Application of protein mutant 5H-PSI and coding gene thereof in preparation of medicine combined with tumor cells

Abstract

The invention provides application of a protein mutant 5H-PSI and a coding gene thereof in preparation of a medicine combined with tumor cells, and belongs to the technical field of molecular biology. According to the protein mutant 5H-PSI, part of glutamic acid and aspartic acid residues in wild type PSI protein are mutated into histidine, specifically, D at the 40th site of the wild type PSI protein is mutated into H, E at the 54th site of the wild type PSI protein is mutated into H, E at the 56th site of the wild type PSI protein is mutated into H, E at the 58th site of the wild type PSI protein is mutated into H, and E at the 64th site of the wild type PSI protein is mutated into H. The number of positive charges on the surface of the PSI protein is increased through mutation, the PSI protein can be combined with cell membranes of tumor cells more easily, and the PSI protein still has very high mediating membrane fusion activity under the condition that the pH is higher than 5.5.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to application of a protein mutant 5H-PSI and a coding gene thereof in preparing a drug combining with tumor cells. Background Art

[0002] Aspartic protease (AP) is an important class of proteolytic enzymes that participate in the body's metabolism and biological regulation. It is widely present in animals, plants, and pathogenic bacteria cells. APs have been found in plant organs and tissues such as flowers, leaves, and stems. In plants, APs are related to physiological processes such as protein precursor processing, protein digestion, and programmed cell apoptosis. Plant APs are mainly composed of three parts: the N-terminal region, PSI, and C-terminal region. The N-terminal region and the C-terminal region have high homology with APs in mammals and microorganisms, but the PSI sequence is unique to plant APs. PSI protein is a plant-specific insert sequence (PSI) located on plant aspartic proteases. Plant APs mainly exist in the form of zymogens, which are converted into mature hydrolases after processing. PSIs are separated from APs in this process.

[0003] However, the tumor microenvironment is weakly acidic, with a pH generally higher than 4.5, mostly in the range of 5.5-6.8. The ability of natural PSI to bind to tumor cells is inhibited under this pH environment. How to modify PSI protein so that it can efficiently bind to tumor cell membranes in the tumor microenvironment has become an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present invention is to provide a protein mutant 5H-PSI and its encoding gene for use in the preparation of a drug that binds to tumor cells. The protein mutant 5H-PSI of the present invention can efficiently bind to the tumor cell membrane in the tumor microenvironment.

[0005] The present invention provides a use of a protein mutant 5H-PSI or a coding gene or a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in preparing a product that binds to tumor cells;

[0006] The amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1;

[0007] The coding gene is inserted into the recombinant vector;

[0008] The recombinant bacteria comprises the recombinant vector.

[0009] Preferably, the tumor cells include tumor cells whose outer membranes contain phosphatidylserine; and the binding with tumor cells preferably includes binding with cell membranes of tumor cells.

[0010] Preferably, the working pH of the product is 5.5-6.5.

[0011] The present invention also provides a use of a protein mutant 5H-PSI or a coding gene or a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in the preparation of a tumor targeting drug and / or a tumor diagnostic agent carrier;

[0012] The amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1;

[0013] The coding gene is inserted into the recombinant vector;

[0014] The recombinant bacteria comprises the recombinant vector.

[0015] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0016] The present invention also provides a tumor diagnostic reagent carrier, comprising dioleoylphosphatidylserine and the protein mutant 5H-PSI described in the above scheme loaded on the dioleoylphosphatidylserine.

[0017] The present invention also provides a method for preparing the tumor diagnostic reagent carrier described in the above scheme, comprising the following steps:

[0018] The protein mutant 5H-PSI described in the above scheme is mixed with dioleoylphosphatidylserine to obtain a tumor diagnostic reagent carrier.

[0019] The present invention also provides the use of the tumor diagnostic reagent carrier described in the above scheme or the tumor diagnostic reagent carrier prepared by the preparation method in the preparation of tumor diagnostic reagents.

[0020] The present invention also provides a tumor diagnostic reagent, comprising a tumor diagnostic reagent carrier and a fluorescent dye loaded on the tumor diagnostic reagent carrier; the tumor diagnostic reagent carrier is the tumor diagnostic reagent carrier described in the above scheme or the tumor diagnostic reagent carrier prepared by the preparation method.

[0021] Preferably, the fluorescent dye comprises DiD.

