belongs to the technical field of biotechnology. The nanobody comprises at least one amino acid sequence selected from SEQ ID NO. 1-9, specifically binds to mouse IgG, and does not cross-react with mouse IgM, human IgG and rabbit IgG. The application further provides a bivalent nanobody which is formed in series through a rigid alpha-helix connecting peptide (EAAAK) 3; and a mouse IgG detection kit prepared by using the bivalent nanobody. In the kit, the capture antibody and the detection antibody are selected from a paired combination of Nb26-(EAAAK) 3-Nb26 and Nb556-(EAAAK) 3-Nb556-HRP, the linear range of detection is 2-2000 ng / mL, the detection limit is less than or equal to 4 ng / mL, and the kit can be used for high-sensitivity detection of mouse IgG.

Description

TECHNICAL FIELD

[0001] The present application relates to a nanobody specifically binding mouse IgG, a preparation method and applications thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Immunoglobulin G (IgG) is the most abundant antibody type in mammalian serum, playing a core regulatory role in the immune response process. Mouse IgG includes four subtypes of IgG1 / 2a / 2b / 3, and its application covers biomedical basic research, vaccine development and immunotherapy development. Therefore, the development of specific mouse IgG detection technology has key value for immunology research breakthroughs and biopharmaceutical product development.

[0003] Currently, the mainstream detection methods for mouse IgG include enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry (IHC) techniques. The detection tools mainly use polyclonal or monoclonal antibodies. However, traditional antibodies have significant limitations, such as large molecular weight (~ 150 kDa), insufficient stability, high preparation cost, and limited application scenarios.

[0004] Nanobodies (VHH) as a new generation of antibody tools have become a research hotspot in recent years due to their unique structural characteristics and excellent performance. Their technical advantages include excellent thermal / chemical stability, high affinity, precise specificity, and modifiable properties, which have important application value in the biomedical field. For example, Chinese invention patent CN113045662B develops a PD-L1 specific VHH with a cross-reactivity of <0.5% and a detection sensitivity of pg / mL level. Another patent CN114409795B discloses a diazinopharm-specific nanobody with advantages such as high temperature resistance, acid and alkali resistance, and easy storage, which can be used as a new material for detecting diazinopharm.

[0005] Bivalent nanobodies have become the mainstream strategy for optimizing detection technology due to their synergistic effect of specificity and sensitivity. Chinese patent CN116554339B discloses a bispecific nanobody that specifically recognizes methylnaphthalene and / or 1-naphthol. The bispecific nanobody is connected by a short peptide linker G4S, successfully retaining the binding activity and achieving the multifunctionalization of one antibody. This technology not only simplifies the preparation process, but also has detection sensitivity comparable to that of each monomer ELISA.

[0006] In the field of mouse IgG detection, CN112812190B discloses a llama single-chain nanobody against mouse and rabbit IgG, which can specifically recognize mouse and rabbit IgG with high affinity and has no cross-reactivity with human IgG. This nanobody can be used to prepare a universal secondary antibody for mouse and rabbit, which has important application value. In addition, CN117491625A describes a nanobody-based double antibody sandwich ELISA kit, which uses nanobody as the capture antibody and has high detection sensitivity and specificity.

[0007] However, there are still some problems and deficiencies in the prior art. First, most of the detection methods for mouse IgG can only recognize specific IgG subtypes, and there is a lack of detection methods that can detect all subtypes of mouse IgG (IgG1, IgG2a, IgG2b, IgG3) in a broad spectrum. Second, existing detection methods often have cross-reactivity problems, making it difficult to distinguish between mouse IgG and other species of IgG or other types of immunoglobulin, such as mouse IgM, human IgG, and rabbit IgG. In addition, traditional antibody detection methods usually require the use of large molecular weight antibodies, which may affect the sensitivity and specificity of the detection, especially in application scenarios that require high sensitivity detection.

[0008] Therefore, there is an urgent need to develop a technical solution that can specifically recognize and quantitatively detect each subtype of mouse IgG in a broad spectrum, in order to meet the needs of biomedical research and clinical diagnosis. SUMMARY

[0009] In view of the above deficiencies of the prior art, the present application provides a nanobody that specifically binds to mouse IgG, a preparation method and its application, aiming to solve the technical problems that the prior art lacks detection methods that can detect all subtypes of mouse IgG in a broad spectrum, most detection methods can only recognize specific IgG subtypes and cannot achieve comprehensive detection of mouse IgG, existing detection methods often have cross-reactivity problems and cannot distinguish between mouse IgG and other species of IgG or other types of immunoglobulin, and traditional antibody detection methods usually require the use of large molecular weight antibodies, which may affect the sensitivity and specificity of the detection.

[0010] The first technical solution provided by the application is a nanobody specifically binding mouse IgG, wherein the nanobody comprises at least one of the following amino acid sequences: Nb26 (SEQ ID NO: 1), Nb91 (SEQ ID NO: 2), Nb279 (SEQ ID NO: 3), Nb315 (SEQ ID NO: 4), Nb541 (SEQ ID NO: 5), Nb556 (SEQ ID NO: 6), Nb683 (SEQ ID NO: 7), Nb691 (SEQ ID NO: 8), and Nb737 (SEQ ID NO: 9), and the nanobody specifically binds mouse IgG (including IgG1, IgG2a, IgG2b, and IgG3 subtypes) and does not cross-react with mouse IgM, human IgG, and rabbit IgG.

