A secondary antibody against mouse IgG and rabbit IgG, and a preparation method and application thereof

Abstract

The application relates to a secondary antibody against mouse IgG and rabbit IgG and a preparation method and application thereof. The secondary antibody against mouse IgG and rabbit IgG is sequentially composed of an anti-mouse IgG nanobody, a biotin site binding peptide segment and an anti-rabbit IgG nanobody from an amino terminal to a carboxyl terminal. In the application, a novel structure of the secondary antibody against mouse IgG and rabbit IgG and a preparation method are designed, high-efficiency expression is realized, the secondary antibody against mouse IgG and rabbit IgG capable of site biotinization is obtained, various modification and transformation of the secondary antibody based on a "biotin-streptavidin" bridge can be further carried out, a preparation method of the secondary antibody against mouse IgG and rabbit IgG with a multienzyme label is further developed, the secondary antibody against mouse IgG and rabbit IgG with similar affinity to mouse IgG and rabbit IgG is obtained, the preparation cycle of the secondary antibody can be shortened, the preparation process of the secondary antibody can be simplified, the batch stability can be improved, and the dependence on experimental animals can be reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a secondary antibody against mouse IgG and rabbit IgG, and a preparation method and application thereof. BACKGROUND

[0002] Immune detection is a technology that uses antigen-antibody specific reaction to detect specific proteins, hormones and enzymes and other biological molecules, and has a wide range of applications in disease diagnosis, treatment monitoring, drug development and other fields. Western blotting, immunohistochemistry and enzyme-linked immunosorbent assay are three commonly used technologies for immune detection, which are respectively applied to qualitative, localization and quantitative detection of analytes, and all of the three methods are highly dependent on secondary antibodies that specifically recognize primary antibodies and enhance signals. Antibodies from rabbits and mice are often used as primary antibodies (primary antibodies), and secondary antibodies (secondary antibodies) are antibodies that can bind to primary antibodies, and their main role is to detect the presence of primary antibodies and amplify the detection signal. Traditional secondary antibodies are polyclonal antibodies, and their preparation is based on the immunogenicity of antibodies as macromolecular proteins, immunization of heterologous animals, and production of immunoglobulins against the antibodies by the immune system of the heterologous animals. The production cycle is long, the process is complex, the controllability between batches is poor, the polyclonal antibodies have strong cross-reactions with other antibodies, which can easily lead to non-specific background staining, and the preparation of polyclonal antibodies inevitably harms experimental animals, which requires consideration of animal ethics.

[0003] The traditional preparation method of enzyme-labeled secondary antibodies usually involves chemical coupling of a polymerase to an antibody molecule. For example, CN119199097A discloses a high-sensitivity polymerase-labeled secondary antibody and a preparation method thereof, which specifically includes the following steps:

[0004] (1) activating the labeled enzyme with a coupling agent; (2) coupling the activated labeled enzyme obtained in step (1) with hyperbranched aminated polyethylene glycol to obtain a hyperbranched complex loaded with an enzyme; and (3) activating the hyperbranched complex loaded with an enzyme obtained in step (2) with a coupling agent, and coupling with a secondary antibody to obtain a polymerase. However, such a process requires complex chemical reactions, which can produce unstable and randomly cross-linked molecules, and the number of polymerases coupled to each antibody is small and the fusion ratio of the antibody and the enzyme cannot be strictly controlled. Such enzyme-labeled antibodies often cannot meet the signal amplification requirements of immunohistochemical detection.

[0005] In summary, it is of great significance to develop a preparation method of enzyme-labeled secondary antibodies with high efficiency, high stability and simple operation, and to reduce the dependence on experimental animals for the field of enzyme-labeled secondary antibody preparation. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the application provides a secondary antibody against mouse IgG and rabbit IgG, a preparation method and application thereof, designs a specific structure of the secondary antibody against mouse IgG and rabbit IgG, and a high-efficiency preparation method, and further develops a novel enzyme-labeled secondary antibody preparation method, optimizes the preparation strategy of the secondary antibody, so as to shorten the preparation period of the secondary antibody, simplify the preparation process of the secondary antibody, improve the stability between batches, and reduce the dependence on experimental animals.

[0007] To achieve the above object, the application adopts the following technical solutions:

[0008] In a first aspect, the application provides a secondary antibody against mouse IgG and rabbit IgG, which comprises, from the amino terminus to the carboxyl terminus, an anti-mouse IgG nanobody, a biotin site-specific binding peptide segment and an anti-rabbit IgG nanobody.

[0009] The application designs a specific structure of the secondary antibody against mouse IgG and rabbit IgG, fuses the two nanobodies with the biotin site-specific binding peptide segment, so that the two nanobody fusion proteins can only bind to a single biotin at a specific site, while maintaining the similar affinity of the two antibodies, and can be used for specific modification and modification, such as coupling with polymerase through the "biotin-streptavidin" bridge, so as to avoid the precipitation of biotin and streptavidin due to high-level cross-linking.

[0010] It can be understood that the specific connection structure designed based on the application can realize the polymerase labeling based on the "biotin-streptavidin" bridge, and in theory, any anti-mouse IgG nanobody, anti-rabbit IgG nanobody, etc. are applicable, and are not limited to specific sequences.

[0011] Preferably, the amino acid sequence of the anti-mouse IgG nanobody comprises the sequence shown in SEQ ID NO. 1.

[0012] Preferably, the amino acid sequence of the biotin site-specific binding peptide segment comprises the sequence shown in SEQ ID NO. 2.