[0022] The present invention provides a protein mutant 5H-PSI or a coding gene or a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in the preparation of a product that binds to tumor cells; the amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1. The protein mutant 5H-PSI of the present invention is a mutation of some glutamic acid and aspartic acid residues in a wild-type PSI protein to histidine, specifically, the D at position 40 of the wild-type PSI protein is mutated to H, the E at position 54 is mutated to H, the E at position 56 is mutated to H, the E at position 58 is mutated to H, and the E at position 64 is mutated to H. The mutation of the present invention increases the number of positive charges on the surface of the PSI protein, making it easier for the PSI protein to bind to the cell membrane of tumor cells, and still has a strong activity of mediating membrane fusion under conditions of pH higher than 5.5. The tumor microenvironment is a special physiological environment composed of tumor cells and surrounding cells and various molecules secreted by cells. In this environment, tumor cells use various means to inhibit some normal physiological functions of normal somatic cells or immune cells, and transform them into supporting tumor cell proliferation and metastasis. The acidic pH range of the tumor microenvironment is mostly concentrated between 5.0 and 6.8. The protein mutant 5H-PSI of the present invention can efficiently bind to the tumor cell membrane in the tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 The results of fluorescence microscopy observation of the U87 MG tumor cell BSA control group;

[0025] Figure 2 The results of the 5H-PSI experimental group of U87 MG tumor cells observed under a fluorescence microscope;

[0026] Figure 3 The results of 293T somatic cell BSA control group observed under fluorescence microscope;

[0027] Figure 4 The results of the 293T somatic cell 5H-PSI experimental group observed under a fluorescence microscope;

[0028] Figure 5 This is the result of observing the fluorescence signal of the LN229 cell control group by flow cytometry under pH 5.5;

[0029] Figure 6The results of fluorescence signal observation by flow cytometry of the LN229 control group with PSI added at pH 5.5;

[0030] Figure 7 The results of fluorescence signal observation by flow cytometry in the experimental group with 5H-PSI added at pH 5.5;

[0031] Figure 8 is the fluorescence signal intensity of the three groups of cells under pH 5.5;

[0032] Fig. 9 This is the result of observing the fluorescence signal of the LN229 control group by flow cytometry under pH 6.5;

[0033] Fig.10 The results of fluorescence signal observation by flow cytometry of the LN229 control group with PSI added at pH 6.5;

[0034] Fig.11 The results of fluorescence signal observation by flow cytometry of the LN229 experimental group with 5H-PSI added at pH 6.5;

[0035] Fig.12 is the fluorescence signal intensity of the three groups of cells under pH 6.5;

[0036] Fig.13 The SDS-PAGE electrophoresis diagram of the supernatant, precipitate and 5H-PSI protein solution control group of 5H-PSI-DOPS vector group after ultracentrifugation;

[0037] Fig.14 The figure is a flow cytometric graph after the 5H-PSI-DOPS vector labeled with DiD fluorescence at a final concentration of 10uM was co-incubated with tumor cells LN229 at pH 6.5 for 4h; the horizontal axis is the cell fluorescence intensity, NC is the LN229 cell control group with an equal amount of PBS solution added, and Vector is the experimental group with the 5H-PSI-DOPS vector labeled with DiD fluorescence added;

[0038] Figure 1 to Figure 4 The left and right images in the middle are the fluorescence-bright field overlay image and fluorescence image of the cells, respectively. The fluorescence image can display the fluorescence signal; the fluorescence-bright field overlay image is a superposition of the cell image under bright field and the fluorescence signal, which can determine whether the fluorescence signal appears on the cell and its location on the cell. DETAILED DESCRIPTION

[0039] The present invention provides an application of a protein mutant 5H-PSI or a coding gene, a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in preparing a product combined with tumor cells; the coding gene is inserted into the recombinant vector; the recombinant bacterium comprises the recombinant vector; the amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1, specifically: IVSMECKTIVSQYGEMIWDLLVSGVRPDQVCSQAGLCFVHGAQHVSSNIKTVVHRHTHGSSVGHAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0040] The protein mutant 5H-PSI of the present invention is a mutant of some glutamic acid and aspartic acid residues in the wild-type PSI protein mutated to histidine, specifically, the D at position 40 of the wild-type PSI protein is mutated to H, the E at position 54 is mutated to H, the E at position 56 is mutated to H, the E at position 58 is mutated to H, and the E at position 64 is mutated to H. The mutation of the present invention increases the number of positive charges on the surface of the PSI protein, making it easier for the PSI protein to bind to the cell membrane of tumor cells, and still has a strong activity of mediating membrane fusion under conditions of pH higher than 5.5.

