CN104450617B - Application of cell sorting system based on specific recognition of endonuclease - Google Patents
Application of cell sorting system based on specific recognition of endonuclease Download PDFInfo
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Abstract
The invention relates to application of a cell sorting system based on specific recognition of endonuclease. The cell sorting system comprises the following components including first single-chain nucleotide, second single-chain nucleotide, an antibody, a cell sorting medium and restriction endonuclease, wherein the first single-chain nucleotide and second single-chain nucleotide are complemented to each other, and in addition, recognition sites of the restriction endonuclease are contained in complementary regions; the first single-chain nucleotide, the second single-chain nucleotide, the antibody and the cell sorting medium are all modified, the modified first single-chain nucleotide can be connected with the modified antibody, and the modified second single-chain nucleotide can be connected with the modified cell sorting medium. The invention also provides a purpose of a kit for sorting DC cells, NK cells and CIK cells from cell samples. With the adoption of the cell sorting technology, various cells are simultaneously sorted, a great amount of cells and fragile cells can be sorted, and the yield and the survival rate of the cells are high.
Description
Technical field
The present invention relates to biomedicine technical field, specifically, is related to a kind of based on Cobra venom endonuclease specific recognition
Cell sorting system application.
Background technology
Existing cell sorting techniques mainly have following two：
1. Flow cytometry
Realized by separating containing single celled drop.Cell to be measured is combined with monoclonal antibody and fluorescent dye
After make suspension specimen, under certain gas pressure, sample enters flow chamber, the single file row under not celliferous buffer parcel
Row.Under the higher-order of oscillation of the piezoquartz of the hyperfrequency of flow chamber, liquid stream is fractured into uniform drop, and cell to be measured is just included
Among drop.Fill these drops with positive or negative electric charge, when charged drop is by electric field, occur in the presence of electric field inclined
Turn, among later falling into corresponding catcher, so as to realize cell sorting.
2. immunomagnetic bead technique
The immunoreactive antibody of magnetic bead surfaces coating tool, is specifically bound with the antigen of cell surface；The latter puts
Under powerful magnetic field, will separate with unconjugated cell；Depart from behind magnetic field can disappearing magnetism immediately, can thus screen
Or the cell of removal institute labelling, so as to reach the purpose for filtering out positive or negative cell.Its flow chart is as shown in Figure 1.
Above technology has the following disadvantages：Although Flow cytometry can be sorted obtains aim cell, 1.
Cell is must be able to by fluorescence molecule labelling；2. the unicellular of suspension can only be detected；3. assorting room is not gentle enough, to cell damage
Greatly；4. may contamination of cells；5. it is unfavorable for the sorting of bulk sample.Although immunological magnetic bead sorting technology can pass through positive and negative
Two kinds of screenings obtain aim cells, but 1. cannot complete " once with reference to " and separate various kinds of cell；2. if necessary to from a sample
Multiple aim cells being screened inside product, needing repeatedly to combine and eluting, the cytoactive for obtaining is poor, yield is low；3. repeatedly sort,
High cost；4. aim cell is not completely separated with magnetic bead, is unfavorable for follow-up study.
At present can be thorough with carrier with regard to aim cell little to cell damage, various kinds of cell being sorted, obtained simultaneously
Separate, it is adaptable to which the technology of a large amount of and fragile cell sorting has not been reported.
The content of the invention
The purpose of the present invention is for deficiency of the prior art, there is provided the cell based on Cobra venom endonuclease specific recognition
The application of separation system.
Another purpose of the present invention is to provide the application of the test kit of sorting DC cells, NK cells and CIK cell.
For realizing above-mentioned first purpose, the present invention is adopted the technical scheme that：
Application of the cell sorting system based on Cobra venom endonuclease specific recognition in sorting cells, described sorting system
System includes following components：First single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium, restricted enzyme；
The first described single-stranded nucleotide and the second single-stranded nucleotide are complementary, and complementary region contains the identification of the restricted enzyme
Site；Described the first single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium are modified, after modification
One single-stranded nucleotide can be connected with the antibody after modification, and the second single-stranded nucleotide after modification can be with the cell sorting after modification
Medium is connected.
