CN113004404A

CN113004404A - Binding proteins against PCT and methods for detecting PCT

Info

Publication number: CN113004404A
Application number: CN201911307720.1A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 马秋燕; 覃婷; 游辉
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2021-06-22
Anticipated expiration: 2039-12-18
Also published as: CN113004404B

Abstract

The invention discloses a binding protein for PCT and a method for detecting PCT, and relates to the technical field of antibodies. The binding protein for PCT disclosed in the present invention includes an antigen-binding domain, and the antigen-binding domain includes at least one of the following complementarity determining regions: CDR-VH1: G-Y-X1-F-T-D-Y-X2 ‑M‑X3, CDR‑VH2: G‑X1‑N‑P‑X2‑N‑G‑G‑X3‑S‑Y‑N‑Q‑X4‑F‑K‑G and CDR‑VH3: X1‑R ‑Y‑P‑X2‑Y‑Y‑G‑P‑X3‑Y‑A‑V. The binding protein can specifically bind to PCT, and has good binding activity and affinity. Using the binding protein of the present invention to detect PCT can improve the sensitivity and specificity of detection, and is used for the detection of PCT and the diagnosis of diseases in which PCT is used as a marker Offers more protein options.

Description

Binding proteins against PCT and methods for detecting PCT

Technical Field

The invention relates to the technical field of antibodies, in particular to binding protein aiming at PCT and a method for detecting the PCT.

Background

The Procalcitonin (PCT) coding gene is positioned on the No. 11 human chromosome, consists of 6 exons and 5 introns, has the total length of 7600bp, has the length of 2800bp, is translated into procalcitonin precursors in the rough endoplasmic reticulum of cells beside thyroid follicles after being transcribed, and is further modified and processed under the action of endogenous polypeptide enzyme to generate a final product PCT of 116 amino acids.

PCT is a new indicator with high sensitivity and specificity in the diagnosis of severe systemic bacterial, fungal, parasitic, acute malaria infection, Systemic Inflammatory Response Syndrome (SIRS), multiple organ failure syndrome (MODS). PCT is mainly produced under the stimulation of bacterial toxin and inflammatory cell factors, serum PCT is not generally increased under a non-infectious inflammation state, the generation of PCT is very fast in the infectious inflammation process, the PCT is increased within 2-6 hours of an endotoxin stimulation reaction, and the blood PCT of a serious systemic infected person can be up to 1000 times within 24 hours. PCT is currently widely accepted as a new marker of infectious inflammation. The kit can not only identify whether the bacterial infection is caused or not at an early stage, but also is an early warning and diagnosis index of septicemia, and PCT is positively correlated with the degree of bacterial infection, and the increase or decrease of PCT directly reflects the trend of disease deterioration or disease improvement, so that the detection of PCT has important reference values in differential diagnosis, prognosis judgment, curative effect observation, reasonable guidance and application of antibacterial agents and the like of bacterial infection, septicemia and MODS.

PCT methods for detecting inflammation have been approved by the U.S. Food and Drug Administration (FDA). In the early days, the detection of PCT mainly uses a gel chromatography method and a high performance liquid chromatography method, and the two methods are time-consuming, have high operation requirements and are difficult to automate. In recent years, the detection of PCT has been performed mostly by an immunological method, and has advantages such as high specificity, high sensitivity, and easy handling, including a double antibody sandwich immunochemical method, a colloidal gold method, and a radioimmunoassay. All the immunology methods are premised on the preparation of specific monoclonal antibodies against PCT, and the PCT content in the plasma of healthy people is extremely low and is below 0.2ng/mL, so that the requirements on parameters such as the affinity of the prepared antibodies are high. The preparation of the PCT specific antibody provides key raw materials for the immunodetection of PCT, and has important significance for expanding the clinical application of PCT and reducing the medical cost.

At present, the monoclonal antibody for detecting PCT in China has some defects in sensitivity, specificity and affinity and has a larger space for improvement, so that the monoclonal antibody for detecting PCT in the field still has strong demand.

