Preparation method and application of cyanine dye-mutant protein composite fluorophore

Abstract

The invention is applicable to the technical field of near-infrared two-region imaging, and provides a preparation method and application of a cyanine dye-mutant protein composite fluorophore. The cyanine dye-mutant protein composite fluorophore is a compound formed by covalent binding of cyanine dye molecules and mutant protein. The mutant protein is single mutant human serum albumin of which cysteine at the 461, 476, 477 or 487 sites is mutated into glycine; and the cyanine dye molecule is CO-1080. Through site-specific mutagenesis treatment, the binding capacity of the human serum albumin and specific cyanine dye molecules under the room temperature condition is greatly improved. The cyanine dye-mutant protein composite fluorophore prepared by the invention shows good biological safety and near-infrared two-region luminescence capability in vivo, can be applied to fluorescence imaging of living animals, and particularly has a remarkable effect in the aspect of noninvasive evaluation of the integrity of a blood-tissue barrier of an organism.

Description

Technical Field

[0001] The present invention belongs to the technical field of near-infrared second-zone imaging, and in particular relates to a preparation method and application of a cyanine dye-mutant protein composite fluorophore. Background Art

[0002] Near-infrared (NIR) fluorescence imaging technology has opened up a new avenue for deep tissue biological imaging. Compared with traditional near-infrared region I (NIR-I, 700-900nm) fluorescence, near-infrared region II (NIR-II, 1000-1700nm) fluorescence exhibits lower absorption and scattering, less biological tissue autofluorescence, and higher tissue penetration depth and spatial resolution of fluorescence imaging in living tissues. At present, NIR-II probes that can bind non-covalently or covalently to albumin have shown great application potential in fields such as vascular imaging, lymphatic system visualization and cancer detection. The development of NIR-II probes that can target endogenous or exogenous albumin in situ and have good biosafety is of great significance for in vivo studies of small animal models. However, the potential of NIR-II imaging in imaging complex tissue barriers (such as the blood-testis barrier) has yet to be fully explored.

[0003] The blood-testis barrier (BTB) is a physical barrier formed by tight junctions between Sertoli cells in the seminiferous tubules of the testis. It is crucial for protecting developing spermatocytes from harmful substances and maintaining a stable environment for spermatogenesis. In scientific research and clinical diagnosis, assessing the integrity of the BTB is crucial for understanding male fertility and the impact of various diseases or treatments on testicular function. However, traditional methods for assessing BTB integrity and associated spermatogenesis disorders have many limitations, such as potential invasiveness, lack of precision, or limited in vitro analysis. These methods include histological examination using hematoxylin-eosin (H&E) staining, visualization of lanthanum nitrate leakage through the tissue barrier using lanthanum tracing, visualization of fluorescence distribution near the tissue barrier using biotin tracing, and Western blotting analysis to measure the expression levels of BTB-related proteins. Therefore, non-invasive, real-time imaging techniques that can monitor BTB integrity in vivo are urgently needed to provide an effective means for studying the testicular microenvironment and understanding testicular physiology and pathology. To this end, the present invention provides a preparation method and application of a cyanine dye-mutant protein composite fluorophore. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a cyanine dye-mutant protein composite fluorophore and its application, aiming to solve the problems raised in the above background technology.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A cyanine dye-mutant protein composite fluorophore is a complex formed by covalently binding a cyanine dye molecule and a mutant protein.

[0007] Furthermore, the mutant protein is a single mutant human serum albumin in which cysteine ​​at position 461, 476, 477 or 487 is mutated to glycine;

[0008] The nucleic acid sequence, amino acid sequence, and primer sequences of wild-type human serum albumin and four single mutant human serum albumins used as templates are shown below:

[0009] (1) Wild-type human serum albumin (Wild-type HSA):

[0010] Nucleic acid sequence:

[0011]

[0012] Amino acid sequence:

[0013] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (as shown in SEQ ID NO.2).

[0014] Primer sequence:

[0015] HSA-F:

[0016] CGAGAAAAGAGAGGCTGAAGCTGATGCTCATAAATCAGAA (as shown in SEQ ID NO.3).

[0017] HSA-R:

[0018] GTCTAAGGCGAATTAATTCGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCAAACC CAAT (as shown in SEQ IDNO.4).

