Method for specifically separating eukaryotic cells from prokaryotic cells in various environments

Abstract

The invention discloses a method for specifically separating eukaryotic cells from prokaryotic cells in various environments. The invention provides a concanavalin A and magnetic bead complex. The concanavalin A and magnetic bead complex is a complex formed by connecting concanavalin A and magnetic beads, the invention also provides a method for separating eukaryotic cells and prokaryotic cells, which comprises the following steps: adding the concanavalin A and magnetic bead complex into a cell resuspension to be separated, and incubating; and separating the eukaryote cells adsorbed by the magnetic beads from the prokaryote cells not adsorbed by the magnetic beads by using a magnetic frame. The raw materials such as the streptavidin magnetic beads and the concanavalin A involved in the method are low in price and simple to prepare; magnetic separation is utilized, and operation is simple and fast; the separation effect is good.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for the specific separation of eukaryotic cells and prokaryotic cells in various environments. Background Technology

[0002] Multi-omics technologies, such as metagenomics and metatranscriptomics, have significant advantages in studying the symbiotic relationships and trophic structures of marine eukaryotes and their symbiotic microorganisms. This is primarily achieved by extracting DNA / RNA from eukaryotes or prokaryotes and performing next-generation sequencing. For example, in 2023, Kshitij Tandon et al. directly extracted total DNA from reef-building corals collected in shallow waters, and then obtained metagenomics by controlling read length quality, pruning, and deleting host-related reads. Other methods, such as grinding, filtering, and centrifugation, were used by Steven J. Robbins et al. in their metagenomic studies of reef-building corals, and by T. Keller-Costa et al. in their studies of octagonal corals, to separate eukaryotic and prokaryotic cells.

[0003] However, these methods all suffer from drawbacks such as cumbersome techniques, poor separation results, and high levels of eukaryotic genetic information contamination in metagenomic sequencing results. Therefore, developing a simpler method that can more effectively separate eukaryotic and prokaryotic cells is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to provide a concanavalin A and magnetic bead complex, and based on this, to provide a method for the specific separation of eukaryotic and prokaryotic cells in various environments.

[0005] In a first aspect, the present invention claims protection for a concanavalin A and magnetic bead complex.

[0006] The concanavalin A and magnetic bead complex claimed in this invention is a complex formed by combining concanavalin A and magnetic beads.

[0007] Furthermore, the concanavalin A and magnetic bead complex can be prepared by a method including the following steps: modifying the surface of magnetic beads with streptavidin, labeling biotin on concanavalin A, and then obtaining the concanavalin A and magnetic bead complex through the binding action of streptavidin and biotin.

[0008] In one embodiment of the present invention, when preparing the concanavalin A and magnetic bead complex, 0.6144 pmol of biotin-labeled concanavalin A (calculated based on the amount of concanavalin A) is added to every 75 million magnetic beads with surface modified by streptavidin.

[0009] Furthermore, the method further includes an incubation step of 26°C for 10 min after mixing magnetic beads with streptavidin-modified surfaces and biotin-labeled concanavalin A.

[0010] Secondly, the present invention claims protection for a product used for separating eukaryotic cells and prokaryotic cells.

[0011] The products claimed in this invention include the concanavalin A and magnetic bead complex described in the first aspect above.

[0012] Furthermore, the product may also include a magnetic rack.

[0013] Thirdly, the present invention claims protection for the use of the concanavalin A and magnetic bead complex described in the first aspect above or the product described in the second aspect above in the isolation of eukaryotic and prokaryotic cells.

[0014] Fourthly, the present invention claims the use of the concanavalin A and magnetic bead complex described in the first aspect above or the product described in the second aspect above in the process of removing host contamination or bacterial contamination during sequencing.

[0015] Fifthly, the present invention claims a method for separating eukaryotic cells and prokaryotic cells.

[0016] The method for separating eukaryotic and prokaryotic cells claimed in this invention may include the following steps: (A1) Resuspend all cells to be separated, and then add the concanavalin A and magnetic bead complex described in the first aspect above to the obtained cell resuspension and incubate. (A2) Using a magnetic frame, eukaryotic cells adsorbed by magnetic beads and prokaryotic cells not adsorbed by magnetic beads are separated.

