CN117604032A - Method for over-expression of genes in crustacean body - Google Patents
Method for over-expression of genes in crustacean body Download PDFInfo
- Publication number
- CN117604032A CN117604032A CN202310613942.6A CN202310613942A CN117604032A CN 117604032 A CN117604032 A CN 117604032A CN 202310613942 A CN202310613942 A CN 202310613942A CN 117604032 A CN117604032 A CN 117604032A
- Authority
- CN
- China
- Prior art keywords
- over
- solution
- crustacean
- blood cells
- collecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 60
- 230000002018 overexpression Effects 0.000 title claims abstract description 58
- 241000238424 Crustacea Species 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 41
- 210000001808 exosome Anatomy 0.000 claims abstract description 135
- 210000000601 blood cell Anatomy 0.000 claims abstract description 83
- 239000013613 expression plasmid Substances 0.000 claims abstract description 25
- 210000004027 cell Anatomy 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims description 41
- 239000013612 plasmid Substances 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 23
- 230000003321 amplification Effects 0.000 claims description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 18
- 210000000170 cell membrane Anatomy 0.000 claims description 17
- 230000001086 cytosolic effect Effects 0.000 claims description 17
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000000338 in vitro Methods 0.000 claims description 15
- 239000011159 matrix material Substances 0.000 claims description 15
- 230000008929 regeneration Effects 0.000 claims description 13
- 238000011069 regeneration method Methods 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 12
- 239000013049 sediment Substances 0.000 claims description 11
- 239000003146 anticoagulant agent Substances 0.000 claims description 10
- 229940127219 anticoagulant drug Drugs 0.000 claims description 10
- 238000003208 gene overexpression Methods 0.000 claims description 10
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 10
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 9
- 239000000600 sorbitol Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- MVMSCBBUIHUTGJ-UHFFFAOYSA-N 10108-97-1 Natural products C1=2NC(N)=NC(=O)C=2N=CN1C(C(C1O)O)OC1COP(O)(=O)OP(O)(=O)OC1OC(CO)C(O)C(O)C1O MVMSCBBUIHUTGJ-UHFFFAOYSA-N 0.000 claims description 5
- MVMSCBBUIHUTGJ-GDJBGNAASA-N GDP-alpha-D-mannose Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=C(NC(=O)C=2N=C1)N)OP(O)(=O)OP(O)(=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O MVMSCBBUIHUTGJ-GDJBGNAASA-N 0.000 claims description 5
- 241001672730 Scylla paramamosain Species 0.000 claims description 5
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 5
- 239000011654 magnesium acetate Substances 0.000 claims description 5
- 229940069446 magnesium acetate Drugs 0.000 claims description 5
- 235000011285 magnesium acetate Nutrition 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 235000011056 potassium acetate Nutrition 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 239000012456 homogeneous solution Substances 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 241000238553 Litopenaeus vannamei Species 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- -1 HEPES-pH 7.2 Chemical compound 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 235000010356 sorbitol Nutrition 0.000 claims description 2
- 230000006378 damage Effects 0.000 abstract description 3
- 238000001890 transfection Methods 0.000 abstract description 3
- 210000003527 eukaryotic cell Anatomy 0.000 abstract description 2
- 230000003612 virological effect Effects 0.000 abstract description 2
- 102100040124 Apoptosis-inducing factor 1, mitochondrial Human genes 0.000 description 58
- 102000004899 14-3-3 Proteins Human genes 0.000 description 47
- 241000238097 Callinectes sapidus Species 0.000 description 47
- 241000238557 Decapoda Species 0.000 description 47
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 38
- 101710112812 14-3-3 protein Proteins 0.000 description 31
- 108020004999 messenger RNA Proteins 0.000 description 24
- 238000002347 injection Methods 0.000 description 21
- 239000007924 injection Substances 0.000 description 21
- 101000723543 Homo sapiens 14-3-3 protein theta Proteins 0.000 description 15
- 238000005119 centrifugation Methods 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000004624 confocal microscopy Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 238000001262 western blot Methods 0.000 description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000006907 apoptotic process Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 210000003370 receptor cell Anatomy 0.000 description 6
- 238000005215 recombination Methods 0.000 description 6
- 230000006798 recombination Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 101100322968 Arabidopsis thaliana ALF4 gene Proteins 0.000 description 3
- 101100500204 Arabidopsis thaliana DTX19 gene Proteins 0.000 description 3
- 101100422776 Arabidopsis thaliana SUR1 gene Proteins 0.000 description 3
- 101100269338 Mus musculus Aff4 gene Proteins 0.000 description 3
- 101100178756 Mus musculus Tcf12 gene Proteins 0.000 description 3
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 3
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 3
- 101150013766 alf1 gene Proteins 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000009109 downstream regulation Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 102000034356 gene-regulatory proteins Human genes 0.000 description 3
- 108091006104 gene-regulatory proteins Proteins 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108700020469 14-3-3 Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- ZNJHFNUEQDVFCJ-UHFFFAOYSA-M sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCS(O)(=O)=O)CC1 ZNJHFNUEQDVFCJ-UHFFFAOYSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention belongs to the field of molecular biology, and particularly relates to a method for over-expressing genes in crustacean bodies, which mainly comprises the following steps: 1) Designing an over-expression gene primer; 2) Constructing an over-expression plasmid; 3) The artificial exosomes encapsulate the over-expression plasmid. The invention connects the gene segment to be over-expressed with the carrier, then uses the artificial exosome to transport it into the crustacean blood cell, to realize the over-expression of the target gene in the crustacean blood cell. The method is non-viral transfection, can effectively enter cells, is simple and practical, has low damage to the cells, and solves the problem that the prior art cannot realize efficient overexpression of specific target genes in crustaceans. Wherein exogenous genes are introduced into eukaryotic cells by exosomes, which promote the cells to express specific genes, with safety and modified properties.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for over-expressing genes in crustacean bodies.
