CN117603322A - Recombinant streptococcal protein G, affinity chromatography resin and application thereof - Google Patents
Recombinant streptococcal protein G, affinity chromatography resin and application thereof Download PDFInfo
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- CN117603322A CN117603322A CN202311588735.6A CN202311588735A CN117603322A CN 117603322 A CN117603322 A CN 117603322A CN 202311588735 A CN202311588735 A CN 202311588735A CN 117603322 A CN117603322 A CN 117603322A
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- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C12R2001/84—Pichia
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Abstract
The invention discloses recombinant streptococcal protein G, affinity chromatography resin and application thereof, and relates to the technical field of recombinant proteins. Through a large number of screening, the amino acid sequence of the streptococcus protein G is optimized and truncated, and the recombinant streptococcus protein G adopting the amino acid sequence shown in SEQ ID NO.1 is found to be easy to express in plasmids, and the expression quantity of the recombinant streptococcus protein G can be improved and the expression can be stabilized by means of glycosylation modification of pichia pastoris. The streptococcus protein G provided by the invention can be specifically combined with IgG, and has higher binding force compared with the existing protein G. The streptococcus protein G can be coupled with agarose, is used for preparing affinity chromatography resin, and is used for separating and purifying biomolecules such as antibodies. The label is coupled to streptococcal protein G, and can be used for luminescence, color development or radioactivity detection of antibodies or functional fragments.
Description
Technical Field
The invention relates to the technical field of recombinant proteins, in particular to recombinant streptococcal protein G, affinity chromatography resin and application thereof.
Background
Streptococcal Protein G (SPG) is a cell wall protein on the surface of streptococci, consisting of approximately 202 amino acids. The N-terminal part is albumin binding domain, and the C-terminal part is IgG binding domain and cell wall binding domain. Thus, the specific structure allows SPG to function in combination with a variety of different antibodies, streptococcal protein G is also known as a superantigen.
Streptococcal protein G has shown great potential in application, mainly in immunology and immunochemistry, due to its broad specificity of binding to antibodies IgG of different species. In ELISA aspect, SPG can replace secondary antibody, has the characteristics of strong specificity, high sensitivity and small dosage, and can well replace SPA [1]. Pei Fu the full et al [2] uses horseradish peroxidase-labeled streptococcal protein G as an enzyme-labeled secondary antibody of a rapid ELISA to detect clonorchis sinensis specific antibodies in human serum, and shows high specificity and sensitivity. In the process of affinity chromatography, SPG can be combined with Sepharose to assemble an affinity chromatography column for separating and purifying various IgG; meanwhile, SPG was used to prepare solid phase antibodies for radioanalysis, showing that SPG is a good solid phase antibody preparation medium [3]. In terms of colloidal gold immunochromatography, bendayan [4] et al combine SPG with colloidal gold, and then complex a plurality of monoclonal or polyclonal antibodies for locating various antigen sites, and the results show that gold-labeled SPG can stably bind to antibodies of various mammals to be tested. Thus, SPG-colloidal gold is a better multifunctional high-resolution probe for cellular immunochemistry. Furthermore, fowler [5] et al have also achieved good results by anchoring specific antibodies to thiolated recombinant SPG scaffolds as part of electrochemical immunosensors for efficient capture of analytes.
At present, the intracellular expression SPG of the escherichia coli has the defects of easy loss of plasmids, difficult purification and the like. SPG is used as a biological macromolecule, has poor stability, is easy to fall off from a carrier in the elution process, reduces the recycling efficiency and the service life of a chromatographic column, and increases the production cost. The SPG molecules have large steric hindrance, the unit carrier coupling ligand density is low, and the steric orientation of protein immobilization in the coupling process is inconsistent, so that the steric hindrance is further increased, the dynamic load of the chromatographic column is greatly reduced, and the large-scale application is difficult. Thus, finding suitable Streptococcal Protein G (SPG) is particularly critical.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide recombinant streptococcal protein G, affinity chromatography resin and application thereof, which are used for solving the technical problems.
In the present invention, streptococcal Protein G is synonymous with Protein G, recombinant Protein G.
The invention is realized in the following way:
in a first aspect, the present invention provides a recombinant streptococcal protein G comprising an amino acid sequence as shown in SEQ ID NO. 1.