Disclosure of Invention

The invention aims to provide a binding protein capable of specifically binding to PCT and application thereof. The binding protein provided by the invention can be specifically bound with PCT, has better binding activity and affinity, can improve the sensitivity and specificity of detection when being used for detecting the PCT, and can be used for diagnosing diseases taking the PCT as a marker.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies, and "antibody functional fragments" include antigen-compound-binding fragments of these antibodies, including Fab, F (ab') 2, Fd, Fv, scFv, diabodies, and minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework region" or "FR" region means the region of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the terms "isolated" or "purified" mean that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention:

in a first aspect, embodiments of the present invention provide a binding protein for PCT, said binding protein comprising an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

a complementarity determining region CDR-VH1 having the amino acid sequence G-Y-X1-F-T-D-Y-X2-M-X3, wherein: x1 is S or T, X2 is Y, T or F, X3 is Q, H or N;

a complementarity determining region CDR-VH2 having the amino acid sequence G-X1-N-P-X2-N-G-X3-S-Y-N-Q-X4-F-K-G, wherein: x1 is I or L, X2 is GG or N, X3 is T or S, X4 is Q, K or N;

a complementarity determining region CDR-VH3 having the amino acid sequence X1-R-Y-P-X2-Y-G-P-X3-Y-a-V, wherein: x1 is G or A, X2 is I or L, X3 is I, V or L;

a complementarity determining region CDR-VL1 having the amino acid sequence R-S-S-X1-S-X2-V-H-S-X3-G-N-T-Y-X4-H, wherein: x1 is N or Q, X2 is I or L, X3 is Q, K or N, X4 is I, V or L;

a complementarity determining region CDR-VL2 having the amino acid sequence X1-X2-S-X3-R-F-S, wherein: x1 is S, K or R, X2 is I, V or L, X3 is N or Q;

a complementarity determining region CDR-VL3 having the amino acid sequence X1-Q-S-X2-H-X3-P-W, wherein: x1 is F or S, X2 is S or T, and X3 is I, V or L.

The binding protein provided by the embodiment of the invention contains an antigen binding domain, the antigen binding domain comprises at least one of the complementarity determining regions, the amino acid sequence of the complementarity determining region is discovered and disclosed for the first time, the binding protein is a novel sequence, the binding protein can be endowed with the capacity of specifically binding PCT antigen, and has better binding activity and affinity.

In alternative embodiments, in the complementarity determining region CDR-VH1, X1 is T; in the complementarity determining region CDR-VH2, X3 is S; in the complementarity determining region CDR-VH3, X1 is G; in the complementarity determining region CDR-VL1, X1 is Q; in the complementarity determining region CDR-VL2, X1 is S; in the complementarity determining region CDR-VL3, X1 is S.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is Y.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is T.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is F.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is H.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X1 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X1 is L.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is GG.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X4 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X4 is K.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X4 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is K.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X3 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X2 is S.

In alternative embodiments, in the complementarity determining region CDR-VL3, X2 is T.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is L.

In alternative embodiments, the similar complementarity determining regions have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the complementarity determining regions described above.

In alternative embodiments, the antigen binding domain has a K with the PCT protein_D≤8.85×10^-9Affinity of mol/L.

In alternative embodiments, the antigen binding domain has a K with the PCT protein_D≤8×10^-9mol/L、7×10^-9mol/L、6×10^-9mol/L、5×10^-9mol/L、4×10^-9mol/L、3×10^-9mol/L、2×10^-9mol/L、1×10^- ⁹mol/L、9×10^-10mol/L、8×10^-10mol/L、7×10^-10mol/L、6×10^-10mol/L、5×10^-10mol/L、4×10^-10mol/L、3×10^-10mol/L、2×10^-10mol/L or 1X 10^-10Affinity of mol/L.

In an alternative embodiment, the antigen binding domain has a 1.11 x 10 affinity to PCT protein^-10≤K_D≤8.85×10^-9Affinity of mol/L.

K_DThe detection of (2) is carried out with reference to the method in the examples of the present invention.

In an alternative embodiment, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 1-62:

in alternative embodiments, in the complementarity determining region CDR-VH1, X1 is S; in the complementarity determining region CDR-VH2, X3 is T; in the CDR-VH3, X1 is A; in the complementarity determining region CDR-VL1, X1 is N; in the complementarity determining region CDR-VL2, X1 is K; in the complementarity determining region CDR-VL3, X1 is F.

