WO2022242500A1 - 一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法 - Google Patents

一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法 Download PDF

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WO2022242500A1
WO2022242500A1 PCT/CN2022/091873 CN2022091873W WO2022242500A1 WO 2022242500 A1 WO2022242500 A1 WO 2022242500A1 CN 2022091873 W CN2022091873 W CN 2022091873W WO 2022242500 A1 WO2022242500 A1 WO 2022242500A1
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ligase
antibody
gene
recognition sequence
specific recognition
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PCT/CN2022/091873
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French (fr)
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陈韬
岳斌
吕爽
张伟
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苏州有诺真生物科技有限公司
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Priority to EP22803822.0A priority Critical patent/EP4298902A1/en
Publication of WO2022242500A1 publication Critical patent/WO2022242500A1/zh
Priority to US18/486,336 priority patent/US20240081302A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of biotechnology, in particular to a preparation method of an antibody carrying a universal site-specific coupling interface based on a genetically modified vertebrate.
  • Antibody is a protein biomacromolecule that binds to the corresponding antigen with strong specificity and affinity. Antibodies are the basis of modern biopharmaceuticals and are widely used in disease treatment, diagnostic reagents and biomedical research. Antibody discovery mainly includes methods such as hybridoma, phage display, and B cell sorting. Vertebrate animals, such as mice, rabbits, sheep, chickens, and camels, are important animal platforms for the development of monoclonal and polyclonal antibodies.
  • the purpose of the present invention is to provide a method for preparing an antibody carrying a universal site-specific coupling interface based on a genetically modified vertebrate.
  • the present invention claims a method for constructing a vertebrate model, the vertebrate model is used to produce an antibody carrying a universal site-specific conjugation interface.
  • the method for constructing a vertebrate model claimed in the present invention may include the following steps: knocking in a specific ligase A at a certain position A of a certain immunoglobulin coding gene A in the genome of the recipient animal.
  • the coding gene of a sex recognition sequence or a coding gene of a certain intein (intein) A is obtained to obtain the vertebrate model.
  • the coding gene A is the Igkc gene (ie, the gene encoding the constant region domain of the antibody kappa light chain), and the position A is the 3' end of the Igkc gene. That is, at the antibody level, the specific recognition sequence of the ligase A will be site-specifically inserted into the C-terminus of the antibody kappa-type light chain.
  • the vertebrate can be selected from any one of the following: mice, rats, rabbits, sheep, chickens, camels, horses, donkeys, hamsters, guinea pigs, alpacas.
  • the vertebrate is a mouse; the coding gene A is the Igkc gene on chromosome 6 in the mouse genome; the position A is located at the Igkc gene on chromosome 6 in the mouse genome Between position 70726754 and position 70726755 (Mouse Genome GRcm38/mm10 version) of the gene (Gene ID: 16071).
  • the ligase A can be selected from any of the following: Sortase staph enzyme, Sortase strep enzyme, Butelase enzyme, oaAEP1 enzyme, Formylglycine generating enzyme (FGE), glutamyl aminotransferase, tubulin tyrosine Ligase (Tubulin Tyrosine Ligase; TTL), Trypsiligase, Sfp phosphopantetheinyl transferase, SpyLigase.
  • Sortase staph enzyme Sortase strep enzyme
  • Butelase enzyme Butelase enzyme
  • oaAEP1 enzyme Formylglycine generating enzyme (FGE)
  • glutamyl aminotransferase glutamyl aminotransferase
  • tubulin tyrosine Ligase Tubulin Tyrosine Ligase
  • Trypsiligase Sfp phosphopantetheinyl transferase
  • SpyLigase
  • the specific recognition sequence can be LPXTG (X is any amino acid); when the ligase A is Sortase strep enzyme, the specific recognition sequence The specific recognition sequence can be LPXTA (X is any amino acid); when the ligase A is Butelase enzyme, the specific recognition sequence can be NHV; when the ligase A is oaAEP1 enzyme, the specific recognition sequence The sequence can be NGL; when the ligase A is a Formylglycine generating enzyme, the specific recognition sequence can be CXPXR (X is any amino acid); when the ligase A is glutamyl transaminase, the specificity The recognition sequence can be LLQGA; when the ligase A is tubulin tyrosine ligase, the specific recognition sequence can be VDSVEGEEEGEE; when the ligase A is Trypsili
  • the site-specific knock-in can be realized by CRISPR/Cas9 technology.
  • target sequence cleaved by Cas9 nuclease is located within the range of 500 bp upstream and downstream of the position A of the coding gene A of immunoglobulin in the genome of the recipient animal, so as to insert specificity through homologous recombination mechanism. Recognition sequence of DNA.
  • the target sequence is specifically SEQ ID No.1 (that is, GGAATGAGTGTTAGAGA*CAAAGG, wherein * is the Cas9 cleavage position) (at this time, the vertebrate is a mouse, and the immunoglobulin
  • the position A of the coding gene A is located at No. 70726754 and No. 70726755 of the Igkc gene (Gene ID: 16071) on chromosome 6 in the mouse genome (mouse genome GRcm38/mm10 version).
  • the homologous recombination vector used as a site-directed knock-in tool contains DNA fragment A; the DNA fragment A is sequentially composed of the 5' homology arm, the coding gene of the specific recognition sequence of the ligase A or The coding gene of intein A, and 3' homology arm composition; The 5' homology arm is located upstream of the position A of the coding gene A of immunoglobulin in the genome of the recipient animal 120bp sequence; the 3' homology arm is a 150bp sequence located downstream of the position A of the immunoglobulin-encoding gene A in the genome of the recipient animal.
  • the 5' homology arm is the 1-120th position of SEQ ID No.2; the 3' homology arm is the 157-306th position of SEQ ID No.2.
  • the coding gene of the specific recognition sequence of the ligase A is the 133-147th position of SEQ ID No.2 (the ligase A is Sortase A enzyme, the 133-147th position of SEQ ID No.2 It is the coding gene of the specific recognition sequence of Sortase A enzyme).
  • nucleotide sequence of the DNA fragment A is shown in SEQ ID No.2.
  • the construction method of the vertebrate model claimed in the present invention may specifically include the following steps:
  • Cas9mRNA, gRNA and the homologous recombination vector described above are injected into the fertilized egg cytoplasm of the recipient animal to obtain F0 generation animals;
  • the sequence of the gRNA is SEQ ID No.4.
  • step (2) The F0 generation animal obtained in step (1) is crossed with the recipient animal, and the position A of the coding gene A of the immunoglobulin in the genome is obtained from the F1 generation animal A heterozygous animal of the gene encoding the specific recognition sequence of the ligase A or the gene encoding the intein A knocked in site-specifically.
  • step (3) may also be included:
  • Both the heterozygous animal in step (2) and the homozygous animal in step (3) can be used as the vertebrate model that can be used to produce antibodies carrying universal site-specific conjugation interfaces.
  • sequence of the Cas9mRNA can be SEQ ID No.3.
  • the homologous recombination vector is specifically the DNA fragment A (SEQ ID No.2).
  • the present invention claims the use of the vertebrate model constructed by the method described in the first aspect above in the production of antibodies carrying a universal site-specific conjugation interface.
  • the present invention claims a method for producing an antibody carrying a universal site-specific conjugation interface.
  • the method for producing an antibody carrying a universal site-specific coupling interface as claimed in the present invention may include the following steps:
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the immunogen is S1 antigen (Dongkang Bio, VISC2-S101), and the vertebrate is a mouse (such as a C57BL/6J mouse).
  • the present invention claims to protect the vertebrate model constructed by using the method described in the first aspect above.
  • the present invention claims to protect the antibody carrying the universal site-specific coupling interface prepared by the method described in the third aspect above.
  • the general site-specific coupling interface specifically refers to the specific recognition sequence of the ligase A or the coding gene of the intein A linked to a specific position of the antibody.
  • the interface is the specific recognition sequence of the ligase A linked to a specific position of the antibody
  • the interface and the linker used to link with the effector group can be used to realize the described
  • the antibody is coupled to the effector group, and the linker can catalyze the linking (such as covalent linking) of the specific recognition sequence with the linker under the action of the ligase A.
  • the ligase A is Sortase A enzyme
  • the specific recognition sequence can be LPXTG (X is any amino acid)
  • the corresponding linker is N-terminal (free end) with oligoglycine (such as 1 - 5 consecutive Gs, another example is 3-5 consecutive G) polypeptide molecules or polypeptide molecules whose N-terminus (free end) is alkylamine.
  • the ligase A is Sortase A enzyme
  • the specific recognition sequence is LPETG
  • the corresponding linker is a polypeptide molecule whose N-terminus (free end) is GGG.
  • the interface is the coding gene of the intein A connected to a specific position of the antibody
  • the antibody is reduced with a reducing agent, and an active group will be generated on it, and the active group can be used to start with the N-terminal Effector molecule or peptide reaction with cysteine as the initiator.
  • the present invention directly introduces a DNA sequence encoding a specific amino acid sequence into the locus of a vertebrate antibody gene, so that the antibody produced by the animal carries a specific site-specific connection site that can be mediated by an enzyme.
  • Figure 1 is a schematic diagram of gene editing in USB mice of the present invention. Schematic diagram of the mouse lgkc locus (the gene is shown from left to right, the full length is 532bp), the gray area represents the open reading frame (ORF), and the white area represents the untranslated region (UTR) of the gene. After successful editing, the wild-type allele in the genome will be replaced by the mutant allele.
  • Figure 2 shows the sequencing results of positive wild-type and F0 generation mice.
  • Upper part wild-type mouse gene sequence
  • lower part mutant mouse gene sequence.
  • the arrow indicates the insertion site of the inserted gene, and gene sequencing of the F0 generation mice showed a mixed sequence after the insertion position, indicating that the insertion was successful.
  • Figure 3 shows the genotype detection results of F0 generation mice and F1 mutant mice.
  • A is the genotype of F0 generation mice detected by PCR, and a total of 6 positive mice (marked numbers) were screened.
  • B is the genotype of F1 mutant mice detected by PCR, and a total of 3 positive mice (marked numbers) were screened.
  • Figure 4 shows the genotypes of mutant homozygous mice detected by PCR and sequencing.
  • Figure 5 is the detection of the immune effect of the S1 antigen.
  • Fig. 6 shows the cross-linking reaction of antibodies and peptides extracted from the sera of USB heterozygous mice (A) and USB homozygous mice (B).
  • Fig. 7 shows the cross-linking reaction between antibodies and polypeptides prepared by culturing hybridoma cells.
  • Figure 8 is the detection result of Sortase A protein with 5-15% SDS-PAGE gradient gel.
  • Figure 9 is the ELISA detection of USB-antibody directly labeled HRP function.
  • Figure 10 is the result of connecting USB-antibody to DNA.
  • A is the sample treated with reducing buffer;
  • B is the sample treated with non-reducing buffer.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
  • the sequence of the gRNA targeting genome of Igkc gene is as follows:
  • Homologous recombination vector DNA chemically synthesized DNA fragment shown in SEQ ID No.2.
  • the 1-120th position of SEQ ID No.2 is the 5' homology arm; the 133-147th position is the coding gene of the specific recognition sequence of Sortase A enzyme, and the 157-306th position is the 3' homology arm.
  • the nucleotide sequence of the Cas9 mRNA obtained by in vitro transcription is SEQ ID No.3.
  • the nucleotide sequence of the gRNA obtained by in vitro transcription is SEQ ID No.4, and its targeting sequence is SEQ ID No.1.
  • the homologous recombination vector constructed in step 2 and the Cas9 mRNA and gRNA obtained by in vitro transcription in step 3 into a 40 ⁇ l system with water, so that the final concentrations of the homologous recombination vector, Cas9 mRNA and gRNA are 100 ng/ ⁇ l, 100 ng/ ⁇ l, and 50 ng/ ⁇ l, respectively. ⁇ l.
  • the prepared samples were injected into the fertilized egg cytoplasm of C57BL/6J mice, cultured at 37°C for 24 hours to the two-cell stage, and then transplanted into surrogate female mice (mouse variety ICR), until the F0 generation mice were born.
  • the PCR reaction mixture was prepared according to the proportions shown in Table 1 below:
  • Element add volume total DNA 1.5 ⁇ l 5' primer (10 ⁇ M) 1 ⁇ l 3' primer (10 ⁇ M) 1 ⁇ l dNTP (2.5mM) 1.5 ⁇ l 10X PCR buffer (add Mg 2+ ) 3 ⁇ l TaKaRa Taq HS (5U/ ⁇ l) 0.2 ⁇ l H 2 O 21.8 ⁇ l
  • Primer sequence 5' Primer: 5'-GCTGATGCTGCACCAACTGTATCC-3';
  • the PCR products were sequenced and identified.
  • the results show that: through microinjection of fertilized eggs, the present invention obtains 23 F0 generation mice in total.
  • the genotypes of the F0 generation mice were identified by PCR+sequencing, and the PCR results were confirmed by sequencing, and a total of 6 positive F0 generation mice with correct homologous recombination were obtained (Fig. 2 and A in Fig. 3).
  • Figure 2 shows the sequencing results of wild-type and positive F0 generation mice (sequencing primer: GGTGCCTCAGTCGTGTGCTTCTTG).
  • F0 generation positive mice were mated with wild-type C57BL/6J mice to obtain F1 generation positive mice.
  • the identification steps are the same as F0 (PCR+sequencing).
  • Female F1 generation positive mice (heterozygous) are mated with male F1 generation positive mice (heterozygous), and homozygous positive mice are obtained from the offspring.
  • the identification steps are the same as F0 (PCR+sequencing).
  • Embodiment 2 USB mouse immune response
  • USB mice Homozygous USB mice can produce normal immunity should be the premise of using USB mice to produce USB antibodies.
  • Antigen immunization was performed on USB mice (S1 antigen, East Antibiotics, VISC2-S101). details as follows:
  • Immune animals 4-6 weeks of homozygous USB mice (i.e. the homozygous positive mice obtained in Step 7 of Example 1) take blood as a control before immunization. On the first day of the immunization cycle, 30 micrograms of antigen plus an equal volume of Freund's complete The adjuvant was mixed and injected subcutaneously into mice (Freund's complete adjuvant, Sigma F5881).
  • the second immunization subcutaneous injection of 30 ⁇ g antigen plus an equal volume of Freund’s incomplete; Freund’s incomplete adjuvant, Thermo, 77140
  • blood collection on the 21st day ELISA detection of serum titer
  • three immunizations on the 35th day (30 ⁇ g Antigen plus an equal volume of Freund's incomplete subcutaneous injection), blood collection on the 42nd day (the third immunization), and ELISA to detect the serum titer.
  • Coating solution for S1 antigen (coating solution: dissolve 1.59g Na 2 CO 3 and 2.94g NaHCO 3 in 990ml pure water, add 10ml 10% (mass fraction) NaN 3 , pH 9.6. Store at 4°C), After dilution, 100 ⁇ l/well was added to a 96-well plate and left overnight at 4°C.
  • USB mouse serum is used for blocking solution (PBS-T containing 2% BSA and 0.1% (mass fraction) NaN 3 . Stored at 4°C) diluted 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000 times , add 100 ⁇ l/well. Incubate at room temperature for 1h.
  • chromogenic solution 4 mg ABTS (azino-di-3-ethyl-benzthiazodinsulphonate) was dissolved in 10 ml 50 mM citric acid, pH 4.0, and 10 ⁇ l 30% H 2 O 2 was added. Add 100 ⁇ l chromogenic solution to each well.
  • ABTS azino-di-3-ethyl-benzthiazodinsulphonate
  • Serum was taken before immunization, after the second immunization, and after the third immunization, and the titer of the serum was detected by ELISA.
  • the results are shown in Table 2 and Figure 5. It can be seen that the antibody titer of the USB mouse gradually increased, which means that it has a normal immune response.
  • the polypeptide was site-specifically linked to the light chain region containing the USB sequence.
  • This embodiment uses the mouse serum extraction of USB heterozygote (being the F1 generation heterozygous positive mouse that the embodiment 1 obtains) and USB homozygote (being the F1 generation homozygous positive mouse that the embodiment 1 obtains) with genetic background respectively
  • the antibody reacted with the polypeptide containing Sortase A recognition sequence, and the connection product was detected by Western blot.
  • the molecular weight becomes larger.
  • the band that appears above the prototype kappa chain is the connection product, and this band does not appear in the control group (Fig. 6 in A). Since not all cells of heterozygous mice carry the USB gene, the secreted antibodies are a mixture of normal antibodies and antibodies carrying the USB interface. After linking to the polypeptide, a considerable part of the antibodies still fail to link to the polypeptide.
  • Bone marrow hybridoma is an important source of raw materials for large-scale production of antibodies.
  • bone marrow hybridoma cells were prepared from B cells extracted from homozygous USB mice (i.e. the F1 generation homozygous positive mice obtained in Example 1), monoclonal antibodies were extracted from the cell culture supernatant, and the monoclonal antibodies were verified.
  • the cloned antibody can cross-link the polypeptide to the antibody light chain under the action of Sortase A enzyme.
  • Bone marrow hybridoma cells prepared from the B cells extracted from the USB mice obtained in Example 1); transpeptidase Sortase A; protein A adsorption beads (Sigma, 5015979001); polypeptide (GGGSYPYDVPDYAGKPIPNPLLGLDS TEQKLISEEDLK, Beijing Zhongkeya Synthesized by Photobiology Technology Co., Ltd.); HRP-labeled rat anti-mouse secondary antibody (Aibosheng, 030604A06H);
  • Embodiment 4 practical application of USB antibody
  • Ndel and XhoI recognition sequences were constructed at the 5' and 3' ends of the Sortase A gene, respectively, and inserted into the corresponding restriction sites of the pET30a(+) vector, and the recombinant plasmid pET30a-Sortase A was obtained after sequencing and verification.
  • the recombinant plasmid pET30a-Sortase A was transformed into BL21(DE3) competent medium, and cultured in Kan + resistant solid LB medium for 16 hours.
  • Sortase A protein was detected with 5-15% SDS-PAGE gradient gel (Fig. 8).
  • USB mice the F1 generation homozygous positive mice obtained in Example 1 and the F1 generation heterozygous positive mice obtained in Example 1.
  • Polypeptide-biotin sequence: GGGSENLYFQAK-biotin, synthesized by Zhongke Yaguang.
  • Fusion cell screening Cell fusion can be performed after the serum antibody titer reaches 1:8000. S1 antigen was injected intravenously three days before fusion. On the day of fusion, mouse splenocytes were fused with Sp2/0 cells, and the hybridoma cells generated by fusion were grown in HAT selective medium.
  • ELISA is used to detect whether the hybridomas secrete specific antibodies.
  • Antigen i.e. S1 antigen
  • hybridoma cell culture supernatant was used as the primary antibody
  • HRP-labeled rat anti-mouse IgG was used as the secondary antibody
  • the stable expression of the specific antibody was screened (denoted as S1-UBS antibody) cell line, expanded culture and cryopreserved.
  • Antibody-polypeptide-biotin was purified by PD-10 purification column.
  • the primary antibody (the "antibody-HRP" obtained in step 6 or the control in step 7) was diluted in PBS-T or blocking solution, and 100 ⁇ l/well was added. Incubate for 1 h at room temperature in a humid chamber.
  • Polypeptide-azide GGGSYPYDVPDYAGKPIPNPLLGLDSTEQKLISE EDLK-azide (C-terminal azide modification), synthesized by Zhongke Yaguang.
  • Step 1 The method of immunizing animals with S1 antigen is the same as Step 1.
  • mice After the immunization process, kill the mice, take 2ml of serum from the immunized mice, incubate with 500 ⁇ l protein A beads and serum for 1 hour, wash protein A beads with 1ml PBS for 3 times, and wash protein A beads with 0.1ml pH 2.0 glycine buffer After elution for 5 minutes, the pH of the eluent was adjusted to 7.4 with 2M Tris-HCl pH 8.0 to obtain a polyclonal antibody against the S1 protein. Quantification was performed using a BCA kit (Thermofisher, 23225).
  • the PD-10 purification column purifies antibody-polypeptide-azide. Quantification was performed using a BCA kit (Thermofisher, 23225).
  • the control was a sample incubated with the same amount of antibody and water.
  • reaction mixture was mixed with reducing loading buffer and non-reducing loading buffer respectively, and boiled for 5 minutes.
  • Mouse antibodies are composed of heavy chains and light chains, and more than 95% of antibody light chains are kappa chains.
  • This invention adds a specific amino acid sequence to the C-terminus of the kappa chain through CRISPR-mediated gene knock-in, making it genetically modified More than 95% of the antibodies produced by mice carry specific enzyme-mediated site-directed attachment sites.
  • the site-specific connection site introduced by the example of the present invention is the Sortase A recognition site.
  • the transformed mice can normally stimulate immune responses and generate polyclonal antibodies and monoclonal antibodies with site-specific connection sites.
  • the generated antibodies with fixed-point connection sites can be connected normally, and various application groups can be added to realize downstream applications of antibodies, such as ELISA, immunofluorescent staining, and immuno-PCR.

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Abstract

一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法。提供了一种脊椎动物模型的构建方法，所述脊椎动物模型用于生产携带通用定点偶联接口的抗体；所述方法包括如下步骤：在受体动物的基因组中免疫球蛋白的某一编码基因A的某一位置A处定点敲入某种连接酶A的特异性识别序列的编码基因或某种内含肽A的编码基因，得到所述脊椎动物模型。被改造小鼠可以正常激发免疫反应，生成带定点连接位点的多克隆抗体与单克隆抗体，带定点连接位点的抗体可以正常进行定点连接，加上各种应用基团，实现抗体下游应用，比如ELISA，免疫荧光染色，免疫PCR。

Description

一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法 技术领域
本发明涉及生物技术领域，具体涉及一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法。
背景技术
抗体是一种蛋白类生物大分子，通过极强的特异性和亲和力与相应的抗原结合。抗体是现代生物制药学的基础，被广泛用于疾病治疗、诊断试剂和生物医学研究。抗体发现主要有杂交瘤、噬菌体展示、B细胞分选等方法。脊椎动物，如鼠，兔，羊，鸡，骆驼等是开发单克隆抗体和多克隆抗体的重要动物平台。
以往研究表明，在抗体定点位置引入特定氨基酸序列，可以实现通过连接酶，如Sortase，butelase，oaAEP1，介导的特异性连接反应，可以定点的引入若干基团。
目前为止，现有技术中未见在动物水平直接对抗体编码基因进行基因编辑，定点敲入接口编码基因，进而获得可以直接生产自带接口的抗体的动物的相关报道。
发明公开
本发明的目的是提供一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法。
第一方面，本发明要求保护一种脊椎动物模型的构建方法，所述脊椎动物模型用于生产携带通用定点偶联接口的抗体。
本发明所要求保护的脊椎动物模型的构建方法，可包括如下步骤：在受体动物的基因组中免疫球蛋白的某一编码基因A的某一位置A处定点敲入某种连接酶A的特异性识别序列的编码基因或者某种内含肽(intein)A的编码基因，得到所述脊椎动物模型。
在所述方法中，所述编码基因A为Igkc基因(即抗体kappa轻链的恒定区结构域编码基因)，所述位置A为Igkc基因的3’端。即在抗体水平，所述连接酶A的特异性识别序列将被定点插入到抗体kappa型轻链的C端。
在所述方法中，所述脊椎动物可选自如下任一：小鼠、大鼠、兔、羊、鸡、骆驼、马、驴、仓鼠、豚鼠、羊驼。
在本发明的具体实施方式中，所述脊椎动物为小鼠；所述编码基因A为小鼠基因组中6号染色体上的Igkc基因；所述位置A位于小鼠基因组中6号染色体上的Igkc基因(Gene ID:16071)的第70726754和第70726755位(小鼠基因组GRcm38/mm10版本)之间。
在所述方法中，所述连接酶A可选自如下任一：Sortase staph酶、Sortase strep酶、Butelase酶、oaAEP1酶、Formylglycine生成酶(FGE)、谷胺酰转氨酶、微管蛋白酪氨酸连接酶(Tubulin Tyrosine Ligase；TTL)、Trypsiligase、 Sfp phosphopantetheinyl转移酶、SpyLigase。
当所述连接酶A为Sortase staph A(以下统称Sortase A)酶时，所述特异性识别序列可为LPXTG(X为任意氨基酸)；当所述连接酶A为Sortase strep酶时，所述特异性识别序列可为LPXTA(X为任意氨基酸)；当所述连接酶A为Butelase酶时，所述特异性识别序列可为NHV；当所述连接酶A为oaAEP1酶时，所述特异性识别序列可为NGL；当所述连接酶A为Formylglycine生成酶时，所述特异性识别序列可为CXPXR(X为任意氨基酸)；当所述连接酶A为谷胺酰转氨酶时，所述特异性识别序列可为LLQGA；当所述连接酶A为微管蛋白酪氨酸连接酶时，所述特异性识别序列可为VDSVEGEEEGEE；当所述连接酶A为Trypsiligase时，所述特异性识别序列可为YRH；当所述连接酶A为Sfp phosphopantetheinyl转移酶时，所述特异性识别序列可为DSLEFIASKLA；当所述连接酶A为Sfp phosphopantetheinyl转移酶时，所述特异性识别序列可为DSLEFIASKLA；当所述连接酶A为SpyLigase时，所述特异性识别序列可为AHIVMVDAYKPTK或ATHIKFSKRD。
在所述方法中，所述定点敲入可通过CRISPR/Cas9技术实现。
进一步地，作为被Cas9核酸酶切割的靶序列位于所述受体动物的基因组中免疫球蛋白的所述编码基因A的所述位置A上下游500bp范围内，以便通过同源重组机制插入特异性识别序列的DNA。
在本发明的具体实施方式中，所述靶序列具体为SEQ ID No.1(即GGAATGAGTGTTAGAGA*CAAAGG，其中*是Cas9切割位置)(此时，所述脊椎动物为小鼠，所述免疫球蛋白的所述编码基因A的所述位置A位于小鼠基因组中6号染色体上的Igkc基因(Gene ID:16071)的第70726754位和第和第70726755位(小鼠基因组GRcm38/mm10版本)。
进一步地，作为定点敲入工具的同源重组载体上含有DNA片段A；所述DNA片段A自上游到下游依次由5’同源臂、所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因，以及3’同源臂组成；所述5’同源臂为位于所述受体动物基因组中免疫球蛋白的所述编码基因A的所述位置A上游的120bp序列；所述3’同源臂为位于所述受体动物基因组中免疫球蛋白的所述编码基因A的所述位置A下游的150bp序列。
在本发明的具体实施方式中，所述5’同源臂为SEQ ID No.2的第1-120位；所述3’同源臂为SEQ ID No.2的第157-306位。
进一步地，所述连接酶A的特异性识别序列的编码基因为SEQ ID No.2的第133-147位(所述连接酶A为Sortase A酶，SEQ ID No.2的第133-147位为Sortase A酶的特异性识别序列的编码基因)。
更进一步地，所述DNA片段A的核苷酸序列如SEQ ID No.2所示。
本发明所要求保护的脊椎动物模型的构建方法，具体可包括如下步骤：
(1)将Cas9mRNA、gRNA和前文所述的同源重组载体注射到所述受体动物 的受精卵胞质中，得到F0代动物；
所述gRNA的序列为SEQ ID No.4。
(2)将步骤(1)获得的所述F0代动物与所述受体动物进行杂交，从F1代动物中获得在基因组中所述免疫球蛋白的所述编码基因A的所述位置A处定点敲入所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因的杂合体动物。
进一步地，在步骤(2)之后，还可包括如下步骤(3)：
(3)将雄性的所述杂合体动物与雌性的所述杂合体动物进行一次或多次杂交，从杂交后代中获得在基因组中所述免疫球蛋白的所述编码基因A的所述位置A处定点敲入所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因的纯合体动物。
步骤(2)中的所述杂合动物和步骤(3)中的所述纯合动物均可作为能够用于生产携带通用定点偶联接口的抗体的所述脊椎动物模型。
其中，所述Cas9mRNA的序列可为SEQ ID No.3。
所述同源重组载体具体为所述DNA片段A(SEQ ID No.2)。
第二方面，本发明要求保护利用前文第一方面中所述方法构建得到的脊椎动物模型在生产携带通用定点偶联接口的抗体中的应用。
第三方面，本发明要求保护一种生产携带通用定点偶联接口的抗体的方法。
本发明所要求保护的生产携带通用定点偶联接口的抗体的方法，可包括如下步骤：
P1、按照前文第一方面中所述方法制备得到脊椎动物模型；
P2、用免疫原对所述脊椎动物模型进行免疫，从而制备得到携带通用定点偶联接口且抗所述免疫原的抗体。
其中，所述抗体为单克隆抗体或多克隆抗体。
在本发明的具体实施方式中，所述免疫原为S1抗原(东抗生物，VISC2-S101)，所述脊椎动物为小鼠(如C57BL/6J小鼠)。
第四方面，本发明要求保护利用前文第一方面中所述方法构建得到的脊椎动物模型。
第五方面，本发明要求保护利用前文第三方面中所述方法制备得到的携带通用定点偶联接口的抗体。
在本发明中，所述通用定点偶联接口具体是指与抗体特定位置相连的所述连接酶A的特异性识别序列或所述内含肽A的编码基因。
当所述接口为与抗体特定位置相连的所述连接酶A的特异性识别序列时，利用该接口和用于与效应基团(如HRP、多聚HRP、DNA)相连的linker可以实现所述抗体与所述效应基团相偶联，所述linker能够在所述连接酶A的作用下催化所述特异性识别序列与所述linker连接(如共价连接)。
在本发明中，所述连接酶A为Sortase A酶，所述特异性识别序列可为LPXTG (X为任意氨基酸)，相应的所述linker为N末端(游离端)为以寡甘氨酸(如1-5个连续的G，再如3-5个连续的G)的多肽分子或者为N末端(游离端)为烷基胺(alkylamine)的多肽分子。
在本发明的具体实施方式中，所述连接酶A为Sortase A酶，所述特异性识别序列为LPETG，相应的所述linker为N末端(游离端)为GGG的多肽分子。
当所述接口为与抗体特定位置相连的所述内含肽A的编码基因时，利用还原剂对所述抗体进行还原，其上会产生活性基团，所述活性基团可用与N端起始分子为半胱氨酸的效应分子或多肽反应。
本发明以小鼠为例，直接在脊椎动物抗体基因座位定点引入编码特定氨基酸序列的DNA序列，使动物生成的抗体携带特定可被酶介导的定点连接位点。
附图说明
图1为本发明USB小鼠基因编辑示意图。小鼠lgkc基因座示意图(基因从左到右显示，全长532bp)，灰色区域代表开放阅读框(ORF)，白色区域代表基因非翻译区域(UTR)。编辑成功后，基因组中的野生型等位基因将被突变型等位基因替代。
图2为阳性野生型和F0代小鼠小鼠的测序结果。上部：野生型小鼠基因序列；下部：突变型小鼠基因序列。箭头所示为插入基因插入位点，F0代小鼠基因测序显示在插入位置后为混合序列，表明插入成功。
图3为F0代小鼠和F1突变型小鼠基因型检测结果。A为PCR检测F0代小鼠基因型，共筛选得到6只阳性小鼠(标数字者)。B为PCR检测F1突变型小鼠基因型，共筛选得到3只阳性小鼠(标数字者)。
图4为PCR和测序检测突变型纯合子小鼠基因型。
图5为S1抗原免疫效果检测。
图6为USB杂合子小鼠(A)和USB纯合子小鼠(B)血清提取抗体与多肽交联反应。
图7为培养杂交瘤细胞制备抗体与多肽交联反应。
图8为Sortase A蛋白以5-15％SDS-PAGE梯度胶检测结果。
图9为ELISA检测USB-抗体直标HRP功能。
图10为USB-抗体与DNA连接结果。A为还原缓冲液处理样品；B为非还原缓冲液处理样品。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、USB小鼠基因编辑
通过CRISPR-Cas9技术，在小鼠受精卵里的6号染色体的Igkc基因(Gene ID:16071)第70726754位-第70726755位之间(小鼠基因组GRcm38/mm10版本)之间通过同源重组插入了“GGAGGTGGATCACTTCCAGAAACTGGTGGAGGAAGT”这段序列，暨“USB”序列，如图1所示。此基因编辑使得将出生的小鼠的Igkc轻链羧基端加上了“GGGSLPETGGGS”的氨基酸，其中，“LPETG”是转肽酶(Sortase A)的特异性识别位点，可以在蛋白质水平上实现定点连接。经过胚胎植入，对出生的小鼠进行是否有“USB序列”的PCR和基因测序，如期待一样，若干出生小鼠有相应的正确序列。通过后续的交配，生成纯系USB小鼠。具体如下：
一、gRNA的设计
Igkc基因的gRNA靶向基因组的序列如下：
5’-GGAATGAGTGTTAGAGA*CAA AGG-3’(SEQ ID No.1)。
带下划线的碱基为PAM，*为切割位点。
二、同源重组载体(donor vector)
同源重组载体(donor vector)：DNA化学合成SEQ ID No.2所示DNA片段。
SEQ ID No.2的第1-120位为5’同源臂；第133-147位为Sortase A酶的特异性识别序列的编码基因，第157-306位为3’同源臂。
三、Cas9mRNA和gRNA的体外转录
经体外转录所得的Cas9mRNA的核苷酸序列为SEQ ID No.3。
经体外转录所得的gRNA的核苷酸序列为SEQ ID No.4，其靶向序列为SEQ ID No.1。
四、注射受精卵获得F0代嵌合体小鼠
用水将步骤二构建的同源重组载体和步骤三体外转录获得的Cas9mRNA和gRNA配制成40μl体系，使其中同源重组载体、Cas9mRNA和gRNA的终浓度分别为100ng/μl，100ng/μl，50ng/μl。将配制好的样品注射到C57BL/6J小鼠的受精卵胞质中，于37℃培养24小时至二细胞期后移植到代孕雌鼠(小鼠品种ICR)中，至F0代小鼠出生。
五、F0代小鼠基因型的鉴定
1、提取小鼠总DNA
(1)使用TaKaRa MiniBEST Universal Genomic DNA Extraction kit(Ver.5.0_Code No.9765)提取总基因组DNA。
(2)剪取2-5mm小鼠尾巴组织，每个样品加180μl GL缓冲液，20μl蛋白酶K和10μl RNase A。56℃孵育过夜。
(3)12,000rpm，2min离心取上清。
(4)加200μl GL缓冲液，200μl无水乙醇，充分混匀。
(5)上述混合物加如试剂盒中的离心柱，12000rpm离心2min，弃离心穿出液。
(6)加500μl WA缓冲液，12000rpm离心1min，弃离心穿出液。
(7)加700μl WB缓冲液，12000rpm离心1min，弃离心穿出液。
(8)重复步骤(7)。
(9)将离心柱转移到收集管中，12,000rpm离心2min。
(10)将离心柱转移到1.5ml EP管中，加50～200μL灭菌水溶解DNA，室温放置5min。
(11)12,000rpm离心2min，得到小鼠基因组DNA。
2、PCR检测
PCR反应混合物按如下表1所示比例配制：
表1、PCR反应混合物配制比例
成分 加入体积
总DNA 1.5μl
5’引物(10μM) 1μl
3’引物(10μM) 1μl
dNTP(2.5mM) 1.5μl
10X PCR缓冲液(加Mg 2+) 3μl
TaKaRa Taq HS(5U/μl) 0.2μl
H 2O 21.8μl
注：引物序列：5’引物：5’-GCTGATGCTGCACCAACTGTATCC-3’；
3’引物：5’-GGACTGCCATGTAGTGGACAGCC-3’。
PCR产物：
野生型:695bp
突变型:731bp
PCR反应条件设置：
Figure PCTCN2022091873-appb-000001
变性-退火-延伸共35个循环。
并对PCR产物进行测序鉴定。
结果显示：经受精卵显微注射，本发明共获得23只F0代小鼠。通过PCR+测序对F0代小鼠的基因型进行鉴定，PCR结果经测序确认，共获得6只正确同源重组的阳性F0代小鼠(图2和图3中A)。图2为野生型和阳性F0代小鼠的测序结果(测序引物：GGTGCCTCAGTCGTGTGCTTCTTG)。
六、F1代小鼠获得及基因型鉴定
F0代阳性小鼠与野生型C57BL/6J小鼠交配，繁育获得F1代阳性小鼠。
鉴定步骤与F0相同(PCR+测序)。
结果显示：F1代中有三只小鼠为突变型(图3中B)。
七、F1代阳性小鼠杂交获得纯合阳性小鼠
雌性的F1代阳性小鼠(杂合)与雄性的F1代阳性小鼠(杂合)交配，从繁育后代中获得纯合阳性小鼠。
鉴定步骤与F0相同(PCR+测序)。
结果显示：所有受检测小鼠均为突变型纯合子(图4)。
实施例2、USB小鼠免疫反应
纯合USB小鼠可以产生正常的免疫应是用USB小鼠产生USB抗体的前提。对USB小鼠进行了抗原免疫(S1抗原，东抗生物，VISC2-S101)。具体如下：
1、免疫动物：4-6周纯合USB小鼠(即实施例1步骤七所得的纯合阳性小鼠)取血作为免疫前对照，免疫周期第一天，30微克抗原加等体积的弗氏完全佐剂混匀皮下注射小鼠(弗氏完全佐剂，Sigma F5881)。第14天二次免疫(30μg抗原加等体积的弗氏不完全皮下注射；弗氏不完全佐剂，Thermo，77140)，第21天采血，ELISA检测血清效价，第35天三次免疫(30μg抗原加等体积的弗氏不完全皮下注射)，第42天采血(第三次免疫)，ELISA检测血清效价。
2、ELISA步骤：
(1)S1抗原用包被液(包被液：溶解1.59g Na 2CO 3和2.94g NaHCO 3于990ml纯净水，加10ml 10％(质量分数)NaN 3，pH 9.6。4℃保存)，稀释后100μl/孔加到96孔板中，4℃过夜。
(2)PBS-T洗4次后封闭1h。
(3)USB小鼠血清用于封闭液(PBS-T含2％BSA和0.1％(质量分数)NaN 3。4℃保存)稀释1000、2000、4000、8000、16000、32000、64000、128000倍，加100μl/孔。室温孵育1h。
(4)PBS-T洗4次。
(5)用PBST稀释HRP标记大鼠抗小鼠二抗(爱博生，030604A06H)1:1000，每孔加100μl显色液。室温孵育1h。
(6)PBS-T洗4次。
(7)配制显色液：4mg ABTS(azino-di-3-ethyl-benzthiazodinsulphonate)溶解于10ml 50mM柠檬酸中，pH 4.0，加10μl 30％H 2O 2。每孔加100μl显色液。
(8)显色后405nm波长读取数值。
取免疫前，二次免疫，和三次免疫后的血清，ELISA检测血清效价。结果如表2和图5所示，可见：USB小鼠抗体效价逐步增高，意味着其有正常的免疫反应。
表2、随着免疫进行USB小鼠抗体效价逐步增高
Figure PCTCN2022091873-appb-000002
实施例3、USB端口功能验证
一、具有USB遗传背景的小鼠血清提取的多克隆抗体与多肽连接反应
为验证USB小鼠体内产生的B细胞分泌的抗体是否可在酶催化作用下，将多肽定点连接到含USB序列的轻链区。本实施例使用基因背景分别为USB杂合子(即实施例1所得的F1代杂合阳性小鼠)和USB纯合子(即实施例1所得的F1代纯合阳性小鼠)的小鼠血清提取的抗体与含Sortase A识别序列的多肽反应，用Western blot检测连接产物。
1、实验材料
经实施例1鉴定得到的USB杂合子小鼠和USB纯合子小鼠的血清；蛋白A珠(Sigma,5015979001)、Sortase A酶(具体制备方法参见实施例4)、多肽(GGGSYPYDVPDYAGKPIPNPLLGLDS TEQKLISEEDLK，北京中科亚光生物科技有限公司合成)、PBS、HRP标记大鼠抗小鼠二抗(爱博生，030604A06H)、ECL发光液(上海天能，180-5001)。
2、实验步骤
(1)分别取USB杂合子小鼠和USB纯合子小鼠血清各20μl，用30μl蛋白A珠提取总抗体检测提纯抗体浓度。
(2)按50倍摩尔浓度将多肽(GGGSYPYDVPDYAGKPIPNPLLGLDSTEQKLISEEDLK)与抗体混合，并加Sortase A酶至终浓度2μM。阴性对照为多肽(GGGSYPYDVPDYAGKPIPNPLLGLDSTEQKLISEEDLK)与抗体混合物，不加Sortase A酶，37℃过夜。
(3)取15μl反应混合物，12％胶上还原样，转膜3h，5％脱脂奶粉封闭过夜。
(4)1:3000(体积比)牛奶+PBST稀释HRP标记大鼠抗小鼠IgG二抗(爱博生，030604A06H)，室温1h，显色。
3、实验结果
USB杂合子小鼠体内天然产生的抗体与多肽交联后，分子量变大，在连接组中，在原型kappa链的上方出现的条带即为连接产物，此条带不出现在对照组(图6中A)。由于杂合子小鼠并非所有的细胞都携带USB基因，因而分泌的抗 体为正常抗体和携带USB接口抗体的混合物，与多肽连接后，仍有相当一部分抗体未能连接多肽。
与之相比，USB纯合子小鼠体内产生的抗体几乎全部携带USB标签，与多肽交联后，绝大部分抗体都能与多肽交联(图6中B)。
该实验证明具有USB遗传背景的小鼠体内的抗体可以在Sortase A酶催化下完成交联反应。
二、具有USB遗传背景的骨髓杂交瘤细胞产生的单克隆抗体与多肽连接反应
骨髓杂交瘤是规模化制备抗体原料的重要来源。本实施例用从纯合USB小鼠(即实施例1所得的F1代纯合阳性小鼠)提取的B细胞制备骨髓杂交瘤细胞，从细胞培养上清液中提取单克隆抗体，验证该单克隆抗体可在Sortase A酶作用下将多肽交联到抗体轻链。
1、实验材料
骨髓杂交瘤细胞(从实施例1所得的USB小鼠提取的B细胞制备骨髓杂交瘤细胞)；转肽酶Sortase A；蛋白A吸附珠(Sigma,5015979001)；多肽(GGGSYPYDVPDYAGKPIPNPLLGLDS TEQKLISEEDLK，北京中科亚光生物科技有限公司合成)；HRP标记大鼠抗小鼠二抗(爱博生，030604A06H)；
2、实验步骤
(1)杂交瘤细胞制备：待血清抗体滴度达到1：8000后即可以进行细胞融合。融合前3天静脉注射S1抗原(东抗生物，VISC2-S101)。融合当天取小鼠脾细胞与Sp2/0细胞融合，融合生成的杂交瘤细胞在HAT选择性培养基中(Sigma，H0262)生长。
(2)筛选抗体滴度较高的细胞株：ELISA检测。方法同上。杂交瘤细胞上清液倍比稀释的前几个浓度，ELISA读数高于1.8，抗体滴度越高越好。
检测时包被S1抗原(东抗生物，VISC2-S101)，取杂交瘤细胞培养上清液作为一抗，HRP标记的大鼠抗小鼠IgG(爱博生，030604A06H)作为二抗，筛选出稳定表达特异性抗体的细胞株，扩大培养并冻存。
(3)取3ml上清液，用100μl蛋白A吸附珠提抗体，并检测提纯抗体浓度。
(4)抗体与多肽交联反应实验组、对照组设置和操作与步骤一2相同。
3、实验结果
选取的三个单克隆抗体与多肽连接后，轻链均发生上移(图7)。该实验证明，通过体外培养杂交瘤细胞制备的携带USB标签的单抗可在Sortase A酶作用下将特定的分子交联到该抗体上。
实施例4、USB抗体实际应用
一、USB小鼠产生的单克隆抗体在ELISA中的应用
(一)、实验材料
1、S1抗原，东抗生物，VISC2-S101；
2、Sortase A蛋白的制备
序列：
Figure PCTCN2022091873-appb-000003
(1)Sortase A基因的5’和3’端分别构建Ndel和XhoI识别序列，插入到pET30a(+)载体的相应酶切位点中，经测序验证正确后得到重组质粒pET30a-Sortase A。将重组质粒pET30a-Sortase A转化到BL21(DE3)感受态中，在Kan +抗性的固体LB培养基培养16小时。
(2)挑取一单克隆菌落于LB培养基(Kan +抗性)培养至细菌OD 6000.6左右，加入IPTG 30℃诱导表达4小时。
(3)表达后的细菌超声裂解，将诱导前，诱导后裂解上清和沉淀进行SDS-PAGE检测，结果显示，Sortase A蛋白以可溶形式表达。
(4)扩大培养细菌，IPTG 30℃诱导表达4小时。
(5)超声细菌，将裂解上清过Ni柱纯化，最终SDS-PAGE检测Sortase A蛋白纯度大于90％。，Sortase A蛋白以5-15％SDS-PAGE梯度胶检测(图8)。
3、USB小鼠：实施例1所得的F1代纯合阳性小鼠和实施例1所得的F1代杂合阳性小鼠。
4、多肽-biotin，序列：GGGSENLYFQAK-biotin，中科亚光合成。
5、PD-10纯化柱，GE Healthcare,17085101。
6、Streptavidin-HRP，Sigma,S5512。
7、HRP标记的大鼠抗小鼠二抗(爱博生，030604A06H)
(二)、实验方法
1、免疫动物：第1天，30微克S1抗原加等体积的弗氏完全佐剂混匀，皮下多点注射4-6周龄USB小鼠。第21、42、63天加强免疫，均为30微克抗原加等体积的弗氏不完全佐剂混匀，皮下多点注射小鼠。第1、28、49、70天尾静脉采血，ELISA检测血清效价(第1天所采血清用于检测免疫前血清抗体滴度)。抗体滴度指的是，血清样品读数比背景值高出0.5时的最大稀释倍数的倒数。
2、融合细胞筛选：待血清抗体滴度达到1：8000后即可以进行细胞融合。融合前三天静脉注射S1抗原。融合当天取小鼠脾细胞与Sp2/0细胞融合，融合生成的杂交瘤细胞在HAT选择性培养基中生长。
3、筛选抗体滴度高的细胞株：杂交瘤是否分泌特异性抗体用ELISA方法检测。检测时包被抗原(即S1抗原)，取杂交瘤细胞培养上清液作为一抗，HRP标记的大鼠抗小鼠IgG(爱博生，030604A06H)为二抗，筛选出稳定表达特异性抗体(记为S1-UBS抗体)的细胞株，扩大培养并冻存。
4、提取S1-UBS抗体，抗体与50倍摩尔浓度的多肽-biotin混合，加Sortase A蛋白至终浓度2μM。37℃连接过夜。连接产物为抗体-多肽-biotin。
5、PD-10提纯柱提纯抗体-多肽-biotin。
6、取等摩尔量的Streptavidin-HRP与抗体-多肽-biotin孵育。得到抗体-HRP。
7、设置对照，即带S1-USB端口的抗体与Streptavidin-HRP的混合物，因为S1-USB抗体未连接多肽-biotin，故理论上不能与Streptavidin-HRP结合，也不能显色。
8、抗体-HRP与对照组做ELISA。ELISA步骤如下：
(1)S1抗原用包被液稀释至0.1-5g/ml，100μl/孔加到96孔板中，4℃湿盒过夜。
(2)PBS-T洗4次。
(3)每孔加160-200μl封闭液，室温湿盒孵育1h。
(4)一抗(步骤6所得的“抗体-HRP”或步骤7的对照)稀释于PBS-T或封闭液中，加100μl/孔。室温湿盒培养1h。
(5)PBS-T洗4次。
(8)每孔加100μl显色液。
(9)显色后405nm波长读取数值。
(三)、实验结果
结果如图9所示。可见，利用USB小鼠免疫的得到的B细胞与骨髓瘤细胞融合得到的杂交瘤细胞可以分泌含有UBS接口的抗体，该抗体可以经过Sortase A催化使之携带biotin基团，并与streptavidin-HRP结合形成携带HRP的S1抗体，该S1-HRP抗体具有正常识别抗原并显色的能力。与之形成对比的是，S1-USB抗体在不含biotin的情况下，不能与streptavidin-HRP结合，因而背景很低。进一步说明USB接口在该模型中的关键作用。
二、USB小鼠产生的多克隆抗体在与DNA连接中的应用
(一)、实验材料：
1、S1抗原，东抗生物，VISC2-S101；
2、Sortase A蛋白的制备，同步骤一。
4、多肽-azide：GGGSYPYDVPDYAGKPIPNPLLGLDSTEQKLISE EDLK-azide(C末端azide修饰)，中科亚光合成。
3、DNA oligo
序列：5’-CAGGTAGTAGTACGTCTGTTTCACGATGAGACTGGATTCG-3’，5’端DBCO 修饰；安徽通用合成。
(二)、实验方法
1、S1抗原免疫动物方法与步骤一种相同。
2、免疫过程结束后，处死小鼠，取免疫小鼠的血清2ml，用500μl蛋白A珠与血清孵育1h，蛋白A珠以1ml PBS洗涤3次，蛋白A珠以0.1ml pH 2.0甘氨酸缓冲液洗脱5min，以2M Tris-HCl pH8.0调节洗脱液pH至7.4，得到抗S1蛋白的多克隆抗体。使用BCA试剂盒(Thermofisher，23225)定量。
3、取100μg多克隆抗体与25倍摩尔浓度的多肽-azide混合，加Sortase A蛋白至终浓度2μM。37℃连接过夜。连接产物为抗体-多肽-azide。
4、PD-10提纯柱提纯抗体-多肽-azide。使用BCA试剂盒(Thermofisher，23225)定量。
5、取2μg多克隆抗体与2倍摩尔浓度的DNA oligo孵育，室温反应过夜。
对照为相同量抗体与水孵育样本。
6、反应混合物分别用还原上样缓冲液和非还原上样缓冲液混和，煮沸5min。
7、以Western blot检测小鼠kappa轻链。使用抗体为大鼠抗小鼠kappa轻链抗体(爱博生生物技术有限公司，116111C10)，HRP标记小鼠抗大鼠二抗(爱博生生物技术有限公司，011003G07H)。
(三)、实验结果：
结果如图10所示，还原缓冲液处理样品抗体与对照相比，轻链位置明显上移(图10中A)。非还原缓冲液处理显示，整个抗体分子量也上移(图10中B)，这些结果显示USB小鼠生成的抗体经过Sortase A催化使之携带azide基团，该抗体与DBCO-DNA oligo连接生成抗体-DNA复合物，该复合物可用于相关检测。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
小鼠的抗体由重链和轻链组成，超过95％的抗体轻链是kappa链，本发明把特定氨基酸序列通过CRISPR介导的基因敲入方式加到了kappa链的C端，使得被基因改造的小鼠生成的超过95％的抗体都携带特定可被酶介导的定点连接位点。本发明示例引入的定点连接位点为Sortase A识别位点，实验表明，被改造的小 鼠可以正常激发免疫反应，生成带定点连接位点的多克隆抗体与单克隆抗体。生成的带定点连接位点的抗体可以正常进行定点连接，加上各种应用基团，实现抗体下游应用，比如ELISA、免疫荧光染色、免疫PCR。

Claims (22)

  1. 一种脊椎动物模型的构建方法，所述脊椎动物模型用于生产携带通用定点偶联接口的抗体；所述方法包括如下步骤：在受体动物的基因组中免疫球蛋白的某一编码基因A的某一位置A处定点敲入某种连接酶A的特异性识别序列的编码基因或者某种内含肽A的编码基因，得到所述脊椎动物模型。
  2. 根据权利要求1所述的方法，其特征在于：所述编码基因A为Igkc基因，所述位置A为Igkc基因的3’端。
  3. 根据权利要求1所述的方法，其特征在于：所述脊椎动物选自如下任一：小鼠、大鼠、兔、羊、鸡、骆驼、马、驴、仓鼠、豚鼠、羊驼。
  4. 根据权利要求3所述的方法，其特征在于：所述脊椎动物为小鼠；所述编码基因A为小鼠基因组中6号染色体上的Igkc基因，所述位置A位于小鼠基因组中6号染色体上的Igkc基因的第70726754和第70726755位之间。
  5. 根据权利要求1-4中任一所述的方法，其特征在于：所述连接酶A可选自如下任一：Sortase staph酶、Sortase strep酶、Butelase酶、oaAEP1酶、Formylglycine生成酶、谷胺酰转氨酶、微管蛋白酪氨酸连接酶、Trypsiligase、Sfp phosphopantetheinyl转移酶、SpyLigase。
  6. 根据权利要求5所述的方法，其特征在于：当所述连接酶A为Sortase staphA酶时，所述特异性识别序列为LPXTG，X为任意氨基酸；
    当所述连接酶A为Sortase strep酶时，所述特异性识别序列为LPXTA，X为任意氨基酸；
    当所述连接酶A为Butelase酶时，所述特异性识别序列为NHV；
    当所述连接酶A为oaAEP1酶时，所述特异性识别序列为NGL；
    当所述连接酶A为Formylglycine生成酶时，所述特异性识别序列为CXPXR，X为任意氨基酸；
    当所述连接酶A为谷胺酰转氨酶时，所述特异性识别序列为LLQGA；
    当所述连接酶A为微管蛋白酪氨酸连接酶时，所述特异性识别序列为VDSVEGEEEGEE；
    当所述连接酶A为Trypsiligase时，所述特异性识别序列为YRH；
    当所述连接酶A为Sfp phosphopantetheinyl转移酶时，所述特异性识别序列为DSLEFIASKLA；
    当所述连接酶A为Sfp phosphopantetheinyl转移酶时，所述特异性识别序列为DSLEFIASKLA；
    当所述连接酶A为SpyLigase时，所述特异性识别序列为AHIVMVDAYKPTK或ATHIKFSKRD。
  7. 根据权利要求1-6中任一所述的方法，其特征在于：所述定点敲入是利用CRISPR/Cas9技术实现的；
  8. 根据权利要求7所述的方法，其特征在于：作为被Cas9核酸酶切割的靶序列位于所述受体动物的基因组中免疫球蛋白的所述编码基因A的所述位置A处上下游500bp范围内。
  9. 根据权利要求8所述的方法，其特征在于：所述靶序列为SEQ ID No.1。
  10. 根据权利要求9所述的方法，其特征在于：作为定点敲入工具的同源重组载体上含有DNA片段A；所述DNA片段A自上游到下游依次由5’同源臂、所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因，以及3’同源臂组成；所述5’同源臂为位于所述受体动物基因组中免疫球蛋白的所述编码基因A的所述位置A上游的120bp序列；所述3’同源臂为位于所述受体动物基因组中免疫球蛋白的所述编码基因A的所述位置A下游的150bp序列。
  11. 根据权利要求10所述的方法，其特征在于：所述5’同源臂为SEQ ID No.2的第1-120位。
  12. 根据权利要求10所述的方法，其特征在于：所述3’同源臂为SEQ ID No.2的第157-306位。
  13. 根据权利要求10所述的方法，其特征在于：所述连接酶A的特异性识别序列的编码基因为SEQ ID No.2的第133-147位。
  14. 根据权利要求10-13中任一所述的方法，其特征在于：所述DNA片段A的核苷酸序列如SEQ ID No.2所示。
  15. 根据权利要求1-14中任一所述的方法，其特征在于：所述方法包括如下步骤：
    (1)将Cas9 mRNA、gRNA和权利要求10-14任一中所述的同源重组载体注射到所述受体动物的受精卵胞质中，得到F0代动物；
    所述gRNA的序列为SEQ ID No.4；
    (2)将步骤(1)获得的所述F0代动物与所述受体动物进行杂交，从F1代动物中获得在基因组中所述免疫球蛋白的所述编码基因A的所述位置A处定点敲入所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因的杂合体动物。
  16. 根据权利要求15所述方法，其特征在于：在步骤(2)之后，还包括如下步骤(3)：
    (3)将雄性的所述杂合体动物与雌性的所述杂合体动物进行一次或多次杂交，从杂交后代中获得在基因组中所述免疫球蛋白的所述编码基因A的所述位置A处定点敲入所述连接酶A的特异性识别序列的编码基因或所述内含肽A的编码基因的纯合体动物。
  17. 根据权利要求15或16所述方法，其特征在于：所述Cas9 mRNA的序列为SEQ ID No.3。
  18. 利用权利要求1-17中任一所述方法构建得到的脊椎动物模型在生产携带通用定点偶联接口的抗体中的应用。
  19. 一种生产携带通用定点偶联接口的抗体的方法，包括如下步骤：
    P1、按照权利要求1-17中任一所述方法制备得到脊椎动物模型；
    P2、用免疫原对所述脊椎动物模型进行免疫，从而制备得到携带通用定点偶联接口且抗所述免疫原的抗体。
  20. 根据权利要求19所述方法，其特征在于：所述抗体为单克隆抗体或多克隆抗体。
  21. 利用权利要求1-17中任一所述方法构建得到的脊椎动物模型。
  22. 利用权利要求19或20所述方法制备得到的携带通用定点偶联接口的抗体。
PCT/CN2022/091873 2021-05-18 2022-05-10 一种基于基因改造脊椎动物的携带通用定点偶联接口的抗体的制备方法 WO2022242500A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110321183A1 (en) * 2009-01-30 2011-12-29 Whitehead Institute For Biomedical Research Methods for ligation and uses thereof
CN105142675A (zh) * 2013-03-15 2015-12-09 恩比伊治疗股份公司 通过序列特异性转肽酶制备免疫配体/效应分子结合物的方法
CN109906030A (zh) * 2016-11-04 2019-06-18 安健基因公司 用于产生仅重链抗体的经基因修饰的非人动物和方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110321183A1 (en) * 2009-01-30 2011-12-29 Whitehead Institute For Biomedical Research Methods for ligation and uses thereof
CN105142675A (zh) * 2013-03-15 2015-12-09 恩比伊治疗股份公司 通过序列特异性转肽酶制备免疫配体/效应分子结合物的方法
CN109906030A (zh) * 2016-11-04 2019-06-18 安健基因公司 用于产生仅重链抗体的经基因修饰的非人动物和方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KHOSHNEJAD MAKAN, BRENNER JACOB S., MOTLEY WILLIAM, PARHIZ HAMIDEH, GREINEDER COLIN F., VILLA CARLOS H., MARCOS-CONTRERAS OSCAR A.: "Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9", SCIENTIFIC REPORTS, vol. 8, 29 January 2018 (2018-01-29), pages 1 - 9, XP093008528 *
KHOSHNEJAD MAKAN, BRENNER JACOB S., PARHIZ HAMIDEH, MUZYKANTOV VLADIMIR R.: "CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation", PROTEIN CHROMATOGRAPHY : METHODS AND PROTOCOLS, vol. 2033, 23 July 2019 (2019-07-23), New York, NY, pages 81 - 93, XP009541359, ISSN: 1064-3745, ISBN: 978-1-4939-6412-3, DOI: 10.1007/978-1-4939-9654-4_7 *
LE GALL CAMILLE M., VAN DER SCHOOT JOHAN M. S., RAMOS-TOMILLERO IVÁN, KHALILY MELEK PARLAK, VAN DALEN FLORIS J., WIJFJES ZACHARIAS: "Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering", BIOCONJUGATE CHEMISTRY, vol. 32, 21 January 2021 (2021-01-21), pages 301 - 310, XP093008511 *
MARUYAMA TAKESHI, DOUGAN STEPHANIE K, TRUTTMANN MATTHIAS C, BILATE ANGELINA M, INGRAM JESSICA R, PLOEGH HIDDE L: "Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining", NATURE BIOTECHNOLOGY, vol. 33, no. 5, 23 March 2015 (2015-03-23), pages 538 - 542, XP055290186 *
VAN DER SCHOOT JOHAN M S; FENNEMANN FELIX L; VALENTE MICHAEL; DOLEN YUSUF; HAGEMANS IRIS M; BECKER ANOUK M D; LE GALL CAMILLE M; V: "Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering", SCIENCE ADVANCES, vol. 5, 28 August 2019 (2019-08-28), pages 1 - 11, XP055859715 *

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