A high-efficiency functional magnetic magnetosome (BMP-CSA)

Abstract

The application mainly adopts a chemical coupling method to directionally couple streptavidin (CSA) with an N terminal added with cysteine expressed by an exogenous gene to a magnetic magnetosome (BMP) to prepare a functional magnetic magnetosome BMP-CSA.The application has the beneficial effects that the application uses a biological nanometer magnetic bead as a solid phase carrier and selects SPDP as a crosslinking agent, so that the poor dispersibility of the magnetic beads caused by the coupling between the magnetic beads and the magnetic beads due to the crosslinking of glutaraldehyde is avoided, meanwhile, the SPDP as the crosslinking agent can directionally couple the CSA on the nanometer magnetic bead, and the function of the CSA in binding biotin is fully reserved.In addition, 1 mg of the SA magnetic bead can bind 5 μg of biotin-dsDNA (600 bp).

Description

Technical Field

[0001] This invention belongs to the field of functional magnetic body technology, specifically relating to a high-efficiency functional magnetic body (BMP-CSA). Background Technology

[0002] Magnetosomes (BMPs) are nanoparticles composed of iron(III) oxide or iron(III) sulfide, with a diameter between 30 and 100 nm, produced by biomineralization by magnetotactic bacteria. They are biomembrane-coated nanoparticles. Due to their advantages such as good biocompatibility, ease of modification, and uniform particle size, magnetosomes are widely used in the biomedical field.

[0003] Streptavidin (SA) is a tetrameric protein secreted by Streptomyces avidinii, with a size of 66 kDa. One molecule of streptavidin can bind with high specificity to four molecules of biotin, with an affinity constant of 10. 15 L / mol. Because SA lacks a glycosyl group and has a low isoelectric point, it exhibits a lower negative background than avidin in detection, significantly improving detection sensitivity. The Sa-Biotin system can couple antigens, antibodies, and nucleic acid molecules, and can also be labeled with various materials such as enzymes. Therefore, the Sa-Biotin system is used as a bioreaction amplification system for detecting antigens or antibodies. Currently, this system is widely used in enzyme immunoassay, immunohistochemistry, and molecular hybridization techniques. Existing technologies report that streptavidin can bind to magnetic particles; however, the coupling between magnetic beads after binding results in poor bead dispersion and reduces the biotin-binding function of streptavidin. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention mainly employs a chemical coupling method to directionally couple exogenously expressed streptavidin (CSA) with cysteine ​​added to its N-terminus to magnetosomes (BMP) to prepare functional magnetosomes BMP-CSA.

[0005] The first aspect of the present invention provides a streptavidin (CSA) with a cysteine ​​residue added to its N-terminus, the base sequence of which is SEQ ID NO:1 and the amino acid sequence of which is SEQ ID NO:2.

[0006] A second aspect of the present invention provides a highly efficient functional magnetosome (BMP-CSA), comprising a magnetosome (BMP) and streptavidin (CSA) with a cysteine ​​residue added to the N-terminus of the magnetosome via a bifunctional reagent, wherein the base sequence of the streptavidin (CSA) with the cysteine ​​residue added to the N-terminus is SEQ ID NO:1 and its amino acid sequence is SEQ ID NO:2.

[0007] In a further technical solution, the bifunctional reagent includes succinimide 3-(2-pyridyldithio)-propionate (SPDP).

[0008] A third aspect of the present invention provides a method for manufacturing a high-efficiency functional magnetic microstructure (BMP-CSA), comprising the following steps:

[0009] Succinimide 3-(2-pyridyldithio)-propionate (SPDP) reacts chemically with the surface of a magnetosome (BMP) to form a BMP-SPDP complex containing pyridinium dithiol.

[0010] A BMP-SPDP complex containing pyridine dithiol was coupled with streptavidin (CSA) with cysteine ​​added to the N-terminus to obtain a BMP-CSA magnetosome.