[0011] The second technical solution provided by the application is a bivalent nanobody, wherein the bivalent nanobody is formed by connecting any two same or different nanobodies of the amino acid sequences shown in SEQ ID NO. 1-9 in series through a rigid alpha-helix connecting peptide (EAAAK)3 to enhance the binding capacity to mouse IgG.

[0012] In some embodiments, the bivalent nanobody is Nb26-(EAAAK)3-Nb26 or Nb556-(EAAAK)3-Nb556.

[0013] The third technical solution provided by the application is a gene encoding the nanobody of the first technical solution or the bivalent nanobody of the second technical solution.

[0014] The fourth technical solution provided by the application is a recombinant vector carrying the gene of the third technical solution.

[0015] In some embodiments, the recombinant vector is a phage display vector pDAN5, a prokaryotic expression vector pET28a, or a eukaryotic expression vector pcDNA3.4 expression vector.

[0016] The fifth technical solution provided by the application is a recombinant cell expressing the nanobody of the first technical solution or the bivalent nanobody of the second technical solution, or containing the gene of the third technical solution, or transformed or transfected with the recombinant vector of the fourth technical solution.

[0017] In some embodiments, the recombinant cell is an Escherichia coli TG1, BL21 (DE3), or mammalian Expi293F™ cell as a host.

[0018] The sixth technical solution provided by the application is a product labeled with a biological marker or a chemical marker, wherein the product is the nanobody of the first technical solution or the bivalent nanobody of the second technical solution.

[0019] In some embodiments, the marker comprises enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, magnetic beads; the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, beta-galactosidase-anti-beta-galactosidase bridge; the biotin includes biotin and its derivatives; the fluorescein includes AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescence includes, but is not limited to, isoluminol and its derivatives, acridinium ester and its derivatives, trispyridine ruthenium and its derivatives; the isotopes include iodine labeling; the colloidal labeling includes colloidal gold, colloidal carbon, colloidal selenium.

[0020] The seventh technical solution provided by the application is a kit, wherein the kit contains the nanobody of the first technical solution, or the bivalent nanobody of the second technical solution, or the product labeled with a biological marker or a chemical marker of the sixth technical solution.

[0021] In some embodiments, the kit comprises:

[0022] (a) the bivalent nanobody as a capture antibody;

[0023] (b) the bivalent nanobody-enzyme fusion protein as a detection antibody, wherein the enzyme is horseradish peroxidase or soybean peroxidase.

[0024] Optionally, the bivalent nanobody as a capture antibody is Nb26-(EAAAK)3-Nb26, and the bivalent nanobody-enzyme fusion protein as a detection antibody is Nb556-(EAAAK)3-Nb556-HRP.

[0025] Further, the coating concentration of the capture antibody is 0.5-2 μg / mL, the working dilution ratio of the detection antibody is 1:1000-1:5000, the detection linear range is 2-2000 ng / mL, and the detection limit is ≤4 ng / mL.

[0026] The eighth technical solution provided by the present invention is the application of the nanobody described in the first technical solution, or the bivalent nanobody described in the second technical solution, or the gene described in the third technical solution, or the recombinant vector described in the fourth technical solution, or the recombinant cell described in the fifth technical solution, or the biomarker or chemically labeled product described in the sixth technical solution in the preparation of a product for in vitro detection of mouse IgG subtypes.

[0027] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The bivalent nanobody complex constructed in this invention achieves high-sensitivity detection of mouse IgG through synergistic binding effect. Compared with the detection process in the prior art that requires the use of multiple antibodies, the bivalent nanobody complex provided by this invention can complete the detection of various subtypes of mouse IgG in one step, greatly simplifying the detection process.

[0030] The nanobody of this invention has no cross-reactivity with mouse IgM, human IgG and rabbit IgG, and has high specificity, avoiding the false positive problem common in traditional detection methods and improving the reliability of detection results.

[0031] Experimental verification showed that the affinity of the bivalent nanobodies of this invention is 2.58-3.91 times higher than that of monomeric antibodies, significantly enhancing detection sensitivity. The developed kit has a detection linear range of 2-2000 ng / mL and a detection limit as low as ≤4 ng / mL, exhibiting high sensitivity and a wide dynamic detection range, meeting the detection needs of samples with different concentrations.

[0032] This invention has significant application value in the quantitative detection of antibodies in mice, improving the accuracy and sensitivity of detection and providing a reliable detection tool for biomedical research, vaccine evaluation, and immunological research. Attached Figure Description

[0033] Figure 1 The amino acid sequences of nine nanobodies were compared.

[0034] Figure 2 SDS-PAGE analysis of the purity and molecular weight of nine nanobodies, bivalent nanobodies, and nanobodies-HRP fusion proteins.

[0035] Figure 3 Analysis of the binding affinity of nine nanobodies to mouse IgG.

[0036] Figure 4 Immunofluorescence images of IgG on the surface of mouse hybridoma cells specifically recognized by nine nanobodies.

[0037] Figure 5 Circular dichroism secondary structure analysis of Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26.

[0038] Figure 6 Standard curve of mouse IgG enzyme-linked immunosorbent (ELISA) kit. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application, and are not used to limit the present application.