Background
The gene overexpression is to clone the target gene CDS onto a corresponding plasmid or virus vector, and the gene can realize a large number of transcription and translation under the condition of manual control by utilizing a regulatory element constructed on a vector framework, thereby realizing the overexpression of the target gene. Overexpression of the target gene and observation of the phenotype produced by the target gene are an indispensable method for exploring the function of the gene. At present, there are two general ways of over-expressing genes in animals: genetic engineering and viral vector-mediated transfection of genes in animals. At present, crustaceans do not have mature cell lines, the overexpression of target genes cannot be realized by a genetic engineering method, and a high-efficiency virus vector-mediated method for in-vivo gene transfection of crustaceans is not developed. Currently, there are two main methods for over-expression studies: recombinant proteins (Gao et al, mhio.2023, 14 (2): e 0291922) or mRNA of the gene of interest (Yang et al, PLoS pathlog.2016, 12 (12): e 1006127) are injected directly into crustaceans, but the efficiency of both approaches remains to be further improved. The realization of high expression of genes in crustaceans plays an important role in researching the gene functions of crustaceans so as to promote the deep research of biological phenomena and functions of crustaceans, and therefore, the research of crustaceans is urgent to need an accurate and efficient in-vivo gene transfection method.
The exosomes are outer membrane vesicles with the diameter of 30-150nm, and play an important role in information communication between cells. Exosomes are similar in size and function to synthetic nanoparticles, have the advantages of low toxicity, no immunogenicity, good permeability and the like as natural endogenous transport carriers, and have been regarded as the most potential drug delivery carriers so far, and are suitable for delivering various chemical substances, proteins, nucleic acids and gene therapeutic agents. One key problem with exosomes as delivery vehicles is how to obtain high-yield, high-purity exosomes, and artificial exosome in vitro synthesis techniques are already available in crustaceans. The artificial exosome is used as a carrier to realize the overexpression of genes in the crustacean body, and has important significance for constructing a crustacean gene function research platform.
Patent application number 202211465018X describes an artificial exosome of crustacean blood cells, but only describes a preparation method of the artificial exosome and an effect of the artificial exosome capable of directly wrapping protein or RNA and keeping corresponding activity, but most proteins cannot be purified in vitro or cannot be purified in vitro to ensure the activity of the protein, but the corresponding nucleic acid cannot be directly wrapped and translated in vivo, so that the method can only be applied to a small amount of proteins or nucleic acids, and the effect generated after the injection of the receptor is extremely limited, and a method which is efficient, wide in application range and capable of enabling the gene in the crustacean to be over-expressed needs to be further developed, so that the method can be truly applied to production research.
Disclosure of Invention
The invention aims to solve the problem that the target gene in the crustacean body cannot be efficiently over-expressed at present, and provides a novel method for over-expressing the gene in the crustacean body. The method utilizes an artificial exosome to wrap plasmids with target genes, and the plasmids are injected into crustacean bodies to realize the overexpression of the target genes. The artificial exosome carrying the plasmid is nontoxic, can be efficiently taken up by the receptor cells, and can bring the plasmid into the receptor cells to realize the overexpression of the target gene.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for gene overexpression in crustacean bodies, comprising the steps of:
A. amplifying a target sequence;
B. loading the target sequence on a carrier to construct an over-expression plasmid;
C. wrapping the over-expression plasmid in an artificial exosome to form an over-expression exosome;
D. the over-expression exosome is injected into crustacean body to over-express the gene.
Preferably, the sequence of interest comprises a nucleic acid.
Preferably, the sequence of interest comprises one or more of 14-3-3, AIF, p53, GFP.
Preferably, the sequence gene of interest may be a crustacean endogenous or exogenous gene.
Preferably, the vectors include all vectors containing promoters that can utilize the transcription and translation system in the crustacean body.
Preferably, the vector comprises one or more of the pIZ-wsv249p-egfp plasmid (Shi et al, developmental and comparative immunology.2018,88,70-76), pcdna3.1 plasmid, pzt/V5-His plasmid; the crustacean comprises one or more of Scylla paramamosain and Penaeus vannamei Boone.
Preferably, in step a, an amplification primer of the target sequence is designed according to the target sequence and the vector, and amplification is performed; in the step B, the over-expression plasmid is constructed by adopting a one-step cloning method; in step C, the over-expression plasmid is not less than 200ng.
Preferably, step C comprises:
c1, collecting blood cells;
c2, separating the cell membrane and cytoplasmic matrix of the blood cells;
and C3, adding the cell membrane and the cytoplasmic matrix into a reaction system, adding the over-expression plasmid, and reacting to obtain the over-expression exosome.
Preferably, the regeneration system is an ATP regeneration system comprising ATP, GDP-mannose, creatine phosphate, HEPEs-pH 7.2, sorbitol, potassium acetate, magnesium acetate; the incubation buffer comprises KCl and CaCl 2 ，HEPES-NaOH-pH 7.4，MgOAc，DTT。
Preferably, the ATP regeneration system comprises 10mM ATP,500mM GDP-mannose, 400mM creatine phosphate, 40mM creatine phosphate, 20mM HEPEs-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate; the incubation buffer included 80mM KCl,20mM CaCl 2 ，12.5mM HEPES-NaOH-pH 7.4，1.5mM MgOAc，1mM DTT。
The artificial exosome of the crustacean blood cells is extremely difficult, and ATP concentration or buffer pH is not suitable for synthesis. According to the invention, through long-term research, the proportion is continuously adjusted and regulated, and finally, a proper ATP regeneration system and incubation buffer solution are determined, so that the crustacean blood cell artificial exosome is successfully synthesized.