Preferably, the first described single-stranded nucleotide and the second single-stranded nucleotide are partial sequence complementarity or complete complementary.
Preferably, the length of the first described single-stranded nucleotide or the second single-stranded nucleotide is 6bp-1kb.
It is highly preferred that the length of the first described single-stranded nucleotide or the second single-stranded nucleotide is 18bp-50bp.
Preferably, described cell sorting medium is magnetic bead, detached dowel or polycation nano particle.
It is highly preferred that the first described single-stranded nucleotide is by sulfydryl modification, the second described single-stranded nucleotide is by biotin
Modification, described antibody are modified by maleimide, and described magnetic bead is modified by Streptavidin.
For realizing above-mentioned second purpose, the present invention is adopted the technical scheme that：
The test kit of sorting DC cells, NK cells and CIK cell answering in sorting DC cells, NK cells and CIK cell
With described test kit includes following components：Three pairs of single-stranded nucleotides of sulfydryl and biotin modification, CD3, CD56 and HLA-DR
Antibody, Streptavidin MagneSphere, restricted enzyme BamH I, EcoR I, Hind III.
The complementary region of three pairs of described single-stranded nucleotides includes restricted enzyme BamH I, EcoR I, Hind respectively
The recognition site of III.
Preferably, the sequence of three pairs of described single-stranded nucleotides is respectively as shown in SEQ ID NO.1-SEQ ID NO.6.
The invention further relates to a kind of method for sorting aim cell from cell sample, described method includes following step
Suddenly：
A) synthesize complementary single-stranded nucleotide, prepare " magnetic bead/single-stranded nucleotide " and " antibody/single-stranded nucleotide " and be combined
Thing；
B) " antibody/single-stranded nucleotide " complex is added in cell sample, formed " single-stranded nucleotide/antibody/cell "
Complex；
C) " magnetic bead/single-stranded nucleotide " complex is added in " single-stranded nucleotide/antibody/cell " complex, is formed
" magnetic bead/double chain nucleotide/antibody/cell " complex；
D) by the effect of externally-applied magnetic field, magnetic absorption aim cell is gone unless aim cell；
E) different aim cells is directed to, is sheared containing specific identification sequence using different restricted enzyme every time
Double chain nucleotide, the gradually different aim cell of eluting.
Preferably, in step b), in the volume of cell sample, cell sample, aim cell number, aim cell are corresponding anti-
The ratio of weight is：1mL cell samples: 106-109Individual aim cell: the corresponding antibody of 5-100 μ g aim cells.
Preferably, " single-stranded nucleotide/antibody/cell " complex described in step c) and " magnetic bead/single-stranded nucleotide "
The number ratio of complex is 1:5-1:100.
Preferably, step b) reaction conditions are 2-8 DEG C, 5-30min.
Preferably, step c) reaction conditions are 15-25 DEG C, 5-30min.
The present invention is separately related to a kind of method for sorting DC cells, NK cells and CIK cell from cell sample, it include with
Lower step：
A) synthesize three pairs of complementary single-stranded nucleotides, prepare three kinds " magnetic beads/single-stranded nucleotide " and three kinds of " antibody/single-stranded
Nucleotide " complex, described antibody are respectively CD3, CD56 and HLA-DR antibody, described three pairs of single stranded nucleotide acid fragments
Sequence is respectively as shown in SEQ ID NO.1-SEQ ID NO.6；
B) three kinds of " antibody/single-stranded nucleotide " complex are added in cell sample, forms " single-stranded nucleotide/antibody/thin
Born of the same parents " complex；
C) three kinds of " magnetic bead/single-stranded nucleotide " complex are added in " single-stranded nucleotide/antibody/cell " complex,
Form " magnetic bead/double chain nucleotide/antibody/cell " complex；
D) by the effect of externally-applied magnetic field, separate and obtain the cell combined with magnetic bead；
E) different aim cells is directed to, is gradually washed using restricted enzyme BamH I, EcoR I, Hind III respectively
It is de-.
Preferably, in step b), in the volume of cell sample, cell sample, aim cell number, aim cell are corresponding anti-
The ratio of weight is：1mL cell samples: 106-109Individual aim cell: the corresponding antibody of 5-100 μ g aim cells.