[0041] The amino acid sequence of the wild-type PSI protein of the present invention is shown in SEQ ID No. 3, specifically: IVSMECKTIVSQYGEMIWDLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0042] In the present invention, the nucleotide sequence of the coding gene is preferably as shown in SEQ ID NO.2, specifically:

[0043] 5'-ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCTGGGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCAGGCAGGTCTGTGCTTCGTGCACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGT TGTTCATCGCCACACTCACGGTTCCAGCGTTGGCCACGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGGAGAAAGTCCTGGAGTACGTTAACCAGCTGTGCGAAAAAATTCCG-3'.

[0044] In the present invention, the recombinant vector preferably includes a starting vector and the coding gene; the starting vector preferably includes pET-32a(+). In the specific implementation of the present invention, it is preferred to insert the coding gene described in the above technical solution between the Msc I and Xho I restriction sites of pET-32a(+), delete the His tag carried by the original plasmid vector itself, and keep the other sequences of pET-32a(+) unchanged to obtain the recombinant vector. The plasmid pET-32a(+) described in the present invention preferably carries ampicillin resistance. The present invention has no strict requirements on the method for constructing the recombinant vector, and a method well known in the art can be used.

[0045] In the present invention, the recombinant bacteria preferably include host cells and the recombinant vector; the host cells preferably include Escherichia coli, more preferably include competent cells of Escherichia coli, and further preferably Rosetta-gamiB (DE3) pLysS. The Rosetta-gamiB (DE3) pLysS of the present invention is purchased from Shanghai Jingkang Biological Co., Ltd.

[0046] In the present invention, the method for preparing the protein mutant comprises the following steps:

[0047] Using pET-32a(+) as a starting vector, inserting the coding gene of the protein mutant 5H-PSI between the MscI and XhoI restriction sites of the pET-32a(+), deleting the coding gene of the His tag carried by the pET-32a(+), and keeping the other sequences of the pET-32a(+) unchanged, to obtain a recombinant vector;

[0048] Transforming host cells with the recombinant vector to obtain recombinant bacteria;

[0049] Cultivating the recombinant bacteria to obtain a culture solution containing the expressed protein;

[0050] The culture fluid containing the expressed protein is recovered and purified to obtain the protein mutant 5H-PSI.

[0051] The present invention first uses pET-32a(+) as a starting vector, inserts the coding gene of the protein mutant 5H-PSI between the MscI and XhoI restriction sites of the pET-32a(+), deletes the coding gene of the His tag carried by the pET-32a(+), and keeps other sequences of the pET-32a(+) unchanged, thereby obtaining a recombinant vector.

[0052] In the present invention, due to the influence of the amino acids introduced by the mutation, if the His tag carried by pET-32a(+) itself is not deleted, the TrxA fusion protein and the target protein after enzyme digestion will be bound to the Ni column and cannot be separated using the Ni-NTA chromatography column.

[0053] After obtaining the recombinant vector, the present invention transforms the recombinant vector into a host cell to obtain a recombinant bacterium.

[0054] The present invention has no strict requirements on the method of transformation, and any method well known in the art can be used.

[0055] After obtaining the recombinant bacteria, the present invention cultured the recombinant bacteria to obtain a culture solution containing the expressed protein.

[0056] The present invention preferably uses LB culture medium containing antibiotics to culture the recombinant bacteria. In the present invention, the antibiotics preferably include kanamycin (Kana), chloramphenicol (Chl), tetracycline (Tet) and ampicillin (Amp); the concentration of kanamycin in the LB culture medium is preferably 15μg / mL, the concentration of chloramphenicol is preferably 34μg / mL, the concentration of tetracycline is preferably 12.5μg / mL, and the concentration of ampicillin is preferably 50μg / mL. In the present invention, the temperature of the culture is preferably 37°C, the time of the culture is preferably 16h, and the rotation speed of the culture is preferably 250rpm. The present invention preferably uses an incubator for the culture.

[0057] After obtaining the culture fluid containing the expressed protein, the present invention recovers and purifies the culture fluid containing the expressed protein to obtain the protein mutant 5H-PSI.

[0058] In the present invention, the recovery and purification preferably use a Ni-NTA column.