[0017] Further, in step (A1), 25 million magnetic beads of the concanavalin A and magnetic bead complex described in the first aspect above are introduced per 10,000 to 100,000 cells.

[0018] Further, in step (A1), the incubation time is 10 minutes, such as incubation at 26°C for 10 minutes.

[0019] Sixthly, the present invention claims the use of the method described in the fifth aspect above for removing host contamination or bacterial contamination during sequencing.

[0020] In NGS sequencing, host contamination or bacterial contamination can waste background noise. The contamination can be removed using the method described in the first part above before proceeding with subsequent sequencing.

[0021] This invention enables efficient removal of host or bacterial contamination during sequencing (with a removal rate greater than 99%).

[0022] In one embodiment of the present invention, the eukaryotic cell is a PBMC cell, and the prokaryotic cell is Escherichia coli.

[0023] In another embodiment of the present invention, the eukaryotic cell is a deep-sea coral, and the prokaryotic cell is a deep-sea coral symbiotic microorganism.

[0024] This invention utilizes the principle that concanavalin A can specifically bind to glycoproteins on the surface of eukaryotic cell membranes / nuclear membranes to establish a method for the specific separation of eukaryotic and prokaryotic cells. This method has the following advantages: 1. Streptavidin magnetic beads and concanavalin A are inexpensive raw materials and simple to prepare; 2. Utilizing magnetic separation, the operation is simple and quick; 3. Excellent separation effect (separation purity > 99%). Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the principle of this invention, which utilizes the specific binding of concanavalin A to glycoproteins on the surface of eukaryotic cell membranes. In this diagram, A represents the modification of the magnetic bead surface with streptavidin, and the concanavalin A labeled with biotin. The binding of biotin and streptavidin enables the connection between concanavalin A and the magnetic beads. B represents the specific binding of the concanavalin A-magnetic bead complex to eukaryotic cells, preventing its binding to prokaryotic cells. C represents the use of a magnetic rack to adsorb the magnetic beads onto one side of the centrifuge tube. At this point, prokaryotic cells remain free within the liquid in the centrifuge tube, while eukaryotic cells are specifically concentrated on one side of the tube, thus achieving the separation of eukaryotic and prokaryotic cells.

[0026] Figure 2 The fluorescence detection method of this invention was used to separate PBMCs and Escherichia coli. In sample A, the conjugation of a complex of concanavalin A and magnetic beads in eukaryotic cells (PBMC samples) was confirmed by fluorescence detection to be effective (i.e., fluorescence was observed on the eukaryotic cells). In sample B, the conjugation of a complex of concanavalin A and magnetic beads in prokaryotic cells (E. coli cells) was confirmed to be ineffective by fluorescence detection (i.e., no fluorescence was observed on the prokaryotic cells).

[0027] Figure 3The fluorescence detection method of this invention was used to separate deep-sea corals and symbiotic microorganisms (prokaryotic cells). In sample A, a complex of concanavalin A and magnetic beads was formed in a eukaryotic cell nucleus (deep-sea coral sample). Fluorescence detection demonstrated that the binding of the magnetic beads to the cell nucleus was effective (flocculent fluorescence was observed under the microscope). Samples B and C showed a complex of concanavalin A and magnetic beads formed in symbiotic microorganisms (prokaryotic cells). Fluorescence detection clearly showed that the binding was ineffective (no fluorescence in sample B), and the presence of the symbiotic microorganisms could be observed under bright field (sample C).

[0028] Figure 4 The genomes of deep-sea coral symbiotic microorganisms, which were decontaminated by the host, were compared with the reference genome of deep-sea corals. The results showed that the genome coverage was as low as about 0.397%, which means that the eukaryotic cell nuclear separation rate was over 99%. Detailed Implementation

[0029] This invention utilizes the principle that concanavalin A can specifically bind to glycoproteins on the surface of eukaryotic cell membranes to establish a method for the specific separation of eukaryotic cells (or eukaryotic cell nuclei) from prokaryotic cells.

