CN116041455A

CN116041455A - High-efficiency functional magnetosome (BMP-CSA)

Info

Publication number: CN116041455A
Application number: CN202211234696.5A
Authority: CN
Inventors: 张金菊; 王红光
Original assignee: Guoke Rongzhi Suzhou Biomedical Technology Co ltd
Current assignee: Guoke Rongzhi Suzhou Biomedical Technology Co ltd
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2023-05-02

Abstract

The invention mainly adopts a chemical coupling method to directionally couple the N-terminal of exogenously expressed streptavidin (CSA) added with cysteine to magnetosome (BMP) to prepare the functional magnetosome BMP-CSA. The invention has the beneficial effects that: according to the invention, the biogenic nano magnetic beads are used as solid phase carriers, and SPDP is used as a cross-linking agent, so that poor magnetic bead dispersibility caused by coupling between magnetic beads due to glutaraldehyde cross-linking is avoided, and meanwhile, CSA can be directionally coupled on the nano magnetic beads by using the SPDP as the cross-linking agent, so that the function of combining CSA with biotin is fully reserved. In addition, 1mg SA magnetic beads of the invention can bind 5. Mu.g of biotin-dsDNA (600 bp).

Description

High-efficiency functional magnetosome (BMP-CSA)

Technical Field

The invention belongs to the technical field of functional magnetosomes, and particularly relates to a high-efficiency functional magnetosomes (BMP-CSA).

Background

Magnetosomes (BMP) are produced by biomineralization of magnetotactic bacteria, a nanoparticle coated with a biofilm and having a particle size between 30 and 100nm, consisting of ferroferric oxide or ferroferric sulfide. The magnetosome has the advantages of good biocompatibility, easy modification, uniform particle size and the like, so that the magnetosome is widely applied to the biomedical field.

Streptavidin (SA) is a tetrameric protein secreted by Streptomyces (Streptomyces avidinii) and has a size of 66kDa. One molecule of streptavidin can be combined with four molecules of biotin with high specificity, and the affinity constant of the streptavidin and the four molecules of biotin is 10 ¹⁵ L/mol. As SA has no glycosyl and low isoelectric point, the SA has lower negative background than avidin in detection, thus greatly improving the detection sensitivity. The Sa-Biotin system (streptavidin-Biotin system) can be coupled with antigen, antibody and nucleic acid molecule, and can be labeled by various materials such as enzyme. Therefore, the Sa-Biotin system is used as a biological reaction amplification system for detecting an antigen or an antibody. At present, the system is widely applied to the technologies of enzyme immunity, immunohistochemistry, molecular hybridization and the like. The prior art reports that streptavidin can bind to magnetic particles, however, the magnetic beads have poor dispersibility due to coupling between the magnetic beads after binding, and the function of streptavidin binding to biotin is also reduced.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention mainly adopts a chemical coupling method to directionally couple the N-terminal of exogenously expressed streptavidin (CSA) added with cysteine to magnetosome (BMP) to prepare the functional magnetosome BMP-CSA.

In a first aspect, the invention provides a streptavidin (CSA) with cysteine added at the N-terminal, wherein the base sequence is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2.

The second aspect of the invention provides a high-efficiency functional magnetosome (BMP-CSA), which comprises a magnetosome (BMP) and streptavidin (CSA) with cysteine added at the N end of the surface of the magnetosome through double-functional reagent coupling, wherein the base sequence of the streptavidin (CSA) with the cysteine added at the N end is SEQ ID NO:1, and the amino acid sequence of the streptavidin (CSA) with the cysteine added at the N end is SEQ ID NO:2.

In a further embodiment, the bifunctional reagent comprises succinimidyl 3- (2-pyridyldithio) -propionate (SPDP).

In a third aspect, the invention provides a method for preparing a highly efficient functional magnetosome (BMP-CSA), comprising the steps of:

the succinimide 3- (2-pyridyldithio) -propionate (SPDP) reacts with the surface of magnetosome (BMP) to generate a BMP-SPDP complex containing pyridine dithiol;

and coupling the pyridine dithiol BMP-SPDP compound with streptavidin (CSA) with cysteine added at the N end to obtain the BMP-CSA magnetosome.

