CN117209600A - SS-B binding proteins, methods of preparation and use - Google Patents
SS-B binding proteins, methods of preparation and use Download PDFInfo
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- CN117209600A CN117209600A CN202311214350.3A CN202311214350A CN117209600A CN 117209600 A CN117209600 A CN 117209600A CN 202311214350 A CN202311214350 A CN 202311214350A CN 117209600 A CN117209600 A CN 117209600A
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Abstract
The invention provides an SS-B binding protein, a preparation method and application thereof, and relates to the technical field of antibodies. The VH of the SS-B binding protein comprises CDRs of 26 th to 33 th, 51 th to 58 th and 97 th to 107 th of a sequence shown in SEQ ID NO.1, and the VL comprises CDRs of 27 th to 38 th, 56 th to 58 th and 95 th to 103 th of a sequence shown in SEQ ID NO. 2; alternatively, VH comprises CDRs at positions 26 to 33, 51 to 57 and 90 to 109 of the sequence shown in SEQ ID NO. 3, and VL comprises CDRs at positions 27 to 37, 55 to 57 and 94 to 102 of the sequence shown in SEQ ID NO. 4. The binding protein has good specific binding capacity with SS-B, and can be used for identifying SS-B antigen and diagnosing and detecting other diseases including Sjogren syndrome and the like.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an SS-B binding protein, a preparation method and application.
Background
An anti-SS-B antibody is one of anti-nuclear antibodies, also called anti-La antibodies, whose target antigen La protein contains RNA recognition sites (RRM), ATP binding sites, and nuclear localization signals, and is associated with multiple small RNAs within a cell.
The anti-SS-B antibody and the anti-SS-A antibody are both marker antibodies for diagnosing Sjogren syndrome, wherein the antibodies have higher specificity than the anti-SS-A antibody, and the positive rate of primary Sjogren syndrome is 65% -85%, so that the antibodies can be used for diagnosing, distinguishing diagnosis and prognosis reference of the Sjogren syndrome. The index can be detected by a clinical selective two-way immunodiffusion method, an enzyme-linked immunosorbent assay, an immunoblotting method and the like, and is used for diagnosing the primary Sjogren syndrome. In addition, the anti-SS-B antibody can also appear in the serum of a few patients with systemic lupus erythematosus or rheumatoid arthritis, and can also be seen in the neonatal lupus syndrome to cause congenital heart block, so that the detection of the index has certain prompt significance for the diseases.
Most of anti-SS-B antibodies in the conventional detection kit are human positive serum, but the serum sources are difficult, and the batch-to-batch differences are large; in addition, the immune animal polyclonal antibody is coupled with human IgG, the operation is complex, and the cost is high. Therefore, the development of the high-affinity anti-SS-B recombinant monoclonal antibody which is simple to obtain, can be produced in a large scale and is widely applied, and particularly can be used in a clinical detection kit, thereby having important significance and value.
In view of this, the present invention has been made.
Disclosure of Invention
The object of the present invention is to provide SS-B binding proteins which have a good specific binding capacity to SS-B. Based on the SS-B binding proteins provided by the present invention, the present invention also aims to provide uses thereof. It is another object of the present invention to provide an SS-B binding protein with a good binding activity, which can be easily prepared, can be used for the identification of SS-B antigen, and can be used for the auxiliary diagnosis and detection of other diseases including SS-B antibody positive sicca syndrome.
Noun definition:
the term "binding protein" as used herein refers to a protein that binds to a particular antigen, and broadly refers to all proteins and protein fragments that contain complementarity determining regions (CDR regions). The protein or protein fragment may be an antibody, "antibody" particularly referring to a full-length antibody. The terms "antibody" and "full length antibody" include polyclonal antibodies and monoclonal antibodies. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the terms "antibodies" and "immunoglobulins" are used interchangeably.
Proteins and protein fragments may also be antigen binding fragments comprising a portion or all of the CDRs of an antibody that lack at least some of the amino acids present in the full-length chain but are still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from but not limited to F (ab') 2 Fab', fab, fv (consisting of VH and VL), scFv (single chain antibody, wherein VH and VL are linked by a linker peptide), dsFv (disulfide stabilized Fv antibody (disulfide stabilized Fv fragments, dsFv)), bispecific antibody, nanobody, and antibody minimal recognition unit. In addition to the functional fragments described above, any fragment whose half-life has been increased is included.
"variable region" or "variable domain" of a binding protein refers to the domain of an antibody that recognizes and binds an antigen at the amino terminus of the heavy or light chain, the composition and arrangement of the amino acids of the segment determining the specificity of an antibody for recognizing an antigen. The heavy chain variable domain may be referred to as a "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) linked by 4 Framework Regions (FR). The framework regions and the ranges of the CDRs have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any of the CDR determination methods well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, and in general the variable regions VL/VH of the heavy and light chains can be obtained by ligating the CDRs numbered from the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combined arrangement.
