A mutant of a cea / afp double tumor marker fusion antigen

Abstract

The application provides a CEA / AFP double tumor marker fusion antigen mutant, and belongs to the technical field of bioengineering and tumor immunology. The fusion mutant comprises, from N terminus to C terminus, a signal peptide, a CEA antigen functional domain, a flexible connecting peptide and an AFP antigen functional domain. The CEA domain comprises any one or more than one of S40L, N76L, N104L and Q137L site-directed mutations, and the AFP domain comprises any one or more than one of N494I, K549Q and G596V site-directed mutations. The mutant is significantly superior to the wild-type fusion protein in terms of thermal stability, antibody binding affinity and long-term storage stability. The fusion antigen mutant can be used as a standard, quality control or calibration for combined detection of CEA and AFP, and is applied to tumor immune diagnosis, specific antibody preparation and immune mechanism research.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the technical fields of bioengineering and tumor immunology, and specifically relates to a CEA / AFP dual tumor marker fusion antigen mutant. BACKGROUND ART

[0002] Liver cancer and gastrointestinal tumors are highly prevalent malignant tumors worldwide, wherein alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are the core dual marker combination for clinical screening, diagnosis and prognosis evaluation. AFP is a specific tumor marker for primary liver cancer, and CEA is widely used in the detection of gastrointestinal tumors such as colorectal cancer and gastric cancer. The combined detection of the two can significantly improve the sensitivity and specificity of tumor screening, effectively reduce the missed diagnosis and misdiagnosis rates of single marker detection, and has been listed as the preferred combination scheme for combined tumor screening in clinical diagnosis and treatment guidelines at home and abroad.

[0003] At the current stage, the supporting antigens for in vitro diagnostic kits are mainly divided into two categories: physical reconstitution of natural antigens and simple fusion of full-length sequences, and both types of products have obvious technical shortcomings: (1) Natural CEA and AFP proteins have different folding characteristics and physicochemical stability. When they are separately prepared and then mixed in equal amounts for use, the activity attenuation of the two antigens is not synchronized, resulting in large batch-to-batch differences of finished products, which is not conducive to the standardized mass production of diagnostic reagents.

[0004] (2) The natural full-length protein contains a large number of disordered flexible fragments and redundant transmembrane anchoring sequences, so the protein is prone to unfolding, agglomeration and sedimentation, with poor overall thermal stability. Conformational unfolding and protein degradation easily occur during in vitro storage, making it difficult to meet the stability requirements of diagnostic reagents, antibody preparation and industrialized batch applications.

[0005] (3) Most of the existing CEA / AFP fusion proteins are directly spliced with full-length sequences, without referring to mature industrialized truncation schemes for region simplification. The redundant sequences cause folding disorder of the fusion protein and spatial shielding of antigen epitopes, which ultimately leads to poor expression solubility and low binding affinity between the antigen and the antibody, failing to meet the high-sensitivity detection requirements for trace samples.

[0006] Antigen tandem fusion technology is a core technical approach to address the issues of uneven compatibility and large batch-to-batch variability in multi-marker detection. Existing research and patents have confirmed that by truncating core epitopes, tandemly connecting peptides for adaptability, and site-directed conformational mutations, integrated expression of multiple antigen single chains can be achieved, balancing the immunogenicity of each antigen with the overall stability of the protein. Based on existing tandem antigen design systems, this invention, through structural biology analysis, bioinformatics screening, and multiple rounds of functional validation, has for the first time obtained a set of site-directed combined mutations that synergistically enhance protein thermal stability and antibody binding affinity. This mutation system effectively constrains the flexible conformation of the dual-domain structure, optimizes the hydrophobic interface and electrostatic microenvironment of the antigen epitope, and ultimately obtains a dual-tumor marker fusion mutant antigen with high conformational stability, high detection sensitivity, and strong anti-degradation ability, thus solving a long-standing technical shortcoming of existing technologies. Summary of the Invention

[0007] To address the technical bottlenecks of existing CEA and AFP tumor antigens, such as poor compatibility, insufficient structural stability, low antibody binding affinity, and easy aggregation and inactivation of fusion proteins, this invention selects a mature antigen truncated fragment validated by industrialization and combines it with a single highly flexible short peptide (GGS)5 as a spacer element to achieve dual antigen tandem. Then, through dual-segment multi-site synergistic site-directed mutagenesis, while fully preserving the specificity of the core antigen epitopes of CEA and AFP, the conformational stability, storage tolerance, antibody binding activity, and expression uniformity of the fusion protein are simultaneously improved. This provides a CEA / AFP dual-target fusion antigen mutant with a reasonable structure, balanced activity, high efficiency of eukaryotic expression, and scalable mass production, as well as its preparation method.

[0008] To achieve the above-mentioned objectives, this invention provides a CEA / AFP dual-target fusion antigen mutant protein, wherein the overall arrangement of the fusion protein is: Igκ secretion signal peptide CEA(35-550aa) (GGS)5 AFP(403-609aa 6×His.

[0009] This invention precisely truncates the natural full-length proteins CEA and AFP, removing signal peptides, transmembrane domains, disordered redundant fragments, and unstable structural regions, retaining only the core functional epitopes that can be specifically recognized by commercially available detection antibodies. It also incorporates adaptable flexible linker peptides to ensure that the two antigen structures fold independently and the epitopes are fully exposed. By fixing the protein conformation through site-directed mutagenesis, the overall performance is comprehensively optimized.

[0010] The present invention achieves the above-mentioned objectives by adopting the following technical solution: In a first aspect, the present invention provides a CEA / AFP dual tumor marker fusion antigen mutant protein, wherein the mutant protein comprises, from the N-terminus to the C-terminus, a signal peptide, a CEA antigen functional domain, a flexible linker peptide, and an AFP antigen functional domain. The CEA antigen functional domain is the amino acid sequence from position 35 to position 550 of the human CEACAM5 protein (Uniprot: P06731), and contains any one or more site-directed amino acid mutations from S40L, N76L, N104L, and Q137L. The AFP antigen functional domain is the amino acid sequence from position 403 to position 609 of the human AFP protein (Uniprot: P02771), and contains any one or more site-directed amino acid mutations of N494I, K549Q, and G596V.

[0011] Furthermore, the mutant protein also includes a purification tag; The mutant protein comprises, from N-terminus to C-terminus, a signal peptide for secretory expression in mammalian cells, a CEA antigen functional domain, a flexible linker peptide, an AFP antigen functional domain, and a purification tag. Optionally, the purification tag is selected from 6×His purification tag, Flag purification tag, Strep-tag II purification tag, GST purification tag, MBP purification tag, HAT purification tag, or SUMO purification tag.

[0012] Furthermore, the signal peptide is a signal peptide that is secreted and expressed by mammalian cells; Optionally, the signal peptide is selected from Igκ signal peptide, human IgG heavy chain signal peptide, human serum albumin signal peptide, human growth hormone signal peptide, mouse Igκ light chain signal peptide, human CD33 signal peptide, human IL-2 signal peptide, human tPA signal peptide, bee venom peptide signal peptide or glucosidase signal peptide. Optionally, the flexible linker peptide is selected from highly flexible linker peptides (GGS)5, (GGS)5, etc. n (G4S) n (G4S)4, (G5S) n , GSGGSG, GGSGGSG, GSG4SG4S, G4SG4SG4SG4S, G5SG5S, GSGGSGGSG, GGSGGSGGSGGSG, GSGGSGGSGGSGGSG, G4SG4SG4S, GSG4SG4SG4S or G5SG5SG5S, where n is any integer from 1 to 10.

[0013] Furthermore, the mutant protein comprises, from the N-terminus to the C-terminus, the following components in sequence: an Igκ signal peptide for secretory expression in mammalian cells, a CEA antigen functional domain, a flexible linker peptide, an AFP antigen functional domain, and a 6×His purification tag. Optionally, the amino acid sequence of the Igκ signal peptide is shown in SEQ ID NO:1; Optionally, the mutant protein is a double mutant protein, wherein the site-directed amino acid mutation combination is selected from CEA-N76L + AFP-G596V, CEA-N76L + CEA-N104L or AFP-N494I + AFP-G596V. Optionally, the mutant protein is a triple mutant protein, which contains the site-directed amino acid mutation combination of CEA-N76L + CEA-N104L + AFP-G596V; Optionally, the mutant protein is a tetramutant protein, which contains the site-directed amino acid mutation combination of CEA-N76L + CEA-N104L + AFP-N494I + AFP-G596V.

