PrA / G-Cys fusion protein and application thereof

Abstract

The invention discloses a PrA / G-Cys fusion protein and application thereof. The structure of the PrA / G-Cys fusion protein is represented by a protein A sequence, a connecting peptide, a protein A sequence, a connecting peptide, a protein B sequence, a connecting peptide, a protein B sequence, a connecting peptide, (10-60) * a cysteine-containing polypeptide sequence, and 6 * a tag sequence, the protein A sequence is a ProA-B protein 1-38aa sequence, and the protein B sequence is a ProG-C2 protein 1-25aa sequence. The PrA / G-Cys fusion protein is high in prokaryotic expression quantity and easy to purify; after the polypeptide is coupled with HRP, one polypeptide can carry a plurality of HRP, so that a signal enhancement effect is achieved, and the polypeptide can be used as a second antibody to be applied to immunohistochemical staining.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a PrA / G-Cys fusion protein and its applications. Background Technology

[0002] Traditional secondary antibody protocols are a core component of immunoassay techniques based on the specific binding of antigen and antibody, widely used in experiments such as Western blotting, ELISA, immunohistochemistry (IHC), and immunofluorescence (IF). Secondary antibodies target and bind to the Fc or Fab fragment of the primary antibody, amplifying the signal and enabling detection through the chromogenic group they carry. Primary antibodies are typically rabbit or mouse antibodies, binding to the target site; secondary antibodies are generally polyclonal antibodies, such as goat anti-rabbit or goat anti-mouse antibodies, coupled with chromogenic groups, such as enzymes (HRP horseradish peroxidase, AP alkaline phosphatase) or fluorescent dyes (FITC, Cy3, Alexa Fluor series). The preparation of secondary antibodies requires animal immunization, blood collection followed by affinity chromatography purification, and conjugation of the chromogenic group.

[0003] Traditional secondary antibodies rely on animal immunization, a process that typically takes 2-3 months, resulting in high feeding costs. Furthermore, not all animals produce high-titer antibodies, and subsequent affinity purification requires specialized chromatography columns, further increasing costs. Immune responses vary naturally among different animals, and even within the same animal, antiserum quality can fluctuate at different immunization stages. These variations can lead to discrepancies in signal intensity and background levels between different batches of secondary antibodies, affecting experimental reproducibility.

[0004] The signal intensity of traditional secondary antibodies depends on the coupling sites on the surface of the secondary antibody molecule, such as amino and carboxyl groups. The number of these sites is limited; typically, one IgG molecule can only couple 2-4 HRPs. For target antigens present in extremely low concentrations in the sample, it is easy to fail to detect the target. Summary of the Invention

[0005] To address at least one of the aforementioned problems, this invention provides a PrA / G-Cys fusion protein and its application; the PrA / G-Cys fusion protein has high prokaryotic expression levels and is easy to purify; after being coupled with HRP, a single polypeptide can carry multiple HRPs, playing a role in signal enhancement, and can be used as a secondary antibody in immunohistochemical staining.

[0006] To achieve the above objectives, the present invention employs the following technical means: The first aspect of the present invention provides a PrA / G-Cys fusion protein, wherein the structure of the PrA / G-Cys fusion protein is represented as: protein A sequence + linker peptide + protein A sequence + linker peptide + protein B sequence + linker peptide + protein B sequence + linker peptide + (10-60) × cysteine-containing polypeptide sequence + 6 × tag sequence; The protein A sequence is the ProA-B protein 1-38aa sequence, and the protein B sequence is the ProG-C2 protein 1-25aa sequence.

[0007] In some embodiments of the present invention, the protein A sequence and protein B sequence may also be one of the full-length sequences of ProA-B protein and ProG-C2 protein.

[0008] In some embodiments of the present invention, the protein A sequence and protein B sequence may also be sequences of other lengths within the full-length sequences of ProA-B protein and ProG-C2 protein.

[0009] In some embodiments of the present invention, protein A sequence and protein B sequence can be protein sequences of the same or different lengths of the same protein.

[0010] In some embodiments of the present invention, the linker peptide is (GGGGS). n In a preferred embodiment of the present invention, n=2, that is, the linker peptide is GGGGSGGGGS.

[0011] In some embodiments of the present invention, the cysteine-containing polypeptide sequence is a Cys-Gly-Ser sequence.

[0012] In some embodiments of the present invention, the cysteine-containing polypeptide sequence may also be a single amino acid repeat of lysine or cysteine, or may be interspersed with other amino acids, such as glycine, glutamic acid, serine, etc.

[0013] In some embodiments of the present invention, the tag sequence is a His tag sequence. In other embodiments of the present invention, the tag sequence may also be other tags, such as a GST tag sequence.

[0014] In some embodiments of the present invention, the amino acid sequence of the PrA / G-Cys fusion protein is shown in SEQ ID No. 6.