The term "constant region" or "constant domain" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination. The heavy chain of an antibody has one variable domain (VH) followed by a number of constant domains or regions, such as one or more of the hinges, CH1, CH2, CH3, and CH4, the CH1 domain being adjacent to the VH domain and amino-terminal to the hinge region of the heavy chain of the antibody, and not forming part of the Fc region of the antibody; the hinge region includes a portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is typically a CH3 domain, which CH3 domain typically forms the C-terminal portion of an antibody, and in some antibody types, the constant region in IgM and IgE, for example, also includes a CH4 domain. The constant regions of antibodies may be derived from IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, as well as subclasses and mutated forms thereof.
The invention is not limited in the manner in which the SS-B binding protein is obtained. In some alternative embodiments, the corresponding antibody is obtained upon expression in a cell after ligation to the vector using a polynucleotide encoding an SS-B binding protein. The above vectors may be introduced into eukaryotic cells, particularly mammalian cells, and constructed to express the SS-B binding protein. In other alternative embodiments, the binding protein may also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as an automated peptide synthesizer (e.g., an automated peptide synthesizer sold by Applied BioSystems, etc.); the antigen binding fragments are also optionally produced by enzymatic cleavage of antigen binding molecules (including intact antibodies), such as pepsin or papain cleavage; or chemical cleavage, e.g., by chemical reduction cleavage of disulfide bonds.
The terms "specifically recognizes," "selectively binds," and "specifically binds" or the like refer to the binding of a binding protein to an epitope on a predetermined antigen. Typically, the binding protein is less than about 10 -5 M, e.g. less than about 10 -5 M、10 -6 M、10 -7 M、10 -8 M、10 -9 M or 10 -10 M or less K d The values are combined. The K of an antibody can be determined using methods well established in the art d Values. Other standard assays for evaluating the binding capacity of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, western blot, RIA, and flow cytometry analysis.
The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide encodes the SS-B binding protein described above, optionally a sense strand or an antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.
The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.
Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).
The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Offspring may differ from primary cells morphologically and/or in genomic DNA by natural, accidental, or deliberate mutations that are not necessarily identical to primary cells. "transformant" and "transformed cell" include primary test cells and cultures derived therefrom.
Herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes polypeptides or nucleic acids that are removed from their original environment, e.g., if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some protein or other cellular component that is normally associated therewith or that is normally admixed therewith or in solution. Isolated polypeptides include naturally produced said polypeptides contained in cell lysates, purified or partially purified forms of said polypeptides, recombinant polypeptides, said polypeptides expressed or secreted by cells, and said polypeptides in heterologous host cells or cultures. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
In order to solve the technical problems, the following technical scheme is provided:
according to one aspect of the present invention, there is provided an SS-B binding protein having a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region comprises complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3;
wherein, the amino sequences of the VH-CDR1, the VH-CDR2 and the VH-CDR3 are respectively shown in the 26 th to 33 th positions (GYTFTNYG), the 51 th to 58 th positions (IDTNTGEP) and the 97 th to 107 th positions (ARGNYGNYFDY) of SEQ ID NO:1, and the amino sequences of the VL-CDR1, the VL-CDR2 and the VL-CDR3 are respectively shown in the 27 th to 38 th positions (QSLLYSSNQKNY), the 56 th to 58 th positions (WAS) and the 95 th to 103 th positions (QQYYSYPLT) of SEQ ID NO: 2;
or (b)
The amino sequences of VH-CDR1, VH-CDR2 and VH-CDR3 are shown in positions 26-33 (GDSITSGY), 51-57 (ISYSGST) and 96-109 (ARWGYYRDYYAMDY) of SEQ ID NO:3, and the amino sequences of VL-CDR1, VL-CDR2 and VL-CDR3 are shown in positions 27-37 (QSLLHSNGKTY), 55-57 (LVS) and 94-102 (LQGTHFPYT) of SEQ ID NO:4, respectively.
The heavy chain variable region or the light chain variable region of the SS-B binding protein may differ in the remainder (e.g., framework region) by the removal of CDR regions.
In alternative embodiments, the SS-B binding protein is selected from the group consisting of SS-B binding protein 1 and SS-B binding protein 2;
wherein the amino acid sequence of the heavy chain variable region of the SS-B binding protein 1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the SS-B binding protein 2 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
In alternative embodiments, the SS-B binding protein is an intact antibody, F (ab') 2 One of Fab', fab, fv, scFv, dsFv, bispecific antibodies and antibody minimal recognition units.