[0014] This invention successfully constructed five combined mutants by combining and superimposing positive sites obtained from single-point mutation screening. Performance evaluation results confirmed that the selected mutation sites exhibited a good synergistic effect, and the combined mutants showed significantly improved thermal stability, affinity, and long-term storage stability compared to single-point mutants and wild-type mutants. Among them, the four-mutant CEA-N76L / N104L-AFP-N494I / G596V demonstrated the best overall performance, fully meeting the stringent requirements of clinical in vitro diagnostic reagents for the thermal stability, affinity, and storage stability of raw material proteins. It can significantly improve the detection sensitivity, accuracy, and shelf life of diagnostic kits, and has extremely high clinical application value.

[0015] In some embodiments, the mutant protein contains a single-site-directed amino acid mutation in the functional domain of the CEA antigen, wherein the single-site-directed amino acid mutation is selected from S40L, N76L, N104L, or Q137L; preferably, the single-site-directed amino acid mutation is N76L, the heat denaturation temperature Tm of the mutant protein is 61.8±0.3℃, which is 3.6℃ higher than that of the wild-type fusion protein without the mutation, the residual activity after being treated at 65℃ for 30 minutes is 28.7±1.5%, and the binding affinity Kd value with the anti-CEA antibody is 2.05±0.09 nM.

[0016] In some embodiments, the mutant protein contains a single-site-directed amino acid mutation in the functional domain of the AFP antigen, wherein the single-site-directed amino acid mutation is selected from N494I, K549Q, or G596V; preferably, the single-site-directed amino acid mutation is G596V, the mutant protein has a thermal denaturation temperature Tm of 61.2±0.2℃, which is 3.0℃ higher than that of the wild-type fusion protein without the mutation, a residual activity of 26.4±1.4% after isothermal treatment at 65℃ for 30 minutes, and a binding affinity Kd of 1.62±0.07 nM to the anti-AFP antibody.

[0017] In some embodiments, the mutant protein is a double mutant protein or a triple mutant protein; the site-directed amino acid mutation combination of the double mutant protein is selected from CEA-N76L+AFP-G596V, CEA-N76L+CEA-N104L, or AFP-N494I+AFP-G596V; the site-directed amino acid mutation combination of the triple mutant protein is CEA-N76L+CEA-N104L+AFP-G596V; preferably, the double mutant protein is CEA-N76L+AFP-G596V, and its thermal denaturation temperature Tm value is 6. The heat denaturation temperature (Tm) of the fusion protein is 4.5±0.2℃, which is 6.3℃ higher than that of the wild-type fusion protein. After being treated at 65℃ for 30 minutes, the residual activity is 45.2±1.8%, and after being stored at 4℃ for 3 months, the residual binding activity is 72.8±1.2%. Preferably, the triple mutant protein is CEA-N76L+CEA-N104L+AFP-G596V, with a heat denaturation temperature (Tm) of 66.2±0.2℃, which is 8.0℃ higher than that of the wild-type fusion protein. After being treated at 65℃ for 30 minutes, the residual activity is 52.4±1.7%, and after being stored at 4℃ for 3 months, the residual binding activity is 79.5±1.0%.

[0018] In some embodiments, the mutant protein is a tetramutant protein, comprising the site-directed amino acid mutation combination CEA-N76L+CEA-N104L+AFP-N494I+AFP-G596V; the tetramutant protein has a heat denaturation temperature (Tm) of 67.8±0.3℃, which is 9.6℃ higher than that of the wild-type fusion protein; after isothermal treatment at 65℃ for 30 minutes, the residual activity is 59.6±1.5%; after isothermal treatment at 70℃ for 30 minutes, the residual activity is 25.1±1.0%; the binding affinity (Kd) with anti-CEA antibody is 1.47±0.06 nM; and the binding affinity (Kd) with anti-AFP antibody is 1.12±0.05 nM. The residual binding activity after storage at 4°C for 3 months was 85.2±0.8% (nM). Preferably, the tetramutant protein contains, from the N-terminus to the C-terminus, an Igκ secretion signal peptide, a CEA antigen functional domain, a (GGS)5 flexible linker peptide, an AFP antigen functional domain, and a 6×His purification tag. The amino acid sequence of the Igκ secretion signal peptide is shown in SEQ ID NO:1.

[0019] In some implementations, the 6×His purification tag is immobilized at the C-terminus of the fusion protein, without interfering with the exposure of CEA and AFP antigenic domain epitopes and the binding activity of specific antibodies.

[0020] In a second aspect, the present invention provides a recombinant gene encoding the mutant protein described in the first aspect of the present invention.

[0021] In some embodiments, the recombinant gene refers to a nucleotide sequence encoding the mutant protein described in the first aspect of the present invention, wherein the nucleotide sequence is codon-optimized according to the selected expression system, and the recombinant gene comprises, from the 5′ end to the 3′ end, the following nucleotide sequences in sequence: a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a CEA antigen functional domain, a nucleotide sequence encoding a flexible linker peptide, a nucleotide sequence encoding an AFP antigen functional domain, and optionally a nucleotide sequence encoding a purification tag; the coding sequences of each functional fragment can be directly linked or linked through restriction enzyme cleavage site sequences.

[0022] In some implementations, the recombinant gene undergoes mammalian cell codon optimization to adapt it for efficient and soluble expression in mammalian cell expression systems. The codon optimization includes synonymous codon substitution of the nucleotide sequence encoding the mutant protein according to the preferences of HEK293 cells, CHO cells, or mammalian cells, and removal of the internal TATA box, polyA signal sequence, splice donor / acceptor site, and ribosome entry site, in order to improve the transcription efficiency, mRNA stability, and translation efficiency of the recombinant gene in mammalian cells, thereby achieving efficient and soluble expression of the fusion protein.

[0023] In some embodiments, the nucleotide sequence of the recombinant gene is selected from any of the following: (1) a nucleotide sequence encoding the Igκ signal peptide shown in SEQ ID NO:1, the CEA antigen functional domain shown in SEQ ID NO:2 (containing any one or more mutations of S40L, N76L, N104L, Q137L), and the AFP antigen functional domain shown in SEQ ID NO:3 (containing any one or more mutations of N494I, K549Q, G596V); (2) a variant nucleotide sequence having at least 95% sequence identity with the above nucleotide sequence and encoding the same mutant protein; or (3) a nucleotide sequence that hybridizes to the full-length complementary sequence of the above nucleotide sequence under strict hybridization conditions and encodes the same mutant protein.

[0024] In some embodiments, the 5′ and / or 3′ ends of the recombinant gene further include restriction enzyme sites for gene cloning and vector construction, wherein the restriction enzyme sites are selected from any one or more of HindIII, XhoI, BamHI, EcoRI, NheI, or NotI recognition sequences; optionally, the 5′ end of the recombinant gene further includes a Kozak concordance sequence (such as GCCACC) to enhance translation initiation efficiency in eukaryotic cells; optionally, the recombinant gene further includes a promoter sequence preceding the signal peptide coding sequence and / or a transcription termination sequence following the coding sequence.

[0025] Thirdly, the present invention provides a recombinant expression vector comprising the recombinant gene described in the second aspect of the present invention.

[0026] In some embodiments, the recombinant expression vector is a eukaryotic expression vector selected from any one of pcDNA3.1, pcDNA3.4, pTT5, pCEP4, pCI-neo, pEAK8, or pBiFC-VN173; preferably, the expression vector is pcDNA3.1 or pcDNA3.4, which contains a CMV promoter, a multiple cloning site, a polyadenylation signal, and a selection marker gene, suitable for efficient transient transfection and stable expression in HEK293 cells or CHO cells.

[0027] In some embodiments, the recombinant expression vector further comprises an expression regulatory element operatively linked to the expression regulatory element, the expression regulatory element being selected from any one or more of the CMV promoter, EF-1α promoter, SV40 promoter, chicken β-actin promoter, RSV promoter, or enhancer sequences of the above promoters; the expression vector further comprises any one of the selection markers selected from neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene, or dihydrofolate reductase gene, for screening positive transfected cells and establishing stable cell lines.

[0028] In some embodiments, the recombinant expression vector is a viral vector, which is selected from any one of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or retroviral vectors; the viral vector carries the recombinant gene described in the second aspect of the present invention, and is used to efficiently transduce the recombinant gene into host cells to achieve transient or stable expression of the mutant protein.