In a further technical scheme, the method comprises the following steps:

extracting streptomycete genome as a template, and carrying out PCR (polymerase chain reaction) by using primers with base sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 to obtain target gene streptavidin;

the primer of SEQ ID NO. 5 is:

PET-28a-SAR：

5’-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3’；

the primer of SEQ ID NO. 6 is:

PET-28a-C-SAF：

5’-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3’；

double-enzyme cutting pET28 (a+) by using restriction endonucleases BamHI and HindIII, cloning streptavidin with cysteine added into the N end of a target gene onto a vector, and obtaining a vector pET28-CSA;

pET28-CSA is transformed into an expression strain E.coli BL21 by a heat shock transformation method, a kanamycin resistance plate is coated, a T7 with a base sequence of SEQ ID NO. 7 and a T7 term.rev colony PCR with a base sequence of SEQ ID NO. 8 are selected for verification, target conditions of about 500bp are amplified, and a correct strain E.coli BL2-CSA is screened;

the base sequence of T7 is:

5’-TAATACGACTCACTATAGGG-3’；

the base sequence of T7 term. Rev is:

5’-TGCTAGTTATTGCTCAGCGG-3’；

culturing the correctly screened strain E.coli BL2-CSA in a shake flask culture medium, adding IPTG with the final concentration of 1mM for induction expression, and observing the protein expression condition by SDS-PAGE;

shake flask expression is carried out on E.coli BL2-CSA, and nickel affinity chromatography column is selected for purification, thus obtaining streptavidin (CSA) with cysteine added at the N-terminal of exogenous expression of escherichia coli.

In a further technical scheme, the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used for PCR:

the PCR reaction (50. Mu.l) was as follows:

the PCR procedure was as follows:

the invention has the beneficial effects that: the invention takes the biogenic nano magnetic beads as solid phase carriers and adopts SPDP as a cross-linking agent, thereby avoiding poor magnetic bead dispersibility caused by coupling between magnetic beads due to glutaraldehyde cross-linking, and simultaneously, CSA can be directionally coupled (the directional coupling mainly uses sulfydryl at the N end of CSA protein and the SPDP as a bifunctional reagent for coupling, so that the CSA is orderly arranged on magnetic corpuscles) on the nano magnetic beads by utilizing the SPDP as the cross-linking agent, and fully retaining the function of combining the CSA with biotin. In addition, 1mg SA magnetic beads of the invention can bind 5. Mu.g of biotin-dsDNA (600 bp).

Drawings

FIG. 1SDS-PAGE electrophoresis to examine the expression of SA and CSA;

in the figure, (1) represents 1mM IPTG induction before, (2) represents 1mM IPTG induction after.

FIG. 2SDS-PAGE electrophoresis to examine purification of SA protein;

FIG. 3SDS-PAGE electrophoresis to examine the purification of CSA protein;

FIG. 4 agarose gel electrophoresis to examine the activity of purified proteins SA and CSA;

in FIG. 4, 1, biotinylated DNA; 2:40. Mu.L SA was incubated with biotinylated DNA; 3:40. Mu.L of CSA were incubated with biotinylated DNA; 4:80. Mu.L SA was incubated with biotinylated DNA; mu.L of CSA was incubated with biotinylated DNA; 6, DNA which is not biotinylated; 7:40. Mu.L SA was incubated with non-biotinylated DNA; 8:40. Mu.L of CSA were incubated with non-biotinylated DNA; mu.L SA was incubated with non-biotinylated DNA; mu.L of CSA was incubated with non-biotinylated DNA.