[0015] A second aspect of the present invention provides a recombinant expression vector for expressing the PrA / G-Cys fusion protein described in the first aspect.

[0016] A third aspect of the present invention provides an engineered bacterium containing the PrA / G-Cys fusion protein described in the first aspect or the recombinant expression vector described in the second aspect.

[0017] A fourth aspect of the invention provides a conjugate obtained by conjugating the PrA / G-Cys fusion protein of the first aspect with HRP.

[0018] The fifth aspect of the invention provides the application of the conjugate described in the fourth aspect as a secondary antibody in immunohistochemical staining detection.

[0019] A sixth aspect of the invention provides the use of the conjugate described in the fourth aspect in the preparation of an immunohistochemical detection kit. In the kit, the conjugate is used to prepare a secondary antibody reagent.

[0020] Beneficial effects of the present invention Compared with existing technologies, the present invention has the following advantages: The present invention utilizes the Fc-terminal characteristics of ProG and ProA antibodies to efficiently bind to the antibodies, selects the specific binding fragments as chimeras, and fuses 60 amino acids at the C-terminus of the fusion protein. At the same time, the C-terminus is fused with a His tag, which facilitates the purification of E. coli after expression. The prokaryotic expression level is high and it is easy to purify. The fusion protein is coupled with HRP, and multiple HRPs can be carried on one polypeptide, which plays a role in signal enhancement. When applied to immunohistochemical staining, its staining effect is similar to that of traditional secondary antibodies, and it can be used as a substitute for secondary antibodies in IHC. Attached Figure Description

[0021] Figure 1 The results of Coomassie Brilliant Blue staining for six PrA / G-Cys fusion proteins are shown. Figure 2 The results of Coomassie Brilliant Blue staining of six PrA / G-Cys fusion protein-HRP conjugates are shown. Figure 3 The results show the ELISA detection of six PrA / G-Cys fusion protein-HRP conjugates; Figure 4 Immunohistochemical assays of six PrA / G-Cys fusion protein-HRP conjugates are shown; Figure 5 The immunohistochemical staining results of PrA / G-Cys fusion protein-HRP and PrA / G-Lys fusion protein-HRP are shown. Detailed Implementation

[0022] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0024] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0025] Example 1: PrA / G-Cys fusion protein gene design In this application, we abbreviate the fusion protein of Protein A and Protein G as ProA / G, and the fusion protein after expressing a cysteine ​​residue containing a thiol group (Cys) as ProA / G-Cys.

[0026] Protein A is a cell wall protein isolated from Staphylococcus aureus type A, with a molecular weight of approximately 42 kDa. It contains five IgG-binding domains (E, D, A, B, and C), among which B and C have the strongest affinity. Protein A has a strong binding affinity for human IgG1, IgG2, and IgG4, and also has good affinity for IgG from rabbits, pigs, dogs, guinea pigs, and other species. However, it has a very weak binding affinity for human IgG3 and mouse IgG1, and almost no binding to human IgM, IgA, IgE, or avian antibodies (IgY).

[0027] Protein G is a cell wall protein isolated from Gram-negative streptococci (Streptococcus sp.). It has a molecular weight of approximately 25 kDa and contains two main binding domains (C1 and C2). It can strongly bind to all human IgG subclasses and has a strong affinity for IgG from various mammals, including mice, rats, goats, cattle, and sheep. In particular, it can effectively bind to mouse IgG1, which has a weak binding affinity to Protein A. However, it still does not bind to other types of antibodies such as human IgM, IgA, and IgE.

[0028] The sequence of Protein A is referenced from UniProt (https: / / www.uniprot.org / ) number P02976; the sequence of Protein G is referenced from UniProt number P19909. When designing the PrA / G-Cys fusion protein, the domains with higher affinity were retained. Protein A retained its B domain (sequence shown in SEQ ID NO.1), and Protein G retained its C2 domain (sequence shown in SEQ ID NO.2). These domains were then expressed in tandem via linkers.

[0029] A thiol-containing cysteine ​​residue (Cys) was fused to the ProA / G peptide as a coupling site with HRP. HRP can be chemically modified to introduce a maleimide group (-MAL) (e.g., using an SMCC crosslinking agent), which then undergoes a specific thioether bond reaction with the thiol group (-SH) of Cys. Cys is a neutral amino acid, which can reduce background staining caused by charge issues in tissues. Furthermore, the specificity of MAL-SH coupling is much higher than that of NHS-NH2 coupling, making it suitable for scenarios with stringent background requirements. Simultaneously, the tandem expression of Cys is interspersed with neutral amino acids glycine (Gly) and serine (Ser) to reduce Cys density and increase peptide flexibility, making HRP more accessible. Finally, an HIS tag was fused to the C-terminus of the PrA / G-Cys peptide for purification after prokaryotic expression.