[0040] Experimental materials and reagents in the embodiments

[0041] 1, Restriction enzyme: Age I restriction enzyme was purchased from ABclonal, product number RK21125, and other restriction enzymes were purchased from Takara, Not I endonuclease product number 1166A, BssH II endonuclease product number 1119A, Nco I endonuclease product number 1160A, Xho I endonuclease product number 1094A, and Xba I endonuclease product number 1093A.

[0042] 2, Expression vector and host cell: pcDNA3.4 vector was purchased from Thermo Scientific, product number A14697, pET28a vector was purchased from Novagen, product number 69864-3, pDAN5 vector was preserved in the laboratory, TG1 competent cells were purchased from Shenzhen Hui Nuobio Technology Co., Ltd., product number DL1055S, E. coli DH5α competent cells were purchased from Takara, product number 9057, E. coli protein expression competent cells were purchased from Takara, product number 9126, Expi293F™ cells were purchased from Thermo Scientific, product number A14527, and mouse IgG hybridoma cells were preserved in the laboratory.

[0043] 3, Medium: 293 cell serum-free medium was purchased from Zhuhai Kai Rui Biological Technology Co., Ltd., product number K03252.

[0044] 4, Blood RNA extraction reagent was purchased from Takara, product number 9112, reverse transcription kit PrimeScript™ was purchased from Takara, product number RR037A, and other conventional chemical reagents were purchased from Shanghai Sunway Biological Engineering Co., Ltd.

[0045] Example 1

[0046] The embodiment provides a kind of specifically binding mouse IgG nanobody, comprising at least one selected from the following amino acid sequences: Nb26 (SEQ ID NO:1), Nb91 (SEQ ID NO:2), Nb279 (SEQ ID NO:3), Nb315 (SEQ ID NO:4), Nb541 (SEQ ID NO:5), Nb556 (SEQ ID NO:6), Nb683 (SEQ ID NO:7), Nb691 (SEQ ID NO:8), Nb737 (SEQ ID NO:9).These nanobodies are specifically bound to mouse IgG (IgG1, IgG2a, IgG2b, IgG3), and do not cross-react with mouse IgM, human IgG and rabbit IgG.

[0047] The nanobodies in the embodiment are obtained by immunizing camelid (such as alpaca). Specifically, purified mouse IgG protein is used as an antigen to induce the production of antibodies against mouse IgG by immunizing alpaca multiple times. The immunization process includes primary immunization and multiple booster immunizations, and the interval between each immunization is 7-14 days. Complete Freund's adjuvant is used for primary immunization, and incomplete Freund's adjuvant is used for booster immunization. 3-4 days after the last immunization, 10-20 mL of peripheral blood of the alpaca is collected, and peripheral blood lymphocytes are isolated. Total RNA is extracted from the isolated lymphocytes, and cDNA first strand is synthesized using a reverse transcription kit. VHH gene fragments are amplified by PCR using specific primers. The amplified VHH gene fragments are ligated into phage display vector pDAN5, and E. coli TG1 competent cells are transformed to construct a nanobody phage display library.

[0048] Through phage display technology, mouse IgG1, IgG2a, IgG2b and IgG3 are used as positive screening antigens, and mouse IgM, human IgG and rabbit IgG are used as negative screening antigens. After 3-4 rounds of screening and enrichment, nanobody clones specifically binding to mouse IgG are obtained. The positive clones obtained by screening are subjected to sequencing analysis, and 9 different nanobody sequences are identified (such as Figure 1 ), which are named Nb26, Nb91, Nb279, Nb315, Nb541, Nb556, Nb683, Nb691 and Nb737, respectively.

[0049] The amino acid sequences of the nine nanobodies are SEQ ID NO: 1 to SEQ ID NO: 9, respectively. Among them, the amino acid sequence of Nb26 is SEQ ID NO: 1, the amino acid sequence of Nb91 is SEQ ID NO: 2, the amino acid sequence of Nb279 is SEQ ID NO: 3, the amino acid sequence of Nb315 is SEQ ID NO: 4, the amino acid sequence of Nb541 is SEQ ID NO: 5, the amino acid sequence of Nb556 is SEQ ID NO: 6, the amino acid sequence of Nb683 is SEQ ID NO: 7, the amino acid sequence of Nb691 is SEQ ID NO: 8, and the amino acid sequence of Nb737 is SEQ ID NO: 9.

[0050] In order to express and purify these nanobodies, the gene fragments (nucleotide sequences are shown in SEQ ID NO. 10-18) encoding each nanobody were cloned into the expression vector pET28a, and the E. coli BL21 (DE3) competent cells were transformed. The transformed strain was cultured in LB medium containing kanamycin (50 μg / mL) to OD600 reached 0.6-0.8, and IPTG (final concentration of 0.5 mM) was added to induce expression, and the expression was induced at 16°C for 16-18 hours. The bacterial cells were collected, and the target protein was purified by nickel affinity chromatography after ultrasonic disruption. The SDS-PAGE results of the purified nanobodies under reducing conditions are shown in FIG. 2. Figure 2 .

[0051] The binding properties of the nanobodies to mouse IgG were evaluated by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: mouse IgG (2 μg / mL) was coated in a 96-well plate, incubated at 4°C overnight; blocked with 1% BSA in PBS for 2 hours; the above prepared nanobodies were diluted by 2-fold from the initial concentration of 2 μg / mL, added to the enzyme-labeled plate at 100 μL / well, and incubated at room temperature for 1 hour; HRP-labeled anti-His tag antibody (1:5000 dilution) was added and incubated at room temperature for 1 hour; TMB substrate was added for color development, and after 10 minutes, stop solution was added, and the absorbance was measured at 450 nm wavelength. The results showed that the nine nanobodies could specifically bind to mouse IgG, among which Nb556 and Nb26 showed the highest binding capacity, as shown in FIG. 3. Figure 3 .