Preferably, the method comprises the following steps:
A. amplifying a target sequence:
a1, designing an in vitro amplification primer of the target sequence according to the target sequence and an enzyme cutting site behind a promoter of the vector and a one-step cloning method;
a2, amplifying the target sequence by using the in-vitro amplification primer, and purifying and collecting an amplification product;
B. loading the target sequence on a carrier, and constructing an over-expression plasmid:
b1, carrying out double enzyme digestion on the vector according to enzyme digestion sites at two ends of the target sequence, purifying and collecting a double enzyme digested product;
b2, connecting the amplification product with the double-enzyme-digested product, and screening recombinant plasmids which are successfully connected to form the over-expression plasmid;
C. wrapping the over-expression plasmid in an artificial exosome to form an over-expression exosome:
c1, collecting blood cells:
c1-1, drawing blood from crustacean, mixing with anticoagulant, centrifuging and collecting precipitate;
c1-2, re-suspending the sediment obtained in the step A1 by using an anticoagulant, and centrifugally collecting the sediment to obtain the blood cells;
c2, separating the cell membrane and cytoplasmic matrix of the blood cells:
c2-1, re-suspending the blood cells with a homogeneous solution, and carrying out ultrasonic vibration on ice to crush the blood cells to obtain a crushed cell solution;
c2-2, centrifuging the crushed cell solution, and collecting supernatant;
c2-3, centrifuging the supernatant obtained in the step C2-2, and collecting the supernatant and the precipitate;
c2-4, centrifuging the supernatant obtained in the step C2-3, and collecting the supernatant to obtain a solution of the cytoplasmic matrix;
c2-5, re-suspending the sediment obtained in the step C2-3 by using the homogenized liquid and LiCl solution, centrifuging, and re-suspending by using the homogenized liquid to obtain a solution of the cell membrane;
c3, adding the solution of the cytoplasmic matrix and the solution of the cell membrane into an ATP regeneration system and an incubation buffer solution, adding the over-expression plasmid, reacting to obtain the over-expression exosome, and freezing for preservation;
D. the over-expression exosome is injected into crustacean body to over-express the gene.
Preferably, the homogenized solution comprises 250mM sorbitol, 137mM NaCl,10mM PMSF, dissolved with Tris-HCl-pH 7.4; in step C3, the volume to mass ratio of the solution of the cytoplasmic matrix, the solution of the cell membrane, the ATP regeneration system, the incubation buffer, and the over-expression plasmid is 17uL:10uL:8uL:200ng.
Too little ATP can result in the inability of artificial exosomes to be synthesized successfully.
Preferably, the in vitro amplification primers are designed according to the sequence of the target gene and the sequence of the selected vector, including the target gene without the optional restriction sites on the vector.
Preferably, in the step A1, the crustacean includes one or more of scylla paramamosain and penaeus vannamei boone, exosomes are prepared from blood of corresponding species in different species, the volume ratio of the exosomes to the anticoagulant is 1:1 after blood is drawn, and the centrifugation operation is 1000×g for 10min; in the step A2, the volume ratio of the sediment obtained in the step A1 is 1000:1 by using an anticoagulant, and the centrifugation operation is 1000 Xg for 10min; in the step B1, the volume ratio of the homogeneous solution to the blood cells is 10:1; in the step B1, the ultrasonic operation is that ultrasonic is conducted for 3s, and the ultrasonic operation is conducted for 2s, and the ultrasonic operation lasts for 3min; in the step B2, the centrifugation operation is 1500 Xg for 10min; in the step B3, the centrifugation operation is 20000 Xg for 30min; in the step B4, the centrifugation operation is 60000 Xg centrifugation for 30min; in the step B5, the volume ratio of the homogenized liquid to the LiCl solution to the sediment is 2:1:1 before centrifugation, and the volume ratio of the homogenized liquid to the sediment is 5:1 after centrifugation; in the step B5, the centrifugation operation is 20000 Xg for 30min; in step C, the temperature of the cryopreservation was-80 ℃.
Preferably, the homogenized solution comprises 250mM sorbitol, 137mM NaCl,10mM PMSF, dissolved with Tris-HCl-pH 7.4.
The method for over-expressing the genes in the crustacean body can obtain over-expressed exosomes.
The recombinant plasmid of the artificial exosome coated over-expressed gene realizes the over-expression of the gene in the crustacean.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
by adopting the production method of the invention, the exosomes carrying bioactive substances can be produced simply and efficiently; the exosome can be easily absorbed by the receptor cells to play the role of bioactive substances, so that the plasmid carried by the exosome can be ensured to efficiently enter the receptor cells, the expression efficiency of the plasmid in the receptor cells is improved, and the exosome has no obvious immune rejection reaction and can not cause injury to the receptor cells. Exosomes carrying biologically active substances can be widely used as a novel delivery tool for immunological research and treatment of various diseases.
The invention connects the gene segment to be over-expressed with the carrier, then uses the artificial exosome to transport it into the crustacean blood cell, to realize the over-expression of the target gene in the crustacean blood cell. The method is non-viral transfection, can effectively enter cells, is simple and practical, has low damage to the cells, and solves the problem that the prior art cannot realize efficient overexpression of specific target genes in crustaceans. Wherein exogenous genes are introduced into eukaryotic cells by exosomes, which promote the cells to express specific genes, with safety and modified properties.
Drawings
FIG. 1 is a flow chart of a method embodying the present invention;
FIG. 2A shows PCR detection of 14-3-3mRNA, AIF mRNA and p53mRNA loaded in artificial exosomes prepared from blue crab blood cells after the artificial exosomes were coated with 14-3-3mRNA, AIF mRNA and p53mRNA, respectively; b is to use blue crab blood cells to prepare artificial exosomes to wrap 14-3-3 protein, AIF protein and p53 protein respectively, and then Western blot is used for detecting whether the artificial exosomes are loaded with 14-3-3 protein, AIF protein and p53 protein.