The inventor optimizes and truncates the amino acid sequence of the streptococcus protein G through a large number of screening, and discovers that the recombinant streptococcus protein G adopting the amino acid sequence shown in SEQ ID NO.1 is easy to express in plasmids, and can improve the expression quantity of the recombinant streptococcus protein G and stably express by means of glycosylation modification of pichia pastoris. The prior art adopts escherichia coli for expression, and has the problems of no glycosylation modification, low expression quantity and unstable expression.
The recombinant streptococcal protein G described above may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
The amino acid sequence shown in SEQ ID NO.1 is as follows:
TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEHHHHHH*。
in a second aspect, the present invention provides a nucleic acid molecule encoding a recombinant streptococcal protein G, which encodes a recombinant streptococcal protein G as described above.
In an alternative embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.
The nucleotide sequence of the nucleic acid molecule is as follows:
ACCTACAAGCTGATCCTGAACGGTAAGACCTTGAAGGGTGAGACTACTACTGAAGCTGTTGACGCTGCTACTGCCGAGAAGGTTTTTAAGCAATACGCCAACGACAACGGTGTTGACGGTGAATGGACTTACGATGACGCTACTAAGACCTTCACCGTTACTGAGAAGCCAGAGGTTATTGACGCTTCCGAATTGACTCCAGCTGTCACCACTTACAAGCTGGTGATCAACGGAAAGACTCTGAAGGGCGAAACAACTACAGAGGCAGTTGATGCTGCAACCGCTGAAAAGGTGTTCAAGCAGTACGCTAATGATAACGGCGTCGATGGTGAGTGGACCTATGACGATGCTACCAAGACTTTCACTGTCACCGAAAAGCCAGAAGTGATCGACGCATCTGAGTTGACCCCTGCTGTTACTACCTACAAGTTGGTCATTAACGGCAAAACCCTTAAAGGCGAGACTACCACTAAGGCTGTCGATGCTGAGACTGCTGAGAAGGCCTTTAAACAGTATGCAAACGACAATGGCGTGGACGGCGTTTGGACATACGACGATGCAACAAAAACCTTTACCGTCACCGAACATCACCATCACCACCACTAA。
taking the degeneracy of codons into consideration, the sequence of the gene encoding the recombinant streptococcal protein G can be modified in the coding region thereof under the condition of not changing the amino acid sequence to obtain the gene encoding the same recombinant streptococcal protein G amino acid sequence; the modified gene can also be artificially synthesized according to the codon preference of a host for expressing the recombinant streptococcal protein G so as to improve the expression efficiency of the recombinant streptococcal protein G.
In a third aspect, the invention provides a recombinant vector comprising a nucleic acid molecule as described above encoding recombinant streptococcal protein G.
The vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce the DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.
In a fourth aspect, the invention provides a recombinant cell comprising a nucleic acid molecule encoding recombinant streptococcal protein G as described above or a recombinant vector as described above.
The recombinant cells are selected from pichia pastoris cells or escherichia coli.
In a fifth aspect, the present invention provides a method for preparing recombinant streptococcal protein G, comprising: the recombinant cells described above are cultured.
In a preferred embodiment of the present invention, the preparation method comprises: firstly, transforming competent cells with a recombinant vector comprising a nucleic acid molecule for encoding recombinant streptococcal protein G, obtaining a positive strain through identification, extracting plasmids, carrying out plasmid linearization treatment, transforming the linearized plasmids into pichia pastoris competent cells, screening to obtain the positive recombinant pichia pastoris strain, and culturing to obtain the recombinant streptococcal protein G.
In an alternative embodiment, the positive recombinant pichia pastoris strain is subjected to methanol-induced expression.
In a sixth aspect, the present invention also provides an affinity chromatography resin comprising agar to which the recombinant streptococcal protein G described above or the recombinant streptococcal protein G produced by the above production method is coupled.
In an alternative embodiment, the agar is a cyanogen bromide activated agarose matrix.
The processed agarose has a primary structure consisting of alternating D-galactose and 3-anhydrogalactose residues. These sugars provide an uncharged hydrophilic matrix. For most affinity applications requiring harsh activation or use conditions, crosslinked agarose is generally preferred over uncrosslinked agarose. But the increased stability by crosslinking can result in 30-50% loss of potential reactive sites (consumed in the crosslinking chemistry). In addition, the addition of cross-linked agarose to stabilize bead agarose does not significantly reduce porosity. Therefore, cross-linked agarose is preferred. For most affinity applications requiring harsh activation or use conditions, crosslinked agarose is generally preferred over uncrosslinked agarose, and crosslinked agarose is more widely used. The loss of potential reaction sites by 30-50% does not affect the normal use thereof.