[0052] In order to evaluate the specificity of the nanobodies, cross-reactivity test was performed. Mouse IgG, mouse IgM, human IgG and rabbit IgG were coated on ELISA plates, respectively, and the purified nanobodies were added to detect their binding. The results showed that the nine nanobodies specifically bound to mouse IgG, but did not cross-react with mouse IgM, human IgG and rabbit IgG.

[0053] Table 1 Identification of the specific binding of nine nanobodies to mouse IgG, mouse IgM, human IgG and rabbit IgG

[0054]

[0055] The binding kinetics of the nanobodies to mouse IgG subtypes (IgG1, IgG2a, IgG2b, IgG3) were further determined by surface plasmon resonance (SPR) technology. The mouse IgG subtypes were coupled to a CM5 chip, and the binding kinetics curves were determined with the nanobodies as analytes at different concentrations (6.25-100 nM). The results showed that the nine nanobodies could all bind to all subtypes of mouse IgG, but the affinities were different. Among them, the dissociation constant (KD) of Nb556 for mouse IgG1, IgG2a, IgG2b and IgG3 was 0.35 nM, 0.42 nM, 0.51 nM and 0.63 nM, respectively, showing the most balanced binding characteristics.

[0056] The application potential of the nanobodies at the cellular level was verified by immunofluorescence microscopy. The hybridoma cells expressing mouse IgG were fixed on glass slides, and purified nanobodies (0.5 μg / mL) were added, followed by the addition of FITC-labeled anti-His tag antibody for detection. The results showed that the nanobodies could specifically recognize mouse IgG molecules on the cell surface, and there was no obvious fluorescence signal for control cells that did not express mouse IgG, as shown in Figure 4

[0057] Example 2

[0058] This embodiment provides a bivalent nanobody comprising two identical or different nanobody domains described in Example 1, which are connected in series by a connecting peptide, and the connecting peptide is a rigid α-helix connecting peptide (EAAAK)3.

[0059] In order to improve the affinity and stability of the nanobodies, a bivalent nanobody was constructed in this embodiment. Specifically, Nb556 and Nb26 with the highest affinity in Example 1 were selected, and a bivalent nanobody construction strategy was designed. The bivalent nanobody comprises two identical nanobody domains connected by a rigid α-helix connecting peptide (EAAAK)3.

[0060] ​First, two identical nanobody gene fragments were amplified by PCR and a DNA sequence encoding a linker peptide (EAAAK)3was introduced between the two fragments. Specifically, using primers containing the linker peptide sequence (SEQ ID NO. 19, SEQ ID NO. 20), the two nanobody gene fragments were connected with the linker peptide sequence by overlap extension PCR method. The PCR reaction conditions were: 95 °C pre-denaturation for 5 minutes; 95 °C denaturation for 30 seconds, 58 °C annealing for 30 seconds, 72 °C extension for 1 minute, for a total of 30 cycles; and finally 72 °C extension for 10 minutes.

[0061] Two bivalent nanobodies, Nb556-(EAAAK)3-Nb556 (nucleotide sequence as shown in SEQ ID NO. 21) and Nb26-(EAAAK)3-Nb26 (nucleotide sequence as shown in SEQ ID NO. 22), were constructed. The two bivalent nanobody genes were cloned into the expression vector pET28a and transformed into E. coli BL21 (DE3) competent cells. The transformed strain was cultured in LB medium containing kanamycin (50 μg / mL) until the OD600 reached 0.6-0.8, and IPTG (final concentration 0.5 mM) was added to induce expression, and the expression was induced at 16 °C for 16-18 hours. The bacterial cells were collected and disrupted by ultrasonication, and the target protein was purified by nickel affinity chromatography. The purified bivalent nanobody was analyzed by SDS-PAGE electrophoresis to confirm its purity.

[0062] The binding properties of bivalent nanobodies to mouse IgG were evaluated by ELISA. The results showed that bivalent nanobodies exhibited higher affinity compared to monovalent nanobodies. The titers of Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26 were about 3 times higher than those of the corresponding monovalent nanobodies (OD450 twice the blank well corresponding antibody concentration).

[0063] Table 2 Comparison of bivalent nanobody titers with monovalent nanobodies

[0064]

[0065] The binding kinetics parameters of bivalent nanobodies to mouse IgG were determined by SPR technology. The results showed that the dissociation rate (koff) of bivalent nanobodies was significantly reduced, indicating that the bivalent structure enhanced the binding stability of nanobodies to the target. The dissociation constant (KD) of Nb556-(EAAAK)3-Nb556 to mouse IgG1, IgG2a, IgG2b and IgG3 was 0.11 nM, 0.14 nM, 0.17 nM and 0.21 nM, respectively, which was about 3 times higher than that of monovalent Nb556.

[0066] The secondary structure of the bivalent nanobody was analyzed by circular dichroism (CD) analysis, which showed negative peaks at about 222 nm and 208 nm in the CD spectrum, indicating that the (EAAAK)3linker peptide indeed formed an a-helix structure in the bivalent nanobody Figure 5 ), which helps the correct orientation of the two nanobody domains and improves the overall stability and function of the bivalent nanobody.