[0013] Preferably, the amino acid sequence of the anti-rabbit IgG nanobody comprises the sequence shown in SEQ ID NO. 3.

[0014] Preferably, the anti-mouse IgG nanobody, the biotin site-specific binding peptide segment and the anti-rabbit IgG nanobody are connected (or hinged) through a connecting polypeptide.

[0015] Preferably, the amino acid sequence of the connecting polypeptide is independently selected from the sequence shown in SEQ ID NO. 4 or the sequence shown in SEQ ID NO. 5.

[0016] Preferably, the amino acid sequence of the secondary antibody against mouse IgG and rabbit IgG comprises the sequence shown in SEQ ID NO. 6.

[0017] SEQ ID NO. 1:

[0018] QVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGREW VAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCAKT YSGNYYSNYTVANYGTTGRGTLVTVSS.

[0019] SEQ ID NO. 2:

[0020] GLNDIFEAQKIEWHE.

[0021] SEQ ID NO. 3:

[0022] QVQLVESGGGLAQPGGSLRLSCAVSGFRFSFYQMTWVRQAPGKGLEWV ADINSAGGTTYYADSVKGRFAISRDNAKNTLYLQMNSLKPEDTAVYYCAKGK FPVESRRHGGTAQWDEYDYWGQGTRVTVSS.

[0023] SEQ ID NO. 4:

[0024] GGGGSGGGGSGGGGS.

[0025] SEQ ID NO. 5:

[0026] PKSCDKTHTCPPCPAPELLGG.

[0027] SEQ ID NO. 6:

[0028] MGQVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGREWVAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCAKTYSGNYYSNYTVANYGTTGRGTLVTVSSGGGGSGGGGSGGGGSGLNDIFEAQKIEWHEPKSCDKTHTCPPCPAPELLGGQVQLVESGGGLAQPGGSLRLSCAVSGFRFSFYQMTWVRQAPGKGLEWVADINSAGGTTYYADSVKGRFAISRDNAKNTLYLQMNSLKPEDTAVYYCAKGKFPVESRRHGGTAQWDEYDYWGQGTRVTVSS.

[0029] It can be understood that, based on the antibodies designed in the present application, functionally similar antibodies obtained by using genetic modification means in the art to make amino acid substitutions, deletions or additions, etc. should be within the scope of the present application. The number of amino acids substituted, deleted or added can be any value, such as 1, 5, 10, 15 or more, so that the sequence identity of the changed amino acid sequence to its respective original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In the art, when performing conservative substitution with amino acids with similar or similar properties, the function of the protein is usually not changed, such as substituting amino acids with similar properties in the CDR region and / or the FR region. The amino acid residues that can be substituted conservatively are well known in the art. Such substituted amino acid residues are or are not encoded by the genetic code, and therefore, the antibodies obtained by conservative substitution with amino acids with similar or similar properties are also within the scope of the present application.

[0030] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-mouse IgG and rabbit IgG secondary antibody of the first aspect.

[0031] Preferably, the nucleic acid sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID NO. 7.

[0032] SEQ ID NO. 7:

[0033] ATGGGCCAGGTTCAGCTGGTGGAAAGTGGTGGCGGTTGGGTTCAGCCGGGTGGCAGTCTGCGTCTGAGCTGCGCCGCAAGTGGCTTTACCTTTAGTGATACCGCCATGATGTGGGTTCGCCAGGCACCGGGTAAAGGTCGCGAATGGGTGGCAGCAATTGATACCGGTGGTGGCTATACCTATTATGCAGATAGTGTGAAAGGTCGCTTTACCATTAGCCGTGATAATGCAAAAAATACCCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCACGCTATTATTGTGCAAAAACCTATAGTGGCAATTATTATAGCAACTATACCGTTGCAAACTATGGTACCACCGGTCGTGGCACCCTGGTGACCGTGAGTAGCGGTGGCGGCGGTAGTGGTGGTGGCGGTAGCGGTGGCGGTGGCTCAGGTTTAAATGATATTTTTGAAGCCCAGAAAATCGAATGGCATGAACCGAAAAGTTGCGATAAAACCCATACCTGTCCGCCGTGCCCGGCACCGGAACTGCTGGGTGGTCAGGTGCAGCTGGTTGAAAGTGGTGGTGGTCTGGCACAGCCGGGTGGTAGTCTGCGTTTAAGCTGTGCAGTTAGTGGCTTTCGTTTTAGTTTTTATCAGATGACCTGGGTTCGTCAGGCCCCGGGCAAAGGTCTGGAATGGGTTGCAGATATTAATAGTGCAGGTGGCACCACCTATTATGCGGATAGTGTGAAGGGTCGCTTTGCCATTAGCCGCGATAATGCCAAAAATACCTTATATCTGCAGATGAACAGTCTGAAACCGGAGGATACCGCCGTGTATTATTGTGCCAAAGGCAAATTTCCGGTGGAAAGCCGCCGCCACGGTGGTACCGCACAGTGGGATGAATATGATTATTGGGGTCAGGGTACCCGCGTTACCGTGAGCAGC.

[0034] In the present application, the specific nucleic acid sequence is designed and optimized to adapt to the host cell preference, improve the codon adaptation index, and reduce the translation barrier, thereby significantly improving the expression efficiency of the protein.

[0035] In a third aspect, the present application provides a recombinant vector containing the nucleic acid molecule of the second aspect.

[0036] In one or more embodiments of the present application, the starting vector of the recombinant vector can be a pET expression vector.