FIG. 3A shows the content of 14-3-3 protein in blood cells compared with control group, as observed under confocal microscope after injection of 14-3-3mRNA and exosomes loaded with 14-3-3mRNA into blue crabs for 12 h; b is 14-3-3mRNA and 14-3-3 mRNA-loaded exosomes are injected into prawns for 12 hours, and then the content of 14-3-3 protein in blood cells is compared with that in a control group by observation under a confocal microscope; c is 14-3-3 protein and exosomes loaded with 14-3-3 protein are injected into blue crabs for 12 hours, and the content of 14-3-3 protein in blood cells is compared with that in a control group by observation under a confocal microscope; d is 14-3-3 protein and exosomes loaded with 14-3-3 protein were injected into prawns for 12h, and the content of 14-3-3 protein in blood cells was observed under confocal microscope compared with control group.
FIG. 4A shows AIF mRNA and AIF mRNA-loaded exosomes after injection into blue crabs for 12h, as observed under confocal microscopy, compared to control blood cell levels; b is AIF mRNA and AIF mRNA-loaded exosomes were injected into shrimp for 12h, and observed under confocal microscopy, compared to control group, the content of AIF protein in blood cells; c is AIF protein and AIF protein-loaded exosomes are injected into blue crabs for 12 hours, and then observed under a confocal microscope, and compared with a control group, the AIF protein content in blood cells is obtained; d is the content of AIF protein in blood cells compared with the control group, observed under confocal microscope after injecting AIF protein and AIF protein-loaded exosomes into prawns for 12 h.
FIG. 5A shows the p53mRNA and p53mRNA loaded exosomes injected into blue crabs for 12h, and the p53 protein content in blood cells compared to control group, as observed under confocal microscopy; b is p53mRNA and p53 mRNA-loaded exosomes were injected into prawns for 12h, and observed under confocal microscopy, compared to control, the content of p53 protein in blood cells; c is p53 protein and p53 protein loaded exosomes are injected into blue crabs for 12 hours, and the content of p53 protein in blood cells is observed under a confocal microscope and compared with a control group; d is p53 protein and p53 protein-loaded exosomes were injected into prawns for 12h and observed under confocal microscopy, compared to the control group, the content of p53 protein in blood cells.
FIG. 6A is a graph of test for the inclusion of pIZ-wsv249p-egfp-14-3, pIZ-wsv p-egfp-AIF and pIZ-wsv249p-egfp-p53 in exosomes loaded with pIZ-wsv249p-egfp-14-3, pIZ-wsv249p-egfp-AIF and pIZ-wsv249p-egfp-p53, respectively; b is that after artificial exosomes loaded with pIZ-wsv249p-egfp vectors are injected into blue crabs for 12 hours, the content of GFP protein is observed under a confocal microscope, and the uncoated artificial exosomes are injected into the blue crabs as a control; c is that after artificial exosomes loaded with pIZ-wsv249p-egfp vector are injected into prawns for 12h, the content of GFP protein is observed under a confocal microscope, and the uncoated artificial exosomes are injected into the prawns as a control.
FIG. 7A shows the content of 14-3-3 protein in blood cells after injection of exosomes loaded with 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3, respectively, into blue crabs for 12h under confocal microscopy; b is the content of 14-3-3 protein and its downstream regulatory protein MyD88 and TLR in blue crab blood cells after injection of exosomes loaded with 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 respectively into blue crab for 12h (Sun et al, journal of immunology.2022,209: 710-722); c is the transcription level change of the anti-lipopolysaccharide factors ALF1, ALF4 and ALF5 in blue crab blood cells after the exosomes loaded with 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 respectively are injected into blue crab for 12 h; d is the content of 14-3-3 protein in blood cells after the exosomes loaded with 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 respectively are injected into prawns for 12h, and the content of 14-3-3 protein in blood cells is observed under a confocal microscope; e is the content of 14-3-3 protein and downstream regulatory proteins MyD88 and TLR in prawn blood cells after 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 exosomes are respectively loaded and injected into prawns for 12 h; f is the transcriptional level change of anti-lipopolysaccharide factors ALF1, ALF4 and ALF5 in prawn blood cells after the exosomes loaded with 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 respectively are injected into prawn for 12 h.
FIG. 8A shows the AIF protein content of blood cells after injection of exosomes loaded with AIF mRNA, AIF protein and pIZ-wsv249p-egfp-AIF, respectively, into scylla paramamosain for 12h under confocal microscopy; b is the apoptosis level change of blue crab blood cells detected by a flow cytometer after the exosomes loaded with AIF mRNA, AIF protein and pIZ-wsv249p-egfp-AIF respectively are injected into blue crabs for 12 hours (Gong et al, PLoS pathens.2020, 16 (5), e 1008366); c is the content of AIF protein in blood cells observed under a confocal microscope after injecting exosomes loaded with AIF mRNA, AIF protein and pIZ-wsv249p-egfp-AIF into prawns for 12 h; D. e and F are exosomes loaded with AIF mRNA, AIF protein and pIZ-wsv249p-egfp-AIF, respectively, and after injection into prawns for 12h, the flow cytometer detected changes in apoptosis levels of prawn blood cells.