In alternative embodiments, the species from which the remainder of the sequence of the antibody is derived includes one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.
In alternative embodiments, the SS-B binding protein is an antibody or antigen binding fragment comprising a constant region.
In alternative embodiments, at least a portion of the constant region sequence of the SS-B binding protein is a human consensus constant region sequence.
In alternative embodiments, the constant region of the SS-B binding protein may be selected from the group consisting of the sequences of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, and subclasses and mutant forms thereof. The constant regions may be of a species such as, but not limited to, bovine, equine, dairy cow, porcine, ovine, caprine, rat, mouse, guinea pig, hamster, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In alternative embodiments, the SS-B binding protein comprises a heavy chain constant region and a light chain constant region.
In alternative embodiments, the heavy chain constant region in the SS-B binding protein is of human origin, and more preferably the constant region of human IgG1, including CH1-CH2-CH3. The amino acid sequence of the heavy chain constant region preferably comprises the sequence shown in SEQ ID NO. 5.
In alternative embodiments, the light chain constant region in the SS-B binding protein is murine in origin, and the amino acid sequence of the murine light chain constant region preferably comprises the sequence set forth in SEQ ID No. 6.
In an alternative embodiment, the SS-B binding protein is a human murine chimeric antibody whose heavy chain comprises a human derived heavy chain constant region having the amino acid sequence shown in SEQ ID NO. 5 and whose light chain comprises a murine light chain constant region having the amino acid sequence shown in SEQ ID NO. 6.
The invention provides an SS-B binding protein, which is a human-mouse chimeric antibody, wherein the heavy chain amino acid sequence of the SS-B binding protein is shown as SEQ ID NO. 7, and the light chain amino acid sequence is shown as SEQ ID NO. 8;
alternatively, the heavy chain amino acid sequence of the SS-B binding protein is shown as SEQ ID NO. 9, and the light chain amino acid sequence is shown as SEQ ID NO. 10.
According to another aspect of the present invention, there is also provided a biological material comprising a polynucleotide, a vector, a cell; wherein the polynucleotide encodes the SS-B binding protein described above; the vector carries the polynucleotide; the cells carry the polynucleotide, or contain the vector or are capable of expressing the SS-B binding protein.
After the polynucleotide encoding the SS-B binding protein is ligated to a vector, the vector may be introduced into eukaryotic cells, particularly mammalian cells, and a cell line capable of expressing the SS-B binding protein is constructed, and the corresponding protein is obtained in the cell.
In some alternative embodiments, the host cell used to express the SS-B binding protein is a 293 cell (human kidney epithelial cell line) or CHO cell (chinese hamster ovary cell).
According to another aspect of the present invention, there is also provided a method for preparing an SS-B binding protein, comprising culturing a cell capable of expressing said SS-B binding protein.
In an alternative embodiment, the method of preparation further comprises transforming a host cell with a polynucleotide encoding a polypeptide comprising the SS-B binding protein and expressing, and purifying to obtain the SS-B binding protein.
In alternative embodiments, the polynucleotide comprising the gene encoding the SS-B binding protein is synthesized as desired, and/or a suitable expression vector is prepared as desired, transformed into a desired host cell and expressed, and the SS-B binding protein is obtained by purification.
In an alternative embodiment, the polynucleotide of the SS-B binding protein comprises a heavy chain gene expression plasmid and a light chain gene expression plasmid, the heavy chain gene expression plasmid and the light chain gene expression plasmid are co-transformed into a host cell, and the SS-B binding protein is obtained by purification.
In an alternative embodiment, a complete IgG1 heavy chain expression plasmid is constructed comprising fusing the C-terminal heavy chain variable region to a constant region fragment of human IgG 1.
In alternative embodiments, the host cell is preferably a eukaryotic cell, more preferably a mammalian cell, and even more preferably a 293 cell or CHO cell.
According to another aspect of the present invention, there is also provided the use of the above SS-B binding protein, the above biomaterial as described in any one of (a) to (e) below;
(a) Preparing a reagent or a kit for detecting SS-B;
(b) Preparing a reagent or kit for diagnosing a disease, including SS-B antibody positive disease;
(c) Detection of SS-B for non-diagnostic and therapeutic purposes;
(d) The method is used for preparing an SS-B antibody detection quality control product;
(e) Preparing a composition for tracing;
in alternative embodiments, the disease comprises sjogren's syndrome, systemic lupus erythematosus or rheumatoid arthritis.
According to another aspect of the present invention, there is also provided a reagent or kit comprising the above SS-B binding protein; and/or the above biological material.
In alternative embodiments, the agent is a quality control or a composition for use in a tracer.