FIG. 5 agarose gel electrophoresis to examine the ability of BMP-CSA to bind biotin;

in fig. 5: 1: control: 10 μl;2: control: 5 μl;3: mag-SA 5. Mu.l; 4:Mag-SA 10. Mu.l; 5, silicon-based magnetic beads: 5 μl;6, silicon-based magnetic beads: 10 μl.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

EXAMPLE 1 exogenous expression of streptavidin by E.coli

1. Target gene

The base sequence of streptavidin (CSA) added with cysteine at the N end is SEQ ID NO. 1, the amino acid sequence of which is SEQ ID NO. 2, and the streptavidin (CSA) added with cysteine at the N end is introduced Sulfhydryl (SH) added at the N end of Streptavidin (SA) as a target gene.

The base sequence (SEQ ID NO: 1) of streptavidin (CSA) with cysteine added to the N-terminus is as follows:

5’-TGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGCATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCCGTTCAGCAG-3’。

the amino acid sequence (SEQ ID NO: 2) of streptavidin (CSA) with cysteine added to the N-terminus is as follows:

CGGGGSGGGGSGGGGSDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVK PSAASIDAAKKAGVNNGNPLDAVQQ。

the base sequence of Streptavidin (SA) is SEQ ID NO. 3, which serves as a control gene.

The base sequence of Streptavidin (SA) (SEQ ID NO: 3) is as follows:

5’-GACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGCATCACCGGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCCGTTCAGCAG-3’。

2. primer design

The primers were designed by the molecular cloning method using seamless cloning as follows:

PET-28a-SAF(SEQ ID NO:4)：

5’-TGGACAGCAAATGGGTCGCGGATCCATGGACCCCTCCAAGGACTCG-3’

PET-28a-SAR(SEQ ID NO:5)：

5’-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3’

PET-28a-C-SAF(SEQ ID NO:6)：

5’-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3’

3. plasmid construction

Extracting Streptomyces avidinii genome as a template, carrying out PCR by using the primers to obtain a control gene and a target gene, carrying out double digestion on pET28 (a+) by using restriction endonucleases BamHI and HindIII, cloning the control gene and the target gene onto vectors respectively, and naming the obtained expression vectors as pET28-SA and pET28-CSA respectively.

The PCR reaction (50. Mu.l) was as follows:

the PCR procedure was as follows:

4. strain construction

The control genes and the target genes in the expression vectors pET28-SA and pET28-CSA are sequenced, the expression vectors pET28-SA and pET28-CSA which are sequenced correctly are transformed into the expression strain E.coli BL21 by a heat shock transformation method, a kanamycin resistance flat plate is coated, and T7 term.rev colony PCR is selected for verification, if target conditions of about 500bp are amplified, the target conditions are the correct strains E.coli BL2-SA and E.coli BL2-CSA.

T7(SEQ ID NO:7)：5’-TAATACGACTCACTATAGGG-3’；

T7 term.rev(SEQ ID NO:8)：5’-TGCTAGTTATTGCTCAGCGG-3’。

5. Cultivation and purification

The correctly selected strains were cultured in shake flask medium, and induced by adding IPTG at a final concentration of 1mM, and protein expression was observed by SDS-PAGE, and the results are shown in FIG. 1.

After shake flask expression is carried out on E.coli BL2-SA and E.coli BL2-CSA, a nickel affinity chromatographic column is selected for purification, so that pure target proteins SA and CSA which are soluble proteins are obtained, and the results are shown in figures 2 and 3:

the activity verification of the purified CSA and SA proteins is carried out, a biotin-marked primer is designed firstly, for the convenience of experiments, 16S universal primer is selected to mark biotin, the size of the product is 600bp, meanwhile, 400bp PCR products without biotin marks are set as a reference, and the primer sequences are as follows:

biotin F (SEQ ID NO: 9) 5' -TGCGATAAGCGTCGGTAAGG-3'5' modified Biotin

Biotin R(SEQ ID NO:10):5’-TACCCTGCAACTTAACGCCC-3’

The activity of both recombinant proteins was examined after incubation of the purified SA and CSA proteins with biotinylated DNA, and the results are shown in FIG. 4.