In an alternative embodiment, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 63-72:

in alternative embodiments, the binding protein includes at least 3 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain, or3 complementarity determining regions of a light chain); alternatively, the binding protein comprises at least 6 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain);

in alternative embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In alternative embodiments, the binding protein is a functional fragment of an antibody, such as any one of a nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. As will be readily understood by those skilled in the art based on the teachings of the present invention, functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In an alternative embodiment, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID Nos. 1-4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID Nos. 5-8.

In addition, based on the disclosure of the present invention, the species source of the heavy chain or light chain framework region of the binding protein may be human, so as to constitute a humanized antibody.

In alternative embodiments, the binding protein further comprises an antibody constant region.

In alternative embodiments, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the antibody constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the antibody constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 10.

The sequences of SEQ ID NOS: 1-10 are shown in the following table:

in a second aspect, the present embodiments provide the use of a binding protein according to any one of the preceding embodiments in the manufacture of a reagent or kit for diagnosing an infectious inflammation;

in alternative embodiments, the infectious inflammation is selected from any one of severe systemic bacterial, fungal, parasitic or acute malaria infection, systemic inflammatory response syndrome, multiple organ failure syndrome, and sepsis.

In a third aspect, the embodiments of the present invention provide a reagent or a kit for diagnosing infectious inflammation, which contains the binding protein as described above;

In a fourth aspect, an embodiment of the present invention provides a method for detecting a PCT, including: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above method is for the purpose of non-disease diagnosis.

It should be noted that one skilled in the art can perform qualitative or quantitative detection of PCT protein in a test sample based on the characteristics of immune complex formation by antibody/antigen binding. The method for detecting an antigen or an antibody based on the formation of an immune complex upon binding of the antibody to the antigen comprises:

(1) the detection purpose is realized by a precipitation reaction, which comprises the following steps: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoblotting, and the like;

(2) the detection purpose is realized by marking an indicator for displaying the signal intensity, and the method comprises the following steps: immunofluorescence, radioimmunoassay, chemiluminescence immunoassay, and enzyme-linked immunoassay (e.g., double antibody sandwich, indirect method, or competitive method);

the indicator may be selected appropriately according to different detection methods, including but not limited to the indicators described below:

(1) in the immunofluorescence method, the indicator may be a fluorescent dye, for example, a fluorescein-based dye (including Fluorescein Isothiocyanate (FITC), hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or an analog thereof), a rhodamine-based dye (including red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or an analog thereof), a Cy-series dye (including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or an analog thereof), an Alexa-series dye (including Alexa fluor350, 405, 430, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or an analog thereof), a protein-based dye (including Phycoerythrin (PE), Phycocyanin (PC), allophycocyanin (allophycocyanin), polymetaxanthin-chlorophyll protein (preCP), etc.);

(2) in radioimmunoassays, the indicator may be a radioisotope, for example: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, 18F, and the like.

(3) In enzyme-linked immunoassays, the indicator may be an enzyme that catalyzes the development of a substrate (e.g., horseradish peroxidase, alkaline phosphatase, or glucose oxidase, etc.).

(4) In the chemiluminescent immunoassay, the indicator may be a chemiluminescent liquid such as acridinium ester, horseradish peroxidase and luminol, alkaline phosphatase and AMPPD, electrochemiluminescent agents such as ruthenium terpyridyl and tripropylamine, and the like.

Based on the disclosure of the above binding protein, those skilled in the art can easily think of using any one or a combination of several methods or other methods to realize the quantitative or qualitative detection of PCT in the sample to be detected, and whatever method is selected, so long as the PCT is detected by using the binding protein disclosed in the present invention, it is within the scope of the present invention.

In an alternative embodiment, the binding protein is labeled with an indicator that indicates the strength of the signal, such that complexes of the binding protein bound to PCT protein are detected.

In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding a binding protein according to any one of the preceding embodiments;

in alternative embodiments, the nucleic acid is DNA or RNA.