Preferably, " single-stranded nucleotide/antibody/cell " complex described in step c) and " magnetic bead/single-stranded nucleotide "
The number ratio of complex is 1:5-1:100.
Preferably, step b) reaction conditions are 2-8 DEG C, 5-30min.
Preferably, step c) reaction conditions are 15-25 DEG C, 5-30min.
Herein, described " cell sorting medium " includes all media for sorting cells, as long as sorting can be reached
The medium of purpose be all possible, such as magnetic bead, detached dowel, polycation nano particle etc. other can reach sorting purpose
Medium.Described the first single-stranded nucleotide, the second single-stranded nucleotide, antibody, the modification mode of cell sorting medium are not limited
In the mode that above content is recorded, any mode that combine can these molecules all can be acceptance.Advantage of the present invention exists
In：
The present invention introduces two species specificity recognition mechanism (restricted enzyme on the basis of regular growth sorting technology
The specific recognition of specific recognition, antibody and antigen to Double-stranded nucleotide sequence), realize that separating for several times is obtained primary sorption again
To various purpose products.
The cell sorting techniques of the present invention can make up the deficiencies in the prior art, mutually compare cell wound with Flow Cytometry
Evil is little；Compared with immunomagnetic bead technique, various kinds of cell can be sorted simultaneously, and the aim cell for obtaining can be with cell sorting medium
Separate, thus cell can be used to sort again.The technology of the present invention can carry out the sorting of a large amount of and fragile cell, cell yield and
Survival rate is high.
The technology of the present invention, on the basis of cell sorting, can be that cell therapy is carried as the supporting technology in cell therapy
For high-purity, great-hearted cell, it can also be used to test and analyze field, it is used in combination with micro detection of nucleic acids equipment, to not
Same object carries out detection by quantitative.Which is that the fields such as clinical diagnosises, cell separation, detection, cell therapy bring glad tidings.
Description of the drawings
Fig. 1 is the principle of immunological magnetic bead sorting technology and flow chart.
Fig. 2 is principle and flow chart of the present invention based on the cell sorting system of Cobra venom endonuclease specific recognition.
Fig. 3 is that single-stranded nucleotide is combined schematic diagram with antibody by Thiol/Malemide.Ab：Antibody；Oligo：It is single-stranded
Nucleotide；SH-：Sulfydryl.
Fig. 4 is the NK cell flow cytometry testing results of present invention sorting.
Fig. 5 is the CIK cell Flow cytometry result of present invention sorting.
Fig. 6 is the DC cell flow cytometry testing results of present invention sorting.
Fig. 7 is form after the NK cell culture that two methods are sorted.A. the cellular morphology of selected by flow cytometry apoptosis, B. this
The cellular morphology of bright sorting.
Specific embodiment
Below in conjunction with the accompanying drawings the specific embodiment that the present invention is provided is elaborated.
Cell sorting system of the present invention based on Cobra venom endonuclease specific recognition sorts flow chart as shown in Fig. 2 step
Including：
1. select different antibody to different aim cell surface markers, two complementary single stranded oligonucleotides of synthesis are (about
20bp), single stranded oligonucleotide is end modified Thiol (sulfydryl), and another single stranded oligonucleotide is end modified to be had
Biotin (biotin).
2. two single stranded oligonucleotides respectively with the antibody modified by Maleimide (maleimide) and
The coated cell sorting dielectric surfaces of Streptavidin (Streptavidin) are reacted, formed respectively " cell sorting medium/
Single-stranded nucleotide " and " antibody/single-stranded nucleotide " two kinds of structures.
3. " antibody/single-stranded nucleotide " complex is added in cell sample, antibody is by the surface on identifying purpose cell
Labelling, and combined with cell, form " single-stranded nucleotide/antibody/cell " structure.
4. subsequently " cell sorting medium/single-stranded nucleotide " structure is added in separation system, two single-stranded nucleotides
By base pair complementarity, " cell sorting medium/double chain nucleotide/antibody/cell " structure is formed.