[0011] Further technical solutions include the following steps:

[0012] The target gene streptavidin was obtained by PCR using primers with the base sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as templates extracted from Streptomyces genome;

[0013] The primer for SEQ ID NO:5 is:

[0014] PET-28a-SAR:

[0015] 5'-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3';

[0016] The primer for SEQ ID NO:6 is:

[0017] PET-28a-C-SAF:

[0018] 5'-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3';

[0019] pET28(a+) was double-digested with restriction endonucleases BamHⅠ and HindⅢ, and streptavidin with cysteine ​​added to the N-terminus of the target gene was cloned into the vector to obtain the vector pET28-CSA.

[0020] pET28-CSA was transformed into expression strain E. coli BL21 by heat shock transformation. The cells were plated on kanamycin resistance plates, and colony PCR was performed using T7 with the base sequence SEQ ID NO:7 and T7 term.rev with the base sequence SEQ ID NO:8 to verify the results. Approximately 500 bp of the target condition was amplified, and the correct strain E. coli BL2-CSA was screened.

[0021] The base sequence of T7 is:

[0022] 5'-TAATACGACTCACTATAGGG-3';

[0023] The base sequence of T7 term.rev is:

[0024] 5'-TGCTAGTTATTGCTCAGCGG-3';

[0025] The correctly screened strain E. coli BL2-CSA was cultured in shake flask medium, and IPTG was added to a final concentration of 1 mM to induce expression. The protein expression was observed by SDS-PAGE.

[0026] E. coli BL2-CSA was expressed in shake flasks and purified using a nickel affinity chromatography column to obtain streptavidin (CSA) with an N-terminus cysteine ​​residue added, which was exogenously expressed in E. coli.

[0027] In a further technical solution, the primers shown in SEQ ID NO:3 and SEQ ID NO:4 are used in PCR:

[0028] The PCR reaction system (50 μl) is as follows:

[0029]

[0030] The PCR procedure is as follows:

[0031]

[0032] The beneficial effects of this invention are as follows: This invention uses bio-derived magnetic nanobeads as a solid-phase carrier and selects SPDP as a cross-linking agent, thereby avoiding the poor dispersion of magnetic beads caused by glutaraldehyde cross-linking and resulting in coupling between magnetic beads. Simultaneously, using SPDP as a cross-linking agent allows for the directional coupling of CSA (directional coupling mainly utilizes the thiol group at the N-terminus of the CSA protein and the bifunctional reagent SPDP to couple CSA into an orderly arrangement on the magnetosomes) onto the magnetic nanobeads, fully preserving the biotin-binding function of CSA. Furthermore, 1 mg of SA magnetic beads in this invention can bind 5 μg of biotin-dsDNA (600 bp). Attached Figure Description

[0033] Figure 1 SDS-PAGE electrophoresis was used to examine the expression of SA and CSA;

[0034] In the figure, ① represents before induction with 1mM IPTG, and ② represents after induction with 1mM IPTG.

[0035] Figure 2 SDS-PAGE electrophoresis was used to verify the purification of SA protein;

[0036] Figure 3 SDS-PAGE electrophoresis was used to verify the purification of CSA protein;

[0037] Figure 4 Agarose gel electrophoresis was used to examine the activity of purified proteins SA and CSA;

[0038] Figure 4 In the formula: 1: Biotinylated DNA; 2: 40 μL SA co-incubated with biotinylated DNA; 3: 40 μL CSA co-incubated with biotinylated DNA; 4: 80 μL SA co-incubated with biotinylated DNA; 5: 80 μL CSA co-incubated with biotinylated DNA; 6: Unbiotinylated DNA; 7: 40 μL SA co-incubated with unbiotinylated DNA; 8: 40 μL CSA co-incubated with unbiotinylated DNA; 9: 80 μL SA co-incubated with unbiotinylated DNA; 10: 80 μL CSA co-incubated with unbiotinylated DNA.

[0039] Figure 5 Agarose gel electrophoresis was used to detect the ability of BMP-CSA to bind biotin.