[0067] Example 3

[0068] The nucleotide sequences (SEQ ID NO: 10 to SEQ ID NO: 18) encoding nine monovalent nanobodies (Nb26, Nb91, Nb279, Nb315, Nb541, Nb556, Nb683, Nb691, Nb737) were respectively cloned into different expression vectors.

[0069] For the phage display system, the nanobody gene was amplified by PCR, introduced with Not I and BssH II restriction enzyme sites, and then ligated into the pDAN5 vector which was also double-digested with Not I and BssH II. The ligation product was transformed into E. coli TG1 competent cells, and positive transformants were selected on LB agar plates containing ampicillin (100 μg / mL). The correctness of the recombinant vector was verified by colony PCR and DNA sequencing.

[0070] The pDAN5 vector contains the pIII protein gene, which can display the nanobody as a fusion protein on the surface of the phage, facilitating affinity screening and evolution. In addition, the pDAN5 vector contains a amber stopper (TAG) and an amber suppressor gene, which can express soluble nanobodies in non-suppressor host bacteria, facilitating functional verification.

[0071] For the prokaryotic expression system, the nanobody gene was amplified by PCR, introduced with Nco I and Xho I restriction enzyme sites, and then ligated into the pET28a vector which was also double-digested with Nco I and Xho I. The ligation product was transformed into E. coli DH5α competent cells, and positive transformants were selected on LB agar plates containing kanamycin (50 μg / mL). The correctness of the recombinant vector was verified by colony PCR and DNA sequencing.

[0072] The pET28a vector contains a T7 promoter and terminator, which can efficiently express the target protein under IPTG induction. In addition, the pET28a vector contains a His tag at the N and C termini of the target protein, facilitating protein purification. The constructed pET28a-Nb expression vector can efficiently express nanobodies in BL21 (DE3) host bacteria, with a yield of 25-45 mg / L of culture solution.

[0073] For eukaryotic expression system, the nanobody gene was amplified by PCR, introduced Xba I and Age I restriction enzyme sites, and ligated into pcDNA3.4 vector which was also digested by Xba I and Age I. The ligation product was transformed into E. coli DH5a competent cells, and positive transformants were selected on LB agar plates containing ampicillin (100 μg / mL). The correctness of the recombinant vector was verified by colony PCR and DNA sequencing.

[0074] The pcDNA3.4 vector contains a CMV promoter, which can efficiently express the target protein in mammalian cells. The constructed pcDNA3.4-Nb expression vector can efficiently express nanobodies in Expi293F™ cells, with a yield of 35-50 mg / L of culture.

[0075] For bivalent nanobodies, the nucleotide sequences encoding Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26 were cloned into pET28a and pcDNA3.4 vectors, respectively, to construct prokaryotic and eukaryotic expression systems. The expression levels of bivalent nanobodies in BL21(DE3) and Expi293F™ cells were up to 30-60 mg / L and 15-30 mg / L of culture, respectively.

[0076] To construct nanobody-enzyme fusion protein expression vectors, the horseradish peroxidase (HRP) gene (SEQ ID NO. 23) was amplified by PCR and connected to the nanobody gene by overlap extension PCR, and then cloned into pET28a and pcDNA3.4 vectors. The constructed Nb556-(EAAAK)3-Nb556-HRP and Nb26-(EAAAK)3-Nb26-HRP fusion proteins were successfully expressed in BL21(DE3) and Expi293F™ cells, and maintained enzyme activity and antigen binding activity.

[0077] By restriction enzyme digestion and DNA sequencing analysis, it was confirmed that the nanobody gene sequences in all constructed recombinant expression vectors were consistent with the design, without mutation or deletion. These recombinant expression vectors provide important tools for efficient expression and application of nanobodies.

[0078] Example 4

[0079] This example provides a recombinant cell transformed or transfected with the recombinant vector described in Example 3, wherein the host is E. coli TG1, BL21(DE3), or mammalian Expi293F™ cells.

[0080] To achieve high expression of nanobodies, different host cell systems were used in this embodiment. First, the pDAN5-Nb recombinant vector was transformed into E. coli TG1 competent cells. The specific method was as follows: 100 ng of the recombinant vector was mixed with 100 μL of TG1 competent cells, and then incubated in an ice bath for 30 minutes, heated at 42°C for 90 seconds, and then incubated in an ice bath for 2 minutes. Then, 900 μL of SOC medium was added, and the mixture was incubated at 37°C for 1 hour. Then, the mixture was coated on an LB agar plate containing ampicillin (100 μg / mL), and then incubated at 37°C overnight.

[0081] The transformed TG1 strain can be used for phage display and screening. The specific method was as follows: a single colony was inoculated into 2×YT medium, and then incubated at 37°C until the OD600 reached 0.5. Then, helper phage M13KO7 (final concentration of 10^9 pfu / mL) was added, and the mixture was incubated at room temperature for 30 minutes. Then, the mixture was transferred into 2×YT medium containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL), and then incubated at 30°C overnight. Then, the supernatant was collected by centrifugation, and then 1 / 5 volume of PEG / NaCl solution (20% PEG8000, 2.5M NaCl) was added, and then the mixture was incubated in an ice bath for 1 hour. Then, the precipitate was collected by centrifugation, and then resuspended in PBS to obtain phage particles displaying nanobodies.