[0059] The steps of recovery and purification of the present invention preferably include:

[0060] The culture solution containing the expressed protein is combined with the equilibrated Ni-NTA column, and washed with buffer 1, buffer 2 and buffer 3 in sequence, and the eluate after the buffer 3 column is collected;

[0061] Performing a first dialysis on the eluate after the buffer 3 column to obtain a dialysate;

[0062] After the dialysate is treated with enzyme digestion, it is washed with buffer 1 and buffer 3 in sequence, and the eluate after the buffer 3 column is collected;

[0063] The eluate after the buffer 3 column was dialyzed for the second time to obtain the purified protein.

[0064] In the present invention, the buffer 1 includes Tris and NaCl; the concentration of Tris in the buffer 1 is preferably 20mM, and the concentration of NaCl is 300mM; the pH of the buffer 1 is preferably 7.4. In the present invention, the buffer 2 includes Tris, NaCl and imidazole; the concentration of Tris in the buffer 2 is preferably 20mM, the concentration of NaCl is preferably 300mM, and the concentration of imidazole is preferably 20mM; the pH of the buffer 2 is preferably 7.4. In the present invention, the buffer 3 includes Tris, NaCl and imidazole; the concentration of Tris in the buffer 3 is preferably 20mM, the concentration of NaCl is preferably 300mM, and the concentration of imidazole is preferably 300mM; the pH of the buffer 3 is preferably 7.4.

[0065] In the present invention, the first dialysis preferably uses a dialysis bag with a molecular weight cutoff of 7 kDa; the second dialysis preferably uses a dialysis bag with a molecular weight cutoff of 1 kDa.

[0066] In the present invention, when the dialysate obtained from the first dialysis is subjected to enzymatic cleavage, thrombin is preferably used; the activity unit of the thrombin is 0.5U / μL; the dosage of the thrombin is preferably 1μL / mg protein. The present invention uses thrombin for enzymatic cleavage to remove the TrxA solubilizing tag connected to the protein, avoiding affecting the properties of the protein itself.

[0067] In the present invention, the tumor cells preferably include tumor cells whose outer membranes contain negatively charged phospholipids. In the present invention, the tumor cells preferably include U-87MG cells or LN229 cells. In the present invention, the binding to tumor cells includes binding to the cell membrane of tumor cells.

[0068] In the present invention, the product preferably includes a medicine.

[0069] In the present invention, the dosage form of the drug preferably includes an injection; the injection preferably includes a lyophilized powder injection and / or an intravenous injection.

[0070] In the present invention, the concentration of the protein mutant 5H-PSI in the product is preferably 0.12 to 2.4 mg / mL.

[0071] In the present invention, the working pH of the product is preferably 5.5 to 6.5.

[0072] Due to the influence of the amino acids introduced by the mutation, if the tag is not deleted, the TrxA fusion protein and the target protein after enzyme digestion will be bound to the Ni column and cannot be separated using the Ni-NTA chromatography column.

[0073] The present invention also provides a use of a protein mutant 5H-PSI or a coding gene or a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in the preparation of a tumor targeting drug and / or a tumor diagnostic reagent carrier; the coding gene is inserted into the recombinant vector; the recombinant bacterium comprises the recombinant vector; the amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1.

[0074] In the present invention, the dosage form of the tumor targeting drug and / or tumor diagnostic agent carrier includes an injection; the injection preferably includes a lyophilized powder injection and / or an intravenous injection.

[0075] The present invention also provides a tumor diagnostic reagent carrier, comprising dioleoylphosphatidylserine (DOPS) and the protein mutant 5H-PSI described in the above scheme loaded on the dioleoylphosphatidylserine.

[0076] In the present invention, DOPS in the tumor diagnostic reagent carrier exists in the state of a single phospholipid layer. In the present invention, multiple amino acids in the protein mutant 5H-PSI participate in the binding with the dioleoylphosphatidylserine phospholipid layer, which are Ile1, Val2, Met4, Glu5, Thr8, Gln12, Glu35, Leu36, Phe38, Glu64, Ala65, Pro66, Leu67, Thr69, Glu72, Met73, Val76, Trp77, Asn80, Leu98, Lys101, Ile102, Pro103.

[0077] In the present invention, the molar ratio of DOPS to the protein mutant 5H-PSI is preferably 4:1.