[0037] In a fourth aspect, the present application provides a recombinant cell containing the nucleic acid molecule of the second aspect.

[0038] The recombinant cell of the present application expresses the anti-mouse IgG and rabbit IgG secondary antibody of the first aspect, which can contain a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule, and the host cell can be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell, such as a bacterial cell, a yeast cell, or a mammalian cell. A representative example is Escherichia coli. In one or more embodiments, the E. coli cell can be E. coli Rosetta-gami2 (DE3) pLysS.

[0039] In a fifth aspect, the present application provides a method for preparing the anti-mouse IgG and rabbit IgG secondary antibody of the first aspect, which comprises:

[0040] The recombinant vector of the third aspect is introduced into a host cell to obtain a recombinant cell, which is cultured, and the cell is mixed with a lysis buffer to obtain a lysis product; the lysis product is subjected to purification treatment to obtain the anti-mouse IgG and rabbit IgG secondary antibody.

[0041] The transformation of the vector into the host cell can be performed by conventional methods well known to those skilled in the art. For example, CaCl2 method, electroporation method, calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging. The obtained transformants can be cultured by conventional methods well known to those skilled in the art, and the culture medium can be a conventional culture medium. The antibody produced by the transformants can be separated and purified by physical and chemical methods, and conventional methods well known to those skilled in the art such as salting out, centrifugation, cell disruption, and chromatography can be used.

[0042] In one or more embodiments, the culture utilizes a self-induction culture medium, and the self-induction culture medium does not contain a chemical inducer such as IPTG; preferably, the self-induction culture medium comprises proteose peptone, yeast extract, glycerol, lactose, glucose, Na2HPO4, NH4Cl, KH2PO4, Na2SO4, and MgSO4.

[0043] In one or more embodiments, the auto-induction medium comprises: tryptone 10 g / L, yeast extract 5 g / L, glycerol 5 g / L, lactose 2 g / L, glucose 0.5 g / L, Na2HPO425 mmol / L, NH4Cl 50 mmol / L, KH2PO425 mmol / L, Na2SO45 mmol / L, MgSO42 mmol / L, wherein the amount of each component can be floated within 30%, within 20%, within 10% or within 5% up and down.

[0044] Preferably, the culture conditions comprise: after being cultured at 34-40°C (preferably 37±2°C, more preferably 37±1°C) for 4-8h (preferably 6±1h, more preferably 6±0.5h), adjusting to 17-23°C (preferably 20±2°C, more preferably 20±1°C) for 17-27h (preferably 22±4h, more preferably 22±3h).

[0045] In the present application, specific culture conditions are designed, and specific temperature and culture time are controlled, which can improve the growth and metabolic efficiency of cells, promote the correct folding of proteins, improve the biological activity and stability of recombinant proteins, and improve the expression amount of recombinant proteins.

[0046] In one or more embodiments, the culture is a shaker culture, and the culture is carried out at 220±100 rpm, preferably 220±60 rpm, more preferably 220±30 rpm.

[0047] Preferably, the lysis buffer contains phosphate, sodium chloride, glycerol and phenylmethylsulfonyl fluoride (PMSF).

[0048] In the present application, a specific component lysis buffer is designed, which can improve the protein extraction efficiency and protect the protein structure and activity.

[0049] In one or more embodiments, the lysis buffer comprises:

[0050] Phosphate: 20±5mM (preferably 20±3mM, more preferably 20±2mM);

[0051] Sodium chloride: 500±100mM (preferably 500±70mM, more preferably 500±30mM);

[0052] Glycerol: 5±2% (v / v) (preferably 5±0.5%, more preferably 5±1% after 5±0.5%);

[0053] PMSF: 1±0.6mM (preferably 1±0.5mM, more preferably 1±0.3mM).

[0054] In one or more embodiments, after the lysis buffer treatment, physical disruption of the cells is performed; more preferably, ultrasonication is performed under ice bath conditions.

[0055] Preferably, the ultrasonication is performed at a power of 150-250 W (e.g., 160, 170, 180, 190, 200, 210, 220, 230, or 240 W, etc.), for 3-5 s (e.g., 3.5, 4, or 4.5 s, etc.), with a pause of 5-7 s (e.g., 5.5, 6, or 6.5 s, etc.), for a total of 10-20 min (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19 min, etc.).

[0056] In the present application, the specific ultrasonication conditions are controlled, which can accelerate cell rupture, improve protein extraction efficiency, reduce protein degradation, improve protein quality, and improve experimental repeatability.

[0057] In one or more embodiments, the recombinant vector construction step is as follows: 1) optimizing the gene sequence of the secondary antibody against mouse IgG and rabbit IgG for expression in E. coli, and synthesizing the optimized gene fragment; 2) linking the gene fragment to an expression vector; 3) transforming E. coli DH5a competent cells with the ligation product, screening for positive transformants, and expanding the culture after Sanger sequencing verification, and extracting the recombinant plasmid.

[0058] In one or more embodiments, the recombinant cell preparation step is as follows: 1) transforming E. coli competent cells with the recombinant vector; 2) picking at least 3 single colonies into a small amount of LB medium for culture, and then inoculating into a small amount of autoinduction medium for culture, and screening for strains that efficiently express the secondary antibody against mouse IgG and rabbit IgG; 3) inoculating the secondary antibody against mouse IgG and rabbit IgG high-efficiency expression strain into LB medium for culture to the logarithmic growth phase, and then freezing as seed bacteria after adding glycerol.