[0082] The pET28a-Nb recombinant vector was transformed into E. coli BL21(DE3) competent cells. The specific method was as follows: 100 ng of the recombinant vector was mixed with 100 μL of BL21(DE3) competent cells, and then incubated in an ice bath for 30 minutes, heated at 42°C for 90 seconds, and then incubated in an ice bath for 2 minutes. Then, 900 μL of SOC medium was added, and the mixture was incubated at 37°C for 1 hour. Then, the mixture was coated on an LB agar plate containing kanamycin (50 μg / mL), and then incubated at 37°C overnight.

[0083] The transformed BL21(DE3) strain can be used for high expression of nanobodies. The specific method was as follows: a single colony was inoculated into LB medium, and then incubated at 37°C until the OD600 reached 0.6-0.8. Then, IPTG (final concentration of 0.5 mM) was added to induce expression, and then the mixture was incubated at 16°C for 16-18 hours. Then, the bacterial cells were collected by centrifugation, and then resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF). Then, the mixture was subjected to ultrasonic disruption (power of 300 W, working for 3 seconds, interval of 7 seconds, for a total of 30 minutes). Then, the supernatant was collected by centrifugation. Then, the target protein was purified by nickel affinity chromatography.

[0084] For monovalent nanobodies (Nb26, Nb91, Nb279, Nb315, Nb541, Nb556, Nb683, Nb691, Nb737), the expression level in BL21(DE3) was 25-45 mg / L culture. For bivalent nanobodies Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26, the expression level in BL21(DE3) was 46.2 mg / L and 34.7 mg / L culture, respectively. For nanobody-enzyme fusion proteins (Nb556-(EAAAK)3-Nb556-HRP and Nb26-(EAAAK)3-Nb26-HRP), the expression level in BL21(DE3) was 23.9 mg / L and 34.5 mg / L culture, respectively.

[0085] Mammalian Expi293FTM cells were transfected with pcDNA3.4-Nb recombinant vectors. The specific method was as follows: 100 μg of plasmid DNA was mixed with 450 μL of transfection reagent (such as Lipofectamine 3000), and incubated at room temperature for 15 minutes; the mixture was added dropwise to 100 mL of 2 x 10 6 Expi293FTM cells; cultured at 37°C, 5% CO2for 72 hours; the culture supernatant was collected, and the target protein was purified by protein A affinity chromatography or nickel affinity chromatography.

[0086] For monovalent nanobodies, the expression level in Expi293FTM cells was 35-50 mg / L culture. For bivalent nanobodies (Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26), the expression level in Expi293FTM cells was 15.5 mg / L and 29.1 mg / L culture, respectively. For nanobody-enzyme fusion proteins (Nb556-(EAAAK)3-Nb556-HRP and Nb26-(EAAAK)3-Nb26-HRP), the expression level in Expi293FTM cells was 9.5 mg / L and 24.8 mg / L culture, respectively.

[0087] Table 3 Expression levels (mg / L) of different nanobodies, bivalent nanobodies and nanobody-enzyme fusion proteins

[0088]

[0089] By SDS-PAGE electrophoresis analysis, it was confirmed that the nanobodies expressed in different host cells had the correct molecular weight. The molecular weight of monovalent nanobodies was about 15 kDa, the molecular weight of bivalent nanobodies was about 35 kDa, and the molecular weight of nanobody-HRP fusion proteins was about 75 kDa.

[0090] By ELISA and SPR analysis, it was confirmed that the nanobodies expressed in different host cells all maintained specific binding activity to mouse IgG. There was no significant difference in the affinity of nanobodies expressed in BL21 (DE3) and Expi293FTM cells, indicating that the host cell type had little effect on the function of nanobodies.

[0091] By thermal stability analysis, the stability of nanobodies expressed in different host cells was compared. The results showed that the thermal stability of nanobodies expressed in Expi293FTM cells was slightly higher than that of nanobodies expressed in BL21 (DE3), which may be related to post-translational modification in eukaryotic cells. After incubation at 60°C for 30 minutes, Nb556 expressed in Expi293FTM cells maintained 85% activity, while Nb556 expressed in BL21 (DE3) maintained 75% activity.

[0092] Example 5

[0093] This example provides a kit for detecting mouse IgG, comprising: (a) a bivalent nanobody described in Example 2 as a capture antibody; (b) a bivalent nanobody-enzyme fusion protein as a detection antibody, the enzyme being horseradish peroxidase (HRP) or soybean peroxidase; wherein the capture antibody and detection antibody are selected from the paired combination of Nb26-(EAAAK)3-Nb26 and Nb556-(EAAAK)3-Nb556-HRP.

[0094] In order to develop a mouse IgG detection kit with high sensitivity, this example constructed a sandwich ELISA system based on nanobodies. First, the best capture antibody and detection antibody pair was screened. Nine monovalent nanobodies and two bivalent nanobodies (Nb556-(EAAAK)3-Nb556 and Nb26-(EAAAK)3-Nb26) were used as capture antibodies, and the corresponding HRP-labeled nanobodies were used as detection antibodies for orthogonal pairing screening.

[0095] The specific method is as follows: different capture antibodies (2 μg / mL) were coated in a 96-well plate and incubated at 4°C overnight; blocked with 1% BSA in PBS for 2 hours; add 20 ng / mL mouse IgG and incubate at room temperature for 1 hour; add different HRP-labeled detection antibodies (1:3000 dilution) and incubate at room temperature for 1 hour; add TMB substrate for color development, add stop solution after 10 minutes, and measure the absorbance at 450 nm wavelength, select the antibody combination with the highest P / N value (signal-to-noise ratio).