[0078] The present invention also provides a method for preparing the tumor diagnostic reagent carrier described in the above scheme, comprising the following steps:

[0079] The protein mutant 5H-PSI described in the above scheme is mixed with dioleoylphosphatidylserine to obtain a tumor diagnostic reagent carrier.

[0080] Before mixing the protein mutant 5H-PSI and DOPS described in the above scheme, the present invention preferably further includes dissolving DOPS in chloroform and then blowing the chloroform dry with N2 gas; DOPS is initially a powder, and the chloroform dissolution and air drying are to form a uniform phospholipid layer for subsequent vector construction operations. After blowing the chloroform dry with N2 gas and before the mixing, the present invention preferably further includes placing the chloroform-dried DOPS in a vacuum environment to convert the phospholipid layer into a single phospholipid layer. The present invention has no special restrictions on the mixing operation, which is subject to uniform mixing. After the mixing, the present invention preferably further includes ultrasonic oscillation of the mixed material; the time of the ultrasonic oscillation is preferably 30 minutes.

[0081] The present invention also provides the use of the tumor diagnostic reagent carrier described in the above scheme or the tumor diagnostic reagent carrier prepared by the preparation method in the preparation of tumor diagnostic reagents.

[0082] The present invention also provides a tumor diagnostic reagent, comprising a tumor diagnostic reagent carrier and a fluorescent dye loaded on the tumor diagnostic reagent carrier; the tumor diagnostic reagent carrier is the tumor diagnostic reagent carrier described in the above scheme or the tumor diagnostic reagent carrier prepared by the preparation method.

[0083] In the present invention, the tumor diagnostic reagent is preferably prepared by the following method:

[0084] The tumor diagnostic reagent carrier and the fluorescent dye are mixed to obtain a tumor diagnostic reagent.

[0085] In the present invention, based on the amount of the protein mutant 5H-PSI in the tumor diagnostic reagent carrier being 100 μM, the amount of the fluorescent dye is preferably 5 μL, and this amount of fluorescent dye is excessive. In the present invention, the mixing temperature is preferably 37° C.; the mixing time is preferably 30 min. After mixing the tumor diagnostic reagent carrier and the fluorescent dye, the present invention preferably further includes chromatography of the mixed material using superdex G25 to remove free fluorescent dye that is not bound to the carrier, thereby preventing cells from being stained by free dye, resulting in false positive results.

[0086] In the present invention, the fluorescent dye is preferably a far-infrared fluorescent dye; the fluorescent dye is preferably a DiD fluorescent dye; the DiD is preferably Vybrant TMDiD cell-labeling solution. DiD is a lipophilic fluorescent dye. DiD is a long-chain dialkyl carbocyanine compound that inserts into the hydrophobic layer of the phospholipid membrane through its long alkane chain, thereby marking the phospholipid DOPS layer in the carrier, and has low toxicity to cells. After DiD binds to phospholipids, the dye will diffuse laterally across the entire membrane, staining the entire membrane, and the fluorescence of the dye will also be greatly enhanced after binding to phospholipids. The present invention performs DiD staining on the phospholipid DOPS on the carrier. After the carrier reaches the lesion, it can fuse with the tumor cells and transfer the fluorescent signal to the tumor cells, thereby achieving tumor cell staining.

[0087] The tumor diagnostic reagent of the present invention can effectively mark far-infrared fluorescent dye on tumor cells. The fluorescent signal of the fluorescent dye can pass through the skin and tissues and can be detected in the in vivo imaging device to help tumor diagnosis and positioning.

[0088] To further illustrate the present invention, the application of a protein mutant 5H-PSI and its encoding gene provided by the present invention in the preparation of a drug that binds to tumor cells is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1

[0090] Construction of recombinant vector pET-32a(+)-5H-PSI

[0091] (1) Site-directed mutagenesis

[0092] Using a plasmid containing a gene encoding a wild-type PSI protein (specific nucleotide sequence: SEQ ID NO.4: 5'-ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCTGGGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCTCAGGCAGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGTTGTTGAGCGCGAGACTGAAGGTTCCAGCGTTGGCGAGGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGGAGAAAGTCCTGGAGTACGTTAACCAGCTGTGCGAAAAAATTCCG-3') as a template, site-directed mutagenesis was performed to obtain a gene encoding a wild-type PSI protein.

[0093] (2) The coding gene shown in SEQ ID NO.2 was inserted between the Msc I and Xho I restriction sites of PET-32a(+), and the His tag carried by the original plasmid vector was deleted to obtain the recombinant vector pET-32a(+)-5H-PSI.