SA and CSA are respectively incubated with biotinylated DNA and non-biotinylated DNA, and the incubated DNA is subjected to a nucleic acid gel electrophoresis experiment, so that the result shows that the SA and CSA show band hysteresis after being incubated with the biotinylated DNA, and no hysteresis band appears with non-biotin, thereby indicating that SA and CSA can be combined with the biotinylated DNA.

EXAMPLE 2 efficient functional magnetosome (BMP-CSA) preparation

Proteins and lipids on the biological membrane of the magnetosome cause the surface of the magnetosome to contain a large number of amino groups, and studies have shown that a single magnetosome contains about ten thousand amino groups, which provides powerful conditions for modification of the magnetosome. The method comprises the following steps of (1) directionally fixing CSA on the surface of a magnetosome by taking a bifunctional reagent succinimidyl 3- (2-pyridyldithio) -propionate (SPDP) as a cross-linking agent of the two, reacting a succinimidyl ester (NHS-) active group in the SPDP with an-NH 2 group on the surface of the magnetosome to generate a BMP-SPDP complex containing pyridine dithiol, and then directionally coupling the CSA on the surface of the magnetosome by generating disulfide bonds through a-SH reaction with the surface of the CSA to obtain the high-efficiency functional magnetosome (BMP-CSA), wherein the specific operation steps are as follows:

1) The above treated magnetosomes were resuspended in 0.01M HEPES (ph=7.4) and washed, and this step was repeated three times.

2) A proper amount of succinimidyl 3- (2-pyridyldithio) -propionate SPDP is weighed and dissolved in 100 μl DMSO, 900 μl of 0.01M HEPES is added after complete dissolution, the final concentration is adjusted to 1mM, and the ultrasonic cleaner is operated for 1min (ultrasonic vibration centrifuge tube is carried out while ultrasonic), intermittent for 1min, and repeated for 30 times.

3) Placing the magnetosome on a magnetic frame for adsorption, discarding the supernatant, adding 500 mu.l of 0.01M HEPES for cleaning six times, washing off unbound SPDP, adding 2mg/ml CSA (dissolved in HEPES solution) into an EP tube, performing ultrasonic operation in an ultrasonic cleaner for 1min, intermittently for 1min, and repeating for 30 times to obtain the functional magnetosome BMP-CSA.

Test example 1 preliminary test of streptavidin magnetic beads bound to biotin

30 microgram of high-efficiency functional magnetosome (BMP-CSA) is incubated with biotin-labeled PCR (5 microliter, 10 microliter) products, and the incubated supernatant is subjected to agarose gel electrophoresis to detect the binding capacity of streptavidin magnetic beads to biotin, and the detection result is shown in figure 5.

Picture description: 1: control: 10 μl;2: control: 5 μl;3: mag-CSA 5. Mu.l; 4:Mag-CSA, 10 μl;5, silicon-based magnetic beads: 5 μl;6, silicon-based magnetic beads: 10 μl;

as can be seen from the figure, the nucleic acid gel electrophoresis showed no DNA in the supernatant after incubation with biotinylated DNA, whereas the blank (1, 2) and negative control (5, 6) had distinct DNA bands, which is a good indication of total binding of biotin DNA to BMP-CSA.

Test example 2 streptavidin magnetic beads combined with biotinylated DNA assay

The biotin-labeled 16s universal primer is used for amplification by taking escherichia coli as a template, the amplified product is used for detecting the content of the streptavidin magnetic beads combined with biotin, and the amplification of the 16s without the biotin label is used as a control.

The primer sequences were as follows:

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

600-R(SEQ ID NO:12)：TACCCTGCAACTTAACGCCC

and (3) recovering the amplified PCR product by using a Takara gel recovery kit to obtain a pure PCR product for detecting the content of the streptavidin magnetic bead coupled biotin-dsDNA fragment.

The specific operation steps are as follows:

1. placing 1mg of magnetic beads into an EP tube, and re-suspending and magnetic-sucking three times with PBS

2. Purified biotin-dsDNA fragments (mass ratio of magnetic beads: 1:100) were added to EP tube containing magnetic beads, the magnetic beads were incubated in a shaker (25 ℃ C., 200 rpm) for 10min, and the DNA content of the supernatant was detected with a micro-UV spectrophotometer after magnetic adsorption.