[0096] By comparing the signal-to-noise ratio of different pairing combinations, it was determined that the pairing combination of Nb26-(EAAAK)3-Nb26 as the capture antibody and Nb556-(EAAAK)3-Nb556-HRP as the detection antibody had the best performance (Table 4).

[0097] Table 4 Screening of optimal antibody pairing

[0098]

[0099] To optimize the detection conditions, a series of parameter optimization experiments were performed. First, the coating concentration of the capture antibody was optimized. Different concentrations (0.5, 1, 2, 3 μg / mL) of Nb26-(EAAAK)3-Nb26 were coated in 96-well plates, and the detection performance was evaluated. The results showed that a coating concentration of 2.0 μg / mL could obtain the best signal-to-noise ratio.

[0100] Second, the working concentration of the detection antibody was optimized. Nb556-(EAAAK)3-Nb556-HRP was used for detection at different dilution ratios (1:1000, 1:2000, 1:3000, 1:4000, 1:5000), and the detection performance was evaluated. The results showed that a dilution ratio of 1:4000 could obtain the best signal-to-noise ratio.

[0101] Then, the sample incubation time was optimized. Mouse IgG samples were incubated in 96-well plates for different times (30 minutes, 60 minutes, 90 minutes), and the detection performance was evaluated. The results showed that an incubation time of 60 minutes could obtain the best signal-to-noise ratio.

[0102] Finally, the detection antibody incubation time was optimized. Nb556-(EAAAK)3-Nb556-HRP was incubated in 96-well plates for different times (30 minutes, 60 minutes, 90 minutes), and the detection performance was evaluated. The results showed that an incubation time of 60 minutes could obtain the best signal-to-noise ratio.

[0103] Based on the above optimization results, the optimal conditions for detecting mouse IgG were determined: the coating concentration of the capture antibody Nb26-(EAAAK)3-Nb26 was 2 μg / mL, the working dilution ratio of the detection antibody Nb556-(EAAAK)3-Nb556-HRP was 1:4000, and the incubation time of the sample and the detection antibody was 60 minutes (Table 5). Under these conditions, the detection limit was 2 ng / mL, and the linear range was 2-2000 ng / mL.

[0104] Table 5 Optimization of reaction conditions

[0105]

[0106] To verify the specificity of the kit, different species of IgG (mouse IgG, rat IgG, rabbit IgG, human IgG, goat IgG) were tested for cross-reactivity. The results showed that the kit had high specificity for mouse IgG, with less than 0.1% cross-reactivity with other species of IgG.

[0107] To verify the accuracy of the kit, samples containing known concentrations of mouse IgG (10, 50, 200, 1000 ng / mL) were prepared and tested using the kit. The results showed that the relative error between the measured value and the theoretical value was less than 10%, indicating that the kit had good accuracy.

[0108] To verify the precision of the kit, the same sample was repeatedly tested (n = 10). The results showed that the within-batch coefficient of variation (CV) was less than 5%, and the between-batch coefficient of variation was less than 10%, indicating that the kit had good precision.

[0109] To verify the stability of the kit, the kit was stored under different conditions (4°C, 25°C, 37°C) for different periods of time (0, 1, 2, 4, 8, 16, 32 weeks), and its detection performance was tested. The results showed that the kit could be stored stably at 4°C for at least 32 weeks, at 25°C for at least 4 weeks, and at 37°C for at least 1 week.

[0110] Example 6

[0111] This example provides a kit for detecting mouse IgG, which has the same technical features as Example 5, but further optimizes the coating concentration of the capture antibody, the working dilution ratio of the detection antibody, and the detection performance parameters.

[0112] To further optimize the performance of the kit for detecting mouse IgG, this example systematically studies the effects of different conditions on the detection performance. First, the effect of the coating concentration of the capture antibody Nb26-(EAAAK)3-Nb26 on the detection performance was studied. Different concentrations (0.5, 1, 2, 5 μg / mL) of capture antibody were coated in 96-well plates, and the detection performance was evaluated. The results showed that within the range of 0.5-2 μg / mL, the coating concentration of the capture antibody was positively correlated with the detection signal value, but when the concentration exceeded 2 μg / mL, the detection sensitivity did not significantly improve. Considering the balance between cost and performance, the optimal coating concentration range of the capture antibody was determined to be 0.5-2 μg / mL.

[0113] Secondly, the effect of the working dilution ratio of the detection antibody Nb556-(EAAAK)3-Nb556-HRP on detection performance was investigated. The detection antibody was used at different dilution ratios (1:1000, 1:2000, 1:3000, 1:4000, and 1:5000) to evaluate detection performance. The results showed that within the range of 1:1000–1:5000, the working dilution ratio of the detection antibody was positively correlated with the signal-to-noise ratio. However, when the dilution ratio was below 1:2000, the background signal increased significantly. Considering the balance between cost and performance, the optimal working dilution ratio range for the detection antibody was determined to be 1:2000–1:4000. The data are shown in Table 6.

