CN114891834B - Method for preparing high-yield antibody rabbit based on CRISPR technology - Google Patents
Method for preparing high-yield antibody rabbit based on CRISPR technology Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
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- A01K2227/107—Rabbit
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- A—HUMAN NECESSITIES
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
A method for preparing high-yield antibody rabbits based on a CRISPR technology belongs to the technical field of biology. The invention aims to create a method for preparing high-yield antibody rabbits by adopting a CRISPR technology for developing rabbit monoclonal antibodies. The method comprises the following steps: obtaining enough rabbit fertilized eggs, microinjection of embryos and genotyping of rabbits. The invention provides a means for developing rabbit monoclonal antibodies, promotes the antibody preparation technology to play an unprecedented important role in diagnosis, pharmacodynamics and clinical application, and leads to a new era of new therapeutic antibody research.
Description
Technical Field
The invention belongs to the field of biotechnology.
Background
Since the mouse immune system cannot recognize some immunogens, especially those of murine origin, and rabbits can produce a strong immune response to foreign proteins and have high affinity, rabbit polyclonal antibodies are widely used in numerous immunological experiments. In recent years, CRISPR/Cas9 gene editing technology has opened up a new idea for high-efficiency gene editing, greatly promotes the development of genetically modified animals, and is successfully applied to a plurality of species such as zebra fish, mice, rats, rabbits, pigs, monkeys and the like.
Disclosure of Invention
The invention aims to create a method for preparing high-yield antibody rabbits by adopting a CRISPR technology for developing rabbit monoclonal antibodies.
The method comprises the following steps:
step 1, obtaining enough rabbit fertilized eggs: selecting female New Zealand white rabbits, injecting follicle stimulating hormone 50 IU into the female New Zealand white rabbits in estrus to promote follicular development and maturation, injecting once every 12 hours, continuously injecting for 3 days, mating the female donor rabbits with male rabbits after the last injection, injecting 100 IU human chorionic gonadotropin into the female donor rabbits through the ear margin, taking out the ovaries and oviducts after injecting for 18-20 hours, placing the ovaries and oviducts into a culture dish, flushing fertilized embryos by using egg flushing liquid DPBS-BSA, transferring the embryos into culture liquid EBSS, and placing the embryos into a culture box with saturated humidity of 5% CO2 at 38.5 ℃ for incubation for standby;
step 2, embryo microinjection: centrifuging in vitro synthesized IGHG-sgRNA of 50ng/UL, single-stranded DNA and Cas9mRNA of 200ng/UL, mixing, sucking 3 mu L into an injection needle, injecting embryo cell nuclei, transplanting 30-50 fertilized eggs after injection into oviduct of recipient female rabbits in the same period, providing sufficient feed and drinking water for the female rabbits in the same period, maintaining a clean feeding environment, standardizing feeding, and transferring to a delivery room for feeding to a pre-delivery period after 20 days of gestation;
step 3, identifying the genotype of the rabbit: extracting DNA of the tissue, performing PCR and sequencing, and determining genotype;
PCR primer: an upstream primer: SEQ ID NO. 4: GACCACCATCACCATCTTCA
A downstream primer: SEQ ID NO. 3: GGGTGGACGACAGATGC
PCR reaction system
Template 1ul
1ul of upstream primer
1ul of downstream primer
2X taqplus 12.5 ul
9.5ul of distilled water
Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s;35 cycles; extending at 72 ℃ for 5min; the PCR product was sequenced and if the G at position 19 of the sgRNA sequence was mutated to A, it was confirmed that a single base mutation was obtained.
The single-stranded design of the invention is to add 50bp to the left and right of the sgRNA sequence on the IGHG gene sequence according to the sgRNA sequence, mutate the 19 th G on the sgRNA into A, and the specific oligonucleotide single-stranded sequence is SEQ ID NO. 2.
The invention designs sgRNA for mutating tail tyrosine motif aiming at rabbit IGHG sequence, and the specific sgRNA sequence is SEQ ID NO. 1.