FIG. 9A shows the p53 protein content of blood cells after injection of exosomes loaded with p53-mRNA, p53-protein and pIZ-wsv249p-egfp-p53, respectively, into blue crabs for 12h under confocal microscopy; b is that exosomes loaded with P53mRNA, P53 protein and pIZ-wsv249P-egfp-P53 respectively are injected into blue crabs for 12 hours, and a flow cytometer detects the apoptosis level change of blue crab blood cells (Gong et al, journal of virology.2022,96 (6), e 0202921); c is the content of P53 protein in blood cells observed under a confocal microscope after exosomes loaded with P53mRNA, P53 protein and pIZ-wsv249P-egfp-P53 respectively are injected into prawns for 12 h; D. e and F are exosomes loaded with P53mRNA, P53 protein and pIZ-wsv249P-egfp-P53, respectively, and after injection into prawns for 12h, the flow cytometer detected changes in apoptosis levels of prawn blood cells.
Detailed Description
The present invention will be further described in detail below with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, so that those skilled in the art can better understand the present invention and implement the present invention, but the present invention is not limited thereto. 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. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
All components used in the examples below were obtained by purchasing or self-making.
Example 1
The embodiment of the invention provides a method for over-expressing genes in crustacean bodies, which is shown in a figure 1, and is specifically realized by the following steps:
s1, designing an over-expression gene primer;
s2, constructing an overexpression plasmid;
s3, cell collection: extracting blood from scylla paramamosain or prawn with 5mL syringe, preparing exosomes in different species by using blood of corresponding species, mixing 1mL blood with 1mL anticoagulant, centrifuging 1000g for 10min, centrifuging the obtained precipitate by anticoagulant resuspension centrifuging 1000g for 10min again, and collecting cell precipitate;
s4, cell disruption: the above cell pellet was resuspended in 10 volumes of homogenized solution (250 mM sorbitol, 137mM NaCl,10mM PMSF, dissolved with Tris-HCl-pH 7.4) and broken on ice at 5% power for 3min (ultrasound on 3s, off 2 s);
s5, low-speed centrifugation: centrifuging 1500 Xg of the solution crushed in the step S2 for 10min;
s6, high-speed centrifugation: centrifuging the supernatant after S3 centrifugation again by 20000 Xg for 30min;
s7, collecting cytoplasmic solution: centrifuging 60000 Xg of supernatant after S4 centrifugation for 30min and collecting the supernatant after centrifugation;
s8, cell membrane solution collection: the precipitate after S4 centrifugation was resuspended with twice the volume of the homogenized solution and one time the volume of LiCl solution, centrifuged for 30min at 20000 Xg and resuspended with 5 times the volume of the homogenized solution;
s9, in vitro reaction: the following reaction system was prepared: 17uL of cytoplasmic solution obtained in S7, 10uL of cell membrane solution obtained in S8, 4uL of ATP regenerating system (10mM ATP,500mM GDP-mannose, 400mM creatine phosphate, 40mM creatine phosphate, 20mM HEPES-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate); 8uL incubation buffer (80mM KCl,20mM CaCl) 2 12.5mM HEPES-NaOH, pH 7.4,1.5mM,MgOAc,1mM DTT)), and finally adding 200ng of the over-expression plasmid, blowing and mixing by a pipetting gun, and reacting for 20min at 30 ℃ in a metal bath;
s10, ultracentrifugation is carried out to collect exosomes carrying over-expression plasmids. Centrifugation at 20000 Xg for 30min, pellet collection and resuspension with 50uL PBS gave the over-expressed exosomes, which were detected by agarose gel electrophoresis, the results are shown in FIG. 6A. The results indicate that the artificial exosomes can encapsulate the over-expression plasmid.
S11, injecting the over-expression exosome into crustacean body to make the gene over-expressed.
Example 2
S1, adding 14-3-3mRNA into an artificial exosome synthesis system, and detecting whether the artificial exosome is successfully loaded with the 14-3-3mRNA by agarose gel electrophoresis;
the results are shown in FIG. 2A. The results showed that the synthetic artificial exosomes successfully encapsulate 14-3-3mRNA.
S2, injecting the artificial exosome wrapped with 14-3-3mRNA and 14-3-3mRNA into blue crabs or prawns respectively, collecting blue crabs and prawn blood cells respectively after 12 hours, and observing the 14-3-3 protein content in the blood cells under a confocal microscope;
the results are shown in FIGS. 3A and 3B. The results show that compared with the direct injection of 14-3-3mRNA, the content of 14-3-3 in blue crab and prawn blood cells is obviously improved after the injection of the artificial exosome coated with 14-3-3mRNA.
S3, adding 14-3-3 protein (14-3-3 recombinant protein) into an artificial exosome synthesis system, and detecting whether the artificial exosome is successfully loaded with the 14-3-3 protein by Western blot;
the results are shown in FIG. 2B. The results show that the synthesized artificial exosomes successfully encapsulate 14-3-3 proteins.
S4, respectively injecting the artificial exosome coated with the 14-3-3 protein and the 14-3-3 protein into the blue crabs or the prawns, respectively collecting the blue crabs and the prawn blood cells after 12 hours, and observing the content of the 14-3-3 protein in the blood cells under a confocal microscope;
the results are shown in FIGS. 3C and 3D. The results show that compared with the direct injection of 14-3-3 recombinant protein, the content of 14-3-3 in blue crab and prawn blood cells is obviously improved after the injection of the artificial exosome coated with 14-3-3 protein.
S5.14-3-3 primer design: according to the primer design of pIZ-wsv249p-egfp, the gene sequence of 14-3-3 and the EasyGeno rapid recombination kit, 14-3-3 in-vitro amplification primers are designed;
s6, construction of an overexpression plasmid: the 14-3-3 in vitro amplification primer designed as above obtains a large number of 14-3-3 fragments by PCR, simultaneously cuts the pIZ-wsv249p-egfp vector according to the selected cleavage site, and ligates the 14-3-3 fragment to the pIZ-wsv249p-egfp vector according to the instructions of the day root easy geno rapid recombination kit;
s7, adding pIZ-wsv249p-egfp-14-3-3 recombinant plasmid in the synthetic process of the artificial exosome, and detecting whether the artificial exosome is successfully loaded with pIZ-wsv249p-egfp-14-3-3 by agarose gel electrophoresis;
the results are shown in FIG. 6A. The results showed that the synthesized artificial exosomes were successfully loaded with pIZ-wsv249p-egfp-14-3-3.