Based on the disclosure of the amino acid sequence of the binding protein, one skilled in the art can easily obtain the nucleic acid sequence encoding the binding protein according to the codon corresponding to the amino acid, and obtain various nucleic acid sequences encoding the binding protein according to the degeneracy of the codon, which are within the protection scope of the present invention as long as they encode the binding protein.

In a sixth aspect, embodiments of the invention provide a vector comprising a nucleic acid according to the previous embodiments.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the nucleic acid sequence is linked to the regulatory sequence in a manner that allows expression. Regulatory sequences, including promoters, enhancers and other expression control elements, are selected to direct the expression of the protein of interest in a suitable host cell.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, which may be propagated and in which the genetic information may be present or absent) or an expression vector (i.e., a vector which comprises the necessary genetic elements to permit expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the invention are capable of autonomous replication and thus provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a signal peptide sequence encoding for secretion or integration of the protein expression product into a membrane, and optionally a nucleic acid sequence encoding for a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In a seventh aspect, embodiments of the present invention provide a host cell comprising a vector according to the previous embodiments.

The expression vectors of the invention are useful for transforming host cells. Such transformed host cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the binding proteins of the invention. Host cells of the present invention include microorganisms such as bacteria (e.g., Escherichia coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells.

In an eighth aspect, embodiments of the invention provide a method of producing a binding protein of any one of the preceding embodiments, comprising:

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions such that the binding protein is expressed. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The bound protein may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a reduced SDS-PAGE of the PCT monoclonal antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

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 skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of activities or events, as some activities may occur in different orders and/or concurrently with other activities or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

This example provides a method for preparing recombinant antibodies against PCT

1 construction of recombinant plasmid

(1) Primer and method for producing the same

Amplification of Heavy Chain and Light Chain 5' RACE primers:

(2) antibody variable region gene cloning and sequencing

RNA is extracted from a hybridoma cell line secreting a monoclonal antibody against PCT, first strand cDNA synthesis is carried out by using an SMARTERTM RACE cDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and an obtained first strand cDNA product is used as a PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CHR primers. The primer pair of Light Chain can amplify a target band about 0.8KB, and the primer pair of Heavy Chain can amplify a target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH 5. alpha. competent cells, and after colonies were grown, the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen for sequencing.

(3) Sequence analysis of variable region Gene of anti-PCT monoclonal antibody

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 339bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 366bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(4) Construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the anti-PCT antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and the primers are as follows:

a0.75 KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

The coating solution diluted the recombinant PCT protein (produced by Fengcheng) to 1. mu.g/ml, 100. mu.l per well, and overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 30min-1 h; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results show that the OD of the reaction after the cell supernatant is diluted 1000 times is still larger than 1.0, and the OD of the reaction without the cell supernatant is smaller than 0.1, which indicates that the antibodies generated after the plasmid is transiently transformed are all active on the recombinant PCT protein.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme, and sterile water to 500 mul, water bath enzyme digestion overnight at 37 ℃; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml, mixing 100 μ l plasmid with 700 μ l cell in a centrifuge tube, transferring into an electric rotating cup, performing electric rotation, and counting the next day; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody (i.e., PCT monoclonal antibody) was subjected to reducing SDS-PAGE, and the electrophoretogram thereof was shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (i.e., heavy chain, SEQ ID NO:14) and 28kD of Mr (i.e., light chain, SEQ ID NO: 12).

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Further analysis revealed that the variable region of the heavy chain of the PCT monoclonal antibody (WT) of example 1 is shown in SEQ ID NO 13, wherein the amino acid sequences of the complementarity determining regions of the heavy chain are as follows:

CDR-VH1：G-Y-S(X1)-F-T-D-Y-F(X2)-M-Q(X3)；

CDR-VH2：G-L(X1)-N-P-GG(X2)-N-G-G-T(X3)-S-Y-N-Q-Q(X4)-F-K-G；

CDR-VH3：A(X1)-R-Y-P-I(X2)-Y-Y-G-P-V(X3)-Y-A-V；

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：R-S-S-N(X1)-S-I(X2)-V-H-S-Q(X3)-G-N-T-Y-V(X4)-H；