In the detection process, three repeats are carried out on each sample, meanwhile, biotin-dsDNA fragments without biotin marks are set for comparison, a proper amount of DNA is reserved before incubation with the magnetic beads, the concentration is detected, and the capacity of the magnetic beads to bind biotin is calculated through the concentration difference before and after coupling of the streptavidin magnetic beads.

The test results are shown in the following table:

the results show that: 1mg SA beads bind 5. Mu.g biotin-dsDNA (600 bp).

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

The N-terminal cysteine added streptavidin (CSA) is characterized in that the base sequence of the N-terminal cysteine added streptavidin (CSA) is SEQ ID NO. 1, and the amino acid sequence thereof is SEQ ID NO. 2.
2. The magnetosome is characterized by comprising a magnetosome (BMP) and streptavidin (CSA) with cysteine added at the N end of the surface of the magnetosome, wherein the base sequence of the streptavidin (CSA) with the cysteine added at the N end is SEQ ID NO:1, and the amino acid sequence of the streptavidin (CSA) with the cysteine added at the N end is SEQ ID NO:2.
3. The BMP-CSA magnetosome as in claim 2, wherein the bifunctional reagent comprises succinimidyl 3- (2-pyridyldithio) -propionate (SPDP).
4. A method of preparing a magnetosome according to any one of claims 2 to 3, wherein said BMP-CSA magnetosome is prepared by the steps of:

the succinimide 3- (2-pyridyldithio) -propionate (SPDP) reacts with the surface of magnetosome (BMP) to generate a BMP-SPDP complex containing pyridine dithiol;

and coupling the pyridine dithiol BMP-SPDP compound with streptavidin (CSA) with cysteine added at the N end to obtain the BMP-CSA magnetosome.
5. The method for producing streptavidin (CSA) having a cysteine added to the N-terminus according to any one of claims 2-4, comprising the steps of:

extracting streptomycete genome as a template, and carrying out PCR (polymerase chain reaction) by using primers with base sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 to obtain target gene streptavidin;

the primer of SEQ ID NO. 5 is:

PET-28a-SAR：

5’-GGTGCTCGAGTGCGGCCGCAAGCTTCTACTGCTGAACGGCGTC-3’；

the primer of SEQ ID NO. 6 is:

PET-28a-C-SAF：

5’-TGGACAGCAAATGGGTCGCGGATCCATGTGCGGTGGCGGAGGGTCTGGTGGCGGAGGGTCCGGTGGCGGAGGGTCAGACCCCTCCAAGGACTCG-3’；

double-enzyme cutting pET28 (a+) by using restriction endonucleases BamHI and HindIII, cloning target gene streptavidin to a vector, and obtaining a vector pET28-CSA;

pET28-CSA is transformed into an expression strain E.coli BL21 by a heat shock transformation method, a kanamycin resistance plate is coated, a T7 with a base sequence of SEQ ID NO. 7 and a T7 term.rev colony PCR with a base sequence of SEQ ID NO. 8 are selected for verification, target conditions of about 500bp are amplified, and a correct strain E.coli BL2-CSA is screened;

the base sequence of T7 is:

5’-TAATACGACTCACTATAGGG-3’；

the base sequence of T7 term. Rev is:

5’-TGCTAGTTATTGCTCAGCGG-3’；

culturing the correctly screened strain E.coli BL2-CSA in a shake flask culture medium, adding IPTG with the final concentration of 1mM for induction expression, and observing the protein expression condition by SDS-PAGE;

shake flask expression is carried out on E.coliBL2-CSA, and nickel affinity chromatography column is selected for purification, thus obtaining streptavidin (CSA) with cysteine added at the N-terminal of exogenous expression of escherichia coli.
6. The method for preparing a BMP-CSA magnetosome according to claim 5, wherein the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used in PCR:

the PCR reaction (50. Mu.l) was as follows:

the PCR procedure was as follows:

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