WO2022048604A1 - 一种非人哺乳动物或其子代的制备方法及其应用 - Google Patents

一种非人哺乳动物或其子代的制备方法及其应用 Download PDF

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WO2022048604A1
WO2022048604A1 PCT/CN2021/116282 CN2021116282W WO2022048604A1 WO 2022048604 A1 WO2022048604 A1 WO 2022048604A1 CN 2021116282 W CN2021116282 W CN 2021116282W WO 2022048604 A1 WO2022048604 A1 WO 2022048604A1
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heavy chain
constant region
encoding
chain constant
mouse
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PCT/CN2021/116282
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French (fr)
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王皓毅
叶建华
高晖
杜小明
仵毅
周鹏
项光海
王天姿
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北京仁源欣生生物科技有限公司
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Priority to EP21863668.6A priority Critical patent/EP4209591A1/en
Priority to CN202180054115.7A priority patent/CN116194586A/zh
Priority to JP2023515139A priority patent/JP2023539785A/ja
Publication of WO2022048604A1 publication Critical patent/WO2022048604A1/zh
Priority to US18/177,298 priority patent/US20240032514A1/en

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Definitions

  • the present application relates to the field of biotechnology, in particular to a method for preparing a non-human mammal or its progeny and its application, and the non-human mammal or its progeny can be used to produce heavy chain antibodies.
  • Antibody heavy chain includes heavy chain constant region ( CH ) and heavy chain variable region ( VH ); wherein: heavy chain constant region of IgD, IgG, IgA includes 4 domains: CH1, hinge region Hinge, CH2, CH3 ; IgM and IgE include 4 domains: CH1, CH2, CH3, CH4 (Janeway's Immunobiology , 9th Edition); the CH1 domain of the heavy chain constant region is connected to the light chain constant domain by disulfide bonds; the heavy chain can be
  • the variable region includes the hypervariable regions of the complementarity determining regions (CDRs) and relatively conserved regions called framework regions (FRs), the heavy chain variable region includes 3 CDRs and 4 FRs, from the amino terminus to the carboxy terminus In the following order: FR1, CDR1, FR2, CDR2, FR3, C
  • antibodies include the following five classes: IgM, IgD, IgG, IgE, and IgA.
  • the same type of antibody still has some differences in antigen specificity in the constant region of the heavy chain, so they can be divided into several subclasses, such as: human IgG includes IgG1, IgG2, IgG3, IgG4, etc.; human IgA includes IgA1, IgA2 etc.; the subclasses of antibodies may vary between different mammals (eg, humans, alpacas, and mice), and between different strains of mice (eg, C57BL/6 mice and BALB/c mice).
  • the genes related to antibody heavy chain include L, V, D, J, C five types of gene fragments, wherein: heavy chain variable region is encoded by V, D, J gene fragments, heavy chain constant region is encoded by C gene fragment .
  • Human antibody heavy chain genes include functional about 40 V genes, 23 D genes, 6 J genes and 9 C genes (source: Janeway's Immunobiology , 9th Edition, Page 177).
  • the gene encoding the IgM heavy chain constant region is the Ighm gene (Immunoglobulin heavy constant mu), the gene encoding the IgD heavy chain constant region is the Ighd gene (Immunoglobulin heavy constant delta), and the gene encoding the IgG heavy chain constant region is Ighg (Immunoglobulin heavy constant mu) gamma), the gene encoding the IgA heavy chain constant region is Igha (Immunoglobulin heavy constant alpha), etc.; correspondingly, the gene encoding the IgG1 heavy chain constant region is Ighg1 (Immunoglobulin heavy constant gamma 1), the gene encoding the IgG3 heavy chain constant region Because of Ighg3 (Immunoglobulin heavy constant gamma 3), the gene encoding the constant region of the IgG2b heavy chain is Ighg2b (Immunoglobulin heavy constant gamma 2b).
  • Mature B cells generate secreted antibody IgM after antigen stimulation, and a second rearrangement occurs after re-antigen stimulation, and the type of immunoglobulin expressed and secreted on the membrane will be converted from IgM with low affinity to IgG with high affinity.
  • IgA, IgE and other classes or subclasses of immunoglobulins this phenomenon is called class switch (Class switch) or isotype switch (Isotype switch).
  • Heavy chain antibody also called heavy chain only antibody, refers to an immunoglobulin antibody composed of only two heavy chains. Naturally occurring heavy chain antibodies are found in camelids and sharks in nature. The heavy chain locus in the camelid germline contains the gene segment encoding the heavy chain constant region, and during maturation, the rearranged VDJ binding region is spliced to the 5' end of the gene segment encoding the IgG hinge region, making it IgG2 and IgG3 heavy chain antibodies are produced in the absence of the CH1 region that mediates binding to the light chain and cannot bind to the light chain. Heavy chain antibodies can also be produced by genetically modifying animals, and the way to classify the produced heavy chain antibodies is also based on the antigen specificity of the heavy chain constant region of the antibody.
  • Heavy chain antibodies are much smaller, a heavy chain of IgG2c heavy chain antibody is about 40kDa, and the variable region of the heavy chain that determines the specificity of antigen recognition is only about 15kDa.
  • Heavy chain antibodies are characterized by their small molecular weight and can bind to some hidden antigenic epitopes, and are especially suitable for targets that are difficult to obtain antibodies.
  • the purpose of this application is to provide a method for preparing a non-human mammal or its progeny and its application.
  • a first aspect of the present application provides a method for preparing a non-human mammal or a progeny thereof, comprising the following steps:
  • the steps of not expressing or incorrectly expressing the CH1 domain of the IgM heavy chain constant region in the non-human mammal are:
  • one, two, three, four or more than four genes encoding IgG heavy chain constant regions in a non-human mammal are not expressed or incorrectly expressed CH1 during expression
  • the steps to construct a domain are:
  • the step of making the first gene encoding the constant region of an IgG heavy chain in a non-human mammal not express or not correctly express the CH1 domain when expressed;
  • the first gene encoding the IgG heavy chain constant region in the non-human mammal not express or incorrectly express the IgG heavy chain constant region it encodes when expressed, and make the second or the first gene encoding the IgG heavy chain constant region in the non-human mammal. a step in which one, two or three of the three or fourth genes encoding the IgG heavy chain constant region do not express or incorrectly express the CH1 domain when expressed;
  • the present application also provides a non-human mammal that does not express or incorrectly expresses the CH1 domain of the IgM heavy chain constant region in vivo, and whose in vivo one, two, three, four or more encoding IgG heavy chain constant The genes in the region do not express or incorrectly express the CH1 domain.
  • the CH1 domain of the IgM heavy chain constant region that is not expressed or incorrectly expressed in vivo is:
  • the IgM heavy chain constant regions and the IgD heavy chain constant regions are not expressed or are incorrectly expressed.
  • one, two, three, four or more than four genes encoding the constant region of the IgG heavy chain do not express or incorrectly express the CH1 domain as follows:
  • the first gene encoding the constant region of an IgG heavy chain does not express or incorrectly express the CH1 domain
  • the first gene encoding an IgG heavy chain constant region does not express or incorrectly expresses the IgG heavy chain constant region it encodes, and the second or third or fourth gene encoding an IgG heavy chain constant region One, two or three of the CH1 domains are not expressed or incorrectly expressed;
  • the first and second genes encoding the IgG heavy chain constant region do not express or incorrectly express the IgG heavy chain constant region they encode, and the third or fourth gene encoding the IgG heavy chain constant region One or both of the CH1 domains are not expressed or incorrectly expressed;
  • the first, second and third genes encoding the IgG heavy chain constant region do not express or incorrectly express the IgG heavy chain constant region they encode, and the fourth gene encoding the IgG heavy chain constant region does not Expression or incorrect expression of the CH1 domain;
  • the first gene encoding the IgG heavy chain constant region correctly expresses the IgG heavy chain constant region it encodes, and one or both of the second or third or fourth genes encoding the IgG heavy chain constant region are correctly expressed. one or three unexpressed or incorrectly expressed CH1 domains;
  • the first and second IgG heavy chain constant region-encoding genes correctly express the IgG heavy chain constant region they encode, and one or both of the third or fourth IgG heavy chain constant region encoding genes One does not express or incorrectly express the CH1 domain;
  • the first, second and third genes encoding the IgG heavy chain constant region correctly express the IgG heavy chain constant region they encode and the fourth gene encoding the IgG heavy chain constant region is not expressed or incorrectly Express the CH1 domain.
  • a second aspect of the present application provides a method for preparing a non-human mammal or a progeny thereof, comprising the following steps:
  • the target genes include: one, two, three, four or more than four nucleotide sequences encoding the CH1 domain on the genes encoding the constant region of the IgG heavy chain .
  • the application also provides a non-human mammal whose genome includes a nucleotide sequence encoding the CH1 domain of the IgM heavy chain constant region and a target gene knocked out, the target gene comprising:
  • the nucleotide sequence on the genome of the non-human mammal including the CH1 domain encoding the IgM heavy chain constant region is knocked out:
  • the target gene is:
  • nucleotide sequence encoding the CH1 domain from the first gene encoding the IgG heavy chain constant region to the second gene encoding the IgG heavy chain constant region;
  • nucleotide sequence encoding the CH1 domain on the fourth gene encoding the constant region of the IgG heavy chain.
  • the non-human mammal is a rodent; optionally, the rodent is a rat or a mouse; further optionally, the The rodent is a mouse; further, the mouse is a C57BL/6 mouse or a BALB/c mouse.
  • the first gene encoding the constant region of the IgG heavy chain is Ighg3.
  • the non-human mammal when the non-human mammal is a C57BL/6 mouse, the first gene encoding the constant region of the IgG heavy chain is Ighg3, and the second encoding the IgG heavy chain is Ighg3.
  • the chain constant region gene Ighg1 the third gene Ighg2b encoding the IgG heavy chain constant region, the fourth gene Ighg2c encoding the IgG heavy chain constant region;
  • the first gene encoding the constant region of the IgG heavy chain is Ighg3, the second gene encoding the constant region of the IgG heavy chain, Ighg1, and the third gene encoding the constant region of the IgG heavy chain
  • the gene Ighg2b, the fourth gene Ighg2a encoding the constant region of the IgG heavy chain.
  • the genome of the non-human mammal includes a complete gene encoding ⁇ light chain and/or ⁇ light chain; alternatively, the non-human mammal Kappa light chains and/or lambda light chains are normally expressed in animals.
  • the gene knockout method includes one or more of the following: gene targeting technology, CRISPR/Cas9 method, zinc finger nuclease method, transcription activator-like Effector Nuclease Method.
  • the non-human mammal or its progeny are used to produce heavy chain antibodies.
  • a third aspect of the present application provides a method for preparing a C57BL/6 mouse or its progeny, comprising the following steps:
  • the application also provides a C57BL/6 mouse, the nucleotide sequence encoding the heavy chain constant region of the antibody IgM, IgD, IgG1, IgG2b, IgG3 in the genome, and the gene encoding the heavy chain constant region of the antibody IgG2c in the genome
  • the nucleotide sequence encoding the CH1 domain was knocked out.
  • the genome of the C57BL/6 mouse includes a complete gene encoding a ⁇ light chain and/or a ⁇ light chain; optionally, the C57BL /6 mice can express ⁇ light chain and/or ⁇ light chain normally.
  • the gene knockout method includes one or more of the following: gene targeting technology, CRISPR/Cas9 method, zinc finger nuclease method, transcription activator like effector nuclease method.
  • the exon 1 of the gene encoding the IgM heavy chain constant region of the mouse to the exon 1 of the gene encoding the IgG2c heavy chain constant region is knocked out. the entire nucleotide sequence of the child.
  • sgRNA and target targeting the upstream of exon 1 of the gene encoding the IgM heavy chain constant region of the mouse are used.
  • sgRNA downstream of exon 1 of the mouse gene encoding the IgG2c heavy chain constant region are used.
  • the targeting sequence upstream of exon 1 of the mouse encoding IgM heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.1 and SEQ ID NO.1 ID NO.2; and/or, the targeting sequences downstream of exon 1 of the mouse encoding IgG2c heavy chain constant region gene targeted by sgRNA include SEQ ID NO.3 and SEQ ID NO.4.
  • the sgRNA upstream of the exon No. 1 of the gene targeting the mouse encoding the IgM heavy chain constant region is SEQ ID NO.9 and SEQ ID NO.10 and/or, the sgRNAs targeting the downstream of exon 1 of the gene encoding the constant region of the IgG2c heavy chain in mice are SEQ ID NO.11 and SEQ ID NO.12.
  • the C57BL/6 mice or their progeny are used to produce heavy chain IgG2c antibodies.
  • the fourth aspect of the present application provides a preparation method of a C57BL/6 mouse or its progeny, comprising the following steps:
  • the application also provides a C57BL/6 mouse, the nucleotide sequence encoding the CH1 domain on the gene encoding the IgM heavy chain constant region in its genome, and the gene encoding the IgG3 heavy chain constant region in its genome The nucleotide sequence encoding the CH1 domain starting from the gene encoding the IgG2c heavy chain constant region was knocked out.
  • the genome of the C57BL/6 mouse includes a complete gene encoding a ⁇ light chain and/or a ⁇ light chain; optionally, the C57BL /6 mice can express ⁇ light chain and/or ⁇ light chain normally.
  • the gene knockout method includes one or more of the following: gene targeting technology, CRISPR/Cas9 method, zinc finger nuclease method, transcription activator like effector nuclease method.
  • the exon 1 of the gene encoding the IgM heavy chain constant region of the mouse is knocked out, and the gene from the mouse is also knocked out.
  • sgRNA targeting the upstream and downstream of exon 1 of the gene encoding the IgM heavy chain constant region of the mouse is used , sgRNAs targeting the upstream of exon 1 of the mouse gene encoding the constant region of the IgG3 heavy chain and sgRNAs targeting the downstream of exon 1 of the mouse encoding IgG2c were also used.
  • the targeting sequence upstream of exon 1 of the mouse encoding IgM heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.1 and SEQ ID NO.1 ID NO.2;
  • the targeting sequence downstream of exon 1 of the mouse encoding IgM heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.5 and SEQ ID NO.6;
  • the targeting sequence upstream of exon 1 of the mouse encoding IgG3 heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.7 and SEQ ID NO.8;
  • the targeting sequence downstream of exon 1 of the mouse encoding IgG2c heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.3 and SEQ ID NO.4.
  • the sgRNA upstream of the exon No. 1 of the gene targeting the mouse encoding the IgM heavy chain constant region is SEQ ID NO.9 and SEQ ID NO.10 ;
  • mice are SEQ ID NO.13 and SEQ ID NO.14;
  • the sgRNA targeting the upstream of exon 1 of the gene encoding the constant region of the IgG3 heavy chain in mice is SEQ ID NO.15 and SEQ ID NO.16;
  • the sgRNAs targeting the downstream of exon 1 of the mouse encoding the constant region of the IgG2c heavy chain are SEQ ID NO.11 and SEQ ID NO.12.
  • the C57BL/6 mice or their progeny are used to produce heavy chain IgG2c antibodies.
  • the fifth aspect of the present application provides a method for preparing a non-human mammal or its progeny, comprising the step of knocking out the nucleotide sequence encoding the CH1 domain of the IgM heavy chain constant region on the genome of the non-human mammal .
  • the present application also provides a non-human mammal whose genome is knocked out of the nucleotide sequence encoding the CH1 domain of the IgM heavy chain constant region.
  • the non-human mammal is a rodent; optionally, the rodent is a rat or a mouse; further optionally, the The rodent is a mouse; further, the mouse is a C57BL/6 mouse or a BALB/c mouse.
  • the non-human mammal or its progeny are used to construct the above non-human mammal or its progeny.
  • the sixth aspect of the present application provides a non-human mammal or its progeny derived from the above-mentioned preparation method, a C57BL/6 mouse or its progeny derived from the above-mentioned preparation method, the above-mentioned non-human mammal, The application of the above-mentioned C57BL/6 mice in screening target heavy chain antibodies.
  • the method of phage display is used when screening the target heavy chain antibody.
  • the screening target heavy chain antibody is screening C-reactive protein, coronavirus S protein or coronavirus N protein antigen-specific IgG2c heavy chain antibody.
  • a seventh aspect of the present application provides a method for screening a target heavy chain antibody, comprising using a non-human mammal or its progeny constructed from the preparation method described in the first aspect above, derived from the second aspect above
  • the non-human mammal or its progeny constructed by the preparation method, the C57BL/6 mouse or its progeny constructed by the preparation method described in the third aspect, or derived from the preparation described in the fourth aspect above The constructed C57BL/6 mice or their progeny were screened as immunized animals.
  • the method of phage display is used when screening the target heavy chain antibody.
  • the screening target heavy chain antibody is screening C-reactive protein, coronavirus S protein or coronavirus N protein antigen-specific IgG2c heavy chain antibody.
  • the eighth aspect of the present application provides a non-human mammalian cell or cell line or primary cell culture
  • the non-human mammalian cell or cell line or primary cell culture is derived from the non-human mammalian cell or cell line or primary cell culture constructed by the above preparation method.
  • the ninth aspect of the present application provides an isolated tissue or isolated organ or a culture thereof, the isolated tissue or isolated organ or its culture derived from the non-human mammal constructed by the above preparation method or its offspring generation, or derived from the C57BL/6 mice constructed by the above-mentioned preparation method or their progeny.
  • a tenth aspect of the present application provides an sgRNA composition, the sgRNA composition comprising: an sgRNA targeting the upstream of exon 1 of a gene encoding an IgM heavy chain constant region in mice and an sgRNA targeting mouse encoding IgG2c sgRNA downstream of exon 1 of the heavy chain constant region gene;
  • the targeting sequence upstream of the exon 1 of the mouse encoding IgM heavy chain constant region gene targeted by the sgRNA includes SEQ ID NO.1 and SEQ ID NO.2;
  • the targeting sequence downstream of exon 1 of the mouse encoding IgG2c heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.3 and SEQ ID NO.4;
  • the targeting sequence downstream of exon 1 of the mouse encoding IgM heavy chain constant region gene targeted by sgRNA includes SEQ ID NO.5 and SEQ ID NO.6;
  • the targeting sequences upstream of the exon 1 of the mouse encoding the constant region of the IgG3 heavy chain targeted by the sgRNA include SEQ ID NO.7 and SEQ ID NO.8.
  • the sgRNA upstream of the exon No. 1 of the gene targeting the mouse encoding the IgM heavy chain constant region is SEQ ID NO.9 and SEQ ID NO.10;
  • mice are SEQ ID NO.13 and SEQ ID NO.14;
  • the sgRNA targeting the upstream of exon 1 of the gene encoding the constant region of the IgG3 heavy chain in mice is SEQ ID NO.15 and SEQ ID NO.16;
  • the sgRNAs targeting the downstream of exon 1 of the mouse encoding the constant region of the IgG2c heavy chain are SEQ ID NO.11 and SEQ ID NO.12.
  • the eleventh aspect of the present application provides a knockout vector, which comprises: one or more DNA sequences encoding sgRNA, and the targeting sequence of the sgRNA is selected from the following one:
  • the backbone of the knockout vector is an sgRNA expression vector.
  • a twelfth aspect of the present application provides a cell comprising the above knockout vector.
  • Incorrect expression is opposite to correct expression, and both correct expression and incorrect expression in this application actually have the meaning commonly understood in the art. If the CH1 domain is incorrectly expressed, it refers to the incorrect expression of the CH1 domain of the heavy chain constant region caused by the mutation or deletion at the genome level, resulting in the loss of the ability of the CH1 domain to bind to the light chain; for example, encoding the heavy chain constant region Mutations or deletions within 10 nucleotides upstream and downstream of the exons of the CH1 domain result in the loss of the ability of the CH1 domain to bind to the light chain.