[0059] In one or more embodiments, the step of culturing the recombinant cells to express the secondary antibody against mouse IgG and rabbit IgG is as follows: 1) inoculating the recombinant bacterial seed bacteria into LB liquid medium for 8-20 h; 2) inoculating the culture into autoinduction medium at an inoculation amount of 1‰-1%; 3) culturing in a constant-temperature horizontal shaker at 200-300 rpm, first at 37°C for 1-6 h, and then at 17-30°C for 10-24 h; and 4) collecting the bacterial cells by filtration or centrifugation, and removing the liquid medium.

[0060] In one or more embodiments, the purification treatment includes: 1) resuspending the recombinant cell culture with lysis buffer, lysing the bacterial cells, and separating the supernatant and the precipitate; 2) filtering the lysed supernatant and then loading it onto a chromatography column for purification.

[0061] In one or more embodiments, the lysis product is further added with a nuclease and MgCl2, and incubated at 25±3℃ (preferably, 25±2℃ or 25±1℃).

[0062] Preferably, the purification method comprises affinity purification.

[0063] Preferably, the purification buffer for the affinity purification comprises phosphate, sodium chloride, glycerol and imidazole.

[0064] In the present application, specific components of the affinity purification buffer are designed to improve the purification efficiency, protect the activity of the protein, and reduce the interference of impurities.

[0065] In one or more embodiments, the purification buffer comprises: 20±6mM (preferably, 20±4mM, more preferably, 20±2mM) phosphate, 500±100mM (preferably, 500±80mM, more preferably, 500±400mM) sodium chloride, 5±1% (preferably, 5±0.8%, more preferably, 5±0.4%) glycerol, and 10±5mM (preferably, 10±3mM, more preferably, 10±2mM) imidazole.

[0066] In a sixth aspect, the present application provides a use of the anti-mouse IgG and rabbit IgG secondary antibody of the first aspect in the preparation of a polymeric enzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody.

[0067] In the present application, two nanobodies are fused with a biotin site-specific binding peptide segment, so that the two nanobody fusion proteins can bind to a single biotin at a specific site. The two nanobody fusion proteins are coupled with a polymeric enzyme through a "biotin-streptavidin" bridge, so as to obtain a polymeric enzyme-labeled mouse and rabbit universal secondary antibody with similar affinity to mouse IgG and rabbit IgG.

[0068] In a seventh aspect, the present application provides a polymeric enzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody, which comprises the anti-mouse IgG and rabbit IgG secondary antibody of the first aspect, wherein the anti-mouse IgG and rabbit IgG secondary antibody is connected with a biotin at the biotin site-specific binding peptide segment, and the biotin is connected with a streptavidin coupled with a polymeric horseradish peroxidase.

[0069] In an eighth aspect, the present application provides a preparation method of the polymeric enzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody of the seventh aspect, which comprises:

[0070] The anti-mouse IgG and rabbit IgG secondary antibody is prepared by the preparation method of the anti-mouse IgG and rabbit IgG secondary antibody of the fifth aspect, and the anti-mouse IgG and rabbit IgG secondary antibody is biotinylated to obtain a biotinylated antibody, and the biotinylated antibody is mixed with streptavidin coupled with polyhorseradish peroxidase to obtain the polyenzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody.

[0071] The anti-mouse IgG and rabbit IgG secondary antibody is prepared by the preparation method of the anti-mouse IgG and rabbit IgG secondary antibody of the fifth aspect, and the anti-mouse IgG and rabbit IgG secondary antibody is biotinylated to obtain a biotinylated antibody, and the biotinylated antibody is mixed with streptavidin coupled with polyhorseradish peroxidase to obtain the polyenzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody.

[0072] Preferably, the biotinylation process comprises mixing the anti-mouse IgG and rabbit IgG secondary antibody with biotin ligase, biotin, ATP and MgCl2.

[0073] In one or more embodiments, the preparation of the polyenzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody comprises: replacing the anti-mouse IgG and rabbit IgG secondary antibody dissolving buffer with a linking buffer and concentrating to a required concentration; adding the anti-mouse IgG and rabbit IgG secondary antibody, biotin ligase BirA, biotin, ATP, MgCl2 into the linking buffer to prepare a reaction system; removing excess biotin by ultrafiltration to obtain the site-specific biotinylated anti-mouse IgG and rabbit IgG secondary antibody; mixing the site-specific biotinylated anti-mouse IgG and rabbit IgG secondary antibody with streptavidin coupled with polyenzyme in a proportion, and incubating in a constant-temperature horizontal shaker; adding excess free biotin for continuous incubation, and adding a preservation solution to obtain the polyenzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody.

[0074] Compared with the prior art, the present application has at least the following beneficial effects:

[0075] In the present application, a novel structure of anti-mouse IgG and rabbit IgG secondary antibody is designed, the anti-mouse IgG nanobody, the biotin site-specific binding peptide segment and the anti-rabbit IgG nanobody are specifically connected, and a specific preparation method is designed to realize efficient expression, obtain the site-specific biotinylated anti-mouse IgG and rabbit IgG secondary antibody, and further modify the secondary antibody based on the "biotin-streptavidin" bridge, which has a wide application prospect. The present application further develops the preparation method of the polyenzyme-labeled anti-mouse IgG and rabbit IgG secondary antibody, which can shorten the preparation period of the secondary antibody, simplify the preparation process of the secondary antibody, improve the batch-to-batch stability, and reduce the dependence on experimental animals. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Structure diagram of the poly-HRP-labeled mouse and rabbit universal secondary antibody.