S8, respectively injecting the 14-3-3mRNA, 14-3-3 protein and pIZ-wsv249p-egfp-14-3-3 artificial exosomes into blue crabs or prawns, respectively collecting blue crabs and prawn blood cells after 12 hours, detecting the content of the 14-3-3 protein in the blood cells through a confocal microscope and a Western blot, detecting the content change of 14-3-3 downstream regulatory proteins MyD88 and TLR through the Western blot, and detecting the expression change of 14-3-3 downstream regulatory anti-lipopolysaccharide factors ALF1, ALF4 and ALF5 through qPCR;
the results are shown in FIGS. 7A, 7B, 7C, 7D, 7E and 7F. The results show that compared with the artificial exosome coated with 14-3-3mRNA or 14-3-3 protein, the content of 14-3-3 in blue crabs and prawn blood cells and the influence of the content on the downstream regulation and control channels are more obvious after the artificial exosome coated with pIZ-wsv249p-egfp-14-3-3 is injected.
Example 3
S1, adding AIF mRNA into an artificial exosome synthesis system, and detecting whether the artificial exosome successfully loads the AIF mRNA by agarose gel electrophoresis;
the results are shown in FIG. 2A. The results showed that the synthetic artificial exosomes successfully encapsulate AIF mRNA.
S2, injecting the artificial exosome coated with AIF mRNA and AIF mRNA into blue crabs or prawns respectively, collecting blue crabs and prawn blood cells after 12 hours respectively, and observing the AIF protein content in the blood cells under a confocal microscope;
the results are shown in FIGS. 4A and 4B. The results show that compared with the direct injection of AIF mRNA, the AIF is obviously improved in blue crab and prawn blood cells after the injection of the artificial exosome wrapping the AIF mRNA.
S3, adding AIF protein (recombinant protein of AIF) into an artificial exosome synthesis system, and detecting whether the artificial exosome successfully loads the AIF protein through Western blot;
the results are shown in FIG. 2B. The results show that the synthetic artificial exosomes successfully encapsulate AIF proteins.
S4, injecting the artificial exosome coated with the AIF protein and the AIF protein into the blue crabs or the prawns respectively, collecting blue crabs and prawn blood cells respectively after 12 hours, and observing the AIF protein content in the blood cells under a confocal microscope;
the results are shown in FIGS. 4C and 4D. The results show that compared with the direct injection of AIF protein, the AIF is obviously improved in blue crab and prawn blood cells after the injection of the artificial exosome coated with the AIF protein.
S5, AIF primer design: designing an AIF in vitro amplification primer according to the cleavage site on pIZ-wsv249p-egfp, the gene sequence of AIF and the design specification of a primer of a day root easy Geno rapid recombination kit;
s6, construction of an overexpression plasmid: according to the AIF in vitro amplification primer designed as above, a large number of AIF fragments are obtained by PCR, while the pIZ-wsv249p-egfp vector is cut according to the selected cleavage site, and the AIF fragments are connected to pIZ-wsv249p-egfp vector according to the use instructions of the EasyGeno rapid recombination kit;
s7, adding pIZ-wsv249p-egfp-AIF recombinant plasmid in the synthetic process of the artificial exosome, and detecting whether the artificial exosome is successfully loaded with pIZ-wsv249p-egfp-AIF by agarose gel electrophoresis;
the results are shown in FIG. 6A. The results showed that the synthetic artificial exosomes were successfully loaded with pIZ-wsv249p-egfp-AIF.
S8, respectively injecting artificial exosomes wrapping AIF mRNA, AIF protein and pIZ-wsv249p-egfp-AIF into blue crabs or prawns, respectively collecting blue crabs and prawn blood cells after 12 hours, detecting the AIF protein content in the blood cells by a confocal microscope and Western blot, and detecting the apoptosis rate by a flow cytometry;
the results are shown in FIGS. 8A, 8B, 8C, 8D, 8E and 8F. The results show that compared with the artificial exosome coated with AIF mRNA or AIF protein, the content of AIF in blue crabs and prawn blood cells and the influence of AIF on the downstream regulation and control channel are more obvious after the artificial exosome coated with pIZ-wsv249p-egfp-AIF is injected.
Example 4
S1, adding p53mRNA into an artificial exosome synthesis system, and detecting whether the artificial exosome is successfully loaded with the p53mRNA by agarose gel electrophoresis;
the results are shown in FIG. 2A. The results showed that the synthetic artificial exosomes successfully encapsulate p53 mRNA.
S2, injecting the artificial exosome wrapped with the p53mRNA and the p53mRNA into the blue crabs or the prawns respectively, collecting blue crabs and prawn blood cells respectively after 12 hours, and observing the p53 protein content in the blood cells under a confocal microscope;
the results are shown in FIGS. 5A and 5B. The results show that compared with the direct injection of the P53mRNA, the content of the P53 in the blue crab and the prawn blood cells is obviously improved after the injection of the artificial exosome which wraps the P53 mRNA.
S3, adding p53 protein (p 53 recombinant protein) into an artificial exosome synthesis system, and detecting whether the artificial exosome successfully loads the p53 protein through Western blot;
the results are shown in FIG. 2B. The results show that the synthetic artificial exosomes successfully encapsulate the p53 protein.