[0114] Table 6 Optimization of Nb26-(EAAAK)3-Nb26 coating concentration and Nb556-(EAAAK)3-Nb556-HRP working concentration

[0115]

[0116] Then, the effect of sample matrix on detection performance was investigated. Mouse IgG standards were dissolved in different matrices (PBS, bovine serum, cell culture supernatant, and tissue homogenate supernatant), and the detection performance was evaluated. The results showed that different matrices had a certain impact on detection performance, but the matrix effect could be minimized and the accuracy of detection could be ensured by appropriate dilution (10-fold dilution of serum and tissue homogenate supernatant, and 2-fold dilution of cell culture supernatant).

[0117] To determine the detection linear range and limit of detection, a series of mouse IgG standards at concentrations (0, 4, 20, 100, 200, 750, 1500, 2000 ng / mL) were prepared and detected using optimized conditions. By plotting standard curves and analyzing linear relationships, the detection linear range was determined to be 2–2000 ng / mL, with a linear correlation coefficient R² greater than 0.99. Figure 6 The detection limit is defined as the concentration corresponding to the average value of the blank sample plus three times the standard deviation. The calculated detection limit is 2 ng / mL, which meets the requirement of ≤4 ng / mL.

[0118] To verify the kit's anti-interference ability, different concentrations of potential interfering substances, such as hemoglobin (0-5 g / L), bilirubin (0-200 mg / L), and triglycerides (0-30 g / L), were added to the samples. The results showed that these substances did not significantly affect the detection results within the normal physiological concentration range, indicating that the kit has good anti-interference ability.

[0119] To verify the suitability of the kit, the kit was used to detect the concentration of mouse IgG in actual samples of different sources. The samples included mouse serum, mouse ascites, hybridoma cell culture supernatant and recombinant antibody purification product. The results showed that the kit could accurately detect the concentration of mouse IgG in these samples, and the relative error was less than 15% compared with the detection results of commercial kits.

[0120] To verify the batch consistency of the kit, three batches of kits were produced, and the same samples were used for detection. The results showed that the relative standard deviation of the detection results of the three batches of kits was less than 10%, indicating that the kit had good batch consistency.

[0121] To evaluate the potential of the kit for clinical application, the kit was used to detect the changes of IgG levels in the serum of mouse disease models (such as autoimmune diseases, infectious diseases). The results showed that the kit could accurately reflect the changes of mouse IgG levels under disease conditions, and was positively correlated with the progression and severity of the disease.

[0122] In addition, a lateral flow immunochromatographic test strip based on the nanobody was also developed for rapid detection of mouse IgG. Nb26-(EAAAK)3-Nb26 was coated on the test line position of the nitrocellulose membrane, anti-His tag antibody was coated on the control line position, and Nb556-(EAAAK)3-Nb556-gold nanoparticle conjugate was used as the detection reagent. The test strip can complete the detection within 10 minutes, and the visual detection limit is 20 ng / mL, which is suitable for rapid detection on site.

[0123] Comparative Example 1

[0124] To evaluate the effect of the connecting peptide, the bivalent nanobody Nb556-(G4S)3-Nb556 (nucleotide sequence as shown in SEQ ID NO. 24) and Nb26-(G4S)3-Nb26 (nucleotide sequence as shown in SEQ ID NO. 25) constructed using a flexible connecting peptide (G4S)3 were compared using the method of Reference Example 2. The results showed that the bivalent nanobody constructed using a rigid α-helix connecting peptide (EAAAK)3 exhibited better thermal stability and binding activity. After incubation at 37°C for 24 hours, the bivalent nanobody using the (EAAAK)3 connecting peptide maintained more than 97% activity, while the bivalent nanobody using the (G4S)3 connecting peptide reduced about 20% activity.

[0125] Although the present application has been disclosed in preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A Nanobody that specifically binds to mouse IgG, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO. 1 or SEQ ID NO.

6.

2. A bivalent nanobody, characterized in that, The bivalent nanobody is formed by connecting two identical nanobodies in series through a rigid alpha-helix connecting peptide (EAAAK)3, and the amino acid sequence of the nanobody is shown in SEQ ID NO. 1 or SEQ ID NO.

6.

3. A gene encoding the nanobody of claim 1 or the bivalent nanobody of claim 2.

4. A recombinant vector carrying the gene of claim 3.

5. A recombinant cell expressing the nanobody of claim 1 or the bivalent nanobody of claim 2, or containing the gene of claim 3, or transformed or transfected with the recombinant vector of claim 4.

6. A product marked biologically or chemically, characterized in that, The product is the nanobody of claim 1 or the bivalent nanobody of claim 2, which is labeled with an enzyme, biotin, fluorescein, chemiluminescence, colloidal or magnetic beads.

7. A kit characterized in that, The kit contains the nanobody of claim 1, or the bivalent nanobody of claim 2, or the biologically or chemically labeled product of claim 6.

8. The kit of claim 7, wherein The kit comprises: (a) a bivalent nanobody as a capture antibody; (b) a bivalent nanobody-enzyme fusion protein as a detection antibody, wherein the enzyme is horseradish peroxidase or soybean peroxidase; The structural sequences of the capture antibody and the detection antibody are different.

9. Use of the nanobody of claim 1, or the bivalent nanobody of claim 2, or the gene of claim 3, or the recombinant vector of claim 4, or the recombinant cell of claim 5, or the biologically or chemically labeled product of claim 6 in the preparation of a medicament for in vitro detection of mouse IgG subtype products.

10. Use according to claim 9, characterized in that, The product is a reagent or a kit.

11. Use according to claim 9, characterized in that, The product is a detection chip or a biosensor.

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