Second antibody for resisting mouse IgG and rabbit IgG as well as preparation method and application of second antibody

Abstract

The invention relates to a second antibody for resisting mouse IgG and rabbit IgG as well as a preparation method and application of the second antibody. The second antibody of the anti-mouse IgG and the rabbit IgG sequentially comprises an anti-mouse IgG nano antibody, a biotin fixed-point binding peptide fragment and an anti-rabbit IgG nano antibody from an amino terminal to a carboxyl terminal. According to the design of the second antibody of the anti-mouse IgG and rabbit IgG with a brand-new structure and the preparation method, high-efficiency expression is realized, the second antibody of the anti-mouse IgG and rabbit IgG capable of realizing fixed-point biotinylation is obtained, the second antibody can be further subjected to various modification reformation based on a biotin-streptavidin bridge, the preparation method of the second antibody of the polymerase labeled anti-mouse IgG and rabbit IgG is further developed, and the application of the second antibody of the polymerase labeled anti-mouse IgG and rabbit IgG is expanded. According to the method, the polymerase-labeled mouse-rabbit universal secondary antibody with similar affinity to mouse IgG and rabbit IgG is obtained, the preparation period of the secondary antibody can be shortened, the preparation process of the secondary antibody is simplified, the stability between batches is improved, and meanwhile, the dependence on experimental animals is reduced.

Description

Technical Field

[0001] The invention 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 Art

[0002] Immunoassay is a technique that uses antigen-antibody specific reactions to detect specific proteins, hormones, enzymes and other biological molecules. It has a wide range of applications in disease diagnosis, treatment monitoring, drug development and other fields. Protein immunoblotting, immunohistochemistry and enzyme-linked immunosorbent assay are the three most commonly used techniques for immunoassays, which are used for qualitative, localized and quantitative detection of analytes, respectively. All three methods are highly dependent on secondary antibodies that specifically recognize primary antibodies and enhance signals. Rabbit and mouse antibodies are often used as the first antibody (primary antibody). The second antibody (secondary antibody) is an antibody that can bind to the first antibody. Its main function is to detect the presence of the first antibody and amplify the detection signal. The traditional secondary antibody is a polyclonal antibody, which is prepared by using the immunogenicity of antibodies as large molecular proteins to immunize xenogeneic animals. The immune system of xenogeneic animals produces immunoglobulins against this antibody. The production cycle is long, the process is complicated, and the controllability of production between batches is poor. Polyclonal antibodies have strong cross-reactions with other antibodies, which can easily lead to nonspecific background staining. In addition, the preparation of polyclonal antibodies inevitably harms experimental animals, and animal ethics issues need to be considered.

[0003] The conventional method for preparing enzyme-labeled secondary antibodies is usually to chemically couple polymerase to antibody molecules. For example, CN119199097A discloses a highly sensitive polymerase-labeled secondary antibody and a preparation method thereof, which specifically includes the following steps:

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

[0005] In summary, developing an efficient, highly stable and easy-to-operate method for preparing enzyme-labeled secondary antibodies while reducing dependence on experimental animals is of great significance in the field of enzyme-labeled secondary antibody preparation. Summary of the invention

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides a secondary antibody against mouse IgG and rabbit IgG, and its preparation method and application. A secondary antibody against mouse IgG and rabbit IgG with a specific structure and an efficient preparation method are designed, and a new method for preparing enzyme-labeled secondary antibody is further developed, and the preparation strategy of the secondary antibody is optimized to shorten the preparation cycle of the secondary antibody, simplify the preparation process of the secondary antibody, improve the stability between batches, and reduce the dependence on experimental animals at the same time.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a secondary antibody against mouse IgG and rabbit IgG. The secondary antibody against mouse IgG and rabbit IgG is, from the amino terminus to the carboxyl terminus, a nanobody against mouse IgG, a biotin site-specific binding peptide segment, and a nanobody against rabbit IgG in sequence.

[0009] The present invention designs a secondary antibody against mouse IgG and rabbit IgG with a specific structure, fuses two nanobodies with a biotin site-specific binding peptide segment, which can enable the two nanobody fusion proteins to bind a single biotin only at specific sites, while maintaining similar affinities of the two antibodies, and can be used for specific modification and transformation. For example, by coupling with a polymerase through a "biotin-streptavidin" bridge, precipitation formed by the high-level crosslinking of biotin and streptavidin can be avoided.

[0010] It can be understood that based on the specific connection structure designed by the present invention, polymerase labeling based on the "biotin-streptavidin" bridge can be achieved. In theory, any nanobody against mouse IgG, nanobody against rabbit IgG, etc. are applicable, and are not limited to specific sequences.