CDR-VL2：K(X1)-I(X2)-S-N(X3)-R-F-S；

CDR-VL3：F(X1)-Q-S-T(X2)-H-I(X3)-P-W。

based on the PCT monoclonal antibody of example 1, mutations were made in the complementarity determining regions at sites involved in antibody activity, wherein X1, X2, X3, and X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

And (3) detecting the binding activity:

the recombinant PCT protein (purchased from Fipeng, 141220) was diluted with coating solution (PBS) to 1. mu.g/ml for microplate coating, 100. mu.l per well, overnight at 4 ℃; the next day, washing with washing solution (PBS) for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding the diluted PCT monoclonal antibody in the table 1, 100 mu l/hole, 37 ℃, 30min-1 h; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding developing solution A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L peroxide)Urea), adding developing solution B (50 μ L/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10 min; stop solution (50. mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added₂SO₄) (ii) a OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2.

TABLE 2 Activity data of antibodies and mutants thereof

Antibody concentration (ng/ml)	37.037	12.345	4.1152	1.3717	0.4572	0
							WT	2.001	1.135	0.476	0.271	0.145	0.016
Mutation 1	2.299	1.254	0.547	0.323	0.158	0.042
							Mutation 2	2.107	1.261	0.565	0.334	0.146	0.035
Mutation 3	2.216	1.195	0.500	0.361	0.156	0.048
							Mutation 4	2.153	1.248	0.537	0.299	0.148	0.020
Mutation 5	2.051	1.035	0.436	0.228	0.135	0.010
							Mutation 6	0.334	0.143	-	-	-	-
Mutation 7	0.345	0.145	-	-	-	-
							Mutation 8	0.265	0.128	-	-	-	-
Mutation 9	0.227	0.113	-	-	-	-

As can be seen from the data in Table 2, WT, as well as mutations 1-5, had better binding activity, while mutations 6-9 had poorer binding activity, indicating that binding activity after mutation in the manner of mutation at the mutation sites in Table 1 was not predictable.

(2) Example 1 affinity assays for antibodies and mutants thereof

Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

And (3) affinity detection:

performing enzyme immunoassay in the same manner as activity identification, and performing four gradients of 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml and 0.0625. mu.g/ml; the antibody was diluted in a 2-fold gradient starting at 100ng/ml to 0.195ng/ml loading. And obtaining the OD values corresponding to different antibody concentrations under the conditions of no coating concentration. Under the same coating concentration, the antibody concentration is used as an abscissa and the OD value is used as an ordinate, logarithmic mapping is carried out, and the antibody concentration at 50% of the maximum OD value is calculated according to a fitting equation; substitution into the formula: k ═ n-1)/(2 × (n × Ab '-Ab)) the reciprocal of the affinity constant was calculated, where Ab and Ab' respectively represent the antibody concentration at 50% of maximum OD value at the corresponding coating concentration (Ag, Ag '), and n ═ Ag/Ag'; every two coating concentrations can be combined to calculate a K value, finally, the average value of all K values is obtained, the average value is obtained, and the reciprocal value is obtained to obtain the affinity constant K_D. The results are shown in Table 4 (K)_DIndicating the equilibrium dissociation constant, i.e. affinity).

Table 4 affinity assay data

As can be seen from the data in Table 4, K for mutation 1, mutation 1-1 to mutation 1-61_DAll the mutations are lower, which indicates that the affinity of the mutants is higher, and also indicates that the antibodies with better affinity can be obtained by carrying out mutation according to the mutation sites and mutation modes shown in the table 3.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 results of affinity assay of mutations with WT as backbone

As can be seen from the data in Table 6, K for WT 1, WT 1-1 to WT 1-9_DAll the mutations were low, indicating that the affinity of these mutants was good, and antibodies with good affinity were obtained by mutating the mutants according to the mutation sites and mutation patterns shown in Table 5.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., Ltd

<120> binding protein for PCT and method for detecting PCT

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Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys

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Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

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Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

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Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

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Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser

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Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

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Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Ile Val Lys Pro Gly Ala

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Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Gly

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Tyr Met Glu Leu Arg Ser Ile Thr Ser Glu Asp Ser Ala Thr Tyr Ser