[0094] The above steps were entrusted to Nanjing GenScript Biotech Co., Ltd.

[0095] (3) Preparation of competent cells: 500 μL of Rosetta-gami B (DE3) pLysS strain was inoculated into 50 mL LB liquid medium and cultured at 37°C, 220 rpm in a shaking incubator for 4 h. 600 Between 0.4 and 0.5, transfer the bacterial solution to a 50mL centrifuge tube, place on ice for 20 minutes, centrifuge at 4°C, 4000rpm for 10 minutes, and discard the supernatant. Add 10mL of pre-cooled 0.1M CaCl2 to the precipitate, resuspend the cell precipitate, centrifuge at 4°C, 4000rpm for 10 minutes, discard the supernatant, and repeat twice. Finally, add 3mL of pre-cooled 0.1M CaCl2, resuspend the cells again, use a pipette to distribute the competent cells, add 100μL to each 1.5mL EP tube, add an equal volume of 50% glycerol, and store at -80°C.

[0096] (4) Transformation: Take out 4 μg of the recombinant vector pET-32a(+)-5H-PSI dry powder obtained in step (2) and add 80 μL of ddH2O to dissolve. Take 10 μL of the plasmid aqueous solution and add it to the competent cells prepared in step (1), gently shake and mix, and ice bath for 25 minutes. Then place the bacterial solution in a 42°C water bath, heat shock for 45 seconds, and ice bath the bacterial solution for 2 minutes after heat shock. Then add 2 mL of LB liquid culture medium to the bacterial solution, place it in a shaker at 37°C and 220 rpm for 60 minutes. Take 50 μL of the bacterial solution and spread it on the LB solid culture medium to which 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp have been added, and culture overnight.

[0097] (5) Sequencing and preservation: Take a single colony after overnight culture, extract the plasmid, and sequence it. The sequencing results show that there is a nucleotide sequence consistent with SEQ ID NO.2, and the recombinant bacteria are obtained; the recombinant bacteria are inoculated into LB liquid culture medium to which 15μg / mL Kana, 34μg / mL Chl, 12.5μg / mL Tet and 50μg / mL Amp have been added, and cultured in a shaking incubator at 250rpm and 37℃ for 16h. Take 700μL of the bacterial solution and inject it into a cryopreservation tube, add 50% glycerol at a volume ratio of 1:1, and the glycerol bacteria are prepared.

[0098] (6) Cultivation of recombinant bacteria: Take 1 mL of glycerol bacteria and add it to 50 mL of LB liquid culture medium (containing 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp) supplemented with antibiotics, and culture it in a shaker at 37°C and 220 rpm for 8 h. Then, add 50 mL of the bacterial solution to 900 mL of LB liquid culture medium (containing 15 μg / mL Kana, 34 μg / mL Chl, 12.5 μg / mL Tet and 50 μg / mL Amp) supplemented with antibiotics, and culture it in a shaker at 37°C and 220 rpm for 3 h. Use a UV spectrophotometer to measure the OD600 of the bacterial solution to be between 0.6 and 0.8 (recorded as the bacterial solution before induction). Add 500 μL of 1M IPTG solution to the pre-induction bacterial solution, ice bath for 10 minutes, transfer to a 30°C, 200rpm shaker for 14 hours to obtain the post-induction bacterial solution, centrifuge the post-induction bacterial solution at 4°C, 4000rpm for 10 minutes, and collect the cell pellet. Resuspend the cell pellet with 50mL of 20mM tris buffer (pH 7.4). Add the resuspended bacterial solution to a high-pressure homogenizer precooled at 4°C, and break the bacteria for 3 minutes at 820bar and 860bar. After the high-pressure homogenizer is broken, the bacterial solution is recovered, centrifuged at 4°C, 12000rpm for 30 minutes, and the supernatant is collected.

[0099] (5) The supernatant after centrifugation in step (4) was added to a Ni-NTA column (which had been rinsed with a 20 mM tris buffer at pH 7.4), and placed in a shaker at 4°C and 200 rpm for 3 h of shaking. After the binding was completed, 50 mL of buffer 1 (composed of an aqueous solution of Tris and NaCl, pH 7.4, wherein the Tris concentration was 20 mM and the NaCl concentration was 300 mM), buffer 2 (composed of an aqueous solution of Tris, NaCl and imidazole, pH 7.4, wherein the Tris concentration was 20 mM, the NaCl concentration was 300 mM, and the imidazole concentration was 20 mM), and buffer 3 (composed of an aqueous solution of Tris, NaCl and imidazole, pH 7.4, wherein the Tris concentration was 20 mM, the NaCl concentration was 300 mM, and the imidazole concentration was 300 mM) were used for flow washing, and the post-column eluates were collected respectively.

[0100] (6) The eluate containing the target protein (i.e., the eluate after washing with buffer 3) was transferred to a dialysis bag with a molecular weight cutoff of 7 kDa, and dialyzed overnight in 3 L of dialysate (20 mM Tris, 100 mM NaCl) at pH 8.0. The dialyzed protein was then transferred to a 50 mL centrifuge tube, and 1 μL of thrombin (0.5 U / μL) was added per 1 mg of protein, and the protein solution was obtained after enzymatic digestion overnight;

[0101] (7) The protein solution after enzyme digestion and the washed Ni-NTA column were shaken in a shaker at 16°C and 200 rpm for 2 h, and the flow-through was collected. The Ni-NTA column after binding was washed with buffer 1 and buffer 3 in step (5) in sequence, and the eluate after washing with buffer 1 and the eluate after washing with buffer 3 were collected respectively;

[0102] (8) The eluate after washing with buffer 3 in step (7) was transferred to a 1 kDa dialysis bag and dialyzed in 3 L of pure water for 24 h to obtain an aqueous solution of the protein, which was recorded as an aqueous solution of 5H-PSI protein.

[0103] (9) Isoelectric point detection

[0104] The online prediction software ExPASy Compute pI / Mw was used to input the amino acid sequence corresponding to the coding gene shown in SEQ ID NO.2, and the theoretical isoelectric point was calculated, i.e., 6.56, indicating that the isoelectric point of the 5H-PSI protein obtained in the present invention is 6.56.

[0105] (10) Verification of cell binding effect

[0106] The aqueous solution of 5H-PSI protein separated in step (8) was concentrated to 2.4 mg / mL using a 3kD ultrafiltration tube, and 1 mL of the concentrated 5H-PSI protein solution and a bovine serum albumin (BSA) solution of equal mass concentration were taken respectively, and fluorescently labeled using a FITC kit. The two protein solutions were added to the culture medium of somatic cells 293T and tumor cells U87 MG, respectively, to a final concentration of 0.12 mg / mL of the added protein. After co-culturing with the cells for 2 hours at pH 6.5, the culture medium was removed, and the cells were washed three times with 500 μL of PBS solution, followed by adding 200 μL of pancreatic enzyme to react with the cells for 3 minutes, and the cells were resuspended with PBS buffer. Photographs were taken using an inverted fluorescence microscope, and the results are as follows: Figure 1 to Figure 4 shown.

[0107] Depend on Figure 1 and Figure 2 It can be seen that the experimental results show that compared with the control group (BSA), the tumor cells in the experimental group (5H-PSI protein solution) have stronger fluorescence, indicating that under this pH condition, 5H-PSI has better selectivity for tumor cells. For the same amount of tumor cells, compared with BSA protein, 5H-PSI binds to more cells, that is, it has stronger binding ability.

[0108] Depend on Figure 3 and Figure 4It can be seen that in somatic cell 293T, there is no obvious difference in the fluorescence intensity of the two, that is, 5H-PSI does not have a stronger binding ability to somatic cell 293T.

[0109] (11) Comparison of the binding effects of PSI and 5H-PSI on tumor cells

[0110] PSI (nucleotide sequence as shown in SEQ ID No. 3) and 5H-PSI labeled with FITC fluorescence were added to the tumor cells LN229 to a final protein concentration of 10 μM, and co-cultured with the tumor cells at pH 5.5 and pH 6.5 for 2 h, respectively. The fluorescence signal was observed using a flow cytometer. The results are as follows: Figure 5 to Figure 12 shown.

[0111] Depend on Figures 5 to 8 It can be seen that under pH 5.5 conditions, the fluorescence signal intensity of LN229 cells in the 5H-PSI experimental group was significantly stronger than that of LN229 cells in the PSI control group.

[0112] Depend on Figures 9 to 12 It can be seen that under pH 6.5 conditions, the fluorescence signal intensity of LN229 cells in the 5H-PSI experimental group was significantly stronger than that of LN229 cells in the PSI control group.