[0040] Figure 5 In the following samples: 1: Control: 10 μl; 2: Control: 5 μl; 3: Mag-SA: 5 μl; 4: Mag-SA: 10 μl; 5: Silicon-based magnetic beads: 5 μl; 6: Silicon-based magnetic beads: 10 μl. Detailed Implementation

[0041] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0042] Example 1: Exogenous expression of streptavidin in Escherichia coli

[0043] 1. Target gene

[0044] The base sequence of streptavidin (CSA) with a cysteine ​​residue added to its N-terminus is SEQ ID NO:1, and its amino acid sequence is SEQ ID NO:2. This streptavidin (CSA) with a cysteine ​​residue added to its N-terminus introduces a thiol group (SH) as the target gene.

[0045] The base sequence (SEQ ID NO:1) of streptavidin (CSA) with a cysteine ​​residue added to its N-terminus is as follows:

[0046] 5'--3'.

[0047] The amino acid sequence (SEQ ID NO:2) of streptavidin (CSA) with a cysteine ​​residue added to its N-terminus is as follows:

[0048] CGGGGSGGGGSGGGGSDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVK PSAASIDAAKKAGVNNGNPLDAVQQ.

[0049] The base sequence of streptavidin (SA) is SEQ ID NO:3, which serves as the control gene.

[0050] The base sequence of streptavidin (SA) (SEQ ID NO:3) is as follows:

[0051] 5'-GACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGCATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAA GAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGG AAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCCGTTCAGCAG-3'.

[0052] 2. Primer design

[0053] Primers were designed using a seamless cloning molecular cloning method as follows:

[0054] PET-28a-SAF (SEQ ID NO:4):

[0055] 5'-TGGACAGCAAATGGGTCGCGGATCCATGGACCCCTCCAAGGACTCG-3'

[0056] PET-28a-SAR (SEQ ID NO:5):

[0057] 5'-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3'

[0058] PET-28a-C-SAF (SEQ ID NO:6):

[0059] 5'-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3'

[0060] 3. Plasmid construction

[0061] The genome of Streptomyces avidinii was extracted as a template and PCR was performed using the above primers to obtain the control gene and the target gene. At the same time, pET28(a+) was double-digested with restriction endonucleases BamHⅠ and HindⅢ. The control gene and the target gene were cloned into the vectors respectively, and the resulting expression vectors were named pET28-SA and pET28-CSA respectively.

[0062] The PCR reaction system (50 μl) is as follows:

[0063]

[0064] The PCR procedure is as follows:

[0065]

[0066] 4. Strain construction

[0067] The control and target genes in expression vectors pET28-SA and pET28-CSA were sequenced. The correctly sequenced expression vectors pET28-SA and pET28-CSA were transformed into expression strain E. coli BL21 by heat shock transformation. The cells were plated on kanamycin resistance plates and verified by T7 and T7 term.rev colony PCR. If the target gene was amplified to approximately 500 bp, it was considered to be the correct strain E. coli BL2-SA or E. coli BL2-CSA.

[0068] T7 (SEQ ID NO:7): 5'-TAATACGACTCACTATAGGG-3';

[0069] T7 term.rev (SEQ ID NO:8): 5'-TGCTAGTTATTGCTCAGCGG-3'.

[0070] 5. Cultivation and purification

[0071] The selected strains were cultured in shake flasks, and IPTG was added to a final concentration of 1 mM to induce protein expression. SDS-PAGE was used to observe protein expression, and the results are as follows: Figure 1 As shown.

[0072] After shake-flask expression of E. coli BL2-SA and E. coli BL2-CSA, purification was performed using a nickel affinity chromatography column to obtain pure, soluble target proteins SA and CSA. Figure 2 , Figure 3 As shown:

[0073] The purified CSA and SA proteins were then used for activity verification. First, biotin-labeled primers were designed. For ease of experimentation, 16S universal primers were used to label biotin, producing a 600bp product. A 400bp unlabeled PCR product was also used as a control. The primer sequences are as follows:

[0074] Biotin F(SEQ ID NO:9):5'-TGCGATAAGCGTCGGTAAGG-3'5' Modified Biotin

[0075] Biotin R(SEQ ID NO:10):5'-TACCCTGCAACTTAACGCCC-3'

[0076] The purified SA and CSA proteins were incubated with biotinylated DNA, and the activities of the two recombinant proteins were then tested. The results showed... Figure 4 As shown.