  • the gene encoding the IgG heavy chain constant region does not correctly express the encoded IgG heavy chain constant region during expression, which means that the immunoglobulin antibody obtained by the expression of the gene does not have antibody efficacy;
  • the correct expression of the IgG heavy chain constant region encoded by the gene of the chain constant region means that the immunoglobulin antibody obtained by the expression of the gene has antibody efficacy.
  • the first gene encoding IgG heavy chain constant region, the second gene encoding IgG heavy chain constant region, the third gene encoding IgG heavy chain constant region, the fourth gene encoding IgG heavy chain constant region refers to At the locus encoding the IgG heavy chain constant region, the genes encoding the IgG heavy chain constant region are arranged in order from upstream to downstream.
  • the present application passes through the steps of causing non-human mammals to not express or incorrectly express the CH1 domain of the IgM heavy chain constant region and causing one or more genes encoding the IgG heavy chain constant region to not express or incorrectly express the CH1 domain when expressed Steps to prepare non-human mammals or their progeny, the obtained non-human mammals or their progeny can be used to produce heavy chain antibodies.
  • the non-human mammal obtained by the preparation method of the present application does not introduce any foreign genes encoding the variable region and constant region of the antibody heavy chain, and can directly use all the VDJ genes encoding the variable region of the antibody heavy chain in its own genome, so as to pass Heavy chain variable region rearrangement produces more diverse heavy chain antibodies.
  • IgM In immunology, IgM is generally considered to be extremely important for the development of B cells, which in turn is extremely important for the production of antibodies.
  • the present application has verified through experiments that even if the CH1 domain encoding the IgM heavy chain constant region is not expressed, or even deleting the gene encoding the IgM heavy chain constant region and the gene encoding the IgD heavy chain constant region, it does not significantly affect the immune maturation of non-human mammals , all the mice survived normally and still produced high-titer immune responses, and the inventors also screened heavy chain antibodies with strong specificity and high affinity.
  • a specific subclass of heavy chain IgG antibodies can be obtained by selecting a genetically engineered gene encoding the constant region of an IgG heavy chain.
  • FIG. 1 is a schematic diagram of the gene loci of the variable region and constant region of the mouse antibody heavy chain involved in Example 1 of the present application.
  • FIG. 2 is a schematic diagram of the sgRNA targeting strategy for preparing Ighm-d-g mice in Example 1 of the present application.
  • the boxes in the second row represent exons, the white boxes represent the knockout gene segments, and the numbers are the exon numbers.
  • the five-pointed star indicates the target position of the sgRNA.
  • the arrows indicate the positions against which the primers for genotyping were directed.
  • Figure 3 is a schematic diagram of a PCR gel image for genotype identification of Ighm-d-g mice in Example 1 of the present application, wt: wild type; +/-: heterozygote; -/-: knockout homozygote.
  • Example 4 is a schematic diagram of the detection results of serum titers after immunizing Ighm-dg homozygous mice with CRP (human C-reactive protein) as antigenic protein in Example 2 of the present application. Low, the two lines coincide.
  • CRP human C-reactive protein
  • Figure 5A is a colony PCR diagram of the VH segment of the heavy chain gene of the Ighm-dg homozygous mouse IgG2c antibody in Example 5 of the present application
  • Figure 5B is the IgG2c antibody expressed by the Ighm-dg homozygous mouse in Example 5 of the present application
  • FIG. 6 is the sequencing comparison of the positive clones screened by the heavy chain antibody phage library in Example 6 of the present application.
  • Figure 7 is the ELISA result of specific recognition of the antigen CRP by purified D4-12 in Example 7 of the present application.
  • Figure 8 is the ELISA result of the specific recognition of the antigen CRP by purified 5S-12 in Example 8 of the present application, wherein the OD450 values of the two lines of OVA-D5S-12 and BSA-D5S-12 are very low, and the lines overlap.
  • FIG. 9 is the result of affinity determination of D5S-12 heavy chain antibody in Example 8 of the present application.
  • Figure 10 is a schematic diagram of the sgRNA targeting strategy for preparing IghM mice in Example 9 of the present application. Black boxes indicate exons, and the numbers in them are exon numbers. The five-star symbol indicates the target position of the sgRNA. Arrows indicate primers for genotyping.
  • Figure 11 is an example of PCR identification of IghM mouse genotype in Example 9 of the present application.
  • the Ighm wild-type gene produced a band of about 600 bp
  • the Ighm knockout gene produced a band of about 300 bp.
  • Numbers represent mouse sample numbers, +: wild-type control, -: blank control.
  • Figure 12 is the amino acid sequence diagram of the heavy chain of the IgM antibody expressed by the IghM homozygous mouse in Example 10 of the present application.
  • Figure 13 is a schematic diagram of the sgRNA targeting strategy for preparing IghM-3G3 mice in Example 11 of the present application. Black boxes indicate exons, and the numbers in them are exon numbers. The five-star symbol indicates the target position of the sgRNA. Arrows indicate primers for genotyping.
  • Figure 14 is a schematic diagram of the PCR gel image of the genotype identification of IghM-3G3 mice in Example 11 of the present application, wt: wild type; +/-: heterozygote; -/-: knockout homozygote.
  • Figure 15 is a schematic diagram of the detection results of serum titers after immunizing IghM-3G3 homozygous mice with CRP (human C-reactive protein) as antigenic protein in Example 12 of the present application, the serum titers after the first immunization and after the second immunization were very high Low, the two lines coincide.
  • CRP human C-reactive protein
  • Figure 16A is a colony PCR image of the VH segment of the heavy chain gene of the IghM-3G3 homozygous mouse IgG2c antibody in Example 13 of the present application
  • Figure 16B is the IgG2c antibody expressed by the IghM-3G3 homozygous mouse in Example 13 of the present application
  • Figure 17 is mentioned in Example 15 of the present application, in the case of immunizing IghM-3G3 homozygous mice with the coronavirus S protein antigen, respectively, in the case of unimmunized, one week after the second immunity, one week after the third immunity, and one week after the fourth immunity After blood collection, the serum titer was detected by ELISA after doubling dilution with PBS.
  • Figure 18A is a diagram of the colony PCR identification of the IgG2c antibody heavy chain gene of the IghM-3G3 homozygous mouse immunized with the coronavirus S protein antigen mentioned in Example 16 of the present application; Mentioned, the CDR region amino acid sequence alignment diagram of the positive clone obtained after the construction of the phage display library and the antibody panning of the IgG2c antibody heavy chain gene of the IghM-3G3 homozygous mouse immunized with the coronavirus S protein antigen.
  • Fig. 19 is mentioned in the embodiment 17 of this application, detects whether antibody S-9, S-19, S-27 and S-47 can specifically recognize and bind antigen coronavirus S protein by ELISA method.
  • Figure 20A shows in Example 18 of the present application, in the case of immunizing IghM-DG1 homozygous mice with coronavirus S protein antigen, blood was collected respectively in unimmunized, one week after second immunity, one week after third immunity and one week after fourth immunity, After doubling the dilution of serum with PBS, the results of detecting serum titer by ELISA;
  • Figure 20B is a phage display library construction and antibody panning for the above-mentioned immunization of IghM-DG1 homozygous mice with coronavirus S protein antigen The amino acid sequence alignment of the CDR regions of the positive clones obtained later.
  • Figure 21 is mentioned in Example 18 of the present application, whether the antibodies S-1, S-7, S-12, S-17, S19, S-25, S-51 and S-65 can be detected by ELISA method.
  • Figure 22A is mentioned in Example 19 of the present application, in the case of immunizing IghM-DG1 homozygous mice with the coronavirus N protein antigen, respectively in the case of unimmunized, one week after the second immunity, one week after the third immunity, and one week after the fourth immunity After blood collection, after doubling dilution of serum with PBS, the result diagram of serum titer was detected by ELISA;
  • Figure 22B is for the above-mentioned immunization of IghM-DG1 homozygous mice with coronavirus N protein antigen, constructed by phage display library Alignment with the amino acid sequence of the CDR region of the positive clone obtained after antibody panning.
  • Figure 23 is mentioned in Example 19 of the present application, by ELISA to detect whether antibodies N-1, N-2, N-3, N-5 and N-23 can specifically recognize and bind antigen coronavirus S protein Result graph.
  • FIG 24 is a comparison of the relative expression levels of each immunoglobulin gene in the bone marrow of MDG1 mice and wild-type mice mentioned in Example 20 of the present application.
  • the AD graphs are the relative expression levels of ⁇ gene, ⁇ 2c gene, and ⁇ 2c gene in the bone marrow of the two genotypes compared with ⁇ gene and ⁇ gene.
  • MDG1 is a homozygous knockout mouse
  • WT is a wild-type mouse
  • BM bone marrow
  • the housekeeping gene Gapdh gene is used as an internal reference
  • n 4
  • the data is analyzed by the calculation method of 2- ⁇ Ct
  • the two-tailed T test is used for statistics.
  • Scientific analysis *** represents p ⁇ 0.01.
  • Fig. 25 As mentioned in Example 20 of the present application, the relative expression levels of each immunoglobulin gene in the spleen of MDG1 mice and wild-type mice were compared.
  • the AD graphs show the relative expression levels of ⁇ gene, ⁇ 2c gene, and ⁇ 2c gene in the spleen of the two genotypes compared with ⁇ gene and ⁇ gene.
  • MDG1 is a homozygous knockout mouse
  • WT is a wild-type mouse
  • the housekeeping gene Gapdh gene was used as an internal reference
  • Fig. 26 As mentioned in Example 20 of the present application, the relative expression levels of each immunoglobulin gene in the small intestine of MDG1 mice and wild-type mice were compared.
  • the AD graphs show the relative expression levels of ⁇ gene, ⁇ 2c gene and ⁇ 2c gene in the small intestine of the two genotypes compared to the ⁇ gene and ⁇ gene.
  • MDG1 is a homozygous knockout mouse
  • WT is a wild-type mouse
  • SI the small intestine
  • the housekeeping gene Gapdh gene was used as an internal reference
  • FIG. 27 Western Blot detects the expression form of IgG2c in MDG1 mouse serum.
  • WT represents wild-type mice and MDG1 represents homozygous knockout mice.
  • Picture A is the detection of the expression form of IgG2c under reducing conditions
  • Picture B is the detection of the expression form of IgG2c under non-reducing conditions
  • the serum of wild-type mice was diluted 20 times
  • the serum of MDG1 mice was diluted 100 times
  • Goat Anti-Mouse IgG2c heavy chain (HRP) was diluted 10000 times.
  • Figure 28 is a comparison of the expression levels of various immunoglobulins in the serum of MDG1 mice and wild-type mice in resting state as mentioned in Example 22 of the present application.
  • Figures A-F are the expression levels of IgM, IgG2c, IgM in wild-type mice and IgG2c, total IgG, IgA, and IgE in MDG1 mice in the serum of two genotype mice, respectively.
  • n 20, statistical analysis was performed using a two-tailed T test, * represents p ⁇ 0.05, **** represents p ⁇ 0.01.
  • Figure 29 shows the development of B cells in the bone marrow of MDG1 mice mentioned in Example 23 of the present application.
  • Figures A-C show the grouping status of bone marrow B cells, immature B cells/mature B cells, and progenitor B cells/pre-B cells, respectively.
  • Figures D-F show the proportion and number of bone marrow B cells, progenitor B cells, and pre-B cells, respectively.
  • WT is a wild-type mouse
  • MDG1 is a homozygous knockout mouse
  • pro-B cells are pro-B cells
  • pre-B cells are pre-B cells.
  • n 6, statistical analysis was performed using a two-tailed T test, * represents p ⁇ 0.05, *** represents p ⁇ 0.01.
  • FIG. 30 shows the development of B cells in the spleen of MDG1 mice mentioned in Example 23 of the present application.
  • the AD maps show the grouping status of spleen B cells, IgG2c+/IgM+ B cells, follicular B cells/marginal B cells/transitional B cells, and plasma cells, respectively. Ratio and number of plasma cells, follicular B cells/marginal B cells/transitional B cells.
  • WT is a wild-type mouse
  • MDG1 is a homozygous knockout mouse
  • FOB is a follicular B cell
  • TB is a transitional B cell
  • MZB is a marginal B cell
  • plasma is a plasma cell.
  • Statistical analysis was performed by tailed T test, * represents p ⁇ 0.05, ** represents p ⁇ 0.01.
  • Figure 31 shows the development of B cells in the peritoneal cavity of MDG1 mice mentioned in Example 23 of the present application.
  • Panel A and Panel B are the grouping status of B cells and B1a/B1b/B2 cells in the peritoneal cavity, respectively, and
  • Panel C is the ratio of B cells, B1a cells, B1b cells and B2 cells in the peritoneal cavity, respectively.
  • WT is a wild-type mouse
  • MDG1 is a homozygous knockout mouse.
  • n 7, statistical analysis was performed using a two-tailed T test, * represents p ⁇ 0.05, ** represents p ⁇ 0.01.
  • Figure 32 shows the changing trend of the relative expression levels of antigen-specific antibodies in the serum of wild-type mice and MDG1 mice after chloramphenicol immunization as mentioned in Example 25 of the present application.
  • Figure 33 shows the changing trend of the relative expression levels of antigen-specific antibodies in the serum of wild-type mice and MDG1 mice after atrazine immunization mentioned in Example 25 of the present application.
  • Figure 34 shows the development of germinal center B cells and plasma cells in the spleen of wild-type mice and MDG1 mice after antigen immunization mentioned in Example 26 of the present application.
  • AE diagrams show the development of spleen B cells, germinal center B cells, IgG2c+ germinal center B cells, plasma cells, and IgG2c+ plasma cells after immunization, respectively
  • FJ diagrams show spleen B cells, germinal center B cells, and IgG2c+ germinal center B cells after immunization The proportion and number of cells, plasma cells and IgG2c+ plasma cells.
  • WT is a wild-type mouse
  • MDG1 is a homozygous knockout mouse
  • * represents p ⁇ 0.05
  • px330 plasmid vector purchased from Addgene, plasmid number #58778;
  • pEASY-T5 Zero Cloning vector purchased from Quanshijin Biotechnology Co., Ltd., item number: CT501-01;
  • TOP10 competent cells were purchased from Tiangen Biochemical Technology Co., Ltd., item number: CB104;
  • PET28 carrier purchased from Wuhan Miaoling Biotechnology Co., Ltd., item number: P31003;
  • HiPure Total RNA Plus Mini Kit is sourced from Meijimei, item number R4121;
  • HiPure Gel Pure Micro Kit comes from Meijimei, item number D2110;
  • HiPure Tissue DNA Mini Kit is sourced from Meijimei, item number D3121;
  • the DNA marker comes from Dongsheng, item number M1061/M1062);
  • the protein marker comes from Thermo, Cat. No. 26617;
  • the anti-CD23 antibody comes from Abcam, item number ab25457;
  • CD43 Monoclonal Antibody (eBioR2/60), PE source eBioscience, Cat. No. 85-12-0431-81;
  • CD23 Monoclonal Antibody (B3B4), PE source eBioscience, Cat. No. 85-12-0232-82;
  • Anti-CD5 antibody[53-7.3] (Phycoerythrin) from Abcam, Cat. No. ab114078;
  • CD21/CD35 Monoclonal Antibody eBio8D9(8D9)
  • PE-Cyanine7 from eBioscience, Cat. No. 85-25-0211-80;
  • IgM Monoclonal Antibody (eB121-15F9), PE-Cyanine7 from eBioscience, Cat. No. 85-25-5890-82;
  • Mouse IgG ELISA Quantitation Set source bethyl, Cat. No. E90-131;
  • Ighm-dg mice refer to C57BL/6 mice with knockout of Ighm, Ighd, Ighg3, Ighg1, Ighg2b and the first exon of Ighg2c, which encodes IgG2c CH1 domain of the heavy chain.
  • Ighm-d-g homozygous mice were prepared by the following procedure.
  • the schematic diagram of the gene loci of the heavy chain variable region and constant region of the C57BL/6 mouse antibody is shown in Figure 1.
  • the targeting strategy shown in Figure 2 was designed to knock out the mouse Ighm, Ighd, Ighg3, Ighg1, Ighg2b genes and Nucleotide sequence encoding CH1 domain in Ighg2c.
  • the inventors selected sgRNA targeting sequences (SEQ ID NO: 1, SEQ ID NO: 2) upstream of the first exon of the mouse Ighm gene, and selected downstream of the first exon of mouse Ighg2c.
  • sgRNA targeting sequence SEQ ID NO:3, SEQ ID NO:4
  • the sgRNA sequence was designed according to the targeting sequence, as shown in Table 1.
  • the synthesized forward and reverse DNA oligo encoding the sgRNA sequence was annealed to form complementary double strands, and then ligated to the sgRNA expression vector (px330) using T4 ligase. After the ligation, it was sequenced and verified by a professional sequencing company. Plasmids were further obtained by in vitro transcription to obtain sgRNA.
  • the above-mentioned fertilized egg cells are implanted into surrogate female mice to produce F0 generation chimeric mice.
  • F0 generation chimeric mice Through extraction of mouse tail genomic DNA and PCR detection, individuals with knockout in F0 generation mice were detected.
  • the knockout mice were sequenced to confirm deletion of the target sequence.
  • the F0 generation chimeric mice with the correct gene knockout were selected for subsequent breeding and identification.
  • PCR primers Ighm-dg-1F and Ighm-dg-1R can detect knockout genes (indicated by arrows in Figure 2), and primers Ighd-2F and Ighd-2R can detect wild-type genes (indicated by arrows in Figure 2) , the sequence of each primer is shown in Table 2, and the schematic diagram of the PCR results when detecting individuals with knockout in Ighm-dg mice is shown in Figure 3 .
  • Ighm-dg homozygous knockout gene was amplified with primers Ighm-dg-1F and Ighm-dg-1R, and the target band size was about 700bp (as shown in the left figure), but it could not be amplified with primers Ighd-2F and Ighd-2R A band (as shown on the right); Ighm-dg wild-type gene with primers Ighm-dg-1F and Ighm-dg-1R can not amplify a band (as shown on the left), with primers Ighd-2F and Ighd-2R amplified the target band size of about 600bp (as shown in the right figure).
  • the F0 generation mice with the target gene knockout were mated with wild-type mice to obtain the F1 generation mice.
  • the gene knockout positive F1 generation heterozygous mice that can be stably inherited were selected.
  • the F1 generation heterozygous mice were mated with each other to obtain gene knockout positive F2 generation homozygous mice, namely Ighm-d-g homozygous mice.
  • the method for genotyping the obtained F1 generation heterozygous or F2 generation homozygous mice is the same as step (4).
  • Embodiment 2 antigen immune response and titer detection
  • CRP human C-reactive protein
  • the immunization method is as follows: select 6-8 week-old male mice, the antigen human C-reactive protein (CRP, A-5172, Baiqiao Ruijing) plus an equal volume of Freund's complete adjuvant (F5881, Sigma), emulsified to the dripping water State, that is, for the primary immunization of mice by subcutaneous multi-point injection, the initial immunization injection dose is 100 ⁇ g/mouse, after the primary immunization, follow-up subcutaneous immunization is carried out every 2 weeks, CRP antigen plus equal volume of incomplete Freund’s adjuvant (F5506 , Sigma) after emulsification, the mice were injected subcutaneously at multiple points, and the dose of each injection was 100 ⁇ g/mouse.
  • CRP human C-reactive protein
  • A-5172 Baiqiao Ruijing
  • F5881 emulsified to the dripping water State
  • the serum titer detection method is as follows: Dilute the CRP antigen to 2 ⁇ g/mL, add 100 ⁇ l to the polystyrene enzyme-linked detection plate, and use HRP-goat anti-mouse IgG-Fc (Jackson 115-035-071) to detect the serum titer. A specific IgG2c antibody heavy chain that specifically binds to the CRP antigen.