5. when described cell sorting medium is magnetic bead, then by the effect of externally-applied magnetic field, aim cell will be by magnetic
Absorption, so as to go unless aim cell；When described cell sorting medium be magnetic bead beyond other media when, then using corresponding
Absorption the medium method to go unless aim cell.
6. different aim cells is directed to, every time using different restricted enzyme shearing containing specific identification sequence
Nucleotide, can the gradually different aim cell of eluting, complete once to combine and separate various different aim cells.
Wherein, in above step, it is also possible to first by " antibody/single-stranded nucleotide " complex and " cell sorting medium/single-stranded
Nucleotide " is combined, and is subsequently then added in cell sample.
Embodiment 1
With immune cell therapy very big, the immune cell therapy that is that leading cell therapy plays the role of in terms for the treatment of of cancer
In the major cell types used have：DC cells, NK cells and CIK cell.These cells are different because of condition of culture, so needing
Carry out again cultivating one's ability after separating and obtain good expanding effect.The purpose of the present embodiment is these three in first separation blood
Cell, specific separation scheme are as follows：
1 experiment material
1. CD3, CD56 and HLA-DR antibody, is purchased from Biolegend companies；
2. sulfydryl and the single stranded nucleotide acid fragment of biotin modification, synthesize in Jin Weizhi companies；
3. Cobra venom endonuclease BamH I, EcoR I, Hind III, are purchased from NEB companies；
4. Streptavidin MagneSphere, is purchased from Dongguan City Han Nuo Bioisystech Co., Ltd；
5. cell culture medium X-VIVOTM15Chemically Defined,Serum-free Hematopoietic Cell
Medium, is purchased from Lonza companies of the U.S.；
6. (reaction system of restriction endonuclease is the general of NEB companies Cobra venom endonuclease to elution buffer
Buffer)；
7. human peripheral blood mononuclear cell, extracting method are as follows：
A collector's peripheral blood samples 10mL, with the PBS dilute samples of 2-4 times of volume；15mL is added to drench in 50mL centrifuge tubes
Bar cell separation liquid；
B is slowly added into the blood sample of dilution on lymphocyte separation medium, makes blood sample be maintained at lymph as far as possible
Cell separation liquid upper strata；
C room temperatures × 400g × 30min, collect the PBMC cells of cloud and mist layer；
The resuspended PBMC cells of d 40mL aseptic PBS, room temperature × 200g × 10min remove supernatant；
E 40mL aseptic PBS is resuspended once, room temperature × 200g × 10min, frozen with the resuspended subpackage of frozen stock solution.
2 experimental techniques
CD3, CD5 and HLA-DR antibody (antibody of various cell surfaces such as table 1) respectively from different single-stranded nucleotide pieces
Section is combined, while being combined with different magnetic beads from the single stranded nucleotide sequence which matches, (nucleotide sequence and Cobra venom endonuclease are such as
2), the Cobra venom endonuclease of specificity is configured in elution buffer table.
The single-stranded nucleotide of biotin modification with the coated magnetic bead association reaction condition of Streptavidin is：The two is blended in
37 DEG C of reaction 30min.
The single-stranded nucleotide of sulfydryl modification uses Thermo companies with the antibodies reaction of maleimide modification
Imject Maleimide-Activated Mariculture KLH and Kit test kits.Concrete principle is：sulfo-
The Malemide of SMCC cross-linking agent modifieds is with the characteristic combined closely with sulfydryl and very stable；Antibody and single-stranded nucleotide
Join dependency antibody protein amino and sulfo-SMCC cross-linking agent SO3 -Interaction；Malemide can with carry
The single-stranded nucleotide of sulfydryl interacts to form stable thioether bond (referring to Fig. 3).