[0077] Figure 2 SDS-PAGE detection map of mouse rabbit universal secondary antibody expressed and purified in prokaryotes.

[0078] Figure 3 ELISA result map of purified mouse rabbit universal secondary antibody Nb M&R IgG.

[0079] Figure 4 ELISA result map of purified rabbit mouse universal secondary antibody Nb R&M IgG.

[0080] Figure 5 ELISA detection result map of poly-HRP labeled mouse rabbit universal secondary antibody.

[0081] Figure 6 Immunohistochemical detection result map of poly-HRP labeled mouse rabbit universal secondary antibody. DETAILED DESCRIPTION

[0082] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0083] If a specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0084] Example 1

[0085] In this embodiment, a mouse rabbit universal secondary antibody protein expression recombinant plasmid is constructed.

[0086] The recombinant mouse rabbit universal secondary antibody (named Nb M&R IgG) is composed of anti-mouse IgG nanobody (Anti-Mouse), linker polypeptide (Linker), biotin site binding peptide segment (Avi-tag), hinge (Hinge), and anti-rabbit IgG nanobody (Anti-Rabbit), and the structure is as shown in Figure 1 The protein amino acid sequence is shown in SEQ ID NO. 6, and the underlined parts are the anti-mouse IgG nanobody sequence, the biotin site binding peptide segment sequence, and the anti-rabbit IgG nanobody sequence, respectively.

[0087] The nucleic acid sequence encoding the recombinant mouse rabbit universal secondary antibody is optimized to realize efficient expression of the antibody in prokaryotic cells. The optimized gene fragment is synthesized, and Nco I / Xho I enzyme digestion site sequences are added at both ends. The recombinant mouse rabbit universal secondary antibody protein coding gene has a size of about 927 bp, and the nucleotide sequence is shown in SEQ ID NO. 7.

[0088] Meanwhile, a nucleic acid sequence of another optimized encoding recombinant mouse rabbit universal secondary antibody was synthesized, as shown in SEQ ID NO. 8, which was used as a control to prepare an expression vector and subsequent expression experiments.

[0089] SEQ ID NO. 8:

[0090] ATGGGCCAGGTTCAGCTGGTGGAAAGTGGTGGCGGTTGGGTTCAGCCGGGTGGCAGTCTGCGTCTGAGCTGCGCCGCAAGTGGCTTTACCTTTAGTGATACCGCCATGATGTGGGTTCGCCAGGCACCGGGTAAAGGTCGCGAATGGGTGGCAGCAATTGATACCGGTGGTGGCTATACCTATTATGCAGATAGTGTGAAAGGTCGCTTTACCATTAGCCGTGATAATGCAAAAAATACCCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCACGCTATTATTGTGCAAAAACCTATAGTGGCAATTATTATAGCAACTATACCGTTGCAAACTATGGTACCACCGGTCGTGGCACCCTGGTGACCGTGAGTAGCGGTGGCGGCGGTAGTGGTGGTGGCGGTAGCGGTGGCGGTGGCTCAGGTTTAAATGATATTTTTGAAGCCCAGAAAATCGAATGGCATGAACCGAAAAGTTGCGATAAAACCCATACCTGTCCGCCGTGCCCGGCACCGGAACTGCTGGGTGGTCAGGTTCAGCTGGTGGAAAGTGGCGGTGGCCTGGCACAGCCGGGCGGTAGCTTACGTCTGAGCTGCGCCGTTAGCGGCTTTCGCTTTAGCTTTTATCAGATGACCTGGGTGCGTCAGGCCCCGGGCAAAGGTCTGGAATGGGTGGCAGATATTAATAGTGCCGGCGGCACCACCTATTATGCAGATAGTGTTAAAGGTCGCTTTGCCATTAGCCGTGATAATGCCAAAAATACCCTGTATCTGCAGATGAATAGTCTGAAACCGGAAGATACCGCAGTGTATTATTGCGCAAAAGGCAAATTTCCGGTTGAAAGCCGTCGCCACGGTGGTACCGCCCAGTGGGATGAATATGATTATTGGGGTCAGGGTACCCGTGTGACCGTTAGTAGC.

[0091] The pET28b vector and the synthetic recombinant mouse rabbit universal secondary antibody protein coding gene fragment were digested with Nco I / Xho I, gel electrophoresis was performed, and the DNA fragments were recovered and purified, and the two gene fragments were connected by DNA ligase.

[0092] The ligation product was transformed into E. coli DH5a competent cells and plated on LB plates, and after overnight incubation, positive transformants were selected, and after sequencing to confirm accuracy, the plasmid was extracted, and the recombinant plasmid expressing the recombinant mouse rabbit universal secondary antibody (named pET28b-Nb M&R IgG) was obtained.

[0093] Example 2

[0094] In this embodiment, a recombinant bacterium (Rosetta-gami2(DE3)pLysS / pET28b-Nb M&R IgG) was constructed.

[0095] The recombinant plasmid pET28b-Nb M&R IgG prepared in Example 1 was transformed into E. coli Rosetta-gami2(DE3)pLysS competent cells, and 3 single colonies were selected and cultured in a small amount of LB medium, and then inoculated into a small amount of autoinduction medium for culture, and a strain with high expression of recombinant mouse rabbit universal secondary antibody protein was screened.

[0096] The recombinant mouse rabbit universal secondary antibody protein high expression strain was inoculated into LB medium and cultured to the logarithmic growth phase, and then glycerol was added for freezing as a seed bacterium.