  • Example 3 Examination of spleen, thymus, lymph nodes and organs of unimmunized Ighm-d-g homozygous mice and Ighm-d-g homozygous mice immunized with CRP antigen
  • mice were euthanized by carbon dioxide asphyxiation.
  • the size and morphology of thymus, spleen, mesenteric lymph nodes, submandibular lymph nodes, and heart, liver, lung, kidney and other organs were observed by dissection.
  • the thymus, mesenteric lymph nodes, submandibular lymph nodes and major organs of dg homozygous mice were not abnormal.
  • the dimerized molecular weights of the chains were consistent and included dimers that were not bound by the HRP-goat anti-mouse Ig light chain (Jackson, 115-035-174) antibody.
  • mice were euthanized and spleen cells were taken, lysed by Trizol, total RNA was extracted and reverse transcribed to obtain cDNA, using The following IgG2c subtype antibody specific primers amplified the heavy chain variable region and the heavy chain constant region linked thereto by PCR.
  • MHV1 ATGAAATGCAGCTGGGGGCATSTTCTTC (SEQ ID NO: 21);
  • MHV2 ATGGGATGGAGCTRTATCATSYTCTT (SEQ ID NO: 22);
  • MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT (SEQ ID NO: 23);
  • MHV4 ATGRACTTTGGGYTCAGCTTGRTTT (SEQ ID NO: 24);
  • MHV5 ATGGGACTCCAGGCTTCAATTTAGTTTTCCTT (SEQ ID NO: 25);
  • MHV6 ATGGCTTGTCYTTRGSGCTRCTCTTCTGC (SEQ ID NO: 26);
  • MHV7 ATGGRATGGAGCKGGRGTCTTTMTCTT (SEQ ID NO: 27);
  • MHV8 ATGAGAGTGCTGATTCTTTTGTG (SEQ ID NO: 28);
  • MHV9 ATGGMTTGGGTGTGGAMCTTGCTTATTCCTG (SEQ ID NO: 29);
  • MHV10 ATGGGCAGACTTACCATTCTCATTCCTG (SEQ ID NO:30);
  • MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG (SEQ ID NO: 31);
  • MHV12 ATGATGGTGTTAAGTCCTTCTGTACC (SEQ ID NO: 32);
  • PCR reaction program 98°C, 2min—30 cycles (each cycle is 98°C, 10s—50°C, 20s—72°C, 40s)—72°C, 5min—16°C.
  • the resulting PCR amplification product was connected to the pEASY-T5 Zero Cloning vector, transformed into TOP10 competent cells and coated with LB plates, and 11 clones were randomly picked. band is a positive band).
  • Example 6 Construction and panning of CRP antigen-specific IgG2c heavy chain antibody variable region phage display library
  • the PCR process of the first round is exactly the same as the PCR process mentioned in Example 5;
  • the primers, reaction system and reaction procedure used in the second round of PCR are as follows:
  • MHVF1-SfiI ATGCCATGACTGTggcccaggcggcc GAG GTG AAG CTT CTC GAG TCT GG (SEQ ID NO: 34);
  • MHVF2-SfiI CATGCCATGACTGTggcccaggcggcc SAG GTS CAG CTG MAG GAG TCW GG (SEQ ID NO: 35);
  • MHVF3-SfiI CATGCCATGACTGTggcccaggcggcc GAG GTC CAG CTG CAA CAA TCT GG (SEQ ID NO:36);
  • MHVF4-SfiI CATGCCATGACTGTggcccaggcggcc SAG GTY CAR CTK CAG CAG YCT GG (SEQ ID NO: 37);
  • MHVF5-SfiI CATGCCATGACTGT ggcccaggcggcc GAR GTG AAG CTT GWG GAG TCT GG (SEQ ID NO: 38).
  • B6-IgG2c-Hin-sfi1 ACTCGCGGCCGGCCTGGCCTGTTATGGGCACTCTGGG (SEQ ID NO: 39).
  • PCR reaction program 98°C, 2min—30 cycles (each cycle is 98°C, 10s—65°C, 20s—72°C, 40s)—72°C, 5min—16°C.
  • the PCR amplification product was digested with SfiI (FD1824, Thermo), and the target product was gel-recovered by agarose gel electrophoresis, and the target fragment recovered from the gel was cloned into the SfiI-digested pComb3XSS vector.
  • R2738 electrocompetent cells were electroporated to prepare an IgG2c heavy chain antibody library and perform multiple rounds of panning. A number of positive clones were selected for sequencing and identification, and the results are shown in Figure 6.
  • Coating Dilute the CRP antigen with coating solution to 100 ⁇ g/mL, add it to 2 wells of an ELISA plate (100 ⁇ L/well), and coat overnight at 4°C.
  • Blocking Aspirate the coating solution, wash the plate three times with PBS, block with 300 ⁇ L of 4% skim milk (the second, third, fourth and fifth rounds of blocking solution are 4% BSA), incubate at 37°C for 2h.
  • Binding Aspirate the blocking solution, wash the plate three times with PBS, add 100 ⁇ L of the phage display library, and incubate at 37° C. for 1 h.
  • Washing suck out the unbound phage and wash with PBST (five rounds of panning were carried out in this application, the first and second rounds were washed 3 times with PBST respectively, and the third, fourth and fifth rounds were washed 10 times with PBST respectively) .
  • helper phage M13KO7 (2 ⁇ 10 9 cfu) (purchased from NEB Company, the product number is N03158), and mix well;
  • Example 8 Eukaryotic expression and biological activity identification of variable region of CRP antigen-specific IgG2c heavy chain antibody
  • Example 6 the sequenced sequence was further analyzed, and the most repeated sequence D5S-12 (nucleotide sequence and amino acid sequence shown in Table 7) was selected for eukaryotic expression and biological activity identification. After transfecting KOP293 cells (Kairui, Zhuhai), the supernatant of the cultured cells was collected on the 6th day and subjected to nickel affinity column to purify the target protein.
  • the second part the preparation of IghM-3G3 pure-type mice and their immunization results
  • IghM-3G3 mice refer to C57BL/6 mice with knockout of the first exon on the Ighm gene and knockout of the Ighg3 gene, Ighg1 gene, Ighg2b gene and the first exon located on the Ighg2c gene.
  • IghM pure-type mice ie, C57BL/6 mice with the first exon on the Ighm gene knocked out
  • IghM-3G3 homozygous mice were prepared based on IghM pure-type mice.
  • the preparation of IghM homozygous mice includes the following steps:
  • FIG. 1 The schematic diagram of the gene loci of the heavy chain variable region and constant region of the C57BL/6 mouse antibody is shown in Figure 1.
  • the targeting strategy shown in Figure 10 was designed to knock out the nucleotide encoding the CH1 domain in the mouse Ighm gene. sequence.
  • the target was selected upstream and downstream of the first exon of the mouse Ighm gene.
  • the sgRNA targeting sequence (SEQ ID NO: 1, SEQ ID NO: 2) was selected upstream, and the sgRNA targeting sequence (SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 2) were selected downstream of the first exon of the mouse Ighm gene. NO: 6), and designed the sgRNA sequence according to the targeting sequence, as shown in Table 8.
  • RNA (2) construct the nucleic acid molecule into the backbone plasmid and transcribe in vitro to obtain RNA:
  • the forward and reverse DNA oligos of the synthesized sgRNA sequences were annealed to form complementary double strands, which were ligated to the sgRNA expression vector (px330) using T4 ligase. After ligation, they were sequenced and verified by a professional sequencing company. , and further obtained sgRNA by in vitro transcription.
  • Mouse ovulation is induced, fertilized in vitro, and fertilized eggs are cultivated, and then sgRNA and Cas9 protein are mixed, and the mouse fertilized eggs are electroporated, or the Cas9 protein (or Cas9 mRNA, which is commercially available) is injected into the microinjection together with sgRNA by microinjection. Rat fertilized eggs.
  • the above-mentioned fertilized egg cells are implanted into surrogate female mice to produce F0 generation chimeric mice.
  • F0 generation chimeric mice Through extraction of mouse tail genomic DNA and PCR detection, individuals with knockout in F0 generation mice were detected.
  • the knockout mice were sequenced to confirm deletion of the target sequence.
  • the F0 generation chimeric mice with the correct gene knockout were selected for subsequent breeding and identification.
  • PCR primers Ighm-F and Ighm-R can detect knockout genes (as shown by the arrows in Figure 10).
  • the primer sequences are shown in Table 9:
  • the test results are shown in Figure 11.
  • the Ighm wild-type gene can amplify a band of about 600 bp
  • the Ighm CH1 knockout gene can amplify a band of about 300 bp.
  • clones #1, #2, #3 and #6 contain the Ighm CH1 knockout gene.
  • the knockout mice were sequenced to confirm that the first exon of the Ighm gene in the knockout mice was deleted.
  • the F0 generation mice with the target gene knockout were mated with wild-type mice to obtain the F1 generation mice.
  • the gene knockout positive F1 generation heterozygous mice that can be stably inherited were selected.
  • the F1 generation heterozygous mice were mated with each other to obtain gene knockout positive F2 generation homozygous mice, namely IghM homozygous mice.
  • the method for genotyping the obtained F1 generation heterozygous or F2 generation homozygous mice is the same as step 4.
  • PCR reaction program 98°C, 2min—30 cycles (each cycle is 98°C, 10s—50°C, 20s—72°C, 40s)—72°C, 5min—16°C.
  • the PCR amplification product was ligated into pEASY-T5 Zero Cloning vector, transformed into TOP10 competent cells and spread on LB plate (ampicillin resistance), and clones were picked for colony PCR.
  • the positive clones were sent for sequencing, and the sequencing results showed that the variable region FR4 of the heavy chain of the IgM antibody expressed in the IghM homozygous mice was directly connected to CH2 (the starting amino acid sequence AVAEMN).
  • the results are shown in Figure 12.
  • the genomic IgM CH1 Exon knockout was successful. .
  • a method for preparing IghM-3G3 mouse homozygous comprising the following steps:
  • the schematic diagram of the gene locus of the heavy chain variable region and constant region of the C57BL/6 mouse antibody is shown in Figure 1, and the targeting strategy shown in Figure 13 is designed. Based on the IghM homozygous mouse obtained in Example 9 On the above, the Ighg3 gene, the Ighg1 gene, the Ighg2b gene and the first exon located on the Ighg2c gene were further knocked out.
  • the inventors In order to knock out the first exon encoding the CH1 domain on the mouse Ighm, the Ighg3, Ighg1, Ighg2b genes and the first exon encoding the CH1 domain on Ighg2c, the inventors based on the IghM homozygous mouse On the above, the targeting sequence of sgRNA (SEQ ID NO:7, SEQ ID NO:8) was selected upstream of the first exon of the mouse Ighg3 gene, and downstream of the first exon of the mouse Ighg2c gene was selected The sgRNA targeting sequence (SEQ ID NO:3, SEQ ID NO:4) was obtained, and the sgRNA sequence was designed according to the targeting sequence, as shown in Table 11.
  • RNA (2) construct the nucleic acid molecule into the backbone plasmid and transcribe in vitro to obtain RNA:
  • the forward and reverse DNA oligos of the synthesized sgRNA sequences were annealed to form complementary double strands, and were connected to the sgRNA expression vector (px330) using T4 ligase. , and further obtained sgRNA by in vitro transcription.
  • Mouse ovulation is induced, fertilized in vitro, and fertilized eggs are cultivated, and then sgRNA and Cas9 protein are mixed, and the mouse fertilized eggs are electroporated, or the Cas9 protein (or Cas9 mRNA, which is commercially available) is injected into the microinjection together with sgRNA by microinjection. Rat fertilized eggs.
  • the above-mentioned fertilized egg cells are implanted into surrogate female mice to produce F0 generation chimeric mice.
  • F0 generation chimeric mice Through extraction of mouse tail genomic DNA and PCR detection, individuals with knockout in F0 generation mice were detected.
  • the knockout mice were sequenced to confirm deletion of the target sequence.
  • the F0 generation chimeric mice with the correct gene knockout were selected for subsequent breeding and identification.
  • PCR primers Ighg-1F and Ighg-1R can detect the knockout gene, and primers Ighg-2R and Ighg-1F can detect the wild-type gene (arrow in Figure 13).
  • the sequences of primers are shown in Table 12.
  • the genotype of IghM-3G3 mice was identified by PCR ( Figure 14), and the Ighm genotype was identified with primers Ighm-F and Ighm-R: the Ighm wild-type gene can amplify the target product of about 600 bp, and the knockout gene can expand The target product of about 300bp was added.
  • Ighg genotype was identified by primers Ighg-1F, Ighg-1R and Ighg-2R: Ighg knockout gene can be amplified with primers Ighg-1F and Ighg-2R to amplify the target product of about 500 bp, using primers Ighg-1F and Ighg-1R No band could be amplified; Ighg wild-type gene could not be amplified with primers Ighg-1F and Ighg-2R, but the target product of about 460bp could be amplified with primers Ighg-1F and Ighg-1R. +/-: heterozygotes; wt: wild type; -/-: knockout homozygotes.
  • the F0 generation mice with the target gene knockout were mated with wild-type mice to obtain the F1 generation mice.
  • the gene knockout positive F1 generation heterozygous mice that can be stably inherited were selected.
  • the F1 generation heterozygous mice were mated with each other to obtain gene knockout positive F2 generation homozygous mice, namely IghM-3G3 homozygous mice.
  • the method for genotype identification of the obtained F1 generation heterozygous or F2 generation homozygous mice is the same as step 4.
  • Embodiment 12 IghM-3G3 homozygous mouse antigen immune response and titer detection
  • IghM-3G3 homozygous mice were immunized with human C-reactive protein (CRP).
  • CRP human C-reactive protein
  • the immunization method is as follows: select 6-8 week-old male mice, the antigen human C-reactive protein (CRP, A-5172, Baiqiao Ruijing) plus an equal volume of Freund's complete adjuvant (F5881, Sigma), emulsified to the dripping water After the initial immunization, follow-up subcutaneous immunization is carried out every 2 weeks, CRP antigen plus equal volume of incomplete Freund's adjuvant ( After emulsification of F5506, Sigma), the mice were subcutaneously injected at multiple points, and the dose of each injection was 100 ⁇ g/mouse.
  • the serum titer detection method is as follows: Dilute the CRP antigen to 2 ⁇ g/mL, add 100 ⁇ L to the polystyrene enzyme-linked detection plate, and detect the serum with HRP-goat anti-mouse IgG-Fc (Jackson, 115-035-071). A specific IgG antibody that specifically binds to the CRP antigen.
  • blood was collected from unimmunized, one week after one immunization, and one week after three immunizations.
  • the serum was diluted with PBS, and the doubling dilution started from 1:500 dilution.
  • the serum titer detected by ELISA showed that unimmunized and one There was no specific antibody binding to the antigen CRP in the immunized mice, and the serum titer was about 1:32000 after three immunizations.
  • mice After immunizing IghM-3G3 pure-type mice with CRP antigen and testing the serum titer, the mice were euthanized and spleen cells were collected, lysed by Trizol, extracted total RNA, and reverse transcribed to obtain cDNA, using the following IgG2c subtype antibodies Specific primers amplify the heavy chain variable region and its linked heavy chain constant region by PCR.
  • the resulting PCR amplification product was connected to the pEASY-T5 Zero Cloning carrier, transformed into TOP10 competent cells and coated with LB plates, and 7 clones were randomly picked, and the colony PCR was identified as positive and sent for sequencing (as shown in Figure 16A, a 650bp size band). positive band).
  • the structure is VH-CH1-Hinge-CH2-CH3, and the VH region can be divided into FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in detail, and the IgG2c antibody produced in IghM-3G3 pure and type mice is heavy.
  • the FR4 region of the chain variable region is directly connected to the Hinge region (antibody hinge region), indicating that the CH1 exon of IgG2c was successfully knocked out.
  • Embodiment 14 CRP antigen-specific IgG2c heavy chain antibody variable region phage library display construction and panning;
  • Embodiment 15 IghM-3G3 homozygous mouse antigen immune response and titer detection
  • IghM-3G3 homozygous mice were immunized with the coronavirus S protein antigen.
  • the immunization method is as follows: select 6-8 week old male mice, antigen coronavirus S protein (SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein, 40591-V08H, Yiqiao Shenzhou) plus equal volume of Freund's complete Adjuvant (F5881, Sigma), emulsified to the state of not dripping water, can be used for subcutaneous multi-point injection in the initial immunization of mice, the initial immunization injection dose is 100 ⁇ g/mouse, after the initial immunization, follow-up subcutaneous injection every 2 weeks For immunization, the dose of each injection was 100 ⁇ g/mouse.
  • SARS-CoV-2 2019-nCoV
  • F5881 Freund's complete Adjuvant
  • the serum titer detection method is as follows: Dilute the coronavirus S protein antigen to 2 ⁇ g/mL, add 100 ⁇ L to the polystyrene enzyme-linked detection plate, and use HRP-goat anti-mouse IgG-Fc (Jackson 115-035-071) Detection of specific IgG antibodies that specifically bind to CRP antigen in serum.
  • the blood was collected after no immunization, one week after the second immunization, one week after the third immunization, and one week after the fourth immunization.
  • the serum was diluted with PBS, and the 1:500 dilution was started to do doubling dilution, and the serum titer was detected by ELISA.
  • Figure 17 shows that no specific antibody bound to the S protein of the antigenic coronavirus appeared in the unimmunized mice, and the serum titer was about 1:64000 after the second immunization.
  • mice after immunizing IghM-3G3 homozygous mice with the coronavirus S protein antigen and detecting the serum titer, the mice were euthanized and their splenocytes were taken, and the splenocytes were split by Trizol to extract total RNA, The cDNA was obtained by reverse transcription, and the following IgG2c subtype antibody-specific primers were used to amplify the heavy chain variable region and the heavy chain constant region connected thereto by PCR.
  • the resulting PCR amplification product was connected to the pEASY-T5 Zero Cloning carrier, transformed into TOP10 competent cells and coated with LB plates, and 23 clones were randomly picked, and the clones identified as positive by colony PCR were sent for sequencing (PCR results are shown in Figure 18A, wherein The 650bp size band is a positive band).
  • Embodiment 17 coronavirus S protein antigen-specific IgG2c heavy chain antibody variable region phage library display construction and panning;
  • Example 16 Following the above Example 16, the steps of phage display library construction and antibody panning were exactly the same as those in Example 6. A total of 5 pannings were carried out, and the output phages were significantly enriched each time. Positive clones were selected by PHAGE-ELISA experiment and sent for delivery. Sequencing identification, as shown in Table 15. The similarities and differences in the CDR regions of these sequences were analyzed, and the results are shown in Figure 18B.
  • sequenced sequences were analyzed, and S-9, S-19, S-27 and S-47 (amino acid sequences shown in Table 15) that appeared in the first 5 rounds of panning were selected to be transfected into BL21 E. coli, and added at 30°C. Prokaryotic induction expression was carried out at the concentration of IPTG, and the supernatant was collected by sonication and purified by nickel affinity column.
  • Embodiment 18 IghM-DG1 homozygous mouse antigen immune response and titer detection
  • IghM-DG1 homozygous mice were immunized with the coronavirus S protein antigen.
  • mice antigen immunization and serum titer detection are exactly the same as in Example 15, and the results of serum titer detection are shown in Figure 20A; the steps of phage display library construction and antibody panning are exactly the same as in Example 6, A total of 5 pannings were performed, and each output phage was significantly enriched. Positive clones were selected by PHAGE-ELISA experiment and sent for sequencing identification, as shown in Table 16. The similarities and differences in the CDR regions of these sequences were analyzed, and the results are shown in Figure 20B.
