Recombinant streptococcus protein g, affinity chromatography resin, and use thereof

Abstract

A recombinant streptococcus protein G, an affinity chromatography resin, and a use thereof, relating to the technical filed of recombinant proteins. By means of extensive screening, the amino acid sequence of a streptococcus protein G is optimized and truncated, and it has been found that a recombinant streptococcus protein G having an amino acid sequence as set forth in SEQ ID NO. 1 is easily expressed in a plasmid; in addition, by means of glycosylation modification in Pichia pastoris, the recombinant streptococcus protein G can be stably expressed in increased amount. The recombinant streptococcus protein G can specifically bind with IgG and has a higher binding capability compared with existing protein G. The recombinant streptococcus protein G can be coupled with agarose for preparing an affinity chromatography resin used for separating and purifying biomolecules such as antibodies. A recombinant streptococcus protein G coupled with a marker can be used for luminescence, color development, or radioactivity detection of an antibody or a functional fragment.

Description

Recombinant streptococcal protein G, affinity chromatography resin and application thereof Technical Field

[0001] The present invention relates to the technical field of recombinant proteins, in particular to a recombinant streptococcal protein G, an affinity chromatography resin and applications thereof. Background Art

[0002] Streptococcal protein G (SPG) is a cell wall protein on the surface of Streptococci, composed of approximately 202 amino acids. Its N-terminus is divided into an albumin-binding domain, while the C-terminus contains an IgG-binding domain and a cell wall-binding domain. This unique structure enables SPG to bind to a variety of different antibodies, earning it the name a superantigen.

[0003] Streptococcal protein G has shown great potential in applications due to its ability to specifically bind to IgG antibodies from different species. It is mainly used in immunology and immunochemistry. In ELISA, SPG can replace secondary antibodies and has the characteristics of strong specificity, high sensitivity, and low dosage. It can also be a good substitute for SPA[1]. Pei Fuquan et al.[2] used horseradish peroxidase-labeled streptococcal protein G as an enzyme-labeled secondary antibody for rapid ELISA to detect Clonorchis sinensis-specific antibodies in human serum, showing high specificity and sensitivity. In affinity chromatography, SPG can also be combined with Sepharose (agarose) to assemble into affinity chromatography columns for separation and purification of various IgGs; at the same time, SPG was used to prepare solid-phase antibodies for radioanalysis, showing that SPG is a good solid-phase antibody preparation medium[3]. In colloidal gold immunochromatography, Bendayan et al.[4] combined SPG with colloidal gold and then compounded it with multiple monoclonal or polyclonal antibodies to locate various antigenic sites. The results showed that gold-labeled SPG can stably bind to antibodies from various test mammals. Therefore, SPG-colloidal gold is a better multifunctional high-resolution probe for cell immunochemistry. In addition, Fowler et al. [5] anchored specific antibodies on the thiolated recombinant SPG scaffold as part of the electrochemical immunoreceptor to efficiently capture the analyte, and also achieved good results.

[0004] Currently, intracellular expression of SPG in E. coli suffers from drawbacks such as easy plasmid loss and difficulty in purification. As a biomacromolecule, SPG has poor stability and is easily detached from the carrier during elution, which also reduces the recycling efficiency and service life of the chromatography column and increases production costs. SPG molecules have large steric hindrance and a low density of coupled ligands per unit carrier. Furthermore, the inconsistent spatial orientation of protein immobilization during the coupling process further increases steric hindrance, significantly reducing the dynamic loading capacity of the chromatography column and making large-scale application difficult. Therefore, finding a suitable Streptococcal protein G (SPG) is particularly critical.

[0005] In view of this, the present invention is proposed.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a recombinant streptococcal protein G, an affinity chromatography resin and applications thereof to solve the above technical problems.

[0008] In the present invention, Streptococcal protein G is synonymous with Protein G and recombinant protein G.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a recombinant Streptococcus protein G comprising the amino acid sequence shown in SEQ ID NO.1.

[0011] Through extensive screening, the inventors optimized and truncated the amino acid sequence of Streptococcus protein G and discovered that recombinant Streptococcus protein G using the amino acid sequence shown in SEQ ID NO. 1 is easily expressed in a plasmid. Glycosylation modification in Pichia pastoris can increase the expression level of recombinant Streptococcus protein G and ensure stable expression. Existing technologies using Escherichia coli for expression suffer from the lack of glycosylation, low expression levels, and unstable expression.

[0012] The above-mentioned recombinant streptococcal protein G can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as the automatic peptide synthesizer sold by Applied BioSystems and the like.

[0013] The amino acid sequence shown in SEQ ID NO.1 is as follows:

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding a recombinant Streptococcus protein G, which encodes the above-mentioned recombinant Streptococcus protein G.

[0015] In an optional embodiment, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0016] The nucleotide sequence of the nucleic acid molecule is as follows:

[0017] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned recombinant streptococcal protein G can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same recombinant streptococcal protein G amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the recombinant streptococcal protein G to improve the expression efficiency of the recombinant streptococcal protein G.

[0018] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecule encoding recombinant Streptococcus protein G.

[0019] The vector is an expression vector or a cloning vector, preferably an expression vector, and can refer to any recombinant polynucleotide construct that can directly or indirectly (such as packaging into a virus) introduce the target DNA fragment into the host cell by transformation, transfection or transduction to express the target gene.

[0020] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule encoding recombinant Streptococcus protein G or the aforementioned recombinant vector.

[0021] The recombinant cell is selected from Pichia pastoris or Escherichia coli.

[0022] In a fifth aspect, the present invention provides a method for preparing recombinant Streptococcus protein G, comprising: culturing the above-mentioned recombinant cells.

[0023] In a preferred embodiment of the present invention, the preparation method comprises: first transforming a recombinant vector comprising a nucleic acid molecule encoding recombinant Streptococcus protein G into competent cells, obtaining a positive strain through identification, extracting the plasmid, linearizing the plasmid, transforming the linearized plasmid into Pichia competent cells, screening to obtain a positive recombinant Pichia strain, and culturing to obtain the recombinant Streptococcus protein G.

[0024] In an alternative embodiment, the positive recombinant Pichia pastoris strain is subjected to methanol-induced expression.

[0025] In a sixth aspect, the present invention further provides an affinity chromatography resin comprising agar, and the agar is coupled with the above-mentioned recombinant Streptococcus protein G or the recombinant Streptococcus protein G prepared by the above-mentioned preparation method.

[0026] In an alternative embodiment, the agar is a cyanogen bromide activated agarose matrix.

[0027] Processed agarose has a primary structure composed of alternating D-galactose and 3-anhydrogalactose residues. These sugars provide an uncharged, hydrophilic matrix. Cross-linked agarose is generally preferred over uncross-linked agarose for most affinity applications requiring harsh activation or usage conditions. However, the increased stability achieved through cross-linking results in a 30–50% loss of potential reactive sites (consumed during the cross-linking chemistry). Furthermore, the addition of cross-linked agarose to stabilize beaded agarose does not significantly reduce porosity. Therefore, cross-linked agarose is preferred. For most affinity applications requiring harsh activation or usage conditions, cross-linked agarose is generally preferred over uncross-linked agarose and is widely used. The 30–50% loss of potential reactive sites does not affect its performance.

[0028] Cross-linked agarose is a micron-sized particle made from agarose and a cross-linking agent. Agarose is a natural polysaccharide with excellent biocompatibility and biodegradability. It can react with a cross-linking agent through chemical, physical, or covalent cross-linking to form a cross-linked structure, thereby immobilizing the agarose molecules and enhancing their stability and shape retention. Cross-linked agarose microspheres have a large specific surface area and abundant surface functional groups, making them suitable for applications in adsorption, separation, catalysis, and drug delivery.

[0029] In a seventh aspect, the present invention further provides a protein conjugate, which comprises the above-mentioned recombinant Streptococcus protein G or the recombinant Streptococcus protein G prepared by the above-mentioned preparation method, and the recombinant Streptococcus protein G is labeled with a detectable marker.

[0030] A detectable marker is a substance that has properties that can be observed directly by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0031] In an alternative embodiment, the detectable label is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle labels.

[0032] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0033] Fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0034] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphate glucose deoxidase.

[0035] In an alternative embodiment, radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re、 211 At 125 I. 188 Re、 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0036] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0037] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0038] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0039] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0040] In an eighth aspect, the present invention further provides the use of the above affinity chromatography resin in purifying antibodies;

[0041] In an alternative embodiment, the antibody is an IgG antibody;

[0042] In an alternative embodiment, the antibody is an anti-cytokeratin 8 antibody, an anti-cluster of differentiation 274 antibody, or an anti-human epidermal growth factor receptor-2 antibody.

[0043] The present invention has the following beneficial effects:

[0044] (1) The present invention optimizes and truncates the amino acid sequence of Streptococcus protein G through extensive screening and finds that recombinant Streptococcus protein G with the amino acid sequence shown in SEQ ID NO.1 is easily expressed in a plasmid. With the help of glycosylation modification of Pichia pastoris, the expression level of recombinant Streptococcus protein G can be increased and stably expressed.

[0045] (2) The Streptococcal protein G provided by the present invention can specifically bind to IgG and has a higher binding force than the existing Protein G.

[0046] (3) The streptococcal protein G of the present invention can be coupled to agarose to prepare affinity chromatography resin, which can be used for the separation and purification of biological molecules such as antibodies.

[0047] (4) Labels are coupled to streptococcal protein G, which can be used for luminescent, colorimetric, or radioactive detection of antibodies or functional fragments.

[0048] (5) Streptococcal protein G can also be used to prepare electrochemical immunosensors, thereby achieving rapid and accurate detection of specific substances in samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0050] Figure 1 is the electrophoresis diagram of the recombinant plasmid before and after linearization (M: DNA molecular weight standard, 1: after linearization of the recombinant plasmid, 2: before linearization of the recombinant plasmid);

[0051] Figure 2 is a PCR verification electrophoresis diagram of Pichia pastoris positive transformants (M: DNA molecular weight standard, 1-6: positive transformants, positive: positive control);

[0052] Figure 3 is a small-scale SDS-PAGE verification diagram of the recombinant protein (M: protein molecular weight standard, 1-7: expression test strains, Yin: negative control);

[0053] Figure 4 is a Western blot image of a small test of the recombinant protein (M: protein molecular weight standard, 1-7: expression test strains, positive: positive control);

[0054] Figure 5 is a graph showing the SDS-PAGE results of recombinant protein G eluted with 20mM, 50mM, and 500mM Tris-HCl buffer (M: protein molecular weight standard, flow-through: miscellaneous proteins);

[0055] FIG6 is a graph showing the affinity statistics of goat IgG and recombinant protein G in an ELISA experiment;

[0056] FIG7 is a graph showing the statistical results of the affinity of mouse IgG and recombinant protein G in an ELISA experiment;

[0057] FIG8 is a graph showing the statistical results of the affinity of rabbit IgG and recombinant protein G in an ELISA experiment;

[0058] Figure 9 is a SDS-PAGE comparison of the purity of recombinant protein G and a company's protein G-purified antibody;

[0059] Figure 10 is a staining image of cytokeratin 8 antibody. A is cytokeratin 8 antibody purified by recombinant protein G, and B is cytokeratin 8 antibody purified by protein G of a certain company;

[0060] FIG11 is a diagram of a recombinant vector for recombinant expression of Streptococcus protein G. DETAILED DESCRIPTION

[0061] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0062] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0063] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0064] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0065] Example 1

[0066] This example provides a method for preparing recombinant bacteria producing recombinant Streptococcus protein G.

[0067] 1. Construction of recombinant plasmid

[0068] The nucleotide sequence shown in SEQ ID NO. 2 was inserted into the vector to obtain the vector map shown in FIG11 .

[0069] The recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0070] 2. Preparation of Competent Pichia Pastoris

[0071] Select a single Pichia competent single spot into 50ml YPD medium, shake at 30℃, 220rpm overnight; when the OD value of the bacterial solution is (1.3-1.5), pour the bacterial solution into a 50ml tube in a clean bench, centrifuge, and remove the supernatant; add 7ml LiAc and 70ml DTT (dithiothreitol) to resuspend the cells; incubate on a shaker for 30min, balance, centrifuge, and remove the supernatant; wash once with sterile water, balance, centrifuge, and remove the supernatant; wash again with 7ml sterile water, balance, centrifuge, and remove the supernatant; add 1ml sorbitol, balance, centrifuge, and remove the supernatant (all subsequent operations are performed on an ice box); take a 1.5ml EP tube, add 200μL of bacterial solution, then add 8-10μL of the excised plasmid, blow to mix, and add it to the pre-cooled electric transfer cup along the side; take 1.5ml Add 1ml of sorbitol to the EP tube and set aside. Place the electroporation cuvette containing the bacterial solution into the electroporator. Remove the cuvette, open the lid, add 1ml of sorbitol, close the lid, and return to the clean bench. Transfer the bacterial solution from the electroporation cuvette to a new 1.5ml EP tube and incubate at 30°C for 1 hour on a shaker. Spread the incubated competent bacterial solution onto the corresponding plate and incubate for 48-72 hours. This will produce competent Pichia pastoris GS115.

[0072] 3. Extraction, Transformation and PCR Verification of Recombinant Plasmid

[0073] The correctly identified recombinant plasmid was transformed into Escherichia coli BL21 competent cells, and a single colony was picked for identification and inoculated into LB liquid medium containing 50 μg / mL ampicillin and cultured with shaking until OD 600 The bacteria were harvested when the p-value reached 0.6. Plasmids were extracted using a plasmid extraction kit from Sangon Biotech (Shanghai) Co., Ltd. The recombinant plasmid was linearized with the restriction endonuclease Scl1 (as shown in Figure 1) and transformed into the competent Pichia pastoris GS115 prepared in step 2 by electroporation. Transformed clones were screened on MD plates and verified by PCR in the bacterial culture, ultimately obtaining a recombinant strain with high expression levels.

[0074] 4. PCR Verification of Positive Transformants of Pichia pastoris

[0075] His+ positive transformants grown on MD plates were verified by PCR in the bacterial culture. During the experiment, competent cells transformed with an empty plasmid were added as a blank control, and the linearized plasmid was used as a positive control. PCR verification was performed in the bacterial culture using both forward and reverse AOX primers.

[0076] Reverse AOX primer: 5′-GGC AAA TGG CAT TC TGA CAT-3′;

[0077] Forward AOX primer: 5'GAC TGG TTC CAA TTG ACA AGC-3'.

[0078] The PCR procedure for bacterial solution includes: using the Taq PCR Mix premix (2X, containing red dye) of the Sangon Biotech kit, prepare 20ul of PCR system according to the instructions, take 19ul of the system + 1ul of bacterial solution, and operate according to the PCR temperature and time specified in the instructions.

[0079] The results are shown in FIG2 . The positive transformants can all be amplified to obtain target bands of the same size as the positive control.

[0080] Example 2

[0081] This example conducts a small-scale test to verify the recombinant protein.

[0082] In order to screen for recombinant strains that efficiently express protein G, a methanol-induced expression experiment was performed on a verified recombinant Pichia pastoris, and pPIC9K-GS115 was used as a blank control (i.e., a negative control). The positive control was a linearized plasmid.

[0083] (1) Add 15 ml of BMGY medium to each shake flask, and then add 200 μL of bacterial solution (30°C, 220 rpm, overnight) for the expression test (including blank control); the next day, centrifuge and remove the supernatant; add BMMY medium; add methanol every 12 hours to 0.5% of the final volume; after 3 days, collect the shake flasks and store them at 4°C.

[0084] (2) The fermentation supernatant was subjected to TCA precipitation (trichloroacetic acid precipitation) to obtain samples; the centrifuged supernatant of the fermentation broth was analyzed by SDS-PAGE (as shown in Figure 3), and further confirmed by Western-blot (as shown in Figure 4) whether the expressed recombinant protein was recombinant protein G. The results showed that the molecular weight of the recombinant protein obtained from the seven test strains (22.0510 kDa) was consistent with the expectation.

[0085] Example 3

[0086] Large-scale fermentation production and separation and purification of recombinant protein G.

[0087] (1) Inoculate the expressed strain into YPD medium and culture at 30°C, 220 rpm, and shake until the OD of the bacterial solution reaches 600 When the nm reaches between 4 and 8, transfer the bacterial solution to 1L of BMGY medium. After the glycerol in the medium is completely consumed, add glycerol to the fermentation solution OD 600 When nm reaches between 300-350, stop glycerol feeding and start methanol feeding induction. Culture for 48-72 hours and collect the supernatant by centrifugation.

[0088] (2) Isolation and purification of recombinant protein G

[0089] The fermentation broth supernatant after centrifugation was placed in a dialysis bag and kept overnight in Tris-HCl buffer; the fermentation broth was combined with a nickel column and shaken at 15°C, 140 rpm, for 2-3 hours; the column was loaded to remove small molecules such as pigments and salts, and the protein buffer environment was replaced with the loading buffer for ion exchange chromatography, and eluted with 20mM, 50mM, and 500mM Tris-HCl buffers; and samples of each eluate were collected.

[0090] The SDS-PAGE graph of proteins in the eluate after elution with Tris-HCl buffer of different concentration gradients is shown in Figure 5. The results show that recombinant Streptococcus protein G of the target size was obtained.

[0091] Example 4

[0092] In this example, the ELISA method was used to determine the affinity constants between recombinant protein G and IgG from different species.

[0093] The concentration of protein G was determined by the BCA method. Recombinant protein G was serially diluted starting from 10 μg / mL with coating solution for a total of 8 dilutions, and 100 μL / well was coated on a 96-well plate with 3 replicates for each concentration. The plates were incubated at 4°C overnight. After washing with pure water, the plates were blocked with 5% skim milk powder in PBST solution (150 μL / well) at 37°C for 2 h. After washing with PBST, three secondary antibodies, goat anti-mouse, rabbit anti-mouse, and mouse anti-rabbit, were serially diluted at 1:500, 1:1000, 1:2000, and 1:4000, respectively. 100 μL was added to each well and incubated at 37°C for 2 h. After washing with pure water, TMB was used for color development, the reaction was allowed to react at room temperature for 15 min, terminated with 2 mol / L H2SO4, and the OD450 value was read. The curve was fitted using Graphpad Prism 5 software, and the affinity constant K was calculated by entering the formula: K = (n-1) / (n[Ab]T'-[Ab]T).

[0094] The results are shown in Table 1 and Figures 6, 7, and 8, where the abscissas in Figures 6 and 8 represent the antibody concentration in 1g and the ordinates represent the OD value (OD450) of the corresponding antibody. The results show that the recombinant protein G provided by the present invention has a high affinity for IgG from various sources.

[0095] Table 1 Affinity statistical results

[0096] Example 5

[0097] This embodiment provides an affinity chromatography column.

[0098] Cyanogen bromide-activated agarose matrix can be used to prepare affinity chromatography resins.

[0099] Processed agarose has a primary structure composed of alternating D-galactose and 3-anhydrogalactose residues. These sugars provide an uncharged, hydrophilic matrix. Cross-linked agarose is generally preferred over non-cross-linked agarose for most affinity applications requiring harsh activation or use conditions. However, the increased stability achieved through cross-linking results in a 30-50% loss of potential reactive sites (consumed during the cross-linking chemistry). Adding cross-links to stabilize beaded agarose does not significantly reduce porosity.

[0100] The preparation method of affinity chromatography column is as follows:

[0101] (1) Dissolve protein G in 0.1 M NaHCO3 / 0.5 M NaCl (5.10 mg protein per ml gel);

[0102] (2) Activate agar in pre-cooled 1 mM HCl (200 ml per gram of agar);

[0103] (3) Wash the resin with 5-10 column volumes of distilled water, then wash the resin with 0.1 M NaHCO3 / 0.5 M NaCl (5 ml per gram of dry gel) and immediately transfer it to the ligand in coupling buffer; the coupling buffer consists of: 0.5 M NaCl + 0.1 M NaHCO3, adjusted to pH 8.4;

[0104] (4) Mix protein G with gel at room temperature for 2 h or overnight at 2-8°C;

[0105] (5) Use 0.1M NaHCO3 / 0.5M NaCl to wash away the unreacted ligand;

[0106] (6) Block unreacted genes with 0.2 M glycine or 1 M ethanolamine at room temperature for 2 h or at 2-8 °C for 6 h;

[0107] (7) Wash thoroughly to remove the blocking solution, first with alkaline coupling buffer, then with 0.1 M NaAc / 0.5 M NaCl (pH: 4.0); wash with pH buffer (0.1 M NaAc / 0.5 M NaCl (pH: 4.0)) for 4-5 cycles;

[0108] (8) The resin was stored in 1.0 M NaCl at 2-8°C and 20% ethanol was added.

[0109] Example 6

[0110] The antibody was purified using the affinity chromatography column prepared in Example 5.

[0111] (1) Column loading: Place a 10ml gravity column on a purification rack and install a cap at the bottom. Mix the affinity chromatography resin (protein G filler) prepared in Example 5, take out 1ml of the filler and place it in the gravity column. Fill the column with antibody purification equilibrium solution (MERCK's cyanogen bromide-activated agarose gel TM4B item number: C9142). Take another sieve plate to seal the top of the filler so that the distance between the sieve plate and the filler is 0.5cm. Remove the cap at the bottom of the purification column, let the liquid flow out naturally, and add 10 to 20 times the column volume of antibody purification equilibrium solution to balance the column so that the filler reaches the optimal binding state.

[0112] (2) Sample preparation: 1 ml of mouse ascites (ascites containing cytokeratin 8 antibody and ascites containing human epidermal growth factor receptor-2 antibody) was centrifuged (5000 rpm, 5 min) to obtain the supernatant, and 5 to 10 times the volume of PB was added. After mixing, the supernatant was filtered through a sieve plate and the filtrate was collected for later use.

[0113] The preparation method of mouse ascites is as follows: the monoclonal cell line is expanded and the collected approximately 1*10 6 The cells are injected into selected mice (the mice need to be injected with paraffin oil in the abdominal cavity one week in advance). After waiting for 7-10 days, the mice produce ascites, which is collected for antibody purification.

[0114] (3) Add the filtered filtrate to the equilibrated purification column, collect the effluent, pass it through the column again, collect the effluent again, and temporarily store it in a refrigerator at 4°C. (This is used to detect whether the purification column has reached saturation or the life of the purification column has expired.)

[0115] (4) After loading, flush the purification column with 10 to 20 column volumes of PB to remove unbound proteins.

[0116] (5) Pre-elution: Rinse with 10 to 20 column volumes of antibody purification pre-elution buffer (pH 5.0) to remove loosely bound foreign proteins.

[0117] (6) Elution: Add 10 times column volume of antibody purification eluate (pH 2.7), collect the eluate, 1 ml / tube, generally collect 5 to 8 tubes, and pre-add 100 ml of neutralizing solution (pH 7.0) to the collection tube; the eluted antibody is temporarily stored in a 4°C refrigerator until the concentration is determined and then aliquoted for storage.

[0118] (7) Pass 10 Cv (column volume) of deionized water filtered through a 0.22 mm filter membrane through the column to make the column neutral.

[0119] (8) Pass 5-10 Cv (column volume) of 20% ethanol through the column. When the column is filled with ethanol, disassemble the column and store it at 4°C.

[0120] The SDS-PAGE result of the purified antibody is shown in Figure 9, where M is a marker, 1 is a cytokeratin 8 antibody purified by protein G of Changzhou Tiandirenhe Company, 2 is a cytokeratin 8 antibody purified by recombinant protein G in this example, 3 is a human epidermal growth factor receptor-2 antibody purified by protein G of Changzhou Tiandirenhe Company, and 4 is a human epidermal growth factor receptor-2 antibody purified by recombinant protein G in this example.

[0121] Antibody purity testing revealed that the concentration of the cytokeratin 8 antibody purified by recombinant protein G in this example was 0.43 mg / ml, while the concentration of a company's protein G-purified cytokeratin 8 antibody was 0.3 mg / ml. Therefore, the recombinant protein G provided by the present invention enhances the binding affinity of streptococcal proteins to IgG.

[0122] Example 7

[0123] In this example, immunohistochemistry experiments were performed.

[0124] (1) Slice processing: Place the lung adenocarcinoma tissue pathological sections in a 60℃ constant temperature box and bake for 60 minutes. Soak the sections in xylene I for 15 minutes, then replace with xylene II and soak for 15 minutes. Soak in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes respectively; soak in ddH2O for 5 minutes, and wash 3 times; use a pressure cooker to perform antigen repair (boiling method). Add 10mmol / L citrate buffer (pH6.0) enough to submerge the sections into the pressure cooker, heat to boiling, place the sections on a heat-resistant material section rack, put them into the pot, cover the pot, buckle the pressure valve, continue heating, set the pressure to maintain for 4 minutes, open the vent valve to release the air after the time is up, open the pot lid after the pressure returns to zero, take out the inner pot and let it cool at room temperature. After the solution cooled to room temperature, the sections were removed (approximately 40 minutes); soaked in ddH2O for 5 minutes, washed twice, and soaked in PBST for 5 minutes, washed twice; placed the sections in 20 ml of 3% H2O2-methanol solution, protected from light, and treated at room temperature for 10 minutes; soaked in PBST for 5 minutes, washed three times; added one drop of goat serum blocking solution (approximately 25 μl) to each tissue group, incubated in a wet box at room temperature for 45 minutes; soaked in PBST for 5 minutes, washed three times.

[0125] (2) Incubation of tissue sections with antibodies:

[0126] The treated lung adenocarcinoma tissue sections were added with cytokeratin 8 antibodies (cytokeratin 8 antibodies purified by recombinant protein G provided by the present invention and cytokeratin 8 antibodies purified by protein G from a certain company) and incubated in a 4°C wet box overnight; taken out from the 4°C refrigerator and incubated at room temperature for 60 minutes; gently rinsed with PBST and soaked for 5 minutes, washed 3 times; 25ul of HRP-labeled Changdao Company secondary antibody was added to each tissue group and incubated at room temperature for 45 Prepare the DAB colorimetric solution and allow it to react in the dark for 10-15 minutes before adding it dropwise to the sections and developing for 1-5 minutes. Terminate the colorimetric reaction with distilled water. Add 50 μl of hematoxylin solution to each tissue group and stain for 5-10 minutes. Rinse thoroughly with distilled water. Decolorize the sections in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove and decolorize in distilled water. Debluing is then stopped in PBST (pH 8.0) for 5-10 minutes. Soak in 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then switch to xylene and soak for another 10 minutes. Seal the sections with neutral gum and cover slips. Photograph the sections using a microscope.

[0127] As can be seen from FIG10 , there is no significant difference in the staining effect of the cytokeratin 8 antibody (A) purified by recombinant protein G provided by the present invention and the cytokeratin 8 antibody (B) purified by protein G of a certain company on lung adenocarcinoma tissue.

[0128] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A recombinant streptococcal protein G, characterized in that: It includes the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding a recombinant Streptococcus protein G, characterized in that: It encodes the recombinant Streptococcus protein G according to claim 1; Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: It comprises the nucleic acid molecule encoding recombinant streptococcal protein G as claimed in claim 2.

4. A recombinant cell, characterized in that It comprises the nucleic acid molecule encoding recombinant streptococcal protein G according to claim 2 or the recombinant vector according to claim 3; Preferably, the recombinant cell is selected from Pichia pastoris cells or Escherichia coli.

5. A method for preparing recombinant streptococcal protein G according to claim 1, characterized in that: It includes: Cultivate the recombinant cell according to claim 4.

6. The method for preparing recombinant streptococcal protein G according to claim 5, characterized in that: The preparation method comprises: firstly transforming a recombinant vector including a nucleic acid molecule encoding a recombinant streptococcal protein G into competent cells, obtaining a positive strain through identification, extracting a plasmid, performing a plasmid linearization treatment, transforming the linearized plasmid into a Pichia competent cell, screening to obtain a positive recombinant Pichia strain, and culturing to obtain the recombinant streptococcal protein G; Preferably, the positive recombinant Pichia pastoris strain is subjected to methanol-induced expression.

7. An affinity chromatography resin, characterized in that The invention comprises agar, and the recombinant streptococcal protein G according to claim 1 or the recombinant streptococcal protein G prepared by the preparation method according to any one of claims 5 to 6 is coupled to the agar.

8. A protein conjugate, characterized in that The protein conjugate comprises the recombinant streptococcal protein G according to claim 1 or the recombinant streptococcal protein G prepared by the preparation method according to any one of claims 5 to 6, and the recombinant streptococcal protein G is marked with a detectable marker.

9. The protein conjugate according to claim 8, characterized in that The detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers; Preferably, the nanoparticle marker is selected from any one of nanoparticles and colloids.

10. Use of the affinity chromatography resin according to claim 7 in purifying antibodies; Preferably, the antibody is an IgG antibody; Preferably, the antibody is an anti-cytokeratin 8 antibody, an anti-cluster of differentiation 274 antibody or an anti-human epidermal growth factor receptor-2 antibody.

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