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

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

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

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

[0015] Preferably, the amino acid sequences of the linking polypeptides are each 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 is understood that based on the antibody designed in the present invention, antibodies with similar functions obtained by means of genetic modification in the art, such as amino acid substitution, deletion or addition, etc., should all be within the protection scope of the present invention. The number of amino acids substituted, deleted or added as described above can be any value, such as 1, 5, 10, 15 and above, etc., so that the sequence identity between the changed amino acid sequence and 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 above. In the art, when conservative substitution is carried out with amino acids having similar or similar properties, the function of the protein is usually not changed. For example, when amino acids with similar properties are substituted in the CDR region and / or FR region, the amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues are or are not encoded by the genetic code. Therefore, antibodies obtained by conservative substitution with amino acids having similar or similar properties are also within the protection scope of the present invention.

[0030] In a second aspect, the present invention provides a nucleic acid molecule, and the nucleic acid molecule encodes a secondary antibody against murine IgG and rabbit IgG described in the first aspect.

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

[0032] SEQ ID NO.7:

[0033] ATGGGCCAGGTTCAGCTGGTGGAAAGTGGTGGCGGTTGGGTTCAGCCGGGTGGCAGTCTGCGTCTGAGCTGCGCCGCAAGTGGCTTTACCTTTAGTGATACCGCCATGATGTGGGTTCGCCAGGCACCGGGTAAAGGTCGCGAATGGGTGGCAGCAATTGATACCGGTGGTGGCTATACCTATTATGCAGATAGTGTGAAAGGTCGCTTTACCATTAGCCGTGATAATGCAAAAAATACCCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCACGCTATTATTGTGCAAAAACCTATAGTGGCAATTATTATAGCAACTATACCGTTGCAAACTATGGTACCACCGGTCGTGGCACCCTGGTGACCGTGAGTAGCGGTGGCGGCGGTAGTGGTGGTGGCGGTAGCGGTGGCGGTGGCTCAGGTTTAAATGATATTTTTGAAGCCCAGAAAATCGAATGGCATGAACCGAAAAGTTGCGATAAAACCCATACCTGTCCGCCGTGCCCGGCACCGGAACTGCTGGGTGGTCAGGTGCAGCTGGTTGAAAGTGGTGGTGGTCTGGCACAGCCGGGTGGTAGTCTGCGTTTAAGCTGTGCAGTTAGTGGCTTTCGTTTTAGTTTTTATCAGATGACCTGGGTTCGTCAGGCCCCGGGCAAAGGTCTGGAATGGGTTGCAGATATTAATAGTGCAGGTGGCACCACCTATTATGCGGATAGTGTGAAGGGTCGCTTTGCCATTAGCCGCGATAATGCCAAAAATACCTTATATCTGCAGATGAACAGTCTGAAACCGGAGGATACCGCCGTGTATTATTGTGCCAAAGGCAAATTTCCGGTGGAAAGCCGCCGCCACGGTGGTACCGCACAGTGGGATGAATATGATTATTGGGGTCAGGGTACCCGCGTTACCGTGAGCAGC。

[0034] In the present invention, specific nucleic acid sequences are designed and optimized to be able to adapt to the host cell preference, improve the codon adaptation index, reduce translation obstacles, and thus significantly improve the protein expression efficiency.

[0035] In a third aspect, the present invention provides a recombinant vector, and the recombinant vector contains the nucleic acid molecule described in the second aspect.

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

[0037] In a fourth aspect, the present invention provides a recombinant cell, and the recombinant cell contains the nucleic acid molecule described in the second aspect.

[0038] The recombinant cell of the present invention expresses the secondary antibody against mouse IgG and rabbit IgG described in the first aspect, which may comprise a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule. The host cell may be a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell. Examples of prokaryotic cells are bacterial cells, examples of lower eukaryotic cells are yeast cells, and examples of higher eukaryotic cells are mammalian cells. A representative example is Escherichia coli. In one or more embodiments, the Escherichia coli cell may be Escherichia coli Rosetta-gami2(DE3)pLysS.

[0039] In a fifth aspect, the present invention provides a method for preparing the secondary antibody against mouse IgG and rabbit IgG described in the first aspect, and the preparation method includes:

[0040] Introducing the recombinant vector described in the third aspect into a host cell to obtain a recombinant cell, culturing the recombinant cell, collecting the cells and mixing them with a lysis buffer to obtain a lysis product; purifying the lysis product to obtain the secondary antibody against mouse IgG and rabbit IgG.

[0041] The transformation of the vector into the host cell can be carried out by conventional methods well-known to those skilled in the art. For example, the CaCl 2 method, the electroporation method, the calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. 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, chemical and other methods, and can be carried out by conventional methods well-known to those skilled in the art such as salting out, centrifugation, cell disruption, chromatography, etc.