[0097] Example 3

[0098] In this embodiment, the expression of recombinant mouse rabbit universal secondary antibody protein was carried out.

[0099] Glycerol bacterium activation: 10 μL of glycerol-stored recombinant bacteria (prepared in Example 2) was taken into 2 mL of liquid LB medium, and Kana antibiotic was added to a final concentration of 100 μg / mL, and the mixture was incubated at 37°C, 220 rpm, overnight.

[0100] Autoinduction expression: inoculate 80 mL of autoinduction medium with 1% inoculation amount, incubate at 37°C, 220 rpm, for 6 h, then transfer to 20°C, 220 rpm, and incubate for 22 h.

[0101] The autoinduction medium is prepared as follows: proteose peptone 10 g / L, yeast extract 5 g / L, glycerol 5 g / L, lactose 2 g / L, glucose 0.5 g / L, Na2HPO4 25 mmol / L, NH4Cl 50 mmol / L, KH2PO4 25 mmol / L, Na2SO4 5 mmol / L, MgSO4 2 mmol / L.

[0102] In addition, the expression culture temperature and time of different self-induction are adjusted for expression culture (the rest conditions are the same) as a control group, specifically including:

[0103] (1) 37℃ culture for 6h, then 19℃ or 21℃ culture for 22h;

[0104] (2) 37℃ culture for 6h, then 17℃, 25℃ or 30℃ culture for 22h;

[0105] (3) 37℃ culture for 6h, then 20℃ culture for 21h or 23h;

[0106] (4) 37℃ culture for 6h, then 20℃ culture for 18h or 26h;

[0107] The amount of target protein in the lysis supernatant of control groups (1) and (3) is slightly lower than that of 37℃ culture for 6h, then 20℃ culture for 22h, and the amount of target protein in the lysis supernatant of control groups (2) and (4) is lower than that of control groups (1) and (3), which indicates that the control of specific temperature and culture time can improve the growth and metabolic efficiency of cells, promote the correct folding of proteins, improve the biological activity and stability of recombinant proteins, and further improve the expression amount of recombinant proteins.

[0108] Example 4

[0109] This example carries out cell collection, lysis and protein purification.

[0110] 10000g centrifugal 10min collection of example 3 culture bacteria, washed once with 10mL PBS, resuspended in 4mL lysis buffer (containing 50mM phosphate, 300mM NaCl, 10mM imidazole, 5% glycerol, 1mM PMSF, pH 7.0).

[0111] Ultrasonic crushing in ice bath, power 150W, ultrasonic 4s, stop 8s, total ultrasonic 18min.

[0112] Add 30U / mL nuclease, 3mM MgCl2 in lysis product, 25℃ water bath for 10min.

[0113] 10000g low temperature centrifugal 10min to separate supernatant and precipitate, the supernatant is filtered by 0.22μm filter head and used for protein purification.

[0114] The filtered supernatant was loaded at a low flow rate into the equilibrated nickel column, washed with 10 mM imidazole solution (containing 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM PMSF, pH 7.0) and collected the flow-through, the nickel column was washed with 50, 80 mM imidazole solution, the Nb M&R IgG protein was eluted with 200 mM imidazole solution, the protein concentration was determined by Bradford method, each collected component was sampled for SDS-PAGE detection, and after determining the protein concentration, it was aliquoted and stored at -80°C.

[0115] The SDS-PAGE results are shown in Figure 2 It can be seen that the recombinant rabbit universal secondary antibody Nb M&R IgG protein is mainly expressed in a soluble form, accounting for more than 90% of the total expression amount of the target protein, can be effectively combined with the nickel column, and is fully eluted by 200 mM imidazole eluent. The purity of the purified protein was calculated, and the total amount of the protein was calculated.

[0116] The protein purity was analyzed by using Imag J software to analyze the SDS-PAGE detection graph, and the proportion of the gray scale of the corresponding protein band to the total protein band gray scale of the whole lane was obtained.

[0117] The total amount of protein was calculated by using the Bradford method to measure the protein concentration x protein volume.

[0118] The results show that 15 mL of Nb M&R IgG protein solution is obtained by purification, the protein purity is more than 95%, the Nb M&R IgG protein concentration measured by Bradford method is 0.93 mg / mL, and the total amount of Nb M&R IgG protein obtained by purification is 13.95 mg, that is, 80 mL of culture product is purified to obtain 13.95 mg of Nb M&R IgG protein, and the yield is high.

[0119] However, using other optimized nucleic acid sequences (SEQ ID NO. 8) encoding recombinant rabbit universal secondary antibody to construct recombinant bacteria and express antibodies, the amount of target protein expressed by the recombinant bacteria is too small, only a weak band can be detected by WB, and it is difficult to be detected by Coomassie brilliant blue staining, and it is difficult to obtain Nb M&R IgG protein.

[0120] Example 5

[0121] In this example, the expressed and purified Nb M&R IgG was detected by enzyme-linked immunosorbent assay.

[0122] The recombinant mouse-rabbit universal secondary antibody purified in Example 4 was verified by enzyme-linked immunosorbent assay. 1 μg / mL of rabbit IgG and mouse IgG were coated on ELISA microplates at 4°C overnight, and 1% BSA was used for blocking at 37°C for 1 h. The Nb M&R IgG biotinylated purified in Example 4 was diluted to 6 μg / mL as the first hole concentration, 5-fold gradient dilution, the last hole was blank, and 37°C incubation for 1 h. After 5 times of PBST plate washing and drying, alkaline phosphatase AP-labeled streptavidin was used as the secondary antibody, 37°C incubation for 1 h, 5 times of PBST plate washing and drying. 200 μL of AP enzyme substrate solution was added to each well for reaction for 4 min, and the chemiluminescence signal value was read by an enzyme label instrument.