[0113] In summary, the experiments show that under the conditions of pH 5.5 and pH 6.5, 5H-PSI has a stronger binding ability with tumor cells than WT-PSI.

[0114] (12) 5H-PSI and DOPS (dioleoylphosphatidylserine) construct vectors and perform fluorescent labeling of tumor cells

[0115] Take 0.32 mg of DOPS powder and dissolve it in chloroform, then blow dry the chloroform with N2 gas. Place the sample in a vacuum environment for 3 hours, add 5H-PSI (dissolved in PBS buffer at pH 5.5) at a molar ratio of 4:1, mix thoroughly, and oscillate ultrasonically for 30 minutes. Add 5 μL DiD fluorescent dye (Vybrant TM DiD cell-labeling solution) was incubated at 37°C for 30 minutes. The incubated solution was separated using a Superdex G25 chromatography column. The specific elution process was carried out on an AKTA device under the following conditions: PBS solution with pH 5.5 was used for elution at a flow rate of 0.5 mL / min, and the post-column eluate was collected from the time when the UV detector showed an absorption peak at 280 nm to obtain a 5H-PSI-DOPS carrier.

[0116] Take 10mL of 5H-PSI-DOPS vector and 5H-PSI protein solution with the same protein concentration (both protein concentrations are 10μM), centrifuge at 180000g for 1h, and it is found that the vector group has precipitation, but the protein solution group has no precipitation. The supernatant, precipitation and protein solution of the vector group after centrifugation are taken for SDS-PAGE electrophoresis experiment. The results show that there is no protein band in the supernatant of the vector group, and the target protein band appears in the precipitation, that is, the protein and liposome complete the vector assembly. Fig.13 ).

[0117] The constructed 5H-PSI-DOPS vector was added to the culture medium of tumor cells LN229 until the final concentration of the protein 5H-PSI in the vector was 10μM. The tumor cell group added with PBS buffer was used as the negative control group (NC). The flow cytometry experiment was performed after co-culture for 4 hours under pH 6.5. The results showed that compared with the negative group, under pH 6.5, the vector group (Vector) could effectively label the tumor cells with far-infrared fluorescent dye. The fluorescent signal of this fluorescent dye can pass through the skin and tissues and can be detected in the in vivo imaging device, which helps tumor diagnosis and positioning ( Fig.14 ).

[0118] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a protein mutant 5H-PSI or a coding gene, a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in the preparation of a product that binds to tumor cells; The amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1; The coding gene is inserted into the recombinant vector; The recombinant bacteria comprises the recombinant vector.

2. The use according to claim 1, characterized in that: The tumor cells include tumor cells whose outer membranes contain phosphatidylserine; the binding with tumor cells preferably includes binding with the cell membrane of tumor cells.

3. The use according to claim 1, characterized in that: The working pH of the product is 5.5-6.

5.

4. Use of a protein mutant 5H-PSI or a coding gene or a recombinant vector or a recombinant bacterium expressing the protein mutant 5H-PSI in the preparation of tumor targeting drugs and / or tumor diagnostic reagent carriers; The amino acid sequence of the protein mutant 5H-PSI is shown in SEQ ID NO.1; The coding gene is inserted into the recombinant vector; The recombinant bacteria comprises the recombinant vector.

5. The use according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

6. A tumor diagnostic reagent carrier, characterized in that: The invention comprises dioleoylphosphatidylserine and the protein mutant 5H-PSI according to claim 1 loaded on the dioleoylphosphatidylserine.

7. The method for preparing the tumor diagnostic reagent carrier according to claim 6, characterized in that: The following steps are involved: The protein mutant 5H-PSI according to claim 1 and dioleoylphosphatidylserine are mixed to obtain a tumor diagnostic reagent carrier.

8. Use of the tumor diagnostic reagent carrier according to claim 6 or the tumor diagnostic reagent carrier prepared by the preparation method according to claim 7 in preparing a tumor diagnostic reagent.

9. A tumor diagnostic reagent, characterized in that: It comprises a tumor diagnostic reagent carrier and a fluorescent dye loaded on the tumor diagnostic reagent carrier; the tumor diagnostic reagent carrier is the tumor diagnostic reagent carrier according to claim 6 or the tumor diagnostic reagent carrier prepared by the preparation method according to claim 7.

10. The tumor diagnostic reagent according to claim 9, characterized in that: The fluorescent dye includes DiD.

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