[0019] (2) Single mutant human serum albumin with cysteine ​​at position 461 mutated to glycine (rHSA-C461G):

[0020] Nucleic acid sequence:

[0021]

[0022] Amino acid sequence:

[0023] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLGVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (as shown in SEQ ID NO.6).

[0024] Primer sequence:

[0025] HSA-F:

[0026] CGAGAAAAGAGAGGCTGAAGCTGATGCTCATAAATCAGAA (as shown in SEQ ID NO.3).

[0027] HSA-R:

[0028] GTCTAAGGCGAATTAATTCGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCAAACC CAAT (as shown in SEQ IDNO.4).

[0029] 461-F: TGAACCAATTGGGTGTTCTTCA (as shown in SEQ ID NO. 7).

[0030] 461-R: TGAAGAACACCCAATTGGTTC (as shown in SEQ ID NO. 8).

[0031] (3) Single mutant human serum albumin with cysteine ​​at position 476 mutated to glycine (rHSA-C476G):

[0032] Nucleic acid sequence:

[0033] GATGCTCATAAATCAGAAGTTGCTCACAGATTCAAGGATTTGGGTGAAGAGAACTTCAAGGCTTTGGTCTTGATCGCTTTCGCACAGTACCTGCAACAATGTCCATTTGAAGATCACGTCAAGCTGGTGAATGAAGTTACCGAATTTGCTAAGACCTGCGTTGCTGATG AGTCTGCTGAGAATTGTGATAAATCACTGCATACTTTGTTTGGAGACAAATTGTGCACTGTCGCTACTCTGAGAGAGACTTACGGTGAGATGGCTGACTGTTGTGCTAAACAAGAGCCTGAGAGAAATGAGTGTTTCTTGCAGCATAAAGATGATAATCCAAATTTGCCT