[0123] The results are shown in Table 1. Figure 3 As shown in Table 1, Nb M&R IgG binds to rabbit IgG and mouse IgG, and the detection signal value increases with the increase of the concentration of Nb M&R IgG until reaching the plateau. According to the principle of ELISA saturation concentration method for determining affinity, the EC 50 value, i.e., the dissociation equilibrium constant (KD value), is measured. The smaller the EC 50 value, the stronger the affinity of the antibody to the antigen. The EC 50 value of Nb M&R IgG for detecting rabbit IgG is 0.138 μg / mL (i.e., 4.06 x 10 -9 M), and the EC - value for detecting mouse IgG is 0.145 μg / mL (i.e., 4.25 x 10 9 M), and the affinity reaches 10 -9 M level, which belongs to strong antigen-antibody binding. The affinity of Nb M&R IgG to rabbit IgG and mouse IgG is similar, and it is suitable for preparing mouse-rabbit universal secondary antibody.

[0124] Comparative Example 1

[0125] Another structure of recombinant mouse-rabbit universal secondary antibody was designed in this comparative example. Compared with the recombinant mouse-rabbit universal secondary antibody in Example 1, the difference is that the recombinant mouse-rabbit universal secondary antibody has, in order from the amino terminus to the carboxyl terminus, an anti-rabbit IgG nanobody, a connecting polypeptide, an anti-mouse IgG nanobody, a hinge, and a biotin site binding peptide segment, and the sequence of each part is unchanged. The methods of Examples 1-5 were referred to for expression vector construction, recombinant bacteria construction, protein expression and purification, and enzyme-linked immunosorbent assay.

[0126] The results of enzyme-linked immunosorbent assay are shown in Table 2. Figure 4 As shown in Table 2, Nb R&M IgG binds to rabbit IgG and mouse IgG, and the detection signal value increases with the increase of the concentration of Nb R&M IgG until reaching the plateau. According to the principle of ELISA saturation concentration method for determining affinity, the EC 50The value is the dissociation equilibrium constant (KD value), EC 50 The smaller the value, the stronger the affinity of the antibody to the antigen. The EC 50 value of the Nb R&MIgG for detecting rabbit IgG was 0.105 μg / mL (i.e. 3.08 x 10 -9 M), and the EC 50 value of the Nb R&MIgG for detecting mouse IgG was 0.163 μg / mL (i.e. 4.78 x 10 -9 M), both of which reached the 10 -9 M level, belonging to strong antigen-antibody binding. However, the affinity of the Nb R&MIgG to rabbit IgG and mouse IgG was slightly different, and when used to prepare rabbit-mouse universal secondary antibody, it showed a preference for rabbit primary antibody, and the color development signal was stronger in the rabbit primary antibody detection group and slightly weaker in the mouse primary antibody detection group.

[0127] Compared with the antibody structure of Comparative Example 1, the recombinant Nb M&R IgG protein designed in Example 1 has, from the amino terminus to the carboxyl terminus, an anti-mouse IgG nanobody, a connecting polypeptide, a biotin site binding peptide segment, a hinge, and an anti-rabbit IgG nanobody. The universal secondary antibody prepared by using this specific arrangement has the advantage of similar affinity to mouse IgG and rabbit IgG, and is more suitable for preparing a rabbit-mouse universal secondary antibody.

[0128] Example 6

[0129] In this example, the Nb M&R IgG prepared in Example 5 was used to prepare a poly-HRP labeled rabbit-mouse universal secondary antibody.

[0130] According to the Avi-tag protein biotin labeling kit operation instruction, the purified Nb M&R IgG was site-specific biotin labeled. Specifically, the kit was taken out from the refrigerator 30 min before the experiment and equilibrated to room temperature (18-25°C). The Nb M&R IgG, biotin ligase BirA, biotin, ATP, and MgCl2 were added to the reaction system prepared by the connection buffer, incubated at 30°C for 30 min, and ultrafiltration was performed to remove excess biotin, thereby obtaining site-specific biotinylated Nb M&R IgG. The site-specific biotinylated Nb M&R IgG was mixed with poly-HRP-conjugated streptavidin (PolyHRP-SA) at a certain ratio, incubated at 37°C in a constant temperature horizontal shaker at 500 rpm for 2 h, and then excess free biotin was added for further incubation for 1 h, thereby obtaining a poly-HRP labeled rabbit-mouse universal secondary antibody Nb M&R IgG-BS-pHRP.

[0131] The enzyme-labeled plate was coated with rabbit IgG and mouse IgG, respectively, with 0.1 μg / mL in the first well, three-fold gradient dilution, and blank in the last well, overnight at 4°C. The next day, wash 5 times with PBST, dry, and add 200 μL of 1% BSA to each well, block at 37°C for 1 h, wash 5 times with PBST, dry, and add 100 μL of Nb M&R IgG-BS-pHRP (0.3 mg / mL, diluted 1:10,000 for use) to each well, incubate at 37°C for 1 h, wash 5 times with PBST, dry, and add 100 μL of HRP substrate luminescent solution to each well, immediately read the chemiluminescence signal value with an enzyme-labeled instrument.

[0132] The results, as shown in Figure 5 , showed that Nb M&R IgG-BS-pHRP was combined with rabbit IgG and mouse IgG, and the signal value increased with the increase of the coating concentration of rabbit IgG and mouse IgG. Nb M&R IgG-BS-pHRP can be used as a mouse and rabbit universal secondary antibody.

[0133] Paraffin sections of tissues positive for CK5&6, Ki67, PSA, BAP1, PD-L2, c-Met, P40, CRP, FOXL2, and SDHA were selected for immunohistochemical detection. After baking, dewaxing, hydration, and repair, the corresponding primary antibodies were added for incubation. Mouse-derived primary antibodies were used for detection of CK5&6, Ki67, PSA, BAP1, and PD-L2, and rabbit-derived primary antibodies were used for detection of c-Met, P40, CRP, FOXL2, and SDHA. After incubation and washing, the poly-HRP-labeled mouse and rabbit universal secondary antibody Nb M&R IgG-BS-pHRP prepared in the application was added for incubation and color development.

[0134] The results showed that the poly-HRP-labeled mouse and rabbit universal secondary antibody Nb M&R IgG-BS-pHRP could be used for detection of CK5&6, Ki67, PSA, BAP1, PD-L2, c-Met, P40, CRP, FOXL2, and SDHA, with accurate target positioning and significant differences between positive sites and surrounding negative tissues. The detection results of CK5&6, Ki67, c-Met, and P40 are shown in Figure 6 , which showed that CK5&6 was mainly located in the cytoplasm, Ki67 was mainly located in the nucleus, c-Met was mainly located in the cell membrane or cytoplasm, and P40 was mainly located in the nucleus.

[0135] To sum up, the application designs a new structure of anti-mouse IgG and rabbit IgG secondary antibody and a preparation method, realizes high-efficiency expression, obtains the anti-mouse IgG and rabbit IgG secondary antibody capable of site-specific biotinylation, only binds a single biotin at a specific site, can be further modified based on the "biotin-streptavidin" bridge, further develops a preparation method of the anti-mouse IgG and rabbit IgG secondary antibody with a multimeric enzyme label, obtains the multimeric enzyme label mouse and rabbit universal secondary antibody with similar affinity to mouse IgG and rabbit IgG, can shorten the secondary antibody preparation period, simplify the secondary antibody preparation process, improve the batch stability, and simultaneously reduce the dependence on experimental animals.

[0136] The applicant declares that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the application, which can be easily thought of by any person skilled in the art, falls within the protection scope and disclosure scope of the application.

Claims

1. A secondary antibody against mouse IgG and rabbit IgG, characterized in that, The amino acid sequences of the secondary antibodies against mouse IgG and rabbit IgG are those shown in SEQ ID NO.

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the secondary antibodies against mouse IgG and rabbit IgG as described in claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleic acid sequence of the nucleic acid molecule is shown in SEQ ID NO.

7.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules as described in claim 2 or 3.

6. The method for preparing secondary antibodies against mouse IgG and rabbit IgG as described in claim 1, characterized in that, The preparation method includes: The recombinant vector of claim 4 is introduced into host cells, cultured, and the cells are collected and mixed with lysis buffer to obtain lysis products; The lysis products were purified to obtain the secondary antibodies against mouse IgG and rabbit IgG. The cultivation conditions include: incubating at 34~40℃ for 4~8 h, then adjusting to 17~23℃ for 17~27 h.

7. The method for preparing secondary antibodies against mouse IgG and rabbit IgG as described in claim 6, characterized in that, The lysis buffer contains phosphate, sodium chloride, glycerol, and benzyl sulfonyl fluoride; The mixing process also includes an ultrasonic treatment step; The ultrasonic treatment power is 150~250 W, ultrasonic treatment lasts for 3~5 seconds, pauses for 5~7 seconds, and ultrasonic treatment lasts for a total of 10~20 minutes; The purification method includes affinity purification; The affinity purification buffer contains phosphate, sodium chloride, glycerol, and imidazole.

8. The use of the secondary antibodies against mouse IgG and rabbit IgG as described in claim 1 in the preparation of polymerase-labeled secondary antibodies against mouse IgG and rabbit IgG.

9. A polymerase-labeled secondary antibody against mouse IgG and rabbit IgG, characterized in that, The polymerase-labeled anti-mouse IgG and rabbit IgG secondary antibodies include the anti-mouse IgG and rabbit IgG secondary antibodies as described in claim 1, wherein the anti-mouse IgG and rabbit IgG secondary antibodies are linked to biotin at the biotin-binding peptide, and the biotin is linked to streptavidin conjugated with polyhortradiction peroxidase.

10. The method for preparing polymerase-labeled anti-mouse IgG and rabbit IgG secondary antibodies as described in claim 9, characterized in that, The preparation method includes: Secondary antibodies against mouse IgG and rabbit IgG are prepared using the method for preparing secondary antibodies against mouse IgG and rabbit IgG as described in claim 6 or 7. The secondary antibodies against mouse IgG and rabbit IgG are then subjected to biotinylation to obtain biotinylated antibodies. The biotinylated antibodies are then mixed with streptavidin conjugated with polyhortradiction peroxidase to obtain polymerase-labeled secondary antibodies against mouse IgG and rabbit IgG.

11. The method for preparing polymerase-labeled secondary antibodies against mouse IgG and rabbit IgG according to claim 10, characterized in that, The biotinylation process includes mixing the secondary antibodies against mouse IgG and rabbit IgG with biotin ligase, biotin, ATP and MgCl2.

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