[0029] In some embodiments, the recombinant expression vector further comprises a signal peptide coding sequence located at the 5′ end of the recombinant gene coding sequence and operatively linked to the 5′ end of the CEA antigen functional domain coding sequence. The signal peptide is selected from any one of Igκ signal peptide, human IgG heavy chain signal peptide, human serum albumin signal peptide, human growth hormone signal peptide, mouse Igκ light chain signal peptide, human CD33 signal peptide, human IL-2 signal peptide, human tPA signal peptide, bee venom peptide signal peptide, or glucosidase signal peptide. Optionally, a protease recognition site coding sequence is included between the 3′ end of the signal peptide coding sequence and the 5′ end of the CEA antigen functional domain coding sequence for precise cleavage of the signal peptide by a signal peptidase during protein maturation.

[0030] In some embodiments, the recombinant expression vector further comprises a fusion tag coding sequence located at the 3′ end of the recombinant gene coding sequence and operatively linked to the 3′ end of the AFP antigen functional domain coding sequence. The fusion tag is selected from any one of the following: a 6×His purification tag, a Flag purification tag, a Strep-tag II purification tag, a GST purification tag, an MBP purification tag, a HAT purification tag, or a SUMO purification tag coding sequence. Optionally, a linker peptide coding sequence is further included between the fusion tag coding sequence and the AFP antigen functional domain coding sequence to ensure that the fusion tag does not interfere with the antigen epitope exposure and antibody binding activity of the mutant protein.

[0031] In some embodiments, the recombinant expression vector further comprises a signal peptide coding sequence and a fusion tag coding sequence, wherein the recombinant expression vector comprises, from the 5′ end to the 3′ end, a promoter, a signal peptide coding sequence, a CEA antigen functional domain coding sequence, a flexible linker peptide coding sequence, an AFP antigen functional domain coding sequence, a fusion tag coding sequence, and a transcription termination signal; preferably, the signal peptide is an Igκ signal peptide, and the fusion tag is a 6×His purification tag.

[0032] Fourthly, the present invention provides a recombinant host cell comprising the recombinant expression vector described in the third aspect of the present invention.

[0033] In some embodiments, the host cell is a mammalian cell selected from any one of HEK293 cells, HEK293F cells, HEK293T cells, CHO cells (including CHO-K1, CHO-S, and CHO-DG44), COS-7 cells, Vero cells, BHK cells, Per.C6 cells, CAP cells, or NSO cells; preferably, the host cell is a HEK293F suspension cell or a CHO-S suspension cell, suitable for large-scale serum-free suspension culture to achieve efficient soluble secretory expression of the fusion antigen mutant protein.

[0034] In some embodiments, the host cell is a transiently transfected host cell, and the recombinant expression vector is introduced into the host cell by any one of the following methods: calcium phosphate coprecipitation, liposome transfection, cationic polymer transfection (such as PEI), electroporation, or microinjection. The transiently transfected host cell expresses the mutant protein within 24-72 hours after transfection, which is suitable for rapid small-scale preparation of the mutant protein and functional screening of candidate mutants.

[0035] In some implementations, the host cell is a stably transfected host cell, and the recombinant expression vector is integrated into the host cell genome through homologous recombination, lentiviral infection, or a transposon system. A monoclonal cell line stably expressing the mutant protein is obtained by screening with a selection marker. The stably transfected host cell enables long-term, continuous, and large-scale production of the mutant protein, which is suitable for industrial-scale batch preparation of antigen raw materials for diagnostic reagents.

[0036] In some embodiments, the host cell is a eukaryotic expression system host cell, and the recombinant expression vector contains an inducible promoter operatively linked to the recombinant gene, wherein the inducible promoter is selected from any one of a tetracycline inducible promoter, a metallothionein promoter, or a heat shock protein promoter; the expression timing and expression level of the mutant protein are regulated by adding appropriate inducers to optimize protein folding efficiency, reduce aggregate formation, and increase soluble protein yield.

[0037] Fifthly, the present invention provides a method for preparing the mutant protein described in the first aspect of the present invention, the method comprising the following steps: culturing the recombinant host cell described in the fourth aspect of the present invention, inducing it to express the mutant protein, collecting the culture and purifying the mutant protein therefrom.

[0038] In some embodiments, the preparation method includes the following steps: (1) The codon-optimized recombinant fusion gene was cloned into a eukaryotic expression vector to construct a recombinant expression plasmid; (2) Transfect or transform the recombinant expression plasmid into host cells and screen to obtain positive expression strains / cell lines; (3) Induce positive host cells to express fusion protein efficiently and collect cell culture supernatant; (4) The supernatant was purified step by step by nickel affinity chromatography, ion exchange chromatography and gel filtration chromatography, and then dialyzed and sterilized to obtain high-purity dual-target fusion antigen mutant protein.

[0039] Sixthly, the present invention provides any of the following products: (1) An isolated site-directed amino acid mutation site, wherein the mutation site is selected from any one or more of S40L, N76L, N104L and / or Q137L of human CEACAM5 protein (Uniprot: P06731) and N494I, K549Q and / or G596V of human AFP protein (Uniprot: P02771); (2) A protein lyophilized formulation, wherein the protein lyophilized formulation comprises the mutant protein and lyophilization protectant described in the first aspect of the present invention; (3) A protein conjugate comprising the mutant protein described in the first aspect of the present invention and a tracer conjugate thereto; (4) A detection kit comprising the mutant protein described in the first aspect of the present invention; (5) A combined CEA and AFP detection kit, wherein the combined detection kit contains the mutant protein described in the first aspect of the present invention as a standard, quality control or calibrator.

[0040] In some embodiments, the lyophilization protectant in the protein lyophilized formulation is selected from any one or more of sucrose, trehalose, glucose, mannitol, sorbitol, glycine, arginine, monosodium glutamate, polyvinylpyrrolidone, polyethylene glycol, or dextran; preferably, the lyophilization protectant is trehalose and / or a combination of sucrose and mannitol. After reconstitution, the protein conformation and immunomodulatory activity remain stable, making it suitable for long-term storage and transportation of diagnostic kits.

[0041] In some embodiments, the tracer label is selected from any one of enzyme labels, fluorescent labels, chemiluminescent labels, electrochemiluminescent labels, radioisotope labels, biotin labels, colloidal gold labels, fluorescent microsphere labels, magnetic bead labels, or quantum dot labels; preferably, the tracer label is horseradish peroxidase or alkaline phosphatase, and the protein conjugate is prepared by any one of the following methods: glutaraldehyde crosslinking method, periodate oxidation method, succinimide ester conjugation method, or click chemistry method. The protein conjugate retains the binding activity of the mutant protein with anti-CEA antibody and / or anti-AFP antibody, while generating a detectable signal, suitable for signal amplification and quantitative analysis in immunoassay.

[0042] In some embodiments, the detection kit is a CEA detection kit, an AFP detection kit, or a CEA / AFP combined detection kit; the detection kit further includes any one or more components selected from the group consisting of: a solid-phase carrier coated with anti-CEA antibody and / or anti-AFP antibody, an enzyme-labeled antibody, a chemiluminescent substrate, a chromogenic solution, a stop solution, a washing solution, a diluent, and a positive control; the mutant protein serves as a calibrator, control, or standard of the detection kit to establish a standard curve for quantitative detection of CEA and / or AFP, or to evaluate the intra- and inter-batch detection precision and accuracy of the kit.

[0043] In some implementations, the combined detection kit is a dual-marker combined detection kit, wherein the kit is simultaneously coated with anti-CEA antibody and anti-AFP antibody on the same solid-phase carrier, or is coated with anti-CEA antibody and anti-AFP antibody separately in a dual-well parallel detection manner; the detection platform of the combined detection kit is selected from any one of the following: chemiluminescence immunoassay platform, enzyme-linked immunosorbent assay platform, electrochemiluminescence immunoassay platform, time-resolved fluorescence immunoassay platform, immunochromatography platform, or immunoturbidimetric platform; the mutant protein serves as a universal calibrator for the combined detection kit, which can simultaneously calibrate both CEA and AFP detection channels, eliminating systematic errors caused by individual calibration of the dual markers, and improving the accuracy and consistency of the combined detection results of the dual markers.