[0034] CGTTTGGTTAGACCTGAAGTTGATGTTATGTGTACCGCCTTTCATGATAACGAAGAA

[0035] ACCTTTCTGAAGAAGTACCTGTATGAGATCGCTAGGCGTCACCCTTATTTCTACGCA

[0036] CCAGAATTGCTGTTCTTCGCTAAGAGATACAAGGCAGCTTTCACCGAATGTTGTCAA

[0037] GCTGCAGACAAGGCTGCTTGTTTGTTGCCAAAGTTGGACGAGTTGAGAGACGAAGG

[0038] TAAGGCTTCTTCCGCTAAACAGAGACTTAAATGCGCTAGTTTGCAGAAATTCGGAGA

[0039] AAGAGCCTTAAGGCTTGGGCTGTTGCCAGACTGTCTCAAAGATTTCCAAAGGCTGA

[0040] GTTCGCTGAGGTTAGTAAACTGGTTACTGACCTTACTAAGGTTCACACTGAATGCTG

[0041] TCATGGAGACTTGCTTGAGTGTGCTGATGATAGAGCTGATTTGGCCAAGTATATCTG

[0042] TGAGAATCAAGATTCTATCTCTTCTAAGCTGAAAGAGTGTTGTGAGAAGCCATTGTT

[0043] GGAGAAGTCTCACTGTATCGCAGAGGTTGAGAACGATGAAATGCCTGCAGATTTGC

[0044] CTTCATTGGCCGCTGACTTTGTCGAATCTAAAGATGTTTGCAAGAACTACGCTGAAG

[0045] CTAAGGACGTCTTTCTGGGTATGTTTCTATACGAGTATTGCTAGACGTCATCCAGATT

[0046] ACTCTGTCGTCTTGTTGTTGCGTTTGGCTAAGACTTACGAAACTACACTGGAGAAAT

[0047] GTTGTGCTGCTGCTGATCCACATGAATGCTACGCTAAGGTGTTTGACGAGGTTCAAAC

[0048] CATGGTCGAAGAACCACAGAACCTTATCAAGCAGAACTGTGAACTGTTTGAACAA

[0049] TTGGGTGAGTACAAGTTTCAGAACGCATTGCTGGTTAGATACACTAAGAAAGTGCC

[0050] ACAAGTTTCTACTCCAACTCTTGTTGAAGTCTCAAGAAACTTGGGTAAGGTTGGTTC

[0051] TAAGTGCTGTAAGCATCCAGAGGCTAAACGTATGCCATGTGCTGAGGATTACCTGTC

[0052] TGTTGTTCTGAACCAATTGTGTGTTCTTCATGAGAAAACGCCTGTTTCTGACAGAGT

[0053] GACTAAAGGCTGTACTGAATCTTTGGTGAATAGAAGGCCTTGCTTTAGTGCTTTGGA

[0054] AGTTGACGAAACCTACGTGCCTAAAGAGTTTAACGCCGAAACCTTTACCTTTCATGC

[0055] TGACATTTGTACTTTGTCCGAGAAAGAGAGACAAATCAAGAAGCAGACTGCTTTGG

[0056] TTGAATTGGTGAAACATAAGCCTAAAGCTACTAAAGAGCAACTGAAGGCCGTCATG

[0057] GACGATTTCGCTGCATTTGTCGAGAAGTGTTGTAAGGCTGATGACAAAGAGACTTGT

[0058] TTCGCTGAAGAGGGTAAGAAGTTGGTCGCTGCATCTCAAGCTGCATTGGGTTTG (as shown in SEQ ID NO.9).

[0059] Amino acid sequence:

[0060] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKGCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (as shown in SEQ ID NO.10).

[0061] Primer sequences:

[0062] HSA-F:<...>

[0063] CGAGAAAAGAGAGGCTGAAGCTGATGCTCATAAATCAGAA (as shown in SEQ ID NO.3).

[0064] HSA-R:

[0065] GTCTAAGGCGAATTAATTCGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCAAACC CAAT (as shown in SEQ IDNO.4).

[0066] 476-F: AGAGTGACTAAAGGCTGTACTGAATC (as shown in SEQ ID NO.11).

[0067] 476-R: TTCAGTACAGCCTTTAGTCACTCT (as shown in SEQ ID NO. 12).

[0068] (4) Single mutant human serum albumin with cysteine ​​at position 477 mutated to glycine (rHSA-C477G):

[0069] Nucleic acid sequence:

[0070]

[0071] Amino acid sequence:

[0072] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCGTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (as shown in SEQ ID NO.14).

[0073] Primer sequence:

[0074] HSA-F:

[0075] CGAGAAAAGAGAGGCTGAAGCTGATGCTCATAAATCAGAA (as shown in SEQ ID NO.3).

[0076] HSA-R:

[0077] GTCTAAGGCGAATTAATTCGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCAAACC CAAT (as shown in SEQ IDNO.4).

[0078] 477-F: AGAGTGACTAAATGCGGTACTGAATC (as shown in SEQ ID NO. 15).

[0079] 477-R: GATTCAGTACCGCATTTAGTCACTCT (as shown in SEQ ID NO. 16).

[0080] (5) Single mutant human serum albumin with cysteine ​​at position 487 mutated to glycine (rHSA-C487G):

[0081] Nucleic acid sequence:

[0082]

[0083] Amino acid sequence:

[0084] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPGFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (as shown in SEQ ID NO. 18).

[0085] Primer sequence:

[0086] HSA-F:

[0087] CGAGAAAAGAGAGGCTGAAGCTGATGCTCATAAATCAGAA (as shown in SEQ ID NO. 3).

[0088] HSA-R:

[0089] GTCTAAGGCGAATTAATTCGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCAAACC CAAT (as shown in SEQ ID NO. 4).

[0090] 487-F: GAATAGAAGGCCTGGCTTTAGTGCT (as shown in SEQ ID NO. 19).

[0091] 487-R: AGCACTAAAGCCAGGCCTTCTATTC (as shown in SEQ ID NO. 20).

[0092] Furthermore, the cyanine dye molecule is CO-1080.

[0093] Furthermore, the complex is a covalently bound complex obtained by reacting a single mutant human serum albumin with a chlorine atom on the six-membered ring in the middle of a cyanine dye molecule according to its own specific site; the outer layer of the covalently bound complex is the single mutant human serum albumin, that is, the cyanine dye molecule is wrapped inside the single mutant human serum albumin.

[0094] A method for preparing the cyanine dye-mutant protein composite fluorophore as described above, comprising the steps of extracting the mutant protein and constructing the cyanine dye-mutant protein composite fluorophore;

[0095] The mutant protein extraction step comprises the following steps:

[0096] Step 1: plasmid construction;

[0097] The sequences of the human serum albumin mutants were amplified from the codon-optimized human serum albumin sequence, with mutations or substitutions in key regions mapped to the primers. Following polymerase chain reaction (PCR), the human serum albumin fragments were completely religated and inserted into the pPIC9K plasmid linearized with EcoRI and NotI using a seamless cloning kit. All plasmids were sequenced prior to transfection.