The composition for tracer comprises the SS-B binding protein and a label; the label comprises one or more of enzyme, luminescent label, fluorescent microsphere, color microsphere, latex microsphere, colloidal gold, quantum dot, biotin or streptavidin, radionuclide, radiocontrast agent, paramagnetic ion, metal and photosensitizer. The SS-B binding protein and the label in the composition for labelling in the reagent or kit may or may not be linked; when the connection is not needed, the connection is carried out when the connection is needed. The attachment may be, but is not limited to, chemical attachment or physical adsorption.
Examples of enzymes are, for example, but not limited to, alkaline phosphatase or horseradish peroxidase, and luminescent labels are, for example, but not limited to, fluorescent proteins, synthetic class-IIMolecular or polymeric dyes, etc., specific examples include, but are not limited to, alexa 350, alexa 405, alexa430, alexa 488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxy fluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosin, phthalocyanine, cyanine, alternet-2-oxa-3-diazole azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyl diamine europium, europium cryptate or chelate, diamine, bis anthocyanin, la Jolla Blue dye, allophycocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine and texas red. The fluorescent microspheres, the colored microspheres and the latex microspheres are each independently selected from the group of products acceptable in the art, such as from commercially available products. Radionuclides include, but are not limited to 110 In、 111 In、 177 Lu、 18 F、 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc、 94 Tc、 99 mTc、 120 I、 123 I、 124 I、 125 I、 131 I、 154-158 Gd、 32 P、 11 C、 13 N、 15 O、 186 Re、 188 Re、 51 Mn、 52 mMn、 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and is provided with 83 One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).
It will be appreciated that the reagents or kits provided herein remove the above antibody compositions, and optionally solid phase carriers, and optionally further comprise reagents and/or consumables well known to those skilled in the art for detecting reactions, such as, but not limited to, one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking fluids, washes, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality controls and labels.
In alternative embodiments, the reagents or kits of any of the above aspects may be used in a general immunoassay method acceptable in the art, including but not limited to immunoblotting, immunohistochemistry, ELISA, immunochromatography or immunomagnetic beads, and those skilled in the art may formulate the reagents or other reagents in the kit according to the corresponding detection means, which is not limited in the present invention.
In alternative embodiments, the reagent or kit is se:Sup>A multiplex assay reagent or kit, including antinuclear antibody assay kits, and other assay indicators include at least one of dsDNA, nucleosomes, histones, sm, ribosomal P proteins, PCNA, SS-A-52, SS-A-60, CENP-B, AMA M2, scl-70, jo-1, PM-Scl, mi-2, ku, RNP/Sm.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses an SS-B binding protein which specifically recognizes and binds to an SS-B antigen, and further prepares a human-mouse chimeric recombinant monoclonal antibody. The technology of the invention has the following advantages:
1) As the monoclonal antibody sequence can be expressed at any time by 293F cells, the preparation process is simple to operate, the time consumption is short, the expression quantity is high, the production process is controllable, and the product batch-to-batch difference is small.
2) Compared with the direct use of human serum, the preparation method can avoid the problems of difficult sample sources and high cost.
3) The SS-B binding protein disclosed by the invention can be used as a quality control product in a detection kit, is more convenient to use compared with immune polyclonal antibody without coupling human IgG, can avoid the problems of complicated operation and low subsequent coupling efficiency of the immune polyclonal antibody, reduces the production cost, stabilizes the product quality and can obviously improve the reaction value;
4) The SS-B binding protein disclosed by the invention can be specifically combined with an SS-B antigen, can be coupled with gel particles to carry out immunoaffinity chromatography purification of the SS-B antigen, can be used for preparing high-purity antigen protein, and can also be used as a coupling marker for detecting the SS-B antigen.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a nucleic acid electrophoretogram of the heavy chain (Fd) and light chain (kappa, lambda) used to construct the library of example 1;
FIG. 2 is a nucleic acid electrophoresis of Fab gene fragments used in the construction of the library of example 1;
FIG. 3 is an SDS-PAGE protein electrophoresis of the anti-SS-B recombinant monoclonal antibody of example 2;
FIG. 4 is an analysis of binding between the recombinant monoclonal antibodies and the SS-B protein by ELISA in example 3, wherein 1-SSB and 2-SSB are the results of binding activities between two recombinant monoclonal antibodies and the SS-B antigen, respectively, and 1-control and 2-control are the results of binding activities between two recombinant monoclonal antibodies and the control protein BSA, respectively.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 screening of SS-B binding proteins
Preparation of phage display library:
(1) Spleen was taken from mice immunized with SS-B antigen, and lymphocytes were isolated using a mouse lymphocyte isolate.