[0030] Based on the above analysis, different gene designs were performed on the PrA / G-Cys fusion protein to determine the optimal molecular design scheme. The molecular design is as follows: i, (ProA-B+GGGGSGGGGS)×2+(ProG-C2+GGGGSGGGGS)×2+Cys-Gly-Ser×20+6×His, totaling 316 aa, approximately 35.1 kDa, amino acid sequence is SEQ ID No.3.

[0031] ii. (ProA-B(1-38aa)+GGGGSGGGGS)×2+(ProG-C2+GGGGSGGGGS)×2+Cys-Gly-Ser×20+6×His, totaling 276aa, approximately 30.7kDa, amino acid sequence is SEQ ID No.4.

[0032] iii. (ProA-B+GGGGSGGGGS)×2+(ProG-C2(1-25aa)+GGGGSGGGGS)×2+Cys-Gly-Ser×20+6×His, totaling 272aa, approximately 30.1kDa, amino acid sequence is SEQ ID No.5.

[0033] iv. (ProA-B(1-38aa)+GGGGSGGGGS)×2+(ProG-C2(1-25aa)+GGGGSGGGGS)×2+Cys-Gly-Ser×20+6×His, totaling 232aa, approximately 25.7kDa, amino acid sequence is SEQ ID No.6.

[0034] v, (ProG-C2(1-25aa)+GGGGSGGGGS)×2+(ProA-B(1-38aa)+GGGGSGGGGS)×2+Cys-Gly-Ser×20+6×His, totaling 232aa, approximately 25.7kDa, amino acid sequence is SEQ ID No.7.

[0035] vi, (ProA-B(1-38aa)+GGGGSGGGGS)×2+(ProG-C2(1-25aa)+GGGGSGGGGS)×2+Cys-Gly-Ser×40+6×His, totaling 292aa, approximately 32.3kDa, amino acid sequence is SEQ ID No.8.

[0036] Example 2 Synthesis of 6 PrA / G-Cys fusion protein particles The designed amino acid sequences were reverse-translated into DNA sequences and optimized for the codon bias of *E. coli* to improve expression efficiency. EcoRI and HindIII restriction enzyme sites were added to both ends of the six molecular designs to provide cloning sites for the pET-28a(+) vector. The final sequences were synthesized by a gene synthesis company and cloned into pET-28a(+) according to the EcoRI and HindIII restriction sites, resulting in plasmids designed with six different sequences, i-vi.

[0037] The obtained i-vi6 plasmid dry powders were transformed into Escherichia coli DH5α for scale-up culture. (1) Centrifuge the dry plasmid powder at 5000 rpm for 1 min, and add 20 μL of ddH2O deionized water to dissolve the plasmid; (2) Take one 100 μL competent cell and thaw it on ice for 10 min, add 2 μL plasmid, gently shake to mix, incubate on ice for 30 min, then heat shock at 42℃ for 60 s without stirring, and incubate on ice for 5 min. (3) Add 400 μL of antibiotic-free LB liquid medium, shake at 180 rpm and incubate at 37°C for 45 min; (4) Centrifuge at 6000 rpm for 5 min, and resuspend the bacterial pellet in 150 μL of supernatant. Spread 50 μL and 100 μL of the supernatant onto kanamycin-resistant LB plates, respectively. (5) Incubate the plate upright for 1 hour, then invert it and incubate at 37°C for 14 hours; (6) Pick a single colony into LB liquid medium, add kanamycin antibiotic, shake at 220 rpm for 14 h, and extract plasmid according to the instructions of the plasmid extraction kit.

[0038] The extracted plasmids were sequenced and verified by a gene sequencing company to ensure the correctness of the sequence.

[0039] Example 3: Expression of PrA / G-Cys fusion protein in a prokaryotic system (1) Transform the correct plasmid, which has been verified by sequencing, into Escherichia coli expression strain BL21 Star (DE3) (Invitrogen™, C601003).

[0040] (2) Streak the platter on an LB plate containing 50 µg / mL kanamycin, incubate it upside down at 37°C overnight, and obtain a single colony.

[0041] (3) Pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing 50 µg / mL kanamycin. Incubate overnight at 37°C with shaking at 200-250 rpm.

[0042] (4) Transfer the overnight bacterial culture to 1L of fresh 50 µg / mL kanamycin LB medium at a ratio of 1:100.

[0043] (5) Incubate at 37℃ and 250 rpm, and monitor the OD600 value regularly. When the OD600 reaches 0.6-0.8, add the optimal concentration of 1mM IPTG and incubate at 25℃ and 200-250 rpm for 20 hours with shaking.

[0044] (6) After induction, collect the bacterial culture at 4°C, centrifuge at 6000×g for 15 min, and freeze at -80°C for later use.

[0045] Example 4: Purification of PrA / G-Cys fusion protein (1) The bacterial cells were resuspended in binding buffer (50mM Tris-HCl, pH 8.0, 500mM NaCl, 10mM imidazole, 1× protease inhibitor) and sonicated in an ice bath for 10s, 5s intervals, for a total of 2min. Centrifuged at 12000-15000×g for 30min at 4℃ and the supernatant was collected.

[0046] (2) Equilibrate the nickel ion affinity resin column (Ni-NTA Agarose, Qiagen, 30210) with 10 column volumes of binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10-20 mM imidazole, pH 8.0).

[0047] (3) Load the supernatant into a well-equilibrated nickel ion affinity resin column at a flow rate of 1 mL / 5 min.

[0048] (4) Rinse the column with 10 column volumes of binding buffer to remove unbound contaminants.

[0049] (5) Elution was performed using elution buffers containing different concentrations of imidazole (40 mM, 80 mM, and 250 mM). The target protein was competitively eluted at 250 mM imidazole. The eluted fractions were collected, and A280 absorbance was measured. Fractions containing high-purity target protein were combined.

[0050] (6) Rinse the column with 5 column volumes of deionized water to remove residual salt and imidazole.

[0051] (7) Pass the column through a column with 5 column volumes of 20 mM Tris-HCl, 500 mM NaCl, pH 8.0.

[0052] (8) Rinse with 10 column volumes of deionized water to completely remove EDTA.

[0053] (9) Pass the column through a 5-column volume of 0.2 M nickel sulfate (NiSO4) solution. The resin will recombine Ni. 2+ It will return to its light blue color.

[0054] (10) Equilibrate the column with 10 times the CV binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10-20 mM imidazole, pH 8.0).

[0055] (11) Pass the column through 5 column volumes of storage buffer (20% ethanol) and store at 4°C.

[0056] The purified expression levels of the six PrA / G-Cys fusion proteins are shown in Table 1.

[0057] Table 1. Expression levels of six molecular designs for the PrA / G-Cys fusion protein

[0058] The results showed that the molecular weights of the six molecular designs ranged from 25 to 35 kDa, and the molecular weight affected the expression level in prokaryotic expression. The corresponding expression level differences, from highest to lowest, were iv>ii>iii>vi>i>v; the expression levels of schemes ii and iv were relatively high; the expression level of scheme v was the lowest, possibly because the molecular design of v interfered with the synthesis or folding of proteins in prokaryotic cells, thus failing to achieve high expression.

[0059] Example 5: Coomassie Brilliant Blue staining of six PrA / G-Cys fusion proteins (1) SDS-PAGE: Prepare a 4%~20% gradient gel, pour it into a gel applicator, and insert a 15-well comb. After the gel solidifies, place it in the electrophoresis buffer, and add protein markers and different molecularly designed PrA / G-Cys fusion proteins to the 15-well comb. Add SDS-PAGE protein loading buffer (5×) (Sangon Biotech, C506032) to each of the six proteins, with a protein concentration of 1 mg / ml and a loading volume of 10 μg. Perform electrophoresis at 80V for 40 min; then at 120V for 80 min.

[0060] (2) Coomassie Brilliant Blue Staining: Add sufficient Coomassie Brilliant Blue staining solution (Genstar, E154-01) to the staining box and stain at room temperature on a shaker at 60 rpm for 1 hour; add destaining solution to the staining box and destain on a shaker at room temperature, changing the destaining solution every 20 minutes until the background is clear and the bands are distinct. Expose the stained gel in a gel imaging system.

[0061] The Coomassie Brilliant Blue staining results of the six molecularly designed PrA / G-Cys fusion proteins are shown in the figure. Figure 1 .

[0062] The left side of the diagram shows the protein markers. Lanes 1-6 correspond to the six PrA / G-Cys fusion proteins designed from i to vi. Molecular design i has a molecular weight of approximately 35.1 kDa, molecular design ii approximately 30.7 kDa, molecular design iii approximately 30.1 kDa, molecular designs iv and v both approximately 25.7 kDa, and molecular design vi approximately 32.3 kDa. In Coomassie brilliant blue staining, the six PrA / G-Cys fusion proteins match their theoretical molecular weights, and their band intensities are similar.

[0063] Example 6: Conjugation of six PrA / G-Cys fusion proteins with HRP (1) Reduction of PrA / G-Cys fusion protein 1. The concentration of the 6 purified PrA / G-Cys fusion proteins was adjusted to 1 mg / mL, and different concentrations of TCEP were added: 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, and incubated at room temperature for 30 min.

[0064] 2. After the reaction is complete, centrifuge at 4000×g for 10 min using a 10kD ultrafiltration tube (Millipore, UFC8010) to remove excess TCEP.

[0065] 3. Take a small amount of the ultrafiltration sample and use Ellman's reagent (Thermo Scientific, 22582) to detect the free thiol content. 1 mol of fusion protein theoretically contains 20 mol (40 mol for vi molecule design) -SH, but in practice it needs to reach ≥15 mol -SH (≥40 mol for vi molecule design), otherwise it needs to be reduced again.

[0066] (2) SMCC activates HRP 1. Dissolve HRP in 0.1M PBS + 5mM EDTA at pH 7.2 and adjust to 10mg / mL.

[0067] 2. Prepare the reaction system with SMCC and HRP in a molar ratio of 10:1. Add 25 μL of 10 mg / mL SMCC solution to 10 mg HRP, mix gently, and react at room temperature in the dark for 30 min.

[0068] 3. After the reaction is complete, centrifuge at 4000×g for 10 min using a 10kD ultrafiltration tube (Millipore, UFC8010) to remove excess SMCC.

[0069] (3) Fusion protein coupled with activated HRP 1. Activate HRP: Fusion protein = 25:1 (35:1 for vi molecule design) (molar ratio) mix. For example, for a 30kDa fusion protein (1mg / mL≈33.3nmol / mL), 33.3×25=832.5nmol of activated HRP needs to be added (44kDa HRP≈832.5×44≈36.6mg).

[0070] 2. Slowly add activated HRP to the fusion protein solution, gently invert to mix, and react at room temperature in the dark for 2 hours.

[0071] 3. Add L-cysteine ​​to a final concentration of 10 mM and incubate at room temperature for 15 min to block unreacted maleimide groups.

[0072] 4. After the reaction is complete, centrifuge at 4000×g for 10 min using a 100kD ultrafiltration tube (Millipore, UFC9100) to remove excess uncoupled fusion protein and free HRP.

[0073] 5. Measure the absorbance of the conjugate at the characteristic absorption peaks of HRP at 403 nm and 280 nm, and calculate the HRP loading of the PrA / G-Cys fusion protein according to Formula 1.

[0074] Formula 1: HRP / fusion protein molar ratio = (A403 × 0.62) / (A280 - A403 × 0.41) The loading amounts of PrA / G-Cys fusion protein coupled with HRP are shown in Table 2.

[0075] Table 2. Loading amounts of PrA / G-Cys fusion protein and HRP conjugation

[0076] The results showed that the HRP loading values ​​of molecular design iv were similar, while the loading value of molecular design vi was 14.329, significantly higher than the other groups. This is because molecular design vi fused and expressed more Cys that could be used for coupling. None of the six molecular designs reached the theoretical number of Cys fused and expressed. Theoretically, iv can carry 20 HRPs and vi can carry 40 HRPs, but in actual coupling, protein folding may prevent the exposure of some Cys thiol groups, or the molecular weight of HRPs may be relatively large compared to the PrA / G-Cys fusion proteins, resulting in insufficient steric hindrance for coupling to smaller proteins.

[0077] To determine the molecular weight of the six PrA / G-Cys fusion protein-HRP conjugates and whether there was any free fusion protein or free HRP residue, the six conjugates were stained with Coomassie Brilliant Blue, as described in Example 6. The results are shown below. Figure 2 In the figure, the left side is the protein marker, lane 1 is free HRP, lanes 2 and 3 are molecular design i before and after the fusion protein is coupled with HRP, lanes 3 and 4 correspond to molecular design ii, lanes 5 and 6 correspond to molecular design iii, lanes 7 and 8 correspond to molecular design iv, lanes 9 and 10 correspond to molecular design v, and lanes 11 and 12 correspond to molecular design vi, respectively, comparing the size of each fusion protein before and after coupling.

[0078] The results showed that the PrA / G-Cys fusion proteins in all six groups were located around 30 kDa before coupling, with a loading amount of 10 μg and clear bands. After HRP coupling, the main bands were located above 250 kDa and the bands were diffuse, indicating that the coupling products were not homogeneous. A slight band was observed at 44 kDa in all six PrA / G-Cys fusion protein-HRP conjugates, indicating a small amount of HRP residue. No obvious bands were observed around 30 kDa, indicating that the free fusion protein had been completely removed. Although molecular design vi detected more HRP coupling, it could not be distinguished from other groups due to insufficient gradient gel separation.

[0079] Example 7: ELIASA detection of six PrA / G-Cys fusion protein-HRP conjugates (1) Coating: Mouse IgG (Hangzhou Bailing Biotechnology, cat#BX50141) or rabbit IgG (Hangzhou Bailing Biotechnology, cat#BX50204) were coated onto the microplate (catalog number), 100 μL per well, at a concentration of 1 μg / mL, diluted with PBS, and incubated overnight at 4℃.

[0080] (2) Wash the plate three times with PBST in a plate washer and pat dry any residual liquid.

[0081] (3) Blocking: Add 200 μL of blocking solution to each well, dissolve 5% skim milk powder in PBS, and incubate at 37°C for 1 hour.

[0082] (4) Wash the plate three times with PBST in a plate washer and pat dry any residual liquid.

[0083] (5) Sample loading: A blank control was set up, and free HRP was used as a negative control. Serum-purified goat anti-polyclonal antibody (Yisheng Bio, 34851ES60) and goat anti-rabbit polyclonal antibody (Yisheng Bio, 34850ES60) were used as positive controls. The samples to be tested were 6 PrA / G-Cys fusion protein-HRP conjugates serially diluted 10-fold, for a total of 8 gradients, and diluted in 5% skim milk. 100 μL per well, incubated at 37℃ for 1 hour.

[0084] (6) Wash the plate three times with PBST in a plate washer and pat dry any residual liquid.

[0085] (7) Color development: Add 100 μL of TMB color development solution to each well and incubate at 30°C in the dark for 15 min.

[0086] (8) Termination: Add 50 μL of 2M H2SO4 to each well to terminate the reaction, and immediately measure the absorbance at 450 nm on an ELISA reader to calculate IC50.

[0087] See results Figure 3 In Figure A, the source of the antibody is mouse antibody, and in Figure B, the source of the antibody is rabbit antibody.

[0088] Column C shows the IC50 values, reflecting the binding affinity between the antibody / protein and the coating source. A lower IC50 value indicates stronger binding affinity. Free HRP does not bind to mouse or rabbit antibodies, exhibiting an extremely high IC50 value (1.09 x 10⁻⁶). 4 ; 1.37X10 3 The IC50 values ​​of serum-purified goat anti-mouse and goat anti-rabbit polyclonal antibodies against mice and rabbits were 8.12 and 12.1 ng / mL, respectively, and they did not bind to non-corresponding coating sources. The IC50 of the fusion protein-HRP conjugate group was on the same order of magnitude as that of the goat anti-mouse and goat anti-rabbit control groups, indicating similar IgG binding activity, and that HRP retained its corresponding activity after conjugation. Comparing the IC50 values, group i was higher than the positive control; group ii retained part of the ProA-B binding domain, with an increased IC50 against rabbit anti-coated sources, but a slightly decreased IC50 against mouse anti-coated sources; group iii retained part of the ProG-C2 binding domain, with the IC50 against rabbit / mouse anti-coated sources remaining essentially unchanged; group iv retained part of the front core of the ProA-B and ProG-C2 binding domains, with IC50 against both rabbit and mouse anti-coated sources weaker than group i and similar to the control; group v had the strongest IC50 among all groups; and group vi was close to group i but slightly higher.

[0089] The results showed that the fusion peptides expressing ProA-B (1-38aa) and ProG-C2 (1-25aa) in tandem (group iv) had a stronger affinity for IgG than the complete binding domains of ProA-B and ProG-C2 (group i), and also stronger than the fusion peptides using ProA-B (1-38aa) or ProG-C2 (1-25aa) alone (groups ii and iii). This indicates that the tandem combination of ProA-B (1-38aa) and ProG-C2 (1-25aa) is more suitable for the expression and folding of the PrA / G-Cys fusion protein, and can better exert the binding function of ProA-B and ProG-C2 for IgG, which is better than using the binding domain fragments alone.

[0090] When the expression order of ProA-B (1-38aa) and ProG-C2 (1-25aa) was reversed (group v), the expression level of the fusion protein was relatively low (Table 1), and its binding affinity to IgG was the lowest among all groups. This indicates that the fusion expression order of ProA-B and ProG-C2 is equally important for yield and titer. This may be because different polypeptide fragments correspond to different folding patterns, which can affect the expression efficiency and protein folding function of E. coli.

[0091] When the amount of Cys was increased, leading to more HRP conjugation (group vi) (Table 2), the IC50 did not show a significantly higher signal despite carrying more HRP. Conjugating more HRP to the fusion peptide may result in the IgG binding domain being blocked by HRP, leading to reduced IgG binding efficiency; or although more HRP may be bound, steric hindrance may prevent all HRP from exerting their catalytic effect on the substrate. In summary, increasing the amount of Cys expression in the fusion peptide did not improve the HRP amplification effect.

[0092] Example 8 Immunohistochemical staining detection of six PrA / G-Cys fusion protein-HRP conjugates Immunohistochemical staining was performed on three target sites: cell membrane, cytoplasm, and nucleus, using serum-purified goat anti-mouse + goat anti-rabbit and i-vi group fusion protein-HRP conjugates, respectively.

[0093] The cell membrane target was selected as CD105 (Hangzhou Bailing Biotechnology, cat#BX50332, rabbit monoclonal antibody). CD105 is a cell membrane protein that is mainly expressed in the vascular endothelial cells of the placenta. Placental tissue was selected for IHC staining.

[0094] The target cell nucleus was Ki-67 (Hangzhou Bailing Biotechnology, cat#BX50204, rabbit monoclonal antibody). Ki-67 is a nucleoprotein related to cell proliferation, mainly expressed in the cell nucleus. It shows weak to moderate staining intensity in B cells of the germinal center of the tonsils. Tonsillar tissue was selected for IHC staining.

[0095] The cytoplasmic target was selected as Actin (Hangzhou Bailing Biotechnology, cat#BX50141, mouse monoclonal antibody). Actin is one of the most abundant and functionally core cytoskeletal proteins in eukaryotic cells. When staining endometrial tissue, it should show diffuse positive staining in the cytoplasm of endometrial epithelial cells.

[0096] The immunohistochemical staining process is as follows: (1) Baking slices: Cut the tissue into slices of 1-2 μm and bake them in an oven at 60℃ for about 1 hour; (2) Antigen retrieval: After dewaxing and hydration, the sections were subjected to high-temperature and normal-pressure alkaline retrieval (pH 9.0) and boiled at 100℃ for 15 minutes; (3) Blocking: After cooling to room temperature, rinse with tap water 3 times, then block endogenous peroxidase with 3% H2O2 for about 10 minutes at room temperature; (4) Drawing circles: After rinsing with tap water 3 times, draw circles around the cell slices with an immunohistochemistry pen and immerse them in PBST for 3-5 minutes. (5) Primary antibody incubation: Remove excess liquid from the slide, add about 100 μL of 1:200 diluted primary antibody to the slide, and incubate at room temperature for 30 min; (6) Secondary antibody incubation: Rinse the slides three times with PBST, remove excess liquid from the slides, and add about 100 μL of HRP Polymer Conjugated Goat Anti Mouse + Rabbit IgG (Hangzhou Bailing Biotechnology, cat#BX10001) or a gradient dilution of i-vi group fusion protein-HRP conjugate to the slides. Incubate at room temperature for 30 min. (7) Color development: Rinse the sections three times with PBST, and prepare DAB color development solution using DAB chromogen (Hangzhou Bailing Biotechnology, cat#BX10002) and DAB buffer (Hangzhou Bailing Biotechnology, cat#BX10003). Add about 100 μL of color development solution to the sections and develop for 2 min; (8) Counterstaining: After rinsing the sections with tap water 3 times, counterstain with hematoxylin for 3-6 minutes. After rinsing with tap water, immerse the sections in PBST for 1 minute to return to blue. (9) The slides were subjected to routine immunohistochemical dehydration, clearing, and mounting; (10) Place the stained slides under a microscope for observation and photographing.

[0097] The experimental results are shown in Figure 4 As shown.

[0098] The first line shows the staining of the control secondary antibody on the placenta, tonsils, and endometrium: CD105 positive staining of the placenta is located on the surface of the cell membrane epithelial cells, with a small amount of residue in the cytoplasm; Ki-67 positive staining of the tonsils is located in the cytoplasm of germinal center B cells, showing strong positive staining with a clean background; Actin on the endometrium shows diffuse positive staining in the cytoplasm of endometrial epithelial cells.

[0099] Rows two through seven show the staining of the six PrA / G-Cys fusion protein-HRP conjugates as secondary antibodies on the placenta, tonsils, and endometrium. Ki-67 and Actin staining was significantly weaker than the control in groups i, ii, iii, and v, but groups iv and vi achieved similar intensities to the control. The staining of CD105 by the six PrA / G-Cys fusion protein-HRP conjugates was relatively similar to the control, with group v showing the weakest staining, while groups iv and vi showed slightly stronger positive staining.

[0100] The results showed that the PrA / G-Cys fusion protein-HRP conjugates designed in groups iv and vi could achieve the same effect as the control (serum-purified goat anti-rabbit / mouse conjugated HRP) as a secondary antibody in IHC staining. However, group vi showed slight background staining. Therefore, it is recommended that the PrA / G-Cys fusion protein-HRP conjugates designed in group iv can be used for IHC.

[0101] Example 9: Comparison of immunohistochemical staining of PrA / G-Cys fusion protein-HRP and PrA / G-Lys fusion protein-HRP. Referring to the iv molecular design scheme in Example 1, Cys was replaced with Lys, and plasmid synthesis, prokaryotic expression (actual, fusion protein purification, the operation method is the same as in Examples 2 to 4), and then the PrA / G-Lys fusion protein was coupled with HRP.

[0102] (1) Take 1 mL of HRP solution (1 mg / mL), add 1 mL of 0.1 M MES buffer (pH 6.0), and mix well.

[0103] (2) Add crosslinking reagent according to the molar ratio of HRP:EDC:NHS=1:10:2: Add 20μL EDC solution (10 mg / mL) and mix gently; immediately add 40μL NHS solution (10 mg / mL) and mix gently.

[0104] (3) Incubate at room temperature (25℃) in the dark for 15-30 minutes.

[0105] (4) Slowly add the activated HRP solution to 1 mL of PrA / G-Lys fusion protein solution (1 mg / mL), with the molar ratio of fusion protein to HRP being 1:4, and mix gently.

[0106] (5) Incubate at room temperature in the dark for 2 hours, gently mixing once every 30 minutes during the period.

[0107] (6) Add 50 μL of 0.05 M Tris-HCl buffer (pH 7.5), mix well, and incubate at room temperature for 15 min. The coupling reaction is terminated by blocking the unreacted activation sites through the amino group of Tris.

[0108] (7) The reaction solution was placed in a dialysis bag and dialyzed in 0.01 M PBS (pH 7.2) at 4°C for 24 hours, with the buffer solution changed once during the process.

[0109] (8) Add 50% glycerol and 1% BSA to the purified conjugate, dispense and freeze at -20°C.

[0110] Referring to Example 8, PrA / G-Cys fusion protein-HRP and PrA / G-Lys fusion protein-HRP were simultaneously subjected to immunohistochemical staining. The results are shown in [Figure 8]. Figure 5 .

[0111] The first row shows the staining of PrA / G-Cys fusion protein-HRP on target sites CD105, Ki-67, and Actin tissues.

[0112] The second row shows the PrA / G-Lys fusion protein-HRP. Both fusion proteins showed positive staining on the cell membrane (CD105), cell nucleus (Ki-67), and cytoplasm (Actin), respectively. However, the positive staining of PrA / G-Lys fusion protein-HRP was weaker, while the background staining was significantly stronger than that of PrA / G-Cys fusion protein-HRP.

[0113] The results showed that the positive staining specificity of PrA / G fusion with Cys was stronger and the background was weaker than that of Lys fusion. Fusion expression of polylysine after ProA / G can provide a large number of amino groups to couple with the carboxyl group of HRP via EDC / NHS. However, lysine (Lys) is positively charged, and a large amount of Lys will react with negatively charged groups in the tissue during immunohistochemical staining, causing background staining or non-specific staining. Furthermore, HRP activated by EDC / NHS, carrying NHS groups, is prone to coupling with its own amino groups, causing HRP self-polymerization or ProA / G carrying too much HRP with masked active sites, resulting in no signal amplification or even a weaker signal.

[0114] In summary, the recombinant protein IV molecular design, which fuses ProA-B (1-38 aa) and ProG-C2 (1-25 aa) with 20 Cys-Gly-Ser and His tags, can achieve high expression in *E. coli* and can be purified using a nickel-affinity resin column. By conjugating the free thiol group of the melted protein Cys with SMCC-activated HRP, the recombinant protein IV can carry 11 HRPs and maintains an IC50 similar to that of serum-purified goat anti-rabbit / mouse antibodies in ELISA, indicating that it retains high IgG affinity and HRP catalytic activity after HRP conjugation. In immunohistochemical staining, it achieves similar positive staining localization and intensity to the control secondary antibody for CD105 in the placenta, Ki-67 in the tonsils, and Actin in the endometrium. Finally, we recommend the molecular design of recombinant protein IV group to fusion express ProA-B (1-38aa) and ProG-C2 (1-25aa) as well as 20 groups of Cys-Gly-Ser and His tags. Because of its high prokaryotic expression level, easy purification, and IHC staining effect similar to that of traditional secondary antibodies, it is recommended as an alternative product for secondary antibodies in IHC.

[0115] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A PrA / G-Cys fusion protein, characterized in that: The structure of the PrA / G-Cys fusion protein is represented as: protein A sequence + linker peptide + protein A sequence + linker peptide + protein B sequence + linker peptide + protein B sequence + linker peptide + (10-60) × cysteine-containing polypeptide sequence + 6 × tag sequence. The protein A sequence is the ProA-B protein 1-38aa sequence, and the protein B sequence is the ProG-C2 protein 1-25aa sequence.

2. The PrA / G-Cys fusion protein according to claim 1, characterized in that: The linker peptide is GGGGSGGGGS.

3. The PrA / G-Cys fusion protein according to claim 1, characterized in that: The cysteine-containing polypeptide sequence is a Cys-Gly-Ser sequence.

4. The PrA / G-Cys fusion protein according to claim 1, characterized in that: The tag sequence is the His tag sequence.

5. The PrA / G-Cys fusion protein according to claim 1, characterized in that: The amino acid sequence of the PrA / G-Cys fusion protein is shown in SEQ ID No.

6.

6. A recombinant expression vector for expressing the PrA / G-Cys fusion protein according to any one of claims 1-5.

7. An engineered bacterium containing the PrA / G-Cys fusion protein of any one of claims 1-5 or the recombinant expression vector of claim 6.

8. A coupling agent, characterized in that: The conjugate is obtained by conjugating the PrA / G-Cys fusion protein according to any one of claims 1-5 with HRP.

9. The application of the conjugate according to claim 8 as a secondary antibody in immunohistochemical staining detection.

10. The use of the conjugate according to claim 8 in the preparation of an immunohistochemical detection kit.

Images