[0098] Step 2: Pichia pastoris expression;

[0099] The coding sequence of the variant was inserted into the pPIC9K vector backbone and transfected into Pichia pastoris GS115. The transformed GS115 was first cultured in BMGY medium until the absorbance reached 1. The medium was then replaced with BMMY medium to induce protein expression for 72 h at 30°C and 250 rpm.

[0100] Step 3: Purification of mutant protein;

[0101] After secretion of histidine-tagged recombinant albumin into BMMY culture medium, the target protein was purified by affinity chromatography using a 1 mL nickel column packing. The pH of the culture supernatant was adjusted to approximately 7, and the supernatant and nickel column were then vertically mixed at 4°C for 2 hours. The column was then washed sequentially with 20 mL of phosphate-buffered saline (PBS) and 10 mL of PBS containing 10 mM imidazole. After removing impurities through the above steps, the purified mutant protein was eluted with 5 mL of PBS containing 300 mM imidazole.

[0102] The step of constructing the cyanine dye-mutant protein composite fluorophore comprises the following steps:

[0103] A PBS solution of single mutant human serum albumin is solution A, and a cyanine dye molecule CO-1080 is dissolved in DMSO to obtain solution B. Solution A and solution B are mixed at room temperature to obtain a stable cyanine dye-mutant protein complex fluorophore solution; wherein the molar ratio of the cyanine dye molecule to the single mutant human serum albumin is 1:1, and the final concentration of the cyanine dye-mutant protein complex fluorophore is 2 μM or 10 μM.

[0104] A use of the cyanine dye-mutant protein composite fluorophore as described above in preparing a fluorescent imaging reagent for evaluating the integrity of the blood-testis barrier in living animals.

[0105] Compared with the prior art, the present invention has the following beneficial effects:

[0106] Considering that BTB disruption in vivo can lead to albumin leakage and accumulation in the seminiferous tubules, the present invention selected a composite fluorophore formed by covalently binding the cyanine dye CO-1080, which has albumin covalent targeting properties, to a single mutant human serum albumin as a biomarker for detecting BTB integrity. Compared to other cyanine dyes with emission windows in the near-infrared region I, such as IR-780, IR-783, and IR-808, the composite fluorophore formed by covalently binding the cyanine dye CO-1080, which emits in the near-infrared region II, to endogenous mouse serum albumin accumulates in testes with BTB damage after intravenous injection and exhibits a higher signal-to-noise ratio. Furthermore, compared to endogenous serum albumin, the fluorophore constructed from exogenous human serum albumin and CO-1080 can more quickly distinguish testes with BTB damage from normal testes and exhibits lower skin absorption. Because CO-1080 and exogenous human serum albumin rarely covalently bind to form a stable and bright fluorophore at room temperature, the present invention significantly improves the binding efficiency of CO-1080 to exogenous single mutant human serum albumin at room temperature by mutating the disulfide bond sites of wild-type human serum albumin. The resulting cyanine dye-mutant protein composite fluorophore has good in vivo biosafety and exhibits superior luminescence performance in the near-infrared region II. It also enables faster and more stable visualization of BTB damaged areas, demonstrating high imaging contrast. This discovery also reveals that protein mutation can be an effective strategy for modulating the affinity between small molecules and proteins. The present invention combines non-invasive near-infrared region II imaging technology with BTB integrity assessment, providing a new and information-rich perspective for studying the testicular microenvironment and the physiological or pathological processes that affect BTB integrity. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 This is a schematic diagram showing that the cavity of the mutant protein extracted in Examples 1 and 2 (taking the single mutant human serum albumin in which cysteine ​​at position 476 is mutated to glycine as an example) can be used as a high-efficiency microreactor to combine with the cyanine dye CO-1080.