[0044] In some embodiments, the combined detection kit uses the mutant protein described in the first aspect of the present invention as a positive control or quality control, and the mutant protein is provided in the kit in liquid or lyophilized form; preferably, the mutant protein is provided in lyophilized form, and the kit is suitable for quantitative detection of CEA and AFP in clinical serum and plasma samples, as well as for early tumor screening, auxiliary diagnosis, efficacy monitoring, and recurrence early warning.

[0045] In some embodiments, the test kit is an immunochromatographic test strip, which includes a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a backing plate; the nitrocellulose membrane has a test line and a control line, the test line is coated with anti-CEA antibody and / or anti-AFP antibody, and the conjugate pad is loaded with a marker-labeled detection antibody; the mutant protein is provided separately or pre-coated on the control line as a quality control or calibrator for the test strip.

[0046] In a seventh aspect, the present invention provides any of the following applications: (1) The application of the mutant protein described in the first aspect of the present invention, the recombinant gene described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention in the preparation of a combined detection product for CEA and AFP; (2) The use of the mutant protein described in the first aspect of the present invention in the preparation of CEA-specific antibodies or AFP-specific antibodies; (3) The application of the mutant protein described in the first aspect of the present invention in the study of tumor immune mechanisms; (4) The use of the mutant protein described in the first aspect of the present invention in the preparation of tumor screening, diagnosis, prognostic assessment or recurrence monitoring products; (5) The use of the mutant protein described in the first aspect of the present invention in the preparation of diagnostic or auxiliary diagnostic products for liver cancer, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer or breast cancer; (6) The use of the mutant protein described in the first aspect of the present invention in the preparation of positive control, negative control, calibration, quality control, calibrator or reference for in vitro diagnostic kits; (7) The use of the mutant protein described in the first aspect of the present invention in the preparation of chemiluminescent immunoassay kits, enzyme-linked immunosorbent assay kits, immunochromatographic test strips, electrochemiluminescent immunoassay kits, time-resolved fluorescence immunoassay kits or immunoturbidimetric assay kits; (8) The application of the site-directed amino acid mutation sites described in the sixth aspect of the present invention in the preparation of CEA / AFP dual tumor marker fusion antigen mutant protein.

[0047] In some implementations, the CEA and AFP combined detection product is used for the auxiliary diagnosis, efficacy monitoring, or recurrence early warning of liver cancer, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, or breast cancer. The combined detection product uses the mutant protein described in the first aspect of the present invention as a universal antigen raw material, and simultaneously coats or labels it with anti-CEA antibody and anti-AFP antibody to achieve simultaneous quantitative detection of CEA and AFP dual biomarkers in a single sample, effectively improving the sensitivity and specificity of tumor screening and reducing the missed diagnosis rate and misdiagnosis rate of single biomarker detection.

[0048] In some embodiments, the CEA-specific antibody or AFP-specific antibody is a polyclonal antibody, a monoclonal antibody, or a genetically engineered antibody. The genetically engineered antibody is selected from any one of chimeric antibodies, humanized antibodies, fully human antibodies, nanobodies, or single-chain antibodies. The antibody is prepared by immunizing animals with the mutant protein described in the first aspect of the present invention as an immunogen or by any one of phage display, yeast display, or B-cell single-cell sorting technology. The antibody is used for the immunodetection, targeted therapy, or immunohistochemical analysis of CEA or AFP.

[0049] In some embodiments, the study of tumor immune mechanisms includes functional studies of CEA and / or AFP in tumor occurrence, development, invasion, metastasis, immune escape, and regulation of the tumor microenvironment; the study uses the mutant protein described in the first aspect of the present invention as a molecular probe or immunogen to detect the level of anti-CEA autoantibodies and / or anti-AFP autoantibodies in the sample, or to screen antagonists that can block the binding of CEA and / or AFP to their natural ligands or receptors, or to study the cross-presentation and immune recognition mechanism of CEA and AFP double-positive tumor cell surface antigens.

[0050] In some implementations, the tumor screening, diagnosis, prognostic assessment, or recurrence monitoring product is a protein chip or microarray chip product. The product immobilizes the mutant protein described in the first aspect of this invention on the surface of a solid-phase carrier for high-throughput detection of CEA, AFP, and their autoantibody levels in samples. The product co-coats the mutant protein with any one or more tumor marker proteins selected from AFP, CEA, CA19-9, CA125, CA15-3, PSA, HE4, or SCC on the chip surface to achieve joint detection of multiple tumor markers. The mutant protein serves as a universal quality control antigen for the detection system, used to standardize the signal intensity of different tumor marker detection channels, eliminate batch-to-batch differences, and improve the reliability and comparability of multi-indicator joint detection results.

[0051] In some implementations, the diagnostic or auxiliary diagnostic products for liver cancer, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, or breast cancer use the mutant protein described in the first aspect of the present invention as a standard or quality control product. The products diagnose diseases by detecting the concentration levels of CEA and AFP in the subject's biological samples and combining the combined judgment threshold of CEA and AFP. The combined judgment threshold is determined using the ROC curve analysis method.

[0052] In some embodiments, the positive control, negative control, calibration, quality control, calibrator, or reference of the in vitro diagnostic kit uses the mutant protein described in the first aspect of the present invention as a core ingredient. The product contains a solution or lyophilized form of the mutant protein at a predetermined concentration. The concentration of the mutant protein is traceable to international or national standards. The product is used for kit factory inspection, inter-batch quality control, internal quality control, inter-laboratory quality assessment, or routine calibration in clinical laboratories. Preferably, the calibration or calibrator contains at least five different concentration gradients of the mutant protein, covering the linear range of CEA and AFP detection, for establishing a standard curve for quantitative detection. Preferably, the quality control contains three concentration levels of the mutant protein (low, medium, and high) for evaluating the intra-batch and inter-batch precision of the kit.

[0053] In some embodiments, the chemiluminescent immunoassay kit, enzyme-linked immunosorbent assay kit, immunochromatographic test strip, electrochemiluminescent immunoassay kit, time-resolved fluorescence immunoassay kit, or immunoturbidimetric assay kit uses the mutant protein described in the first aspect of the present invention as a core raw material, and the mutant protein is used in the kit as a coating antigen, labeling antigen, competitive antigen, or calibrator; the test sample of the kit is selected from any one of human serum, plasma, whole blood, tissue fluid, or urine.

[0054] In some embodiments, the application of the site-directed amino acid mutation site described in the sixth aspect in the preparation of CEA / AFP dual tumor marker fusion antigen mutant protein includes introducing the mutation site into the wild-type CEA antigen functional domain and / or wild-type AFP antigen functional domain without the mutation, to improve any one or more properties of the resulting fusion antigen mutant protein, such as thermostability, antibody binding affinity, long-term storage stability, conformational stability, folding efficiency, protease degradation tolerance, or expression yield; the mutation site is introduced into the fusion protein by any one of the following methods: chemical synthesis, site-directed mutagenesis PCR, homologous recombination, oligonucleotide-mediated gene synthesis, or gene editing technology.

[0055] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention, for the first time, through structural biology analysis, bioinformatics screening, and multiple rounds of functional validation, obtained a set of site-directed mutations that synergistically enhance protein performance in the functional domains of CEA antigen (S40L, N76L, N104L, Q137L) and AFP antigen (N494I, K549Q, G596V). The synergistic effect of the seven mutation sites is revealed for the first time in this invention, with no antagonistic effect among the mutation sites and significant cross-domain synergistic gain. Furthermore, this invention employs an industrially validated non-full-length precise truncation scheme (CEA 35-550aa, AFP...). (403-609aa) Removes signal peptides, transmembrane domains, disordered redundant fragments, and unstable structural regions from the natural protein, retaining only the core functional epitopes. This effectively avoids the folding disorder of fusion proteins and spatial masking of antigen epitopes caused by full-length sequence splicing. Combined with a single highly flexible short peptide (GGS)5 as a spacer element to buffer the spatial conflict of the globular domains at both ends, it ensures that the CEA and AFP antigen activities are independently and synchronously preserved. Based on this, the mutant protein of this invention can be efficiently and solublely expressed through a mammalian cell secretory expression system, with a purity ≥95% after purification. No inclusion body refolding treatment is required, and the preparation process is simple and cost-controllable. Performance evaluation results show that the optimal four mutants (CEA-N76L + CEA-N104L + AFP-N494I + ... The AFP-G596V fusion protein exhibits a heat denaturation temperature (Tm) of 67.8℃, which is 9.6℃ higher than that of the wild-type fusion protein. Its residual activity at 65℃ is increased by 47.1 percentage points. The binding affinity (Kd) with anti-CEA and anti-AFP antibodies is 1.47 nM and 1.12 nM, respectively, representing increases of 37.4% and 40.1% compared to the wild-type. Even after storage at 4℃ for 3 months, the residual binding activity remains above 85%. Its overall performance is significantly superior to existing natural antigen physical compound products and conventional fusion protein products. It can serve as a universal calibrator, quality control product, or standard for the combined detection of CEA and AFP dual biomarkers. It is suitable for various detection platforms, including chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), immunochromatography, and electrochemiluminescence immunoassay, significantly improving the detection sensitivity, accuracy, and shelf life of diagnostic kits. It possesses extremely high clinical application value and industrialization prospects. Detailed Implementation

[0056] The overall protein arrangement of the CEA / AFP dual-target fusion antigen mutant provided by this invention is as follows: Igκ secretion signal peptide CEA(35-550aa) (GGS)5 AFP(403-609aa The design basis and stability and affinity optimization mutation design for each functional segment are as follows: 1. Design basis for each functional segment (1) Truncated CEA functional fragment: Based on the human CEA reference sequence Uniport: P06731, the core region of 35-550aa is extracted, covering the recognition epitopes of all currently commercial CEA detection antibodies. The redundant N-terminal sequence and the unstable flexible C-terminal segment are removed, taking into account both protein soluble expression and epitope integrity, and adapting to various immunological detection platforms.

[0057] (2) Truncated AFP functional fragment: Based on the human AFP reference sequence Uniport: P02771, the core antigen region of 403-609aa was truncated. This region is the core recognition region of AFP-specific antibodies. It enriches dominant antigenic epitopes, removes non-functional disordered structures at the N-terminus, and greatly improves the overall folding efficiency of the fusion protein.

[0058] (3) Intermediate flexible linker peptide: The (GGS)5 highly flexible linker peptide is used. It has no fixed secondary structure and can effectively buffer the spatial conflict between the front CEA globular domain and the back AFP domain, avoid the two antigen structures from entangled and the epitope is blocked, and ensure that the CEA and AFP antigen activities are independently and synchronously preserved.

[0059] (4) N-terminal Igκ signal peptide: The mammalian highly active secretory signal peptide can mediate the efficient extracellular secretory expression of the fusion protein, achieve correct protein cleavage and mature folding, avoid intracellular retention and degradation, and be compatible with mainstream eukaryotic expression systems such as HEK293 and CHO to improve protein expression level and solubility.

[0060] (5) C-terminal 6×His purification tag: Located at the end of the fusion protein, it does not interfere with the epitope exposure and antibody binding activity of any antigen domain. It can achieve efficient, high-purity, and specific purification of the protein through nickel affinity chromatography, simplifying the industrial preparation process.

[0061] 2. Stability and affinity optimization mutation design To overcome the technical shortcomings of natural CEA, AFP, and conventional fusion proteins, such as high conformational flexibility, poor thermal stability, low antibody binding affinity, and easy degradation, this invention introduces a combination of specific site-directed mutations not disclosed in existing technologies into two core functional regions. Through a multi-dimensional synergistic mechanism involving solidifying the flexible backbone, enhancing intramolecular hydrophobic stacking, optimizing the epitope electrostatic microenvironment, and filling internal hydrophobic cavities, protein performance is optimized. All mutation sites strictly avoid core antigen anchoring residues, endogenous disulfide bond sites, and key epitope residues for direct antibody binding, achieving a simultaneous and significant improvement in thermal stability and antibody binding affinity without compromising antigen specificity and immune activity.

[0062] (1) Site-directed mutations in the CEA functional region: N76L, N104L, S40L, Q137L Natural CEA proteins suffer from several structural defects, including excessive flexibility in interdomain linker regions, large conformational fluctuations in epitope loops, numerous suspended polar residues on the surface, and uneven distribution of isolated charged residues. These defects lead to poor overall thermal stability, irregular antibody-binding interfaces, and limited binding affinity. This invention addresses these structural deficiencies by precisely introducing the following four cooperating mutations: S6L (position 6 of the mature chain, corresponding to position 40 of the full length): The wild-type serine side chain hydroxyl group is suspended and exposed, lacking intramolecular hydrogen bond acceptors, resulting in a weak epitope hydrophobic interface. Mutating it to leucine thickens the antibody-binding hydrophobic interface, enhances the antigen-antibody hydrophobic interaction, and significantly improves the binding affinity of broad-spectrum CEA monoclonal antibodies; simultaneously, it eliminates conformational fluctuations in the polar side chain, reduces local flexibility, and slightly improves protein thermal stability.

[0063] N42L (position 42 of the mature chain, corresponding to position 76 of the full length): The amino group on the side chain of wild-type asparagine is suspended, forming a polar protrusion on the surface, which hinders the tight adhesion between the antibody and the epitope. After mutation to leucine, the epitope surface can be smoothed, eliminating the steric hindrance caused by the polar protrusion and optimizing the antibody binding interface; at the same time, it increases local hydrophobic accumulation and improves the conformational stability of the epitope.

[0064] N70L (position 70 in mature chain, corresponding to position 104 in full length): This site is located on the flexible epitope loop of the NV domain. Wild-type asparagine enhances the polarity of the loop region and results in high conformational dynamics, making it prone to local unfolding. Mutating to leucine increases the hydrophobic rigidity of the loop region, tightens the epitope loop conformation, inhibits loop unfolding and protease degradation, and simultaneously improves the accuracy of antibody recognition and binding stability.

[0065] Q103L (position 103 of the mature chain, corresponding to position 137 of the full length): The wild-type glutamine side chain amide group is suspended, resulting in no intramolecular interactions, leading to strong hydration around the epitope and large conformational fluctuations. Mutating it to leucine can reduce the degree of hydration around the epitope, reducing the interference of water molecules on antibody binding; at the same time, it can strengthen intramolecular hydrophobic interactions and improve the overall folding stability of the NV domain.

[0066] (2) Site-directed mutations in the AFP functional region: K549Q, G596V, N494I The natural AFP 403-609 region (D3 domain) suffers from epitope electrostatic interference, easy degradation of the disordered C-terminal region, and insufficient filling of the internal hydrophobic cavity, resulting in poor folding stability, low antibody binding efficiency, and easy inactivation during storage. This invention specifically introduces the following three complementary mutations: K549Q (AFP D3 domain core epitope region): This site is located at the center of the AFP-specific monoclonal antibody recognition epitope. The wild-type lysine side chain is exposed with a positive charge, resulting in local electrostatic repulsion and hindering tight adhesion between the antibody and the epitope. Mutation to glutamine eliminates this local electrostatic interference, optimizes the physicochemical microenvironment of the antibody-binding interface, and significantly enhances the binding affinity of the AFP monoclonal antibody. Simultaneously, it stabilizes the local folding conformation of the D3 domain, reducing the risk of epitope unfolding.

[0067] G596V (C-terminal disordered region of the AFP D3 domain): This site is located in the highly flexible disordered degradation hotspot of the AFP C-terminus. Wild-type glycine makes this region extremely loosely conformated, making it a preferred recognition and cleavage site for proteases, leading to easy terminal degradation and rapid activity decline during protein storage. Mutating it to valine significantly increases the rigidity of the C-terminal backbone, shortens the flexible terminal length, significantly inhibits protease degradation, and improves the protein's stability at room temperature and tolerance to repeated freeze-thaw cycles.

[0068] N494I (hydrophobic core region of AFP D3 domain): This site is deeply embedded in the hydrophobic core of the AFP D3 domain. Wild-type asparagine residues are polar residues, and filling the hydrophobic cavity leads to loose core stacking, poor folding stability, and susceptibility to core unfolding, resulting in overall protein inactivation. Mutating to isoleucine strengthens the hydrophobic interactions within the molecule, fills the core cavity, improves protein structural compactness and folding stability, and fundamentally enhances the thermal stability and conformational integrity of the AFP fragment.

[0069] The dual-target fusion antigen mutant finally optimized in this invention contains seven site-directed mutations, combined as follows: CEA region (N76L, N104L, S40L, Q137L) and AFP region (K549Q, G596V, N494I). To verify the synergistic effect of the mutations, this invention constructed wild-type fusion proteins and single- and multi-site mutants. Under completely uniform experimental conditions, multi-dimensional performance screening was performed, and the seven-site global synergistic mutant was determined to be the combination with the best overall performance. There was no antagonistic effect between the mutation sites, and there was a significant cross-domain and cross-functional synergistic effect.

[0070] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields. The invention is further illustrated below with specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0071] Example 1: Construction and expression of wild-type fusion antigen 1.1 Wild-type fusion gene design and synthesis Based on the standard reference amino acid sequences of human CEA (Uniport: P06731) and AFP (Uniport: P02771), the core functional fragments were extracted: amino acids 35–550 of CEA and amino acids 403–609 of AFP. The Igκ secretion signal peptide was used. CEA(35-550aa) (GGS)5 AFP(403-609aa) The optimized sequence of 6×His was tandemly constructed. Full-length codons were optimized based on the codon bias of HEK293F cells, removing the internal TATA box, polyA signal, and splicing sites. An optimized wild-type dual-target fusion gene without site-directed mutations was artificially synthesized. An Igκ light chain signal peptide was fused to the N-terminus, and a 6×His purification tag was fused to the C-terminus. HindIII and XhoI restriction sites were introduced at both ends of the gene, respectively.

[0072] The amino acid sequence of the Igκ secretion signal peptide is: METDTLLLWVLLLWVPGSTGD (SEQ ID NO:1).

[0073] The CEA amino acid sequence from 35 to 550 (a total of 516 amino acids) is as follows: KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQE LFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDP TISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLS (SEQ ID NO:2).

[0074] The amino acid sequence of AFP 403-609 (a total of 207 amino acids) is: KYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGV (SEQ ID NO:3).

[0075] 1.2 Construction of Recombinant Expression Vectors (1) The optimized wild-type fusion gene sequence designed above (containing N-terminal Igκ light chain signal peptide, various functional fragments, flexible linker peptide and C-terminal 6×His purification tag, with HindⅢ and XhoⅠ restriction sites introduced at both ends) was chemically synthesized in full length by Suzhou Genewise Biotechnology Co., Ltd. and the recombinant expression vector was constructed.

[0076] (2) Plasmid verification: After receiving the recombinant plasmid delivered by Suzhou Genewiz, 1 μL of the plasmid was taken for preliminary identification by agarose gel electrophoresis. At the same time, the plasmid was sent to a third-party sequencing company for full-length sequencing verification to confirm that the sequence was completely consistent with the design.

[0077] (3) Plasmid transformation and amplification: 1 μL of the verified recombinant plasmid was transformed into DH5α competent Escherichia coli cells, plated on LB solid plates containing 100 μg / mL ampicillin, and incubated upside down at 37℃ for 12-16 h; single colonies were picked and inoculated into 500 mL of LB liquid medium containing ampicillin, and cultured at 37℃ and 200 rpm for 16 h with shaking; plasmids were extracted using an endotoxin-free plasmid extraction kit for subsequent cell transfection experiments.

[0078] 1.3 Eukaryotic cell suspension expression The positive recombinant plasmid was transfected into HEK293F suspension expression cells, and cultured in a serum-free suspension system at 37℃, 5% CO2, and 120 rpm. Cells were passaged every 2–3 days at a 1:3 ratio to maintain a cell density of 0.5 × 10⁶ cells / year. 6 ~3×10 6 cells / mL. Adjust cell density to 1.5 × 10⁶ cells / mL during transfection. 6 Cells / mL (viable cell rate ≥95%) were used to prepare the transfection complex at a plasmid:PEI mass ratio of 1:3. After standing at room temperature for 15 min, the complex was added to the cell culture system. 24 h after transfection, the temperature was lowered to 33℃. On day 3, a final concentration of 0.5 mM sodium butyrate was added, and the cells were cultured until day 10 to achieve efficient and soluble expression of the fusion protein. The cell culture supernatant was then collected.

[0079] 1.4 Protein chromatography purification (1) Supernatant pretreatment: The collected cell culture supernatant was centrifuged at 4℃ and 8000×g for 30 min to remove cell debris and then filtered through a 0.22 μm filter membrane for sterilization.

[0080] (2) Ni-NTA affinity chromatography: Equilibration: Equilibrate the Ni-NTA agarose gel column (GE Healthcare 17-5318-02) with 5 column volumes of equilibration buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) at a flow rate of 5 mL / min. Sample loading: Load the pretreated supernatant at a flow rate of 2 mL / min; Washing: Wash the column with 10 column volumes of washing buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 8.0) until the UV absorbance (A280) drops to baseline. Elution: Elute the target protein with 5 column volumes of elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0) and collect the elution peak.

[0081] (3) Ion exchange chromatography: The Ni-NTA elution product was dialyzed overnight at 4°C with dialysis buffer (20 mM Tris-HCl, pH 8.0), and the dialysate was changed 3 times. Equilibrate the Q Sepharose FastFlow ion exchange column (GE Healthcare 17-0510-01) with 5 column volumes of equilibration buffer (20 mM Tris-HCl, pH 8.0) at a flow rate of 3 mL / min. Sample loading: Load the dialyzed protein sample at a flow rate of 2 mL / min; Elution: Elute with a linear gradient of 0-1 M NaCl and collect the target protein peak.

[0082] (4) Gel filtration chromatography: Equilibrate the Superdex 200 Increase 10 / 300 GL gel filter column (GE Healthcare 28-9909-44) with 5 column volumes of PBS buffer (pH 7.4) at a flow rate of 0.5 mL / min; Sample loading: Concentrate the ion exchange elution product to 1 mL and load it at a flow rate of 0.5 mL / min; Collect the monomer peaks, concentrate them to 1 mg / mL using an ultrafiltration tube, aliquot them, and store them at -80°C.

[0083] (5) According to SDS-PAGE analysis, the purity of the purified wild-type fusion protein is ≥95%, with no obvious aggregation, degradation and contamination by other proteins. It has good solubility and uniformity, and meets the standards for immunological experiments and biological raw material applications.

[0084] Example 2: Computer-based preliminary screening experiment for candidate mutation sites 2.1 Three-dimensional structural modeling and parameter extraction Using CEA crystal structure PDB 1E07 and AFP crystal structure PDB 1MRK as templates, three-dimensional homologous structures of the truncated fusion protein specific to this invention were constructed. After energy optimization and conformational correction, stable models were obtained. The flexibility parameters of factor B in the whole protein were extracted using ChimeraX software. Flexible loops and disordered interdomain connections with factor B significantly higher than the overall protein mean were screened to determine the core region for thermal stability modification.

[0085] 2.2 Analysis of antigen interface residues Solvent-accessible surface area (SASA) of each amino acid residue was calculated using PDBePISA software. Key epitope residues exposed on the protein surface that directly participate in antibody recognition and binding were screened, and candidate sites for affinity optimization were identified.

[0086] 2.3 Stability Prediction of Folding Free Energy Using the FireProt and Dynamut professional protein mutation prediction platforms, single-point mutation free energy simulations were performed on all candidate sites to screen for post-mutation folding free energy ΔG < 0. A beneficial stable site of 0.5 kcal / mol.

[0087] 2.4 Evolutionary Conservation Selection Multiple sequence alignment (MSA) was constructed using human CEACAM family homologous sequences and multi-species AFP homologous sequences. Highly conserved disulfide bond forming residues and core backbone anchoring residues were removed, while non-conserved safe sites with no risk of folding and modifiability were retained.

[0088] 2.5 Summary of Candidate Sites By integrating the results of flexibility characteristics, epitope distribution, energy parameters, and conservation analysis, an initial candidate mutation site library with both thermal stability optimization and affinity optimization potential was obtained.

[0089] Example 3 Construction and preliminary screening of single-point mutants 3.1 Construction of single-point mutants Based on the wild-type fusion gene backbone obtained in Example 1, seven single-point mutants were constructed using site-directed mutagenesis PCR technology, specifically including: M1 (CEA-N76L), M2 (CEA-N104L), M3 (CEA-S40L), M4 (CEA-Q137L), M5 (AFP-K549Q), M6 (AFP-G596V), and M7 (AFP-N494I). All mutants had identical backbone structure, fragment length, and linker peptide sequences, with only single-point mutation differences. The primer sequences are shown in Table 1 below.

[0090] Table 1 Primer Sequences

[0091] (1) Site-directed mutagenesis PCR reaction system (50 μL): Phusion High-Fidelity DNA polymerase 0.5 μL, 5×Phusion Buffer 10 μL, dNTPs (2.5 mM each) 4 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, wild-type recombinant plasmid template 1 μL (about 50 ng), ddH2O 30.5 μL.

[0092] (2) PCR reaction procedure: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 5 min, for a total of 18 cycles; 72℃ final extension for 10 min.

[0093] (3) DpnI digestion: Add 1 μL of DpnI restriction endonuclease (NEB R0176S) to the PCR product, digest in a water bath at 37℃ for 1 h, and remove the template plasmid.

[0094] (4) Take 10 μL of digestion product and transform it into DH5α competent cells. The subsequent steps are the same as 1.2 in Example 1. Extract plasmid and sequence to verify the correctness of the mutation.

[0095] 3.2 Expression and purification of single-point mutants Following the same expression and purification process as in Example 1, the single-point mutant proteins were prepared. All purified proteins had a purity ≥95%, meeting the requirements for subsequent performance testing.

[0096] SDS-PAGE analysis showed that all fusion proteins exhibited distinct protein bands at the expected molecular weight position (approximately 85 kDa), with purities exceeding 90%. BCA quantification revealed expression levels of 15-25 mg / L for each fusion protein. The purified proteins were quantified using the BCA method, aliquoted, and stored at -80°C for later use. The purified yields of wild-type and mutant proteins are summarized in Table 2.

[0097] Table 2. Purification yields of wild-type and mutant proteins

[0098] 3.3 Preliminary performance screening of single-point mutants Standardized experimental conditions and sample parameters were used, with three parallel replicates for each group, and the average value was taken as the final experimental data. Key detection indicators included: heat denaturation temperature (Tm value), residual activity rate after 7 days at 37℃, and average antibody affinity for the three antigens.

[0099] Preliminary screening results show that each single-point mutation only achieves a slight improvement in protein performance, with Tm values ​​increasing by 0.7–1.3℃, residual activity at 37℃ / 7 days increasing by 0.7–1.3%, and average affinity increasing by 1.28–1.35 times. The limited gain per site cannot meet the requirements for industrialization and detection applications of high-stability, high-affinity antigen raw materials.

[0100] Example 4: Evaluation of the thermal stability of single-point mutants 4.1 Stability Screening – Differential Scanning Fluorescence Method The thermostability of each mutant protein was determined using differential scanning fluorometry (DSF). A StepOnePlus real-time quantitative PCR system was used, with the following reaction mixture: 10 μL protein sample (0.5 mg / mL), 10 μL SYPROOrange dye (final concentration 5×), total volume 20 μL. The temperature was increased from 25℃ to 95℃ at a rate of 1.0℃ / min, with fluorescence signals recorded every 0.5℃ increase. Fluorescence signals were acquired, and the midpoint temperature (Tm) of thermal denaturation for each protein was calculated. Three technical replicates were performed for each sample.

[0101] 4.2 Isothermal residual activity determination All fusion protein samples were adjusted to the same concentration (0.5 mg / mL) and incubated in water baths at 55℃, 60℃, 65℃, and 70℃ for 30 min each. Immediately after incubation, they were cooled on ice for 10 min. The residual activity of the proteins after heat treatment was determined using an indirect ELISA method. The residual activity of each heat-treated sample was calculated with the activity of the untreated protein sample as 100%. Each sample was measured three times.

[0102] The results of the thermal stability evaluation of each mutant Tm value and wild type (WT) are shown in Table 3.

[0103] Table 3. Thermal stability evaluation results of Tm values ​​for each mutant and wild type (WT)

[0104] *: P<0.05 compared to wild type.

[0105] The results showed that some mutants not only improved thermal stability but also increased affinity for the corresponding antibodies. Among them, the N76L and G596V mutants showed the most significant increase in affinity, with Kd values ​​decreasing by 12.8% and 9.8% with anti-CEA antibodies, and by 11.8% and 13.4% with anti-AFP antibodies, respectively.

[0106] Example 5 Affinity evaluation of single-point mutants 5.1 Determining the relative affinity of the fusion protein using an indirect ELISA method The specific steps are as follows: (1) Dilute each fusion protein to 2 μg / mL with coating buffer, coat 100 μL per well of a 96-well microplate, and incubate overnight at 4°C.

[0107] (2) Discard the coating solution, add 200 μL of blocking solution to each well, and seal at 37℃ for 1 h.

[0108] (3) Discard the blocking solution and wash with PBST 3 times, 3 min each time.

[0109] (4) The anti-CEA monoclonal antibody and the anti-AFP monoclonal antibody were serially diluted 2-fold, with a concentration range of 0.01 ng / mL to 10 μg / mL. 100 μL of antibody solution of different concentrations was added to each well and incubated at 37°C for 1 h.

[0110] (5) Discard the antibody solution and wash with PBST 3 times, 3 min each time.

[0111] (6) Add 100 μL of HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution, Beyotime, catalog number: A0216) to each well and incubate at 37°C for 1 h.

[0112] (7) Discard the secondary antibody solution and wash with PBST 5 times, 3 min each time.

[0113] (8) Add 100 μL of TMB colorimetric solution to each well and develop the color at 37°C in the dark for 10 min.

[0114] (9) Add 50 μL of 2 M H2SO4 to each well to stop the reaction, and measure the absorbance value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0115] Plot a binding curve with antibody concentration on the x-axis and absorbance on the y-axis. Calculate the antibody concentration (EC50) at which half of the maximum absorbance value is reached. 50 The value (EC) is used to represent the relative affinity of the fusion protein. 50 The smaller the value, the higher the affinity. Each sample was measured three times.

[0116] 5.2 Experimental Results The affinity evaluation results of each mutant to wild type (WT) are shown in Table 4.

[0117] Table 4. Affinity evaluation results of each mutant to wild type (WT)

[0118] *: P<0.05 compared to wild type.

[0119] The results showed that some mutants not only improved thermal stability but also increased affinity for the corresponding antibodies. Among them, the N76L and G596V mutants showed the most significant increase in affinity, with Kd values ​​decreasing by 12.8% and 9.8% with anti-CEA antibodies, and by 11.8% and 13.4% with anti-AFP antibodies, respectively.

[0120] Example 6: Functional evaluation of CEA-AFP fusion protein combinatorial mutants 6.1 Construction of CEA-AFP fusion protein combinatorial mutant Based on the screening results of the five single-point mutants in Example 4, four positive mutation sites (CEA-N76L, CEA-N104L, AFP-N494I, and AFP-G596V) with the most significant improvement in thermal stability and affinity were selected for combined mutation to construct double, triple, and quadruple mutants. The synergistic effect between mutation sites was verified, and the fusion protein mutant with the best overall performance was screened.

[0121] Following the principle of "gradual superposition and priority preservation of core dominant sites", the following 5 combined mutants were designed: M8 (double mutation): CEA-N76L + AFP-G596V (a combination of sites with dual advantages in thermal stability and affinity); M9 (double mutation): CEA-N76L + CEA-N104L (two-site combination within the CEA domain). M10 (double mutation): AFP-N494I + AFP-G596V (two sites within the AFP domain). M11 (triple mutation): CEA-N76L + CEA-N104L + AFP-G596V (core three-point combination); M12 (quadruple mutation): CEA-N76L + CEA-N104L + AFP-N494I + AFP-G596V (all positive site combination). The construction steps are exactly the same as in Example 3.

[0122] 6.2 Evaluation of the thermostability of the CEA-AFP fusion protein combinatorial mutant Using the same method as in Example 4, the thermal denaturation temperature (Tm value) and isothermal residual activity at different temperatures were determined for each combination of mutants, while wild type (WT) and single-point optimal mutants M2 (N76L) and M7 (G596V) were used as controls.

[0123] The thermal denaturation temperature (Tm value) and the evaluation results of isothermal residual activity at different temperatures for each combination mutant are shown in Table 5.

[0124] Table 5. Thermal denaturation temperature (Tm value) and isothermal residual activity of each mutant combination at different temperatures.

[0125] *: P < 0.05 compared to wild type; #: P < 0.05 compared to single-point best M2; △: P < 0.05 compared to double-mutant M8; ▲: P < 0.05 compared to triple-mutant M11.