[0042] In one or more embodiments, the culturing is carried out using a self-inducing medium that does not contain a chemical inducer such as IPTG; preferably, the self-inducing medium includes: peptone, yeast extract, glycerol, lactose, glucose, Na 2 HPO 4, NH 4 Cl, KH 2 PO 4 , Na 2 SO 4 and MgSO 4 .

[0043] In one or more embodiments, the auto-induction medium comprises: peptone 10 g / L, yeast extract 5 g / L, glycerol 5 g / L, lactose 2 g / L, glucose 0.5 g / L, Na 2 HPO 4 25 mmol / L, NH 4 Cl 50 mmol / L, KH 2 PO 4 25 mmol / L, Na 2 SO 4 5 mmol / L, MgSO 4 2 mmol / L, wherein the dosage of each component can fluctuate within 30%, 20%, 10% or 5% up and down.

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

[0045] In the present invention, by designing specific culturing conditions and controlling specific temperature and culturing time, the growth and metabolic efficiency of cells can be improved, the correct folding of proteins can be promoted, the biological activity and stability of recombinant proteins can be enhanced, and the expression level of recombinant proteins can be increased.

[0046] In one or more embodiments, the culturing is shaking flask culturing, and culturing is carried out under the conditions of 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 invention, by designing a specific component lysis buffer, the protein extraction efficiency can be improved, and the protein structure and activity can be protected.

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

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

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

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

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

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

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

[0056] In the present invention, by controlling specific sonication conditions, cell rupture can be accelerated, the protein extraction efficiency can be improved, protein degradation can be reduced, the protein quality can be improved, and the experimental reproducibility can be enhanced.

[0057] In one or more embodiments, the steps for constructing the recombinant vector are as follows: 1) Optimize the gene sequences for expressing secondary antibodies against mouse IgG and rabbit IgG in Escherichia coli, and synthesize the optimized gene fragments; 2) ligate the gene fragments to the expression vector; 3) transform the ligation product into competent Escherichia coli DH5α cells, screen for positive transformants, and after verification by Sanger sequencing, perform enlarged culture and extract the recombinant plasmid.

[0058] In one or more embodiments, the steps for preparing the recombinant cells are as follows: 1) Transform the recombinant vector into competent Escherichia coli cells; 2) Pick at least 3 single colonies and culture them in a small amount of LB medium, then inoculate them into a small amount of self-inducing medium for culture to screen for strains that highly express secondary antibodies against mouse IgG and rabbit IgG; 3) Inoculate the strains that highly express secondary antibodies against mouse IgG and rabbit IgG into LB medium and culture until the logarithmic growth phase, add glycerol and then freeze and store them as seed bacteria.

[0059] In one or more embodiments, the steps for culturing the recombinant cells to express the secondary antibody against murine IgG and rabbit IgG are as follows: 1) inoculating the recombinant bacterial seed bacteria into an LB liquid medium and culturing for 8-20 h; 2) inoculating the culture into a self-inducing medium at an inoculation amount of 1‰-1%; 3) culturing at 200-300 rpm in a constant-temperature horizontal oscillator, first culturing at 37°C for 1-6 h, and then culturing at 17-30°C for 10-24 h; 4) filtering or centrifuging to collect the bacterial cells and removing the liquid medium.

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

[0061] In one or more embodiments, nuclease and MgCl 2 are also added to the lysis product, and incubated at 25±3°C (preferably 25±2°C or 25±1°C).

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

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

[0064] In the present invention, by designing specific components of the affinity purification buffer, the purification efficiency can be improved, the protein activity can be protected, and the interference of impurities can be reduced.

[0065] In one or more embodiments, the purification buffer includes: 20±6 mM (preferably 20±4 mM, more preferably 20±2 mM) phosphate, 500±100 mM (preferably 500±80 mM, more preferably 500±40 mM) sodium chloride, 5±1% (preferably 5±0.8%, more preferably 5±0.4%) glycerol, 10±5 mM (preferably 10±3 mM, more preferably 10±2 mM) imidazole.

[0066] In a sixth aspect, the present invention provides the use of the secondary antibody against murine IgG and rabbit IgG described in the first aspect in the preparation of a polymerase-labeled secondary antibody against murine IgG and rabbit IgG.

[0067] In the present invention, two nanobodies are fused with a biotin site-specific binding peptide segment, so that the two-nanobody fusion protein binds to a single biotin only at a specific site. Then, the two-nanobody fusion protein is coupled with a polymerase through a "biotin-streptavidin" bridge to obtain a polymerase-labeled murine-rabbit universal secondary antibody with similar affinities for murine IgG and rabbit IgG.

[0068] In a seventh aspect, the present invention provides a secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG. The secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG includes the secondary antibody with an anti-mouse IgG and anti-rabbit IgG described in the first aspect. The secondary antibody with an anti-mouse IgG and anti-rabbit IgG is conjugated with biotin at the biotin site-specific binding peptide segment, and the biotin is linked to streptavidin conjugated with poly-horseradish peroxidase.