[0030] (1) Preparation of concanavalin A and magnetic bead complex: Streptavidin was modified on the surface of magnetic beads, and biotin was labeled with concanavalin A. Through the binding of biotin and streptavidin, concanavalin A and magnetic beads were interconnected. Figure 1 A.

[0031] (2) The concanavalin A-magnetic bead complex specifically binds to eukaryotic cells or nuclei: Various environmental samples are often rich in microorganisms (mainly prokaryotic cells) and some host cells. All cells in the environmental sample are resuspended, and the concanavalin A-magnetic bead complex is added for incubation. This complex can specifically bind to glycoproteins on the surface of the eukaryotic cell membrane or nucleus, but will not bind to prokaryotic cells. For example... Figure 1 B.

[0032] (3) Magnetic separation of samples: After incubation, magnetic beads can be adsorbed onto one side of the centrifuge tube using a magnetic rack. At this time, prokaryotic cells are free in the liquid of the centrifuge tube, while eukaryotic cells are specifically enriched on one side of the centrifuge tube, thus achieving the separation of eukaryotic cells (or eukaryotic cell nuclei) and prokaryotic cells. Figure 1 C.

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1: Determination of the separation efficiency of the concanavalin A-magnetic bead complex for eukaryotic cells. I. Preparation of Concanavalin A-Magnetic Bead Complex (1) Take 75 million naked magnetic beads (Dynabeads™ M-280 streptomycin avidin, catalog number: 11205D) (110μL), remove the supernatant with a magnetic rack, and wash twice with 200μL D-PBS (calcium magnesium).

[0036] (2) Based on the ratio of magnetic beads to concanavalin A, add 38.4 μL of biotin-ConA protein solution (biotin-labeled concanavalin A, solvent: 50% glycerol) with a concentration of 16 nM (concentration is calculated based on the amount of concanavalin A), and make up the volume to 100 μL with D-PBS (calcium magnesium).

[0037] (3) Place it on a mixer and incubate at 26°C for 10 min.

[0038] (4) Remove the supernatant with a magnetic rack and wash twice with 500 μL PBS.

[0039] (5) After removing the supernatant, add 30 μL of D-PBS (calcium magnesium) to resuspend the magnetic beads to obtain the concanavalin A and magnetic bead complex.

[0040] II. Separation of eukaryotic cells using magnetic beads (1) Take 200,000 PBMCs and add 20 μL of the concanavalin A and magnetic bead complex prepared in step one according to the cell input ratio (10 μL of concanavalin A and magnetic bead complex prepared in step one for every 10,000-100,000 cells).

[0041] (2) After gently blowing and sucking to mix, place it on a mixer and incubate at 26°C for 10 min, then examine and count under a microscope.

[0042] (3) The magnetic rack is used, and the supernatant contains unadsorbed eukaryotic cells.

[0043] (4) Collect the supernatant and count the contents under a microscope. (5) Calculate the number of cells in the supernatant to obtain the separation rate of the magnetic bead complex for eukaryotic cells. Separation rate = (1 - number of cells in supernatant / amount added) × 100%.

[0044] The results showed that after adding 200,000 eukaryotic cells (PBMCs), no eukaryotic cells were found in the supernatant, and all the cells were bound to the magnetic beads. Therefore, the separation rate of eukaryotic cells by the magnetic beads was 100%.

[0045] Example 2: Fluorescence detection of the binding effect of concanavalin A on PBMCs and Escherichia coli in the method of the present invention. I. Combining PBMCs and Escherichia coli using the method of this invention (1) For example Figure 1 As shown in Figure A, a FITC fluorescent group is attached to concanavalin A to indicate whether the concanavalin A binds to eukaryotic (PBMC) / prokaryotic (E. coli) cells.

[0046] (2) The luciferin linked with fluorescent groups was incubated with eukaryotic / prokaryotic cells for 15 min, centrifuged at 500g for 20 min, the precipitate was collected, resuspended in 0.04% BSA in PBS, and observed under a fluorescence microscope.

[0047] The results showed that concanavalin A ligation in eukaryotic cells (PBMC samples) was effective, as confirmed by fluorescence detection (i.e., fluorescence was observed on the eukaryotic cells). Figure 2 (A); ligation of concanavalin A in prokaryotic cells (E. coli cells) was confirmed to be invalid by fluorescence detection (i.e., no fluorescence was observed on the prokaryotic cells). Figure 2 (B). The above fluorescence experiments show that the concanavalin A of the present invention can attach to the surface of eukaryotic cells, but cannot attach to the surface of prokaryotic cells.

[0048] Example 3: Isolation of deep-sea corals and their symbiotic microorganisms Symbiotic microorganisms of deep-sea corals affect coral energy utilization and nutrient metabolism. The isolation, purification, and metagenomic sequencing of symbiotic microorganisms are common methods for studying them. However, contamination of the host (deep-sea coral) cell nuclear DNA affects the integrity of metagenomic assembly.

[0049] The following details the method of the present invention for separating deep-sea corals (eukaryotic cells) and symbiotic microorganisms (prokaryotic cells), thereby effectively removing contamination from the host (deep-sea coral) cell nucleus during the isolation, purification, and metagenomic sequencing of symbiotic microorganisms of deep-sea corals.

[0050] 1. Preparation before the experiment Clean bench preparation: Turn on the clean bench fan, first wipe the entire surface with 75% alcohol, then use DNA-OFF to thoroughly wipe the inside of the clean bench and instruments, especially the surfaces of metal and plastic products. Turn off the fan and sterilize with UV irradiation for 30 minutes. Before processing tissues, use RNAse-ZAP to thoroughly wipe the inside of the clean bench using the same method. After wiping, experiments can begin directly.

[0051] Low-temperature horizontal centrifuge parameter settings: temperature 4 ℃, rotation speed 500×g, centrifugation time 5 min. Before the experiment, ensure the centrifuge operating temperature reaches a low temperature of 4 ℃. Low temperature must be maintained throughout the entire operation; it can be done in an ice bath.

[0052] Tissue homogenizer preparation: Clean the tissue homogenizer with 75% alcohol, DNA-OFF, and RNAse-ZAP, rinse it thoroughly with dd H2O, and sterilize it with UV light along with the laminar flow hood.

[0053] 2. Obtaining coral tissue homogenate and cell nuclei Insert the tissue homogenizer into ice and add 500 μL of pre-chilled Homogenization Buffer (HB).

[0054] A coral tissue sample of approximately 1 cm³ was removed from liquid nitrogen and immediately placed in a 30 mm culture dish containing 1 mL of pre-cooled HB.

[0055] Use a sterile scalpel to mince the tissue into 1 mm³ pieces, transfer the tissue suspension to a tissue grinder, and incubate for 2 min.

[0056] Grind the tissue block vertically with a grinding rod A until the resistance is significantly reduced (about 10-15 times when grinding coral tissue). At this point, you can first take 2 μL of the grinding homogenate and mix it with 8 μL of DAPI staining solution, and observe the lysis status under a fluorescence microscope.

[0057] The tissue homogenate was filtered using a 100 μm cell sieve. The tissue homogenizer and cell sieve were washed with 500 μL of HB. The filtrate was collected in a 50 mL centrifuge tube. This step removed larger coral tissue impurities.

[0058] The cells were filtered again using a 30 μm cell sieve, and the sieve was washed with 500 μL of HB. The filtrate was then collected into a 15 mL centrifuge tube. This step removed smaller coral tissue fragments and impurities.

[0059] 3. Collecting and enriching coral symbiotic microorganisms (1) Isolation of symbiotic microorganisms and coral cell nuclei Centrifuge the filtrate at 4 ℃ and 500 g for 5 min, and collect the supernatant (which mainly contains symbiotic microorganisms and a small amount of eukaryotic cell nuclear contamination) into a 2 mL centrifuge tube.

[0060] Centrifuge at 14,000 rpm for 10 min at room temperature to precipitate the symbiotic microorganisms, discard the supernatant, and resuspend the precipitate in 90 μL of NF-H2O.

[0061] (2) Purification of deep-sea coral symbiotic microorganisms The ConA bean protein coupled to the surface of ConA Beads (i.e., the concanavalin A and magnetic bead complex prepared in step one of Example 1) can bind to nuclear surface glycoproteins, thereby eluting eukaryotic cell nuclear contamination. First, the ConA Beads need to be activated: Invert or gently vortex to mix the prepared ConA Beads. Take 40 μL of 1×DPBS in a PCR tube, add 10 μL of ConA Beads, and gently pipette from the bottom of the tube until completely mixed, avoiding liquid adhesion to the sidewalls. Place the PCR tube on a magnetic rack until the liquid becomes clear, then discard the supernatant. Remove the PCR tube, add 50 μL of 1×DPBS to resuspend the magnetic beads, and gently pipette from the bottom of the tube until completely mixed, avoiding liquid adhesion to the sidewalls. Place the PCR tube on a magnetic rack until the liquid becomes clear, then discard the supernatant. Remove the PCR tube, add 10 μL of 1×DPBS to resuspend the magnetic beads, and gently pipette the mixture from the bottom of the tube until completely mixed, avoiding liquid sticking to the sidewalls.

[0062] Add 10 μL of activated ConA Beads to the 90 μL resuspended microbial precipitate obtained in step (1), and place the centrifuge tube on a vertical mixer to mix and react for 10 min.

[0063] After the reaction was completed, the mixture was placed on a magnetic rack, and the symbiotic microorganisms in the supernatant were transferred to a new 2 mL centrifuge tube. The magnetic beads were then resuspended with an appropriate amount of 0.04% BSAI, and the supernatant and resuspended magnetic beads were stained with DAPI to observe the purification status.

[0064] 4. Fluorescence detection separation effect DAPI staining was observed under a fluorescence microscope, and the results are as follows: Figure 3 As shown, a complex of concanavalin A and magnetic beads was formed in the nucleus of a eukaryotic cell (deep-sea coral sample). Fluorescence detection demonstrated that the binding of the magnetic beads to the cell nucleus was effective. Figure 3 In step A, the magnetic beads were resuspended in 0.04% BSAI in the previous step and stained with DAPI, exhibiting flocculent fluorescence under a microscope. When symbiotic microorganisms (prokaryotic cells) attempt to connect the concanavalin A and magnetic bead complexes, fluorescence detection clearly indicates that the connection is invalid. Figure 3 In step B, the supernatant obtained after separation by ConA Beads in the previous step showed no fluorescence after DAPI staining, and the presence of symbiotic microorganisms could be observed under bright field. Figure 3 (C, transparent or gray strip-shaped and spherical structures). The above fluorescence experiments show that the concanavalin A and magnetic bead complex of the present invention can attach to the surface of eukaryotic cell nuclei, but cannot attach to the surface of deep-sea symbiotic microbial cells (prokaryotic cells). The method of the present invention can effectively separate deep-sea symbiotic microbial cells (prokaryotic cells) and eukaryotic cell nuclei (deep-sea coral samples).

[0065] 5. Evaluate the isolation effect by genome sequencing (1) Extracting DNA from symbiotic microorganisms The CTAB (hexadecyltrimethylammonium bromide) method is used, taking advantage of the cationic detergent properties of CTAB to remove protein and polysaccharide impurities, thereby achieving the purpose of extracting and separating nucleic acids.

[0066] The supernatant of the symbiotic microorganisms purified in step 3 using the complex of concanavalin A and magnetic beads was centrifuged at 14,000 rpm for 10 min, and the microbial precipitate was collected.

[0067] Add 1 mL of CTAB extraction buffer and 10 μL of lysozyme, mix thoroughly, and then react in a 37 ℃ metal bath at 800 rpm for 30 min, inverting the container every 5 min to mix.

[0068] After the reaction was complete, 40 μL of proteinase K (10 mg / mL) was added and placed in a metal bath at 55 ℃. The reaction was carried out at 800 rpm for 20 min, and the mixture was inverted and mixed every 5 min.

[0069] After the reaction was complete, add an equal volume of nucleic acid extraction reagent A (phenol:chloroform:isoamyl alcohol = 25:24:1, volume ratio), gently invert and mix, then centrifuge at 8000 rpm for 10 min to separate the layers, and transfer the supernatant to a new 2 mL centrifuge tube.

[0070] Add an equal volume of nucleic acid extraction reagent B (chloroform:isoamyl alcohol = 24:1, volume ratio), gently invert to mix, and centrifuge at 8000 rpm for 10 min to allow the system to separate into layers again. Transfer the supernatant to a new 1.5 mL centrifuge tube.

[0071] Add 0.7 times the volume of isoamyl alcohol, mix thoroughly by inverting the container, and place in a -20 ℃ freezer overnight to precipitate DNA.

[0072] Centrifuge at 8000 rpm for 10 min, discard the supernatant, add 500 μL of freshly prepared 75% ethanol solution to the DNA precipitate, gently mix with a pipette, centrifuge at 8000 rpm for 10 min, and discard the supernatant.

[0073] Repeat the previous step, centrifuge at 8000 rpm for 30 s, and discard the supernatant.

[0074] Open the lid and let the alcohol dry. Add 30 μL of TE buffer (pH=8.0), mix well by pipetting, and let stand for 10 min to re-dissolve the DNA precipitate.

[0075] The concentration of DNA from symbiotic microorganisms was tested using the Qubit dsDNA HS Assay kit and the Qubit fluorescence quantitative instrument, and the total amount of DNA extracted was estimated.

[0076] (2) Sequencing Based on the MGIASy Fast DNA library preparation kit and the MGIASy Double Barcode circularization kit manufactured by BGI Genomics, the DNA of symbiotic microorganisms was used to construct and circularize the library, and the whole genome of the black coral symbiotic microorganisms was sequenced on the MGISEQ-2000RS gene sequencer.

[0077] (3) Off-machine data analysis Metagenomic sequencing data were compared and analyzed to calculate the metagenomic and reference genome coverage. The *Bathypathes* genome was selected as the reference genome, and the CoverM correlation method was used to calculate genome coverage. It was found that the metagenomic and reference genome coverage was only about 0.397% (…). Figure 4 This demonstrates that the isolated symbiotic microorganisms have high purity and are basically free from contamination by the coral host genome, further illustrating the effectiveness of the ConA Beads method in isolating symbiotic microorganisms from deep-sea black corals.

[0078] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A concanavalin A and magnetic bead complex, characterized in that: The concanavalin A and magnetic bead complex is a complex formed by combining concanavalin A and magnetic beads.

2. The concanavalin A and magnetic bead complex according to claim 1, characterized in that: The concanavalin A and magnetic bead complex is prepared by a method comprising the following steps: modifying the surface of magnetic beads with streptavidin, labeling biotin on concanavalin A, and then obtaining the concanavalin A and magnetic bead complex through the binding action of streptavidin and biotin.

3. A product for separating eukaryotic and prokaryotic cells, comprising the concanavalin A and magnetic bead complex as described in claim 1 or 2.

4. The product according to claim 3, characterized in that: The product also includes a magnetic rack.

5. The use of the concanavalin A and magnetic bead complex according to claim 1 or 2, or the product according to claim 3 or 4, in the separation of eukaryotic and prokaryotic cells.

6. Use of the concanavalin A and magnetic bead complex of claim 1 or 2, or the product of claim 3 or 4, for removing host contamination or bacterial contamination during sequencing.

7. A method for separating eukaryotic cells and prokaryotic cells, comprising the following steps: (A1) Resuspend all cells to be separated, and then add the concanavalin A and magnetic bead complex as described in claim 1 or 2 to the resulting cell resuspension and incubate. (A2) Using a magnetic frame, eukaryotic cells adsorbed by magnetic beads and prokaryotic cells not adsorbed by magnetic beads are separated.

8. The method according to claim 7, characterized in that: In step (A1), 25 million magnetic beads of the concanavalin A and magnetic bead complex are introduced per 10,000 to 100,000 cells.

9. Use of the method of claim 7 or 8 for removing host contamination or bacterial contamination during sequencing.

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