S4, injecting the artificial exosome coated with the p53 protein and the p53 protein into the blue crabs or the prawns respectively, collecting blue crabs and prawn blood cells respectively after 12 hours, and observing the p53 protein content in the blood cells under a confocal microscope;
the results are shown in FIGS. 5C and 5D. The results show that compared with the direct injection of the p53 protein, the content of the p53 in the blue crab and the prawn blood cells is obviously improved after the injection of the artificial exosome which wraps the p53 protein.
S5.P53 primer design: according to the enzyme cutting site on pIZ-wsv249p-egfp, the gene sequence of p53 and the design description of the primers of the quick recombination kit of the root of heaven easy Geno, the primers for in vitro amplification of p53 are designed;
s6, construction of an overexpression plasmid: according to the P53 in vitro amplification primer designed as above, a large number of P53 fragments are obtained by PCR, meanwhile, the pIZ-wsv249P-egfp vector is cut according to the selected enzyme cutting site, and the P53 fragments are connected to the pIZ-wsv249P-egfp vector according to the usage instructions of the EasyGeno rapid recombination kit;
s7, adding pIZ-wsv249p-egfp-p53 recombinant plasmid in the synthetic process of the artificial exosome, and detecting whether the artificial exosome is successfully loaded with pIZ-wsv249p-egfp-p53 by agarose gel electrophoresis;
the results are shown in FIG. 6A. The results showed that the synthetic artificial exosomes were successfully loaded with pIZ-wsv249p-egfp-p53.
S8, respectively injecting artificial exosomes wrapping p53mRNA, p53 protein and pIZ-wsv249p-egfp-p53 into blue crabs or prawns, respectively collecting blue crabs and prawn blood cells after 12 hours, detecting the content of p53 protein in the blood cells through a confocal microscope and Western blot, and detecting the apoptosis rate of the blood cells through a flow cytometer;
the results are shown in FIGS. 9A, 9B, 9C, 9D, 9E and 9F. The results show that compared with the artificial exosome coated with p53mRNA or p53 protein, the artificial exosome coated with pIZ-wsv249p-egfp-p53 has more obvious influence on the content improvement of p53 in blue crab and prawn blood cells and the downstream regulation and control channel.
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent changes may be made in the claims herein without departing from the scope of the invention.
Claims (11)
1. A method for gene overexpression in crustacean bodies, comprising the steps of:
A. amplifying a target sequence;
B. loading the target sequence on a carrier to construct an over-expression plasmid;
C. wrapping the over-expression plasmid in an artificial exosome to form an over-expression exosome;
D. the over-expression exosome is injected into crustacean body to over-express the gene.
2. The method of gene overexpression in crustacean according to claim 1, wherein said sequence of interest comprises a nucleic acid.
3. The method of gene overexpression in crustacean according to claim 1, wherein said sequence of interest comprises one or more of 14-3-3, AIF, p53, GFP.
4. The method of gene overexpression in crustacean according to claim 1, wherein said vector comprises one or more of pIZ-wsv249p-egfp plasmid, pcdna3.1 plasmid, pitt/V5-His plasmid; the crustacean comprises one or more of Scylla paramamosain and Penaeus vannamei Boone.
5. The method for gene overexpression in crustacean body according to claim 1, wherein in step a, amplification is performed by designing amplification primers for the target sequence based on the target sequence and the vector; in the step B, the over-expression plasmid is constructed by adopting a one-step cloning method; in step C, the over-expression plasmid is not less than 200ng.
6. The method of claim 1, wherein step C comprises:
c1, collecting blood cells;
c2, separating the cell membrane and cytoplasmic matrix of the blood cells;
and C3, adding the cell membrane and the cytoplasmic matrix into a reaction system, adding the over-expression plasmid, and reacting to obtain the over-expression exosome.
7. The artificial exosome according to claim 1, the method for preparing the artificial exosome, comprising the steps of:
A. collecting the blood cells:
a1, drawing blood from crustacean, mixing with anticoagulant, centrifuging and collecting precipitate;
a2, resuspending the sediment obtained in the step A1 by using an anticoagulant, and centrifugally collecting the sediment to obtain the blood cells;
B. isolating the cell membrane and the cytoplasmic matrix:
b1, re-suspending the blood cells by using a homogeneous solution, and carrying out ultrasonic vibration on ice to crush the blood cells to obtain a crushed cell solution;
b2, centrifuging the crushed cell solution, and collecting supernatant;
b3, centrifuging the supernatant obtained in the step B2, and collecting supernatant and precipitate;
b4, centrifuging the supernatant obtained in the step B3, and collecting the supernatant to obtain a solution of the cytoplasmic matrix;
b5, re-suspending the precipitate obtained in the step B3 by using the homogenized solution and LiCl solution, centrifuging, and re-suspending by using the homogenized solution to obtain a solution of the cell membrane
C. Adding the solution of the cytoplasmic matrix and the solution of the cell membrane into an ATP regeneration system and an incubation buffer solution, adding a supported body, reacting to obtain the artificial exosome, and freezing and preserving.