Crosslinked agarose is a micron-sized particle prepared from agarose and a crosslinking agent. Agarose is a natural polysaccharide with good biocompatibility and biodegradability. The agarose gel can react with a crosslinking agent through chemical crosslinking, physical crosslinking or covalent crosslinking and the like to form a crosslinked structure, so that agarose molecules are immobilized, and the stability and shape retention capacity of the agarose gel are enhanced. The cross-linked agarose microsphere has larger specific surface area and rich surface functional groups, and can be used in the fields of adsorption, separation, catalysis, drug delivery and the like.
In a seventh aspect, the present invention also provides a protein conjugate, the protein conjugate comprising the recombinant streptococcal protein G described above or the recombinant streptococcal protein G prepared by the preparation method described above, and the recombinant streptococcal protein G being labelled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In an alternative embodiment, the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.
In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is 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 Ru、 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。
in alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids; nanoparticles include, but are not limited to: organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an eighth aspect, the invention also provides the use of an affinity chromatography resin as described above for purifying antibodies;
in an alternative embodiment, the antibody is an IgG-type antibody;
in an alternative embodiment, the antibody is an anti-cytokeratin 8 antibody, an anti-surface antigen cluster 274 antibody, or an anti-human epidermal growth factor receptor-2 antibody.
The invention has the following beneficial effects:
(1) Through a large number of screening, the amino acid sequence of the streptococcus protein G is optimized and truncated, and the recombinant streptococcus protein G adopting the amino acid sequence shown in SEQ ID NO.1 is found to be easy to express in plasmids, and the expression quantity of the recombinant streptococcus protein G can be improved and the expression can be stabilized by means of glycosylation modification of pichia pastoris.
(2) The streptococcus protein G provided by the invention can be specifically combined with IgG, and has higher binding force compared with the existing protein G.
(3) The streptococcus protein G can be coupled with agarose, is used for preparing affinity chromatography resin, and can be used for separating and purifying biomolecules such as antibodies.
(4) The label is coupled to streptococcal protein G, and can be used for luminescence, color development or radioactivity detection of antibodies or functional fragments.
(5) Streptococcal protein G can also be used for preparing an electrochemical immunosensor, so that the rapid and accurate detection of specific substances in a sample is realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electropherogram before and after linearization of a recombinant plasmid (M: DNA molecular weight standard, 1: after linearization of the recombinant plasmid, 2: before linearization of the recombinant plasmid);
FIG. 2 is a PCR-validated electrophoresis of Pichia pastoris positive transformants (M: DNA molecular weight standard, 1-6: positive transformants, positive: positive control);
FIG. 3 is a schematic diagram of SDS-PAGE validation of a recombinant protein (M: protein molecular weight standard, 1-7: strain expressing the test, negative: negative control);
FIG. 4 is a Western-blot plot of a small sample of recombinant protein (M: protein molecular weight standard, 1-7: strain expressing small sample, positive: positive control);
FIG. 5 is a SDS-PAGE result of 20mM,50mM, 500mM Tris-HCl buffer eluting recombinant protein G (M: protein molecular weight standard, flow through: hetero protein);
FIG. 6 is a graph showing the statistical result of ELISA experimental affinity between goat IgG and recombinant protein G;
FIG. 7 is a graph showing the results of ELISA experiment affinity statistics of mouse IgG and recombinant protein G;
FIG. 8 is a graph showing the statistical result of ELISA experimental affinity between rabbit IgG and recombinant protein G;
FIG. 9 is a diagram showing SDS-PAGE comparing the purity of antibodies purified from recombinant protein G and protein G from a company;
FIG. 10 is a staining chart of cytokeratin 8 antibodies. A is a recombinant protein G purified cytokeratin 8 antibody, and B is a protein G purified cytokeratin 8 antibody of a certain company;
FIG. 11 is a diagram of a recombinant vector for recombinant expression of streptococcal protein G.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can 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 can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The present example provides a method for preparing recombinant bacteria of recombinant streptococcal protein G.
1. Construction of recombinant plasmids
The nucleotide sequence shown in SEQ ID No.2 was inserted into a vector to obtain a vector map as shown in FIG. 11.
Recombinant plasmids were synthesized by the division of biological engineering (Shanghai).