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Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro

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Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser

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Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val

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Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe

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Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr

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Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala

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His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly

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Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys

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Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro

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Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr

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Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser

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Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His

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Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile

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Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn

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Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys

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Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu

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Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe

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Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu

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Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr

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Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr

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Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr

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Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys

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Claims

1. a binding protein for PCT, it is characterized in that, described binding protein comprises antigen-binding domain; Described antigen-binding domain comprises at least one in following complementarity determining region, or comprises and following at least one complementarity determining region The sequences have at least 80% sequence identity with similar complementarity determining regions:

Complementarity determining region CDR-VH1, its amino acid sequence is G-Y-X1-F-T-D-Y-X2-M-X3, wherein: X1 is S or T, X2 is Y, T or F, X3 is Q, H or N;

Complementarity determining region CDR-VH2, its amino acid sequence is G-X1-N-P-X2-N-G-G-X3-S-Y-N-Q-X4-F-K-G, wherein: X1 is I or L, X2 is GG or N, X3 is T or S, X4 is Q, K or N;

Complementarity determining region CDR-VH3, its amino acid sequence is X1-R-Y-P-X2-Y-Y-G-P-X3-Y-A-V, wherein: X1 is G or A, X2 is I or L, X3 is I, V or L;

Complementarity determining region CDR-VL1, its amino acid sequence is R-S-S-X1-S-X2-V-H-S-X3-G-N-T-Y-X4-H, wherein: X1 is N or Q, X2 is I or L, X3 is Q, K or N , X4 is I, V or L;

Complementarity determining region CDR-VL2, its amino acid sequence is X1-X2-S-X3-R-F-S, wherein: X1 is S, K or R, X2 is I, V or L, X3 is N or Q;

The complementarity determining region CDR-VL3 has an amino acid sequence of X1-Q-S-X2-H-X3-P-W, wherein: X1 is F or S, X2 is S or T, and X3 is I, V or L.

2. The binding protein of claim 1, wherein

In the complementarity determining region CDR-VH1, X1 is T;

In the complementarity determining region CDR-VH2, X3 is S;

In the complementarity determining region CDR-VH3, X1 is G;

In the complementarity determining region CDR-VL1, X1 is Q;

In the complementarity determining region CDR-VL2, X1 is S;

In the complementarity determining region CDR-VL3, X1 is S;

Preferably, in the complementarity determining region CDR-VH1, X2 is Y;

Preferably, in the complementarity determining region CDR-VH1, X2 is T;

Preferably, in the complementarity determining region CDR-VH1, X2 is F;

Preferably, in the complementarity determining region CDR-VH1, X3 is Q;

Preferably, in the complementarity determining region CDR-VH1, X3 is H;

Preferably, in the complementarity determining region CDR-VH1, X3 is N;

Preferably, in the complementarity determining region CDR-VH2, X1 is 1;

Preferably, in the complementarity determining region CDR-VH2, X1 is L;

Preferably, in the complementarity determining region CDR-VH2, X2 is GG;

Preferably, in the complementarity determining region CDR-VH2, X2 is N;

Preferably, in the complementarity determining region CDR-VH2, X4 is Q;

Preferably, in the complementarity determining region CDR-VH2, X4 is K;

Preferably, in the complementarity determining region CDR-VH2, X4 is N;

Preferably, in the complementarity determining region CDR-VH3, X2 is 1;

Preferably, in the complementarity determining region CDR-VH3, X2 is L;

Preferably, in the complementarity determining region CDR-VH3, X3 is 1;

Preferably, in the complementarity determining region CDR-VH3, X3 is V;

Preferably, in the complementarity determining region CDR-VH3, X3 is L;

Preferably, in the complementarity determining region CDR-VL1, X2 is 1;

Preferably, in the complementarity determining region CDR-VL1, X2 is L;

Preferably, in the complementarity determining region CDR-VL1, X3 is Q;

Preferably, in the complementarity determining region CDR-VL1, X3 is K;

Preferably, in the complementarity determining region CDR-VL1, X3 is N;

Preferably, in the complementarity determining region CDR-VL1, X4 is 1;