  • the sequencing sequences were analyzed, and the S-1, S-2, S-7, S-12, S-17, S19 and S-65 antibodies (amino acid sequences shown in Table 16) that appeared in the first 5 rounds of panning were selected.
  • the DNA sequence was cloned into PET28 vector and transfected into BL21 Escherichia coli. Different concentrations of IPTG were added at 16°C to induce prokaryotic expression. The supernatant was collected by ultrasonication and purified by nickel affinity column.
  • MDG1 homozygous mice were immunized with the coronavirus N protein antigen.
  • the immunization method and serum titer detection method are exactly the same as those in Example 15, and the serum titer detection results after immunization are shown in Figure 22A.
  • the antigen used in this example is the coronavirus N protein (SARS-CoV-2 (2019-nCoV) Nucleocapsid-His recombinant Protein, 40588-V08B-B, Yiqiao Shenzhou).
  • phage display library construction and antibody panning are exactly the same as those in Example 6. A total of 5 pannings were performed, and each output phage was significantly enriched. Positive clones were selected by PHAGE-ELISA experiment and sent for sequencing identification, as shown in Table 17 shown. The similarities and differences in the CDR regions of these sequences were analyzed, as shown in Figure 22B.
  • the DNA sequence of the positive clone was cloned into the PET28 vector, transformed into BL21 bacteria, and the expression was induced. Then, the target protein was purified with a nickel column, and the specificity and binding force of the antibody were detected by ELISA. The results are shown in Figure 23. , the results in Figure 23 show that the variable region of the heavy chain antibody extracted this time can specifically recognize and bind to the S protein of the antigen coronavirus, and shows a good concentration dependence.
  • Example 20 Fluorescence quantitative PCR detects the difference in the transcription level of each immunoglobulin gene in mice of two genotypes
  • Embodiment 21 Western Blot detects the expression of IgG2c in MDG1 mouse serum
  • the sera of 8-week-old wild-type mice and MDG1 mice under the same genetic background were used for Western Blot detection.
  • the serum of wild-type mice was diluted 20 times with PBS, and the serum of MDG1 mice was diluted 100 times with PBS.
  • the intramolecular disulfide bonds were opened with 1 mM DTT, and the Goat Anti-Mouse IgG2c heavy chain (HRP) antibody was diluted 10,000-fold.
  • the results of Western Blot detection are shown in Figure 27.
  • Example 22 ELISA detects the expression of each immunoglobulin in the serum of MDG1 mice
  • mice and MDG1 mice under the same genetic background were detected by double-antibody sandwich ELISA.
  • Wild-type mouse IgM was diluted 2000 times, IgG2c was diluted 4000 times, IgG was diluted 10000 times, IgA was diluted 5000 times, and IgE was diluted 20 times;
  • IgM of MDG1 mice was diluted 2000 times and IgG2c was diluted 8000 times , IgG was diluted 10,000 times, IgA was diluted 5,000 times, and IgE was diluted 20 times.
  • Example 23 Development of B cells in bone marrow, spleen and peritoneal cavity of MDG1 mice detected by flow cytometry
  • Bone marrow B cells
  • the flow cytometry results of B cells in the bone marrow are shown in Figure 29.
  • the results in Figure 29 show that the proportion and number of B cells in the bone marrow of MDG1 mice were not statistically different from those of wild-type mice.
  • the proportion of B cells was not statistically different from wild-type mice, but was significantly reduced in number.
  • IgM was highly expressed in mature B cells and low in immature B cells, since MDG1 mice lacked IgM-expressing cells. ⁇ gene, so the development of immature B cells and mature B cells in vivo cannot be determined, but there are a large number of IgG2c+ B cells in MDG1 mice, and the proportion of this group of B cells in wild-type mice is extremely low.
  • the flow cytometry results of B cells in the spleen are shown in Figure 30.
  • the results in Figure 30 show that the proportion of B cells in the spleen of MDG1 mice was significantly lower than that of wild-type mice, but there was no statistical difference in the number;
  • the proportion and number of IgG2c+B cells in the spleen of mice were significantly higher than those of IgM+B cells in wild-type mice, which may be due to the class-switching recombination of IgM in wild-type mice to other immunoglobulin subtypes.
  • the flow cytometry results of B cells in the peritoneal cavity are shown in Figure 31.
  • the results in Figure 31 show that the proportion of B cells in the peritoneal cavity of MDG1 mice is significantly higher than that of wild-type mice, and the proportion of IgM+B1a cells is significantly higher in MDG1 mice. decreased, while the ratio of B1b and B2 cells was significantly increased in MDG1 mice.
  • Example 25 ELISA detects the changing trend of the relative expression of antigen-specific antibodies in mouse serum after immune stimulation
  • this ELISA experiment used chloramphenicol and atrazine coupled with OVA as the coating antigen, and the antigen was used as the coating antigen.
  • the coating amount is 200ng/100 ⁇ L/well
  • the dilution factor of serum in antigen-specific IgM, IgG2c, IgG and IgA detection is 4000 times
  • the dilution factor of serum in IgE detection is 100 times.
  • the reaction time was strictly controlled at 20min, and then the absorbance value at 450nm was read by a microplate reader, the individuals without immune reaction were excluded, and the change trend diagram of the relative expression of antigen-specific antibodies was drawn.
  • Example 26 Developmental detection of germinal center B cells and plasma cells in the spleen after antigen immunization
  • mice Take 6 wild-type mice and 6 MDG1 mice each after antigen immunization, place a 70 ⁇ m filter in a 60 mm dish, add 1 mL of ACK, take the spleen and gently grind it on the filter, and collect the ground cell suspension. After a brief centrifugation, the supernatant was discarded, and 1 mL of FACS was added to resuspend the cells. 20 ⁇ L of the cells were used for staining, and 10 ⁇ L of the cells were diluted 80-fold for counting.
  • the staining scheme of spleen B cells after immunization is shown in Table 21.
  • Table 21 Staining protocol for splenic B cells after immunization
  • Figure 34 shows the development of spleen B cells, germinal center B cells and plasma cells in mice after antigen immunization. There was no statistical difference in number; the proportion and number of germinal center B cells in MDG1 mice were significantly lower than those in wild-type mice, and the proportion of IgG2c+GC B was significantly higher than that in wild-type mice, and no statistics were found in number There was no statistical difference in the proportion and number of plasma cells and IgG2c+ plasma cells between MDG1 mice and wild-type mice after antigen immunization.
  • the non-human mammal or its progeny obtained by the method for preparing the non-human mammal or its progeny of the present application does not introduce any foreign genes encoding the variable region and constant region of the heavy chain of the antibody, and can directly utilize its own genome. All VDJ genes encoding the variable region of the heavy chain of an antibody are rearranged to produce a more diverse heavy chain antibody.

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Abstract

提供了一种非人哺乳动物或其子代的制备方法及其应用，该制备方法包括以下步骤：使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤；以及，使得非人哺乳动物体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤。所得非人哺乳动物或其子代可以用于生产重链抗体。采用所述制备方法获得的非人哺乳动物不引入任何编码抗体重链可变区和恒定区的外源基因，可以直接利用其自身基因组中编码抗体重链可变区的所有VDJ基因，从而通过重链可变区重排产生多样性更好的重链抗体。

Description

一种非人哺乳动物或其子代的制备方法及其应用
交叉引用
本申请要求于2020年9月4日提交的、申请号为202010924095.1、发明名称为“一种非人哺乳动物或其子代的制备方法及其应用”的发明专利申请的优先权益，其全部内容通过引用并入本文。
技术领域
本申请涉及生物技术领域，具体涉及一种非人哺乳动物或其子代的制备方法及其应用，该非人哺乳动物或其子代可以用于生产重链抗体。
背景技术
抗体是由两条相同的重链(H链)和两条相同的轻链(L链)通过链间非共价键或二硫键连接而成的四条肽链结构。抗体重链包括重链恒定区(C H)和重链可变区(V H)；其中：IgD、IgG、IgA的重链恒定区包括4个结构域：CH1，铰链区Hinge，CH2，CH3；IgM和IgE包括4个结构域：CH1，CH2，CH3，CH4(Janeway’s Immunobiology，9 th Edition)；重链恒定区的CH1结构域通过二硫键与轻链恒定结构域进行连接；重链可变区包括互补决定区(CDR)的高度可变区和相对保守的、称为框架区(FR)的区域中，重链可变区包括3个CDR和4个FR，从氨基端至羧基端依下述顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
根据抗体重链恒定区抗原特异性的不同，抗体包括以下5类：IgM、IgD、IgG、IgE、IgA。同一类抗体因其重链恒定区内抗原特异性仍有某些差异，所以又可将它们分为若干亚类，如：人IgG包括IgG1、IgG2、IgG3、IgG4等；人IgA包括IgA1、IgA2等；不同哺乳动物之间(如人、羊驼和小鼠)、不同品系的小鼠之间(如C57BL/6小鼠和BALB/c小鼠)的抗体的亚类种类可能不同。
与抗体重链相关的基因包括L、V、D、J、C五类基因片段，其中：重链可变区由V、D、J三种基因片段编码，重链恒定区由C基因片段编码。人的抗体重链基因中包括有功能的约40个V基因、23个D基因、6个J基因以及9个C基因(来源：Janeway’s Immunobiology，9 th Edition，Page 177)。编码IgM重链恒定区的基因为Ighm基因(Immunoglobulin heavy constant mu)，编码IgD重链恒定区的基因为Ighd基因(Immunoglobulin heavy constant delta)，编码IgG重链恒定区的基因为Ighg(Immunoglobulin heavy constant gamma)，编码IgA重链恒定区的基因为Igha (Immunoglobulin heavy constant alpha)等；相应的，编码IgG1重链恒定区的基因为Ighg1(Immunoglobulin heavy constant gamma 1)，编码IgG3重链恒定区的基因为Ighg3(Immunoglobulin heavy constant gamma 3)，编码IgG2b重链恒定区的基因为Ighg2b(Immunoglobulin heavy constant gamma 2b)。通过重链可变区重排产生具有多样性的抗体。成熟B细胞经抗原刺激后生成分泌型抗体IgM，再次抗原刺激后会发生第二次重排，膜上表达和分泌的免疫球蛋白的种类会从亲和力较低的IgM转换成亲和力高的IgG、IgA、IgE等其他类别或亚类的免疫球蛋白，这种现象称为类别转换(Class switch)或同种型转换(Isotype switch)。
重链抗体(heavy chain antibody)，也叫仅有重链的抗体(heavy chain only antibody)，是指仅由两条重链组成的免疫球蛋白抗体。天然存在的重链抗体存在于自然界骆驼科动物和鲨鱼体内。骆驼科动物胚系中的重链基因座包含编码重链恒定区的基因片段，在成熟期间，经重排的VDJ结合区被剪接到编码IgG铰链区的基因片段的5'末端，从而使得其缺乏介导与轻链结合的CH1区而不能与轻链结合，产生IgG2和IgG3重链抗体。利用基因修饰动物的方法也能生成重链抗体，对生成的重链抗体分类的方式也是根据抗体重链恒定区抗原特异性的不同。
与传统抗体的分子量(150-160kDa)相比，重链抗体要小得多，IgG2c重链抗体一条重链大约40kDa，其决定抗原识别特异性的重链可变区只有约15kDa。重链抗体的特点是分子量小，能够结合一些隐蔽的抗原表位，特别适用于比较难得到抗体的靶点。
公开于该背景技术部分的信息仅仅旨在增加对本申请的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本申请的目的在于提供一种非人哺乳动物或其子代的制备方法及其应用。
解决方案
为实现本申请目的，本申请提供了以下技术方案：
本申请的第一方面，提供了一种非人哺乳动物或其子代的制备方法，其包括以下步骤：
使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤；
以及，使得非人哺乳动物体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤。
上述制备方法在一种可能的实现方式中，使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤为：
仅使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤；
或，使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区和IgD重链恒定区的步骤。
上述制备方法在一种可能的实现方式中，使得非人哺乳动物体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤为：
使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个和第二个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第三个或第四个编码IgG重链恒定区的基因中的一个或两个在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个和第二个和第三个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第四个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个和第二个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第三个或第四个编码IgG重链恒定区的基因中的一个或两个在表达时不表达或不正确表达CH1结构域的步骤；
或，使得非人哺乳动物体内第一个和第二个和第三个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第四个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤。本申请还提供了一种非人哺乳动物，其体内不表达或不正确表达IgM重链恒定区CH1结构域，其体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因不表达或不正确表达CH1结构域。
上述非人哺乳动物在一种可能的实现方式中，其体内不表达或不正确表达IgM重链恒定区CH1结构域为：
仅不表达或不正确表达IgM重链恒定区CH1结构域；
或，不表达或不正确表达IgM重链恒定区和IgD重链恒定区。
上述非人哺乳动物在一种可能的实现方式中，其体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因不表达或不正确表达CH1结构域为：
第一个编码IgG重链恒定区的基因不表达或不正确表达CH1结构域；
或，第一个编码IgG重链恒定区的基因不表达或不正确表达其所编码的IgG重链恒定区、并 且第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个不表达或不正确表达CH1结构域；
或，第一个和第二个编码IgG重链恒定区的基因不表达或不正确表达其所编码的IgG重链恒定区、并且第三个或第四个编码IgG重链恒定区的基因中的一个或两个不表达或不正确表达CH1结构域；
或，第一个和第二个和第三个编码IgG重链恒定区的基因不表达或不正确表达其所编码的IgG重链恒定区、并且第四个编码IgG重链恒定区的基因不表达或不正确表达CH1结构域；
或，第一个编码IgG重链恒定区的基因正确表达其所编码的IgG重链恒定区、并且第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个不表达或不正确表达CH1结构域；
或，第一个和第二个编码IgG重链恒定区的基因正确表达其所编码的IgG重链恒定区、并且第三个或第四个编码IgG重链恒定区的基因中的一个或两个不表达或不正确表达CH1结构域；
或，第一个和第二个和第三个编码IgG重链恒定区的基因正确表达其所编码的IgG重链恒定区、并且第四个编码IgG重链恒定区的基因不表达或不正确表达CH1结构域。
本申请的第二方面，提供了一种非人哺乳动物或其子代的制备方法，包括以下步骤：
敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列的步骤；
以及，敲除以下目标基因的步骤，所述目标基因包括：一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列。
本申请还提供了一种非人哺乳动物，其基因组上包括编码IgM重链恒定区CH1结构域的核苷酸序列以及目标基因被敲除，所述目标基因包括：
一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列。
上述制备方法或非人哺乳动物在一种可能的实现方式中，敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列为：
仅敲除非人哺乳动物基因组上的、编码IgM重链恒定区CH1结构域的核苷酸序列；
或，敲除非人哺乳动物基因组上的、编码IgM重链恒定区和IgD重链恒定区。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述目标基因为：
第一个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
或，从第一个编码IgG重链恒定区的基因开始至第二个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，从第一个编码IgG重链恒定区的基因开始至第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，从第一个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上 的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，从第一个编码IgG重链恒定区的基因开始至最后一个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，第二个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
或，从第二个编码IgG重链恒定区的基因开始至第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，从第二个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
或，从第三个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
或，第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列。
上述制备方法在一种可能的实现方式中，以下步骤在同一操作步骤中完成或在不同的操作步骤中完成：
敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列的步骤；
敲除目标基因的步骤。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述非人哺乳动物为啮齿类动物；可选地，所述啮齿类动物为大鼠或小鼠；进一步可选地，所述啮齿类动物为小鼠；更进一步，所述小鼠为C57BL/6小鼠或BALB/c小鼠。
上述制备方法或非人哺乳动物在一种可能的实现方式中，第一个编码IgG重链恒定区的基因为Ighg3。
上述制备方法或非人哺乳动物在一种可能的实现方式中，当非人哺乳动物为C57BL/6小鼠时，第一个编码IgG重链恒定区的基因为Ighg3、第二个编码IgG重链恒定区的基因Ighg1、第三个编码IgG重链恒定区的基因Ighg2b、第四个编码IgG重链恒定区的基因Ighg2c；
当非人哺乳动物为BALB/c小鼠时，第一个编码IgG重链恒定区的基因为Ighg3、第二个编码IgG重链恒定区的基因Ighg1、第三个编码IgG重链恒定区的基因Ighg2b、第四个编码IgG重链恒定区的基因Ighg2a。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述非人哺乳动物基因组上包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述非人哺乳动物中能正常表达κ轻链和/或λ轻链。
上述制备方法或非人哺乳动物在一种可能的实现方式中，基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效 应因子核酸酶方法。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述非人哺乳动物或其子代用于生产重链抗体。
本申请的第三方面，提供了一种C57BL/6小鼠或其子代的制备方法，其包括以下步骤：
敲除C57BL/6小鼠的基因组中编码抗体IgM重链恒定区和IgD重链恒定区的基因的步骤；
以及，敲除C57BL/6小鼠的基因组中从编码IgG3重链恒定区的基因开始至编码IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤。
本申请还提供了一种C57BL/6小鼠，其基因组中编码抗体IgM、IgD、IgG1、IgG2b、IgG3的重链恒定区的核苷酸序列，以及基因组中编码抗体IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列被敲除。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，所述C57BL/6小鼠基因组中包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述C57BL/6小鼠能正常表达κ轻链和/或λ轻链。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效应因子核酸酶方法。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，敲除了小鼠编码IgM重链恒定区的基因的第1号外显子至编码IgG2c重链恒定区的基因的第1号外显子的全部核苷酸序列。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，基因敲除的步骤中，使用了靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游的sgRNA和靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，靶向小鼠编码IgM重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；和//或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.11、SEQ ID NO.12。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，所述C57BL/6小鼠或其子代用于生产重链IgG2c抗体。
本申请的第四方面，提供了一种C57BL/6小鼠或其子代的制备方法，其包括以下步骤：
敲除C57BL/6小鼠的基因组中编码IgM重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤；
敲除C57BL/6小鼠的基因组中从编码IgG3重链恒定区的基因开始至编码IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤。
本申请还提供了一种C57BL/6小鼠，其基因组中编码IgM重链恒定区的基因上的、编码CH1结构域的核苷酸序列，以及其基因组中从编码IgG3重链恒定区的基因开始至编码IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列被敲除。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，所述C57BL/6小鼠基因组中包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述C57BL/6小鼠能正常表达κ轻链和/或λ轻链。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效应因子核酸酶方法。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，基因敲除的步骤中，敲除了小鼠编码IgM重链恒定区的基因的第1号外显子，还敲除了小鼠从编码IgG3重链恒定区的基因第1号外显子至编码IgG2c重链恒定区的基因第1号外显子的全部核苷酸序列。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，基因敲除的步骤中，使用了靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游和下游的sgRNA，还使用了靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA和靶向小鼠编码IgG2c第1号外显子下游的sgRNA。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；
和/或，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.5和SEQ ID NO.6；
和/或，sgRNA靶向的小鼠编码IgG3重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.7和SEQ ID NO.8；
和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，靶向小鼠编码IgM重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；
和/或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.13和SEQ ID NO.14；
和/或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.15 和SEQ ID NO.16；
和/或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.11和SEQ ID NO.12。
上述制备方法或C57BL/6小鼠在一种可能的实现方式中，所述C57BL/6小鼠或其子代用于生产重链IgG2c抗体。
本申请的第五方面，提供了一种非人哺乳动物或其子代的制备方法，其包括敲除非人哺乳动物基因组上的、编码IgM重链恒定区CH1结构域的核苷酸序列的步骤。
本申请还提供了一种非人哺乳动物，其基因组上的、编码IgM重链恒定区CH1结构域的核苷酸序列被敲除。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述非人哺乳动物为啮齿类动物；可选地，所述啮齿类动物为大鼠或小鼠；进一步可选地，所述啮齿类动物为小鼠；更进一步，所述小鼠为C57BL/6小鼠或BALB/c小鼠。
上述制备方法或非人哺乳动物在一种可能的实现方式中，所述非人哺乳动物或其子代用于构建上述非人哺乳动物或其子代。
本申请的第六方面，提供了一种来源于上述制备方法构建的非人哺乳动物或其子代、来源于上述制备方法构建的C57BL/6小鼠或其子代、上述非人哺乳动物、上述C57BL/6小鼠在筛选目标重链抗体中的应用。
上述应用在一种可能的实现方式中，筛选目标重链抗体时采用噬菌体展示的方法。
上述应用在一种可能的实现方式中，所述筛选目标重链抗体为筛选C反应蛋白、冠状病毒S蛋白或冠状病毒N蛋白抗原特异性IgG2c重链抗体。
本申请的第七方面，提供了一种筛选目标重链抗体的方法，其包括使用来源于上述第一方面所述的制备方法构建的非人哺乳动物或其子代、来源于上述第二方面所述的制备方法构建的非人哺乳动物或其子代、来源于上述第三方面所述的制备方法构建的C57BL/6小鼠或其子代、或来源于上述第四方面所述的制备方法构建的C57BL/6小鼠或其子代作为免疫动物进行筛选。
上述方法在一种可能的实现方式中，筛选目标重链抗体时采用噬菌体展示的方法。
上述方法在一种可能的实现方式中，所述筛选目标重链抗体为筛选C反应蛋白、冠状病毒S蛋白或冠状病毒N蛋白抗原特异性IgG2c重链抗体。
本申请的第八方面，提供了一种非人哺乳动物细胞或细胞系或原代细胞培养物，所述非人哺乳动物细胞或细胞系或原代细胞培养物来源于上述制备方法构建的非人哺乳动物或其子代、或来源于上述的制备方法构建的C57BL/6小鼠或其子代。
本申请的第九方面，提供了一种离体组织或离体器官或其培养物，所述离体组织或离体器官或其培养物来源于上述制备方法构建的非人哺乳动物或其子代、或来源于上述 的制备方法构建的C57BL/6小鼠或其子代。
本申请的第十方面，提供了一种sgRNA组合物，所述sgRNA组合物包括：靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游的sgRNA和靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA；
或，靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游和下游的sgRNA；
或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA和靶向小鼠编码IgG2c第1号外显子下游的sgRNA。
上述sgRNA组合物在一种可能的实现方式中，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；
和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4；
和/或，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.5和SEQ ID NO.6；
和/或，sgRNA靶向的小鼠编码IgG3重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.7和SEQ ID NO.8。
上述sgRNA组合物在一种可能的实现方式中，靶向小鼠编码IgM重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；
和/或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.13和SEQ ID NO.14；
和/或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.15和SEQ ID NO.16；
和/或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.11和SEQ ID NO.12。
本申请的第十一方面，提供了一种敲除载体，其包括：编码sgRNA的一段或多段DNA序列，所述sgRNA的靶向序列选自以下的一种：
靶向小鼠编码IgM重链恒定区的基因第1号外显子上游；
或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游；
或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游；
或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游。
上述敲除载体在一种可能的实现方式中，所述敲除载体的骨架是sgRNA表达载体。
本申请的第十二方面，提供了一种细胞，包含上述敲除载体。
不正确表达：与不正确表达相对的是正确表达，本申请中的正确表达和不正确表达实际上都具有本领域通常所理解的含义。如不正确表达CH1结构域是指：基因组水平的突变或缺失引 起的重链恒定区CH1结构域的不正确表达，从而导致CH1结构域失去与轻链结合的能力；如：编码重链恒定区CH1结构域的外显子的上下游各10个核苷酸范围内的突变或缺失，从而导致CH1结构域失去与轻链结合的能力。又如编码IgG重链恒定区的基因在表达时不正确表达其所编码的IgG重链恒定区是指该基因表达所获得的免疫球蛋白抗体不具有抗体功效；与之相对的，编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区是指该基因表达所获得的免疫球蛋白抗体具有抗体功效。
第一个编码IgG重链恒定区的基因、第二个编码IgG重链恒定区的基因、第三个编码IgG重链恒定区的基因、第四个编码IgG重链恒定区的基因：是指在编码IgG重链恒定区的基因座上，编码IgG重链恒定区的各基因按照从上游到下游的顺序进行排列的顺序。
有益效果
本申请通过使得非人哺乳动物不表达或不正确表达IgM重链恒定区CH1结构域的步骤以及使得一个或多个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤，制备非人哺乳动物或其子代，所得非人哺乳动物或其子代可以用于生产重链抗体。采用本申请制备方法获得的非人哺乳动物不引入任何编码抗体重链可变区和恒定区的外源基因，可以直接利用其自身基因组中编码抗体重链可变区的所有VDJ基因，从而通过重链可变区重排产生多样性更好的重链抗体。
免疫学中通常认为IgM对于B细胞的发育极为重要，而B细胞的发育又对抗体的产生极为重要。本申请通过实验验证了即使不表达编码IgM重链恒定区CH1结构域，甚至删除编码IgM重链恒定区的基因和编码IgD重链恒定区的基因，也没有显著影响非人哺乳动物的免疫成熟，所有小鼠正常存活，并仍能产生高效价的免疫反应，并且发明人还从中筛选得到了特异性强、亲和力高的重链抗体。
通过选择基因改造的编码IgG重链恒定区的基因，可以获得特定亚类的重链IgG抗体。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1是本申请实施例1中涉及的小鼠抗体重链可变区和恒定区的基因位点示意图。
图2是本申请实施例1中制备Ighm-d-g小鼠的sgRNA打靶策略示意图。第二行中的方框表示外显子，白色方框表示被敲除基因片段，数字是外显子序号。五角星表示sgRNA的靶点位置。箭头表示基因型鉴定的引物针对的位置。
图3是本申请实施例1中对Ighm-d-g小鼠进行基因型鉴定的PCR胶图示意图，wt：野生型；+/-：杂合子；-/-：敲除纯合子。
图4是本申请实施例2中用CRP(人C反应蛋白)作为抗原蛋白免疫Ighm-d-g纯合型小鼠后的血清效价检测结果示意图，一免后和未免疫时血清效价均很低，两条线重合。
图5A是本申请实施例5中Ighm-d-g纯合型小鼠IgG2c抗体重链基因VH段的菌落PCR图；图5B是本申请实施例5中Ighm-d-g纯合型小鼠表达的IgG2c抗体的重链氨基酸序列比对图。
图6是本申请实施例6中重链抗体噬菌体文库筛选阳性克隆测序比对。
图7是本申请实施例7中纯化D4-12对抗原CRP的特异性识别ELISA结果。
图8是本申请实施例8中纯化5S-12对抗原CRP特异性识别的ELISA结果，其中OVA-D5S-12和BSA-D5S-12两条线OD450值很低，线条重合。
图9是本申请实施例8中D5S-12重链抗体亲和力测定结果。
图10是本申请实施例9中制备IghM小鼠的sgRNA打靶策略示意图。黑色方框表示外显子，其中的数字是外显子序号。五星符号表示sgRNA的靶点位置。箭头表示基因型鉴定的引物。
图11是本申请实施例9中PCR鉴定IghM小鼠基因型示例。Ighm野生型基因产生约600bp条带，Ighm敲除基因产生约300bp条带。数字代表小鼠样品编号，+：野生型对照，-：空白对照。
图12是本申请实施例10中IghM纯合型小鼠表达的IgM抗体重链氨基酸序列图。
图13是本申请实施例11中制备IghM-3G3小鼠的sgRNA打靶策略示意图。黑色方框表示外显子，其中的数字是外显子序号。五星符号表示sgRNA的靶点位置。箭头表示基因型鉴定的引物。
图14是本申请实施例11中对IghM-3G3小鼠进行基因型鉴定的PCR胶图示意图，wt：野生型；+/-：杂合子；-/-：敲除纯合子。
图15是本申请实施例12中用CRP(人C反应蛋白)作为抗原蛋白免疫IghM-3G3纯合型小鼠后的血清效价检测结果示意图，一免后和二免后血清效价均很低，两条线重合。
图16A是本申请实施例13中IghM-3G3纯合型小鼠IgG2c抗体重链基因VH段的菌落PCR图；图16B是本申请实施例13中IghM-3G3纯合型小鼠表达的IgG2c抗体的重链氨基酸序列比对图。
图17是本申请实施例15中提到的，用冠状病毒S蛋白抗原免疫IghM-3G3纯合型小鼠的情况下，分别在未免疫、二免一周后、三免一周后和四免一周后采血，用PBS对血清进行倍比稀释后，通过ELISA检测血清效价的结果图。
图18A是本申请实施例16中提到的，以冠状病毒S蛋白抗原免疫的IghM-3G3纯合型小鼠的IgG2c抗体重链基因的菌落PCR鉴定图；图18B是本申请实施例17中提到的，以冠状病毒S蛋白抗原免疫的IghM-3G3纯合型小鼠的IgG2c抗体重链基因经噬菌体展示 库构建和抗体淘选后获得的阳性克隆的CDR区氨基酸序列比对图。
图19是本申请实施例17中提到的，通过ELISA法检测抗体S-9、S-19、S-27和S-47能否特异性识别并结合抗原冠状病毒S蛋白的结果图。
图20A是本申请实施例18中，用冠状病毒S蛋白抗原免疫IghM-DG1纯合型小鼠的情况下，分别在未免疫、二免一周后、三免一周后和四免一周后采血，用PBS对血清进行倍比稀释后，通过ELISA检测血清效价的结果图；图20B是针对上述用冠状病毒S蛋白抗原免疫IghM-DG1纯合型小鼠，通过噬菌体展示库构建和抗体淘选后获得的阳性克隆的CDR区氨基酸序列比对图。
图21是本申请实施例18中提到的，通过ELISA法检测抗体S-1、S-7、S-12、S-17、S19、S-25、S-51和S-65能否特异性识别并结合抗原冠状病毒S蛋白的结果图。
图22A是本申请实施例19中提到的，用冠状病毒N蛋白抗原免疫IghM-DG1纯合型小鼠的情况下，分别在未免疫、二免一周后、三免一周后和四免一周后采血，用PBS对血清进行倍比稀释后，通过ELISA检测血清效价的结果图；图22B是是针对上述用冠状病毒N蛋白抗原免疫IghM-DG1纯合型小鼠，通过噬菌体展示库构建和抗体淘选后获得的阳性克隆的CDR区氨基酸序列比对图。
图23是本申请实施例19中提到的，通过ELISA法检测抗体N-1、N-2、N-3、N-5和N-23能否特异性识别并结合抗原冠状病毒S蛋白的结果图。
图24是本申请实施例20中提到的，MDG1小鼠和野生型小鼠骨髓中各免疫球蛋白基因的相对表达量比较。A-D图分别为两基因型小鼠骨髓中μ基因、γ2c基因、γ2c基因相较于μ基因、α基因的相对表达量。MDG1为纯合子敲除小鼠，WT为野生型小鼠，BM为骨髓，以持家基因Gapdh基因作为内参，n＝4，利用2 -ΔΔCt的计算方法进行数据分析，采用双尾T检验进行统计学分析，***代表p＜0.01。
图25本申请实施例20中提到的，MDG1小鼠和野生型小鼠脾脏中各免疫球蛋白基因的相对表达量比较。A-D图分别为两基因型小鼠脾脏中μ基因、γ2c基因、γ2c基因相较于μ基因、α基因的相对表达量。MDG1为纯合子敲除小鼠，WT为野生型小鼠，以持家基因Gapdh基因作为内参，n＝4，利用2 -ΔΔCt的计算方法进行数据分析，采用双尾T检验进行统计学分析，**代表p＜0.01。
图26本申请实施例20中提到的，MDG1小鼠和野生型小鼠小肠中各免疫球蛋白基因的相对表达量比较。A-D图分别为两基因型小鼠小肠中μ基因、γ2c基因、γ2c基因相较于μ基因、α基因的相对表达量。MDG1为纯合子敲除小鼠，WT为野生型小鼠，SI为小肠，以持家基因Gapdh基因作为内参，n＝4，利用2 -ΔΔCt的计算方法进行数据分析，采用双尾T检验进行统计学分析。
图27本申请实施例21中提到的，Western Blot检测MDG1小鼠血清中IgG2c的表达 形式。WT代表野生型小鼠，MDG1代表纯合子敲除小鼠。A图为还原条件下IgG2c的表达形式检测，B图为非还原条件下IgG2c的表达形式检测，野生型小鼠的血清进行20倍稀释，MDG1小鼠的血清进行100倍稀释，Goat Anti-Mouse IgG2c heavy chain(HRP)进行10000倍稀释。
图28是本申请实施例22中提到的，MDG1小鼠和野生型小鼠静息状态下血清中各免疫球蛋白表达量比较。A-F图分别为两基因型小鼠血清中IgM、IgG2c、野生型小鼠的IgM与MDG1小鼠的IgG2c、总IgG、IgA、IgE的表达量。n＝20，采用双尾T检验进行统计学分析，*代表p＜0.05，****代表p＜0.01。
图29是本申请实施例23中提到的，MDG1小鼠骨髓中B细胞的发育情况。A-C图分别为骨髓B细胞、未成熟B细胞/成熟B细胞、祖B细胞/前B细胞的分群状态，D-F图分别为骨髓B细胞、祖B细胞和前B细胞的比例和数目。WT为野生型小鼠，MDG1为纯合子敲除小鼠，pro-B cell为祖B细胞，pre-B cell为前B细胞。n＝6，采用双尾T检验进行统计学分析，*代表p＜0.05，***代表p＜0.01。
图30是本申请实施例23中提到的，MDG1小鼠脾脏中B细胞的发育情况。A-D图分别为脾脏B细胞、IgG2c+/IgM+B细胞、滤泡B细胞/边缘B细胞/过渡期B细胞、浆细胞的分群状态，E-H图分别为脾脏B细胞、IgG2c+/IgM+B细胞、浆细胞、滤泡B细胞/边缘B细胞/过渡期B细胞的比例和数目。WT为野生型小鼠，MDG1为纯合子敲除小鼠，FO B为滤泡B细胞，T B为过渡期B细胞，MZ B为边缘B细胞，plasma为浆细胞，n＝4，采用双尾T检验进行统计学分析，*代表p＜0.05，**代表p＜0.01。
图31是本申请实施例23中提到的，MDG1小鼠腹膜腔中B细胞的发育情况。A图和B图分别为腹膜腔B细胞、B1a/B1b/B2细胞的分群状态，C图分别为腹膜腔B细胞、B1a细胞、B1b细胞和B2细胞的比例。WT为野生型小鼠，MDG1为纯合子敲除小鼠。n＝7，采用双尾T检验进行统计学分析，*代表p＜0.05，**代表p＜0.01。
图32是本申请实施例25中提到的，氯霉素免疫后野生型小鼠和MDG1小鼠血清中抗原特异性抗体相对表达量的变化趋势。A-E分别为氯霉素特异性的IgM、IgG2c、IgG、IgA、IgE的相对表达量，横坐标表示免疫次数，LMS表示氯霉素，MDG1为纯合子敲除小鼠，WT为野生型小鼠，n＝7。
图33是本申请实施例25中提到的，阿特拉津免疫后野生型小鼠和MDG1小鼠血清中抗原特异性抗体相对表达量的变化趋势。A-E分别为阿特拉津特异性的IgM、IgG2c、IgG、IgA、IgE的相对表达量，横坐标表示免疫次数，ATLJ表示阿特拉津，MDG1为纯合子敲除小鼠，WT为野生型小鼠，n＝3。
图34是本申请实施例26中提到的，抗原免疫后野生型小鼠和MDG1小鼠脾脏中生发中心B细胞和浆细胞的发育。A-E图分别为免疫后脾脏B细胞、生发中心B细胞、IgG2c+ 生发中心B细胞、浆细胞、IgG2c+浆细胞的发育情况，F-J图分别为免疫后脾脏B细胞、生发中心B细胞、IgG2c+生发中心B细胞、浆细胞、IgG2c+浆细胞的比例及数目。WT为野生型小鼠，MDG1为纯合子敲除小鼠，n＝6，采用双尾T检验进行统计学分析，*代表p＜0.05，**代表p＜0.01。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
另外，为了更好的说明本申请，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本申请同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本申请的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
以下实施例所用实验材料及其来源如下：
px330质粒载体，购自Addgene，质粒编号#58778；
pEASY-T5 Zero Cloning载体，购自全式金生物技术有限公司，货号：CT501-01；
TOP10感受态细胞，购自天根生化科技有限公司，货号：CB104；
PET28载体，购自武汉淼灵生物科技有限公司，货号：P31003；
HiPure Total RNA Plus Mini Kit来源美基美，货号R4121；
HiPure Gel Pure Micro Kit来源美基美，货号D2110；
HiPure Tissue DNA Mini Kit来源美基美，货号D3121；
2×M5 Hiper plus Taq HiFi PCR mix来源聚合美，货号MF002-plus；
反转录试剂5X All-In-One RT MasterMix来源abmgood，货号490；
DNA marker来源东盛，货号M1061/M1062)；
蛋白marker来源Thermo，货号26617；
Hieff qPCR SYBR Green Master Mix来源Yeasen，货号11201ES08；
APC/Cy7 anti-mouse CD38 Antibody来源Biolegend，货号102727；
APC/Cy7 anti-mouse IgM Antibody来源Biolegend，货号406515；
CD45R(B220)Monoclonal Antibody(RA3-6B2),APC来源eBioscience，货号 85-17-0452-82；
anti-CD23 antibody(Allophycocyanin)来源Abcam，货号ab25457；
CD45R(B220)Monoclonal Antibody(RA3-6B2),eFluor 450来源eBioscience，货号85-48-0452-82；
BV421 Rat Anti-Mouse CD138来源BD Horizon，货号562610；
CD19 Monoclonal Antibody(eBio1D3(1D3)),eFluor 506来源eBioscience，货号85-69-0193-80；
CD43 Monoclonal Antibody(eBioR2/60),PE来源eBioscience，货号85-12-0431-81；
CD23 Monoclonal Antibody(B3B4),PE来源eBioscience，货号85-12-0232-82；
Anti-CD5 antibody[53-7.3](Phycoerythrin)来源Abcam，货号ab114078；
CD21/CD35 Monoclonal Antibody(eBio8D9(8D9)),PE-Cyanine7来源eBioscience，货号85-25-0211-80；
PE-Cy TM7 Hamster Anti-Mouse CD95来源BD Pharmingen，货号553653；
CD19 Monoclonal Antibody PE-Cyanine7来源eBioscience，货号25-0193-82；
IgM Monoclonal Antibody(eB121-15F9),PE-Cyanine7来源eBioscience，货号85-25-5890-82；
CD43 Monoclonal Antibody(eBioR2/60),FITC来源eBioscience，货号85-11-0431-85；
Mouse IgG2c Antibody-FITC Conjugated来源Aviva system biology，货号OASA06628；
7-AAD Viability Staining Solution来源eBioscience，货号85-00-6993-50；
Goat Anti-Mouse IgG2c heavy chain(HRP)来源Abcam，货号ab97255；
ECL Prime Western Blot Dtection reagent来源GE，货号RPN2236
Mouse IgM ELISA Quantitation Set来源bethyl，货号E90-101；
Mouse IgG2c ELISA Quantitation Set来源bethyl，货号E90-136；
Mouse IgG ELISA Quantitation Set来源bethyl，货号E90-131；
Mouse IgA ELISA Quantitation Set来源bethyl，货号E90-103；
Mouse IgE ELISA Quantitation Set来源bethyl，货号E90-115；
TMB Substrate Set来源Biolegend，货号421101；
Freund's Adjuvant complete来源sigma，货号F5881；
Freund's Adjuvant incomplete来源sigma，货号F5506。
第一部分、Ighm-d-g纯合型小鼠及其免疫结果
实施例1、制备Ighm-d-g纯合型小鼠(本文中也称为MDG1小鼠)
Ighm-d-g小鼠是指被敲除了Ighm基因、Ighd基因、Ighg3基因、Ighg1基因、Ighg2b基因以及位于Ighg2c基因上的第一个外显子的C57BL/6小鼠，该外显子负责编码IgG2c重链的CH1结构域。通过以下步骤，制备Ighm-d-g纯合型小鼠。
(1)核酸分子的获得：
C57BL/6小鼠抗体重链可变区和恒定区的基因位点示意图如图1所示，设计如图2所示的打靶策略以敲除小鼠Ighm、Ighd、Ighg3、Ighg1、Ighg2b基因和Ighg2c中的编码CH1结构域的核苷酸序列。发明人在小鼠Ighm基因的第一个外显子上游选择了sgRNA的靶向序列(SEQ ID NO:1、SEQ ID NO:2)，在小鼠Ighg2c的第1个外显子下游选择了sgRNA靶向序列(SEQ ID NO:3、SEQ ID NO:4)，并根据靶向序列设计了sgRNA序列，具体如表1所示。
表1
Figure PCTCN2021116282-appb-000001
(2)将核酸分子构建入骨架质粒并体外转录得到sgRNA：
将合成的编码sgRNA序列的正向和反向DNA oligo通过退火形成互补的双链，利用T4连接酶连接在sgRNA表达载体(px330)上，连接后经专业测序公司测序验证，结果表明获得了目的质粒，进一步通过体外转录获得sgRNA。
(3)向宿主动物受精卵导入所述sgRNA和Cas9蛋白：
小鼠促排卵、体外受精并培育受精卵，然后将sgRNA和Cas9蛋白混合、电转小鼠受精卵或者采用显微注射的方法将Cas9蛋白(或Cas9mRNA，可商购)与sgRNA一起注 射进入小鼠受精卵。
(4)将含有上述sgRNA和Cas9蛋白的细胞植入宿主动物体内：
将上述受精卵细胞植入代孕母鼠体内，可生产F0代嵌合体鼠。通过提取鼠尾基因组DNA和PCR检测，检测F0代小鼠中发生了敲除的个体。对基因敲除小鼠进行测序，确认删除了靶序列。挑选基因正确敲除的F0代嵌合鼠用于后续繁殖和鉴定。
PCR引物Ighm-d-g-1F和Ighm-d-g-1R能检测敲除基因(如图2中箭头所示)，引物Ighd-2F和Ighd-2R能检测野生型基因(如图2中箭头所示)，各引物的序列如表2，检测Ighm-d-g小鼠中发生敲除的个体时的PCR结果示意图如图3所示。Ighm-d-g纯合子敲除基因用引物Ighm-d-g-1F和Ighm-d-g-1R扩增出目的条带大小约700bp(如左图所示)，用引物Ighd-2F和Ighd-2R不能扩增出条带(如右图所示)；Ighm-d-g野生型基因用引物Ighm-d-g-1F和Ighm-d-g-1R不能扩增出条带(如左图所示)，用引物Ighd-2F和Ighd-2R扩增出目的条带大小约600bp(如右图所示)。
表2
Figure PCTCN2021116282-appb-000002
(5)繁殖杂合、纯合的基因敲除小鼠：
将靶基因敲除的F0代小鼠与野生型鼠交配获得F1代鼠，通过提取鼠尾基因组和PCR检测，挑选可以稳定遗传的基因敲除阳性F1代杂合子小鼠。再将F1代杂合小鼠互相交配即可获得基因敲除阳性F2代纯合子鼠，即Ighm-d-g纯合型小鼠。对获得的F1代杂合或F2代纯合鼠进行基因型的方法与步骤(4)相同。
实施例2、抗原免疫反应和效价检测
用人C反应蛋白(CRP)免疫Ighm-d-g纯合型小鼠。
免疫方法如下：选择6-8周龄雄性鼠，抗原人C反应蛋白(CRP，A-5172,百桥瑞景)加等体积弗氏完全佐剂(F5881，Sigma)，乳化到滴水不化的状态，即用于小鼠初次免疫皮下多点注射，初次免疫注射剂量为100μg/只小鼠，初次免疫后，每2周进行后续皮下免疫，CRP抗原加等体积弗氏不完全佐剂(F5506，Sigma)乳化后，对小鼠皮下多点注射，每次注射剂量为100μg/只小鼠。
血清效价检测方法如下：将CRP抗原稀释成2μg/mL，取100μl加入聚苯乙烯酶联检测板包板，用HRP-山羊抗小鼠IgG-Fc(Jackson 115-035-071)检测血清中与CRP抗原特 异性结合的特异性IgG2c抗体重链。
如图4所示，分别在未免疫、一免一周后、二免一周后、三免一周后采血，血清用PBS稀释，从1：500稀释度开始做倍比稀释，ELISA检测血清效价显示，未免疫和一免小鼠体内未出现结合抗原CRP的特异性抗体，从二免后，小鼠体内出现特异性与抗原结合的IgG2c抗体，三免后效价没有进一步提升，其血清效价在1：8000左右，可用于下一步的抗体基因调取实验。
实施例3、未免疫Ighm-d-g纯合型小鼠及CRP抗原免疫后的Ighm-d-g纯合型小鼠的脾脏、胸腺、淋巴节及器官检查
继以上实施例2，小鼠经二氧化碳窒息法安乐处死。解剖观察胸腺、脾脏、肠系膜淋巴结、颌下淋巴结等及心、肝、肺、肾脏等多个器官的大小及形态，解剖显示，未免疫Ighm-d-g纯合型小鼠及抗原免疫后的Ighm-d-g纯合型小鼠的胸腺、肠系膜淋巴结、颌下淋巴结、主要脏器均无明显异常。
实施例4、抗原免疫产生特异性IgG2c重链抗体
继以上实施例2，进行CRP抗原免疫小鼠血清蛋白印迹：取免疫后小鼠血清2μL加入100μL PBS，与10μL CRP抗原-Sepharose填料室温反应60分钟后，6000rpm离心30秒，弃上清。用PBS清洗填料3次，用10μL PBS重悬煮沸，经12％SDS-PAGE电泳，转到PVDF膜后，分别用HRP-山羊抗小鼠IgG-Fc(Jackson 115-035-071，用于检测重链)抗体和HRP-山羊抗小鼠Ig轻链(Jackson，115-035-174)抗体反应并进一步显色。
按照本申请的设计，由于小鼠IgM、IgD、IgG1、IgG2b、IgG3的基因被敲除，用抗原蛋白免疫Ighm-d-g纯合型小鼠只会产生IgG2c亚类抗体，并且由于IgG2c重链的CH1基因被敲除，用抗原蛋白免疫Ighm-d-g纯合型小鼠可以产生IgG2c重链抗体(不含CH1结构域)，IgG2c单独一条重链分子量大约在40KD左右。抗原蛋白免疫后的Ighm-d-g纯合型小鼠产生的抗体，经过分离、电泳、显色等步骤后，在80KD大小条带左右出现特异性与抗原CRP结合的条带，这与理论IgG2c重链的二聚体分子量相符合，并且包括未与HRP-山羊抗小鼠Ig轻链(Jackson，115-035-174)抗体结合的二聚体。
实施例5、IgG2c重链抗体基因调取
继以上实施例2，CRP抗原免疫Ighm-d-g纯和型小鼠并经血清效价检测后，将小鼠进行安乐死并取脾细胞，经Trizol裂解，提取总RNA并反转录得到cDNA，使用如下IgG2c亚型抗体特异性引物通过PCR扩增重链可变区及与之相连接的重链恒定区。
MHV1：ATGAAATGCAGCTGGGGCATSTTCTTC(SEQ ID NO:21)；
MHV2：ATGGGATGGAGCTRTATCATSYTCTT(SEQ ID NO:22)；
MHV3：ATGAAGWTGTGGTTAAACTGGGTTTTT(SEQ ID NO:23)；
MHV4：ATGRACTTTGGGYTCAGCTTGRTTT(SEQ ID NO:24)；
MHV5：ATGGGACTCCAGGCTTCAATTTAGTTTTCCTT(SEQ ID NO:25)；
MHV6:ATGGCTTGTCYTTRGSGCTRCTCTTCTGC(SEQ ID NO:26)；
MHV7:ATGGRATGGAGCKGGRGTCTTTMTCTT(SEQ ID NO:27)；
MHV8:ATGAGAGTGCTGATTCTTTTGTG(SEQ ID NO:28)；
MHV9:ATGGMTTGGGTGTGGAMCTTGCTTATTCCTG(SEQ ID NO:29)；
MHV10:ATGGGCAGACTTACCATTCTCATTCCTG(SEQ ID NO:30)；
MHV11:ATGGATTTTGGGCTGATTTTTTTTATTG(SEQ ID NO:31)；
MHV12:ATGATGGTGTTAAGTCCTTCTGTACC(SEQ ID NO:32)；
联合B6_IgG2c_CH2R1:5’-TGGTCCACCCAAGAGGTCTG-3’(SEQ ID NO:33)；
PCR反应体系如表3所示：
表3
Figure PCTCN2021116282-appb-000003
PCR反应程序：98℃，2min——30个循环(每个循环98℃，10s——50℃，20s——72℃，40s)——72℃，5min——16℃。
将所得PCR扩增产物连接pEASY-T5 Zero Cloning载体，转化TOP10感受态细胞并涂LB平板，随机挑取11个克隆，菌落PCR鉴定为阳性的克隆送测序(如图5A所示，650bp大小条带为阳性条带)。
阳性克隆的测序结果显示(如图5B所示)，Ighm-d-g纯和型小鼠中产生的IgG2c抗体重链可变区FR4区直接与IgG2c Hinge区(抗体铰链区)相连，由于抗体重链的结构是VH-CH1-Hinge-CH2-CH3，其中VH区域又可以详细的分为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，通过图5B可知，Ighm-d-g纯和型小鼠中产生的IgG2c抗体重链可变区FR4区直接与Hinge区(抗体铰链区)相连，说明IgG2c CH1外显子敲除成功。
实施例6、CRP抗原特异性IgG2c重链抗体可变区噬菌体展示库构建和淘选
I、噬菌体展示库构建
继以上实施例2，从CRP免疫的Ighm-d-g纯和型小鼠的脾脏中提取总RNA，使用oligo(Dt)制备cDNA(TAKAR 6110A cDNA合成试剂盒)，采用巢式PCR进行了两轮PCR扩增出重链抗体基因。
第一轮PCR过程与实施例5中提到的PCR过程完全相同；
第二轮PCR所用引物、反应体系、反应程序如下所示：
MHVF1-SfiI：ATGCCATGACTGTggcccaggcggcc GAG GTG AAG CTT CTC GAG TCT GG(SEQ ID NO:34)；
MHVF2-SfiI：CATGCCATGACTGTggcccaggcggcc SAG GTS CAG CTG MAG GAG TCW GG(SEQ ID NO:35)；
MHVF3-SfiI：CATGCCATGACTGTggcccaggcggcc GAG GTC CAG CTG CAA CAA TCT GG(SEQ ID NO:36)；
MHVF4-SfiI：CATGCCATGACTGTggcccaggcggcc SAG GTY CAR CTK CAG CAG YCT GG(SEQ ID NO:37)；
MHVF5-SfiI：CATGCCATGACTGT ggcccaggcggcc GAR GTG AAG CTT GWG GAG TCT GG(SEQ ID NO:38)。
B6-IgG2c-Hin-sfi1：ACTCGCGGCCGGCCTGGCCTGTTATGGGCACTCTGGG(SEQ ID NO:39)。
反应体系如表4所示：
表4
Figure PCTCN2021116282-appb-000004
PCR反应程序：98℃，2min——30个循环(每个循环98℃，10s——65℃，20s——72℃，40s)——72℃，5min——16℃。
将PCR扩增产物用SfiI(FD1824，Thermo)进行酶切，琼脂糖凝胶电泳并将目的产物胶回收，再将胶回收的目的片段克隆到SfiI酶切的pComb3XSS载体上。电转R2738电感受态细胞，制备IgG2c重链抗体文库并进行多轮淘选。挑选多个阳性克隆送测序鉴定，结果如图6所示。
II、淘选
1.淘选
1)包被：将CRP抗原用包被液稀释到100μg/mL，加入酶标板(100μL/孔)2孔，4℃包被过夜。
2)封闭：吸出包被液，用PBS洗板3次，用300μL，4％脱脂牛奶封闭(第二，三，四，五轮封闭液为4％BSA)，37℃孵育2h。
3)结合：吸出封闭液，用PBS洗板3次，加入100μL噬菌体展示文库，37℃孵育1h。
4)洗涤：吸出未结合的噬菌体，PBST洗涤(本申请中共进行了五轮淘选，第一、 第二轮分别用PBST洗3遍，第三、四、五轮分别用PBST洗10遍)。
5)洗脱：加入100μL Gly-HCl(pH3.0)，37℃,5min，用移液器轻柔吸打数次。
6)中和：吸出孔中液体至离心管，预先加入15μL中和缓冲液(1M Tirs-Hcl，PH＝8.8)并混匀。
7)取10μL中和后的洗脱液测定滴度，剩余洗脱液扩增培养后用于下一轮淘选。
2.噬菌体扩增与纯化
1)中和后的洗脱液加入5mL ER2738菌液中(商购菌株，OD 600约为0.5-0.7)，混匀；
2)37℃恒温箱静置30min，然后37℃条件下，180rpm培养1h；
3)将培养物加入20mL LB培养基中，37℃条件下，180rpm培养2h；
4)加入20μL辅助噬菌体M13KO7(2×10 9cfu)(购自NEB公司，货号是N03158)，混匀；
5)37℃恒温箱静置30min，然后37℃条件下，180rpm培养1h后加入氨苄抗生素；
6)37℃，180rpm培养1h后8000rpm，离心5min；
7)用25mL LB培养基/Amp/kan重悬沉淀，30℃条件下，180rpm过夜(约14h)。
8)4℃，8000rpm，离心10min，取上清；
9)加入1/5体积PEG-NaCl溶液，4℃静置约4-6h；
10)4℃，12000rpm，离心20min，弃去上清；
11)沉淀用1mL重悬，取10μL测滴度。
3.滴度测定
1)用PBS稀释待测噬菌体(10μL噬菌体加入到990μL PBS中为10 -2)，分别取10μL稀释后的噬菌体加入200μL的ER2738菌液中，混匀。
2)将感染培养物在37℃静置培养30min；
3)涂布于LB+Amp +抗性平板；
4)37℃倒置过夜培养；
5)计数菌落，计算滴度；
III、制备IgG2c重链抗体可变区文库并进行进行了5次淘选后的结果如以下表5：
表5、CRP-MDG1筛选数据表
Figure PCTCN2021116282-appb-000005
Figure PCTCN2021116282-appb-000006
总共进行了5次淘选，如上表可知输出噬菌体有明显富集，通过PHAGE-ELISA实验挑选阳性克隆送测序鉴定。
实施例7、CRP抗原特异性IgG2c重链抗体可变区的原核表达与生物学活性鉴定
继以上实施例6，对测序序列进行分析，选择前5轮淘选重复出现的D4-12(核苷酸序列和氨基酸序列如表6所示)转染BL21大肠杆菌，并在16℃加入不同浓度IPTG进行原核诱导表达，经超声破碎收集上清并进行镍亲和柱纯化。
包被特异性抗原CRP和无关蛋白OVA(2μg/mL)，用纯化D4-12进行梯度稀释，ELISA检测，结果显示，纯化D4-12重链抗体可变区能特异性识别并结合抗原CRP，并呈现出较好的浓度依赖性，如图7所示。
表6
Figure PCTCN2021116282-appb-000007
实施例8、CRP抗原特异性IgG2c重链抗体可变区的真核表达与生物学活性鉴定
继以上实施例6，对测序序列进行进一步分析，选择重复出现最多的序列D5S-12(核苷酸序列和氨基酸序列如表7所示)进行真核表达和生物学活性鉴定。经转染KOP293细胞(珠海凯瑞)，于第6天收集培养细胞的上清液并进行镍亲和柱纯化目的蛋白。
表7
Figure PCTCN2021116282-appb-000008
Figure PCTCN2021116282-appb-000009
1、包被特异性抗原CRP和无关蛋白OVA或BSA(2μg/mL)，将纯化的D5S-12抗体进行梯度稀释，ELISA检测，结果显示，纯化D5S-12重链抗体能特异性识别并结合抗原CRP，并呈现出较好的浓度依赖性，结果如图8所示。
2、D5S-12重链抗体亲和力测定：用FORTEBIO-OCTET测定纯化D5S-12重链抗体对抗原CRP的亲和力，抗体稀释为10μg/mL，抗原CRP稀释成200，100，50，25，12.5，6.25，3.13nM，经测定，其亲和力常数K D(M)＝9.87E-10,K on(1/Ms)＝8.55E+04,K off(1/s)＝8.44E-05，重链抗体D5S-12对抗原CRP有较高的亲和力，结果如图9所示。
第二部分、制备IghM-3G3纯和型小鼠及其免疫结果
IghM-3G3小鼠是指被敲除了Ighm基因上的第一个外显子以及敲除了Ighg3基因、Ighg1基因、Ighg2b基因以及位于Ighg2c基因上的第一个外显子的C57BL/6小鼠。先制备IghM纯和型小鼠(即敲除了Ighm基因上的第一个外显子的C57BL/6小鼠)，再基于IghM纯和型小鼠制备IghM-3G3纯合型小鼠。
实施例9、制备IghM纯和型小鼠
IghM纯合型小鼠的制备，包括以下步骤：
(1)核酸分子的获得：
C57BL/6小鼠抗体重链可变区和恒定区的基因位点示意图如图1所示，设计如图10所示的打靶策略以敲除小鼠Ighm基因中编码CH1结构域的核苷酸序列。
为了敲除小鼠Ighm基因中编码CH1结构域的核苷酸序列，在小鼠Ighm基因的第1个外显子上下游选取靶点，发明人在小鼠Ighm基因的第一个外显子上游选择了sgRNA的靶向序列(SEQ ID NO:1、SEQ ID NO:2)，在小鼠Ighm基因的第一个外显子下游选择了sgRNA靶向序列(SEQ ID NO:5、SEQ ID NO:6)，并根据靶向序列设计了sgRNA序列，具体如表8所示。
表8
Figure PCTCN2021116282-appb-000010
Figure PCTCN2021116282-appb-000011
(2)将核酸分子构建入骨架质粒并体外转录得到RNA：
将合成的sgRNA序列的正向和反向DNA oligo通过退火形成互补的双链，利用T4连接酶连接在sgRNA表达载体(px330)上，连接后经专业测序公司测序验证，结果表明获得了目的质粒，进一步通过体外转录获得sgRNA。
(3)向宿主动物受精卵导入所述sgRNA和Cas9蛋白：
小鼠促排卵、体外受精并培育受精卵，然后将sgRNA和Cas9蛋白混合、电转小鼠受精卵，或者采用显微注射的方法将Cas9蛋白(或Cas9mRNA，可商购)与sgRNA一起注射进入小鼠受精卵。
(4)将含有上述sgRNA和Cas9蛋白的细胞植入代孕动物体内：
将上述受精卵细胞植入代孕母鼠体内，可生产F0代嵌合体鼠。通过提取鼠尾基因组DNA和PCR检测，检测F0代小鼠中发生了敲除的个体。对基因敲除小鼠进行测序，确认删除了靶序列。挑选基因正确敲除的F0代嵌合鼠用于后续繁殖和鉴定。
PCR引物Ighm-F和Ighm-R能检测敲除基因(如图10中箭头所示)，引物序列见表9：
表9
Figure PCTCN2021116282-appb-000012
检测结果见图11。使用以上引物进行PCR检测时，Ighm野生型基因能扩增出约600bp的条带，Ighm CH1敲除基因能扩增出约300bp的条带。由图11可知，克隆#1，#2，#3和#6含有Ighm CH1敲除基因。对基因敲除小鼠进行测序，确认敲除小鼠Ighm基因的第1个外显子被删除。
(5)繁殖杂合、纯合的基因敲除小鼠：
将靶基因敲除的F0代小鼠与野生型鼠交配获得F1代鼠，通过提取鼠尾基因组和PCR 检测，挑选可以稳定遗传的基因敲除阳性F1代杂合子小鼠。再将F1代杂合小鼠互相交配即可获得基因敲除阳性F2代纯合子鼠，即IghM纯合型小鼠。对获得的F1代杂合或F2代纯合鼠进行基因型的方法与步骤4相同。
实施例10、IgM重链抗体基因调取
按照以上实施例2进行CRP抗原免疫后，将IghM纯和型小鼠进行安乐死并取脾细胞，经Trizol裂解、提取总RNA，并反转录得到cDNA，使用IgM亚型抗体特异性引物通过PCR扩增重链可变区及与之相连接的重链恒定区，所用引物及其序列如下：
MHV1、MHV2、MHV3、MHV4、MHV5、MHV6、MHV7、MHV8、MHV9、MHV10、MHV11、MHV12均见实施例5；
联合B6IghM CH2R4：GTTCATCTCTGCGACAGC(SEQ ID NO:46)；
PCR反应体系如表10所示：
表10
Figure PCTCN2021116282-appb-000013
PCR反应程序：98℃，2min——30个循环(每个循环98℃，10s——50℃，20s——72℃，40s)——72℃，5min——16℃。
将PCR扩增产物连接至pEASY-T5 Zero Cloning载体，转化TOP10感受态细胞并涂LB平板(氨苄抗性)，挑取克隆进行菌落PCR。将阳性克隆送测序，由测序结果可知，IghM纯合型小鼠中表达的IgM抗体重链可变区FR4直接与CH2(起始氨基酸序列AVAEMN)相连，结果如图12所示，基因组IgM CH1外显子敲除成功。。
实施例11、制备IghM-3G3纯和型小鼠
一种制备IghM-3G3小鼠纯合型的方法，包括以下步骤：
(1)核酸分子的获得：
C57BL/6小鼠抗体重链可变区和恒定区的基因位点示意图如图1所示，设计如图13所示的打靶策略，在实施例9所获得的IghM纯合型小鼠的基础上，进一步敲除Ighg3基因、Ighg1基因、Ighg2b基因以及位于Ighg2c基因上的第一个外显子。
为了敲除小鼠Ighm上编码CH1结构域的第一外显子、Ighg3、Ighg1、Ighg2b基因和Ighg2c上编码CH1结构域的第一个外显子，发明人在IghM纯合型小鼠的基础上，在小鼠Ighg3基因的第一个外显子上游选择了sgRNA的靶向序列(SEQ ID NO:7、SEQ ID NO:8)，在小鼠Ighg2c基因的第一个外显子下游选择了sgRNA靶向序列(SEQ ID NO:3、SEQ ID NO:4)，并根据靶向序列设计了sgRNA序列，具体如表11所示。
表11
Figure PCTCN2021116282-appb-000014
(2)将核酸分子构建入骨架质粒并体外转录得到RNA：
将合成的sgRNA序列的正向和反向DNA oligo通过退火形成互补的双链，利用T4连接酶连接到sgRNA表达载体(px330)上，连接后经专业测序公司测序验证，结果表明获得了目的质粒，进一步通过体外转录获得sgRNA。
(3)向宿主动物受精卵导入所述sgRNA和Cas9蛋白：
小鼠促排卵、体外受精并培育受精卵，然后将sgRNA和Cas9蛋白混合、电转小鼠受精卵，或者采用显微注射的方法将Cas9蛋白(或Cas9mRNA，可商购)与sgRNA一起注射进入小鼠受精卵。
(4)将含有上述sgRNA和Cas9蛋白的细胞植入宿主动物体内：
将上述受精卵细胞植入代孕母鼠体内，可生产F0代嵌合体鼠。通过提取鼠尾基因组DNA和PCR检测，检测F0代小鼠中发生了敲除的个体。对基因敲除小鼠进行测序，确认删除了靶序列。挑选基因正确敲除的F0代嵌合鼠用于后续繁殖和鉴定。
PCR引物Ighg-1F和Ighg-1R能检测敲除基因，引物Ighg-2R和Ighg-1F能检测野生型基因(图13的箭头)。引物的序列如表12。
表12
Figure PCTCN2021116282-appb-000015
通过PCR对IghM-3G3小鼠进行基因型鉴定(图14)，Ighm基因型用引物Ighm-F和Ighm-R鉴定：Ighm野生型基因可扩增出约600bp的目的产物，敲除基因可扩增出约300bp的目的产物。Ighg基因型用引物Ighg-1F、Ighg-1R和Ighg-2R鉴定：Ighg敲除基因用引物Ighg-1F和Ighg-2R可扩增出约500bp的目的产物，用引物Ighg-1F和Ighg-1R扩增不出条带；Ighg野生型基因用引物Ighg-1F和Ighg-2R扩增不出条带，用引物Ighg-1F和Ighg-1R可扩增出约460bp的目的产物。+/-：杂合子；wt：野生型；-/-：敲除纯合子。
(5)繁殖杂合、纯合的基因敲除小鼠：
将靶基因敲除的F0代小鼠与野生型鼠交配获得F1代鼠，通过提取鼠尾基因组和PCR检测，挑选可以稳定遗传的基因敲除阳性F1代杂合子小鼠。再将F1代杂合小鼠互相交配即可获得基因敲除阳性F2代纯合子鼠，即IghM-3G3纯合型小鼠。对获得的F1代杂合或F2代纯合鼠进行基因型鉴定的方法与步骤4相同。
实施例12、IghM-3G3纯合型小鼠抗原免疫反应和效价检测
用人C反应蛋白(CRP)免疫IghM-3G3纯合型小鼠。
免疫方法如下：选择6-8周龄雄性鼠，抗原人C反应蛋白(CRP，A-5172,百桥瑞景)加等体积弗氏完全佐剂(F5881，Sigma)，乳化到滴水不化的状态，即可做小鼠初次免疫皮下多点注射用，初次免疫注射剂量为100μg/只小鼠，初次免疫后，每2周进行后续皮下免疫，CRP抗原加等体积弗氏不完全佐剂(F5506，Sigma)乳化后，对小鼠皮下多点注射，每次注射剂量为100μg/只小鼠。
血清效价检测；
血清效价检测方法如下：将CRP抗原稀释成2μg/mL，取100μL加入聚苯乙烯酶联检测板包板，用HRP-山羊抗小鼠IgG-Fc(Jackson，115-035-071)检测血清中与CRP抗原特异性结合的特异性IgG抗体。
如图15所示，分别在未免疫、一免一周后、三免一周后采血，血清用PBS稀释，从1：500稀释度开始做倍比稀释，ELISA检测血清效价显示，未免疫和一免小鼠体内未出现结合抗原CRP的特异性抗体，三免后其血清效价在1：32000左右。
实施例13 IgG2c重链抗体基因调取
CRP抗原免疫IghM-3G3纯和型小鼠并经血清效价检测后，将小鼠进行安乐死并取脾细胞，经Trizol裂解、提取总RNA，并反转录得到cDNA，使用如下IgG2c亚型抗体特异性引物通过PCR扩增重链可变区及与之相连接的重链恒定区。
MHV1、MHV2、MHV3、MHV4、MHV5、MHV6、MHV7、MHV8、MHV9、MHV10、MHV11、MHV12、联合B6_IgG2c_CH2R1均见实施例5；
PCR反应体系、PCR反应程序如实施例5所示：
将所得PCR扩增产物连接pEASY-T5 Zero Cloning载体，转化TOP10感受态细胞并涂 LB平板，随机挑取7个克隆，菌落PCR鉴定为阳性并送测序(如图16A所示，650bp大小条带为阳性条带)。
阳性克隆的测序结果显示(如图16B所示)，IghM-3G3纯和型小鼠中产生的IgG2c抗体重链可变区FR4区直接与IgG2c Hinge区(抗体铰链区)相连，由于抗体重链的结构是VH-CH1-Hinge-CH2-CH3，其中VH区域又可以详细的分为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，IghM-3G3纯和型小鼠中产生的IgG2c抗体重链可变区FR4区直接与Hinge区(抗体铰链区)相连，说明IgG2c的CH1外显子敲除成功。
实施例14、CRP抗原特异性IgG2c重链抗体可变区噬菌体库展示构建和淘选；
继以上实施例13，噬菌体展示库构建和抗体淘选的步骤与实施例6中完全相同，仅针对表13中的参数进行调整，进行了5次淘选后的结果如以下表13所示：
表13：CRP筛选数据表
Figure PCTCN2021116282-appb-000016
总共进行了5次淘选，如表13可知输出噬菌体有明显富集，通过PHAGE-ELISA实验挑选阳性克隆送测序鉴定，如表14所示。
表14：阳性克隆氨基酸序列
Figure PCTCN2021116282-appb-000017
实施例15、IghM-3G3纯合型小鼠抗原免疫反应和效价检测
本实施例用冠状病毒S蛋白抗原免疫IghM-3G3纯合型小鼠。
免疫方法如下：选择6-8周龄雄性鼠，抗原冠状病毒S蛋白(SARS-CoV-2(2019-nCoV)Spike S1-His Recombinant Protein，40591-V08H,义翘神州)加等体积弗氏完全佐剂 (F5881，Sigma)，乳化到滴水不化的状态，即可做小鼠初次免疫皮下多点注射用，初次免疫注射剂量为100μg/只小鼠，初次免疫后，每2周进行后续皮下免疫，每次注射剂量均为100μg/只小鼠。
血清效价检测；
血清效价检测方法如下：将冠状病毒S蛋白抗原稀释成2μg/mL，取100μL加入聚苯乙烯酶联检测板包板，用HRP-山羊抗小鼠IgG-Fc(Jackson 115-035-071)检测血清中与CRP抗原特异性结合的特异性IgG抗体。
分别在未免疫、二免一周后、三免一周后和四免一周后采血，血清用PBS稀释，从1：500稀释度开始做倍比稀释，ELISA检测血清效价，其结果如图17所示，图17显示，未免疫小鼠体内未出现结合抗原冠状病毒S蛋白的特异性抗体，二免后其血清效价在1：64000左右。
实施例16、IgG2c重链抗体基因调取
按照以上实施例15，以冠状病毒S蛋白抗原免疫IghM-3G3纯合型小鼠并经血清效价检测后，将小鼠进行安乐死并取其脾细胞，脾细胞经Trizol裂解、提取总RNA，并反转录得到cDNA，使用如下IgG2c亚型抗体特异性引物，通过PCR扩增重链可变区及与之相连接的重链恒定区。
MHV1、MHV2、MHV3、MHV4、MHV5、MHV6、MHV7、MHV8、MHV9、MHV10、MHV11、MHV12、联合B6_IgG2c_CH2R1均见实施例5；
PCR反应体系、PCR反应程序如实施例5所示：
将所得PCR扩增产物连接pEASY-T5 Zero Cloning载体，转化TOP10感受态细胞并涂LB平板，随机挑取23个克隆，菌落PCR鉴定为阳性的克隆送测序(PCR结果如图18A所示，其中650bp大小条带为阳性条带)。
实施例17、冠状病毒S蛋白抗原特异性IgG2c重链抗体可变区噬菌体库展示构建和淘选；
继以上实施例16，噬菌体展示库构建和抗体淘选的步骤与实施例6中完全相同，共进行了5次淘选，每次输出噬菌体有明显富集，通过PHAGE-ELISA实验挑选阳性克隆送测序鉴定，如表15所示。并分析这些序列的CDR区异同，其结果如图18B所示。
表15：阳性克隆氨基酸序列
Figure PCTCN2021116282-appb-000018
Figure PCTCN2021116282-appb-000019
对测序序列进行分析，选择前5轮淘选出现的S-9、S-19、S-27和S-47(氨基酸序列如表15所示)转染BL21大肠杆菌，并在30℃加入不同浓度IPTG进行原核诱导表达，经超声破碎收集上清液并进行镍亲和柱纯化。
包被特异性抗原冠状病毒S蛋白和无关蛋白OVA(2μLg/mL)，将纯化后的S-9、S-19、S-27和S-47抗体进行梯度稀释，通过ELISA法进行检测，结果显示，本次调取的重链抗体可变区能特异性识别并结合抗原冠状病毒S蛋白，并呈现出较好的浓度依赖性，如图19所示。
实施例18、IghM-DG1纯合型小鼠抗原免疫反应和效价检测
本实施例用冠状病毒S蛋白抗原免疫IghM-DG1纯合型小鼠。
小鼠抗原免疫操作和血清效价检测的步骤与实施例15中完全相同，其血清效价检测结果如图20A所示；噬菌体展示库构建和抗体淘选的步骤与实施例6中完全相同，共进行了5次淘选，每次输出噬菌体有明显富集，通过PHAGE-ELISA实验挑选阳性克隆送测序鉴定，如表16所示。并分析这些序列的CDR区异同，其结果如图20B所示。
表16：阳性克隆氨基酸序列
Figure PCTCN2021116282-appb-000020
Figure PCTCN2021116282-appb-000021
对测序序列进行分析，选择前5轮淘选出现的S-1、S-2、S-7、S-12、S-17、S19和S-65抗体(氨基酸序列如表16所示)的DNA序列克隆到PET28载体，并转染BL21大肠杆菌，在16℃加入不同浓度IPTG进行原核诱导表达，经超声破碎收集上清并进行镍亲和柱纯化。
包被特异性抗原冠状病毒S蛋白和无关蛋白OVA(2μg/mL)，将纯化的S-1、S-7、S-12、S-17、S19、S-25、S-51和S-65抗体进行梯度稀释，通过ELISA法进行检测，结果显示，本次调取的重链抗体可变区能特异性识别并结合抗原冠状病毒S蛋白，并呈现出较好的浓度依赖性，如图21所示。
实施例19、IgG2c重链抗体基因调取
本实施例用冠状病毒N蛋白抗原免疫MDG1纯合型小鼠。
免疫方法和血清效价检测方法与实施例15中的完全相同，免疫后血清效价检测结果如图22A所示。本实施例中所用抗原为冠状病毒N蛋白(SARS-CoV-2(2019-nCoV)Nucleocapsid-His recombinant Protein，40588-V08B-B,义翘神州)。
噬菌体展示库构建和抗体淘选的步骤与实施例6中完全相同，共进行了5次淘选，每次输出噬菌体有明显富集，通过PHAGE-ELISA实验挑选阳性克隆送测序鉴定，如表17所示。并分析这些序列的CDR区异同，如图22B所示。
表17：阳性克隆氨基酸序列
Figure PCTCN2021116282-appb-000022
将阳性克隆的DNA序列克隆到PET28载体上，转化到BL21细菌中，进行诱导表达，然后用镍柱纯化目的蛋白，并通过ELISA法检测抗体的特异性及结合力，其结果如图23所示，图23结果显示：本次调取的重链抗体可变区能特异性识别并结合抗原冠状病毒S蛋白，并呈现出较好的浓度依赖性。
实施例20、荧光定量PCR检测两基因型小鼠中各免疫球蛋白基因转录水平的差异
取MDG1小鼠和野生型小鼠的脾脏提取RNA，反转录成cDNA，将cDNA稀释5倍后取5μL进行荧光定量PCR实验，每个基因型选择4只个体，每只个体的每个基因进行2个技术重复，实验中所用到的引物以及扩增片段长度如表18所示，荧光定量PCR扩增体系如表19所示。
表18:荧光定量PCR实验所用引物序列及扩增产物长度
Figure PCTCN2021116282-appb-000023
Figure PCTCN2021116282-appb-000024
表19:荧光定量PCR扩增体系
2×SYBR Green mix 10μL
上游引物(10μM) 1μL
下游引物(10μM) 1μL
cDNA 5μL
ddH 2O 3μL
扩增程序：
95℃：15min
Figure PCTCN2021116282-appb-000025
溶解曲线
65℃：1min
以0.11℃/s的速度持续升温至95℃
程序结束后根据溶解曲线判断数值的可信度，随后采用2 -ΔΔCt方法计算各免疫球蛋白基因转录本的相对表达水平，利用双尾T检验进行统计学分析，统计结果如图24、图25和图26所示，由这些结果可见：MDG1小鼠骨髓、脾脏和小肠中μ基因的转录本表达量低于野生型小鼠，γ2c基因的转录本表达量高于野生型小鼠，且MDG1小鼠三个免疫组织中γ2c基因转录本的表达量都高于野生型小鼠中μ基因转录本的表达量，α基因转录本的表达量在两基因型小鼠中未见统计学上的差异。
实施例21、Western Blot检测MDG1小鼠血清中IgG2c的表达形式
取相同遗传背景下8周龄的野生型小鼠和MDG1小鼠的血清进行Western Blot检测，野生型小鼠的血清用PBS稀释20倍，MDG1小鼠的血清用PBS稀释100倍，还原条件下使用1mM DTT打开分子内二硫键，Goat Anti-Mouse IgG2c heavy chain(HRP)抗体稀释10000倍。Western Blot检测结果如图27所示，由图27可见：MDG1小鼠血清中的IgG2c在还原条件下的分子量大小约为45kDa，非还原条件下的分子量大小约为95kDa，符合CH1结构域和轻链缺失的分子量大小。
实施例22、ELISA检测MDG1小鼠血清中各免疫球蛋白的表达量
取相同遗传背景下8周龄的野生型小鼠和MDG1小鼠的血清使用双抗夹心法ELISA进行检测。野生型小鼠的IgM进行2000倍稀释，IgG2c进行4000倍稀释，IgG进行10000倍稀释，IgA进行5000倍稀释，IgE进行20倍稀释；MDG1小鼠的IgM进行2000倍稀释，IgG2c进行8000倍稀释，IgG进行10000倍稀释，IgA进行5000倍稀释，IgE进行20倍稀释。使用酶标仪读取各样品孔在450nm处的吸光值，使用ELISA Calc根据四参 数拟合得到标准曲线并计算各孔免疫球蛋白的含量，采用双尾T检验进行统计分析。
结果如图28所示，由图28可见：MDG1小鼠血清中确实不表达IgM，其IgG2c的表达水平显著高于野生型小鼠，和野生型小鼠中IgM的表达水平相当，由于野生型小鼠中存在其它IgG亚型，因此其血清中总IgG的表达水平显著高于MDG1小鼠，MDG1小鼠血清中IgE的表达水平显著低于野生型小鼠，IgA的表达水平与野生型小鼠相比并未出现统计学上的差异。
实施例23、流式细胞术检测MDG1小鼠骨髓、脾脏和腹膜腔B细胞的发育
骨髓B细胞：
准备同一遗传背景下8周龄的野生型小鼠和MDG1小鼠各6只，取其下肢的两根股骨和胫骨，用FACS将骨髓细胞冲至离心管中，短暂离心后使用ACK重悬并用70μm的滤网过滤细胞悬液，离心后弃上清，加入1mL FACS重悬细胞，取其中50μL细胞用于染色，取10μL细胞稀释40倍进行计数，骨髓B细胞的染色方案如表20所示。
脾脏B细胞：
准备同一遗传背景下8周龄的野生型小鼠和MDG1小鼠各4只，将70μm滤网放于60mm皿中，加入1mL ACK，取其脾脏放在滤网上轻轻研磨，收集研磨后的细胞悬液，短暂离心后弃上清，加入1mL FACS重悬细胞，取其中50μL细胞用于染色，取10μL细胞稀释40倍进行计数，脾脏B细胞的染色方案如表20所示。
腹膜腔B细胞：
准备同一遗传背景下8周龄的野生型小鼠和MDG1小鼠各7只，将其腹部朝上固定在泡沫板上，剪开腹部表皮露出腹膜部分，用注射器吸取5mL FACS注入腹膜腔中，使用移液枪吹吸2-3次后将细胞悬液收集至离心管中，短暂离心后使用ACK重悬并用70μm的滤网过滤细胞悬液，离心后弃上清，加入200μL FACS重悬细胞，取其中100μL细胞用于染色，腹膜腔B细胞的染色方案如表20所示。
表20:流式细胞实验染色方案
  APC V450 V500 PE PE‐Cy7 PerCP APC‐Cy7 FITC
骨髓   B220   CD43 IgM     IgG2c
脾脏 B220 CD138 CD19 CD23 CD21 7‐AAD IgM IgG2c
腹膜腔 CD23     CD5 CD19 7‐AAD    
骨髓中B细胞的流式结果如图29所示，图29结果显示：MDG1小鼠骨髓中B细胞的比例和数目与野生型小鼠相比未见统计学上的差异，祖B细胞和前B细胞的比例与野生型小鼠相比未见统计学上的差异，但在数目上显著降低，IgM在成熟B细胞中高表达，未成熟B细胞中低表达，由于MDG1小鼠缺失表达IgM的μ基因，所以无法确定其体内未成熟B细胞和成熟B细胞的发育情况，但MDG1小鼠中存在着大量IgG2c+B细胞，这群B细胞在野生型小鼠中的比例极低。
脾脏中B细胞的流式结果如图30所示，图30结果显示：MDG1小鼠脾脏中B细胞的比例显著低于野生型小鼠，但在数目上未见统计学上的差异；MDG1小鼠脾脏中IgG2c+B细胞的比例和数目显著高于野生型小鼠中IgM+B细胞的比例和数目，这可能与野生型小鼠中的IgM发生类别转换重组到其它免疫球蛋白亚型所导致；由于本次流式实验选择的是未免疫刺激状态下的小鼠，因此野生型小鼠和MDG1小鼠脾脏内浆细胞的比例和数目均较低，且未见统计学上的差异；MDG1小鼠脾脏中滤泡B细胞、过渡期B细胞以及边缘B细胞的比例和数目与野生型小鼠相比未见统计学上的差异。
腹膜腔中B细胞的流式结果如图31所示，图31结果显示：MDG1小鼠腹膜腔中B细胞的比例显著高于野生型小鼠，IgM+B1a细胞的比例在MDG1小鼠中显著降低，而B1b和B2细胞的比例在MDG1小鼠中显著升高。
实施例24、使用特定抗原对野生型小鼠和MDG1小鼠进行免疫刺激
准备同一遗传背景下6周龄的野生型小鼠和MDG1小鼠各6-7只，使用偶联BSA的氯霉素和阿特拉津进行免疫实验，每只小鼠每次免疫100μg/100μL。初次免疫使用弗氏完全佐剂，背部皮下注射50μL，腹腔注射50μL，加强免疫使用弗氏不完全佐剂，腹腔注射100μL，共加强免疫4次，初次免疫前3天，每次加强免疫后3天进行内眦采血。
实施例25、ELISA检测免疫刺激后小鼠血清中抗原特异性抗体相对表达量的变化趋势
继以上实施例24，为了避免免疫刺激后血清中大量存在的BSA特异性的抗体对检测结果造成干扰，本次ELISA实验采用偶联OVA的氯霉素和阿特拉津作为包被抗原，抗原包被量为200ng/100μL/孔，抗原特异性IgM、IgG2c、IgG以及IgA检测中血清的稀释倍数为4000倍，IgE检测中血清的稀释倍数为100倍，为保证各样品孔的一致性，加入TMB显色液后，反应时间严格控制在20min，之后使用酶标仪读取450nm处的吸光值，剔除未发生免疫反应的个体，绘制抗原特异性抗体相对表达量的变化趋势图。
氯霉素和阿特拉津特异性抗体相对表达量的变化分别如图32和图33所示，其结果显示：从整体免疫应答情况来看，氯霉素的免疫效果优于阿特拉津。MDG1小鼠中抗原特异性IgG2c的变化趋势与野生型小鼠中IgG2c的变化趋势相似，不同于野生型小鼠中持续低水平表达的IgM，由于野生型小鼠中存在其它IgG亚型，因此野生型小鼠中抗原特异性总IgG的含量高于MDG1小鼠，此外MDG1小鼠中抗原特异性IgA和IgE的相对表达量稍高于野生型小鼠。
实施例26、抗原免疫后，脾脏中生发中心B细胞以及浆细胞的发育检测
取抗原免疫后的野生型小鼠和MDG1小鼠各6只，将70μm滤网放于60mm皿中，加入1mL ACK，取其脾脏放在滤网上轻轻研磨，收集研磨后的细胞悬液，短暂离心后弃上清，加入1mL FACS重悬细胞，取其中20μL细胞用于染色，取10μL细胞稀释80 倍进行计数，免疫后脾脏B细胞的染色方案如表21所示。
表21:免疫后脾脏B细胞的染色方案
  FITC PerCP APC PE‐Cy7 APC‐Cy7 V450 V500
脾脏 IgG2c 7‐AAD B220 Fas CD38 CD138 CD19
抗原免疫后小鼠脾脏B细胞、生发中心B细胞和浆细胞的发育情况如图34所示，图34结果显示：MDG1小鼠免疫后脾脏B细胞的比例显著低于野生型小鼠，但在数目上未见统计学上的差异；MDG1小鼠中生发中心B细胞的比例和数目都显著低于野生型小鼠，IgG2c+GC B的比例显著高于野生型小鼠，数目上未见统计学上的差异；抗原免疫后MDG1小鼠和野生型小鼠中浆细胞以及IgG2c+浆细胞的比例和数目都未见统计学上的差异。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
工业实用性
采用本申请的非人哺乳动物或其子代的制备方法获得的非人哺乳动物或其子代不引入任何编码抗体重链可变区和恒定区的外源基因，可以直接利用其自身基因组中编码抗体重链可变区的所有VDJ基因，从而通过重链可变区重排产生多样性更好的重链抗体。

Claims (43)

  1. 一种非人哺乳动物或其子代的制备方法，其特征在于，包括以下步骤：
    使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤；
    以及，使得非人哺乳动物体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤。
  2. 根据权利要求1所述的制备方法，其特征在于：使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤为：
    仅使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区CH1结构域的步骤；
    或，使得非人哺乳动物体内不表达或不正确表达IgM重链恒定区和IgD重链恒定区的步骤。
  3. 根据权利要求1或2所述的制备方法，其特征在于：使得非人哺乳动物体内一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤为：
    使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个和第二个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第三个或第四个编码IgG重链恒定区的基因中的一个或两个在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个和第二个和第三个编码IgG重链恒定区的基因在表达时不表达或不正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第四个编码IgG重链恒定区的基因在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第二个或第三个或第四个编码IgG重链恒定区的基因中的一个、两个或三个在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个和第二个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第三个或第四个编码IgG重链恒定区的基因中的一个或两个在表达时不表达或不正确表达CH1结构域的步骤；
    或，使得非人哺乳动物体内第一个和第二个和第三个编码IgG重链恒定区的基因在表达时正确表达其所编码的IgG重链恒定区、并且使得非人哺乳动物体内第四个编码IgG重链恒定区的基 因在表达时不表达或不正确表达CH1结构域的步骤。
  4. 一种非人哺乳动物或其子代的制备方法，其特征在于，包括以下步骤：
    敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列的步骤；
    以及，敲除以下目标基因的步骤，所述目标基因包括：一个、两个、三个、四个或四个以上编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列。
  5. 根据权利要求4所述的制备方法，其特征在于：敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列的步骤为：
    仅敲除非人哺乳动物基因组上的、编码IgM重链恒定区CH1结构域的核苷酸序列的步骤；
    或，敲除非人哺乳动物基因组上的、编码IgM重链恒定区和IgD重链恒定区的步骤。
  6. 根据权利要求4或5所述的制备方法，其特征在于：所述目标基因为：
    第一个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
    或，从第一个编码IgG重链恒定区的基因开始至第二个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，从第一个编码IgG重链恒定区的基因开始至第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，从第一个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，从第一个编码IgG重链恒定区的基因开始至最后一个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，第二个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
    或，从第二个编码IgG重链恒定区的基因开始至第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，从第二个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，第三个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列；
    或，从第三个编码IgG重链恒定区的基因开始至第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列的全部核苷酸序列；
    或，第四个编码IgG重链恒定区的基因上的、编码CH1结构域的核苷酸序列。
  7. 根据权利要求4-6之一所述的制备方法，其特征在于：以下步骤在同一操作步骤中完成或在不同的操作步骤中完成：
    敲除非人哺乳动物基因组上的、包括编码IgM重链恒定区CH1结构域的核苷酸序列的步骤；
    敲除目标基因的步骤。
  8. 根据权利要求1-7之一所述的制备方法，其特征在于，所述非人哺乳动物为啮 齿类动物；可选地，所述啮齿类动物为大鼠或小鼠；进一步可选地，所述啮齿类动物为小鼠；更进一步，所述小鼠为C57BL/6小鼠或BALB/c小鼠。
  9. 根据权利要求3、6、7或8所述的制备方法，其特征在于：第一个编码IgG重链恒定区的基因为Ighg3。
  10. 根据权利要求3、6、7、8或9所述的制备方法，其特征在于：当非人哺乳动物为C57BL/6小鼠时，第一个编码IgG重链恒定区的基因为Ighg3、第二个编码IgG重链恒定区的基因Ighg1、第三个编码IgG重链恒定区的基因Ighg2b、第四个编码IgG重链恒定区的基因Ighg2c；
    当非人哺乳动物为BALB/c小鼠时，第一个编码IgG重链恒定区的基因为Ighg3、第二个编码IgG重链恒定区的基因Ighg1、第三个编码IgG重链恒定区的基因Ighg2b、第四个编码IgG重链恒定区的基因Ighg2a。
  11. 根据权利要求1-10之一所述的制备方法，其特征在于：所述非人哺乳动物基因组上包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述非人哺乳动物中能正常表达κ轻链和/或λ轻链。
  12. 根据权利要求1-10之一所述的制备方法，其特征在于：基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效应因子核酸酶方法。
  13. 根据权利要求1-10之一所述的制备方法，其特征在于：所述非人哺乳动物或其子代用于生产重链抗体。
  14. 一种C57BL/6小鼠或其子代的制备方法，其特征在于，包括以下步骤：
    敲除C57BL/6小鼠的基因组中编码抗体IgM重链恒定区和IgD重链恒定区的基因的步骤；
    以及，敲除C57BL/6小鼠的基因组中从编码IgG3重链恒定区的基因开始至编码IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤。
  15. 根据权利要求14所述的制备方法，其特征在于：所述C57BL/6小鼠基因组中包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述C57BL/6小鼠能正常表达κ轻链和/或λ轻链。
  16. 根据权利要求14所述的制备方法，其特征在于，基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效应因子核酸酶方法。
  17. 根据权利要求14所述的制备方法，其特征在于，基因敲除的步骤中，敲除小鼠编码IgM重链恒定区的基因的第1号外显子至编码IgG2c重链恒定区的基因的第1号外显子的全部核苷酸序列。
  18. 根据权利要求17所述的制备方法，其特征在于，基因敲除的步骤中，使用了靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游的sgRNA和靶向小鼠编码IgG2c重链恒定区的基因的第1号外显子下游的sgRNA；
    可选地，sgRNA靶向的小鼠编码IgM重链恒定区的基因的第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因的第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4。
  19. 根据权利要求18所述的制备方法，其特征在于，靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；和//或，靶向小鼠编码IgG2c重链恒定区的基因的第1号外显子下游的sgRNA为SEQ ID NO.11、SEQ ID NO.12。
  20. 根据权利要求14-19之一所述的制备方法，其特征在于，所述C57BL/6小鼠或其子代用于生产重链IgG2c抗体。
  21. 一种C57BL/6小鼠或其子代的制备方法，其特征在于，包括以下步骤：
    敲除C57BL/6小鼠的基因组中编码IgM重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤；
    以及，敲除C57BL/6小鼠的基因组中从编码IgG3重链恒定区的基因开始至编码IgG2c重链恒定区的基因上的、编码CH1结构域的核苷酸序列的步骤。
  22. 根据权利要求21所述的制备方法，其特征在于：所述C57BL/6小鼠基因组中包括完整的编码κ轻链和/或λ轻链的基因；可选地，所述C57BL/6小鼠能正常表达κ轻链和/或λ轻链。
  23. 根据权利要求22所述的制备方法，其特征在于，基因敲除的方法包括以下的一种或几种：基因打靶技术、CRISPR/Cas9方法、锌指核酸酶方法、转录激活子样效应因子核酸酶方法。
  24. 根据权利要求21所述的制备方法，其特征在于，基因敲除的步骤中，敲除小鼠编码IgM重链恒定区的基因的第1号外显子，还敲除小鼠从编码IgG3重链恒定区的基因第1号外显子至编码IgG2c重链恒定区的基因第1号外显子的全部核苷酸序列。
  25. 根据权利要求24所述的制备方法，其特征在于，基因敲除的步骤中，使用了靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游和下游的sgRNA，还使用了靶向小鼠编码IgG3重链恒定区的基因的第1号外显子上游的sgRNA和靶向小鼠编码IgG2c重链恒定区的基因的第1号外显子下游的sgRNA。
  26. 根据权利要求25所述的制备方法，其特征在于，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；
    和/或，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.5和SEQ ID NO.6；
    和/或，sgRNA靶向的小鼠编码IgG3重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.7和SEQ ID NO.8；
    和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4。
  27. 根据权利要求26所述的制备方法，其特征在于，靶向小鼠编码IgM重链恒定区的 基因第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；
    和/或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.13和SEQ ID NO.14；
    和/或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.15和SEQ ID NO.16；
    和/或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.11和SEQ ID NO.12。
  28. 根据权利要求20-27之一所述的制备方法，其特征在于，所述C57BL/6小鼠或其子代用于生产重链IgG2c抗体。
  29. 一种非人哺乳动物或其子代的制备方法，其特征在于，包括敲除非人哺乳动物基因组上的、编码IgM重链恒定区CH1结构域的核苷酸序列的步骤。
  30. 根据权利要求29所述的制备方法，其特征在于，所述非人哺乳动物为啮齿类动物；可选地，所述啮齿类动物为大鼠或小鼠；进一步可选地，所述啮齿类动物为小鼠；更进一步，所述小鼠为C57BL/6小鼠或BALB/c小鼠。
  31. 根据权利要求29或30所述的制备方法，其特征在于，所述非人哺乳动物或其子代用于构建权利要求21所述的非人哺乳动物或其子代。
  32. 一种来源于权利要求1-3之一所述的制备方法构建的非人哺乳动物或其子代、来源于权利要求4-13之一所述的制备方法构建的非人哺乳动物或其子代、来源于权利要求14-20之一所述的制备方法构建的C57BL/6小鼠或其子代、或来源于权利要求21-28之一所述的制备方法构建的C57BL/6小鼠或其子代在筛选目标重链抗体中的应用。
  33. 根据权利要求32所述的应用，其特征在于，筛选目标重链抗体时采用噬菌体展示的方法；
    优选地，所述筛选目标重链抗体为筛选C反应蛋白、冠状病毒S蛋白或冠状病毒N蛋白抗原特异性IgG2c重链抗体。
  34. 一种筛选目标重链抗体的方法，其包括使用来源于权利要求1-3之一所述的制备方法构建的非人哺乳动物或其子代、来源于权利要求4-13之一所述的制备方法构建的非人哺乳动物或其子代、来源于权利要求14-20之一所述的制备方法构建的C57BL/6小鼠或其子代、或来源于权利要求21-28之一所述的制备方法构建的C57BL/6小鼠或其子代作为免疫动物进行筛选。
  35. 根据权利要求34所述的方法，其中，筛选目标重链抗体时采用噬菌体展示的方法；
    优选地，所述筛选目标重链抗体为筛选C反应蛋白、冠状病毒S蛋白或冠状病毒N蛋白抗原特异性IgG2c重链抗体。
  36. 一种非人哺乳动物细胞或细胞系或原代细胞培养物，其特征在于，所述非人哺乳动物细胞或细胞系或原代细胞培养物来源于权利要求1-3之一所述的制备方法构建的非人哺乳动物或其子代、权利要求4-13之一所述的制备方法构建的非人哺乳动物或其子代、权利要求14-20之一所述的制备方法构建的C57BL/6小鼠或其子代、权利要求21-28之一所述的制备方法构建的C57BL/6小鼠或其子代、或权利要求29-31之一所述的制备方法构建的非人哺乳动物或其子代。
  37. 一种离体组织或离体器官或其培养物，其特征在于，所述离体组织或离体器官或其培养物来源于权利要求1-3之一所述的制备方法构建的非人哺乳动物或其子代、权利要求4-13之一所述的制备方法构建的非人哺乳动物或其子代、权利要求14-20之一所述的制备方法构建的C57BL/6小鼠或其子代、权利要求21-28之一所述的制备方法构建的C57BL/6小鼠或其子代、或权利要求29-31之一所述的制备方法构建的非人哺乳动物或其子代。
  38. 一种sgRNA组合物，其特征在于，所述sgRNA组合物包括：靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游的sgRNA和靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA；
    或，靶向小鼠编码IgM重链恒定区的基因的第1号外显子上游和下游的sgRNA；
    或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA和靶向小鼠编码IgG2c第1号外显子下游的sgRNA。
  39. 根据权利要求38所述的sgRNA组合物，其特征在于，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.1和SEQ ID NO.2；
    和/或，sgRNA靶向的小鼠编码IgG2c重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.3和SEQ ID NO.4；
    和/或，sgRNA靶向的小鼠编码IgM重链恒定区的基因第1号外显子下游的靶向序列包括SEQ ID NO.5和SEQ ID NO.6；
    和/或，sgRNA靶向的小鼠编码IgG3重链恒定区的基因第1号外显子上游的靶向序列包括SEQ ID NO.7和SEQ ID NO.8。
  40. 根据权利要求38所述的sgRNA组合物，其特征在于，靶向小鼠编码IgM重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.9和SEQ ID NO.10；
    和/或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.13和SEQ ID NO.14；
    和/或，靶向的小鼠编码IgG3重链恒定区的基因第1号外显子上游的sgRNA为SEQ ID NO.15和SEQ ID NO.16；
    和/或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游的sgRNA为SEQ ID NO.11 和SEQ ID NO.12。
  41. 一种敲除载体，其特征在于，包括：编码sgRNA的一段或多段DNA序列，所述sgRNA的靶向序列选自以下的一种：
    靶向小鼠编码IgM重链恒定区的基因第1号外显子上游；
    或，靶向小鼠编码IgG2c重链恒定区的基因第1号外显子下游；
    或，靶向小鼠编码IgM重链恒定区的基因第1号外显子下游；
    或，靶向小鼠编码IgG3重链恒定区的基因第1号外显子上游。
  42. 根据权利要求41所述的敲除载体，其特征在于，所述敲除载体的骨架是sgRNA表达载体。
  43. 一种细胞，其特征在于，包含权利要求41-42之一所述的敲除载体。
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