2. Pichia pastoris competent preparation
Selecting Pichia pastoris competent cell spots into 50ml YPD medium, shaking at 30deg.C and 220rpm overnight; after the OD value of the bacterial liquid is (1.3-1.5), pouring the bacterial liquid into a 50ml tube in an ultra clean bench, centrifuging, and removing the supernatant; 7ml LiAc and 70ml DTT (dithiothreitol) were added to resuspend cells; incubating for 30min, balancing, centrifuging, and removing supernatant; washing with sterile water once, balancing, centrifuging, and removing supernatant; washing with 7ml of sterile water again, balancing, centrifuging, and removing supernatant; adding 1ml sorbitol, balancing, centrifuging, removing supernatant (all operations are performed on ice chest later); taking a 1.5ml EP tube, adding 200 mu L of bacterial liquid, then adding 8-10 mu L of cut plasmid, blowing and mixing uniformly, and adding into an electric rotating cup pre-cooled along the side surface; taking 1.5ml of EP tube, adding 1ml of sorbitol for standby; placing the electric rotating cup with the bacterial liquid into an electric rotating instrument, taking out the electric rotating cup, opening a cover, adding 1ml of sorbitol, closing the cover and taking back to the super clean bench; transferring the bacterial liquid in the electric rotating cup into a new 1.5ml EP tube, incubating for 1h at 30 ℃, and keeping the shaking table from rotating; and (5) coating the incubated competent bacteria liquid into a corresponding plate, and culturing for 48-72h. Pichia pastoris competent GS115 was prepared.
3. Extraction, transformation and PCR verification of recombinant plasmids
Transforming the recombinant plasmid with correct identification into competent cells of escherichia coli BL21, selecting single colony for identification, inoculating into LB liquid medium containing 50 mug/mL ampicillin, and shake culturing to OD 600 And collecting bacteria at 0.6. Extracting plasmid by using a plasmid extraction kit of a manufacturer of a living organism (Shanghai), linearizing the recombinant plasmid by using restriction enzyme Scl1 (shown in figure 1), and transforming the pichia pastoris competent GS115 prepared in the step 2 by using an electric shock method; the transformed clone is subjected to MD plate screening and bacterial liquid PCR verification to finally obtain a high-surfaceAn up-to-date amount of recombinant strain.
4. PCR verification of Pichia pastoris positive transformants
Performing bacterial liquid PCR verification on His+ positive transformants grown on MD plates, adding competence of empty plasmid transformation as a blank control in the experimental process, and taking linearized plasmids as positive controls. And respectively utilizing the forward AOX primer and the reverse AOX primer to carry out bacterial liquid PCR verification.
Reverse AOX primer: 5'-GGC AAA TGG CAT TC TGA CAT-3';
forward AOX primer 5'GAC TGG TTC CAA TTG ACAAGC-3'.
The bacterial liquid PCR process includes: the PCR temperature and time of the kit are as follows, wherein 20ul of PCR system is prepared from Taq PCR Mix premix (2X, containing red dye) of the kit according to the specification, 19ul of system+1 ul of bacterial liquid is taken, and the operation is performed according to the specification.
As a result, referring to FIG. 2, the positive transformants were amplified to obtain the target band of the same size as that of the positive control.
Example 2
This example performs pilot verification of recombinant proteins.
In order to screen recombinant strains which efficiently express the protein G, the verified recombinant pichia pastoris is subjected to a methanol induction expression experiment, and pPIC9K-GS115 is used as a blank control (namely, a negative control). Positive control was linearized plasmid.
(1) 15ml BMGY medium was added to each flask, followed by 200. Mu.L of bacterial liquid (30 ℃,220rpm, overnight) to be used for the expression of the pilot (including the blank); the next day, centrifuging, removing the supernatant; adding BMMY culture medium; methanol was added every 12h to 0.5% of the final volume; and 3 days later, collecting the shake flask, and storing at 4 ℃.
(2) Obtaining a sample from the fermentation supernatant by a TCA precipitation method (trichloroacetic acid precipitation method); the supernatant from the fermentation broth was analyzed by SDS-PAGE (FIG. 3) and further by Western-blot (FIG. 4) to determine whether the expressed recombinant protein was recombinant protein G. The results showed that the molecular weight (22.0510 kDa) of the recombinant protein obtained from the 7 ministrains was consistent with the expectations.
Example 3
And (3) large-scale fermentation production, separation and purification of recombinant protein G.