[0077] SA and CSA were incubated with biotinylated DNA and non-biotinylated DNA, respectively. The incubated DNA was then subjected to nucleic acid gel electrophoresis. The results showed that SA and CSA showed delayed bands in nucleic acid gel electrophoresis after incubation with biotinylated DNA, while no delayed bands were observed with non-biotinylated DNA. This indicates that SA and CSA can bind to biotinylated DNA.

[0078] Example 2: Fabrication of High-Efficiency Functional Magnetoids (BMP-CSA)

[0079] The proteins and lipids on the biomembrane of magnetosomes result in a large number of amino groups on the magnetosome surface. Studies have shown that a single magnetosome contains approximately 10,000 amino groups, providing favorable conditions for magnetosome modification. The bifunctional reagent succinimide 3-(2-pyridyldithio)-propionate (SPDP) is used as a crosslinking agent to directionally immobilize CSA on the magnetosome surface. The succinimide ester (NHS-) active group in SPDP reacts with the -NH2 groups on the magnetosome surface to generate a BMP-SPDP complex containing pyridinium dithiol. Subsequently, the CSA is directionally coupled to the magnetosome surface by reacting with the -SH groups present on the CSA surface to form disulfide bonds, resulting in a highly efficient functional magnetosome (BMP-CSA). The specific operation steps are as follows:

[0080] 1) The treated magnetic particles were resuspended in 0.01M HEPES (pH=7.4) and then washed. This step was repeated three times.

[0081] 2) Weigh an appropriate amount of succinimide 3-(2-pyridyldithio)-propionate (SPDP) and dissolve it in 100 μl of DMSO. After complete dissolution, add 900 μl of 0.01 M HEPES and adjust the final concentration to 1 mM. Ultrasonicate the centrifuge tube for 1 min (while ultrasonicating), then pause for 1 min and repeat 30 times.

[0082] 3) Place the above magnetosomes on a magnetic rack for adsorption, discard the supernatant, add 500 μl of 0.01 M HEPES to wash six times to remove unbound SPDP, add 2 mg / ml CSA (dissolved in HEPES solution) to the EP tube, ultrasonically clean for 1 min, pause for 1 min, repeat 30 times to obtain functional magnetosomes BMP-CSA.

[0083] Experimental Example 1: Preliminary Test of Streptavidin Magnetic Bead Binding to Biotin

[0084] Take 30 micrograms of high-efficiency functional magnetic granules (BMP-CSA) and incubate them together with biotin-labeled PCR products (5 μl, 10 μl). Collect the supernatant after incubation and use agarose gel electrophoresis to detect the ability of streptavidin magnetic beads to bind biotin. The detection results are as follows: Figure 5 As shown.

[0085] Image captions: 1: Control: 10 μl; 2: Control: 5 μl; 3: Mag-CSA: 5 μl; 4: Mag-CSA: 10 μl; 5: Silicon-based magnetic beads: 5 μl; 6: Silicon-based magnetic beads: 10 μl;

[0086] As shown in the figure, after BMP-CSA was co-incubated with biotinylated DNA, nucleic acid gel electrophoresis showed that the supernatant did not contain DNA, while the blank control group (1, 2) and the negative control group (5, 6) had obvious DNA bands. This result fully demonstrates that all the DNA of biotin was bound to BMP-CSA.

[0087] Experimental Example 2: Detection of streptavidin magnetic beads binding to biotinylated DNA

[0088] Biotin-labeled 16S universal primers were used to amplify E. coli as a template. The amplification products were used to test the biotin-binding content of streptavidin magnetic beads. At the same time, unlabeled 16S primers were amplified as a control.

[0089] The primer sequences are as follows:

[0090] 600biotin-F (SEQ ID NO:11): TGCGATAAGCGTCGGTAAGG

[0091] 600-R(SEQ ID NO:12):TACCCTGCAACTTAACGCCC

[0092] The amplified PCR product was recovered using a Takara gel recovery kit to obtain a pure PCR product, which was then used to detect the content of streptavidin-conjugated biotin-dsDNA fragments.

[0093] The specific steps are as follows:

[0094] 1. Place 1 mg of magnetic beads in an EP tube, resuspend with PBS, and wash three times with magnetic adsorption.