The major surfaces labelling of 1. various cells of table
| Cell type | Surface markers |
| NK cells | CD3-、CD56+ |
| DC cells | HLA-DR+ |
| CIK cell | CD3+、CD56+ |
2. nucleotide sequence of table and Cobra venom endonuclease
Experiment flow is as follows：
1. three kinds of antibody/single-stranded nucleotides are added separately in human peripheral blood mononuclear cell, additional proportion behaviour periphery
Blood volume: aim cell number: the corresponding antibody mass of aim cell=1mL human peripherals: 108Individual aim cell: 50 μ g mesh
The corresponding antibody of cell.Using containing 0.5%HAS carry out it is resuspended, mix, react 10min on ice, obtain single-stranded nucleotide/
Antibody/cell；
2. magnetic bead/single-stranded nucleotide is added, which is 50 with the number ratio of single-stranded nucleotide/antibody/cell:1, normal-temperature reaction
5min；
3. separated in being put into magnetic frame, isolated all magnetic beads that can be combined with cell；
4. will be cultivated as CIK cell after the collection of unconjugated cell；
5., in the magnetic bead for combining, the corresponding Cobra venom endonuclease eluents of HLA-DR are added, DC cells is eluted；
6. the corresponding Cobra venom endonuclease eluents of CD56 are added, by CD56+ and the cell of the cell and CD56+ of CD3- is washed
Take off；
7. the corresponding Cobra venom endonuclease eluents of CD3 are added, by CD3+ and under the cell eluting of the cell and CD3+ of CD56+
Come；
8. by the cell centrifugation for eluting, collection, 2 times are cleaned with PBS；
9. by separation after different cells with corresponding culture medium culturing 10d after, identified with flow cytometer sorting knot
Really.
It should be noted that：Step 1. aim cell number, aim cell pair in the volume of middle cell sample, cell sample
The ratio of the antibody mass answered can be in following scope：1mL cell samples: 106-109Individual aim cell: 5-100 μ g aim cells
Corresponding antibody；Step 2. described in " single-stranded nucleotide/antibody/cell " complex and " magnetic bead/single-stranded nucleotide " be combined
The number ratio of thing can be in following scope 1:5-1:100；1. reaction condition can be in following scope for step：2-8 DEG C, 5-30min；
2. reaction condition can be in following scope for step：15-25 DEG C, 5-30min.
2 experimental results
Flow cytometry result is as Figure 4-Figure 6.Data analysiss：Use be can be seen that from up flow type result
The NK cell yields that the inventive method sorting is obtained are 97.4%, and heteroproteose cell content is low, and NK cells have obtained good purification；
CIK cell yield is 63.6%, although heteroproteose cell is more, but can obtain purification in incubation；DC cell yields are
5.4%, be that ordinary stream cell instrument (1.7%) is sorted more than 3 times.
Embodiment 2
1 experimental technique
1. it is thin NK to be isolated from human peripheral blood mononuclear cell with flow cytometer and the method for separating of embodiment 1 respectively
Born of the same parents, are cultivated, observation of cell form.
2. mtt assay detects the survival rate of cell, and step is as follows：
A) inoculating cell：By the cell suspension of debita spissitudo, according to the volume in 100 μ L～200 μ L/ holes, 96 holes are seeded to
Plate, if 10 multiple holes.In addition, arranging blank control wells with culture fluid.
B) cultured cells：5%CO2, 37 DEG C of incubation cultured cells.
C) colour generation：The MTT solution (5mg/mL) of nutrient solution volume 10% in hole after culture, is added, 2～4h is incubated.
D) terminate culture, after centrifugation (250g × 4min), discard supernatant.
E) add 100 μ L DMSO per hole, be positioned over 5～10min of vibration on plate shaker, crystal is fully melted.With two
Methyl sulfoxide arranges zeroing hole；Control wells are returned to zero with blank well.
F) colorimetric：550nm wavelength is selected, each hole absorbance value is determined on enzyme linked immunological monitor.
2 experimental results
Cellular morphology after culture 4d is as shown in fig. 7, the survival ratio of cell is as shown in table 3.Data analysiss：Fluidic cell
The NK cells of instrument sorting form the homogeneous colony of fraction, and the present invention is then most of homogeneous colony, present invention sorting training
Cell of the foster cell colony yield apparently higher than selected by flow cytometry apoptosis；The cytoactive extinction that selected by flow cytometry apoptosis is obtained
Value (0.4941), hence it is evident that less than the method for separating (0.798) of the use present invention, illustrate the cytoactive that method for separating of the present invention is obtained
It is high.
The activity of the cell of table 3.MTT methods detection
Above example shows：, compared with fluidic cell sorting technology, separation process is more for the cell sorting method of the present invention
Heating and, it is little to cell injury, and a large amount of cell sortings can be carried out；Compared with traditional immunological magnetic bead sorting technology, can be with
Realize once combining the various aim cells of sorting, can also selective elution aim cell as needed, and the cell for obtaining with
Cell sorting medium (such as magnetic bead) is separate.On the whole, cell survival rate and yield are improve, save the time and into
This.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of without departing from the inventive method, can also make some improvement and supplement, and these improve and supplement also should be regarded as
Protection scope of the present invention.
Claims (7)
1. application of the cell sorting system based on Cobra venom endonuclease specific recognition in sorting cells, it is characterised in that institute
The separation system stated includes following components：First single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium, restriction
Property restriction endonuclease；The first described single-stranded nucleotide and the second single-stranded nucleotide are complementary, and complementary region contain it is described it is restricted in
The recognition site of enzyme cutting；Described the first single-stranded nucleotide, the second single-stranded nucleotide, antibody, cell sorting medium are modified,
The antibody that first single-stranded nucleotide of sulfydryl modification is modified with maleimide is connected, and the second of biotin modification is single-stranded
The cell sorting medium that nucleotide is modified with Streptavidin is connected.
2. application according to claim 1, it is characterised in that the first described single-stranded nucleotide and the second single-stranded nucleotide
For partial sequence complementarity or complete complementary.
3. application according to claim 1, it is characterised in that the first described single-stranded nucleotide or the second single-stranded nucleotide
Length be 6bp-100bp.
4. application according to claim 3, it is characterised in that the first described single-stranded nucleotide or the second single-stranded nucleotide
Length be 18bp-50bp.
5. application according to claim 1, it is characterised in that described cell sorting medium is magnetic bead, detached dowel or poly-
Cationic nano-grain.
6. application of the test kit of DC cells, NK cells and CIK cell in sorting DC cells, NK cells and CIK cell is sorted,
Characterized in that, described test kit includes following components：Three pairs of complementary single-stranded nucleotides；Each pair single-stranded nucleotide is by first
Single-stranded nucleotide and the second single-stranded nucleotide composition, the complementary region of three pairs of described single-stranded nucleotides are included in restricted respectively
Enzyme cutting BamH I, EcoR I, the recognition site of Hind III；The first single-stranded nucleotide of sulfydryl modification, biotin modification second are single
Chain nucleotide, CD3, CD56 and HLA-DR antibody, Streptavidin MagneSphere, restricted enzyme BamH I, EcoR I, Hind
III。
7. application according to claim 6, it is characterised in that the sequence of three pairs of described single-stranded nucleotides is respectively such as SEQ
Shown in ID NO.1-SEQ ID NO.6.
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| CN201410737627.5A CN104450617B (en) | 2014-12-05 | 2014-12-05 | Application of cell sorting system based on specific recognition of endonuclease |
| PCT/CN2015/096216 WO2016086861A1 (en) | 2014-12-05 | 2015-12-02 | Cell sorting system and method based on endonuclease specific recognition, and applications thereof |
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| WO2016086861A1 (en) * | 2014-12-05 | 2016-06-09 | 赛业（苏州）生物科技有限公司 | Cell sorting system and method based on endonuclease specific recognition, and applications thereof |
| CN105087788B (en) * | 2015-08-03 | 2018-11-23 | 上海白泽医疗器械有限公司 | It is a kind of sort people's cell immunomagnetic beads, its preparation and cutting method |
| CN114807031A (en) * | 2022-05-13 | 2022-07-29 | 山东赛恩福干细胞工程集团有限公司 | Construction method of human peripheral blood immune cell bank and stem cell bank |
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| CN101165173A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for carrying cell sorting by using gold magnetism particles |
| CN101892196A (en) * | 2010-07-14 | 2010-11-24 | 北京大学 | Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting |
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| CN101165173A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for carrying cell sorting by using gold magnetism particles |
| CN101892196A (en) * | 2010-07-14 | 2010-11-24 | 北京大学 | Cell sorting magnetic bead, synthesis method thereof and application thereof in cell sorting |
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