[0069] In an eighth aspect, the present invention provides a method for preparing the secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG as described in the seventh aspect. The preparation method includes:

[0070] Preparing the secondary antibody with an anti-mouse IgG and anti-rabbit IgG by using the preparation method of the secondary antibody with an anti-mouse IgG and anti-rabbit IgG described in the fifth aspect, biotinylating the secondary antibody with an anti-mouse IgG and anti-rabbit IgG to obtain a biotinylated antibody, and mixing the biotinylated antibody with streptavidin conjugated with poly-horseradish peroxidase to obtain the secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG.

[0071] Based on the designed specific structure of the secondary antibody with an anti-mouse IgG and anti-rabbit IgG, the present invention conducts polymerase labeling, site-specific biotinylation and polymerase connection, which can shorten the preparation cycle of the secondary antibody, simplify the preparation process of the secondary antibody, improve the stability between batches, and reduce the dependence on experimental animals at the same time.

[0072] Preferably, the biotinylation treatment includes: mixing the secondary antibody with an anti-mouse IgG and anti-rabbit IgG with biotin ligase, biotin, ATP and MgCl 2 together.

[0073] In one or more embodiments, the preparation of the secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG includes: replacing the dissolution buffer of the secondary antibody with an anti-mouse IgG and anti-rabbit IgG with a connection buffer and concentrating it to the required concentration; adding the secondary antibody with an anti-mouse IgG and anti-rabbit IgG, biotin ligase BirA, biotin, ATP, and MgCl 2 to the connection buffer to prepare a reaction system; ultrafiltering to remove excess biotin to obtain the site-specific biotinylated secondary antibody with an anti-mouse IgG and anti-rabbit IgG; mixing the site-specific biotinylated secondary antibody with an anti-mouse IgG and anti-rabbit IgG with streptavidin conjugated with a polymerase in proportion and incubating in a constant temperature horizontal oscillator; adding an excess of free biotin and continuing to incubate, and adding a preservation solution to obtain the secondary antibody with a polymerase-labeled anti-mouse IgG and anti-rabbit IgG.

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

[0075] In the present invention, a novel-structured secondary antibody against mouse IgG and rabbit IgG is designed. The anti-mouse IgG nanobody, the biotin site-specific binding peptide segment, and the anti-rabbit IgG nanobody are specifically linked, and a specific preparation method is designed to achieve high-efficiency expression, obtaining a secondary antibody capable of site-specific biotinylation of mouse IgG and rabbit IgG. It can be further modified in various ways based on the "biotin-streptavidin" bridge, showing broad application prospects. The present invention further develops a preparation method for a secondary antibody labeled with a polymerase against mouse IgG and rabbit IgG, which can shorten the preparation cycle of the secondary antibody, simplify the preparation process, improve the stability between batches, and reduce the dependence on experimental animals. Description of the Drawings

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

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

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

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

[0080] Figure 5 ELISA result diagram of the poly-HRP-labeled mouse-rabbit universal secondary antibody detected.

[0081] Figure 6 Immunohistochemistry result diagram of the poly-HRP-labeled mouse-rabbit universal secondary antibody detected. Detailed Embodiments

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

[0083] For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0084] Example 1

[0085] In this example, a recombinant plasmid for expressing the mouse-rabbit universal secondary antibody protein was constructed.

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

[0087] The nucleic acid sequence encoding the recombinant mouse-rabbit universal secondary antibody was optimized to achieve high-efficiency expression of the antibody in prokaryotic cells, and an optimized gene fragment was synthesized. Nco I / Xho I restriction site sequences were added to both ends respectively, and a recombinant mouse-rabbit universal secondary antibody protein-encoding gene with a size of about 927 bp was obtained. Its nucleotide sequence is as shown in SEQ ID NO.7.

[0088] At the same time, other optimized nucleic acid sequences encoding the recombinant mouse-rabbit universal secondary antibody, shown as SEQ ID NO.8, were synthesized and used as a control for the preparation of expression vectors and subsequent expression experiments.

[0089] SEQ ID NO.8:

[0090] ATGGGCCAGGTTCAGCTGGTGGAAAGTGGTGGCGGTTGGGTTCAGCCGGGTGGCAGTCTGCGTCTGAGCTGCGCCGCAAGTGGCTTTACCTTTAGTGATACCGCCATGATGTGGGTTCGCCAGGCACCGGGTAAAGGTCGCGAATGGGTGGCAGCAATTGATACCGGTGGTGGCTATACCTATTATGCAGATAGTGTGAAAGGTCGCTTTACCATTAGCCGTGATAATGCAAAAAATACCCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCACGCTATTATTGTGCAAAAACCTATAGTGGCAATTATTATAGCAACTATACCGTTGCAAACTATGGTACCACCGGTCGTGGCACCCTGGTGACCGTGAGTAGCGGTGGCGGCGGTAGTGGTGGTGGCGGTAGCGGTGGCGGTGGCTCAGGTTTAAATGATATTTTTGAAGCCCAGAAAATCGAATGGCATGAACCGAAAAGTTGCGATAAAACCCATACCTGTCCGCCGTGCCCGGCACCGGAACTGCTGGGTGGTCAGGTTCAGCTGGTGGAAAGTGGCGGTGGCCTGGCACAGCCGGGCGGTAGCTTACGTCTGAGCTGCGCCGTTAGCGGCTTTCGCTTTAGCTTTTATCAGATGACCTGGGTGCGTCAGGCCCCGGGCAAAGGTCTGGAATGGGTGGCAGATATTAATAGTGCCGGCGGCACCACCTATTATGCAGATAGTGTTAAAGGTCGCTTTGCCATTAGCCGTGATAATGCCAAAAATACCCTGTATCTGCAGATGAATAGTCTGAAACCGGAAGATACCGCAGTGTATTATTGCGCAAAAGGCAAATTTCCGGTTGAAAGCCGTCGCCACGGTGGTACCGCCCAGTGGGATGAATATGATTATTGGGGTCAGGGTACCCGTGTGACCGTTAGTAGC。

[0091] The pET28b vector and the synthesized recombinant murine and rabbit universal secondary antibody protein-coding gene fragment were double-digested with Nco I / Xho I, subjected to gel electrophoresis, and then recovered and purified. The two gene fragments were ligated with DNA ligase.

[0092] The ligation product was transformed into Escherichia coli DH5α competent cells and spread on an LB plate. After overnight incubation in an inverted position, positive transformants were picked, and the plasmid was extracted after sequencing to confirm the accuracy, thus obtaining a recombinant plasmid expressing recombinant murine and rabbit universal secondary antibody (named pET28b-Nb M&R IgG).

[0093] Example 2

[0094] In this example, 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 separately transformed into Escherichia coli Rosetta-gami2(DE3)pLysS competent cells. Three single colonies were picked and cultured in a small amount of LB medium, and then inoculated into a small amount of self-inducing medium for culture to screen for strains that highly express recombinant murine and rabbit universal secondary antibody protein.

[0096] The strain highly expressing recombinant murine and rabbit universal secondary antibody protein was inoculated into an LB medium and cultured until the logarithmic growth phase, and then glycerol was added and stored frozen as a seed bacterium.

[0097] Example 3

[0098] In this example, the expression of recombinant murine and rabbit universal secondary antibody protein was carried out.

[0099] Activation of glycerol bacteria: 10 μL of glycerol-preserved recombinant bacteria (prepared in Example 2) was taken and added to 2 mL of liquid LB medium, and Kana antibiotic with a final concentration of 100 μg / mL was added. The culture was carried out overnight at 37°C and 220 rpm.

[0100] Self-inducing expression: Inoculation was carried out at an inoculation amount of 1% into 80 mL of self-inducing medium, and the culture was carried out at 37°C and 220 rpm for 6 h, and then transferred to 20°C and 220 rpm for 22 h.

[0101] Among them, the formula of the self-inducing medium: peptone 10 g / L, yeast extract 5 g / L, glycerol 5 g / L, lactose 2 g / L, glucose 0.5 g / L, Na 2 HPO 4 25 mmol / L, NH 4 Cl 50 mmol / L, KH 2 PO 4 25 mmol / L, Na 2 SO4 5 mmol / L, MgSO 4 2 mmol / L.

[0102] In addition, different self-induced expression culture temperatures and times were adjusted for expression culture (with the remaining conditions being the same) as the control group, specifically including:

[0103] (1) After culturing at 37 °C for 6 h, transfer to 19 °C or 21 °C and continue culturing for 22 h;

[0104] (2) After culturing at 37 °C for 6 h, transfer to 17 °C, 25 °C or 30 °C and continue culturing for 22 h;

[0105] (3) After culturing at 37 °C for 6 h, transfer to 20 °C and continue culturing for 21 h or 23 h;

[0106] (4) After culturing at 37 °C for 6 h, transfer to 20 °C and continue culturing for 18 h or 26 h;

[0107] The amount of the target protein in the lysed supernatant of the control groups (1) and (3) was slightly lower than that after culturing at 37 °C for 6 h and then transferring to 20 °C and continuing culturing for 22 h, while the amount of the target protein in the lysed supernatant of the control groups (2) and (4) was lower than that of the control groups (1) and (3), indicating that by controlling the specific temperature and culture time in the present invention, the growth and metabolic efficiency of cells can be improved, the correct folding of proteins can be promoted, the biological activity and stability of the recombinant protein can be enhanced, and the expression level of the recombinant protein can be further increased.

[0108] Example 4

[0109] In this example, bacterial cells were collected, lysed, and the protein was purified.

[0110] The cultured bacterial cells of Example 3 were collected by centrifugation at 10000 g for 10 min, washed once with 10 mL of PBS, and resuspended in 4 mL of lysis buffer (containing 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM PMSF, pH 7.0).

[0111] Ultrasonic disruption was performed in an ice bath at a power of 150 W, with ultrasonic treatment for 4 s and then stopping for 8 s, for a total of 18 min.

[0112] Add 30 U / mL nuclease and 3 mM MgCl2 to the lysate, and incubate in a water bath at 25 °C for 10 min.

[0113] Centrifuge at 10000 g at low temperature for 10 min to separate the supernatant and precipitate. The supernatant was filtered through a 0.22 μm filter head and used for protein purification.

[0114] The filtered supernatant was loaded onto the equilibrated nickel column at a low flow rate. The flow-through was washed with a 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 nickel column was washed with 50 and 80 mM imidazole solutions, and the Nb M&R IgG protein was eluted with a 200 mM imidazole solution. The protein concentration was determined using the Bradford method. Samples from each collected fraction were taken for SDS-PAGE analysis. After determining the protein concentration, the samples were aliquoted and stored at -80 °C.

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

[0116] Protein purity = Analyze the SDS-PAGE detection map using Image J software to obtain the ratio of the gray value of the corresponding protein band to the total gray value of all protein bands in the entire lane.

[0117] Total protein amount = Protein concentration measured by the Bradford method × Volume of the protein solution.

[0118] The results showed that 15 mL of Nb M&R IgG protein solution was obtained by purification. The protein purity exceeded 95%. The concentration of Nb M&R IgG protein measured by the Bradford method was 0.93 mg / mL. The total amount of Nb M&R IgG protein obtained by purification was 13.95 mg, that is, 13.95 mg of Nb M&R IgG protein was obtained from the purification of 80 mL of culture product, indicating a high yield.

[0119] When other optimized nucleic acid sequences (SEQ ID NO.8) encoding the recombinant murine-rabbit universal secondary antibody were used to construct recombinant bacteria and express the antibody, the amount of the target protein expressed by the recombinant bacteria was too small. Only weak bands could be detected by WB and it was difficult to be detected by Coomassie Brilliant Blue staining, and it was difficult to obtain the Nb M&R IgG protein.

[0120] Example 5

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

[0122] The recombinant murine - rabbit universal secondary antibody obtained by purification in Example 4 was verified by enzyme - linked immunosorbent assay (ELISA). Rabbit IgG and mouse IgG at a concentration of 1 μg / mL were respectively coated onto ELISA microplates and incubated overnight at 4°C. Then, 1% BSA was used to block at 37°C for 1 h. The Nb M&R IgG purified in Example 4 was biotinylated and diluted to 6 μg / mL as the initial well concentration, and then serially diluted 5 - fold. The last well was blank. After incubation at 37°C for 1 h, the plates were washed 5 times with PBST and then patted dry. Streptavidin labeled with alkaline phosphatase AP was used as the secondary antibody, incubated at 37°C for 1 h, and the plates were washed 5 times with PBST and patted dry. 200 μL of AP enzyme substrate solution was added to each well and reacted for 4 min, and the chemiluminescence signal value was read with an enzyme - linked immunosorbent assay reader.

[0123] The results are as Figure 3 shown. Nb M&R IgG binds to both rabbit IgG and mouse IgG, and the detected signal value increases with the increase in the added concentration of Nb M&R IgG until it reaches a plateau. According to the principle of affinity determination by ELISA saturation concentration method, the EC 50 value is the dissociation equilibrium constant (KD value). The smaller the EC 50 value, the stronger the affinity of the antibody for the antigen. The EC 50 value obtained by detecting rabbit IgG with Nb M&R IgG is 0.138 μg / mL (i.e., 4.06×10 -9 M), and the EC50 value obtained by detecting mouse IgG is 0.145 μg / mL (i.e., 4.25×10 - 9 M). The affinities both reach the 10 -9 M level, belonging to strong antigen - antibody binding. The affinities of Nb M&R IgG for rabbit IgG and mouse IgG are similar, which is suitable for preparing a murine - rabbit universal secondary antibody.

[0124] Comparative Example 1

[0125] In this comparative example, another structure of recombinant murine - rabbit universal secondary antibody was designed. Compared with the structure of the recombinant murine - rabbit universal secondary antibody in Example 1, the difference is only that the recombinant murine - rabbit universal secondary antibody is, from the amino - terminal to the carboxyl - terminal, anti - rabbit IgG nanobody, linker polypeptide, anti - mouse IgG nanobody, hinge, and biotin site - specific binding peptide segment. Only the order of each part is changed, and the sequence remains the same. Referring to the methods of Examples 1 - 5, vector construction, recombinant bacteria construction, protein expression and purification, and enzyme - linked immunosorbent assay were carried out.

[0126] The results of the enzyme - linked immunosorbent assay are as Figure 4 shown. Nb R&M IgG binds to both rabbit IgG and mouse IgG, and the detected signal value increases with the increase in the added concentration of Nb R&M IgG until it reaches a plateau. According to the principle of affinity determination by ELISA saturation concentration method, the EC 50The value is the dissociation equilibrium constant (KD value), EC 50 The smaller the value, the stronger the affinity of the antibody for the antigen. The EC value obtained by detecting rabbit IgG with Nb R&MIgG 50 is 0.105 μg / mL (i.e., 3.08×10 -9 M), and the EC value obtained by detecting mouse IgG is 50 0.163 μg / mL (i.e., 4.78×10 -9 M). The affinity reaches the 10 -9 M level, belonging to strong antigen-antibody binding. However, the affinity of Nb R&MIgG for rabbit IgG and mouse IgG has a slightly larger difference. When used to prepare a universal secondary antibody for rabbits and mice, it is prone to show a preference for rabbit primary antibodies. The color development signal of the rabbit primary antibody detection group is stronger, and the color development signal of the mouse primary antibody detection group is slightly weaker.

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

[0128] Example 6

[0129] In this example, the universal secondary antibody for mice and rabbits labeled with poly-HRP was prepared by using the Nb M&R IgG prepared in Example 5.

[0130] According to the operation manual of the Avi-tag protein biotin labeling kit, site-specific biotin labeling of the purified Nb M&R IgG was performed. Specifically, the kit was taken out of the refrigerator 30 min before the experiment and equilibrated to room temperature (18 - 25 °C). Nb M&RIgG, biotin ligase BirA, biotin, ATP, and MgCl 2 were added to the ligation buffer to prepare a reaction system, incubated at 30 °C for 30 min, and ultrafiltered to remove excess biotin, thus obtaining site-specific biotinylated Nb M&R IgG. The site-specific biotinylated Nb M&R IgG was mixed with streptavidin conjugated with poly-horseradish peroxidase (HRP) (PolyHRP-SA) in a certain proportion, incubated at 37 °C and 500 rpm in a constant temperature horizontal oscillator for 2 h, and an excess of free biotin was added and incubated for another 1 h, thus obtaining the poly-HRP-labeled universal secondary antibody for mice and rabbits Nb M&R IgG-BS-pHRP.

[0131] Coat the ELISA plate with rabbit IgG and mouse IgG respectively. The first well is at 0.1 μg / mL, diluted in a three-fold gradient, and the last well is blank. Incubate overnight at 4°C. The next day, wash 5 times with PBST, pat dry, add 200 μL of 1% BSA to each well, block at 37°C for 1 h, wash 5 times with PBST, pat dry, add 100 μL of Nb M&R IgG-BS-pHRP (0.3 mg / mL, diluted 1:10000) to each well, incubate at 37°C for 1 h, wash 5 times with PBST, pat dry, add 100 μL of HRP substrate luminescent solution to each well, and immediately read the chemiluminescence signal value with an ELISA reader.

[0132] The results are as Figure 5 shown. Nb M&R IgG-BS-pHRP binds to both rabbit IgG and mouse IgG, and the signal value increases with the increase in the coating concentration of rabbit IgG and mouse IgG. Nb M&R IgG-BS-pHRP can be used as a universal secondary antibody for mice and rabbits.

[0133] Select tissues positive for CK5&6, Ki67, PSA, BAP1, PD-L2, c-Met, P40, CRP, FOXL2, and SDHA respectively to prepare paraffin sections for immunohistochemical detection. After baking, dewaxing, hydration, and repair, add the corresponding primary antibody for incubation. Among them, mouse-derived primary antibodies are used to detect CK5&6, Ki67, PSA, BAP1, and PD-L2, and rabbit-derived primary antibodies are used to detect c-Met, P40, CRP, FOXL2, and SDHA. After incubation and washing, add the poly-HRP-labeled universal secondary antibody Nb M&R IgG-BS-pHRP prepared in the present invention for incubation and color development.

[0134] The results show that the poly-HRP-labeled universal secondary antibody Nb M&R IgG-BS-pHRP can detect CK5&6, Ki67, PSA, BAP1, PD-L2, c-Met, P40, CRP, FOXL2, and SDHA. The target localization is accurate, and the positive sites are significantly different from the surrounding negative tissues. Exemplary detection results of CK5&6, Ki67, c-Met, and P40 are as Figure 6 shown. CK5&6 is mainly localized in the cytoplasm, Ki67 is mainly localized in the nucleus, c-Met is mainly localized in the cell membrane or cytoplasm, and P40 is mainly localized in the nucleus.

[0135] In summary, in the present invention, a novel-structured secondary antibody against mouse IgG and rabbit IgG and a preparation method thereof are designed to achieve efficient expression, and a secondary antibody capable of site-specific biotinylation of mouse IgG and rabbit IgG is obtained. It binds a single biotin only at specific sites, and various modifications of the secondary antibody can be further carried out based on the "biotin-streptavidin" bridge. Further, a preparation method of a secondary antibody labeled with a polymerase against mouse IgG and rabbit IgG is developed, and a polymerase-labeled mouse-rabbit universal secondary antibody with similar affinities for mouse IgG and rabbit IgG is obtained, which can shorten the preparation cycle of the secondary antibody, simplify the preparation process of the secondary antibody, improve the stability between batches, and reduce the dependence on experimental animals at the same time.

[0136] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A secondary antibody against mouse IgG and rabbit IgG, characterized in that: The secondary antibodies against mouse IgG and rabbit IgG are composed of anti-mouse IgG nano antibodies, biotin site-specific binding peptide segments and anti-rabbit IgG nano antibodies from the amino end to the carboxyl end.

2. The secondary antibody against mouse IgG and rabbit IgG according to claim 1, characterized in that: The amino acid sequence of the anti-mouse IgG nanobody includes the sequence shown in SEQ ID NO.1; Preferably, the amino acid sequence of the biotin site-specific binding peptide segment includes the sequence shown in SEQ ID NO.2; Preferably, the amino acid sequence of the anti-rabbit IgG nanobody includes the sequence shown in SEQ ID NO.3; Preferably, the anti-mouse IgG nanobody, the biotin site-specific binding peptide and the anti-rabbit IgG nanobody are connected by a connecting polypeptide; Preferably, the amino acid sequences of the connecting polypeptides are each independently selected from the sequence shown in SEQ ID NO.4 or the sequence shown in SEQ ID NO.5; Preferably, the amino acid sequence of the secondary antibody against mouse IgG and rabbit IgG includes the sequence shown in SEQ ID NO.

6.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the secondary antibody against mouse IgG and rabbit IgG according to claim 1 or 2.

4. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule of claim 3.

6. The method for preparing a secondary antibody against mouse IgG and rabbit IgG according to claim 1 or 2, characterized in that: The preparation method comprises: Introducing the recombinant vector of claim 4 into host cells, culturing the cells, collecting the cells and mixing them with a lysis buffer to obtain a lysis product; Purifying the lysate to obtain the secondary antibody against mouse IgG and rabbit IgG; Preferably, the culture conditions include: after culturing at 34-40°C for 4-8 hours, adjusting to 17-23°C for 17-27 hours; Preferably, the lysis buffer contains phosphate, sodium chloride, glycerol and phenylmethylsulfonyl fluoride; Preferably, the mixing further comprises a step of ultrasonic treatment; Preferably, the ultrasonic treatment has a power of 150 to 250 W, ultrasonic treatment for 3 to 5 seconds, and a rest period of 5 to 7 seconds, for a total of 10 to 20 minutes; Preferably, the purification method comprises affinity purification; Preferably, the purification buffer for affinity purification contains phosphate, sodium chloride, glycerol and imidazole.

7. Use of the secondary antibody against mouse IgG and rabbit IgG according to claim 1 or 2 in the preparation of polymerase-labeled secondary antibody against mouse IgG and rabbit IgG.

8. A polymerase-labeled secondary antibody against mouse IgG and rabbit IgG, characterized in that: The polymerase-labeled anti-mouse IgG and rabbit IgG secondary antibody comprises the anti-mouse IgG and rabbit IgG secondary antibody according to claim 1 or 2, wherein the anti-mouse IgG and rabbit IgG secondary antibody is connected with biotin at the biotin site-specific binding peptide segment, and the biotin is connected with streptavidin coupled with polymerized horseradish peroxidase.

9. The method for preparing a polymerase-labeled secondary antibody against mouse IgG and rabbit IgG as claimed in claim 8, characterized in that: The preparation method comprises: The method for preparing the secondary antibody against mouse IgG and rabbit IgG according to claim 6 is used to prepare the secondary antibody against mouse IgG and rabbit IgG, the secondary antibody against mouse IgG and rabbit IgG is biotinylated to obtain the biotinylated antibody, and the biotinylated antibody is mixed with streptavidin coupled to polymerized horseradish peroxidase to obtain the polymerase-labeled secondary antibody against mouse IgG and rabbit IgG.

10. The method for preparing a polymerase-labeled secondary antibody against mouse IgG and rabbit IgG according to claim 9, characterized in that: The biotinylation treatment comprises: mixing the secondary antibodies against mouse IgG and rabbit IgG with biotin ligase, biotin, ATP and MgCl2.

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