[0095] 2. Add purified biotin-dsDNA fragments (1:100 mass ratio with magnetic beads) to an EP tube containing magnetic beads. Incubate the magnetic beads in a shaker (25℃, 200rpm) for 10min. After magnetic adsorption, detect the DNA content in the supernatant using a micro-ultraviolet spectrophotometer.

[0096] During the testing process, each sample was tested in triplicate, and a biotin-dsDNA fragment without biotin labeling was set up as a control. An appropriate amount of DNA was collected and its concentration was measured before incubation with the magnetic beads. The biotin binding capacity of the magnetic beads was calculated by the concentration difference before and after streptavidin magnetic bead coupling.

[0097] The test results are shown in the table below:

[0098]

[0099] The results showed that 1 mg of SA magnetic beads could bind 5 μg of biotin-dsDNA (600 bp).

[0100] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Streptavidin with a cysteine ​​residue added to its N-terminus, characterized in that, The base sequence of the gene encoding the streptavidin with a cysteine ​​residue added to its N-terminus is SEQ ID NO:1, and its amino acid sequence is SEQ ID NO:

2.

2. A BMP-CSA magnetic core, characterized in that, The invention includes a magnetosome and streptavidin with a cysteine ​​residue added to the N-terminus of the magnetosome via a bifunctional reagent. The base sequence of the streptavidin with the cysteine ​​residue added to the N-terminus is SEQ ID NO:1, and its amino acid sequence is SEQ ID NO:

2. The bifunctional reagent includes succinimide 3-(2-pyridyldithio)-propionate; The BMP-CSA magnet is prepared by the following steps: Succinimide 3-(2-pyridyldithio)-propionate reacts chemically with the surface of the magnetosome to form a BMP-SPDP complex containing pyridine dithiol; BMP-CSA magnetosomes were obtained by coupling a pyridine dithiol BMP-SPDP complex with streptavidin containing cysteine ​​at the N-terminus.

3. The method for preparing streptavidin with an N-terminus cysteine ​​added as described in claim 1, characterized in that, Includes the following steps: The target gene was obtained by PCR using the primers shown in SEQ ID NO:5 and SEQ ID NO:6 as templates extracted from Streptomyces genome; The primer for SEQ ID NO:5 is: PET-28a-SAR: 5'-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3'; The primer for SEQ ID NO:6 is: PET-28a-C-SAF: 5'-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3'; pET28(a+) was double-digested with restriction endonucleases BamHⅠ and Hind Ⅲ, and the target gene was cloned into the vector to obtain the vector pET28-CSA; pET28-CSA was transformed into expression strain E. coli BL21 by heat shock transformation. The cells were plated on kanamycin resistance plates, and colony PCR was performed using T7 cells with the base sequence SEQ ID NO:7 and T7 term.rev cells with the base sequence SEQ ID NO:8 to verify the results. Approximately 500 bp of the target condition was amplified, and the correct strain E. coli BL2-CSA was screened. The base sequence of T7 is: 5'-TAATACGACTCACTATAGGG-3'; The base sequence of T7 term.rev is: 5'-TGCTAGTTATTGCTCAGCGG-3'; The correctly screened strain E. coli BL2-CSA was cultured in shake flask medium, and IPTG was added to a final concentration of 1 mM to induce expression. The protein expression was observed by SDS-PAGE. E. coli BL2-CSA was expressed in shake flasks and purified using a nickel affinity chromatography column to obtain streptavidin with an N-terminus cysteine ​​residue added to the exogenous expression in E. coli.

4. The manufacturing method as described in claim 3, characterized in that, PCR is being performed on SEQ ID NO:5 and SEQ ID NO:

6. The PCR reaction system is as follows: 5 μl of DNA polymerase; 10×Buffer 5μl; 10μM PET-28a-SAF / PET-28a-C-SAF 2.5μl; 10 μM PET-28a-SAR 2.5 μl; Template 2μl; ddH2O 33μl; The PCR procedure is as follows: 95℃ for 5 minutes; 95℃ 30 s； 60℃ 30s； 72℃ for 1 min; 72℃ for 10 min; Store at 25°C.

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