[0108] Figure 2The figures are gel electrophoresis analysis of the complexes of CO-1080 and human serum albumin (including wild type and four single mutants) in Example 1, and a statistical graph of the ratio of the CO-1080@rHSA band brightness to the CO-1080@HSA band brightness; wherein a is a gel electrophoresis analysis of the complexes of CO-1080 and human serum albumin (including wild type and four single mutants), and b is a statistical graph of the ratio of the CO-1080@rHSA complex (CO-1080 and single mutant human serum albumin complex, i.e., recombinant mutant human serum albumin complex) band brightness to the CO-1080@HSA complex (CO-1080 and wild-type human serum albumin complex, i.e., wild-type human serum albumin complex) band brightness.

[0109] Figure 3 These are comparison images of the whole body and testicles of a C57 male mouse with BTB damage captured by a near-infrared camera at different time points after injection of the cyanine dye-mutant protein complex in Example 2; a is a comparison image of the whole body, and b is a comparison image of the testicles.

[0110] Figure 4 This is a statistical graph of the brightness of the testes and skin of BTB-damaged (BTBDisruption) and normal (Normal) C57 male mice at different time points after injection of cyanine dye-mutant protein complex in Example 2.

[0111] Figure 5 This is a statistical analysis of the differences in testis and skin signals between BTB-injured and normal C57 male mice at different time points after injection of cyanine dye-mutant protein complex in Example 2.

[0112] Figure 6 This is Example 2, which evaluates the biosafety of the cyanine dye-mutant protein complex fluorophore. DETAILED DESCRIPTION

[0113] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0114] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0115] Figure 1 This is a schematic diagram showing that the cavity of the mutant protein extracted in Examples 1 and 2 (taking the single mutant human serum albumin in which cysteine ​​at position 476 is mutated to glycine as an example) can be used as a high-efficiency microreactor to combine with the cyanine dye CO-1080.

[0116] Example 1: This example provides a cyanine dye-mutant protein composite fluorophore, which is a covalently bound complex obtained by reacting a single mutant human serum albumin with a chlorine atom on the six-membered ring in the middle of the cyanine dye molecule according to its own specific site. The single mutant human serum albumin is encapsulated with a cyanine dye molecule, and the cyanine dye molecule is coated by the hydrophobic cavity of the single mutant human serum albumin itself, forming a stable and strong fluorescent probe.

[0117] The preparation method of the cyanine dye-mutant protein composite fluorophore mainly includes two steps: extracting the mutant protein and constructing the cyanine dye-mutant protein composite fluorophore. The mutant protein is a single mutant human serum albumin in which cysteine ​​at position 461, 476, 477 or 487 is mutated to glycine.

[0118] Extraction of mutant proteins mainly includes the following steps:

[0119] Step 1: Plasmid construction.

[0120] The sequences of the human serum albumin mutants were amplified from the codon-optimized human serum albumin sequence, with mutations or substitutions in key regions mapped to the primers. Following polymerase chain reaction (PCR), the human serum albumin fragments were fully religated and inserted into the pPIC9K plasmid linearized with EcoRI and NotI using a seamless cloning kit. All plasmids were sequenced prior to transfection.

[0121] Step 2: Pichia pastoris expression.

[0122] The coding sequence of the variant was inserted into the pPIC9K vector backbone and transfected into Pichia pastoris GS115. The transformed GS115 was first cultured in BMGY medium until the absorbance reached 1. The medium was then replaced with BMMY medium to induce protein expression for 72 hours at 30°C and 250 rpm.

[0123] Step 3: Purification of mutant protein.

[0124] After secretion of histidine-tagged recombinant albumin into BMMY culture medium, the target protein was purified by affinity chromatography using a 1 mL nickel column. The pH of the culture supernatant was adjusted to approximately 7, and the supernatant and nickel column were vertically mixed at 4°C for 2 hours. The column was then washed sequentially with 20 mL of phosphate-buffered saline (PBS) and 10 mL of PBS containing 10 mM imidazole. After removing contaminants, the purified mutant protein was eluted with 5 mL of PBS containing 300 mM imidazole.

[0125] The construction of cyanine dye-mutant protein complex fluorophore mainly includes the following steps:

[0126] A single mutant human serum albumin solution in PBS is designated Solution A, while the cyanine dye CO-1080 is dissolved in DMSO to form Solution B. Mixing Solution A and Solution B at room temperature yields a stable cyanine dye-mutant protein complex fluorophore solution. The molar ratio of cyanine dye to single mutant human serum albumin is 1:1, and the final concentration of the cyanine dye-mutant protein complex fluorophore is 2 μM.

[0127] like Figure 2 As shown in the gel electrophoresis analysis of Figure a, under room temperature incubation conditions, CO-1080 has almost no covalent binding to wild-type human serum albumin, while CO-1080 has obvious covalent binding to four single mutant human serum albumins; Figure 2 As shown in the statistical graph in (b), under room temperature incubation conditions, the brightness of the complexes of CO-1080 and single-mutant human serum albumin (especially the single-mutant human serum albumin with cysteine ​​476 mutated to glycine) is significantly higher than that of the complexes of CO-1080 and wild-type human serum albumin. This indicates that mutating cysteine ​​residues at specific sites can enhance the binding of albumin to CO-1080 at room temperature.

[0128] Example 2: This example provides a cyanine dye-mutant protein composite fluorophore, which is a covalently bound complex obtained by reacting a single mutant human serum albumin with a chlorine atom on the six-membered ring in the middle of the cyanine dye molecule according to its own specific site. The single mutant human serum albumin is encapsulated with a cyanine dye molecule, and the cyanine dye molecule is coated by the hydrophobic cavity of the single mutant human serum albumin itself, forming a stable and strong fluorescent probe.

[0129] The preparation method of the cyanine dye-mutant protein complex mainly includes two steps: extracting the mutant protein and constructing the cyanine dye-mutant protein complex fluorophore. The mutant protein is a single mutant human serum albumin in which the cysteine ​​at position 476 is mutated to glycine.

[0130] Extraction of mutant proteins mainly includes the following steps:

[0131] Step 1: Plasmid construction.

[0132] The sequences of the human serum albumin mutants were amplified from the codon-optimized human serum albumin sequence, with mutations or substitutions in key regions mapped to the primers. Following polymerase chain reaction (PCR), the human serum albumin fragments were fully religated and inserted into the pPIC9K plasmid linearized with EcoRI and NotI using a seamless cloning kit. All plasmids were sequenced prior to transfection.

[0133] Step 2: Pichia pastoris expression.

[0134] The coding sequence of the variant was inserted into the pPIC9K vector backbone and transfected into Pichia pastoris GS115. The transformed GS115 was first cultured in BMGY medium until the absorbance reached 1. The medium was then replaced with BMMY medium to induce protein expression for 72 hours at 30°C and 250 rpm.

[0135] Step 3: Purification of mutant protein.

[0136] After secretion of histidine-tagged recombinant albumin into BMMY culture medium, the target protein was purified by affinity chromatography using a 1 mL nickel column. The pH of the culture supernatant was adjusted to approximately 7, and the supernatant and nickel column were vertically mixed at 4°C for 2 hours. The column was then washed sequentially with 20 mL of phosphate-buffered saline (PBS) and 10 mL of PBS containing 10 mM imidazole. After removing contaminants, the purified mutant protein was eluted with 5 mL of PBS containing 300 mM imidazole.

[0137] The construction of cyanine dye-mutant protein complex fluorophore mainly includes the following steps:

[0138] A single mutant human serum albumin solution in PBS is designated Solution A, while the cyanine dye CO-1080 is dissolved in DMSO to form Solution B. Mixing Solution A and Solution B at room temperature yields a stable cyanine dye-mutant protein complex fluorophore solution. The molar ratio of cyanine dye to mutant human serum albumin is 1:1, and the concentration of the cyanine dye-mutant protein complex fluorophore is 10 μM. The resulting cyanine dye-mutant protein complex fluorophore solution is then concentrated by ultrafiltration to a concentration of 200 μM for in vivo imaging.

[0139] like Figure 3 As shown in Figure a, after intravenous injection of CO-1080@rHSA (recombinant mutant human serum albumin) complex into BTB-injured mice, the metabolic rate of the fluorophores accumulated in the BTB-injured testis was slower than that of the fluorophores in other areas, and the BTB-injured testis gradually became brighter, and the imaging contrast improved. Figure 3 As shown in Figure b, the brightness of the BTB-injured testis injected with the CO-1080@rHSA complex was significantly higher than that of the BTB-injured testis injected with the CO-1080@HSA (wild-type human serum albumin) complex and the normal testis injected with the CO-1080@HSA complex. Figure 4 As shown in Figure 3, only 3 hours after injection of the cyanine dye-mutant protein complex fluorophore, the brightness of the BTB-injured testis injected with the CO-1080@rHSA complex can be clearly distinguished from the brightness of the BTB-injured testis injected with the CO-1080@HSA complex. Figure 5As shown, 3 hours after injection of the complex fluorophore, the brightness of BTB-injured testes injected with the CO-1080@rHSA complex was statistically significantly different from that of BTB-injured testes injected with the CO-1080@HSA complex or normal testes. These results indicate that the CO-1080@rHSA complex is more effective than the CO-1080@HSA complex in assessing BTB damage, even when incubated at room temperature for 2 hours.

[0140] Taking the single mutant human serum albumin with cysteine ​​mutated to glycine at position 476 as an example, the biosafety of the cyanine dye-mutant protein complex fluorophore was investigated. Figure 6 As shown, the composite fluorophore did not cause obvious damage to the main organ structures of mice, indicating that the composite fluorophore exhibited good biosafety.

[0141] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A cyanine dye-mutant protein composite fluorophore, characterized in that: It is a complex formed by the covalent binding of a cyanine dye molecule and a mutant protein.

2. The cyanine dye-mutant protein composite fluorophore according to claim 1, characterized in that: The mutant protein is a single mutant human serum albumin in which the cysteine ​​at position 461, 476, 477 or 487 is mutated to glycine; The amino acid sequences of the wild-type human serum albumin and the single mutant human serum albumin in which the cysteine ​​at position 461, 476, 477 or 487 is mutated to glycine as templates are shown in SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.14 and SEQ ID NO.18, respectively.

3. The cyanine dye-mutant protein composite fluorophore according to claim 1, characterized in that: The cyanine dye molecule is CO-1080.

4. The cyanine dye-mutant protein composite fluorophore according to claim 2, characterized in that: The complex is a covalently bound complex obtained by the reaction of single mutant human serum albumin with the chlorine atom in the middle six-membered ring of the cyanine dye molecule according to its own specific site; the outer layer of the covalently bound complex is the single mutant human serum albumin, that is, the cyanine dye molecule is wrapped inside the single mutant human serum albumin.

5. A method for preparing a cyanine dye-mutant protein composite fluorophore as claimed in any one of claims 1 to 4, characterized in that: The method comprises the steps of extracting mutant proteins and constructing cyanine dye-mutant protein composite fluorophore; The mutant protein extraction step comprises the following steps: Step 1: plasmid construction; The sequence of the human serum albumin mutant was amplified from the human serum albumin sequence after codon optimization, and the mutations or substitutions in the key regions were all arranged on the primers; after polymerase chain reaction, the human serum albumin fragment was completely reconnected and inserted into the pPIC9K plasmid linearized with EcoRI and NotI with the help of a seamless cloning kit, and all plasmids were sequenced and confirmed before transfection; Step 2: Pichia pastoris expression; The coding sequence of the variant was inserted into the pPIC9K vector backbone and transfected into Pichia pastoris GS115; the transformed GS115 was first cultured in BMGY medium until the absorbance was 1, and then the medium was replaced with BMMY medium to induce protein expression for 72 hours, the culture temperature was 30°C, and the shaking frequency was 250 rpm; Step 3: purification of mutant protein; After the recombinant albumin with histidine tag was secreted into BMMY culture medium, the target protein was purified by affinity chromatography using 1 mL nickel column filler; the pH of the culture supernatant was adjusted to 7, and then the supernatant and the nickel column were vertically mixed at 4°C for 2 h, and then the column was washed with 20 mL phosphate buffer and 10 mL PBS containing 10 mM imidazole in sequence; after removing impurities, the purified mutant protein was eluted with 5 mL PBS containing 300 mM imidazole; The step of constructing the cyanine dye-mutant protein composite fluorophore comprises the following steps: The PBS solution of single mutant human serum albumin is solution A, the cyanine dye molecule CO-1080 is dissolved in DMSO to obtain solution B, and solution A and solution B are mixed at room temperature to obtain a stable cyanine dye-mutant protein complex fluorophore solution; wherein the molar ratio of the cyanine dye molecule to the single mutant human serum albumin is 1:1, and the final concentration of the cyanine dye-mutant protein complex fluorophore is 2 μM or 10 μM.

6. Use of the cyanine dye-mutant protein composite fluorophore according to any one of claims 1 to 4 in the preparation of a fluorescent imaging agent for evaluating the integrity of the blood-testis barrier of living animals.

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