[0126] 6.3 Affinity determination of CEA-AFP fusion protein combinatorial mutants Using the exact same method as in Example 5, the affinity constants (Kd values) and relative affinity (EC values) of each mutant combination with anti-CEA monoclonal antibody and anti-AFP monoclonal antibody were determined. 50 (Value), the same as in Example 5.

[0127] Affinity constants (Kd values) and relative affinities (EC values) of each combined mutant with anti-CEA monoclonal antibodies and anti-AFP monoclonal antibodies. 50 The evaluation results (value) are shown in Table 6.

[0128] Table 6. Affinity constants (Kd values) and relative affinities (EC values) of each mutant combination with anti-CEA monoclonal antibody and anti-AFP monoclonal antibody. 50 value)

[0129] *: P < 0.05 compared to wild type; #: P < 0.05 compared to single-point best M2; △: P < 0.05 compared to double-mutant M8; ▲: P < 0.05 compared to triple-mutant M11.

[0130] 6.4 Long-term storage stability evaluation To evaluate the storage performance of mutants in practical applications, wild-type and optimal mutant protein solutions (0.5 mg / mL, PBS pH 7.4) were aliquoted and stored at 4°C. Samples were taken at 0, 1, 2, and 3 months of storage, and the residual binding activity of the proteins was determined by indirect ELISA, with the initial activity considered as 100%.

[0131] The residual binding activities of wild-type and optimal mutant proteins are shown in Table 7. Experimental results indicate that there is a significant synergistic effect among the four positive mutation sites selected in this invention, with no antagonistic effect, specifically as follows: (1) The Tm value of the double mutant M8 was 2.7℃ higher than that of the single-point optimal M2, the residual activity at 65℃ was increased by 16.5 percentage points, and the Kd values ​​of anti-CEA and anti-AFP antibodies were reduced by 13.2% and 15.8%, respectively. The performance improvement was greater than the sum of the improvement of the two single-point mutants.

[0132] (2) The triple mutant M11 further introduces the N104L mutation on the basis of the double mutant M8, which increases the Tm value by 1.7℃ and the affinity by about 10%, and significantly improves the long-term storage stability.

[0133] (3) The quadruple mutant M12 introduced the N494I mutation on the basis of the triple mutation, and the overall performance reached the best. The Tm value was increased by 9.6℃ compared with the wild type, the residual activity at 65℃ was increased by 47.1 percentage points, the affinity with anti-CEA and anti-AFP antibodies was increased by 37.4% and 40.1% respectively, and the residual activity was still above 85% after being stored at 4℃ for 3 months.

[0134] Table 7 Residual binding activity of wild-type and optimal mutant proteins

[0135] *: P < 0.05 compared to wild type; #: P < 0.05 compared to single-point best M2; △: P < 0.05 compared to double-mutant M8; ▲: P < 0.05 compared to triple-mutant M11.

Claims

1. A CEA / AFP dual tumor marker fusion antigen mutant protein, characterized in that, The mutant protein comprises, from N-terminus to C-terminus, a signal peptide, a CEA antigen functional domain, a flexible linker peptide, and an AFP antigen functional domain. The CEA antigen functional domain is the amino acid sequence from position 35 to position 550 of the human CEACAM5 protein (Uniprot: P06731), and contains any one or more site-directed amino acid mutations from S40L, N76L, N104L, and Q137L. The AFP antigen functional domain is the amino acid sequence from position 403 to position 609 of the human AFP protein (Uniprot: P02771), and contains any one or more site-directed amino acid mutations of N494I, K549Q, and G596V.

2. The mutant protein according to claim 1, characterized in that, The mutant protein also includes a purification tag; The mutant protein comprises, from N-terminus to C-terminus, a signal peptide for secretory expression in mammalian cells, a CEA antigen functional domain, a flexible linker peptide, an AFP antigen functional domain, and a purification tag. Optionally, the purification tag is selected from 6×His purification tag, Flag purification tag, Strep-tag II purification tag, GST purification tag, MBP purification tag, HAT purification tag, or SUMO purification tag.

3. The mutant protein according to claim 1 or 2, characterized in that, The signal peptide is a signal peptide that is secreted and expressed by mammalian cells. Optionally, the signal peptide is selected from Igκ signal peptide, human IgG heavy chain signal peptide, human serum albumin signal peptide, human growth hormone signal peptide, mouse Igκ light chain signal peptide, human CD33 signal peptide, human IL-2 signal peptide, human tPA signal peptide, bee venom peptide signal peptide or glucosidase signal peptide. Optionally, the flexible linker peptide is selected from highly flexible linker peptides (GGS)5, (GGS)5, etc. n (G4S) n (G4S)4, (G5S) n , GSGGSG, GGSGGSG, GSG4SG4S, G4SG4SG4SG4S, G5SG5S, GSGGSGGSG, GGSGGSGGSGGSG, GSGGSGGSGGSGGSG, G4SG4SG4S, GSG4SG4SG4S or G5SG5SG5S, where n is any integer from 1 to 10.

4. The mutant protein according to any one of claims 1-3, characterized in that, The mutant protein comprises, from N-terminus to C-terminus, the following components in sequence: an Igκ signal peptide for secretory expression in mammalian cells, a CEA antigen functional domain, a flexible linker peptide, an AFP antigen functional domain, and a 6×His purification tag. Optionally, the amino acid sequence of the Igκ signal peptide is shown in SEQ ID NO:1; Optionally, the mutant protein is a double mutant protein, wherein the site-directed amino acid mutation combination is selected from CEA-N76L + AFP-G596V, CEA-N76L + CEA-N104L or AFP-N494I + AFP-G596V. Optionally, the mutant protein is a triple mutant protein, which contains the site-directed amino acid mutation combination of CEA-N76L + CEA-N104L + AFP-G596V; Optionally, the mutant protein is a tetramutant protein, which contains the site-directed amino acid mutation combination of CEA-N76L+ CEA-N104L+ AFP-N494I+ AFP-G596V.

5. A recombinant gene encoding the mutant protein of any one of claims 1-4.

6. A recombinant expression vector comprising the recombinant gene of claim 5.

7. A recombinant host cell comprising the recombinant expression vector of claim 6.

8. A method for preparing a mutant protein according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: culturing the recombinant host cell of claim 7, inducing it to express the mutant protein, collecting the culture and purifying the mutant protein therefrom.

9. Any one of the following products: (1) A site-specific amino acid mutation site, characterized in that, The mutation sites are selected from any one or more of the following: S40L, N76L, N104L and / or Q137L of human CEACAM5 protein (Uniprot: P06731) and N494I, K549Q and / or G596V of human AFP protein (Uniprot: P02771). (2) A protein lyophilized formulation, characterized in that the protein lyophilized formulation comprises any one of claims 1-4 mutant protein and lyophilization protectant; (3) A protein conjugate, characterized in that the protein conjugate comprises any one of claims 1-4, and a tracer conjugated thereto; (4) A detection kit, characterized in that the detection kit comprises the mutant protein according to any one of claims 1-4; (5) A CEA and AFP combined detection kit, characterized in that the combined detection kit contains the mutant protein of any one of claims 1-4 as a standard, quality control or calibrator.

10. Applied to any of the following aspects: (1) The use of the mutant protein of any one of claims 1-4, the recombinant gene of claim 5, the recombinant expression vector of claim 6, or the host cell of claim 7 in the preparation of a combined detection product for CEA and AFP; (2) The use of the mutant protein of any one of claims 1-4 in the preparation of CEA-specific antibodies or AFP-specific antibodies; (3) The application of the mutant protein according to any one of claims 1-4 in the study of tumor immune mechanisms; (4) The use of the mutant protein of any one of claims 1-4 in the preparation of tumor screening, diagnosis, prognostic assessment or recurrence monitoring products; (5) The use of the mutant protein of any one of claims 1-4 in the preparation of diagnostic or auxiliary diagnostic products for liver cancer, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer or breast cancer; (6) The use of the mutant protein of any one of claims 1-4 in the preparation of positive controls, negative controls, calibrators, quality control products, calibrators or reference products for in vitro diagnostic kits; (7) The use of the mutant protein of any one of claims 1-4 in the preparation of chemiluminescent immunoassay kits, enzyme-linked immunosorbent assay kits, immunochromatographic test strips, electrochemiluminescent immunoassay kits, time-resolved fluorescence immunoassay kits or immunoturbidimetric assay kits; (8) The application of the site-directed amino acid mutation site described in claim 9 in the preparation of CEA / AFP dual tumor marker fusion antigen mutant protein.

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