The sgRNA synthesis method comprises the following steps: adopting a solid-phase phosphoramidite triester method, producing by taking a commercial DNA synthesizer as a main synthesis platform, connecting single nucleotides to form short-chain DNA, synthesizing from the 3' -5' direction, connecting adjacent nucleotides through 3' -5' phosphodiester bonds, and normally combining the first base of the 3' end on CPG; the steps are as follows: the addition of one base is completed through the steps of deprotection, activation condensation, capping and oxidation; upstream: 5'-CTACAGGAACATGATCGGGC-3'
Downstream 5'-GCCCGATCATGTTCCTGTAG-3'.
The invention provides a means for developing rabbit monoclonal antibodies, promotes the antibody preparation technology to play an unprecedented important role in diagnosis, pharmacodynamics and clinical application, and leads to a new era of new therapeutic antibody research.
Drawings
FIG. 1 is a diagram of sequencing peaks for the case of gene knockout of the present invention;
FIG. 2 shows the IgG content of serum from normal rabbits and genetically edited rabbits after immunization with a pestilence in rabbits.
Detailed Description
The antibody drug is important in all medical biotechnology products, and is mainly used for treating tumors, autoimmune diseases and infectious diseases at present, but the antibody titer is lower based on the traditional antibody preparation method.
The method comprises the following steps:
step 1, obtaining enough rabbit fertilized eggs: selecting female New Zealand white rabbits, injecting follicle stimulating hormone 50 IU into the female New Zealand white rabbits in estrus to promote follicular development and maturation, injecting once every 12 hours, continuously injecting for 3 days, mating the female donor rabbits with male rabbits after the last injection, injecting 100 IU human chorionic gonadotropin into the female donor rabbits through the ear margin, taking out the ovaries and oviducts after injecting for 18-20 hours, placing the ovaries and oviducts into a culture dish, flushing fertilized embryos by using egg flushing liquid DPBS-BSA, transferring the embryos into culture liquid EBSS, and placing the embryos into a culture box with saturated humidity of 5% CO2 at 38.5 ℃ for incubation for standby;
step 2, embryo microinjection: centrifuging in vitro synthesized IGHG-sgRNA of 50ng/UL, single-stranded DNA and Cas9mRNA of 200ng/UL, mixing, sucking 3 mu L into an injection needle, injecting embryo cell nuclei, transplanting 30-50 fertilized eggs after injection into oviduct of recipient female rabbits in the same period, providing sufficient feed and drinking water for the female rabbits in the same period, maintaining a clean feeding environment, standardizing feeding, and transferring to a delivery room for feeding to a pre-delivery period after 20 days of gestation;
step 3, identifying the genotype of the rabbit: extracting DNA of the tissue, performing PCR and sequencing, and determining genotype;
PCR primer: an upstream primer: SEQ ID NO. 4: GACCACCATCACCATCTTCA
A downstream primer: SEQ ID NO. 3: GGGTGGACGACAGATGC
PCR reaction system
Template 1ul
1ul of upstream primer
1ul of downstream primer
2X taqplus 12.5 ul
9.5ul of distilled water
Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s;35 cycles; extending at 72 ℃ for 5min; the PCR product was sequenced and if the G at position 19 of the sgRNA sequence was mutated to A, it was confirmed that a single base mutation was obtained.
The single-stranded design of the invention is to add 50bp to the left and right of the sgRNA sequence on the IGHG gene sequence according to the sgRNA sequence, mutate the 19 th G on the sgRNA into A, and the specific oligonucleotide single-stranded sequence is SEQ ID NO. 2.
The invention designs sgRNA for mutating tail tyrosine motif aiming at rabbit IGHG sequence, and the specific sgRNA sequence is SEQ ID NO. 1.
The sgRNA synthesis method comprises the following steps: adopting a solid-phase phosphoramidite triester method, producing by taking a commercial DNA synthesizer as a main synthesis platform, connecting single nucleotides to form short-chain DNA, synthesizing from the 3' -5' direction, connecting adjacent nucleotides through 3' -5' phosphodiester bonds, and normally combining the first base of the 3' end on CPG; the steps are as follows: the addition of one base is completed through the steps of deprotection, activation condensation, capping and oxidation; upstream: 5'-CTACAGGAACATGATCGGGC-3'
Downstream 5'-GCCCGATCATGTTCCTGTAG-3'.
The present invention is described in further detail below:
1. specific sgRNA sequences of the invention: upstream: 5'-CTACAGGAACATGATCGGGC-3'
Downstream 5'-GCCCGATCATGTTCCTGTAG-3' preparation method: according to the on-line site (http:// www.rgenome.net/cas-offinder /), mutations were designed for Its Tail Tyrosine (ITT) motif against the rabbit IGHG sequence, and sgRNA was synthesized by Kirsrui Biotechnology Co.
2. Specific oligonucleotide single strand: the single-chain design is that according to the sgRNA sequence, 50bp is added to the left and right of the sgRNA sequence on the IGHG gene sequence, and the 19 th G on the sgRNA is mutated into A, and the single-chain sequence is synthesized by the Kirschner Biotechnology Co. The synthesis method comprises the following steps: the solid phase phosphoramidite triester method is adopted, a commercial DNA synthesizer is used as a main synthesis platform for production, single nucleotides are connected to form short-chain DNA, the synthesis is carried out from the 3' -5' direction, adjacent nucleotides are connected through 3' -5' phosphodiester bonds, and normally the first base at the 3' end is combined with CPG. The steps are as follows: the addition of one base is generally accomplished through four steps of deprotection, activation condensation, capping and oxidation.
Preparation of Cas9mRNA
(1) Cas9 plasmid linearization
3x FLAG-NLS-SpCas9-NLS vector (Addgene ID: 48137), byNotIAfter enzyme linearization, the enzyme digestion system (Table 1) was digested overnight at 37℃and 1. Mu.L of the digested product was subjected to 1% agarose gel electrophoresis, and after complete linearization by electrophoresis identification using the undigested original plasmid as a control, the digested product was recovered with the DNA gel recovery kit.
TABLE 1Not ISingle enzyme cutting system
。
(2) Cas9 plasmid in vitro transcription
Relevant liquids in the mMessage mMachine SP Kit (Ambion) were thawed on ice, the transcription system was followed in accordance with the system of Table 2, the product was placed on ice, 1. Mu.L of Tubro DNaseIII was added, and placed in a 37℃water bath for 2 hours to remove residual DNA transcripts, and the integrity and stability of in vitro transcripts was identified by 2% agarose gel electrophoresis.
TABLE 2 in vitro transcription System of Cas9 plasmid
。
(3) Cas9 plasmid in vitro transcript purification
Cas9 synthesized by in vitro transcription was purified using a miRNeasy Mini Kit (Qiagen) purification kit, the specific procedures were performed as per the kit instructions:
(1) taking in vitro transcription product, adding RNase-free water to 100 μl, slightly blowing and mixing, adding 350 mL Buffer RTL and pre-cooled 250 μl absolute ethanol, and mixing.
(2) Pouring the mixed liquid in the step (1) into a pink purification column, standing for 2 min, centrifuging at 12000rpm for 15s, discarding the waste liquid in the collecting pipe, and putting the purification column back into the collecting pipe.
(3) To the purification column was added 500. Mu.L Buffer RPE, centrifuged at 12000rpm for 15. 15s, and the solution was discarded. This step is repeated one pass.
(4) The purification column was placed in an empty collection tube, centrifuged at 12000rpm for 1 min, and dried at room temperature for 2 min.
(5) The purification column was placed in an RNase-free1.5mL centrifuge tube, hanging drop 30. Mu.L of RNase-free water, and centrifuged at 12000rpm for 2 min, and the purified Cas9-mRNA was obtained in the centrifuge tube.
(6) mRNA concentration was measured, diluted to 200 ng/. Mu.L, split-packed to 2. Mu.L/tube and placed in a-80℃refrigerator for use.
4. To obtain a sufficient number of rabbit fertilized eggs, the experiment used a superovulation protocol. Female New Zealand white rabbits (6-8 months old) were selected, and Follicle Stimulating Hormone (FSH) 50-IU was intramuscularly injected during estrus to promote follicular development and maturation, once every 12 hours, for 3 consecutive days. After the last injection, a mating is performed with the male rabbit, and the successfully mated donor female rabbit is injected with 100 IU Human Chorionic Gonadotropin (HCG) by ear margin intravenous. After 18-20h of injection, the female rabbit ear margin is killed by intravenous injection of air, the abdominal cavity is dissected, the ovary and the oviduct are taken out, the oviduct are placed in a culture dish, fertilized embryos are flushed out by using egg flushing liquid DPBS-BSA, the embryos are transferred into culture liquid EBSS, and the culture liquid EBSS is placed in an incubator with saturated humidity of 5% CO2 at 38.5 ℃ for incubation for standby.
5. Embryo microinjection: in vitro synthesized IGHG-sgRNA (50 ng/UL), single stranded DNA and Cas9mRNA (200 ng/UL), were briefly centrifuged and mixed, 3. Mu.L was aspirated into the needle, and embryo nuclei were injected. The fertilized eggs (about 30-50) after injection are transplanted into oviducts of recipient female rabbits in the same period of oestrus, so that sufficient feed and drinking water are provided for the female rabbits in the same period of oestrus, a clean feeding environment is maintained, and standardized feeding is realized. After gestation for 20 days, the breeding is transferred to a delivery room for breeding to a pre-delivery period.
6. Identification of Rabbit genotype
DNA of the tissue is extracted, and the extraction method is operated according to the instruction of the kit, PCR and sequencing are carried out, and the genotype is determined.
PCR primer: an upstream primer: GACCACCATCACCATCTTCA
A downstream primer: GGGTGGACGACAGATGC
PCR reaction system
Template 1ul
1ul of upstream primer
1ul of downstream primer
2X taqplus 12.5 ul
9.5ul of distilled water
Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s;35 cycles; extending at 72℃for 5min.
The PCR product was sequenced by Biotechnology, inc., and if the G at position 19 of the sgRNA sequence was mutated to A, it was confirmed that a single base mutation was obtained.
7. Antibody level detection
After the genetically modified rabbits develop to 8 weeks of age, the commercial rabbit pestilence vaccine is used for immunization, immunization is carried out once every 7 days, 1ml of each immunization is measured, serum is collected, the collected serum is used for antibody titer detection, and the commercial rabbit pestivirus ELISA antibody detection kit is used for operation, and the method comprises the following steps:
1) Setting 2 enzyme-labeled wells, and adding 100ul of negative control;
2) 100ul of positive control was added to 2 wells;
3) Diluting a sample to be detected by 1:20, and adding 100ul of diluted sample into a corresponding hole;
4) Incubating for 30min at 37 ℃;
5) Discarding the liquid, adding 250ul of washing liquid into each hole to wash the plate, repeating for 5 times for 30s each time;
6) Adding 100ul of enzyme-labeled conjugate to each well;
7) Incubating for 30min at 37 ℃, and repeating the step 5);
8) Adding 100ul of substrate liquid to each well;
9) Incubation at 37deg.C for 15min (development in dark);