8. The method of claim 7, wherein in step C3, the reaction system comprises a regeneration system and an incubation buffer; the regeneration system is an ATP regeneration system and comprises ATP, GDP-mannose, creatine phosphate, HEPES-pH 7.2, sorbitol, potassium acetate and magnesium acetate; the incubation buffer comprises KCl and CaCl 2 ，HEPES-NaOH-pH 7.4，MgOAc，DTT。
9. The method of gene overexpression in crustacean according to claim 8, wherein said ATP regeneration system comprises 10mM ATP,500mM GDP-mannose, 400mM creatine phosphate, 40mM creatine phosphate, 20mM HEPEs-pH 7.2, 250mM sorbitol, 150mM potassium acetate, 5mM magnesium acetate; the incubation buffer included 80mM KCl,20mM CaCl 2 ，12.5mM HEPES-NaOH-pH 7.4，1.5mM MgOAc，1mM DTT。
10. The method of gene overexpression in crustacean bodies according to claim 1, comprising the steps of:
A. amplifying a target sequence:
a1, designing an in vitro amplification primer of the target sequence according to a one-step cloning method according to the target sequence and an enzyme cutting site behind a promoter of the vector;
a2, amplifying the target sequence by using the in-vitro amplification primer, and purifying and collecting an amplification product;
B. loading the target sequence on a carrier, and constructing an over-expression plasmid:
b1, carrying out double enzyme digestion on the vector according to enzyme digestion sites at two ends of the target sequence, purifying and collecting a double enzyme digested product;
b2, connecting the amplification product with the double-enzyme-digested product, and screening recombinant plasmids which are successfully connected to form the over-expression plasmid;
C. wrapping the over-expression plasmid in an artificial exosome to form an over-expression exosome:
c1, collecting blood cells:
c1-1, drawing blood from crustacean, mixing with anticoagulant, centrifuging and collecting precipitate;
c1-2, re-suspending the sediment obtained in the step A1 by using an anticoagulant, and centrifugally collecting the sediment to obtain the blood cells;
c2, separating the cell membrane and cytoplasmic matrix of the blood cells:
c2-1, re-suspending the blood cells with a homogeneous solution, and carrying out ultrasonic vibration on ice to crush the blood cells to obtain a crushed cell solution;
c2-2, centrifuging the crushed cell solution, and collecting supernatant;
c2-3, centrifuging the supernatant obtained in the step C2-2, and collecting the supernatant and the precipitate;
c2-4, centrifuging the supernatant obtained in the step C2-3, and collecting the supernatant to obtain a solution of the cytoplasmic matrix;
c2-5, re-suspending the sediment obtained in the step C2-3 by using the homogenized liquid and LiCl solution, centrifuging, and re-suspending by using the homogenized liquid to obtain a solution of the cell membrane;
c3, adding the solution of the cytoplasmic matrix and the solution of the cell membrane into an ATP regeneration system and an incubation buffer solution, adding the over-expression plasmid, reacting to obtain the over-expression exosome, and freezing for preservation;
D. the over-expression exosome is injected into crustacean body to over-express the gene.
11. The method of gene overexpression in crustacean according to claim 10, wherein said homogenized solution comprises 250mM sorbitol, 137mM NaCl,10mM PMSF, solubilized with Tris-HCl-pH 7.4; in step C3, the volume to mass ratio of the solution of the cytoplasmic matrix, the solution of the cell membrane, the ATP regeneration system, the incubation buffer, and the over-expression plasmid is 17uL:10uL:8uL:200ng.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310613942.6A CN117604032A (en) | 2023-05-26 | 2023-05-26 | Method for over-expression of genes in crustacean body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310613942.6A CN117604032A (en) | 2023-05-26 | 2023-05-26 | Method for over-expression of genes in crustacean body |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117604032A true CN117604032A (en) | 2024-02-27 |
Family
ID=89950304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310613942.6A Pending CN117604032A (en) | 2023-05-26 | 2023-05-26 | Method for over-expression of genes in crustacean body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117604032A (en) |
-
2023
- 2023-05-26 CN CN202310613942.6A patent/CN117604032A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liang et al. | Chondrocyte-specific genomic editing enabled by hybrid exosomes for osteoarthritis treatment | |
| KR20200088408A (en) | Method for producing exosome derived from MSC | |
| CN110448696B (en) | Preparation method and application of targeted drug delivery carrier based on dunaliella salina exosomes | |
| Xin et al. | In-vivo induced CAR-T cell for the potential breakthrough to overcome the barriers of current CAR-T cell therapy | |
| CN103002879A (en) | Microvesicles derived from cell protoplast, and use thereof | |
| CN114007655A (en) | Circular RNA for cell therapy | |
| US20220025403A1 (en) | Reduced and minimal manipulation manufacturing of genetically-modified cells | |
| CN111110855B (en) | Targeting vesicle medicine prepared from erythrocytes | |
| US20210353769A1 (en) | Surface modified extracellular vesicles | |
| JP2022547570A (en) | Engineered adeno-associated virus capsid | |
| US20210147828A1 (en) | Dna damage response signature guided rational design of crispr-based systems and therapies | |
| CN113215104A (en) | Exosome containing CD10-dm protein and preparation method and application thereof | |
| CN116286996A (en) | Medicine carrier targeting CXCL16+ macrophages and preparation method and application thereof | |
| Laga et al. | Isolation and properties of polysomes from human placenta | |
| CN107648620B (en) | Targeting ultra-sonar rice bubble carrying CAIX aptamer and preparation method thereof | |
| CN117604032A (en) | Method for over-expression of genes in crustacean body | |
| EP3912629A1 (en) | Method for delivering gene in cells | |
| CN115029320A (en) | Engineered exosome for tumor radiotherapy sensitization, preparation method and application | |
| CN101148680B (en) | Method for introducing plasmid vector containing gene cure segment in cell by nano particles | |
| Tasca et al. | High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells | |
| WO2020186237A1 (en) | Microglial progenitors for regeneration of functional microglia in the central nervous system and therapeutics uses thereof | |
| CN112639113A (en) | Nucleic acid delivery vector, nucleic acid delivery vector set, nucleic acid delivery composition, and nucleic acid delivery method | |
| CN116286595A (en) | Preparation method of crustacean blood cell artificial exosome | |
| CN113528519A (en) | Egg duck circular RNA circ _2136 and detection reagent, method and application thereof | |
| CN104288776B (en) | A kind of self assembly polypeptide-apoptin gene composite nanometer particle and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |