WO2020216191A1 - 制备噬菌体文库的方法 - Google Patents

制备噬菌体文库的方法 Download PDF

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WO2020216191A1
WO2020216191A1 PCT/CN2020/085706 CN2020085706W WO2020216191A1 WO 2020216191 A1 WO2020216191 A1 WO 2020216191A1 CN 2020085706 W CN2020085706 W CN 2020085706W WO 2020216191 A1 WO2020216191 A1 WO 2020216191A1
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vector
heavy chain
light chain
linker
polynucleotide
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PCT/CN2020/085706
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English (en)
French (fr)
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周辰
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泷搌（上海）生物科技有限公司
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Priority to JP2021562852A priority Critical patent/JP2022532699A/ja
Priority to CN202080004535.XA priority patent/CN112567080A/zh
Priority to BR112021021261A priority patent/BR112021021261A2/pt
Priority to EP20794500.7A priority patent/EP3960916A4/en
Priority to KR1020217037366A priority patent/KR20210153674A/ko
Priority to US17/605,409 priority patent/US20220228138A1/en
Priority to MX2021012914A priority patent/MX2021012914A/es
Publication of WO2020216191A1 publication Critical patent/WO2020216191A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • This application relates to the field of biomedicine, in particular to a method for generating a phage library displaying antibodies or antibody fragments.
  • the library capacity of the library is too small (for example, it is difficult to obtain a library with a library capacity of more than 109 ), and the library diversity is insufficient, and it is difficult to meet the needs of screening high-quality antibodies.
  • the library capacity of the library is too small (for example, it is difficult to obtain a library with a library capacity of more than 109 ), and the library diversity is insufficient, and it is difficult to meet the needs of screening high-quality antibodies.
  • the quality of the library can only be known after the library is built, with high risk and high probability of failure.
  • This application provides a method for generating a phage library displaying antibodies or antibody fragments, a phage library obtained by using the method, and a method for screening antibodies or antibody fragments with the aid of the phage library.
  • the method for producing a phage library displaying antibodies or antibody fragments described in this application has at least one of the following properties: 1) Effectively reduces the probability of introducing mutations caused by PCR; the phage library described in this application is not constructed using conventional antibody libraries in the art
  • the combined PCR strategy used in the method, each component used to construct the phage library can be amplified by only one PCR, and then the required components are connected to obtain the Fab phage library; 2) It can meet the requirements of industrial mass production Requirements; 3) Quality control can be performed relatively easily; each step required to construct the phage library described in this application can be independently and simply controlled in quality; at the same time, after obtaining the phage library described in this application, you can Conveniently (for example, using gene sequencing,
  • the present application provides a method for generating a phage library displaying antibodies or antibody fragments, the method comprising: 1) providing a first polynucleotide, a second polynucleotide and a third polynucleotide, The first polynucleotide comprises LC, the second polynucleotide comprises a linker and the third polynucleotide comprises HC, wherein the LC comprises a light chain encoding the antibody or antibody fragment or The nucleic acid sequence of the light chain fragment, the HC comprises the nucleic acid sequence encoding the heavy chain or heavy chain fragment of the antibody or antibody fragment; 2) introducing the first polynucleotide into the first bacterium to obtain the light chain assembly Bacterial library, introducing the second polynucleotide into a second bacterium to obtain a linker component bacteria, and introducing the third polynucleotide into a third group of bacteria to obtain a heavy chain component bacterial library;
  • the display ligation product of 6 can express the light chain or light chain fragment and the heavy chain or heavy chain fragment in frame .
  • the first polynucleotide, the second polynucleotide and the third polynucleotide are all linear nucleic acid molecules.
  • the nucleic acid sequence encoding the heavy chain or heavy chain fragment and the nucleic acid sequence encoding the light chain or light chain fragment are in different reading frames.
  • the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof. In some embodiments, the linker is about 50 to about 200 bases in length.
  • the LC is located upstream of the linker, and the linker is located upstream of the HC.
  • 2) includes cryopreserving the light chain component bacterial library and/or the heavy chain component bacterial library.
  • 7) includes cryopreserving the displayed bacterial library.
  • 8) includes thawing and culturing the cryopreserved display bacterial library, and using the cultured display bacterial library to construct the phage library.
  • the first polynucleotide includes the structure R1-LC-R2 in the 5'to 3'direction
  • the second polynucleotide includes the structure R3-linked in the 5'to 3'direction
  • the third polynucleotide includes the structure R5-HC-R6 in the 5'to 3'direction
  • the display vector includes the structure R7-display vector fragment-R8 in the 5'to 3'direction
  • the end produced by the restriction endonuclease cleavage of the R2 is capable of recognizing and connecting with the end produced by the restriction endonuclease cleavage of the R3;
  • the ends of any one of R1, R4, R5, R6, R7, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the end produced by the restriction endonuclease cleavage of the R4 capable of recognizing and connecting with the end produced by the restriction endonuclease cleavage of the R5;
  • the ends of any one of R1, R2, R3, R6, R7, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the end produced by the restriction endonuclease cleavage of the R6 is capable of recognizing and connecting with the end produced by the restriction endonuclease cleavage of the R7;
  • the ends of any one of R1, R2, R3, R4, R5, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the end produced by the restriction endonuclease cleavage of the R8 capable of recognizing and connecting with the end produced by the restriction endonuclease cleavage of the R1;
  • the ends of any one of R2, R3, R4, R5, R6, and R7 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the end of any one of R1, R2, R3, R4, R5, R6, R7, and R8 that is cleaved by a restriction endonuclease is a non-palindromic sequence.
  • two or more of R1, R2, R3, R4, R5, R6, R7, and R8 can be recognized and cleaved by the same restriction endonuclease.
  • said R2 is the same as said R3. In certain embodiments, said R4 is the same as said R5. In certain embodiments, said R6 is the same as said R7. In certain embodiments, said R8 is the same as said R1.
  • the R1, R2, R3, R4, R5, and R8 can be recognized and cleaved by the same restriction endonuclease.
  • the R6 and R7 can be recognized and cleaved by the same restriction endonuclease.
  • the restriction endonuclease is selected from SfiI, BsmBI and Esp3I.
  • the R1, R2, R3, R4, R5, and R8 can be recognized and cut by SfiI.
  • the R6 and R7 can be recognized and cleaved by BsmBI and/or Esp3I.
  • the R1 includes the nucleic acid sequence shown in SEQ ID NO:1.
  • the R2 includes the nucleic acid sequence shown in SEQ ID NO: 2.
  • the R3 includes the nucleic acid sequence shown in SEQ ID NO: 2.
  • the R4 includes the nucleic acid sequence shown in SEQ ID NO: 3.
  • the R5 includes the nucleic acid sequence shown in SEQ ID NO: 3.
  • the R6 includes the nucleic acid sequence shown in SEQ ID NO:4.
  • the R7 includes the nucleic acid sequence shown in SEQ ID NO:4.
  • the R8 includes the nucleic acid sequence shown in SEQ ID NO:1.
  • the light chain or light chain fragment comprises a light chain variable region portion and/or a light chain constant region portion.
  • the heavy chain or heavy chain fragment comprises a heavy chain variable region portion and/or a heavy chain constant region portion.
  • the antibody or fragment thereof includes Fab, Fab', (Fab)2 and/or (Fab')2.
  • 2) includes using a restriction endonuclease that recognizes the R1 and R2 to perform the restriction endonuclease treatment on the first polynucleotide, so that the first polynucleotide after the restriction endonuclease
  • the amino acid and the light chain storage carrier fragment are connected to form a light chain storage connection product, and the light chain storage connection product is introduced into the first bacteria to obtain the light chain component bacterial library; wherein the light chain storage vector comprises The R1 and R2, and the light chain storage vector fragment is obtained by using a restriction endonuclease that recognizes the R1 and R2 to cut the light chain storage vector.
  • the light chain storage vector is derived from a pUC vector.
  • the pUC vector is a pUC19 vector or is derived from a pUC19 vector.
  • the pUC vector is a pMD19-T vector or is derived from a pMD19-T vector.
  • the restriction endonuclease that recognizes the R1 and R2 includes SfiI.
  • 2) includes using a restriction endonuclease that recognizes R5 and R6 to cut the third polynucleotide, so that the cut-treated third polynucleotide
  • the amino acid and the heavy chain storage vector fragment are connected to form a heavy chain storage connection product, and the heavy chain storage connection product is introduced into the third bacteria to obtain the heavy chain component bacterial library; wherein the heavy chain storage vector comprises The R5 and R6, and the heavy chain storage vector fragment is obtained by subjecting the heavy chain storage vector to the restriction endonuclease that recognizes the R5 and R6.
  • the heavy chain storage vector is derived from a pUC vector.
  • the pUC vector is a pUC19 vector or is derived from a pUC19 vector. In some embodiments, the pUC vector is a pMD19-T vector or is derived from a pMD19-T vector.
  • the heavy chain storage vector contains and only contains one recognition site for BsmBI.
  • the restriction endonucleases that recognize the R5 and R6 include SfiI, BsmBI and/or Esp3I.
  • 2) includes ligating the second polynucleotide and the carrier fragment to form a linker storage vector, and introducing the linker storage vector into the second bacterium to obtain the linker Component bacteria.
  • the linker storage carrier contains the R3 and R4.
  • the vector fragment used to construct the linker storage vector is derived from a pUC vector.
  • the pUC vector is a pUC19 vector or is derived from a pUC19 vector.
  • the pUC vector is a pMD19-T vector or is derived from a pMD19-T vector.
  • 4) includes using restriction endonucleases that recognize the R1 and R2 to digest the light chain component plasmid, thereby obtaining the released LC.
  • the restriction endonuclease that recognizes the R1 and R2 includes SfiI.
  • 4) includes using restriction endonucleases that recognize the R5 and R6 to digest the heavy chain component plasmid to obtain the released HC.
  • the restriction endonucleases that recognize the R5 and R6 include SfiI, BsmBI and/or Esp3I.
  • 4) includes using restriction endonucleases that recognize the R3 and R4 to cut the linker storage vector to obtain the released linker.
  • 4) includes obtaining an amplification product from the linker storage vector, the amplification product comprising the linker, the R3 and the R4; and using the linker that recognizes the R3 and R4 Restriction endonuclease digests the amplified product to obtain the released linker.
  • the restriction endonuclease that recognizes the R3 and R4 includes SfiI.
  • 5) includes cutting the display vector with restriction endonucleases that recognize the R7 and R8 to release the display vector fragments.
  • the restriction endonucleases that recognize the R7 and R8 include SfiI, BsmBI and/or Esp3I.
  • the display vector is derived from the pComb3x vector.
  • the display vector is digested with restriction endonucleases that recognize the R1, R2, R3, R4, R5, and R8, three different digested fragments will be obtained.
  • the restriction endonuclease that recognizes the R1, R2, R3, R4, R5, and R8 includes SfiI.
  • the display vector is linearized after the restriction endonuclease that recognizes the R6 and R7 is used to cut the display vector.
  • the restriction endonucleases that recognize R6 and R7 include BsmBI and/or Esp3I.
  • the restriction endonucleases that recognize the R1, R2, R3, R4, R5, R6, R7, and R8 are used to digest the display vector, four different Enzyme digestion fragments.
  • the restriction endonucleases that recognize the R1, R2, R3, R4, R5, R6, R7, and R8 include SfiI, BsmBI and/or Esp3I.
  • 2) includes sampling and testing the light chain component bacterial library, the linker component bacteria and/or the heavy chain component bacterial library to determine the light chain component bacterial library, so The library capacity and/or quality of the linker component bacteria and/or the heavy chain component bacteria library.
  • the light chain component of the bacterial library comprises at least 106 different clones.
  • the heavy chain component of bacterial library comprises at least 107 different clones.
  • the proportion of effective clones in the bacterial library of light chain components is at least about 70%.
  • the proportion of effective clones in the heavy chain component bacterial library is at least about 70%.
  • 2) includes calculating the library of the display bacterial library based on the library capacity of the light chain component bacterial library and/or the heavy chain component bacterial library, the linker component bacteria and/or quality Capacity and/or quality.
  • the display bacterial library contains at least 10 10 different clones.
  • the ratio of effective clones in the display bacterial library is at least about 70%.
  • the first polynucleotide and the third polynucleotide are obtained from sample material.
  • the sample material includes a material derived from a sample of peripheral blood lymphocytes.
  • the peripheral blood lymphocytes are human peripheral blood lymphocytes.
  • the sample material includes total RNA and/or mRNA derived from the sample.
  • 8) comprises contacting the displayed bacterial library with helper phage to prepare the phage library.
  • the present application provides a phage library produced by the method.
  • the phage library contains at least 10 10 different clones.
  • the phage library can display at least 10 10 different antibodies or antibody fragments.
  • the present application provides a method for screening antibodies or antibody fragments, which includes using the phage library.
  • Figure 1 shows a schematic diagram of the pUC19 vector.
  • Figure 2 shows a schematic diagram of the modified pUC19 vector in this application.
  • Figure 3 shows a schematic diagram of the light chain storage carrier described in the present application.
  • Figure 4 shows a schematic diagram of the linker storage carrier described in the present application.
  • FIG. 5 shows a schematic diagram of the heavy chain storage carrier described in this application.
  • Figure 6 shows a schematic diagram of the pCom3x vector.
  • Figure 7 shows a schematic diagram of the display vector described in this application.
  • Figure 8 shows a schematic diagram of a plasmid containing the ligation product for display of the present application.
  • phage library generally refers to a library containing polynucleotides encoding different recombinant polypeptides that can be displayed on phage.
  • the phage library may also include a collection of phage transfected with the polynucleotide library.
  • the library may refer to a diverse collection or mixture of polynucleotides, which may include polynucleotides encoding different recombinant polypeptides.
  • the polynucleotide library collection may contain at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or more different polynucleotides. acid.
  • the polynucleotides in the phage library may be derived from a single species (such as mammals, such as humans), its tissues, organs and/or cells.
  • the library may comprise polynucleotides of a common genus.
  • the genus may be a polynucleotide encoding a certain type or class of immunoglobulin subunit polypeptides.
  • the polynucleotide may encode the light chain or light chain fragment of an antibody.
  • the polynucleotide may encode a heavy chain or heavy chain fragment of an antibody.
  • the term "antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein, which is composed of two identical light chains and two identical heavy chains.
  • each L chain is connected to the H chain through a covalent disulfide bond, while the two H chains are connected to each other through one or more disulfide bonds.
  • the number of disulfide bonds depends on the same species of the H chain. type.
  • Each H and L chain also has regularly spaced intrachain disulfide bonds.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain) or four (for mu and epsilon isotypes) constant domains (CH).
  • VH variable domain
  • CH constant domains
  • the term "antibody fragment” generally refers to a part of a complete antibody.
  • the antibody fragment may comprise the antigen binding region and/or antibody variable region of a complete antibody.
  • the antibody fragments can be obtained by chemical methods and/or genetic engineering methods.
  • proteases including pepsin and papain, can be used to digest the antibody to produce the antibody fragment.
  • the antibody fragment may be Fab.
  • Fab generally refers to the production of two identical antigen-binding fragments after papain digests an antibody with a complete structure (for example, the Fc region and the hinge region are removed).
  • the Fab can be composed of a complete light chain, the variable region of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain.
  • VH variable region of the heavy chain
  • CH1 first constant domain
  • linker generally refers to a reagent capable of connecting two or more polynucleotide molecules or fragments thereof.
  • the linker may be a polynucleotide or a fragment thereof.
  • the linker can have different lengths.
  • the length of the linker may be 40bp or more, 50bp or more, 60bp or more, 70bp or more, 80bp or more, 90bp or more, 100bp or more, 150bp or more, 200bp or more or more.
  • the term "LC” generally refers to a polynucleotide comprising a nucleic acid sequence encoding the light chain or light chain fragment of an antibody or antibody fragment.
  • the light chain or light chain fragment may have the ability to bind to the heavy chain of the same or similar antibody.
  • the light chain or light chain fragment may comprise a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region can be divided into ⁇ type and ⁇ type.
  • the light chain also includes a light chain having a ⁇ variable region (V- ⁇ ) connected to a ⁇ constant region (C- ⁇ ) or a ⁇ variable region (V- ⁇ ) connected to a ⁇ constant region (C- ⁇ ). chain.
  • the LC may be a polynucleotide comprising a nucleic acid sequence encoding the light chain of an antibody or antibody fragment.
  • the term "HC” generally refers to a polynucleotide comprising a nucleic acid sequence encoding a heavy chain or heavy chain fragment of an antibody or antibody fragment.
  • the heavy chain or heavy chain fragment may have the ability to bind to the light chain of the same or similar antibody.
  • the heavy chain or heavy chain fragment may include a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region may include a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.
  • the heavy chain constant region may include a CH4 domain but not a hinge region.
  • the "heavy chain constant region" can be CH1, hinge region, CH2, CH3, CH4 domain or any combination thereof.
  • the HC may be a polynucleotide comprising nucleic acid sequences encoding the VH and CH1 of the antibody or antibody fragment.
  • first polynucleotide generally refers to a polynucleotide comprising LC, which may be a nucleic acid sequence comprising a light chain or light chain fragment encoding the antibody or antibody fragment.
  • the first polynucleotide may also have an endonuclease (for example, restriction endonuclease) recognition site at the 5'end and/or 3'end.
  • the first polynucleotide may comprise the structure R1-LC-R2 in a 5'to 3'direction, wherein the R1 and R2 may be recognition sites of restriction endonucleases.
  • the first polynucleotide is digested with an endonuclease that recognizes the endonuclease recognition site in the first polynucleotide (for example, a restriction endonuclease that recognizes R1 and R2, such as SfiI), the first polynucleotide is digested
  • the processed first polynucleotide may comprise the LC.
  • the term "light chain storage vector fragment” generally refers to a fragment of a light chain storage vector that has been digested with restriction endonuclease.
  • the recognition site of the restriction endonuclease contained in the light chain storage vector may be correspondingly the same as the recognition site of the restriction endonuclease contained in the first polynucleotide .
  • the light chain storage carrier may include the R1 and the R2.
  • the obtained light chain storage vector fragment after being digested with a restriction endonuclease (for example, a restriction endonuclease that recognizes the R1 and the R2), the obtained light chain storage vector fragment can be digested with The first polynucleotide is linked.
  • the light chain storage vector fragment may be derived from a pUC vector.
  • the pUC vector may be a pUC19 vector or be derived from a pUC19 vector; for another example, the pUC vector may be a pMD19-T vector or be derived from a pMD19-T vector.
  • the term "light chain storage ligation product” generally refers to the product formed by ligating the first polynucleotide after the digestion treatment with the light chain storage carrier fragment.
  • the first polynucleotide and the light chain storage vector fragment may contain the same restriction endonuclease recognition site, and may be ligated by a ligase (such as DNA ligase) to obtain The light chain stores the connection product.
  • first bacterium generally refers to the bacterium used to introduce or contain the first polynucleotide.
  • the first bacterium may include the LC.
  • the first bacterium can be introduced into the light chain to store the ligation product.
  • the first bacterium can express, replicate and/or store (for example, cryopreserve) the LC, the first polynucleotide and/or the light chain storage ligation product.
  • the term "light chain component bacterial library” generally refers to a bacterial library obtained by introducing the first polynucleotide into the first bacterium.
  • the light chain component bacterial library may be a bacterial library containing a nucleic acid sequence encoding the light chain or light chain fragment of an antibody or antibody fragment.
  • the assembly of bacterial light chain libraries may comprise from about 105-- about 109 (e.g., may comprise from about 105-- 8 to about 10, from about 105-- 7 to about 10, from about 106-- about 10 7 ) Nucleic acid sequences encoding different light chains or light chain fragments of antibodies or antibody fragments.
  • the bacterial library of light chain components may include about 10 7 to about 10 12 (for example, about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 7 to about 10 109, about 107-- 8 to about 10) of the first bacterium.
  • the term "second polynucleotide” generally refers to a polynucleotide comprising a linker.
  • the second polynucleotide may also have an endonuclease (e.g., restriction endonuclease) recognition site at the 5'end and/or 3'end.
  • the first polynucleotide may include the structure R3-linker-R4 in a 5'to 3'direction, wherein the R3 and R4 may be recognition sites for restriction endonucleases.
  • the second polynucleotide is digested with an endonuclease that recognizes the endonuclease recognition site in the second polynucleotide (for example, a restriction endonuclease that recognizes R3 and R4, such as SfiI).
  • an endonuclease that recognizes the endonuclease recognition site in the second polynucleotide for example, a restriction endonuclease that recognizes R3 and R4, such as SfiI
  • the second polynucleotide is digested
  • the processed second polynucleotide may include the linker.
  • the term "vector fragment” when used together with a second polynucleotide or a linker, generally refers to a vector fragment that can be linked to the second polynucleotide.
  • the vector fragment may be derived from a pUC vector.
  • the pUC vector may be a pUC19 vector or be derived from a pUC19 vector; for another example, the pUC vector may be a pMD19-T vector or be derived from a pMD19-T vector.
  • the term "linker storage vector” generally refers to the product formed by joining the second polynucleotide and the vector fragment.
  • the recognition site of the restriction endonuclease contained in the linker storage vector may be correspondingly the same as the recognition site of the restriction endonuclease contained in the first polynucleotide.
  • the linker storage carrier may include the R3 and the R4. In the present application, the linker storage carrier may include only one kind of the linker.
  • the term "second bacterium” generally refers to a bacterium used to introduce or contain the second polynucleotide.
  • the second bacterium may include the linker.
  • the second bacteria may be introduced into the linker storage vector.
  • the second bacterium may express, replicate and/or store (for example, cryopreserve) the linker and/or the second polynucleotide.
  • linker assembly bacteria generally refers to bacteria obtained by introducing the linker storage vector into the second bacteria.
  • the linker component bacteria may be a single species of bacteria.
  • a single species of the linker component bacteria may only include a single species of the linker.
  • the term "third polynucleotide” generally refers to a polynucleotide comprising HC, which may be a nucleic acid sequence comprising a heavy chain or heavy chain fragment encoding the antibody or antibody fragment.
  • the third polynucleotide may also have an endonuclease (for example, restriction endonuclease) recognition site at the 5'end and/or 3'end.
  • the first polynucleotide may comprise the structure R5-HC-R6 in a 5'to 3'direction, wherein the R5 and R6 may be recognition sites of restriction endonucleases.
  • an endonuclease that recognizes the endonuclease recognition site in the third polynucleotide for example, a restriction endonuclease that recognizes R5, such as SfiI; another example, a restriction endonuclease that recognizes R6
  • Nucleases, such as SfiI, BsmBI and Esp3I) after the digestion treatment, the digested third polynucleotide may contain the HC.
  • the term "heavy chain storage vector fragment” generally refers to a fragment of a heavy chain storage vector that has been digested with restriction endonuclease.
  • the recognition site of the restriction endonuclease contained in the heavy chain storage vector may be correspondingly the same as the recognition site of the restriction endonuclease contained in the third polynucleotide .
  • the heavy chain storage carrier may include the R5 and the R6.
  • the resulting heavy chain storage vector fragment after being digested with a restriction endonuclease (for example, a restriction endonuclease that recognizes the R5 and the R6), the resulting heavy chain storage vector fragment can be digested with The third polynucleotide is linked.
  • the heavy chain storage vector fragment may be derived from a pUC vector.
  • the pUC vector may be a pUC19 vector or be derived from a pUC19 vector; for another example, the pUC vector may be a pMD19-T vector or be derived from a pMD19-T vector.
  • the term “heavy chain storage ligation product” generally refers to the product formed by the ligation of the third polynucleotide after the digestion treatment with the heavy chain storage carrier fragment.
  • the third polynucleotide and the heavy chain storage vector fragment may contain the same restriction endonuclease recognition site, and may be ligated by a ligase (such as DNA ligase) to obtain The heavy chain stores the connection product.
  • the term "third bacterium” generally refers to the bacterium used to introduce or contain the third polynucleotide.
  • the third bacterium may include the HC.
  • the third bacteria can be introduced into the heavy chain to store the ligation product.
  • the third bacterium can express, replicate, and/or store (for example, cryopreserve) the HC, the third polynucleotide, and/or the heavy chain storage ligation product.
  • the term "heavy chain component bacterial library” generally refers to a bacterial library obtained by introducing the third polynucleotide into the third bacteria.
  • the heavy chain component bacterial library may be a bacterial library comprising a nucleic acid sequence encoding a heavy chain or heavy chain fragment of an antibody or antibody fragment.
  • the heavy chain component of bacterial library may comprise from about 105-- about 109 (e.g., may comprise from about 105-- 8 to about 10, from about 105-- 7 to about 10, from about 106-- about 10 7 ) Nucleic acid sequences encoding different heavy chains or heavy chain fragments of antibodies or antibody fragments.
  • the heavy chain component bacterial library may comprise about 10 7 to about 10 12 (for example, it may comprise about 10 7 to about 10 11 , about 10 7 to about 10 10 , about 10 7 to about 10 109, about 107-- 8 to about 10) and the third bacterium.
  • the term "light chain component plasmid” generally refers to a plasmid containing the LC obtained from bacteria in the light chain component bacterial library.
  • the light chain component plasmid may include the LC.
  • the light chain assembly plasmid may include the first polynucleotide.
  • the light chain component plasmid may include restriction endonuclease recognition sites, for example, may include R1 and R2.
  • the term "heavy chain component plasmid” generally refers to a plasmid containing the HC obtained from bacteria in the heavy chain component bacterial library.
  • the heavy chain component plasmid may include the HC.
  • the heavy chain component plasmid may include the third polynucleotide.
  • the heavy chain component plasmid may include restriction endonuclease recognition sites, for example, may include R5 and R6.
  • the term "released LC” generally refers to the LC released after the light chain component plasmid is processed.
  • the treatment may be restriction digestion treatment.
  • a suitable restriction endonuclease for example, a restriction endonuclease that recognizes R1 and R2
  • R1 and R2 a restriction endonuclease that recognizes R1 and R2
  • the term "released linker” generally refers to the linker released after the linker component plasmid is processed.
  • the treatment may be restriction digestion treatment.
  • a suitable restriction endonuclease for example, a restriction endonuclease that recognizes R3 and R4
  • R3 and R4 can be selected for the recognition site of the restriction endonuclease on the linker assembly plasmid, so that The linker is released from the linker assembly plasmid and separated.
  • the linker component plasmid may be used as a template to amplify (for example, use PCR amplification) containing the second polynucleotide, and then the obtained amplified product may be subjected to restriction treatment.
  • a suitable restriction endonuclease for example, a restriction endonuclease that recognizes R3 and R4 can be selected for the recognition site of the restriction endonuclease on the linker assembly plasmid, so that The linker is released from the linker assembly plasmid and separated.
  • the term "released HC" generally refers to the HC released after the heavy chain component plasmid is processed.
  • the treatment may be restriction digestion treatment.
  • a suitable restriction endonuclease such as R5 and R6 can be selected for the recognition site of the restriction endonuclease on the heavy chain assembly plasmid, so that the HC can be removed from the heavy chain assembly.
  • the plasmid is released and isolated.
  • the term "released display vector fragment” generally refers to a fragment of the display vector released after the display vector is processed.
  • the display vector may include the structure R7-display vector fragment-R8 in the 5'to 3'direction, wherein the R7 and R8 may be restriction endonucleases.
  • the treatment may be restriction endonuclease digestion treatment.
  • a suitable restriction endonuclease for example, R7 and R8 can be selected for the recognition site of the restriction endonuclease on the display vector, so that the released display vector fragment can be removed from the The display carrier is released and separated.
  • the term "linked” generally refers to linking two or more polynucleotide molecules together.
  • the ligation can be achieved by a ligase (e.g., DNA ligase).
  • a ligase e.g., DNA ligase
  • the 3'end of one polynucleotide is connected to the 5'end of another polynucleotide to form a complete polynucleotide molecule.
  • the term "ligation product for display” generally refers to a polynucleoside comprising the released LC, the released linker, the released HC, and the released display vector fragment Acid molecule.
  • the display ligation product can be obtained by mixing the released LC, the released linker, the released HC, and the released display vector fragment in proportion.
  • the "display” may refer to allowing cells containing the display ligation product to express the protein (for example, antibody or antibody fragment, for example, Fab) encoded by the display ligation product.
  • the protein for example, antibody or antibody fragment, for example, Fab
  • the term "introduction” generally refers to the process of transferring or introducing exogenous polynucleotides into cells.
  • the cell may be a host cell.
  • the introduced cells include primary cells of the subject and their progeny.
  • the cell may be a prokaryotic cell, for example, a bacterial cell.
  • the term "fourth bacteria” generally refers to bacteria used to introduce or contain the display ligation product, wherein the display ligation product includes the released LC, the released linker , The released HC and the released display vector fragment.
  • the fourth bacterium may be transformed into the ligation product for display.
  • the fourth bacterium can express the light chain or light chain fragment of the antibody or antibody fragment encoded by the released LC; it can express the heavy chain of the antibody or antibody fragment encoded by the released HC. Chain or heavy chain fragments.
  • the fourth bacterium can express, replicate and/or store (for example, cryopreserve) the antibody or antibody fragment.
  • the term "display bacterial library” generally refers to a bacterial library containing the ligation product for display.
  • the bacterial library for display may include the fourth bacteria.
  • the displayed bacterial library may contain more than 10 10 different antibodies or antibody fragments.
  • signal peptide pelB generally refers to the signal peptide of pectinase lyase.
  • the signal peptide pelB is usually used in prokaryotic expression systems.
  • accession number of the signal peptide pelB in GenBank may be ABS75961.1.
  • restriction endonuclease generally refers to an enzyme that cuts double-stranded DNA.
  • the restriction endonuclease can produce sticky ends with protruding single-stranded DNA, which can be bonded with DNA ligase.
  • the restriction endonuclease may have the functions of recognition and restriction cleavage.
  • the cutting site of the restriction endonuclease has a certain distance from the recognition site.
  • the restriction endonuclease can be selected from SfiI, BsmBI and Esp3I.
  • linearization generally means that the vector is linear rather than closed loop.
  • the vector such as T vector
  • the original circular structure of the vector is destroyed, thereby forming a linearized vector.
  • the vector such as T vector
  • a 3'-end T-terminal can be generated.
  • the PCR product with the A-terminal can be directly used by the T4 ligase to generate the PCR
  • the product is connected to the linearized carrier.
  • the term "clone” generally refers to the number of colonies.
  • the clone may be the number of colonies in a bacterial library (eg, the light chain component bacterial library, the heavy chain component bacterial library, the display bacterial library, and/or the phage library).
  • the clone may be the number of different colonies in the bacterial library.
  • the clone may be the number of progeny populations produced by a single clone.
  • polynucleotide generally refers to nucleotides, that is, at least two nucleotides linked together.
  • the polynucleotide may be a polymer of any length, including, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, 100,000, etc.
  • the polynucleotide may contain phosphodiester bonds.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a method for generating a phage library displaying antibodies or antibody fragments, the method comprising: 1) providing a first polynucleotide, a second polynucleotide and a third polynucleotide, so The first polynucleotide comprises LC, the second polynucleotide comprises a linker and the third polynucleotide comprises HC, wherein the LC comprises a light chain or light chain encoding the antibody or antibody fragment.
  • the nucleic acid sequence of the chain fragment, the HC comprises the nucleic acid sequence encoding the heavy chain or heavy chain fragment of the antibody or antibody fragment; 2) introducing the first polynucleotide into the first bacterium to obtain the light chain component bacterium Library, introducing the second polynucleotide into a second bacterium to obtain a linker component bacteria, and introducing the third polynucleotide into a third group of bacteria to obtain a heavy chain component bacterial library; 3)
  • the light chain component bacterial library obtains a light chain component plasmid containing the LC, the linker component bacteria obtains the linker component plasmid containing the linker, and the heavy chain component bacterial library obtains the HC 4) Obtain the released LC from the light chain assembly plasmid, obtain the released linker from the linker assembly plasmid, and obtain the released HC from the heavy chain assembly plasmid; 5 ) Provide a display vector, and obtain a released display vector fragment from the
  • the display ligation product of 6 can express the light chain or light chain fragment and the heavy chain or heavy chain fragment in frame.
  • the first polynucleotide, the second polynucleotide and the third polynucleotide may all be linear nucleic acid molecules.
  • the nucleic acid sequence encoding the heavy chain or the heavy chain fragment and the nucleic acid sequence encoding the light chain or the light chain fragment may be in different reading frames.
  • the reading frame may be a continuous nucleotide sequence starting from the start codon to the end of the stop codon, and it no longer contains a promoter or a terminator inside.
  • the reading frame can encode a protein or polypeptide fragment.
  • the linker may include a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof.
  • the linker may include a nucleic acid sequence encoding a fragment of the signal peptide pelB, and the remaining nucleic acid sequence encoding a fragment of the signal peptide pelB may also be located in the R5.
  • the nucleotide at the 3'end of the nucleic acid sequence encoding the fragment of the signal peptide pelB may be located in the R5.
  • the nucleic acid sequence of the fragment encoding the signal peptide pelB contained in the released linker may be the same as that contained in the released HC.
  • the nucleic acid sequences encoding the fragments of the signal peptide pelB are joined to form the nucleic acid sequence encoding the complete signal peptide pelB.
  • the length of the linker may be about 50 to about 200 bases.
  • the length of the linker may be about 50 to about 200 bases, about 50 to about 180 bases, about 50 to about 160 bases, about 50 to about 140 bases, about 50 to about 120 bases, about 50 to about 100 bases, about 50 to about 90 bases, about 50 to about 80 bases, about 50 to about 75 bases, about 50 to about 70 bases, About 50 to about 60 bases.
  • the LC in the ligation product for display of 6), the LC may be located upstream of the linker, and the linker may be located upstream of the HC.
  • the upstream may be the 5'end of the nucleotide sequence.
  • cryopreservation may refer to storage at a temperature lower than -18°C, for example, storage at a temperature lower than -80°C.
  • 8) may include thawing and culturing the cryopreserved display bacterial library, and using the cultured display bacterial library to construct the phage library.
  • the first polynucleotide may include the structure R1-LC-R2 in the 5'to 3'direction
  • the second polynucleotide may include the structure R3-linked in the 5'to 3'direction
  • the third polynucleotide may include the structure R5-HC-R6 in the 5'to 3'direction
  • the display vector may include the structure R7-display vector fragment- in the 5'to 3'direction R8, wherein the R1, R2, R3, R4, R5, R6, R7 and R8 are recognition sites for restriction endonucleases.
  • the end produced by the restriction endonuclease cleavage of the R2 capable of recognizing and connecting with the end produced by the restriction endonuclease cleavage of the R3;
  • the ends of any one of R4, R5, R6, R7, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the R2 may be the same as the R3.
  • the end produced by the restriction endonuclease cleavage of the R4 can recognize and be connected to the end produced by the restriction endonuclease cleavage of the R5;
  • the ends of any one of R2, R3, R6, R7, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the R4 may be the same as the R5.
  • the end produced by the restriction endonuclease cleavage of the R6 can recognize and connect to the end produced by the restriction endonuclease cleavage of the R7;
  • the ends of any one of R2, R3, R4, R5, and R8 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the R6 may be the same as the R7.
  • the end produced by the restriction endonuclease cleavage of the R8 can recognize and connect to the end produced by the restriction endonuclease cleavage of the R1; and is not connected to the R2
  • the ends of any one of R3, R4, R5, R6, and R7 that are cleaved by restriction endonucleases recognize or connect to each other.
  • the R8 may be the same as the R1.
  • the end generated by any one of R1, R2, R3, R4, R5, R6, R7, and R8 after being cleaved by a restriction endonuclease may be a non-palindromic sequence.
  • variable region of the antibody for example, the light chain variable region or the heavy chain can be selected
  • the sequence that is basically not included in the variable region is to maintain the integrity of the variable region of the antibody as much as possible to prevent the destruction (for example, degradation) of the antibody gene library during digestion.
  • the end formed by the restriction endonuclease cleavage of the nuclease recognition site may be a non-palindromic sequence to prevent self-ligation, thereby reducing non-intended linkage.
  • selection of the recognition site of the endonuclease can make the directional connection of multiple fragments possible to improve the connection efficiency.
  • R1, R2, R3, R4, R5, R6, R7, and R8 can be recognized and cleaved by the same restriction endonuclease.
  • the R1, R2, R3, R4, R5 and R8 can be recognized and cleaved by the same restriction endonuclease.
  • the R6 and R7 can be recognized and cleaved by the same restriction endonuclease.
  • the restriction endonucleases that recognize the R1, R2, R3, R4, R5, and R8 may be different from the restriction endonucleases that recognize the R6 and R7.
  • the restriction endonuclease can be selected from SfiI, BsmBI and Esp3I.
  • the BsmBI and Esp3I can be isoenzymes, which can recognize the same recognition site of restriction endonuclease.
  • the R1, R2, R3, R4, R5, and R8 can be recognized and cut by SfiI.
  • the R6 and R7 can be recognized and cleaved by BsmBI and/or Esp3I.
  • SfiI can recognize the sequence (5' to 3') GGCCNNNN/NGGCC (SEQ ID NO: 99) consisting of 13 bases, which can form an overhang at the 3'end (for example, containing 3 Single-stranded sequence of bases), where N can represent any of the four bases of GATC. Therefore, there are 64 different sequences that can be recognized by SfiI.
  • BsmBI and Esp3I can recognize a sequence consisting of 12 bases (5' to 3') CGTCTCN/NNNNN (SEQ ID NO: 100), which can form an overhang at the 5'end (for example, including Single-stranded sequence of 4 bases), where N can represent any of the four bases of GATC. Therefore, there are 264 different sequences that can be recognized by BsmBI and Esp3I.
  • the R1 may include the nucleic acid sequence shown in SEQ ID NO:1.
  • the R2 may include the nucleic acid sequence shown in SEQ ID NO: 2.
  • the R3 may include the nucleic acid sequence shown in SEQ ID NO: 2.
  • the R4 may include the nucleic acid sequence shown in SEQ ID NO:3.
  • the R5 may include the nucleic acid sequence shown in SEQ ID NO: 3.
  • the R6 may include the nucleic acid sequence shown in SEQ ID NO:4.
  • the R7 may include the nucleic acid sequence shown in SEQ ID NO:4.
  • the R8 may include the nucleic acid sequence shown in SEQ ID NO:1.
  • the light chain or light chain fragment may comprise a light chain variable region part and/or a light chain constant region part.
  • the heavy chain or heavy chain fragment may comprise a heavy chain variable region part and/or a heavy chain constant region part.
  • the LC may comprise a polynucleotide encoding the light chain variable region VL of the light chain or light chain fragment.
  • the first polynucleotide may include a polynucleotide encoding the light chain variable region VL of the light chain or light chain fragment.
  • the 5'end of the first polynucleotide can be connected to the R1 and the 3'end of the first polynucleotide can be connected to the R2; and, the 5'end of the light chain storage vector fragment can be The R3 and the 3'end of the light chain storage vector fragment can be connected to the R8.
  • the HC may include a polynucleotide encoding the VH of the heavy chain variable region of the heavy chain or the heavy chain fragment and the CH1 of the heavy chain constant region.
  • the third polynucleotide may include a polynucleotide encoding the heavy chain variable region VH and the heavy chain constant region CH1 of the heavy chain or heavy chain fragment.
  • the 5'end of the third polynucleotide can be connected to the R5 and the 3'end of the third polynucleotide can be connected to the R6; and, the 5'end of the heavy chain storage vector fragment can be The R7 is connected and the 3'end of the heavy chain storage vector fragment can be connected to the R4.
  • the HC may include a polynucleotide encoding the VH of the heavy chain variable region of the heavy chain or heavy chain fragment.
  • the third polynucleotide may include a polynucleotide encoding the heavy chain variable region VH of the heavy chain or heavy chain fragment.
  • the heavy chain storage vector fragment may include a polynucleotide encoding CH1 of the heavy chain constant region of the heavy chain or the heavy chain fragment.
  • the 5'end of the third polynucleotide can be connected to the R5 and the 3'end of the third polynucleotide can be connected to the R6; and, the 5'end of the heavy chain storage vector fragment can be The R7 is connected and the 3'end of the heavy chain storage vector fragment can be connected to the R4.
  • the antibody or fragment thereof may include Fab, Fab', (Fab)2 and/or (Fab')2.
  • 7) in connection with the conversion efficiency of the display may be connected to the product of at least 108 clones / ⁇ g polynucleotides (for example, / ⁇ g polynucleotide of at least 109 clones, 10 of at least 10 Clones/ ⁇ g polynucleotide, at least 10 11 clones/ ⁇ g polynucleotide, at least 10 12 clones/ ⁇ g polynucleotide).
  • 2) may include using restriction endonucleases that recognize the R1 and R2 to cut the first polynucleotide so that the first polynucleotide that has been cut
  • the acid is connected with the light chain storage carrier fragment to form a light chain storage connection product, and the light chain storage connection product is introduced into the first bacteria to obtain the light chain component bacterial library;
  • the light chain storage vector may comprise The R1 and R2, and the light chain storage vector fragment is obtained by using a restriction endonuclease that recognizes the R1 and R2 to cut the light chain storage vector.
  • the length of the LC may be greater than about 600 bp. For example, it can be greater than about 650 bp, can be greater than about 680 bp, or greater than about 700 bp.
  • the length of the first polynucleotide may be greater than about 600 bp.
  • the length of the light chain storage vector fragment may be greater than about 900 bp, for example, may be greater than about 1000 bp, greater than about 1500 bp, greater than about 1800 bp, greater than about 2100 bp.
  • the length of the light chain storage carrier fragment may be significantly different from the length of the LC.
  • the length of the light chain storage carrier fragment can be significantly distinguished from the length of the LC by means of polyacrylamide gel electrophoresis.
  • the light chain storage vector can be derived from any vector, for example, any vector that can be amplified and/or easily stored.
  • the carrier used as a light chain storage carrier may have properties such as high copy number and low molecular weight.
  • the light chain storage vector may be a pUC vector.
  • the pUC vector can be a pUC19 vector (the schematic diagram of the structure can be shown in Figure 1) or derived from the pUC19 vector.
  • the pUC vector can be a pMD19-T vector or derived from a pMD19-T vector.
  • restriction endonuclease that recognizes the R1 and R2 may include SfiI.
  • 2) may include using restriction endonucleases that recognize the R5 and R6 to cut the third polynucleotide so that the cut-treated third polynucleotide is The acid is connected with the heavy chain storage carrier fragment to form a heavy chain storage connection product, and the heavy chain storage connection product is introduced into the third bacteria to obtain the heavy chain component bacterial library; wherein the heavy chain storage vector contains all The R5 and R6, and the heavy chain storage vector fragment is obtained by subjecting the heavy chain storage vector to the restriction endonuclease that recognizes the R5 and R6.
  • the length of the HC may be greater than about 320 bp. For example, it may be greater than about 350 bp, may be greater than about 380 bp, or greater than about 400 bp.
  • the length of the third polynucleotide may be greater than about 320 bp.
  • the length of the heavy chain storage vector fragment may be greater than about 900 bp, for example, may be greater than about 1000 bp, greater than about 1500 bp, greater than about 1800 bp, greater than about 2100 bp.
  • the length of the heavy chain storage carrier fragment may be significantly different from the length of the HC.
  • the length of the light chain storage carrier fragment can be significantly distinguished from the length of the HC by means of polyacrylamide gel electrophoresis.
  • the heavy chain storage vector can be derived from any vector, for example, any vector that can be amplified and/or easily stored.
  • the carrier used as a heavy chain storage carrier may have properties such as high copy number and low molecular weight.
  • the heavy chain storage vector may be a pUC vector.
  • the pUC vector can be a pUC19 vector (the schematic diagram of the structure can be shown in Figure 1) or derived from the pUC19 vector.
  • the pUC vector can be a pMD19-T vector or derived from a pMD19-T vector.
  • the pUC vector or the vector derived from pUC can be engineered/modified.
  • one or more endonuclease recognition sites in the vector can be removed by site-directed mutagenesis (for example, one or more recognition sites of BsmBI are removed).
  • one or more endonuclease recognition sites can also be added to the vector through site-directed mutagenesis (for example, one or more recognition sites of BsmBI are added at selected positions).
  • one or more BsmBI recognition sites originally contained in the vector can be removed by site-directed mutagenesis, and then one or more additional BsmBI recognition sites can be added to another position in the vector To obtain a modified vector (for example, a modified pUC vector).
  • the heavy chain storage carrier may contain and only contain one recognition site of BsmBI.
  • the restriction endonuclease that recognizes the R5 and R6 may include SfiI, BsmBI and/or Esp3I. In some cases, the restriction endonucleases that recognize R5 and R6 are BsmBI and/or Esp3I.
  • 2) may include ligating the second polynucleotide and the vector fragment to form a linker storage vector (for example, according to the instructions of Takara's TA cloning product), and introducing the linker storage vector into all In the second bacteria to obtain the linker assembly bacteria.
  • the linker storage vector may contain the R3 and R4 sites.
  • the length of the linker may be greater than about 70 bp, for example, it may be about 72 bp, or it may be about 90 bp.
  • the length of the second polynucleotide may be greater than about 80 bp, for example, it may be greater than about 90 bp, may be greater than about 100 bp, and may be greater than about 120 bp.
  • the length of the vector fragment may be greater than about 800 bp.
  • the vector fragment used to construct the linker storage vector may be derived from any vector, for example, any vector that can be amplified and/or easily stored.
  • the vector used to construct the linker storage vector may have properties such as high copy number and small molecular weight.
  • the vector fragment used to construct the linker storage vector may be derived from a pUC vector.
  • the pUC vector can be a pUC19 vector (the schematic diagram of the structure can be shown in Figure 1) or derived from the pUC19 vector.
  • the pUC vector can be a pMD19-T vector or derived from a pMD19-T vector.
  • the pUC19 vector or pMD19-T vector includes vectors derived therefrom but modified/engineered.
  • 4) may include using restriction endonucleases that recognize the R1 and R2 to digest the light chain component plasmid to obtain the released LC.
  • the restriction endonuclease that recognizes the R1 and R2 may include SfiI.
  • 4) may include using restriction endonucleases that recognize the R5 and R6 to digest the heavy chain component plasmid to obtain the released HC.
  • the restriction endonuclease that recognizes the R5 and R6 may include SfiI, BsmBI and/or Esp3I.
  • 4) may include the use of restriction endonucleases that recognize the R3 and R4 to cut the linker storage vector to obtain the released linker .
  • the restriction endonuclease (for example, SfiI) that recognizes the R3 and R4 can be directly used to cut the linker storage vector to obtain the released linker.
  • the length of the linker can be about 72 bp or more, for example, it can be about 90 bp or more.
  • 4) may include obtaining an amplification product from the linker storage vector, the amplification product including the linker, the R3 and the R4; and using the recognition method The restriction endonucleases of R3 and R4 cut the amplified product to obtain the released linker.
  • the amplification product including the linker, the R3 and the R4 can be obtained by methods such as PCR, and the length of the amplification product can be about 800 bp or more. For example, it can be about 900 bp or more, it can be about 1000 bp or more, and it can be about 1200 bp or more.
  • a restriction endonuclease (for example, SfiI) that recognizes the R3 and R4 can be used to digest the amplified product to obtain the released linker.
  • the length of the linker can be about 72 bp or more, and can be about 90 bp or more.
  • the length of the amplification product may be significantly different from the length of the linker. In this way, a single linker can be obtained more easily and efficiently.
  • the length of the amplified product can be significantly distinguished from the length of the linker by means of polyacrylamide gel electrophoresis.
  • restriction endonuclease that recognizes the R3 and R4 may include SfiI.
  • 5) may include cutting the display vector with restriction endonucleases that recognize the R7 and R8 to release the display vector fragments.
  • the restriction endonuclease that recognizes the R7 and R8 may include SfiI, BsmBI and/or Esp3I.
  • the display vector can be derived from any suitable vector, such as the pComb3x vector.
  • the pComb3x vector can be modified to be suitable for the purposes of this application.
  • the pComb3x vector may include a SfiI recognition site at the 5'end and a SfiI recognition site at the 3'end.
  • site-directed mutagenesis can be used to remove the SfiI recognition site at the 3'end of the pComb3x vector. Site.
  • the site-directed mutation may not affect the in-frame expression of the protein (eg, antibody heavy chain fragment and/or antibody light chain fragment) in the vector.
  • the mutation may be a nonsense mutation, such as a mutation that only changes the base sequence but does not change the amino acid sequence.
  • the display vector after the display vector is digested with restriction endonucleases that recognize the R1, R2, R3, R4, R5, and R8, three different digested fragments can be obtained.
  • the display vector may include 3 restriction endonuclease recognition sites that can be recognized by SfiI.
  • SfiI restriction endonuclease recognition sites that can be recognized by SfiI.
  • the restriction endonuclease that recognizes the R1, R2, R3, R4, R5, and R8 may include SfiI.
  • the display vector can be linearized after the restriction endonuclease that recognizes the R6 and R7 is used to cut the display vector.
  • restriction endonucleases that recognize the R6 and R7 may include BsmBI and/or Esp3I.
  • the display vector may include 3 restriction endonuclease recognition sites that can be recognized by SfiI, and 1 restriction endonuclease recognition site that can be recognized by BsmBI and/or Esp3I.
  • SfiI restriction endonuclease recognition sites
  • BsmBI restriction endonuclease recognition site
  • Esp3I restriction endonuclease recognition site
  • restriction endonucleases R1 and R2 to obtain the released LC
  • restriction endonucleases R5 and R6 to obtain the released HC
  • restriction endonucleases that recognize the R1, R2, R3, R4, R5, R6, R7, and R8 may include SfiI, BsmBI and/or Esp3I.
  • the vector may contain or encode one or more suitable markers (for example, markers for purification/recognition/screening), the markers It can be, for example, His tag, Flag Tag, fluorescent protein, selective antibiotic, and/or avidin.
  • suitable markers for example, markers for purification/recognition/screening
  • the markers It can be, for example, His tag, Flag Tag, fluorescent protein, selective antibiotic, and/or avidin.
  • 2) may include sampling and testing the light chain component bacterial library, the linker component bacteria and/or the heavy chain component bacterial library to determine the light chain component bacterial library, The library capacity and/or quality of the linker component bacteria and/or the heavy chain component bacteria library.
  • the assembly of bacterial light chain libraries may comprise at least about 106 (e.g., at least about 107, at least about 108, at least about 109, at least about 1010, at least about 10 11 1, about at least 10 12 or more) different clones.
  • the heavy chain component of bacterial libraries may comprise at least about 107 (e.g., at least about 108, at least about 10 9, at least about 1010, at least about 1011, at least about 10 12 or more) different clones.
  • the ratio of effective clones in the bacterial library of light chain components may be at least about 70% (for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%. , At least about 99%).
  • the proportion of effective clones in the heavy chain component bacterial library may be at least about 70% (for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%. , At least about 99%).
  • 2) may include calculating the library capacity of the display bacterial library according to the library capacity of the light chain component bacterial library and/or the heavy chain component bacterial library, the linker component bacteria and/or quality And/or quality.
  • the displayed bacterial library may comprise about at least 10 10 (for example, about at least 10 11 , about at least 10 12 , about at least 10 13 , about at least 10 14 , about at least 10 15 , About at least 10 16 or more) different clones.
  • the ratio of effective clones in the display bacterial library may be at least about 70% (for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least About 99%).
  • the bacteria in the displayed bacterial library can be cultured in liquid.
  • the culture time of the liquid culture can be no more than about 8 hours, for example, can be no more than about 4 hours, no more than about 5 hours, no more than about 6 hours, or no more than about 7 hours.
  • the liquid culture operation is relatively simple.
  • the bacteria in the display bacterial library can be cultured with a small amount of bacterial solution and then selected colonies.
  • the culture time of the plate culture may be about 12-18 hours, for example, may be about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours.
  • the plate culture can select colonies (for example, select single clones) and then perform sequencing analysis.
  • the plate culture can reduce the number of dominant clones.
  • the first polynucleotide and the third polynucleotide can be obtained from sample materials.
  • the sample material may include materials derived from a sample of peripheral blood lymphocytes.
  • the peripheral blood lymphocytes may be human peripheral blood lymphocytes.
  • the sample material can also be derived from any other tissues and/or cells, and is not limited to the peripheral blood lymphocyte sample.
  • the sample material may include total RNA and/or mRNA derived from the sample.
  • 8) may include contacting the displayed bacterial library with helper phage to prepare the phage library.
  • the present application provides a phage library produced by the method.
  • the phage library may comprise about at least 10 10 (e.g., about at least 10 11 , about at least 10 12 , about at least 10 13 , about at least 10 14 , about at least 10 15 , About at least 10 16 or more) different clones.
  • the phage library can display about at least 10 10 kinds (for example, about at least 10 11 kinds, about at least 10 12 kinds, about at least 10 13 kinds, about at least 10 14 kinds, about at least 10 15 kinds, About at least 10 16 or more) different antibodies or antibody fragments.
  • the present application provides a method for screening antibodies or antibody fragments, which includes using the phage library.
  • the antibody fragment may be Fab.
  • the antibody or antibody fragment can specifically bind to TNF ⁇ , IL-6, IL6R, IL17-FR, CD38, GMCSF and Siglec3.
  • Total RNA is extracted from human peripheral blood lymphocytes, and mRNA is further isolated from total RNA (Takara Cat#Z652N/636592, see product instructions for specific test procedures).
  • the 5'-end of the light chain forward primer contains the nucleotide sequence of R1 GCCCCAGGCGGCC (SEQ ID NO:1)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R2 GGCCACATAGGCC (SEQ ID NO: 2)
  • the 5'-end of the heavy chain variable region forward primer contains the nucleotide sequence of R5 GGCCCAACCGGCC (SEQ ID NO: 3)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R6 GGCCCTCAGCGGCC (SEQ ID NO: 4) .
  • the primers were synthesized by Jin Weizhi Company.
  • the 5'-end of the forward primer contains the nucleotide sequence of R3 GGCCACATAGGCC (SEQ ID NO: 2)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R4 GGCCCAACCGGCC (SEQ ID NO: 3).
  • the specific primer sequence can refer to the following table 1-1:
  • the first step is to use the mRNA obtained in Example 1.1 as a template, and use Promega's MMLV to perform reverse transcription to synthesize cDNA (according to the Promega product instructions, where the primers are ThermoCat#N8080127, and the reverse transcriptase is PromegaCat#M1701) .
  • the second step is to use the cDNA obtained in the first step as a template and use the primers obtained in Example 1.2 to amplify the KLC, LLC and VH gene libraries of the component antibodies by PCR (Takara Cat#RR900A, according to the company’s product instructions) .
  • PCR Tekara Cat#RR900A, according to the company’s product instructions
  • the specific primer sequence can refer to the following table 1-2:
  • PCR was performed using the R1-1kb-R2 forward primer and R1-1kb-R2 reverse primer prepared in Example 1.4.1, After purification and recovery by gel electrophoresis (using Axygen gel recovery kit), the PCR product-R1-1kb-R2 (SEQ ID NO: 88) is obtained.
  • the forward primer of R3-linker-R4 (SEQ ID NO: 83) and the reverse primer of R3-linker-R4 (SEQ ID NO: 84) were used for PCR, purified and recovered by gel electrophoresis Then (using the Axygen gel recovery kit), the PCR product-R3-linker-R4 (SEQ ID NO: 90) is obtained, and the second polynucleotide of the present application is obtained.
  • the length of the linker may be 72 bp, and its nucleotide sequence is shown in SEQ ID NO: 89.
  • PCR was performed using the R5-1kb-R6 forward primer and R5-1kb-R6 reverse primer prepared in Example 1.4.1, After purification and recovery by gel electrophoresis, the PCR product-R5-1kb-R6 (SEQ ID NO: 91) is obtained.
  • TA cloning (TA cloning kit, purchased from Takara), insert the R5-1kb-R6 fragment prepared in 1.4.2 into the pMD19-T vector to obtain a vector containing the R5-1kb-R6 fragment, and then use this vector as Template, use primer mutation to remove the original BsmBI restriction site in this vector to obtain the heavy chain storage vector DDB-R5-1kb-R6 for insertion into the VH gene library.
  • the vector map is shown in Figure 5.
  • the specific primer sequence can refer to the following table 1-3:
  • the linker storage vector can be used to transform TG1 competent bacteria (Lucigen company), cultured overnight at 37°C, send colonies for sequencing, and then collect the colonies to obtain the linker component bacteria.
  • the connecting sub-assembly bacteria can be frozen for later use.
  • the first polynucleotide (including KLC and LLC) prepared in Example 1.3 was digested with restriction endonucleases R1 and R2 to obtain the desired light chain fragment (about 0.65 kb).
  • the light chain storage vector DDB-R1-1kb-R2 prepared in Example 1.4 was digested with restriction endonucleases R1 and R2. A light chain storage vector fragment (approximately 2.7 kb) was obtained.
  • the obtained target light chain fragment and the light chain storage vector fragment are mixed, and then ligated by ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a light chain storage ligation product, and then use the light chain storage ligation product to transform TG1 competent Bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer's instructions), spread ampicillin-resistant plates (Thermo, Cat#240845), cultivate overnight at 37°C, send colonies for sequencing, and then collect all colonies to obtain light chain component bacteria library.
  • the quality of the light chain component bacterial library can be tested and/or the light chain component bacterial library can be frozen for future use.
  • the third polynucleotide prepared in Example 1.3 was digested with restriction endonucleases R5 and R6 to obtain a target heavy chain variable region fragment (about 0.35 kb).
  • the heavy chain storage vector DDB-R5-1kb-R6 prepared in Example 1.4 was digested with restriction endonucleases R5 and R6. A heavy chain storage vector fragment (approximately 2.7 kb) was obtained.
  • the obtained target heavy chain variable region fragment and heavy chain storage vector fragment are mixed, and then ligated with ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a heavy chain storage ligation product, and then use the heavy chain storage ligation product to transform TG1 competent bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer’s instructions), spread ampicillin-resistant plates (Thermo, Cat#240845), culture overnight at 37°C, pick the colonies for sequencing, and then collect all colonies to obtain a heavy Chain component bacterial library.
  • the quality of the heavy chain component bacterial library can be tested and/or the heavy chain component bacterial library can be frozen for future use.
  • the light chain component plasmid prepared in Example 1.5.1 was digested with restriction endonucleases R1 and R2, and purified and recovered by gel electrophoresis to obtain the light chain insert LC.
  • the heavy chain component plasmid prepared in Example 1.5.2 was digested with restriction endonuclease R5 and R6, and after purification and recovery by gel electrophoresis, the heavy chain insert HC was obtained.
  • the linker assembly bacteria prepared in Example 1.4.4 was extracted to obtain the linker assembly plasmid.
  • the linker forward primer (SEQ ID NO: 79) and the linker reverse primer (SEQ ID NO: 80) were used for amplification. Increase the 0.8kb fragment containing the linker, then cut the 0.8kb PCR product with restriction endonucleases R3 and R4, and purify and recover by gel electrophoresis (using a small fragment gel recovery kit, purchased from Laifeng Bio, Cat#DK402) to obtain a 72pb linker fragment.
  • the pComb3x vector was purchased.
  • the vector map is shown in Figure 6, and the map of the modified pComb3x-fab vector used for antibody Fab display is shown in Figure 7.
  • the SfiI restriction site at the 3'end of the Fab gene in the pComb3x-fab vector was removed by nonsense mutation.
  • restriction endonuclease R2 restriction site downstream of the stop codon of the light chain in the nonsense mutant vector add restriction endonuclease at the end of the signal peptide of the heavy chain variable region through nonsense mutation
  • the restriction site of nuclease R5 was cut to obtain the modified display vector DDB-R1R2R5R6, and its map is shown in Figure 8.
  • the display vector DDB-R1R2R5R6 prepared in Example 1.7 was digested with restriction endonuclease R7 and restriction endonuclease R8 to obtain a 3.6 kb display vector fragment.
  • the light chain insert LC (0.65kb), the heavy chain insert HC (0.35kb), the linker fragment (72bp) and the display vector fragment (3.6kb) obtained in Example 1.6 according to 1:1:1:1 Mix the molecules in the proportion of, and use T4 DNA ligase at 20°C for more than 20 hours to obtain the ligation product for display.
  • the ligation product was purified by PCR-Clean-up and transferred to TG1 competent bacteria (Lucigen, Cat#60502-2, operated according to the manufacturer’s instructions), and cultured in 2YT medium without antibiotics at 37°C, shaking at 250rpm for 60 Minutes, spread ampicillin-resistant plates (Thermo, Cat#240845) and grow overnight at 37°C. Select colonies for sequencing, and collect all the colonies grown on the plate to display the bacterial library. The displayed bacterial library can be saved for future use.
  • Example 1.8 An appropriate amount of bacterial liquid was taken from the displayed bacterial library prepared in Example 1.8, and cultured in 2YT medium (containing 100 ⁇ g/ml of ampicillin and 2% glucose) at 37°C until the OD 600 reached 0.5. Then, M13KO7 helper phage (purchased from NEB, Cat#N0315S, MOI is about 10-20) was added to the bacterial solution and mixed, then stood still at 37°C for 30 minutes, then shaken at 37°C, 250rpm for 30 minutes, and centrifuged The supernatant of the culture medium containing the M13KO7 helper phage was discarded, and the bacteria were resuspended in a culture medium (containing ampicillin and kanamycin) 4 times the original volume of the bacterial liquid, and shaken overnight at 30°C and 250 rpm. The next day, the phage was collected by PEG precipitation, the phage concentration was titrated, and stored in aliquots. The display antibody phage library is
  • Example 1.5 The quality of the component bacterial library prepared in Example 1.5 was analyzed. The results are shown in Table 2.
  • the ratio of light chain component bacterial library (KLC) to light chain component bacterial library (LLC) is 2:1. Based on the above quality results, it can be estimated that the effective clone ratio of the display bacterial library will reach more than 84% .
  • the library capacity obtained by random combination of the light chain component bacterial library and the heavy chain component bacterial library can theoretically reach up to 10 15 .
  • Example 1.8 Using 197 ⁇ g of the isolated and purified display ligation product obtained in Example 1.8, 398 TG1 competent bacteria (Lucigen, Cat#60502-2, operated according to the manufacturer’s instructions) were transformed into a 2YT culture medium without antibiotics, 37 Incubate with shaking at 250 rpm for 60 minutes, spread 197 ampicillin-resistant plates (Thermo, Cat#240845), cultivate overnight at 37°C, harvest all colonies, and store them in aliquots (that is, the display bacterial library prepared in Example 1.8 is obtained ).
  • the display bacterial library has a library capacity of 10 11 and a background clone ratio of 2%.
  • the 5'-end of the light chain forward primer contains the nucleotide sequence of R1 GCCCCAGGCGGCC (SEQ ID NO:1)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R2 GGCCACATAGGCC (SEQ ID NO: 2)
  • the 5'-end of the heavy chain variable region forward primer contains the nucleotide sequence of R5 GGCCCAACCGGCC (SEQ ID NO: 3)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R6 GGCCCTCAGCGGCC (SEQ ID NO: 4) .
  • the primers were synthesized by Jin Weizhi Company.
  • the 5'-end of the forward primer contains the nucleotide sequence of R3 GGCCACATAGGCC (SEQ ID NO. 2)
  • the 5'-end of the reverse primer contains the nucleotide sequence of R4 GGCCCAACCGGCC (SEQ ID NO. 3).
  • the specific primer sequence can refer to the following table 4-1:
  • the first step is to use the total RNA obtained in Example 4.1 as a template, and use Promega's MMLV to perform reverse transcription to synthesize cDNA (according to the Promega product instructions, where the primer is ThermoCat#N8080127 and the reverse transcriptase is PromegaCat#M1701 ).
  • the second step is to use the cDNA obtained in the first step as a template, and use the primers obtained in Example 4.2 to amplify the KLC, LLC and VH gene libraries of the component antibodies by PCR (Takara Cat#RR900A, according to the company’s product instructions) .
  • PCR Tekara Cat#RR900A, according to the company’s product instructions
  • PCR was performed using the R1-1kb-R2 forward primer and R1-1kb-R2 reverse primer prepared in Example 4.4.1, After purification and recovery by gel electrophoresis (using Axygen gel recovery kit), the PCR product-R1-1kb-R2 (SEQ ID NO. 88) is obtained.
  • the PCR product—R3-linker-R4 (SEQ ID NO.95) is obtained, and the second polynucleotide of the present application is obtained.
  • the length of the linker may be 90 bp, and its nucleotide sequence is shown in SEQ ID NO. 89.
  • PCR was performed using the R5-1kb-R6 forward primer and R5-1kb-R6 reverse primer prepared in Example 4.4.1, After purification and recovery by gel electrophoresis, the PCR product-R5-1kb-R6 (SEQ ID NO.91) is obtained.
  • TA cloning (TA cloning kit, purchased from Takara), insert the R5-1kb-R6 fragment prepared in 4.4.2 into the pMD19-T vector to obtain a vector containing the R5-1kb-R6 fragment, and then use this vector as Template, use primer mutation to remove the original BsmBI restriction site in this vector to obtain the heavy chain storage vector DDB-R5-1kb-R6 for insertion into the VH gene library.
  • the vector map is shown in Figure 5.
  • the linker storage vector can be used to transform TG1 competent bacteria (Lucigen company), cultured overnight at 37°C, send colonies for sequencing, and then collect the colonies to obtain the linker component bacteria.
  • the connecting sub-assembly bacteria can be frozen for later use.
  • the first polynucleotide (including KLC and LLC) prepared in Example 4.3 was digested with restriction endonucleases R1 and R2 to obtain the desired light chain fragment (about 0.65 kb).
  • the light chain storage vector DDB-R1-1kb-R2 prepared in Example 4.4 was digested with restriction endonucleases R1 and R2. A light chain storage vector fragment (approximately 2.7 kb) was obtained.
  • the obtained target light chain fragment and the light chain storage vector fragment are mixed, and then ligated by ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a light chain storage ligation product, and then use the light chain storage ligation product to transform TG1 competent Bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer's instructions), incubate in 2YT culture medium without antibiotics, 37°C, 250rpm shaking for 60 minutes, and spread ampicillin-resistant plates (Thermo, Cat#240845 ), cultivate overnight at 37°C, send colonies for sequencing, and then collect all colonies to obtain a bacterial library of light chain components.
  • the quality of the light chain component bacterial library can be tested and/or the light chain component bacterial library can be frozen for future use.
  • the third polynucleotide prepared in Example 4.3 was digested with restriction endonuclease R5 and R6 to obtain the target heavy chain variable region fragment (about 0.35 kb).
  • the heavy chain storage vector DDB-R5-1kb-R6 prepared in Example 4.4 was digested with restriction endonucleases R5 and R6. A heavy chain storage vector fragment (approximately 2.7 kb) was obtained.
  • the obtained target heavy chain variable region fragment and heavy chain storage vector fragment are mixed, and then ligated with ligase T4 DNA ligase (purchased from NEB, Thermo) to obtain a heavy chain storage ligation product, and then use the heavy chain storage ligation product to transform TG1 competent bacteria (Lucigen, Cat#60502-2, operate according to the manufacturer’s instructions), cultured in 2YT medium without antibiotics, 37°C, 250rpm shaking for 60 minutes, and spread ampicillin-resistant plates (Thermo, Cat#240845), cultivated overnight at 37°C, picked colonies for sequencing, and then collected all colonies to obtain a heavy chain component bacterial library.
  • the quality of the heavy chain component bacterial library can be tested and/or the heavy chain component bacterial library can be frozen for future use.
  • the light chain component plasmid prepared in Example 4.5.1 was digested with restriction endonucleases R1 and R2, and purified and recovered by gel electrophoresis to obtain the light chain insert LC.
  • the heavy chain component plasmid prepared in Example 4.5.2 was digested with restriction endonuclease R5 and R6, and after purification and recovery by gel electrophoresis, the heavy chain insert HC was obtained.
  • the plasmid in the linker assembly bacteria prepared in Example 4.4.4 was extracted to obtain the linker assembly plasmid.
  • the linker component plasmid or the linker storage vector in Example 4.4.4 as a template, use the forward primer (SEQ ID NO.96 or SEQ ID NO.97) of the linker and the reverse primer (SEQ ID NO.97) of the linker.
  • the pComb3x vector was purchased and its vector map is shown in Figure 6, and the map of the modified pComb3x-fab vector used for antibody Fab display is shown in Figure 7.
  • the SfiI restriction site at the 3'end of the Fab gene in the pComb3x-fab vector was removed by nonsense mutation.
  • restriction endonuclease R2 restriction site downstream of the stop codon of the light chain in the nonsense mutant vector add restriction endonuclease at the end of the signal peptide of the heavy chain variable region through nonsense mutation
  • the restriction site of nuclease R5 was cut to obtain the modified display vector DDB-R1R2R5R6, and its map is shown in Figure 8.
  • the display vector DDB-R1R2R5R6 prepared in Example 4.7 was digested with restriction endonuclease R7 and restriction endonuclease R8 to obtain a 3.6 kb display vector fragment.
  • the light chain insert LC (0.65kb), the heavy chain insert HC (0.35kb), the linker fragment (90bp) and the display vector fragment (3.6kb) obtained in Example 4.6 according to 1:1:1:1 Mix the molecules in the ratio of, and use T4 DNA ligase at 20°C for more than 20 hours to obtain the ligation product for display.
  • the ligation product was purified by PCR-Clean-up, transformed into TG1 competent bacteria (purchased from Lucigen, Cat#60502-2), cultured in 2YT medium without antibiotics at 37°C, 250rpm shaking for 60 minutes, then 1
  • the ratio of :20 was expanded to 2YT culture medium containing ampicillin, cultured at 37°C, 250rpm shaking for 4.5 hours, centrifuged to collect the bacteria and cryopreserved, which is the display bacterial library. Before adding ampicillin to expand the culture, take a small amount of bacterial solution to plate, incubate at 37°C overnight, and pick colonies for sequencing.
  • the library of displayed bacteria can be stored for later use.
  • Example 4.8 An appropriate amount of bacterial liquid was taken from the display bacterial library prepared in Example 4.8, and cultured in 2YT culture medium (containing 100 ⁇ g/ml of ampicillin and 2% glucose) at 37° C. until the OD 600 reached 0.5. Then, M13KO7 helper phage (purchased from NEB, Cat#N0315S, MOI is about 10-20) was added to the bacterial solution and mixed, then stood still at 37°C for 30 minutes, then shaken at 37°C, 250rpm for 30 minutes, and centrifuged The supernatant of the culture medium containing the M13KO7 helper phage was discarded, and the bacteria were resuspended in a culture medium (containing ampicillin and kanamycin) 4 times the original volume of the bacterial liquid, and shaken overnight at 30°C and 250 rpm. The next day, the phage was collected by PEG precipitation, the phage concentration was titrated, and stored in aliquots. The display antibody phage
  • the ratio of the light chain component bacterial library (KLC) to the light chain component bacterial library (LLC) is 2:1. Based on the above quality results, it can be estimated that the effective clone ratio of the display bacterial library will reach 82%.
  • the library capacity obtained by random combination of the light chain component bacterial library and the heavy chain component bacterial library can theoretically reach up to 10 18 .
  • Example 4.8 The separated and purified display ligation product 16 ⁇ g obtained in Example 4.8 was transformed into 25 TG1 competent bacteria. After amplification and culture, the bacteria were collected by centrifugation. The total number of bacteria was 2.4*10 12 and stored in aliquots (that is, Example 4.8 The prepared display bacterial library).
  • the display bacterial library capacity is 4*10 10
  • the background clone ratio is 3%
  • the conversion efficiency per ⁇ g ligation product reaches 2.5*10 9 .
  • the first method Randomly select 90 colonies for sequencing, 56 of them have the correct reading frame and no repetitive sequences. The percentage of clones with the correct reading frame is 62.2%.
  • the second method Pave the plate with anti-human Fab antibody, and use ELISA to detect the Fab expression of the above 90 clones.
  • the readings of the two negative controls were 0.07 and 0.082, respectively.
  • a total of 71 clones with a reading higher than 0.1 were determined to be Fab expression, and the positive ratio was 79%.
  • IL6 secreted cytokines
  • IL6R cell membrane proteins
  • EGFR cell membrane proteins
  • the antigen-specific Fab was screened by the liquid phase screening method of biotinylated antigen-binding avidin-labeled magnetic beads.
  • the above 6 biotin-labeled antigens were purchased from AcroBiosystems (Cat#IL6-H8218, ILF-H82W1, GMF-H8214, CD6-H82E8, EGR-H82E3 and CD3-H82E7), and avidin-labeled magnetic beads were purchased From Thermo (Cat#11206D).
  • the antigen concentrations of the three rounds of liquid phase screening were 5 ⁇ g/ml, 3 ⁇ g/ml and 1 ⁇ g/ml respectively.
  • the phage collected in the first round of screening was eluted and amplified for the second round of screening.
  • the phages collected in the second round of screening were used directly in the third round of screening without amplification.
  • the 12 clones with the highest ELISA readings were selected from each group of antigen-specific positive clones for sequencing analysis, and the amino acid sequences of the 12 clones in each group were compared and analyzed. The results are shown in Table 7. The results in Table 7 indicate that each group has multiple unique amino acid sequences.

Abstract

提供了一种产生展示抗体或抗体片段的噬菌体文库的方法，包括提供包含LC的第一多核苷酸、包含连接子的第二多核苷酸和包含HC的第三多核苷酸，将第一、第二和第三多核苷酸分别导入第一、第二和第三细菌中获得轻链组件细菌文库、连接子组件细菌和重链组件细菌文库，从文库获得轻链组件质粒、连接子组件质粒和重链组件质粒，从中获得经释放的LC、经释放的连接子和经释放的HC，与经释放的展示载体片段连接形成展示用连接产物，导入第四细菌中获得展示细菌文库，使用所述展示细菌文库制备展示所述抗体或抗体片段的噬菌体文库。还提供了根据该方法产生的噬菌体文库。

Description

制备噬菌体文库的方法 技术领域
本申请涉及生物医药领域，具体的涉及一种产生展示抗体或抗体片段的噬菌体文库的方法。
背景技术
本领域存在一些制备噬菌体文库的方法，然而这些方法均存在一定的缺陷。例如，文库的库容量过小(例如，难以获得库容量超过10 9的文库)，并且文库多样性不足，难以满足筛选高质量抗体的需求。同时，为了扩大库容量，需要把多个较小的文库进行积累，这不光需要花费数年的时间，而且很难保证这些较小文库之间的一致性，难以控制所获得文库的质量。此外，利用现有的方法构建文库，难以在文库制备的过程中方便、可靠地进行质量控制，通常只能在文库建成之后才得知其质量如何，风险高且失败的概率大。
此外，由于通过目前的方法建立的文库成本高、周期长，长期保存后质量下降严重，难以满足工业化量产的需求。
因此，亟需能够满足工业化量产需求、质量可控且操作简便的方法来制备大容量的噬菌体文库。
发明内容
本申请提供了一种产生展示抗体或抗体片段的噬菌体文库的方法，以及通过使用该方法获得的噬菌体文库，和借助该噬菌体文库筛选抗体或抗体片段的方法。本申请所述的产生展示抗体或抗体片段的噬菌体文库的方法具备至少一个以下性质：1)有效减少PCR所导致的引入突变的几率；本申请所述的噬菌体文库没有采用本领域常规抗体库构建方法所使用的组合PCR策略，用于构建所述噬菌体文库的每个组件可以仅仅通过1次PCR扩增，然后连接所需的所需组件得到Fab的噬菌体文库；2)能够满足工业化量产的需求；3)能够较容易地进行质量控制；构建本申请所述的噬菌体文库所需的每个步骤都可以独立、简便地进行质量控制；同时，在获得本申请所述的噬菌体文库之后，可以简便地(例如，采用基因测序，或者可溶性Fab表达分析)对其进行质量分析；4)制备过程简便、快捷；构建本申请所述的噬菌体文库可以采用特殊的限制性内切核酸酶的识别位点，既保证定向连接，又防止错误连接，连接时各个组件片段的分子个数可以控制为1∶1，从而提高连接转化效率；同时，采用构建轻链组件细菌文库等细菌文库的策略，提高各个抗体或其片段的连接和转化效率(例如，得 到的连接产物，每μg的转化效率可以为约10 9以上)；通过获得连接子组件质粒，确保得到的经释放的连接子具备合适且完整的黏性末端，提高连接转化效率；此外，本申请所述的噬菌体文库既可以通过铺皿培养，也可以通过液体培养；5)产生的文库库容量大且多样性好；从而有助于快速且高效地筛选抗体或其抗原结合片段；6)特异性高；构建本申请所述的噬菌体文库没有采用本领域常规方法所使用的简并引物，避免了简并引物引起的错误，同时用于构建本申请所述的噬菌体文库的引物所需PCR退火温度较高，能够提高这些引物与其扩增目的片段的结合特异性；以及7)可扩增并容易保存；轻链组件细菌文库等细菌文库以及包含连接子组件质粒的细菌均可以在-80℃条件下长期保存，而在需要使用时则可以随时取用(例如，可以仅需两天就获得本申请所述的噬菌体文库)，并且按需进行不同种类和比例的混合。
一方面，本申请提供了一种产生展示抗体或抗体片段的噬菌体文库的方法，所述方法包括：1)提供第一多核苷酸，第二多核苷酸和第三多核苷酸，所述第一多核苷酸包含LC，所述第二多核苷酸包含连接子且所述第三多核苷酸包含HC，其中所述LC包含编码所述抗体或抗体片段的轻链或轻链片段的核酸序列，所述HC包含编码所述抗体或抗体片段的重链或重链片段的核酸序列；2)将所述第一多核苷酸导入第一细菌中以获得轻链组件细菌文库，将所述第二多核苷酸导入第二细菌中以获得连接子组件细菌，将所述第三多核苷酸导入第三组细菌中以获得重链组件细菌文库；3)由所述轻链组件细菌文库获得包含所述LC的轻链组件质粒，由所述连接子组件细菌获得包含所述连接子的连接子组件质粒，且由所述重链组件细菌文库获得包含所述HC的重链组件质粒；4)由所述轻链组件质粒获得经释放的LC，由所述连接子组件质粒获得经释放的连接子，且由所述重链组件质粒获得经释放的HC；5)提供展示载体，并由所述展示载体获得经释放的展示载体片段；6)使所述经释放的LC、经释放的连接子、经释放的HC以及经释放的展示载体片段连接，以形成展示用连接产物；7)将所述展示用连接产物导入第四组细菌中，以获得所述抗体或抗体片段的展示细菌文库；8)使用所述展示细菌文库制备展示所述抗体或抗体片段的噬菌体文库。
在某些实施方式中，在适于抗体或抗体片段表达的条件下，6)的所述展示用连接产物能够依照阅读框表达所述轻链或轻链片段以及所述重链或重链片段。
在某些实施方式中，所述第一多核苷酸，所述第二多核苷酸和所述第三多核苷酸均为线性核酸分子。
在某些实施方式中，编码所述重链或重链片段的核酸序列与编码所述轻链或轻链片段的核酸序列位于不同的阅读框中。
在某些实施方式中，所述连接子包含编码信号肽pelB或其片段的核酸序列。在某些实施方式中，所述连接子的长度为约50至约200个碱基。
在某些实施方式中，在6)的所述展示用连接产物中，所述LC位于所述连接子的上游，且所述连接子位于所述HC的上游。
在某些实施方式中，2)包括冷冻保存所述轻链组件细菌文库和/或所述重链组件细菌文库。
在某些实施方式中，7)包括冷冻保存所述展示细菌文库。
在某些实施方式中，8)包括融化并培养所述经冷冻保存的展示细菌文库，并使用经培养的所述展示细菌文库构建所述噬菌体文库。
在某些实施方式中，所述第一多核苷酸以5’至3’方向包含结构R1-LC-R2，所述第二多核苷酸以5’至3’方向包含结构R3-连接子-R4，所述第三多核苷酸以5’至3’方向包含结构R5-HC-R6，且所述展示载体中以5’至3’方向包含结构R7-展示载体片段-R8，其中所述R1，R2，R3，R4，R5，R6，R7和R8为限制性内切核酸酶的识别位点。
在某些实施方式中，所述R2经限制性内切核酸酶切割后产生的末端：能够与所述R3经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R4、R5、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
在某些实施方式中，所述R4经限制性内切核酸酶切割后产生的末端：能够与所述R5经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R2、R3、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
在某些实施方式中，所述R6经限制性内切核酸酶切割后产生的末端：能够与所述R7经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R2、R3、R4、R5及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
在某些实施方式中，所述R8经限制性内切核酸酶切割后产生的末端：能够与所述R1经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R2、R3、R4、R5、R6及R7中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
在某些实施方式中，所述R1，R2，R3，R4，R5、R6、R7和R8中的任一项经限制性内切核酸酶切割后产生的末端为非回文序列。
在某些实施方式中，所述R1，R2，R3，R4，R5、R6、R7和R8中的两项或更多项能够被相同的限制性内切核酸酶识别及切割。
在某些实施方式中，所述R2与所述R3相同。在某些实施方式中，所述R4与所述R5相同。在某些实施方式中，所述R6与所述R7相同。在某些实施方式中，所述R8与所述R1相同。
在某些实施方式中，所述R1、R2、R3、R4、R5和R8能够被相同的限制性内切核酸酶 识别及切割。在某些实施方式中，所述R6和R7能够被相同的限制性内切核酸酶识别及切割。
在某些实施方式中，所述限制性内切核酸酶选自SfiI，BsmBI和Esp3I。在某些实施方式中，所述R1、R2、R3、R4、R5和R8能够被SfiI识别及切割。在某些实施方式中，所述R6和R7能够被BsmBI和/或Esp3I识别及切割。
在某些实施方式中，所述R1包含SEQ ID NO:1所示的核酸序列。
在某些实施方式中，所述R2包含SEQ ID NO:2所示的核酸序列。
在某些实施方式中，所述R3包含SEQ ID NO:2所示的核酸序列。
在某些实施方式中，所述R4包含SEQ ID NO:3所示的核酸序列。
在某些实施方式中，所述R5包含SEQ ID NO:3所示的核酸序列。
在某些实施方式中，所述R6包含SEQ ID NO:4所示的核酸序列。
在某些实施方式中，所述R7包含SEQ ID NO:4所示的核酸序列。
在某些实施方式中，所述R8包含SEQ ID NO:1所示的核酸序列。
在某些实施方式中，所述轻链或轻链片段包含轻链可变区部分和/或轻链恒定区部分。在某些实施方式中，所述重链或重链片段包含重链可变区部分和/或重链恒定区部分。在某些实施方式中，所述抗体或其片段包括Fab，Fab’，(Fab)2和/或(Fab’)2。
在某些实施方式中，7)中所述展示用连接产物的连接转化效率为至少10 8个克隆/μg多核苷酸。
在某些实施方式中，2)包括使用识别所述R1和R2的限制性内切核酸酶对所述第一多核苷酸进行酶切处理，使经酶切处理的所述第一多核苷酸与轻链存储载体片段连接形成轻链存储连接产物，并将所述轻链存储连接产物导入所述第一细菌中以获得所述轻链组件细菌文库；其中所述轻链存储载体包含所述R1和R2，并且通过使用识别所述R1和R2的限制性内切核酸酶对所述轻链存储载体进行酶切处理而获得所述轻链存储载体片段。
在某些实施方式中，所述轻链存储载体源自pUC载体。在某些实施方式中，所述pUC载体为pUC19载体或源自pUC19载体。在某些实施方式中，所述pUC载体为pMD19-T载体或源自pMD19-T载体。
在某些实施方式中，所述识别所述R1和R2的限制性内切核酸酶包括SfiI。
在某些实施方式中，2)包括使用识别所述R5和R6的限制性内切核酸酶对所述第三多核苷酸进行酶切处理，使经酶切处理的所述第三多核苷酸与重链存储载体片段连接形成重链存储连接产物，并将所述重链存储连接产物导入所述第三细菌中以获得所述重链组件细菌文库；其中所述重链存储载体包含所述R5和R6，并且通过使用识别所述R5和R6的限制性内切核酸酶对所述重链存储载体进行酶切处理而获得所述重链存储载体片段。在某些实施方式 中，所述重链存储载体源自pUC载体。在某些实施方式中，所述pUC载体为pUC19载体或源自pUC19载体。在某些实施方式中，所述pUC载体为pMD19-T载体或源自pMD19-T载体。
在某些实施方式中，所述重链存储载体中包含且仅包含一个BsmBI的识别位点。
在某些实施方式中，所述识别所述R5和R6的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
在某些实施方式中，2)包括使所述第二多核苷酸与载体片段连接形成连接子存储载体，并将所述连接子存储载体导入所述第二细菌中以获得所述连接子组件细菌。在某些实施方式中，所述连接子存储载体中包含所述R3和R4。在某些实施方式中，用于构建所述连接子存储载体的所述载体片段源自pUC载体。在某些实施方式中，其中所述pUC载体为pUC19载体或源自pUC19载体。在某些实施方式中，所述pUC载体为pMD19-T载体或源自pMD19-T载体。
在某些实施方式中，4)包括使用识别所述R1和R2的限制性内切核酸酶对所述轻链组件质粒进行酶切处理，从而获得所述经释放的LC。在某些实施方式中，所述识别所述R1和R2的限制性内切核酸酶包括SfiI。
在某些实施方式中，4)包括使用识别所述R5和R6的限制性内切核酸酶对所述重链组件质粒进行酶切处理，从而获得所述经释放的HC。在某些实施方式中，所述识别所述R5和R6的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
在某些实施方式中，4)包括使用识别所述R3和R4的限制性内切核酸酶对所述连接子存储载体进行酶切处理，从而获得所述经释放的连接子。在某些实施方式中，4)包括由所述连接子存储载体获得扩增产物，所述扩增产物包含所述连接子、所述R3以及所述R4；并且使用识别所述R3和R4的限制性内切核酸酶对所述扩增产物进行酶切处理，从而获得所述经释放的连接子。在某些实施方式中，所述识别所述R3和R4的限制性内切核酸酶包括SfiI。
在某些实施方式中，5)包括使用识别所述R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理以释放所述展示载体片段。在某些实施方式中，所述识别所述R7和R8的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
在某些实施方式中，所述展示载体源自pComb3x载体。
在某些实施方式中，使用识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，将获得三个不同的酶切片段。在某些实施方式中，所述识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶包括SfiI。在某些实施方式中，使用识别所述R6和R7的限制性内切核酸酶对所述展示载体进行酶切处理后，使所述展示载体线性 化。在某些实施方式中，识别所述R6和R7的限制性内切核酸酶包括BsmBI和/或Esp3I。
在某些实施方式中，使用识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，将获得四个不同的酶切片段。在某些实施方式中，所述识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
在某些实施方式中，2)包括对所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库进行取样检测，以确定所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库的库容量和/或质量。
在某些实施方式中，所述轻链组件细菌文库包含至少10 6个不同的克隆。
在某些实施方式中，所述重链组件细菌文库包含至少10 7个不同的克隆。
在某些实施方式中，所述轻链组件细菌文库中有效克隆的比例为至少约70％。
在某些实施方式中，所述重链组件细菌文库中有效克隆的比例为至少约70％。
在某些实施方式中，2)包括根据所述轻链组件细菌文库和/或所述重链组件细菌文库的库容量，所述连接子组件细菌和/或质量测算所述展示细菌文库的库容量和/或质量。
在某些实施方式中，所述展示细菌文库包含至少10 10个不同的克隆。
在某些实施方式中，所述展示细菌文库中有效克隆的比例为至少约70％。
在某些实施方式中，由样品材料获得所述第一多核苷酸和所述第三多核苷酸。在某些实施方式中，所述样品材料包括源自外周血淋巴细胞样品的材料。在某些实施方式中，所述外周血淋巴细胞为人外周血淋巴细胞。在某些实施方式中，所述样品材料包括源自所述样品的总RNA和/或mRNA。
在某些实施方式中，8)包括使所述展示细菌文库与辅助噬菌体接触来制备所述噬菌体文库。
另一方面，本申请提供了一种通过所述的方法产生的噬菌体文库。在某些实施方式中，所述的噬菌体文库包含至少10 10个不同的克隆。在某些实施方式中，所述的噬菌体文库能够展示至少10 10种不同的抗体或抗体片段。
另一方面，本申请提供了一种筛选抗体或抗体片段的方法，其包括使用所述的噬菌体文库。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限 制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是pUC19载体的示意图。
图2显示的是本申请中改造后的pUC19载体的示意图。
图3显示的是本申请所述的轻链存储载体的示意图。
图4显示的是本申请所述的连接子存储载体的示意图。
图5显示的是本申请所述的重链存储载体的的示意图。
图6显示的pCom3x载体的示意图。
图7显示的是本申请所述的展示载体的示意图。
图8显示的是包含本申请的展示用连接产物的质粒的示意图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“噬菌体文库”通常是指包含编码能够展示在噬菌体上的不同重组多肽的多核苷酸的文库。在某些情形下，所述噬菌体文库还可以包含用多核苷酸文库转染的噬菌体的集合。在本申请中，所述文库可以指多核苷酸多样化的集合体或混合物，其可以包含编码不同重组多肽的多核苷酸。例如，所述多核苷酸文库集合体内可以包含至少10 6、10 7、10 8、10 9、10 10、10 11、10 12、10 13、10 14、10 15或更多种不同的多核苷酸。所述噬菌体文库中的多核苷酸可以来源于单一物种(例如哺乳动物，例如人)，其组织、器官和/或细胞。所述文库可以包含共同属类的多核苷酸。例如，所述属类可以为编码某一型或某一类的免疫球蛋白亚基多肽的多核苷酸。例如，所述多核苷酸可以编码抗体的轻链或轻链片段。例如，所述多核苷酸可以编码抗体的重链或重链片段。
在本申请中，术语“抗体”通常是指能够特异性识别和/或中和特定抗原的多肽分子。基本的四链抗体单元是异四聚体糖蛋白，其由两条相同的轻链和两条相同的重链构成。在IgG的情况中，每个L链通过一个共价二硫键连接到H链，而两条H链通过一个或多个二硫键彼此相连，二硫键的数目取决于H链的同种型。每个H和L链还具有规律间隔的链内二硫键。每 条H链在N-末端具有可变结构域(VH)，接着是三个(对于每条α和γ链)或四个(对于μ和ε同种型)恒定结构域(CH)。
在本申请中，术语“抗体片段”通常是指完整抗体的一部分。例如，所述抗体片段可以包含完整抗体的抗原结合区和/或抗体可变区。所述抗体片段可以通过化学方法和/或基因工程的方法获得。例如，可以采用蛋白酶，包括胃蛋白酶和木瓜蛋白酶，消化抗体后产生所述抗体片段。在本申请中，所述抗体片段可以为Fab。
在本申请中，术语“Fab”通常是指由木瓜蛋白酶消化具有完整结构的抗体后(例如，去除了Fc区和铰链区)而产生两个相同的抗原结合片段。Fab可以由完整的轻链、重链可变区(VH)和重链的第一恒定结构域(CH1)组成。每个Fab可以具有单一的抗原结合位点。
在本申请中，术语“连接子”通常是指能够将两种以上多核苷酸分子或其片段相连接的试剂。所述连接子可以为多核苷酸或其片段。在本申请中，所述连接子可以具备不同的长度。例如，所述连接子的长度可以为40bp以上、50bp以上、60bp以上、70bp以上、80bp以上、90bp以上、100bp以上、150bp以上、200bp以上或更长。
在本申请中，术语“LC”通常是指包含编码抗体或抗体片段的轻链或轻链片段的核酸序列的多核苷酸。在本申请中，所述轻链或轻链片段可具备与同一或类似抗体的重链相结合的能力。在本申请中，所述轻链或轻链片段可包含轻链可变区(VL)和轻链恒定区(CL)。所述轻链恒定区可以分为κ型和λ型。所述轻链还包括具有与κ恒定区(C-κ)相连的λ可变区(V-λ)或与λ恒定区(C-λ)相连的κ可变区(V-κ)的轻链。例如，所述LC可以为包含编码抗体或抗体片段的轻链的核酸序列的多核苷酸。
在本申请中，术语“HC”通常是指包含编码抗体或抗体片段的重链或重链片段的核酸序列的多核苷酸。在本申请中，所述重链或重链片段可具备与同一或类似抗体的轻链相结合的能力。在本申请中，所述重链或重链片段可包含重链可变区(VH)和重链恒定区(CH)。所述重链恒定区可包含CH1结构域、铰链区、CH2结构域和CH3结构域。在IgE、IgM和IgY的情况下，所述重链恒定区可包含CH4结构域但是不具有铰链区。在本申请中，所述“重链恒定区”可以为CH1、铰链区、CH2、CH3、CH4结构域或其任何组合。例如，所述HC可以为包含编码抗体或抗体片段的VH和CH1的核酸序列的多核苷酸。
在本申请中，术语“第一多核苷酸”通常是指包含LC的多核苷酸，所述LC可以为包含编码所述抗体或抗体片段的轻链或轻链片段的核酸序列。在本申请中，所述第一多核苷酸还可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一多核苷酸可以以5’至3’方向包含结构R1-LC-R2，其中所述R1，R2可以为限制性内切核酸酶的识别位点。例如，经识别所述第一多核苷酸中的内切酶识别位点的内切酶(例如，识别R1和 R2的限制性内切核酸酶，例如SfiI)酶切处理后，经酶切处理的所述第一多核苷酸可包含所述LC。
在本申请中，术语“轻链存储载体片段”通常是指经限制性内切核酸酶酶切处理的轻链存储载体的片段。在本申请中，所述轻链存储载体所包含的限制性内切核酸酶的识别位点可以与所述第一多核苷酸所包含的限制性内切核酸酶的识别位点对应地相同。例如，所述轻链存储载体可以包含所述R1和所述R2。在本申请中，经限制性内切核酸酶(例如，识别所述R1和所述R2的限制性内切核酸酶)酶切后，得到的所述轻链存储载体片段可以与经酶切处理的所述第一多核苷酸连接。在本申请中，所述轻链存储载体片段可以源自pUC载体。例如，所述pUC载体可以为pUC19载体或源自pUC19载体；又例如，所述pUC载体可以为pMD19-T载体或源自pMD19-T载体。
在本申请中，术语“轻链存储连接产物”通常是指经酶切处理后的所述第一多核苷酸与所述轻链存储载体片段连接形成的产物。在本申请中，所述第一多核苷酸与所述轻链存储载体片段可以包含相同限制性内切核酸酶的识别位点，可以被连接酶(例如DNA连接酶)相连接，而得到所述轻链存储连接产物。
在本申请中，术语“第一细菌”通常是指用于引入或包含所述第一多核苷酸的细菌。所述第一细菌可以包含所述LC。在本申请中，所述第一细菌可以被导入所述轻链存储连接产物。在本申请中，所述第一细菌可以表达、复制和/或存储(例如，冷冻保存)所述LC、所述第一多核苷酸和/或所述轻链存储连接产物。
在本申请中，术语“轻链组件细菌文库”通常是指通过将所述第一多核苷酸导入所述第一细菌中获得的细菌文库。在本申请中，所述轻链组件细菌文库可以为包含编码抗体或抗体片段的轻链或轻链片段的核酸序列的细菌文库。在本申请中，所述轻链组件细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码抗体或抗体片段的不同的轻链或轻链片段的核酸序列。在本申请中，所述轻链组件细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第一细菌。
在本申请中，术语“第二多核苷酸”通常是指包含连接子的多核苷酸。在本申请中，所述第二多核苷酸还可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一多核苷酸可以以5’至3’方向包含结构R3-连接子-R4，其中所述R3，R4可以为限制性内切核酸酶的识别位点。例如，经识别所述第二多核苷酸中的内切酶识别位点的内切酶(例如，识别R3和R4的限制性内切核酸酶，例如SfiI)酶切处理后，经酶切处理的所述第二多核苷酸可包含所述连接子。
在本申请中，当与第二多核苷酸或连接子共同使用时，术语“载体片段”通常是指能够与所述第二多核苷酸连接的载体片段。在本申请中，所述载体片段可以源自pUC载体。例如，所述pUC载体可以为pUC19载体或源自pUC19载体；又例如，所述pUC载体可以为pMD19-T载体或源自pMD19-T载体。
在本申请中，术语“连接子存储载体”通常是指所述第二多核苷酸和所述载体片段连接形成的产物。在本申请中，所述连接子存储载体所包含的限制性内切核酸酶的识别位点可以与所述第一多核苷酸所包含的限制性内切核酸酶的识别位点对应地相同。例如，所述连接子存储载体可以包含所述R3和所述R4。在本申请中，所述连接子存储载体可仅包含一种所述连接子。
在本申请中，术语“第二细菌”通常是指用于引入或包含所述第二多核苷酸的细菌。所述第二细菌可以包含所述连接子。在本申请中，所述第二细菌可以被导入所述连接子存储载体。在本申请中，所述第二细菌可以表达、复制和/或存储(例如，冷冻保存)所述连接子和/或所述第二多核苷酸。
在本申请中，术语“连接子组件细菌”通常是指通过将所述连接子存储载体导入所述第二细菌中获得的细菌。在本申请中，所述连接子组件细菌可以为单一品种的细菌。在本申请中，单一品种的所述连接子组件细菌可以仅包含单一种类的所述连接子。
在本申请中，术语“第三多核苷酸”通常是指包含HC的多核苷酸，所述HC可以为包含编码所述抗体或抗体片段的重链或重链片段的核酸序列。在本申请中，所述第三多核苷酸还可以在5’端和/或3’端存在内切酶(例如，限制性内切核酸酶)的识别位点。例如，所述第一多核苷酸可以以5’至3’方向包含结构R5-HC-R6，其中所述R5，R6可以为限制性内切核酸酶的识别位点。例如，经识别所述第三多核苷酸中的内切酶识别位点的内切酶(例如，识别R5的限制性内切核酸酶，例如SfiI；又例如，识别R6的限制性内切核酸酶，例如SfiI，BsmBI和Esp3I)酶切处理后，经酶切处理的所述第三多核苷酸可包含所述HC。
在本申请中，术语“重链存储载体片段”通常是指经限制性内切核酸酶酶切处理的重链存储载体的片段。在本申请中，所述重链存储载体所包含的限制性内切核酸酶的识别位点可以与所述第三多核苷酸所包含的限制性内切核酸酶的识别位点对应地相同。例如，所述重链存储载体可以包含所述R5和所述R6。在本申请中，经限制性内切核酸酶(例如，识别所述R5和所述R6的限制性内切核酸酶)酶切后，得到的所述重链存储载体片段可以与经酶切处理的所述第三多核苷酸连接。在本申请中，所述重链存储载体片段可以源自pUC载体。例如，所述pUC载体可以为pUC19载体或源自pUC19载体；又例如，所述pUC载体可以为pMD19-T载体或源自pMD19-T载体。
在本申请中，术语“重链存储连接产物”通常是指经酶切处理后的所述第三多核苷酸与所述重链存储载体片段连接形成的产物。在本申请中，所述第三多核苷酸与所述重链存储载体片段可以包含相同限制性内切核酸酶的识别位点，可以被连接酶(例如DNA连接酶)相连接，而得到所述重链存储连接产物。
在本申请中，术语“第三细菌”通常是指用于引入或包含所述第三多核苷酸的细菌。所述第三细菌可以包含所述HC。在本申请中，所述第三细菌可以被导入所述重链存储连接产物。在本申请中，所述第三细菌可以表达、复制和/或存储(例如，冷冻保存)所述HC、所述第三多核苷酸和/或所述重链存储连接产物。
在本申请中，术语“重链组件细菌文库”通常是指通过将所述第三多核苷酸导入所述第三细菌中获得的细菌文库。在本申请中，所述重链组件细菌文库可以为包含编码抗体或抗体片段的重链或重链片段的核酸序列的细菌文库。在本申请中，所述重链组件细菌文库可以包含约10 5-约10 9个(例如，可以包含约10 5-约10 8个、约10 5-约10 7个、约10 6-约10 7个)编码抗体或抗体片段的不同的重链或重链片段的核酸序列。在本申请中，所述重链组件细菌文库可包含约10 7-约10 12个(例如，可以包含约10 7-约10 11个、约10 7-约10 10个、约10 7-约10 9个、约10 7-约10 8个)所述第三细菌。
在本申请中，术语“轻链组件质粒”通常是指从所述轻链组件细菌文库的细菌中获得的、包含所述LC的质粒。在本申请中，所述轻链组件质粒可以包含所述LC。例如，所述轻链组件组件质粒可以包含所述第一多核苷酸。在本申请中，所述轻链组件质粒可以包含限制性内切核酸酶的识别位点，例如，可以包含R1和R2。
在本申请中，术语“重链组件质粒”通常是指从所述重链组件细菌文库的细菌中获得的、包含所述HC的质粒。在本申请中，所述重链组件质粒可以包含所述HC。例如，所述重链组件质粒可以包含所述第三多核苷酸。在本申请中，所述重链组件质粒可以包含限制性内切核酸酶的识别位点，例如，可以包含R5和R6。
在本申请中，术语“经释放的LC”通常是指所述轻链组件质粒经处理后释放出的所述LC。在本申请中，所述处理可以为酶切处理。例如，可以针对所述轻链组件质粒上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，识别R1和R2的限制性内切核酸酶)，从而使所述LC从所述轻链组件质粒中释放而分离。
在本申请中，术语“经释放的连接子”通常是指所述连接子组件质粒经处理后释放出的所述连接子。在本申请中，所述处理可以为酶切处理。例如，可以针对所述连接子组件质粒上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，识别R3和R4的限制性内切核酸酶)，从而使所述连接子从所述连接子组件质粒中释放而分离。又例如，可以 先以所述连接子组件质粒为模板，扩增(例如，使用PCR扩增)包含所述第二多核苷酸，继而对获得的扩增产物进行酶切处理。例如，可以针对所述连接子组件质粒上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，识别R3和R4的限制性内切核酸酶)，从而使所述连接子从所述连接子组件质粒中释放而分离。
在本申请中，术语“经释放的HC”通常是指所述重链组件质粒经处理后释放出的所述HC。在本申请中，所述处理可以为酶切处理。例如，可以针对所述重链组件质粒上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如R5和R6)，从而使所述HC从所述重链组件质粒中释放而分离。
在本申请中，术语“经释放的展示载体片段”通常是指展示载体经处理后释放出的展示载体的片段。在本申请中，所述展示载体可以以5’至3’方向包含结构R7-展示载体片段-R8，其中所述R7和R8可以为限制性内切核酸酶。在本申请中，所述处理可以为经限制性内切核酸酶酶切处理。例如，可以针对所述展示载体上的限制性内切核酸酶的识别位点，选择合适的限制性内切核酸酶(例如，R7和R8)，从而使所述经释放的展示载体片段从所述展示载体中释放而分离。
在本申请中，术语“连接”通常是指将两个或者两个以上的多核苷酸分子连接在一起。例如，所述连接可以通过连接酶(例如，DNA连接酶)实现。例如，使一个多核苷酸的3’端与另一个多核苷酸的5’端连接，从而形成一个完整的多核苷酸分子。
在本申请中，术语“展示用连接产物”通常是指包含所述经释放的LC、所述经释放的连接子、所述经释放的HC以及所述经释放的展示载体片段的一个多核苷酸分子。在本申请中，所述展示用连接产物可以通过所述经释放的LC、所述经释放的连接子、所述经释放的HC和所述经释放的展示载体片段按比例混合而得到。
在本申请中，所述“展示”可以指在使包含所述展示用连接产物的细胞表达所述展示用连接产物所编码的蛋白(例如，抗体或抗体片段，例如，Fab)。
在本申请中，术语“导入”通常是指将外源多核苷酸转移或导入细胞中的过程。所述细胞可以为宿主细胞。所述导入的细胞包括对象的初级细胞及其后代。所述细胞可以为原核细胞，例如，可以为细菌细胞。
在本申请中，术语“第四细菌”通常是指用于引入或包含所述展示用连接产物的细菌，其中所述展示用连接产物包含所述经释放的LC、所述经释放的连接子、所述经释放的HC以及所述经释放的展示载体片段。在本申请中，所述第四细菌可以通过转化而导入所述展示用连接产物。在本申请中，所述第四细菌可以表达所述经释放的LC所编码的抗体或抗体片段的轻链或轻链片段；可以表达所述经释放的HC所编码的抗体或抗体片段的重链或重链片段。 在本申请中，所述第四细菌可以表达、复制和/或存储(例如，冷冻保存)所述抗体或抗体片段。
在本申请中，术语“展示细菌文库”通常是指包含所述展示用连接产物的细菌文库。在本申请中，所述展示用细菌文库可包含所述第四细菌。在本申请中，所述展示细菌文库可以包含10 10个以上的不同的抗体或抗体片段。
在本申请中，术语“信号肽pelB”通常是指果胶酶裂解酶的信号肽。所述信号肽pelB通常用于原核表达***。例如，所述信号肽pelB在GenBank中的登录号可以为ABS75961.1。
在本申请中，术语“限制性内切核酸酶”通常是指一种将双股DNA切开的酶。所述限制性内切核酸酶可以产生具有突出单股DNA的黏性末端，从而可以与DNA连接酶黏合。在本申请中，所述限制性内切核酸酶可以具备识别和限制性切割的作用。例如，所述限制性内切核酸酶的切割位点距离其识别位点存在一定的距离。例如，所述限制性内切核酸酶可以选自SfiI，BsmBI和Esp3I。
在本申请中，术语“线性化”通常是指载体呈线性而非闭合的环状。例如，所述载体进行酶切处理后，破坏了所述载体原有的环状结构，从而形成线性化的载体。例如，所述载体(例如T载体)经限制性内切酶酶切后，可以产生一个3’端的T末端，此时与具有A末端的PCR产物可以直接通过T4连接酶的作用而将该PCR产物与线性化的载体相连接。
在本申请中，术语“克隆”通常是指菌落的数量。例如，所述克隆可以为细菌文库(例如，所述轻链组件细菌文库、所述重链组件细菌文库、所述展示细菌文库和/或所述噬菌体文库)中菌落的数量。在某些情形下，所述克隆可以为所述细菌文库中不同的菌落的数量。在某些情形下，所述克隆可以为某一单一克隆所产生的子代群体的数量。
在本申请中，术语“多核苷酸”通常是指核苷酸，即连接在一起的至少两个核苷酸。所述多核苷酸可以是任何长度的聚合物，包括例如200、300、500、1000、2000、3000、5000、7000、10,000、100,000等。所述多核苷酸可以含有磷酸二酯键。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
一方面，本申请提供一种产生展示抗体或抗体片段的噬菌体文库的方法，所述方法包括：1)提供第一多核苷酸，第二多核苷酸和第三多核苷酸，所述第一多核苷酸包含LC，所述第二多核苷酸包含连接子且所述第三多核苷酸包含HC，其中所述LC包含编码所述抗体或抗体片段的轻链或轻链片段的核酸序列，所述HC包含编码所述抗体或抗体片段的重链或重链片 段的核酸序列；2)将所述第一多核苷酸导入第一细菌中以获得轻链组件细菌文库，将所述第二多核苷酸导入第二细菌中以获得连接子组件细菌，将所述第三多核苷酸导入第三组细菌中以获得重链组件细菌文库；3)由所述轻链组件细菌文库获得包含所述LC的轻链组件质粒，由所述连接子组件细菌获得包含所述连接子的连接子组件质粒，且由所述重链组件细菌文库获得包含所述HC的重链组件质粒；4)由所述轻链组件质粒获得经释放的LC，由所述连接子组件质粒获得经释放的连接子，且由所述重链组件质粒获得经释放的HC；5)提供展示载体，并由所述展示载体获得经释放的展示载体片段；6)使所述经释放的LC、经释放的连接子、经释放的HC以及经释放的展示载体片段连接，以形成展示用连接产物；7)将所述展示用连接产物导入第四组细菌中，以获得所述抗体或抗体片段的展示细菌文库；8)使用所述展示细菌文库制备展示所述抗体或抗体片段的噬菌体文库。
在本申请中，在适于抗体或抗体片段表达的条件下，6)的所述展示用连接产物可以依照阅读框表达所述轻链或轻链片段以及所述重链或重链片段。
在本申请中，所述第一多核苷酸，所述第二多核苷酸和所述第三多核苷酸可以均为线性核酸分子。
在本申请中，编码所述重链或重链片段的核酸序列与编码所述轻链或轻链片段的核酸序列可以位于不同的阅读框中。在本申请中，所述阅读框可以为从起始密码子开始至终止密码子结束的连续的核苷酸序列，并且其内部不再含有启动子或终止子。例如，所述阅读框可以编码蛋白质或多肽片段。
在本申请中，所述连接子可以包含编码信号肽pelB或其片段的核酸序列。
在本申请中，所述连接子可包含编码信号肽pelB的片段的核酸序列，其余编码信号肽pelB的片段的核酸序列还可以位于所述R5中。例如，所述编码信号肽pelB的片段的核酸序列的3’端的核苷酸可以位于所述R5中。例如，当所述经释放的连接子与所述经释放的HC连接时，所述经释放的连接子中包含的编码信号肽pelB的片段的核酸序列可以与所述经释放的HC中包含的编码信号肽pelB的片段的核酸序列相连接，从而形成编码完整的信号肽pelB的核酸序列。
在本申请中，所述连接子的长度可以为约50至约200个碱基。例如，所述连接子的长度可以为约50至约200个碱基、约50至约180个碱基、约50至约160个碱基、约50至约140个碱基、约50至约120个碱基、约50至约100个碱基、约50至约90个碱基、约50至约80个碱基、约50至约75个碱基、约50至约70个碱基、约50至约60个碱基。
在本申请中，在6)的所述展示用连接产物中，所述LC可以位于所述连接子的上游，且所述连接子可以位于所述HC的上游。在本申请中，所述上游可以为核苷酸序列的5’端。
在本申请中，2)可以包括冷冻保存所述轻链组件细菌文库和/或所述重链组件细菌文库。在本申请中，7)可以包括冷冻保存所述展示细菌文库。在本申请中，所述冷冻保存可以指在低于-18℃的条件下保存，例如，可以在低于-80℃的条件下保存。
在本申请中，8)可以包括融化并培养所述经冷冻保存的展示细菌文库，并使用经培养的所述展示细菌文库构建所述噬菌体文库。
在本申请中，所述第一多核苷酸以5’至3’方向可以包含结构R1-LC-R2，所述第二多核苷酸以5’至3’方向可以包含结构R3-连接子-R4，所述第三多核苷酸以5’至3’方向可以包含结构R5-HC-R6，且所述展示载体中以5’至3’方向可以包含结构R7-展示载体片段-R8，其中所述R1，R2，R3，R4，R5，R6，R7和R8为限制性内切核酸酶的识别位点。
在本申请中，所述R2经限制性内切核酸酶切割后产生的末端：能够与所述R3经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R4、R5、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。在本申请中，所述R2可以与所述R3相同。
在本申请中，所述R4经限制性内切核酸酶切割后产生的末端：能够与所述R5经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R2、R3、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。在本申请中，所述R4可以与所述R5相同。
在本申请中，所述R6经限制性内切核酸酶切割后产生的末端：能够与所述R7经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R1、R2、R3、R4、R5及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。在本申请中，所述R6可以与所述R7相同。
在本申请中，所述R8经限制性内切核酸酶切割后产生的末端：能够与所述R1经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且不与所述R2、R3、R4、R5、R6及R7中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。在本申请中，所述R8可以与所述R1相同。
在本申请中，所述R1，R2，R3，R4，R5、R6、R7和R8中的任一项经限制性内切核酸酶切割后产生的末端可以为非回文序列。
在选择内切核酸酶识别位点时(例如，R1，R2，R3，R4，R5、R6、R7和R8时)，可选择抗体的可变区(例如，轻链可变区或重链可变区)中基本上不包含的序列以尽量保持抗体可变区的完整性，防止酶切时破坏(例如，降解)抗体基因库。此外，所述核酸酶识别位点经限制性内切核酸酶切割后形成的末端可以是非回文序列，以防止其自我连接，进而实现 减低非目的连接。此外，所述内切核酸酶识别位点的选择可使得多个片段的定向连接成为可能，以提高连接效率。
在本申请中，所述R1，R2，R3，R4，R5、R6、R7和R8中的两项或更多项(例如，2项、3项、4项、5项、6项、7项或8项)能够被相同的限制性内切核酸酶识别及切割。
例如，在本申请中，所述R1、R2、R3、R4、R5和R8可以被相同的限制性内切核酸酶识别及切割。例如，所述R6和R7能够被相同的限制性内切核酸酶识别及切割。在某些情形中，识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶可以与识别所述R6和R7的限制性内切核酸酶不同。
在本申请中，所述限制性内切核酸酶可以选自SfiI，BsmBI和Esp3I。在本申请中，所述BsmBI和Esp3I可以为同工酶，其可以识别相同的限制性内切核酸酶的识别位点。
在本申请中，例如，所述R1、R2、R3、R4、R5和R8可以被SfiI识别及切割。在本申请中，例如，所述R6和R7能够被BsmBI和/或Esp3I识别及切割。
例如，SfiI可以识别由13个碱基构成的序列(5’至3’)GGCCNNNN/NGGCC(SEQ ID NO:99)，其经过酶切后可形成3’端的突出序列(overhang，例如包含3个碱基的单链序列)，其中N可以代表GATC四种碱基中的任何一种。因此，有64种不同的序列均能够被SfiI识别。
例如，BsmBI和Esp3I可以识别由12个碱基构成的序列(5’至3’)CGTCTCN/NNNNN(SEQ ID NO:100)，其经过酶切后可形成5’端的突出序列(overhang，例如包含4个碱基的单链序列)，其中N可以代表GATC四种碱基中的任何一种。因此，有264种不同的序列均能够被BsmBI和Esp3I识别。
在本申请中，所述R1可以包含SEQ ID NO:1所示的核酸序列。在本申请中，所述R2可以包含SEQ ID NO:2所示的核酸序列。在本申请中，所述R3可以包含SEQ ID NO:2所示的核酸序列。在本申请中，所述R4可以包含SEQ ID NO:3所示的核酸序列。在本申请中，所述R5可以包含SEQ ID NO:3所示的核酸序列。在本申请中，所述R6可以包含SEQ ID NO:4所示的核酸序列。在本申请中，所述R7可以包含SEQ ID NO:4所示的核酸序列。在本申请中，所述R8可以包含SEQ ID NO:1所示的核酸序列。
在本申请中，所述轻链或轻链片段可以包含轻链可变区部分和/或轻链恒定区部分。在本申请中，所述重链或重链片段可以包含重链可变区部分和/或重链恒定区部分。
例如，所述LC可以包含编码所述轻链或轻链片段的轻链可变区VL的多核苷酸。例如，所述第一多核苷酸可以包含编码所述轻链或轻链片段的轻链可变区VL的多核苷酸。所述第一多核苷酸的5’端可连接所述R1且所述第一多核苷酸的3’端可以连接所述R2；且，所述轻 链存储载体片段的5’端可以连接所述R3且所述轻链存储载体片段的3’端可以连接所述R8。
例如，所述HC可以包含编码所述重链或重链片段的重链可变区VH和重链恒定区的CH1的多核苷酸。例如，所述第三多核苷酸可以包含编码所述重链或重链片段的重链可变区VH和重链恒定区的CH1的多核苷酸。所述第三多核苷酸的5’端可连接所述R5且所述第三多核苷酸的3’端可连接所述R6；且，所述重链存储载体片段的5’端可连接所述R7且所述重链存储载体片段的3’端可连接所述R4。
又例如，所述HC可以包含编码所述重链或重链片段的重链可变区VH的多核苷酸。例如，所述第三多核苷酸可以包含编码所述重链或重链片段的重链可变区VH的多核苷酸。此时，所述重链存储载体片段可以包含编码所述重链或重链片段的重链恒定区的CH1的多核苷酸。所述第三多核苷酸的5’端可连接所述R5且所述第三多核苷酸的3’端可连接所述R6；且，所述重链存储载体片段的5’端可连接所述R7且所述重链存储载体片段的3’端可连接所述R4。
在本申请中，所述抗体或其片段可以包括Fab，Fab’，(Fab)2和/或(Fab’)2。
在本申请中，7)中所述展示用连接产物的连接转化效率可以为至少10 8个克隆/μg多核苷酸(例如，可以为至少10 9个克隆/μg多核苷酸、至少10 10个克隆/μg多核苷酸、至少10 11个克隆/μg多核苷酸、至少10 12个克隆/μg多核苷酸)。
在本申请中，2)可以包括使用识别所述R1和R2的限制性内切核酸酶对所述第一多核苷酸进行酶切处理，使经酶切处理的所述第一多核苷酸与轻链存储载体片段连接形成轻链存储连接产物，并将所述轻链存储连接产物导入所述第一细菌中以获得所述轻链组件细菌文库；其中所述轻链存储载体可包含所述R1和R2，并且通过使用识别所述R1和R2的限制性内切核酸酶对所述轻链存储载体进行酶切处理而获得所述轻链存储载体片段。
在本申请中，所述LC的长度可以为大于约600bp。例如，可以为大于约650bp，可以为大于约680bp，大于约700bp。在本申请中，所述第一多核苷酸的长度可以为大于约600bp。例如，可以为大于约650bp，可以为大于约680bp，大于约700bp。在本申请中，所述轻链存储载体片段的长度可以为大于约900bp，例如，可以为大于约1000bp，大于约1500bp，大于约1800bp，大于约2100bp。在本申请中，所述轻链存储载体片段的长度可以与所述LC的长度显著不同。例如，所述轻链存储载体片段的长度可以与所述LC的长度可以借助聚丙烯酰胺凝胶电泳显著区分。
在本申请中，所述轻链存储载体可以源自任何载体，例如任何可扩增和/或容易保存的载体。在某些情形中，用于作为轻链存储载体的所述载体可具有高拷贝数、分子量小等性质。例如，所述轻链存储载体可以为pUC载体。在本申请中，所述pUC载体可以为pUC19载体(其结构示意图可以如图1所示)或源自pUC19载体。在本申请中，所述pUC载体可以为 pMD19-T载体或源自pMD19-T载体。
在本申请中，所述识别所述R1和R2的限制性内切核酸酶可以包括SfiI。
在本申请中，2)可以包括使用识别所述R5和R6的限制性内切核酸酶对所述第三多核苷酸进行酶切处理，使经酶切处理的所述第三多核苷酸与重链存储载体片段连接形成重链存储连接产物，并将所述重链存储连接产物导入所述第三细菌中以获得所述重链组件细菌文库；其中所述重链存储载体包含所述R5和R6，并且通过使用识别所述R5和R6的限制性内切核酸酶对所述重链存储载体进行酶切处理而获得所述重链存储载体片段。
在本申请中，所述HC的长度可以为大于约320bp。例如，可以为大于约350bp，可以为大于约380bp，大于约400bp。在本申请中，所述第三多核苷酸的长度可以为大于约320bp。例如，可以为大于约350bp，可以为大于约380bp，大于约400bp。在本申请中，所述重链存储载体片段的长度可以为为大于约900bp，例如，可以为大于约1000bp，大于约1500bp，大于约1800bp，大于约2100bp。在本申请中，所述重链存储载体片段的长度可以与所述HC的长度显著不同。例如，所述轻链存储载体片段的长度可以与所述HC的长度可以借助聚丙烯酰胺凝胶电泳显著区分。
在本申请中，所述重链存储载体可以源自任何载体，例如任何可扩增和/或容易保存的载体。在某些情形中，用于作为重链存储载体的所述载体可具有高拷贝数、分子量小等性质。例如，所述重链存储载体可以为pUC载体。在本申请中，所述pUC载体可以为pUC19载体(其结构示意图可以如图1所示)或源自pUC19载体。在本申请中，所述pUC载体可以为pMD19-T载体或源自pMD19-T载体。
在本申请中，为了构建所述重链存储载体，可以对所述pUC载体或源自pUC的载体进行工程化/修饰。例如，可以通过定点突变去除所述载体中的一个或多个内切核酸酶识别位点(例如，去除其中的一个或多个BsmBI的识别位点)。在某些情形中，也可以通过定点突变在所述载体中增加一个或多个内切核酸酶识别位点(例如，在选定的位置增加一个或多个BsmBI的识别位点)。
在一个实例中，可通过定点突变的方法去除所述载体中原本含有的一个或多个BsmBI识别位点，然后可在所述载体中的另一个位置添加一个或多个另外的BsmBI识别位点，以获得经改造的载体(例如，经改造的pUC载体)。
在本申请中，所述重链存储载体中可以包含且仅包含一个BsmBI的识别位点。
在本申请中，所述识别所述R5和R6的限制性内切核酸酶可以包括SfiI，BsmBI和/或Esp3I。在某些情形中，识别所述R5和R6的限制性内切核酸酶为BsmBI和/或Esp3I。
在本申请中，2)可以包括使所述第二多核苷酸与载体片段连接形成连接子存储载体(例 如，根据Takara公司TA克隆产品的说明书)，并将所述连接子存储载体导入所述第二细菌中以获得所述连接子组件细菌。在本申请中，所述连接子存储载体中可以包含所述R3和R4位点。
在本申请中，所述连接子的长度可以为大于约70bp，例如，可以为约72bp，或者可以为约90bp。在本申请中，所述第二多核苷酸的长度可以为大于约80bp，例如，可以为大于约90bp，可以为大于约100bp，可以为大于约120bp。在本申请中，所述载体片段的长度可以为大于约800bp。
在本申请中，用于构建所述连接子存储载体的所述载体片段可以源自任何载体，例如任何可扩增和/或容易保存的载体。在某些情形中，用于构建所述连接子存储载体的载体可具有高拷贝数、分子量小等性质。例如，用于构建所述连接子存储载体的所述载体片段可以源自pUC载体。在本申请中，所述pUC载体可以为pUC19载体(其结构示意图可以如图1所示)或源自pUC19载体。在本申请中，所述pUC载体可以为pMD19-T载体或源自pMD19-T载体。
在本申请中，所述pUC19载体或pMD19-T载体包括源自其但经过修饰/改造的载体。
在本申请中，4)可以包括使用识别所述R1和R2的限制性内切核酸酶对所述轻链组件质粒进行酶切处理，从而获得所述经释放的LC。在本申请中，所述识别所述R1和R2的限制性内切核酸酶可以包括SfiI。
在本申请中，4)可以包括使用识别所述R5和R6的限制性内切核酸酶对所述重链组件质粒进行酶切处理，从而获得所述经释放的HC。在本申请中，所述识别所述R5和R6的限制性内切核酸酶可以包括SfiI，BsmBI和/或Esp3I。
在某些情形下，在本申请中，4)可以包括使用识别所述R3和R4的限制性内切核酸酶对所述连接子存储载体进行酶切处理，从而获得所述经释放的连接子。
例如，可以直接使用识别所述R3和R4的限制性内切核酸酶(例如，SfiI)对所述连接子存储载体进行酶切处理，从而获得所述经释放的连接子。在本申请中，所述连接子的长度可以为约72bp以上，例如可以为约90bp以上。
在某些情形下，在本申请中，4)可以包括由所述连接子存储载体获得扩增产物，所述扩增产物包含所述连接子、所述R3以及所述R4；并且使用识别所述R3和R4的限制性内切核酸酶对所述扩增产物进行酶切处理，从而获得所述经释放的连接子。
例如，可以通过PCR等方法获得包含所述连接子、所述R3以及所述R4的所述扩增产物，该扩增产物的长度可以为约800bp以上。例如，可以为约900bp以上，可以为约1000bp以上，可以为约1200bp以上。在本申请中，可以使用识别所述R3和R4的限制性内切核酸 酶(例如，SfiI)对所述扩增产物进行酶切，从而获得所述经释放的连接子。在本申请中，所述连接子的长度可以为约72bp以上，可以为约90bp以上。在本申请中，所述扩增产物的长度可以与所述连接子的长度显著不同。这样可以更容易、更高效地得到单一的所述连接子。例如，所述扩增产物的长度可以与所述连接子的长度可以借助聚丙烯酰胺凝胶电泳显著区分。
在本申请中，所述识别所述R3和R4的限制性内切核酸酶可以包括SfiI。
在本申请中，5)可以包括使用识别所述R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理以释放所述展示载体片段。在本申请中，所述识别所述R7和R8的限制性内切核酸酶可以包括SfiI，BsmBI和/或Esp3I。
在本申请中，所述展示载体可以源自任何合适的载体，例如pComb3x载体。可对所述pComb3x载体进行改造以适合用于本申请的目的。例如，所述pComb3x载体可包含一个位于5’端的SfiI识别位点以及一个位于3’端的SfiI识别位点，在所述改造过程中，可通过定点突变去除所述pComb3x载体中3’端的SfiI识别位点。在某些情形中，所述定点突变可不影响所述载体中蛋白(例如，抗体重链片段和/或抗体轻链片段)的框内表达。例如，所述突变可以为无义突变，例如仅改变碱基序列但不改变氨基酸序列的突变。
在本申请中，使用识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，可以获得三个不同的酶切片段。例如，所述展示载体可以包含3个可以被SfiI识别的限制性核酸内切酶识别位点。当用SfiI酶切处理所述展示载体时，可以得到三个所述不同的酶切片段。
在本申请中，所述识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶可以包括SfiI。在本申请中，使用识别所述R6和R7的限制性内切核酸酶对所述展示载体进行酶切处理后，可以使所述展示载体线性化。
在本申请中，识别所述R6和R7的限制性内切核酸酶可以包括BsmBI和/或Esp3I。
在本申请中，使用识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，可以获得四个不同的酶切片段。例如，所述展示载体可以包含3个可以被SfiI识别的限制性核酸内切酶识别位点，以及1个可以被BsmBI和/或Esp3I识别的限制性核酸内切酶识别位点。当用SfiI、BsmBI和/或Esp3I酶切处理所述展示载体时，可以得到四个所述不同的酶切片段。
例如，利用所述限制性内切核酸酶R1和R2可以获得所述经释放的LC；利用所述限制性内切核酸酶R5和R6可以获得所述经释放的HC；
在本申请中，所述识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶可以包括SfiI，BsmBI和/或Esp3I。
在本申请中，所述载体(例如，所述展示载体或存储载体)可包含或编码一个或多个合适的标记物(例如，用于纯化/识别/筛选的标记物)，所述标记物可以为，例如His tag，Flag Tag，荧光蛋白，选择性抗生素，和/或亲和素等。
在本申请中，2)可以包括对所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库进行取样检测，以确定所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库的库容量和/或质量。
在本申请中，所述轻链组件细菌文库可以包含约至少10 6个(例如，约至少10 7个、约至少10 8个、约至少10 9个、约至少10 10个、约至少10 11个、约至少10 12个或更多)不同的克隆。
在本申请中，所述重链组件细菌文库可以包含至约少10 7个(例如，约至少10 8个、约至少10 9个、约至少10 10个、约至少10 11个、约至少10 12个或更多)不同的克隆。
在本申请中，所述轻链组件细菌文库中有效克隆的比例可以为至少约70％(例如，至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％)。
在本申请中，所述重链组件细菌文库中有效克隆的比例可以为至少约70％(例如，至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％)。
在本申请中，2)可以包括根据所述轻链组件细菌文库和/或所述重链组件细菌文库的库容量，所述连接子组件细菌和/或质量测算所述展示细菌文库的库容量和/或质量。
在本申请中，所述展示细菌文库可以包含约至少10 10个(例如，约至少10 11个、约至少10 12个、约至少10 13个、约至少10 14个、约至少10 15个、约至少10 16个或更多)不同的克隆。
在本申请中，所述展示细菌文库中有效克隆的比例可以为至少约70％(例如，至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％)。
在本申请中，所述展示细菌文库中细菌可以进行液体培养。所述液体培养的培养时间可以为不大于约8小时，例如，可以为不大于约4小时、不大于约5小时、不大于约6小时或者不大于约7小时。在本申请中，所述液体培养操作较为简便。在某些情形下，所述展示细菌文库中的细菌可以取少量菌液铺皿培养，然后再挑选菌落。所述铺皿培养的培养时间可以为约12-18小时，例如，可以为约12小时、13小时、14小时、15小时、16小时、17小时或18小时。在本申请中，所述铺皿培养可以挑选菌落(例如，挑选单克隆)再进行测序分析。在本申请中，所述铺皿培养可以降低优势克隆的数量。
在本申请中，可以由样品材料获得所述第一多核苷酸和所述第三多核苷酸。在本申请中，所述样品材料可以包括源自外周血淋巴细胞样品的材料。在本申请中，所述外周血淋巴细胞可以为人外周血淋巴细胞。在本申请中，所述样品材料也可以源自其他任意的组织和/或细胞， 并不限于所述外周血淋巴细胞样品。
在本申请中，所述样品材料可以包括源自所述样品的总RNA和/或mRNA。
在本申请中，8)可以包括使所述展示细菌文库与辅助噬菌体接触来制备所述噬菌体文库。
另一方面，本申请提供了一种通过所述的方法产生的噬菌体文库。
在本申请中，所述的噬菌体文库可以包含约至少10 10个(例如，约至少10 11个、约至少10 12个、约至少10 13个、约至少10 14个、约至少10 15个、约至少10 16或更多)不同的克隆。在本申请中，所述的噬菌体文库能够展示约至少10 10种(例如，约至少10 11种、约至少10 12种、约至少10 13种、约至少10 14种、约至少10 15种、约至少10 16种或更多)不同的抗体或抗体片段。
另一方面，本申请提供了一种筛选抗体或抗体片段的方法，其包括使用所述的噬菌体文库。
例如，所述的抗体片段可以为Fab。例如，所述抗体或抗体片段可以特异性结合TNFα，IL-6，IL6R，IL17-FR，CD38，GMCSF和Siglec3。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.构建人PBMC的噬菌体表面抗体(Fab)展示库
1.1 获取免疫材料总RNA/mRNA
从人的外周血淋巴细胞提取总RNA，并且进一步从总RNA分离mRNA(Takara Cat#Z652N/636592，具体试验步骤参见产品说明书)。
1.2 设计合成引物
参照《Phage Display》(A Laboratory Mannual，ISBN 0-87969-546-3)，设计针对人重链可变区VH、轻链KLC(全长Kappa轻链)、轻链LLC(全长Lamda轻链)的引物。其中，轻链正向引物5’-端含有R1的核苷酸序列GGCCCAGGCGGCC(SEQ ID NO:1)，反向引物5’-端含有R2的核苷酸序列GGCCACATAGGCC(SEQ ID NO:2)；重链可变区正向引物5’-端含有R5的核苷酸序列GGCCCAACCGGCC(SEQ ID NO:3)，反向引物5’-端含有R6的核苷酸序列GGCCCTCAGCGGCC(SEQ ID NO:4)。引物由金维智公司合成。
以pComb3x载体为模板，设计扩增连接子的正向和反向引物。其中，正向引物5’-端含有R3的核苷酸序列GGCCACATAGGCC(SEQ ID NO:2)，反向引物5’-端含有R4的核苷酸序列GGCCCAACCGGCC(SEQ ID NO:3)。
具体的引物序列可参照下表1-1：
表1-1 引物序列-1
引物名称 SEQ ID NO:
轻链KLC的正向引物 5-22
轻链KLC的反向引物 23
轻链LLC的正向引物 24-48
轻链LLC的反向引物 49
VH的正向引物 50-73
VH的反向引物 74-78
连接子的正向引物 79
连接子的反向引物 80
1.3 获得第一多核苷酸和第三多核苷酸
通过两步法扩增组件抗体基因库。
第一步，以实施例1.1中获得的mRNA为模板，用Promega的MMLV进行逆转录合成cDNA(按照Promega公司产品的说明书进行，其中引物为Thermo Cat#N8080127、逆转录酶为Promega Cat#M1701)。
第二步，以第一步获得的cDNA为模板，利用实施例1.2中获得的引物，通过PCR(Takara Cat#RR900A，按照公司产品的说明书进行)扩增组件抗体的KLC，LLC和VH基因库。凝胶电泳纯化回收后(利用Axygen的凝胶回收试剂盒，按照《分子克隆实验指南》中的记载操作)分别获得PCR产物——KLC片段(即本申请的第一多核苷酸)、LLC片段(即本申请的第一多核苷酸)和VH片段(即本申请的第三多核苷酸)。
1.4 构建存储载体
1.4.1 设计引物
参照实施例1.2的内容设计引物。
设计合成用于获得存储载体的引物。具体的引物序列可参照下表1-2：
表1-2 引物序列-2
引物名称 SEQ ID NO:
R1-1kb-R2的正向引物 81
R1-1kb-R2的反向引物 82
R5-1kb-R6的正向引物 85
R5-1kb-R6的反向引物 86
1.4.2 PCR扩增
以1kb长的人IgG1的Fc(SEQ ID NO:87)为模板，利用实施例1.4.1制备的引物R1-1kb-R2的正向引物和R1-1kb-R2的反向引物，进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R1-1kb-R2(SEQ ID NO:88)。
以pComb3x载体为模板，利用R3-连接子-R4的正向引物(SEQ ID NO:83)和R3-连接子-R4的反向引物(SEQ ID NO:84)进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R3-连接子-R4(SEQ ID NO:90)，得到本申请的所述第二多核苷酸。其中所述连接子的长度可以为72bp，其核苷酸序列如SEQ ID NO:89所示。
以1kb长的人IgG1的Fc(SEQ ID NO:87)为模板，利用实施例1.4.1制备的引物R5-1kb-R6的正向引物和R5-1kb-R6的反向引物，进行PCR，凝胶电泳纯化回收后，得到PCR产物——R5-1kb-R6(SEQ ID NO:91)。
1.4.3 构建轻链存储载体和重链存储载体
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将1.4.2制备的R1-1kb-R2片段***pMD19-T载体，得到用于***全长轻链基因库的轻链存储载体DDB-R1-1kb-R2，其载体图谱如图3所示。
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将1.4.2制备的R5-1kb-R6片段***pMD19-T载体得到含有R5-1kb-R6片段的载体，然后以这个载体为模板，利用引物突变去除这个载体中原有的BsmBI酶切位点，得到用于***VH基因库的重链存储载体DDB-R5-1kb-R6，其载体图谱如图5所示。
具体的引物序列可参照下表1-3：
表1-3 引物序列-3
引物名称 SEQ ID NO:
载体突变的正向引物 92
载体突变的反向引物 93
1.4.4 构建连接子存储载体并获得连接子组件细菌
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将实施例1.4.2制备的R3-连接子-R4***pMD19-T载体，得到连接子存储载体DDB-R3-连接子-R4，其载体图谱如图4所示。可用所述连接子存储载体转化TG1感受态细菌(Lucigen公司)，铺皿37℃培养过夜，送菌落测序，然后收集菌落得到连接子组件细菌。可将该连接子组件细菌冻存备用。
1.5 获得组件细菌文库
1.5.1 获得轻链组件细菌文库
利用限制性内切核酸酶R1和R2酶切实施例1.3所制得的第一多核苷酸(包括KLC和LLC)获得目的轻链片段(约为0.65kb)。
利用限制性内切核酸酶R1和R2酶切实施例1.4所制得的轻链存储载体DDB-R1-1kb-R2。获得轻链存储载体片段(约为2.7kb)。
将所得的目的轻链片段和轻链存储载体片段混合，再利用连接酶T4 DNA ligase(购自NEB，Thermo)连接得到轻链存储连接产物，然后用所述轻链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，送菌落测序，然后收集全部菌落得到轻链组件细菌文库。可检测该轻链组件细菌文库的质量和/或将该轻链组件细菌文库冻存备用。
1.5.2 获得重链组件细菌文库
利用限制性内切核酸酶R5和R6酶切实施例1.3所制得的第三多核苷酸获得目的重链可变区片段(约为0.35kb)。
利用限制性内切核酸酶R5和R6酶切实施例1.4所制得的重链存储载体DDB-R5-1kb-R6。获得重链存储载体片段(约为2.7kb)。
将所得的目的重链可变区片段和重链存储载体片段混合，再利用连接酶T4 DNA ligase(购自NEB，Thermo)连接得到重链存储连接产物，然后用所述重链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，挑菌落测序，然后收集全部菌落得到重链组件细菌文库。可检测该重链组件细菌文库的质量和/或将该重链组件细菌文库冻存备用。
1.6 获得轻链组件质粒、重链组件质粒和连接子片段
使用质粒提取试剂盒(购自Axygen)，分别提取实施例1.5.1制备的轻链组件细菌文库、实施例1.5.2制备的重链组件细菌文库中的质粒，分别得到轻链组件质粒和重链组件质粒。
利用限制性内切核酸酶R1和R2酶切实施例1.5.1制备的轻链组件质粒，经凝胶电泳纯化回收后，得到轻链***片段LC。
利用限制性内切核酸酶R5和R6酶切实施例1.5.2制备的重链组件质粒，经凝胶电泳纯化回收后，得到重链***片段HC。
使用质粒提取试剂盒(购自Axygen)，提取实施例1.4.4制备的连接子组件细菌中的质粒，得到连接子组件质粒。以该连接子组件质粒或实施例1.4.4中的连接子存储载体为模板，用连接子的正向引物(SEQ ID NO:79)和连接子的反向引物(SEQ ID NO:80)扩增含有连接子的0.8kb的片段，然后用限制性内切核酸酶R3和R4酶切该0.8kb的PCR产物，经凝胶电泳纯化回收(利用小片段凝胶回收试剂盒，购自莱枫生物，Cat#DK402)得到72pb的连接子片段。
1.7 获得展示载体
购买获得pComb3x载体，其载体图谱如图6所示，其中用于抗体Fab展示的经改造的pComb3x-fab载体图谱如图7所示。
通过无义突变除去pComb3x-fab载体中在Fab基因3’端的SfiI酶切位点。
然后在该无义突变后的载体中轻链终止密码子的下游加入限制性内切核酸酶R2的酶切位点，在重链可变区信号肽的末端通过无义突变引入限制性内切核酸酶R5的酶切位点，获得经改造后的展示载体DDB-R1R2R5R6，其图谱如图8所示。
1.8 制备展示细菌文库
利用限制性内切核酸酶R7和限制性内切核酸酶R8酶切实施例1.7制备的展示载体DDB-R1R2R5R6，获得3.6kb展示载体片段。
将实施例1.6获得的轻链***片段LC(0.65kb)、重链***片段HC(0.35kb)、连接子片段(72bp)和所述展示载体片段(3.6kb)按照1:1:1:1的分子比例混合，用T4 DNA连接酶20℃连接20小时以上，得到展示用连接产物。
利用PCR-Clean-up纯化连接产物，转入TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃生长过夜。挑选菌落测序，收集平皿上生长的全部菌落，即为展示细菌文库。可将该展示细菌文库保存备用。
1.9 制备展示型抗体噬菌体文库
从实施例1.8所制备的展示细菌文库中取适量的菌液，在2YT培养液(含有氨苄青霉素100μg/ml和2％葡萄糖)中37℃培养至OD 600达到0.5。然后向该菌液中加入M13KO7辅助噬菌体(购自NEB，Cat#N0315S，MOI约为10-20)混匀后，37℃静止30分钟，接着在37℃，250rpm条件下震摇30分钟，离心弃去含有M13KO7辅助噬菌体的培养液上清部分，以该菌液原始体积4倍的培养液(含有氨苄青霉素和卡那霉素)重悬细菌，30℃，250rpm条件下振摇过夜。第二天用PEG沉淀收集噬菌体，滴定噬菌体浓度，分装保存。即得到展示型抗体噬菌体文库。
实施例2 组件细菌文库的质量分析
分析实施例1.5所制备的组件细菌文库的质量。结果如表2所示，
表2 组件细菌文库的质量
Figure PCTCN2020085706-appb-000001
在构建展示细菌文库时，轻链组件细菌文库(KLC)与轻链组件细菌文库(LLC)的比例 为2:1，根据以上质量结果，可估算展示细菌文库的有效克隆比例将达到84％以上。而所述轻链组件细菌文库和重链组件细菌文库随机组合后得到的库容量理论上最高可以达到10 15。
实施例3 展示细菌文库的质量分析
用实施例1.8获得的分离纯化后的展示用连接产物197μg，转化398支TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)197个，37℃培养过夜，收获全部菌落，分装保存(即得到实施例1.8所制备的展示细菌文库)。展示细菌文库的库容量为10 11，背景克隆比例为2％。
分析该展示细菌文库的质量，结果如表3所示：
表3 展示细菌文库的质量
测序批次 送样测序克隆 可用测序报告 正确读框 重复序列 有效克隆 有效克隆比例
第一批 100 79 65 1 64 81％
第二批 100 79 67 0 67 85％
分析表3中两个批次共158个可用测序结果，其中有131个克隆的具有正确的阅读框(包括同时含有正确的重链可变区和轻链)，可见展示细菌文库的有效克隆比例达到83％，与实施例2中的估算结果相吻合。
实施例4.构建人PBMC的噬菌体表面抗体(Fab)展示库
4.1 提取免疫材料总RNA
从外周血淋巴细胞(购自妙通生物Cat#PB100C)提取总RNA(使用Qiagen的RNA小提试剂盒，Cat#74101，具体试验步骤参见试剂盒说明书)。
4.2 设计合成引物
参照《Phage Display》(A Laboratory Mannual，ISBN 0-87969-546-3)，设计针对人重链可变区VH、轻链KLC(全长Kappa轻链)、轻链LLC(全长Lamda轻链)的引物。其中，轻链正向引物5’-端含有R1的核苷酸序列GGCCCAGGCGGCC(SEQ ID NO:1)，反向引物5’-端含有R2的核苷酸序列GGCCACATAGGCC(SEQ ID NO:2)；重链可变区正向引物5’-端含有R5的核苷酸序列GGCCCAACCGGCC(SEQ ID NO:3)，反向引物5’-端含有R6的核苷酸序列GGCCCTCAGCGGCC(SEQ ID NO:4)。引物由金维智公司合成。
以pComb3x载体为模板，设计扩增连接子的正向和反向引物。其中，正向引物5’-端含有R3的核苷酸序列GGCCACATAGGCC(SEQ ID NO.2)，反向引物5’-端含有R4的核苷酸序列GGCCCAACCGGCC(SEQ ID NO.3)。
具体的引物序列可参照下表4-1：
表4-1 引物序列-1
引物名称 SEQ ID NO:
轻链KLC的正向引物 5-22
轻链KLC的反向引物 23
轻链LLC的正向引物 24-48
轻链LLC的反向引物 49
VH的正向引物 50-73
VH的反向引物 74-78
90bp连接子的正向引物 96-97
90bp连接子的反向引物 98
4.3 获得第一多核苷酸和第三多核苷酸
通过两步法扩增组件抗体基因库。
第一步，以实施例4.1中获得的总RNA为模板，用Promega的MMLV进行逆转录合成cDNA(按照Promega公司产品的说明书进行，其中引物为Thermo Cat#N8080127、逆转录酶为Promega Cat#M1701)。
第二步，以第一步获得的cDNA为模板，利用实施例4.2中获得的引物，通过PCR(Takara Cat#RR900A，按照公司产品的说明书进行)扩增组件抗体的KLC，LLC和VH基因库。凝胶电泳纯化回收后(利用Axygen的凝胶回收试剂盒，按照《分子克隆实验指南》中的记载操作)分别获得PCR产物——KLC片段(即本申请的第一多核苷酸)、LLC片段(即本申请的第一多核苷酸)和VH片段(即本申请的第三多核苷酸)。
4.4 构建存储载体
4.4.1 设计引物
设计用于获得存储载体的引物。具体的引物序列可参照下表4-2：
表4-2 引物序列-2
引物名称 SEQ ID NO:
R1-1kb-R2的正向引物 81
R1-1kb-R2的反向引物 82
R5-1kb-R6的正向引物 85
R5-1kb-R6的反向引物 86
4.4.2 PCR扩增
以1kb长的人IgG1的Fc(SEQ ID NO.87)为模板，利用实施例4.4.1制备的引物R1-1kb-R2的正向引物和R1-1kb-R2的反向引物，进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R1-1kb-R2(SEQ ID NO.88)。
以pComb3x载体为模板，利用R3-连接子-R4的正向引物(SEQ ID NO.83)和R3-连接 子-R4的反向引物(SEQ ID NO.84)进行PCR，凝胶电泳纯化回收后(利用Axygen凝胶回收试剂盒)，得到PCR产物——R3-连接子-R4(SEQ ID NO.95)，得到本申请的所述第二多核苷酸。其中所述连接子的长度可以为90bp，其核苷酸序列如SEQ ID NO.89所示。
以1kb长的人IgG1的Fc(SEQ ID NO.87)为模板，利用实施例4.4.1制备的引物R5-1kb-R6的正向引物和R5-1kb-R6的反向引物，进行PCR，凝胶电泳纯化回收后，得到PCR产物——R5-1kb-R6(SEQ ID NO.91)。
4.4.3 构建轻链存储载体和重链存储载体
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将4.4.2制备的R1-1kb-R2片段***pMD19-T载体，得到用于***全长轻链基因库的轻链存储载体DDB-R1-1kb-R2，其载体图谱如图3所示。
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将4.4.2制备的R5-1kb-R6片段***pMD19-T载体得到含有R5-1kb-R6片段的载体，然后以这个载体为模板，利用引物突变去除这个载体中原有的BsmBI酶切位点，得到用于***VH基因库的重链存储载体DDB-R5-1kb-R6，其载体图谱如图5所示。
具体的引物序列可参照下表4-3：
表4-3 引物序列-3
引物名称 SEQ ID NO:
载体突变的正向引物 92
载体突变的反向引物 93
4.4.4 构建连接子存储载体并获得连接子组件细菌
借助TA cloning(TA cloning试剂盒，购自Takara公司)的方法，将实施例4.4.2制备的R3-连接子-R4***pMD19-T载体，得到连接子存储载体DDB-R3-连接子-R4，其载体图谱如图4所示。可用所述连接子存储载体转化TG1感受态细菌(Lucigen公司)，铺皿37℃培养过夜，送菌落测序，然后收集菌落得到连接子组件细菌。可将该连接子组件细菌冻存备用。
4.5 获得组件细菌文库
4.5.1 获得轻链组件细菌文库
利用限制性内切核酸酶R1和R2酶切实施例4.3所制得的第一多核苷酸(包括KLC和LLC)获得目的轻链片段(约为0.65kb)。
利用限制性内切核酸酶R1和R2酶切实施例4.4所制得的轻链存储载体DDB-R1-1kb-R2。获得轻链存储载体片段(约为2.7kb)。
将所得的目的轻链片段和轻链存储载体片段混合，再利用连接酶T4 DNA ligase(购自 NEB，Thermo)连接得到轻链存储连接产物，然后用所述轻链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，送菌落测序，然后收集全部菌落得到轻链组件细菌文库。可检测该轻链组件细菌文库的质量和/或将该轻链组件细菌文库冻存备用。
4.5.2 获得重链组件细菌文库
利用限制性内切核酸酶R5和R6酶切实施例4.3所制得的第三多核苷酸获得目的重链可变区片段(约为0.35kb)。
利用限制性内切核酸酶R5和R6酶切实施例4.4所制得的重链存储载体DDB-R5-1kb-R6。获得重链存储载体片段(约为2.7kb)。
将所得的目的重链可变区片段和重链存储载体片段混合，再利用连接酶T4 DNA ligase(购自NEB，Thermo)连接得到重链存储连接产物，然后用所述重链存储连接产物转化TG1感受态细菌(Lucigen，Cat#60502-2，按照厂家的说明书操作)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，铺氨苄青霉素抗性的平皿(Thermo，Cat#240845)，37℃培养过夜，挑菌落测序，然后收集全部菌落得到重链组件细菌文库。可检测该重链组件细菌文库的质量和/或将该重链组件细菌文库冻存备用。
4.6 获得轻链组件质粒、重链组件质粒和连接子片段
使用质粒提取试剂盒(购自Axygen)，分别提取实施例4.5.1制备的轻链组件细菌文库、实施例4.5.2制备的重链组件细菌文库中的质粒，分别得到轻链组件质粒和重链组件质粒。
利用限制性内切核酸酶R1和R2酶切实施例4.5.1制备的轻链组件质粒，经凝胶电泳纯化回收后，得到轻链***片段LC。
利用限制性内切核酸酶R5和R6酶切实施例4.5.2制备的重链组件质粒，经凝胶电泳纯化回收后，得到重链***片段HC。
使用质粒提取试剂盒(购自Axygen)，提取实施例4.4.4制备的连接子组件细菌中的质粒，得到连接子组件质粒。以该连接子组件质粒或实施例4.4.4中的连接子存储载体为模板，用连接子的正向引物(SEQ ID NO.96或SEQ ID NO.97)和连接子的反向引物(SEQ ID NO.98)扩增含有连接子的0.8kb的片段，然后用限制性内切核酸酶R3和R4酶切该0.8kb的PCR产物，经凝胶电泳纯化回收(利用小片段凝胶回收试剂盒，购自莱枫生物，Cat#DK402)得到90pb的连接子片段。
4.7 获得展示载体
购买获得pComb3x载体，其载体图谱如图6所示，其中用于抗体Fab展示的经改造的 pComb3x-fab载体图谱如图7所示。
通过无义突变除去pComb3x-fab载体中在Fab基因3’端的SfiI酶切位点。
然后在该无义突变后的载体中轻链终止密码子的下游加入限制性内切核酸酶R2的酶切位点，在重链可变区信号肽的末端通过无义突变引入限制性内切核酸酶R5的酶切位点，获得经改造后的展示载体DDB-R1R2R5R6，其图谱如图8所示。
4.8 制备展示细菌文库
利用限制性内切核酸酶R7和限制性内切核酸酶R8酶切实施例4.7制备的展示载体DDB-R1R2R5R6，获得3.6kb展示载体片段。
将实施例4.6获得的轻链***片段LC(0.65kb)、重链***片段HC(0.35kb)、连接子片段(90bp)和所述展示载体片段(3.6kb)按照1:1:1:1的分子比例混合，用T4 DNA连接酶20℃连接20小时以上，得到展示用连接产物。
利用PCR-Clean-up纯化连接产物，转入TG1感受态细菌(购自Lucigen，Cat#60502-2)，在不含抗菌素的2YT培养液中，37℃，250rpm震摇培养60分钟，然后1：20的比例扩大到含有氨苄青霉素的2YT培养液中，37℃，250rpm震摇培养4.5小时，离心收集细菌冻存，即为展示细菌文库。在加氨苄青霉素扩大培养前，取少量菌液铺皿，37℃培养过夜，挑菌落测序。该展示细菌文库可以被保存备用。
4.9 制备展示型抗体噬菌体文库
从实施例4.8所制备的展示细菌文库中取适量的菌液，在2YT培养液(含有氨苄青霉素100μg/ml和2％葡萄糖)中37℃培养至OD 600达到0.5。然后向该菌液中加入M13KO7辅助噬菌体(购自NEB，Cat#N0315S，MOI约为10-20)混匀后，37℃静止30分钟，接着在37℃，250rpm条件下震摇30分钟，离心弃去含有M13KO7辅助噬菌体的培养液上清部分，以该菌液原始体积4倍的培养液(含有氨苄青霉素和卡那霉素)重悬细菌，30℃，250rpm条件下振摇过夜。第二天用PEG沉淀收集噬菌体，滴定噬菌体浓度，分装保存。即得到展示型抗体噬菌体文库。
实施例5 组件细菌文库的质量分析
分析实施例4.5所制备的组件细菌文库的质量。结果如表5所示。
表5 组件细菌文库的质量
Figure PCTCN2020085706-appb-000002
Figure PCTCN2020085706-appb-000003
在构建展示细菌文库时，轻链组件细菌文库(KLC)与轻链组件细菌文库(LLC)的比例为2:1，根据以上质量结果，可估算展示细菌文库的有效克隆比例将达到82％。而所述轻链组件细菌文库和重链组件细菌文库随机组合后得到的库容量理论上最高可以达到10 18。
实施例6 展示细菌文库的质量分析
将实施例4.8获得的分离纯化后的展示用连接产物16μg转化25支TG1感受态细菌，扩增培养后离心收集细菌，收获细菌总量2.4*10 12个，分装保存(即得到实施例4.8所制备的展示细菌文库)。展示细菌文库库容量为4*10 10，背景克隆比例为3％，每μg连接产物的转化效率达到2.5*10 9。
用两种方法分析该展示细菌文库的质量：
第一种方法：随机挑选90个菌落测序，结果56个有正确阅读框，无重复序列，正确阅读框克隆比例62.2％。
第二种方法：用抗人Fab的抗体铺平板，用ELISA检测上述90个克隆的Fab表达，结果两个阴性对照的读数分别为0.07和0.082。共有71个克隆的读数高于0.1，确定为Fab表达，阳性比例79％。
分析序列错误的34个克隆，有18个有Fab表达，占90个克隆的20％。将测序正确和测序错误但有表达的克隆相加(56+18)，有效克隆比例达到82.2％(74/90)，与实施例5中的估算结果相吻合。
实施例7 展示型抗体噬菌体文库的质量分析
用PEG沉淀实施例4.9中的展示型抗体噬菌体文库，离心沉淀后用280ml PBS重悬，加60ml甘油，混匀后分装冻存。取冻存的所述展示型抗体噬菌体文库，进行噬菌体文库滴度分析，结果表明该噬菌体文库的滴度为2x10 13/ml，该噬菌体文库的噬菌体总量达到6.8x10 15,100μl含有50倍库容量的噬菌体。
为分析是否能够从所构建的噬菌体库筛选到抗原特异性抗体，选择三个分泌性细胞因子(IL6、IL17A/F、GMCSF)和三个细胞膜蛋白(IL6R、EGFR、Siglec3)，每个抗原用100μl所述噬菌体文库进行筛选。
用生物素化抗原结合亲和素标记磁珠吸附的液相筛选法筛选抗原特异性Fab。上述6种生物素标记的抗原购自AcroBiosystems(货号依次为Cat#IL6-H8218、ILF-H82W1、GMF-H8214、CD6-H82E8、EGR-H82E3和CD3-H82E7)，亲和素标记的磁珠购自Thermo (Cat#11206D)。三轮液相筛选的抗原浓度分别为5μg/ml、3μg/ml和1μg/ml。第一轮筛选洗脱收集的噬菌体，经扩增后用于第二轮筛选。第二轮筛选洗脱收集的噬菌体，不经扩增直接用于第三轮筛选。
第三轮筛选洗脱收集的噬菌体，侵染TG1细菌，铺氨苄青霉素平皿，37℃培养过夜。次日，从每个抗原特异性平皿挑96个单克隆培养扩增(所述平皿的培养基中包含2YT、氨苄青霉素100μg/ml和0.2％葡萄糖)至OD 600达到0.6，加IPTG(终浓度1mM)诱导过夜。第三天ELISA分析培养液中的抗原特异性Fab，结果如表6所示。
表6 针对不同抗原的阳性克隆比例
抗原 最高ELISA读数 最低ELISA读数 阳性克隆数 阳性克隆比例
IL6 1.029 0.147 54 56％
IL6R 0.735 0.122 42 44％
IL17A/F 1.395 0.133 49 51％
GMCSF 0.925 0.066 84 87％
EGFR 0.729 0.149 24 25％
Siglec3 0.981 0.114 76 79％
表6中将ELISA读数大于最低ELISA读数两倍的克隆统计为阳性克隆。
表6的结果说明，从所述噬菌体抗体库中可分别筛选获得针对IL-6、IL6R、IL17-FR、GMCSF、EGFR和Siglec3的特异性Fab，并且阳性克隆的比例可以达到约25-87％。
从每组抗原特异性阳性克隆中选择ELISA读数最高的12个克隆进行测序分析，比对分析各组12个克隆的氨基酸序列。结果如表7所示。表7的结果说明，每组均有多个独特的氨基酸序列。
表7 针对不同抗原的具有独特氨基酸序列的单克隆Fab的个数
抗原 IL-6 IL6R IL17A/F GMCSF EGFR Siglec3
独特Fab个数 8 8 7 4 4 5
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (87)

  1. 一种产生展示抗体或抗体片段的噬菌体文库的方法，所述方法包括：
    1)提供第一多核苷酸，第二多核苷酸和第三多核苷酸，所述第一多核苷酸包含LC，所述第二多核苷酸包含连接子且所述第三多核苷酸包含HC，其中所述LC包含编码所述抗体或抗体片段的轻链或轻链片段的核酸序列，所述HC包含编码所述抗体或抗体片段的重链或重链片段的核酸序列；
    2)将所述第一多核苷酸导入第一细菌中以获得轻链组件细菌文库，将所述第二多核苷酸导入第二细菌中以获得连接子组件细菌，将所述第三多核苷酸导入第三细菌中以获得重链组件细菌文库；
    3)由所述轻链组件细菌文库获得包含所述LC的轻链组件质粒，由所述连接子组件细菌获得包含所述连接子的连接子组件质粒，且由所述重链组件细菌文库获得包含所述HC的重链组件质粒；
    4)由所述轻链组件质粒获得经释放的LC，由所述连接子组件质粒获得经释放的连接子，且由所述重链组件质粒获得经释放的HC；
    5)提供展示载体，并由所述展示载体获得经释放的展示载体片段；
    6)使所述经释放的LC、经释放的连接子、经释放的HC以及经释放的展示载体片段连接，以形成展示用连接产物；
    7)将所述展示用连接产物导入第四细菌中，以获得所述抗体或抗体片段的展示细菌文库；
    8)使用所述展示细菌文库制备展示所述抗体或抗体片段的噬菌体文库。
  2. 根据权利要求1所述的方法，其中在适于抗体或抗体片段表达的条件下，6)的所述展示用连接产物能够依照阅读框表达所述轻链或轻链片段以及所述重链或重链片段。
  3. 根据权利要求1-2中任一项所述的方法，其中所述第一多核苷酸，所述第二多核苷酸和所述第三多核苷酸均为线性核酸分子。
  4. 根据权利要求1-3中任一项所述的方法，其中编码所述重链或重链片段的核酸序列与编码所述轻链或轻链片段的核酸序列位于不同的阅读框中。
  5. 根据权利要求1-4中任一项所述的方法，其中所述连接子包含编码信号肽pelB或其片段的核酸序列。
  6. 根据权利要求1-5中任一项所述的方法，其中所述连接子的长度为约50至约200个碱基。
  7. 根据权利要求1-6中任一项所述的方法，其中在6)的所述展示用连接产物中，所述LC位于所述连接子的上游，且所述连接子位于所述HC的上游。
  8. 根据权利要求1-7中任一项所述的方法，其中2)包括冷冻保存所述轻链组件细菌文库和/或所述重链组件细菌文库。
  9. 根据权利要求1-8中任一项所述的方法，其中7)包括冷冻保存所述展示细菌文库。
  10. 根据权利要求9所述的方法，其中8)包括融化并培养所述经冷冻保存的展示细菌文库，并使用经培养的所述展示细菌文库构建所述噬菌体文库。
  11. 根据权利要求1-10中任一项所述的方法，其中所述第一多核苷酸以5’至3’方向包含结构R1-LC-R2，所述第二多核苷酸以5’至3’方向包含结构R3-连接子-R4，所述第三多核苷酸以5’至3’方向包含结构R5-HC-R6，且所述展示载体中以5’至3’方向包含结构R7-展示载体片段-R8，其中所述R1，R2，R3，R4，R5，R6，R7和R8为限制性内切核酸酶的识别位点。
  12. 根据权利要求11所述的方法，其中所述R2经限制性内切核酸酶切割后产生的末端：
    能够与所述R3经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且
    不与所述R1、R4、R5、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
  13. 根据权利要求11-12中任一项所述的方法，其中所述R4经限制性内切核酸酶切割后产生的末端：
    能够与所述R5经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且
    不与所述R1、R2、R3、R6、R7及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
  14. 根据权利要求11-13中任一项所述的方法，其中所述R6经限制性内切核酸酶切割后产生的末端：
    能够与所述R7经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且
    不与所述R1、R2、R3、R4、R5及R8中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
  15. 根据权利要求11-14中任一项所述的方法，其中所述R8经限制性内切核酸酶切割后产生的末端：
    能够与所述R1经限制性内切核酸酶切割后产生的末端彼此识别并连接；并且
    不与所述R2、R3、R4、R5、R6及R7中的任一项经限制性内切核酸酶切割后产生的末端彼此识别或连接。
  16. 根据权利要求11-15中任一项所述的方法，其中所述R1，R2，R3，R4，R5、R6、R7和R8中的任一项经限制性内切核酸酶切割后产生的末端为非回文序列。
  17. 根据权利要求11-16中任一项所述的方法，其中所述R1，R2，R3，R4，R5、R6、R7和R8中的两项或更多项能够被相同的限制性内切核酸酶识别及切割。
  18. 根据权利要求11-17中任一项所述的方法，其中所述R2与所述R3相同。
  19. 根据权利要求11-18中任一项所述的方法，其中所述R4与所述R5相同。
  20. 根据权利要求11-19中任一项所述的方法，其中所述R6与所述R7相同。
  21. 根据权利要求11-20中任一项所述的方法，其中所述R8与所述R1相同。
  22. 根据权利要求11-21中任一项所述的方法，其中所述R1、R2、R3、R4、R5和R8能够被相同的限制性内切核酸酶识别及切割。
  23. 根据权利要求11-22中任一项所述的方法，其中所述R6和R7能够被相同的限制性内切核酸酶识别及切割。
  24. 根据权利要求11-23中任一项所述的方法，其中所述限制性内切核酸酶选自SfiI，BsmBI和Esp3I。
  25. 根据权利要求11-24中任一项所述的方法，其中所述R1、R2、R3、R4、R5和R8能够被SfiI识别及切割。
  26. 根据权利要求11-25中任一项所述的方法，其中所述R6和R7能够被BsmBI和/或Esp3I识别及切割。
  27. 根据权利要求11-26中任一项所述的方法，其中所述R1包含SEQ ID NO:1所示的核酸序列。
  28. 根据权利要求11-27中任一项所述的方法，其中所述R2包含SEQ ID NO:2所示的核酸序列。
  29. 根据权利要求11-28中任一项所述的方法，其中所述R3包含SEQ ID NO:2所示的核酸序列。
  30. 根据权利要求11-29中任一项所述的方法，其中所述R4包含SEQ ID NO:3所示的核酸序列。
  31. 根据权利要求11-30中任一项所述的方法，其中所述R5包含SEQ ID NO:3所示的核酸序列。
  32. 根据权利要求11-31中任一项所述的方法，其中所述R6包含SEQ ID NO:4所示的核酸序列。
  33. 根据权利要求11-32中任一项所述的方法，其中所述R7包含SEQ ID NO:4所示的核酸序列。
  34. 根据权利要求11-33中任一项所述的方法，其中所述R8包含SEQ ID NO:1所示的核酸序列。
  35. 根据权利要求1-34中任一项所述的方法，其中所述轻链或轻链片段包含轻链可变区部分和/或轻链恒定区部分。
  36. 根据权利要求1-35中任一项所述的方法，其中所述重链或重链片段包含重链可变区部分和/或重链恒定区部分。
  37. 根据权利要求1-36中任一项所述的方法，其中所述抗体或其片段包括Fab，Fab’，(Fab)2和/或(Fab’)2。
  38. 根据权利要求1-37中任一项所述的方法，其中7)中所述展示用连接产物的连接转化效率为至少10 8个克隆/μg多核苷酸。
  39. 根据权利要求1-38中任一项所述的方法，其中2)包括使用识别所述R1和R2的限制性内切核酸酶对所述第一多核苷酸进行酶切处理，使经酶切处理的所述第一多核苷酸与轻链存储载 体片段连接形成轻链存储连接产物，并将所述轻链存储连接产物导入所述第一细菌中以获得所述轻链组件细菌文库；其中所述轻链存储载体包含所述R1和R2，并且通过使用识别所述R1和R2的限制性内切核酸酶对所述轻链存储载体进行酶切处理而获得所述轻链存储载体片段。
  40. 根据权利要求39所述的方法，其中所述轻链存储载体源自pUC载体。
  41. 根据权利要求40所述的方法，其中所述pUC载体为pUC19载体或源自pUC19载体。
  42. 根据权利要求40-41中任一项所述的方法，其中所述pUC载体为pMD19-T载体或源自pMD19-T载体。
  43. 根据权利要求39-42中任一项所述的方法，其中所述识别所述R1和R2的限制性内切核酸酶包括SfiI。
  44. 根据权利要求39-43中任一项所述的方法，其中2)包括使用识别所述R5和R6的限制性内切核酸酶对所述第三多核苷酸进行酶切处理，使经酶切处理的所述第三多核苷酸与重链存储载体片段连接形成重链存储连接产物，并将所述重链存储连接产物导入所述第三细菌中以获得所述重链组件细菌文库；其中所述重链存储载体包含所述R5和R6，并且通过使用识别所述R5和R6的限制性内切核酸酶对所述重链存储载体进行酶切处理而获得所述重链存储载体片段。
  45. 根据权利要求44所述的方法，其中所述重链存储载体源自pUC载体。
  46. 根据权利要求45所述的方法，其中所述pUC载体为pUC19载体或源自pUC19载体。
  47. 根据权利要求45-46中任一项所述的方法，其中所述pUC载体为pMD19-T载体或源自pMD19-T载体。
  48. 根据权利要求44-47中任一项所述的方法，其中所述重链存储载体中包含且仅包含一个BsmBI的识别位点。
  49. 根据权利要求44-48中任一项所述的方法，其中所述识别所述R5和R6的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
  50. 根据权利要求1-49中任一项所述的方法，其中2)包括使所述第二多核苷酸与载体片段连接形成连接子存储载体，并将所述连接子存储载体导入所述第二细菌中以获得所述连接子组件细菌。
  51. 根据权利要求50所述的方法，其中所述连接子存储载体中包含所述R3和R4。
  52. 根据权利要求50-51中任一项所述的方法，其中用于构建所述连接子存储载体的所述载体片段源自pUC载体。
  53. 根据权利要求52所述的方法，其中所述pUC载体为pUC19载体或源自pUC19载体。
  54. 根据权利要求52-53中任一项所述的方法，其中所述pUC载体为pMD19-T载体或源自pMD19-T载体。
  55. 根据权利要求1-54中任一项所述的方法，其中4)包括使用识别所述R1和R2的限制性内切核酸酶对所述轻链组件质粒进行酶切处理，从而获得所述经释放的LC。
  56. 根据权利要求55所述的方法，其中所述识别所述R1和R2的限制性内切核酸酶包括SfiI。
  57. 根据权利要求1-56中任一项所述的方法，其中4)包括使用识别所述R5和R6的限制性内切核酸酶对所述重链组件质粒进行酶切处理，从而获得所述经释放的HC。
  58. 根据权利要求57所述的方法，其中所述识别所述R5和R6的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
  59. 根据权利要求1-58中任一项所述的方法，其中4)包括使用识别所述R3和R4的限制性内切核酸酶对所述连接子存储载体进行酶切处理，从而获得所述经释放的连接子。
  60. 根据权利要求1-58中任一项所述的方法，其中4)包括由所述连接子存储载体获得扩增产物，所述扩增产物包含所述连接子、所述R3以及所述R4；并且使用识别所述R3和R4的限制性内切核酸酶对所述扩增产物进行酶切处理，从而获得所述经释放的连接子。
  61. 根据权利要求1-60中任一项所述的方法，其中所述识别所述R3和R4的限制性内切核酸酶包括SfiI。
  62. 根据权利要求1-61中任一项所述的方法，其中5)包括使用识别所述R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理以释放所述展示载体片段。
  63. 根据权利要求62所述的方法，其中所述识别所述R7和R8的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
  64. 根据权利要求1-63中任一项所述的方法，其中所述展示载体源自pComb3x载体。
  65. 根据权利要求11-64中任一项所述的方法，其中使用识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，将获得三个不同的酶切片段。
  66. 根据权利要求65所述的方法，其中所述识别所述R1、R2、R3、R4、R5和R8的限制性内切核酸酶包括SfiI。
  67. 根据权利要求11-66中任一项所述的方法，其中使用识别所述R6和R7的限制性内切核酸酶对所述展示载体进行酶切处理后，使所述展示载体线性化。
  68. 根据权利要求67所述的方法，其中所述识别所述R6和R7的限制性内切核酸酶包括BsmBI和/或Esp3I。
  69. 根据权利要求11-68中任一项所述的方法，其中使用识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶对所述展示载体进行酶切处理后，将获得四个不同的酶切片段。
  70. 根据权利要求69所述的方法，其中所述识别所述R1、R2、R3、R4、R5、R6、R7和R8的限制性内切核酸酶包括SfiI，BsmBI和/或Esp3I。
  71. 根据权利要求1-70中任一项所述的方法，其中2)包括对所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库进行取样检测，以确定所述轻链组件细菌文库，所述连接子组件细菌和/或所述重链组件细菌文库的库容量和/或质量。
  72. 根据权利要求1-71中任一项所述的方法，其中所述轻链组件细菌文库包含至少10 6个不同的克隆。
  73. 根据权利要求1-72中任一项所述的方法，其中所述重链组件细菌文库包含至少10 7个不同的克隆。
  74. 根据权利要求1-73中任一项所述的方法，其中所述轻链组件细菌文库中有效克隆的比例为至少约70％。
  75. 根据权利要求1-74中任一项所述的方法，其中所述重链组件细菌文库中有效克隆的比例为至少约70％。
  76. 根据权利要求1-75中任一项所述的方法，其中2)包括根据所述轻链组件细菌文库，所述连接子组件细菌，和/或所述重链组件细菌文库的容量和/或质量测算所述展示细菌文库的容量和/或质量。
  77. 根据权利要求1-76中任一项所述的方法，其中所述展示细菌文库包含至少10 10个不同的克隆。
  78. 根据权利要求1-77中任一项所述的方法，其中所述展示细菌文库中有效克隆的比例为至少约70％。
  79. 根据权利要求1-78中任一项所述的方法，其中由样品材料获得所述第一多核苷酸和所述第三多核苷酸。
  80. 根据权利要求79所述的方法，其中所述样品材料包括源自外周血淋巴细胞样品的材料。
  81. 根据权利要求80所述的方法，其中所述外周血淋巴细胞为人外周血淋巴细胞。
  82. 根据权利要求79-81中任一项所述的方法，其中所述样品材料包括源自所述样品的总RNA和/或mRNA。
  83. 根据权利要求1-82中任一项所述的方法，其中8)包括使所述展示细菌文库与辅助噬菌体接触来制备所述噬菌体文库。
  84. 通过权利要求1-83中任一项所述的方法产生的噬菌体文库。
  85. 根据权利要求84所述的噬菌体文库，其包含至少10 10个不同的克隆。
  86. 根据权利要求84-85中任一项所述的噬菌体文库，其能够展示至少10 10种不同的抗体或抗体 片段。
  87. 筛选抗体或抗体片段的方法，其包括使用根据权利要求84-86中任一项所述的噬菌体文库。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206868A1 (zh) 2021-03-31 2022-10-06 泷搌（上海）生物科技有限公司 用于筛选功能性抗原结合蛋白的载体和方法
WO2023122303A3 (en) * 2021-12-23 2023-11-16 Generation Bio Co. Scalable and high-purity cell-free synthesis of closed-ended dna vectors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101720368A (zh) * 2007-03-09 2010-06-02 中国抗体制药有限公司 储备多样性最大化的功能性人化抗体文库之构建及应用
CN102732974A (zh) * 2012-07-16 2012-10-17 百泰生物药业有限公司 噬菌体抗体库的构建方法及应用此库筛选到的抗cd6抗体
WO2018002952A2 (en) * 2016-06-26 2018-01-04 Gennova Biopharmaceuticals Limited, Antibody phage display library

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2136092T3 (es) * 1991-09-23 1999-11-16 Medical Res Council Procedimientos para la produccion de anticuerpos humanizados.
WO1995011317A1 (en) * 1993-10-19 1995-04-27 The Scripps Research Institute Synthetic human neutralizing monoclonal antibodies to human immunodeficiency virus
CA2462113C (en) * 2001-10-01 2013-01-29 Dyax Corp. Multi-chain eukaryotic display vectors and uses thereof
CN101210241A (zh) * 2006-12-27 2008-07-02 陕西超英生物科技有限公司 一种噬菌体基因工程抗体库基因组装的方法
US10415033B2 (en) * 2017-07-31 2019-09-17 Academia Sinica Phage-displayed single-chain variable fragment libraries and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101720368A (zh) * 2007-03-09 2010-06-02 中国抗体制药有限公司 储备多样性最大化的功能性人化抗体文库之构建及应用
CN102732974A (zh) * 2012-07-16 2012-10-17 百泰生物药业有限公司 噬菌体抗体库的构建方法及应用此库筛选到的抗cd6抗体
WO2018002952A2 (en) * 2016-06-26 2018-01-04 Gennova Biopharmaceuticals Limited, Antibody phage display library

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. ABS75961.1
CEN X. ET AL.: "Construction of a Large Phage Display Antibody Library by In Vitro Package and In Vivo Recombination", APPL MICROBIOL BIOTECHNOL, vol. 71, no. 5, 13 May 2006 (2006-05-13), XP019422040, DOI: 10.1007/s00253-006-0334-5 *
See also references of EP3960916A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206868A1 (zh) 2021-03-31 2022-10-06 泷搌（上海）生物科技有限公司 用于筛选功能性抗原结合蛋白的载体和方法
WO2023122303A3 (en) * 2021-12-23 2023-11-16 Generation Bio Co. Scalable and high-purity cell-free synthesis of closed-ended dna vectors

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