10 Add 50ul of stop solution per well;
11 Measuring absorbance, i.e. OD, of each well at a wavelength of 450nm 450 Values.
Sequence listing
<110> Jilin university
<120> method for preparing high-yield antibody rabbit based on CRISPR technology
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Rabbit (Rabbit)
<400> 1
ctacaggaac atgatcgggc 20
<210> 2
<211> 123
<212> DNA
<213> Rabbit (Rabbit)
<400> 2
aagtggatct tctcgtccgt ggtggagctg aaacacacca tcgctcccga ctacaggaac 60
atgatcaggc aaggggccta ggcccttcgt tctcacagcc tgcctccctg gccagcagga 120
gcc 123
<210> 3
<211> 17
<212> DNA
<213> Rabbit (Rabbit)
<400> 3
gggtggacga cagatgc 17
<210> 4
<211> 20
<212> DNA
<213> Rabbit (Rabbit)
<400> 4
gaccaccatc accatcttca 20
Claims (1)
1. A method for preparing high-yield antibody rabbits based on a CRISPR technology is characterized by comprising the following steps: the method comprises the following steps:
step 1, obtaining enough rabbit fertilized eggs: selecting female New Zealand white rabbits, intramuscular injection of follicle stimulating hormone 50 IU in oestrus to promote follicular development and maturation, injecting once every 12 hours, continuously injecting for 3 days, mating with male rabbits after the last injection, carrying out intravenous injection of 100 IU human chorionic gonadotropin on the ear margin of a donor female rabbit which is successfully mated, carrying out intravenous injection air on the ear margin of the female rabbit for 18-20 hours, dissecting abdominal cavity to take out ovaries and oviducts, placing the ovaries in a culture dish, flushing fertilized embryos by using egg flushing liquid DPBS-BSA, transferring the embryos into culture liquid EBSS, and placing the embryos in a culture box with saturated humidity of 5% CO2 for incubation for standby;
step 2, embryo microinjection: centrifuging in vitro synthesized IGHG-sgRNA of 50ng/UL, single-stranded DNA and Cas9mRNA of 200ng/UL, mixing, sucking 3 mu L into an injection needle, injecting embryo cell nuclei, transplanting 30-50 fertilized eggs after injection into oviduct of recipient female rabbits in the same period, providing sufficient feed and drinking water for the female rabbits in the same period, maintaining a clean feeding environment, standardizing feeding, and transferring to a delivery room for feeding to a pre-delivery period after 20 days of gestation; the DNA sequence of the guide sequence of the IGHG-sgRNA is shown as a sequence 1, and the sequence of the single-stranded DNA is shown as a sequence 2;
step 3, identifying the genotype of the rabbit: extracting DNA of the tissue, performing PCR and sequencing, and determining genotype;
PCR primer: an upstream primer: SEQ ID NO. 4: GACCACCATCACCATCTTCA
A downstream primer: SEQ ID NO. 3: GGGTGGACGACAGATGC
PCR reaction system
Template 1ul
1ul of upstream primer
1ul of downstream primer
2X taqplus 12.5 ul
9.5ul of distilled water
Reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s;35 cycles; extending at 72 ℃ for 5min; the PCR product was sequenced and if the G at position 17 corresponding to the sgRNA guide was mutated to A, it was confirmed that a genetically mutated rabbit was obtained.
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| CN113832189A (en) * | 2021-09-06 | 2021-12-24 | 中国人民解放军军事科学院军事医学研究院 | gRNA for knocking out pig immunoglobulin heavy chain IGHG region and application thereof |
| CN114134144A (en) * | 2020-09-04 | 2022-03-04 | 北京仁源欣生生物科技有限公司 | Preparation method and application of non-human mammal or progeny thereof |
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| CN114134144A (en) * | 2020-09-04 | 2022-03-04 | 北京仁源欣生生物科技有限公司 | Preparation method and application of non-human mammal or progeny thereof |
| WO2022048604A1 (en) * | 2020-09-04 | 2022-03-10 | 北京仁源欣生生物科技有限公司 | Method for preparing non-human mammal or progeny thereof, and use thereof |
| CN113832189A (en) * | 2021-09-06 | 2021-12-24 | 中国人民解放军军事科学院军事医学研究院 | gRNA for knocking out pig immunoglobulin heavy chain IGHG region and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Human immunoglobulin constant heavy G chain (IGHG) (Fcγ) (GM) genes, defining innate variants of IgG molecules and B cells, have impact on disease and therapy;Vivi-Anne Oxelius等;《Clinical Immunology》;第149卷;全文 * |
| 应用CRISPR/Cas9 介导的基因编辑系统研究B 细胞中转 录因子T-bet 的调控作用;韩夏夏等;《上海交通大学学报（医学版）》;第42卷(第4期);全文 * |
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