(1) Inoculating the strain with expression into YPD culture medium, shake culturing at 30deg.C and 220rpm until OD of the bacterial liquid 600 nm is between 4 and 8; transferring the bacterial liquid into 1L of BMGY culture medium; after the glycerol in the culture medium is completely consumed, the glycerol is supplemented to the fermentation broth OD 600 And (3) finishing glycerol replenishment when the nm reaches 300-350, starting methanol replenishment induction, culturing for 48-72h, and centrifugally collecting the supernatant.
(2) Separation and purification of recombinant protein G
Loading the supernatant of the fermentation broth after centrifugation into a dialysis bag for overnight, wherein buffer is Tris-HCL; combining the fermentation liquor with a nickel column, and shaking the mixture at 15 ℃ and 140rpm for 2-3 hours; loading onto a column, removing small molecular substances such as pigment and salts, replacing the protein buffer solution environment with a loading buffer solution of ion exchange chromatography, and eluting with 20mM,50mM and 500mM Tris-HCl buffer solution; samples of each section of eluate were collected.
SDS-PAGE of proteins in the eluate after elution with Tris-HCl buffer at different concentration gradients is shown in FIG. 5. The results showed that recombinant streptococcal protein G of the desired size was obtained.
Example 4
In this example, ELISA was used to determine the affinity constant of recombinant protein G to IgG from different species.
Protein G concentration was determined by BCA method, recombinant protein G was subjected to double dilution from 10. Mu.g/mL with coating solution, total of 8 dilutions, 100. Mu.L/well coated on 96-well plates, 3 replicates for each concentration, overnight at 4 ℃; after washing with pure water, wells were blocked with 5% nonfat dry milk in PBST solution (150. Mu.L /) for 2h at 37 ℃; PBST washing, namely, performing double dilution on 3 secondary antibodies of goat anti-mouse, rabbit anti-mouse and mouse anti-rabbit according to the ratio of 1:500, 1:1000, 1:2000 and 1:4000, adding 100 mu L of the secondary antibodies into each hole, and incubating for 2 hours at 37 ℃; after washing with pure water, TMB was developed and reacted at room temperature for 15min,2mol/L H 2 SO 4 At the end, the OD450 value was read. Fitting the curve with Graphpad Prism 5 software and bringing into the formula: k= (n-1)/(n [ Ab)]T'-[Ab]T), the affinity constant K value is determined.
The results are shown in Table 1 and FIG. 6, and FIG. 7 and FIG. 8, in which the abscissa in FIG. 6 to FIG. 8 represents the lg antibody concentration and the ordinate represents the OD value (OD 450) of the corresponding antibody. The result shows that the recombinant protein G provided by the invention has higher affinity for IgG from various sources.
Table 1 table of affinity statistics
| Species origin of IgG | Affinity of Protein G |
| Goat | 1.49×10 8 |
| Rat (mouse) | 1.35×10 7 |
| Rabbit | 1.89×10 8 |
Example 5
The present example provides an affinity chromatography column.
Cyanogen bromide activated agarose matrices can be used to prepare affinity chromatography resins.
The processed agarose has a primary structure consisting of alternating D-galactose and 3-anhydrogalactose residues. These sugars provide an uncharged hydrophilic matrix. For most affinity applications requiring harsh activation or use conditions, crosslinked agarose is generally preferred over uncrosslinked agarose. But the increased stability by crosslinking results in a 30-50% loss of potential reactive sites (consumed in the crosslinking chemistry). The addition of cross-links to stabilize the bead agarose does not significantly reduce the porosity.
The preparation method of the affinity chromatography column comprises the following steps:
(1) Protein G was dissolved in 0.1M NaHCO 3 0.5M NaCl (5.10 mg protein per ml gel);
(2) Activating agar (200 ml per gram of agar) in pre-chilled 1mM HCl;
(3) The resin was washed with 5-10 column volumes of distilled water, then with 0.1M NaHCO 3 Resin washed with 0.5M NaCl (5 ml per gram xerogel) and immediately transferred to ligand in coupling buffer; the coupling buffer comprises: 0.5M sodium chloride+0.1M sodium bicarbonate, and adjusting the pH value to 8.4;
(4) Mixing protein G with the gel at room temperature for 2h or overnight at a temperature of 2-8 ℃;
(5) Using 0.1M NaHCO 3 Washing away unreacted ligand with 0.5M NaCl;
(6) Blocking unreacted genes with 0.2M glycine or 1M ethanolamine at room temperature for 2h or 2-8deg.C for 6h;
(7) Thoroughly washing to remove blocking solution, first washing with alkaline coupling buffer, then with 0.1MNaAc/0.5M NaCl (pH: 4.0); washing the mixture with pH buffer (0.1M NaAc/0.5M NaCl (pH: 4.0)) for 4-5 times;
(8) The resin was stored in 1.0M NaCl at 2-8deg.C and 20% ethanol was added.
Example 6
The antibodies were purified using the affinity chromatography column prepared in example 5.
(1) And (3) column loading: the standard is 10ml gravity column, put on the purification frame, the bottom installs the head additional. After mixing the affinity chromatography resin (protease G packing) prepared in example 5, 1ml of the packing was taken out and placed in a gravity column, which was filled with an antibody purification equilibration solution (cyanogen bromide activated agarose gel from MERCK Co., ltd.) TM 4B goods number: c9142 A sieve plate is additionally taken to seal the top of the filler, so that the distance between the sieve plate and the filler is 0.5cm. And (3) taking down the bottom end enclosure of the purification column to allow the liquid to naturally flow out, and adding 10-20 times of column volume of antibody purification balancing liquid to balance the column to ensure that the filler reaches the optimal combination state.
(2) Sample preparation: 1ml of mouse ascites (ascites containing cytokeratin 8 antibody and ascites containing human EGF receptor-2 antibody), centrifuging (5000 rpm,5 min) to obtain supernatant, adding PB of 5-10 times volume, mixing, filtering with a sieve plate, and collecting filtrate for use.
The preparation method of the ascites of the mice comprises the following steps: expanding the monoclonal cell line to about 1×10 cells 6 Individual cells are injected into selected mice (the mice need to be injected with paraffin oil in the abdominal cavity in advance for one week), and after waiting for 7-10 days, the mice generate ascites, and the ascites is collected for antibody purification.
(3) Adding the filtered filtrate into the balanced purification column, collecting the effluent again, and temporarily storing in a refrigerator at 4deg.C. (to detect whether the purification column has reached saturation or that the life of the purification column has been reached).
(4) After the sample is loaded, the purification column is washed by PB with 10-20 times of column volume, and unbound impurity proteins are washed off.
(5) Pre-elution: washing with 10-20 times column volume antibody purification pre-eluent (pH 5.0) to wash out the impurity protein with weak binding.
(6) Eluting: adding 10 times column volume of antibody purification eluent (pH 2.7), collecting eluent, 1 ml/tube, generally 5 to 8 tubes, and adding 100ml of neutralizing solution (pH 7.0) in the collecting tube in advance; the eluted antibody is temporarily stored in a refrigerator at 4 ℃ for concentration measurement and then split charging and storage.
(7) The column was then filled with deionized water filtered through a 0.22mm filter membrane at 10Cv (column volume) to neutrality.
(8) The column was packed with 5-10Cv (column volume) of 20% ethanol, and the column was removed and stored at 4℃when the column was filled with ethanol.
SDS-PAGE results of purified antibodies referring to FIG. 9, M is Marker,1 is human and company protein G purified cytokeratin 8 antibody, 2 is recombinant protein G purified cytokeratin 8 antibody in this example, 3 is human EGF receptor-2 antibody purified by human and company protein G in this example, and 4 is recombinant protein G purified human EGF receptor-2 antibody in this example.
The concentration of the recombinant protein G-purified cytokeratin 8 antibody in this example was 0.43mg/ml, and the concentration of the protein G-purified cytokeratin 8 antibody of a certain company was 0.3mg/ml. Therefore, the recombinant protein G provided by the invention improves the binding force of the streptococcus protein and the IgG.
Example 7
This example was used for immunohistochemical experiments.
(1) Slicing: baking lung adenocarcinoma tissue pathological sections in a 60 ℃ incubator for 60 minutes, soaking the sections in xylene I for 15 minutes, replacing xylene II, and then soaking for 15 minutes, wherein the sections are respectively soaked in absolute ethyl alcohol (1) for 5 minutes, absolute ethyl alcohol (2) for 5 minutes, 95% ethyl alcohol for 5 minutes, 85% ethyl alcohol for 5 minutes and 75% ethyl alcohol for 5 minutes; ddH 2 Soaking in O for 5 minutes, and cleaning for 3 times; adding 10mmol/L citrate buffer solution (pH 6.0) which is enough to submerge slices into a pressure cooker for antigen retrieval (boiling method), heating to boiling, placing the slices on a heat-resistant material slice frame, placing into the cooker, covering a cooker cover, fastening a pressure valve, continuing heating, setting pressure maintaining for 4 minutes, opening a deflation valve for deflation after the time is up, opening the cooker cover after the pressure is zeroed, taking out the inner cooker, and cooling at room temperature. Taking out the slice (about 40 min) after the solution is cooled to room temperature; ddH 2 O is soaked for 5 minutes, washed for 2 times, PBST is soaked for 5 minutes, and washed for 2 times; the sections were placed in 20ml of 3% H 2 O 2 -in methanol solution, protected from light, treated at room temperature for 10 minutes; PBST is soaked for 5 minutes and is washed for 3 times; one drop of goat serum blocking solution was added to each tissue group and incubated in a wet box for 45 minutes at room temperature; PBST was soaked for 5 minutes and washed 3 times.
(2) Tissue sections were incubated with antibody mix:
adding the treated lung adenocarcinoma tissue slice into cytokeratin 8 antibody (recombinant protein G purified cytokeratin 8 antibody and cytokeratin 8 antibody purified by protein G of certain company provided by the invention), and incubating overnight in a wet box at 4 ℃; taking out from the refrigerator at the temperature of 4 ℃ and incubating for 60 minutes at room temperature; PBST is gently washed and then soaked for 5 minutes, and the PBST is washed for 3 times; adding 25ul of HRP-labeled secondary antibody of Changdao company into each tissue group, and incubating for 45 minutes at room temperature; washing; preparing DAB color development liquid, reacting for 10-15 min in dark, then dripping the DAB color development liquid onto a slice, and developing for 1-5 min; terminating the color reaction with distilled water; dropping 50ul hematoxylin dye solution into each tissue group, dyeing for 5-10 minutes, and washing with distilled water; the slices are put into 1 percent hydrochloric acid-ethanol for decoloration for 2 to 3 seconds, then are quickly taken out and put into distilled water for termination, and then are put into PBST (pH 8.0) for blue reflection for 5 to 10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min: soaking in absolute ethanol for 5 minutes. Soaking in xylene for 10 min, and soaking for 10 min after changing xylene; dripping a neutral resin sealing piece, and covering a glass slide sealing piece; microscope imaging.
As can be seen from FIG. 10, the recombinant protein G-purified cytokeratin 8 antibody (A) provided by the present invention has no significant difference in staining effect on lung adenocarcinoma tissues as compared with the cytokeratin 8 antibody (B) purified by a certain company protein G.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Recombinant streptococcal protein G, characterized in that it comprises an amino acid sequence as shown in SEQ ID NO. 1.
2. A nucleic acid molecule encoding recombinant streptococcal protein G, characterized in that it encodes recombinant streptococcal protein G according to claim 1;
preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.
3. A recombinant vector comprising the nucleic acid molecule of claim 2 encoding recombinant streptococcal protein G.
4. A recombinant cell comprising 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 cell or escherichia coli.
5. A method of preparing recombinant streptococcal protein G as claimed in claim 1, comprising: culturing the recombinant cell of claim 4.
6. The method for producing recombinant streptococcal protein G according to claim 5, wherein the method comprises: firstly, transforming competent cells with a recombinant vector comprising a nucleic acid molecule for encoding recombinant streptococcal protein G, obtaining a positive strain through identification, extracting plasmids, carrying out plasmid linearization treatment, transforming the linearized plasmids into pichia pastoris competent cells, screening to obtain the positive recombinant pichia pastoris 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 comprising agar to which recombinant streptococcal protein G according to claim 1 or produced by the production method according to any one of claims 5-6 is coupled.
8. A protein conjugate comprising recombinant streptococcal protein G according to claim 1 or prepared by a method according to any one of claims 5-6, wherein the recombinant streptococcal protein G is labelled with a detectable label.
9. The protein conjugate of claim 8, wherein said detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;
preferably, the nanoparticle-based label is selected from any one of nanoparticles and colloids.
10. Use of an affinity chromatography resin according to claim 7 for purifying antibodies;
preferably, the antibody is an IgG-type antibody;
preferably, the antibody is an anti-cytokeratin 8 antibody, an anti-surface antigen cluster 274 antibody, or an anti-human epidermal growth factor receptor-2 antibody.
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