Preferably, in the complementarity determining region CDR-VL1, X4 is V;

Preferably, in the complementarity determining region CDR-VL1, X4 is L;

Preferably, in the complementarity determining region CDR-VL2, X2 is 1;

Preferably, in the complementarity determining region CDR-VL2, X2 is V;

Preferably, in the complementarity determining region CDR-VL2, X2 is L;

Preferably, in the complementarity determining region CDR-VL2, X3 is N;

Preferably, in the complementarity determining region CDR-VL2, X3 is Q;

Preferably, in the complementarity determining region CDR-VL3, X2 is S;

Preferably, in the complementarity determining region CDR-VL3, X2 is T;

Preferably, in the complementarity determining region CDR-VL3, X3 is 1;

Preferably, in the complementarity determining region CDR-VL3, X3 is V;

Preferably, in the complementarity determining region CDR-VL3, X3 is L;

Preferably, the antigen binding domain has an affinity with PCT protein of K _D ≤8.85×10 ^-9 mol/L;

Preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following mutation combinations 1-62:

.

3. The binding protein of claim 1, wherein

In the complementarity determining region CDR-VH1, X1 is S;

In the complementarity determining region CDR-VH2, X3 is T;

In the complementarity determining region CDR-VH3, X1 is A;

In the complementarity determining region CDR-VL1, X1 is N;

In the complementarity determining region CDR-VL2, X1 is K;

In the complementarity determining region CDR-VL3, X1 is F;

Preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following mutation combinations 63-72:

.

4. The binding protein of any one of claims 1-3, wherein the binding protein comprises at least 3 complementarity determining regions; or, the binding protein comprises at least 6 complementarity determining regions;

Preferably, the binding protein is an antibody or a functional fragment thereof;

Preferably, the binding protein is selected from any one of Nanobody, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody and antibody minimum recognition unit;

Preferably, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 whose sequences are shown in SEQ ID NOs: 1-4, and/or whose sequences are shown in SEQ ID Heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 shown in NO: 5-8;

Preferably, the binding protein further comprises an antibody constant region;

Preferably, the antibody constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

Preferably, the species source of the antibody constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , turkey, cockfight, or man;

Preferably, the antibody constant region is derived from mouse;

Preferably, the light chain constant region sequence of the antibody constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the antibody constant region is shown in SEQ ID NO:10.

5. the application of the binding protein of any one of claims 1-4 in the preparation of a reagent or a kit for diagnosing infectious inflammation;

Preferably, the infectious inflammation is selected from any one of severe systemic bacterial, fungal, parasitic or acute malaria infection, systemic inflammatory response syndrome, multiple organ failure syndrome and sepsis.

6. A reagent or kit for diagnosing infectious inflammation, characterized in that it contains the binding protein according to any one of claims 1-4;

7. A method for detecting PCT, characterized in that it comprises: mixing the binding protein according to any one of claims 1-4 with a sample to be tested;

Preferably, the method is a method for realizing PCT detection by a precipitation reaction or a method for realizing PCT detection by marking an indicator showing signal intensity;

Preferably, the method for realizing PCT detection by precipitation reaction is selected from any one or more of the following methods: one-way immunodiffusion test, two-way immunodiffusion test, immunoturbidimetric method, counter-immunoelectrophoresis, immunoelectrophoresis and immunoassay blotting;

Preferably, the method for realizing PCT detection by marking an indicator showing signal intensity is selected from any one or more of the following methods: immunofluorescence, radioimmunoassay, chemiluminescence immunoassay and enzyme-linked immunoassay Law;

Preferably, the indicated indicator is selected from any one of fluorescent dyes, radioisotopes, chemiluminescent liquids and enzymes that catalyze the coloration of substrates.

8. A vector, characterized in that it contains a nucleic acid encoding the binding protein according to any one of claims 1 to 4; preferably, the nucleic acid is DNA or RNA.

9 . A host cell comprising the vector of claim 8 . 10 .

10. A method for producing the binding protein according to any one of claims 1 to 4, characterized in that it comprises:

The host cell according to claim 9 is cultured, and the binding protein is obtained by separating and purifying from the culture medium or from the cultured host cell.