(2) Extracting RNA: taking 1×10 6 And extracting total RNA of the lymphocyte by the cell.
(3) Reverse transcription: and (3) carrying out reverse transcription on the extracted total RNA to synthesize cDNA.
(4) Antibody gene fragment amplification: the VH-CH1 (Fd) regions of the kappa, lambda light and heavy chains of the antibody were amplified by specific amplification primers using cDNA as template.
The reaction system and the reaction procedure were as follows:
20. Mu.L of reaction system: 1. Mu.L of cDNA, 0.8. Mu.L of LPrime F, 0.8. Mu.L of LPrime R, 10. Mu.L of 2X phanta max master mix, 7.4. Mu.L of nucleic-Free Water;
the reaction procedure: melting 95 ℃ for 30s, annealing 55 ℃ for 30sec, extension 72 ℃ for 45s,30 cycles.
After the reaction is finished, loading buffer is added into the system, 1% agarose gel electrophoresis identification is carried out, an electrophoresis diagram is shown in figure 1, and the target band is about 750bp.
The target band was excised, and antibody heavy chain (Fd) gene fragment and light chain gene fragment (κ, λ) were recovered, respectively.
(5) The antibody light and heavy chain gene fragments were combined into complete Fab gene fragments by overlapping PCR.
The reaction system and reaction procedure were as follows:
25 μl reaction system and procedure: light chain 30ng,linker 4.3ng,30ng heavy chain, 0.5. Mu.l sfiF (upstream primer), 0.5. Mu.l sfiR (downstream primer), 12.5. Mu.l 2X phanta max master mix, nucleic-Free Water was added to the total system of 25. Mu.l.
The reaction procedure: melting 95 ℃ for 30s, annealing 55 ℃ for 30sec, extension 72 ℃ for 90s,30 cycles.
After the reaction is finished, loading buffer is added into the system, 1% agarose gel electrophoresis identification is carried out, an electrophoresis diagram is shown in figure 2, and the target band is about 1500bp. The target band was excised and the antibody Fab fragment was recovered.
The upstream primer sfi if: 5'> GAGCAGGAGCATAGGAGGATCGGGCGGCGGCC <3' (SEQ ID NO. 11);
downstream primer sfi ir: 5'> CCATGGCAATGGTGATTCTGCTGCGCGGCCTGGCC <3' (SEQ ID NO. 12).
(6) Constructing a plasmid: the antibody Fab fragment and pComb3xSS plasmid were digested with sfi I, respectively, and the digested fragments were mixed in a molar ratio of 3:1, and then T4 DNA library was added thereto, followed by ligation overnight at 16 ℃.
(7) Library construction: recovering the ligation product from the previous step. Taking 1.5 mu l of recovered product, adding TG1 competence, uniformly mixing, transferring to an electric rotating cup for electric rotating, and carrying out parameter selection: bac-Ec1. After the completion of the electrotransformation, the culture was activated at 37℃for 1 hour, the activated bacterial liquid was transferred to 200ml of a 2 XYT medium, and 1/1000 of ampicillin and a glucose solution having a final concentration of 2% were added thereto, and the culture was carried out at 37℃and 220rpm until OD600 = 0.6. Helper phage M13K07 was added in 20-fold bacterial counts. Mixing, standing in shaking table at 37deg.C for 45min. Centrifugation is carried out for 15min, and 200mL of new 2 XYT+Amp+Kana culture medium is used for resuspension precipitation, 30 ℃ and 220rpm shaking is carried out for 14h for phage amplification. The following day the supernatant was collected by centrifugation, phage were settled by adding 1/4 of the volume of 20% PEG6000 and resuspended in PBS to give phage display library.
(II) panning of anti-SS-B antibodies Using phage display libraries
(1) After SS-B protein coupled with biotin is used as a target antigen and incubated for 1h together with SA magnetic beads, 1X 10 is added into a reaction system 12 The phages obtained in the above procedure were incubated for 1h, and specific phages were captured by the antigen. Wash 10 times with PBST (PBS+0.05% Tween-20).
(2) The antigen-binding magnetic beads were added to TG1 bacteria solution, allowed to stand at 37℃for 45min for infection, and then subjected to activation culture at 220rpm at 37℃for 1h. And (3) taking a proper amount of bacterial liquid subjected to standing infection, carrying out gradient dilution, coating an ampicillin plate, adding glucose (the final concentration is 2%) and 1/1000 of ampicillin antibiotics into the rest bacterial liquid, and shaking at 37 ℃ and 220rpm for 3 hours.
(3) 10 mu L M K07 is added into the bacterial liquid, and the bacterial liquid is subjected to standing and infection at 37 ℃ for 45min.
(4) 6000 Xg, centrifugal 10min, with 10mL 2 XYT+Amp+kana medium heavy suspension, 30 degrees, 220rpm, shaking 14h amplification phage. The following day the supernatant was collected by centrifugation, 1/4 of the volume of 20% PEG6000 settled phage were added and resuspended in PBS to give phage for the first round of screening.
(5) And (3) repeating the steps 1-4 by using the phage obtained by the first round of screening, and carrying out the 2 nd round of screening.
(6) Monoclonal identification: the second round of screening coated plates was used to select for single clones in 96-well deep well plates and phage were amplified overnight by shaking.
And (3) wrapping the plate: the SS-B antigen was coated onto ELISA plates, while BSA was used as a control protein coated plate, overnight at 4 ℃.
Closing: the next day the coating was discarded, the plates were washed 3 times with PBST, patted dry, 3% milk added and blocked at 37℃for 2h.
Preparation of an antibody: and (3) centrifuging the 96-well deep hole plate, diluting the supernatant in a final concentration of 1% milk, and uniformly mixing to serve as a primary antibody for later use.
Incubation resistance: the blocking solution was discarded, the plates were washed 3 times with PBST, the plates were dried by pipetting, and primary antibodies were added and incubated for 2h at 37 ℃.
Secondary antibody incubation: the primary Antibody was discarded, the plates were washed 5 times with PBST, and 1:5000 dilution of Anti-M13 Antibody (HRP) secondary Antibody was added and incubated for 1h at 37 ℃. Discarding the secondary antibody, washing the plate for 5 times by PBST, beating, adding a chromogenic substrate for color development, adding a stop solution after 15min to stop the reaction, and reading by an enzyme-labeling instrument.
(7) Clones with high read (SS-B antigen wells OD > 1) and low non-specific binding (BSA control protein wells OD < 0.5) were selected for sequencing.
Light chain sequencing primer: bomp:5'> GTGTGGAATTGTGAGCGG <3' (SEQ ID NO. 13);
heavy chain sequencing primer: PELB:5'> ACCTATTGCCTACGGCAGCCG <3' (SEQ ID NO. 14).
Sequencing resulted in two SS-B binding protein sequences, SS-B binding protein 1 and SS-B binding protein 2, comprising antigen binding domains.
The heavy chain variable region sequence of the SS-B binding protein 1 is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO. 2:
SEQ ID NO:1
QIQLAQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWIDTNTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGNYGNYFDYWGQGTTLTVSS。
SEQ ID NO:2
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLEIK。
further analysis resulted in SS-B binding protein 1 having the following complementarity determining regions (i.e., the underlined portions of the sequences described above):
heavy chain:
VH-CDR1 is GYTFTNYG (amino acid residues 26 to 33 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 1);
VH-CDR2 is IDTNTGEP (amino acid residues 51-58 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 1);
VH-CDR3 is ARGNYGNYFDY (amino acid residues 97-107 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 1);
light chain:
VL-CDR1 is QSLLYSSNQKNY (amino acid residues 27 to 38 of the N-terminal end of the amino acid sequence shown in SEQ ID NO: 2);
VL-CDR2 is WAS (56 th to 58 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO: 2);
VL-CDR3 is QQYYSYPLT (amino acid residues 95 to 103 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 2).
The heavy chain variable region sequence of the SS-B binding protein 2 is shown as SEQ ID NO. 3, and the light chain variable region sequence is shown as SEQ ID NO. 4:
SEQ ID NO:3
EVQLQQSGPSLVKPSQTLSLTCSVTGDSITSGYLNWIRKFPGNKLEYMGYISYSGSTYCNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARWGYYRDYYAMDYWGQGTSVTVSS。
SEQ ID NO:4
DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSNGKTYLNWLQQRPGQAPKRLFYLVSKLDPGIPDSFSGSGSETDFTLKISRVEAEDLGVYYCLQGTHFPYTFGGGTKLEIK。
further analysis resulted in SS-B binding protein 2 having the following complementarity determining regions (i.e., the underlined portions of the sequences described above):
heavy chain:
VH-CDR1 is GDSITSGY (amino acid residues 26-33 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 3);
VH-CDR2 is ISYSGST (amino acid residues 51-57 of the N end of the amino acid sequence shown in SEQ ID NO: 3);
VH-CDR3 is ARWGYYRDYYAMDY (96 th to 109 th amino acid residues of the N end of the amino acid sequence shown in SEQ ID NO: 3);
light chain:
VL-CDR1 is QSLLHSNGKTY (amino acid residues 27 to 37 of the N-terminal end of the amino acid sequence shown in SEQ ID NO: 4);
VL-CDR2 is LVS (amino acid residues 55-57 of the N-terminal of the amino acid sequence shown in SEQ ID NO: 4);
VL-CDR3 is LQGTHFPYT (amino acid residues 94-102 of the N-terminal end of the amino acid sequence shown in SEQ ID NO: 4).
EXAMPLE 2 expression purification of anti-SS-B recombinant monoclonal antibodies
Sequencing to obtain the Fab region sequence of candidate antibody, and synthesizing gene:
the vector selects PTT5 plasmid, takes EcoRI and BamHI as cloning sites, inserts light chain gene fragments, and the light chain constant region sequence is shown as SEQ ID NO. 6 and is a murine sequence. The CH1 of the heavy chain is replaced by the CH1 of human IgG1, the Fc of the human IgG1 is fused at the C end, the constant region sequence of the heavy chain is shown as SEQ ID NO. 5, the human sequence takes EcoRI and BamHI as cloning sites, and the constructed complete heavy chain fragment is inserted into the PTT5 plasmid.
SS-B binding protein 1: the complete sequence of the light chain is shown as SEQ ID NO. 8, and the complete sequence of the heavy chain is shown as SEQ ID NO. 7;
SS-B binding protein 2: the complete sequence of the light chain is shown as SEQ ID NO. 10, and the complete sequence of the heavy chain is shown as SEQ ID NO. 9.
(II) expression by using a mammalian cell 293F expression system:
(1) Plasmid extraction: the plasmids synthesized by the genes are respectively transformed into TOP10 competence, activated for 1h, transferred into 200ml LB culture medium, cultured overnight at 37 ℃, and extracted by using an endotoxin-free plasmid extraction kit the next day to obtain the corresponding heavy chain plasmids and light chain plasmids.
(2) 120mL of 293F cells were inoculated into 500mL suspension cell culture flasks 1 day before transfection, and the cell density was controlled at 1X 10 6 And each mL.
(3) The next day 40. Mu.g of heavy chain plasmid and 80. Mu.g of light chain plasmid were diluted and gently mixed in 6mL transfection buffer, 480. Mu.L PEI (polyethylenimine) was added, gently mixed, and incubated at room temperature for 20 minutes. Dropwise adding into cells, placing cells into an incubator at 98rpm at 37deg.C with 5% CO 2 And (5) suspension culture.
(4) After 6 days, the culture supernatant was collected, igG was purified with rProtein A as a filler, eluted with 0.1M Glycine (pH 3.0), and neutralized by the addition of 1M Tris (pH 8.0). After the completion of elution, the ultrafiltration tube was replaced with PBS buffer and concentrated, and the protein concentration was measured, and the purity was confirmed by SDS-PAGE. As in fig. 3.
EXAMPLE 3 ELISA determination of the binding Activity of recombinant monoclonal antibody to SS-B antigen
The binding activity of the SS-B binding proteins 1, 2 to the SS-B antigen of example 2 was examined as follows:
(1) SS-B protein (2. Mu.g/mL) was coated on ELISA plates, while BSA was coated as a non-specific binding control, and incubated overnight at 4 ℃.
(2) The coating was discarded, washed 3 times with PBST, patted dry, 3% milk added and blocked for 2h at 37 ℃.
(3) The blocking solution was discarded, PBST was washed 3 times, patted dry, and 1. Mu.M of the antibody to be tested was added for 3-fold gradient dilution, and incubated at 37℃for 1.5h.
(4) The primary antibody was discarded, PBST was washed 5 times, patted dry, and horseradish peroxidase (HRP) -labeled murine anti-human IgG secondary antibody was added and incubated for 1h at 37 ℃.
(5) Discarding the secondary antibody, washing with PBST for 5 times, drying, adding a chromogenic substrate for color development, adding a stop solution after 15min to stop the reaction, and measuring the OD value by an enzyme-labeled instrument.
(6) Nonlinear fitting was performed with OD values on the ordinate and the logarithm of the molar concentration of antibody on the abscissa.
As a result, as shown in FIG. 4, it was found that both the SS-B binding protein 1 and the SS-B binding protein 2 antibodies specifically bind to the SS-B protein without binding to the control protein BSA, and that both antibodies had EC50 values of less than 1nM.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
- An ss-B binding protein comprising a light chain variable region and a heavy chain variable region;the heavy chain variable region comprises complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region comprises complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3;the heavy chain variable region of the SS-B binding protein comprises a VH-CDR1 of an amino acid sequence shown in SEQ ID NO 1 from 26 th to 33 th amino acid residues, a VH-CDR2 of 51 th to 58 th amino acid residues and a VH-CDR3 of 97 th to 107 th amino acid residues at the N terminal;the light chain variable region comprises VL-CDR1 of 27 th to 38 th amino acid residues, VL-CDR2 of 56 th to 58 th amino acid residues and VL-CDR3 of 95 th to 103 th amino acid residues of the amino acid sequence shown in SEQ ID NO. 2 from the N terminal;or,the heavy chain variable region of the SS-B binding protein comprises a VH-CDR1 of 26 th to 33 th amino acid residues, a VH-CDR2 of 51 th to 57 th amino acid residues and a VH-CDR3 of 96 th to 109 th amino acid residues of an amino acid sequence shown in SEQ ID NO 3 from the N terminal;the light chain variable region comprises VL-CDR1 of the amino acid sequence shown in SEQ ID NO. 4 from amino acid residues 27-37, VL-CDR2 of amino acid residues 55-57 and VL-CDR3 of amino acid residues 94-102 of the N-terminal.
- 2. The SS-B binding protein according to claim 1, wherein the heavy chain variable region amino acid sequence of the SS-B binding protein is shown in SEQ ID No.1 and the light chain variable region amino acid sequence is shown in SEQ ID No. 2;alternatively, the amino acid sequence of the heavy chain variable region of the SS-B binding protein is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
- 3. The SS-B binding protein according to claim 1 or 2, wherein the SS-B binding protein is an intact antibody, F (ab') 2 One of Fab', fab, fv, scFv, dsFv, bispecific antibody and antibody minimal recognition unit;preferably, the species from which the remainder of the sequences of the antibodies are derived include one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans.
- 4. The SS-B binding protein according to claim 1 or 2, further comprising a constant region;preferably, at least a portion of the constant region sequence is a human consensus constant region sequence;preferably, the constant region sequence is selected from the sequence of part or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD.
- 5. The SS-B binding protein of claim 4, wherein the SS-B binding protein comprises a heavy chain constant region and a light chain constant region;preferably, the heavy chain constant region in the SS-B binding protein is derived from human, further preferably from the constant region of human IgG 1;preferably, the amino acid sequence of the human derived heavy chain constant region is shown in SEQ ID NO. 5.
- 6. The SS-B binding protein of claim 5, wherein the SS-B binding protein is a human murine chimeric antibody;the heavy chain amino acid sequence of the SS-B binding protein is shown as SEQ ID NO. 7, and the light chain amino acid sequence is shown as SEQ ID NO. 8;alternatively, the heavy chain amino acid sequence of the SS-B binding protein is shown as SEQ ID NO. 9, and the light chain amino acid sequence is shown as SEQ ID NO. 10.
- 7. A biological material comprising a polynucleotide, vector, or cell;the polynucleotide encodes the SS-B binding protein of any one of claims 1-6;the vector carries the polynucleotide;the cell carrying the polynucleotide or containing the vector or being capable of expressing the SS-B binding protein according to any one of claims 1 to 6.
- A method for producing an ss-B binding protein comprising culturing the cell of claim 7;preferably, the cell is produced by transforming a host cell with a polynucleotide encoding an SS-B binding protein comprising the SS-B binding protein of any one of claims 1-6, wherein the polynucleotide comprises a heavy chain gene expression plasmid and a light chain gene expression plasmid, and wherein the cell expressing the SS-B binding protein is produced by co-transforming the heavy chain gene expression plasmid and the light chain gene expression plasmid into a host cell;preferably, the host cell is a eukaryotic cell, preferably a mammalian cell;preferably, the mammalian cells comprise 293 cells or CHO cells, more preferably 293F cells.
- 9. Use of the SS-B binding protein according to any one of claims 1 to 6, or the biomaterial according to claim 7, in any one of the following (a) to (e);(a) Preparing a reagent or a kit for detecting SS-B;(b) Preparing a reagent or kit for diagnosing a disease, including SS-B antibody positive disease;(c) Detection of SS-B for non-diagnostic and therapeutic purposes;(d) The method is used for preparing an SS-B antibody detection quality control product;(e) Preparing a composition for tracing;preferably, the disease comprises sjogren's syndrome, systemic lupus erythematosus or rheumatoid arthritis.
- 10. A reagent or kit comprising the SS-B binding protein of any one of claims 1-6; and/or the biomaterial of claim 7;preferably, the agent is a quality control or a composition for use in a tracer;the composition for tracer comprising the SS-B binding protein according to any one of claims 1 to 5 and a label;the label comprises one or more of enzyme, luminescent label, fluorescent microsphere, color microsphere, latex microsphere, colloidal gold, quantum dot, biotin or streptavidin, radionuclide, radiocontrast agent, paramagnetic ion, metal and photosensitizer.
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