WO2016019888A1 - 筛选纳米抗体的方法及系统 - Google Patents

筛选纳米抗体的方法及系统 Download PDF

Info

Publication number
WO2016019888A1
WO2016019888A1 PCT/CN2015/086253 CN2015086253W WO2016019888A1 WO 2016019888 A1 WO2016019888 A1 WO 2016019888A1 CN 2015086253 W CN2015086253 W CN 2015086253W WO 2016019888 A1 WO2016019888 A1 WO 2016019888A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
antibody
gene
optionally
sequencing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2015/086253
Other languages
English (en)
French (fr)
Inventor
刘晓
聂超
张伟
任哲
张瑞芳
曾晓静
李新洋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BGI Shenzhen Co Ltd
Original Assignee
BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to US15/502,438 priority Critical patent/US10954550B2/en
Priority to CN201580042224.1A priority patent/CN106661629B/zh
Publication of WO2016019888A1 publication Critical patent/WO2016019888A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • the invention relates to the field of biotechnology, and in particular to a method and system for screening nanobodies.
  • VHH variable domain of heavy chain of heavy-chain antibody
  • Nanobodies Since its discovery, Nanobodies have evolved into highly versatile molecules with a wide range of biological applications and clinical applications. Nanobodies have the smallest functional antigen-binding fragments derived from HCAbs in adult camelids. They have high stability and high affinity for antigen binding, and can interact with protein fissures and enzyme active sites to make them act similarly to inhibition. Agent. Therefore, Nanobodies can provide new ideas for designing small molecule enzyme inhibitors from peptide mimetic drugs. Because of the heavy chain only, the production of Nanobodies is easier than that of mAb. The unique properties of Nanobodies, such as stability in extreme temperature and pH environments, can produce large yields at low cost. Therefore, nano-antibodies have great value in the treatment and diagnosis of diseases, and also have great development prospects in the targeted diagnosis and treatment of tumors.
  • Phage display technology mainly extracts a certain amount of peripheral blood or immune tissue from immune camels, isolates lymphocytes, extracts mRNA, and inserts CDNA by RT-PCR. To the appropriate position of the phage coat protein structural gene, the foreign gene is expressed along with the expression of the coat protein, and a monoclonal antibody is selected for antibody library screening.
  • immunization objects are human, mouse, and rabbit, and the camel is rarely immunized, and the experimental period is long. It takes about 77 days to screen for conventional antibodies, phage, etc. in which heavy and light chains are paired with each other. Display technology is not only time-consuming, labor-intensive, but also technically difficult.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent.
  • one of the present invention The aim is to propose a method for screening highly specific mature Nanobodies with short experimental period, high efficiency and no need to analyze the random pairing of heavy and light chains.
  • the invention provides a method of screening for Nanobodies.
  • the method comprises: (1) extracting a nucleic acid sample from tissue or peripheral blood of an immunized animal; (2) obtaining a sequencing result comprising the antibody sequence based on the nucleic acid sample; (3) based on the The sequencing result including the antibody sequence is used to construct an antibody database; (4) the antibody database is used for information analysis, and the nano-antibody sequence is obtained by screening.
  • the method of the invention adopts immunosense library sequencing technology, namely polymerase chain reaction (PCR) and cDNA 5' end rapid amplification (5'-RACE technology) to screen antibodies, which can be directly and efficiently screened to obtain no light chain.
  • the heavy-chain nano-antibody has a test cycle of only about 21 days, which greatly shortens the screening and preparation time of the nano-antibody, greatly improves the efficiency, and does not require heavy and light chain random pairing analysis for screening nano-antibodies, effectively eliminating heavy-light chain errors. Matching and causing error analysis, the obtained nano-antibodies have broad application prospects in disease diagnosis and treatment, drug development and food testing.
  • the method of screening a Nanobody of the present invention further comprises: (5) performing protein mass spectrometry on the serum of the immunized animal to obtain a protein mass spectrometry result; (6) the antibody database Information is integrated with the protein mass spectrometry results to obtain the Nanobody sequence.
  • the method of screening a Nanobody of the present invention further comprises: (7) expressing the Nanobody by a protein expression system based on the Nanobody sequence to identify the Nanobody.
  • the animal is a camelid.
  • the camelid is at least one of a dromedary, a bactrian camel, a guanaco, an alpaca, a llama and an alpaca.
  • the antibody sequence comprises a hypervariable region sequence and a framework region sequence.
  • the step (2) further comprises: (2-1) amplifying the antibody sequence in the nucleic acid sample to obtain an amplification product; (2-2) performing the amplification product Sequencing to obtain the sequencing results of the antibody-containing sequences.
  • the nucleic acid sample is DNA or RNA.
  • the antibody sequence in the nucleic acid sample is amplified by PCR; when the nucleic acid sample is RNA, by 5'-RACE or PCR pair The antibody sequence in the nucleic acid sample is amplified.
  • the PCR is at least one of multiplex PCR, linear amplification mediated PCR and nested PCR.
  • the amplification product is sequenced by a high throughput sequencing device.
  • the high throughput sequencing device is at least selected from the group consisting of Illumina HiSeq2000, HiSeq 2500, MiSeq, MiSeqDx, NextSeq500, Hiseq X Ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, and single molecule sequencing devices.
  • the sequencing throughput is high and the cost is low.
  • the step (3) further comprises: (3-1) aligning the sequencing result of the antibody-containing sequence with a reference sequence to determine an immune-related gene sequence; (3-2) based on the An immunologically related gene sequence, the nucleotide sequence of the antibody is determined; (3-3) a nucleotide sequence of the antibody is translated into an amino acid sequence; (3-4) a VHH sequence is obtained by screening based on the amino acid sequence, The antibody database is constructed based on the VHH sequence.
  • the immune-related gene is at least one of a V gene, a D gene, a J gene, and a C gene.
  • the reference sequence is a pre-rearranged germline sequence of at least one of the known V gene, D gene, J gene and C gene.
  • the amino acid sequence is an indication that the VHH sequence is one of the following:
  • the framework region has one of the following four conserved amino acids: 37F, 44E, 45R, 47G.
  • 37F means that the amino acid at position 37 is phenylalanine (F)
  • 44E means that the amino acid at position 44 is glutamic acid (E)
  • 45R means that the amino acid at position 45 is arginine ( R)
  • 47G means that the amino acid at position 47 is glycine (G);
  • the amino acid at position 37 is valine (V)
  • the amino acid at position 44 is glycine ( G)
  • the amino acid at position 45 is leucine (L)
  • W tryptophan
  • the hinge region has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2:
  • Aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag (SEQ ID NO: 1)
  • Ggaacgaatgaagtatgcaagtgtcccaaatgtcca (SEQ ID NO: 2)
  • the average length of CDR3 is abnormal. Specifically, the length of CDR3 is a large range of variation, whether it is a normal antibody or VHH, but in terms of the average length of CDR3, the average length of CDR3 of VHH is longer, and the average length of CDR3 of a normal antibody is shorter.
  • the step (5) further comprises: (5-1) performing IgG enrichment of the serum of the immunized animal using Protein A/G to obtain an enriched product; (5-2) Purifying the antibody from the enriched product by affinity chromatography using a chromatography coupled to the antigen; (5-3) denaturation and reductive alkylation of the purified product obtained in step (5-2) Enzymatically digesting with a protease to obtain an enzymatically decomposed peptide; (5-4) using the mass spectrometer to decompose the peptide The segment was subjected to protein mass spectrometry to obtain a mass spectrometric detection result of the enzymatic peptide.
  • the protease is pepsin, chymotrypsin, elastase, trypsin, intracellular protease Lys-C, metal endopeptidase Lys-N, endonuclease Glu-C, aspartic acid At least one of the endopeptidase Asp-N and the Clostridin Arg-C.
  • the invention provides a system for screening a Nanobody, according to an embodiment of the invention, the system comprising: a nucleic acid sample extraction device for use in an animal immunized with an antigen a tissue or peripheral blood extraction nucleic acid sample; a sequencing device for obtaining a sequencing result comprising an antibody sequence based on the nucleic acid sample; an antibody database construction device for using the antibody-containing device The sequencing result of the sequence constructs an antibody database; the information analysis device is configured to perform information analysis based on the antibody database, and screen and obtain the nano-antibody sequence.
  • a protein spectrum analysis device for performing protein mass spectrometry on serum of the immunized animal to obtain protein mass spectrometry results; integrating an analysis device, the integration An analysis device is used to integrate the information of the antibody database with the protein mass spectrometry results to obtain the Nanobody sequence.
  • an identification device for expressing the Nanobody by a protein expression system based on the Nanobody sequence to identify the Nanobody.
  • the animal is a camelid.
  • the camelid is at least one of a dromedary, a bactrian camel, a guanaco, an alpaca, a llama and an alpaca.
  • the antibody sequence comprises a hypervariable region sequence and a framework region sequence.
  • the sequencing device further comprises: an amplification unit for amplifying the antibody sequence in the nucleic acid sample to obtain an amplification product; a sequencing unit, the sequencing A unit is used to sequence the amplified product to obtain the sequencing result of the antibody-containing sequence.
  • the nucleic acid sample is DNA or RNA.
  • the antibody sequence in the nucleic acid sample is amplified by PCR; when the nucleic acid sample is RNA, by 5'-RACE or PCR pair The antibody sequence in the nucleic acid sample is amplified.
  • the PCR is at least one of multiplex PCR, linear amplification mediated PCR and nested PCR.
  • the sequencing unit is a high throughput sequencing platform.
  • the high throughput sequencing platform is selected from the group consisting of Illumina HiSeq2000, HiSeq 2500, At least one of MiSeq, MiSeqDx, NextSeq500, Hiseq X ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, and single molecule sequencing platforms.
  • the antibody database construction device further comprises: a aligning unit configured to align the sequencing result of the antibody-containing sequence with a reference sequence to determine an immune-related gene sequence; Determining a unit for determining a nucleotide sequence of an antibody based on the immune-related gene sequence; a translation unit for translating a nucleotide sequence of the antibody into an amino acid sequence; The screening unit is configured to screen for a VHH sequence based on the amino acid sequence, and construct the antibody database based on the VHH sequence.
  • the immune-related gene is at least one of a V gene, a D gene, a J gene, and a C gene.
  • the reference sequence is a pre-rearranged germline sequence of at least one of the known V gene, D gene, J gene and C gene.
  • the screening unit screens for obtaining the indication of the VHH sequence as one of the following:
  • the framework region has one of the following four conserved amino acids: 37F, 44E, 45R, 47G;
  • the hinge region has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;
  • the protein profile analysis device further includes: an enrichment unit that performs IgG enrichment on serum of the immunized animal using Protein A/G to obtain an enriched product An affinity purification unit that affinity-purifies the antibody from the enriched product using a chromatography column coupled to the antigen to obtain a purified product; an enzymatic unit, the enzymatic unit After denaturation and reductive alkylation of the purified product, enzymatic hydrolysis with a protease to obtain a digested peptide; a mass spectrometry detecting unit for performing protein mass spectrometry on the enzymatic peptide Detection is performed to obtain a mass spectrometric detection result of the enzymatic peptide.
  • the protease is pepsin, chymotrypsin, elastase, trypsin, intracellular protease Lys-C, metal endopeptidase Lys-N, endonuclease Glu-C, aspartic acid At least one of the endopeptidase Asp-N and the Clostridin Arg-C.
  • FIG. 1 shows a schematic flow diagram of a method of screening a Nanobody according to an embodiment of the present invention
  • FIG. 2 shows a schematic flow diagram of a method of screening a Nanobody according to another embodiment of the present invention
  • FIG. 3 is a schematic flow chart showing a method of screening a Nanobody according to still another embodiment of the present invention.
  • Figure 4 shows a schematic diagram of a primer design in accordance with an embodiment of the present invention
  • Figure 5 shows a flow chart for analyzing sequencing results in accordance with an embodiment of the present invention
  • Figure 6 shows a schematic flow diagram of a method for screening nanobodies using immunohistochemistry techniques in accordance with an embodiment of the present invention
  • FIG. 7 is a flow chart showing a method for screening a Nanobody using an immunobanking technique in combination with a protein profiling technique according to an embodiment of the present invention
  • Figure 8 shows a schematic structural view of a system for screening Nanobodies in accordance with one embodiment of the present invention
  • Figure 9 is a schematic view showing the structure of a system for screening Nanobodies according to still another embodiment of the present invention.
  • Figure 10 is a schematic view showing the structure of a system for screening Nanobodies according to still another embodiment of the present invention.
  • Figure 11 is a graph showing statistical results of obtaining data of conventional antibodies according to an embodiment of the present invention.
  • Figure 12 is a graph showing statistical results of obtaining data for each nanobody according to an embodiment of the present invention.
  • Figure 13 shows statistical results of obtaining antibody peptides, antibody variable region coverage and CDR3 region coverage, in accordance with one embodiment of the present invention
  • Figure 14 is a graph showing the results of screening for the molecular weight of a Nanobody by SDS-PAGE gel according to still another embodiment of the present invention.
  • the invention provides a method of screening for Nanobodies. According to an embodiment of the invention, referring to Figure 1, the method comprises the following steps:
  • a nucleic acid sample is extracted from tissue or peripheral blood of an immunized animal.
  • the immunized animal can be obtained by immunizing an animal with an antigen.
  • the kind of the antigen is not particularly limited and may be any known antigen.
  • the antigen includes, but is not limited to, epithelial keratin 18 (CK18), nucleoside diphosphate kinase A (NDKA863-17).
  • the dose of the antigen is also not particularly limited.
  • the animal can be immunized at a dose of 10 mg/time.
  • the kind of the animal is not particularly limited as long as the nano-antibody can be effectively screened, and those skilled in the art can flexibly select according to actual conditions.
  • the animal is Camel Things.
  • the camelid may be at least one of a dromedary, a bactrian camel, a guanaco, an alpaca, an alpaca, and an alpaca. Thereby, the efficiency of obtaining a Nanobody can be effectively improved.
  • the animal is a camel.
  • step (2) further comprises the steps of:
  • the nucleic acid sample is DNA or RNA.
  • the antibody sequence comprises a hypervariable region sequence and a framework region sequence.
  • the nano-antibody can be efficiently screened.
  • the antibody sequence in the nucleic acid sample is amplified by PCR; when the nucleic acid sample is RNA, the pair is described by 5'-RACE or PCR The antibody sequence in the nucleic acid sample is amplified.
  • PCR or 5'-RACE technology it is only necessary to design specific primers in the C region, which can effectively and comprehensively enrich antibody sequences, and can count the gene family expression frequency, VJ pairing and base insertion, deletion and CDR region diversity. , length distribution characteristics, can also fully observe the entire antigen-specific antibody library and clone high-specificity mature Nanobody.
  • the PCR is at least one of multiplex PCR, linear amplification mediated PCR and nested PCR.
  • the amplification product is sequenced by a high throughput sequencing device.
  • a sequencing library comprising the amplification product is obtained, and the sequencing library is sequenced by a high-throughput sequencing device.
  • the high throughput sequencing device is at least selected from the group consisting of Illumina HiSeq2000, HiSeq2500, MiSeq, MiSeqDx, NextSeq500, Hiseq X ten, Life SOLiD, Ion TorrentPGM, Proton, Roche 454, and single molecule sequencing devices.
  • the nucleotide sequence of the nucleic acid sample can be obtained quickly and efficiently, and the sequencing throughput is high and the cost is low.
  • sequencing results obtained by sequencing can be filtered to remove low quality data, depending on the situation.
  • step (3) further comprises the steps of:
  • (3-1) Aligning the sequencing result of the antibody-containing sequence with a reference sequence to determine an immune-related gene sequence.
  • the reference sequence is a pre-rearranged germline sequence of at least one of the known V gene, D gene, J gene and C gene.
  • the immune-related gene is at least one of a V gene, a D gene, a J gene, and a C gene.
  • sequencing results including antibody sequences can be filtered to remove low-quality data and remove contaminant-contaminated data, such as sequencing results of the antibody sequence, prior to alignment with the reference sequence, as needed.
  • the part with the lower base quality of the sequence at the end of the sequence is removed, and the average base mass value of the sequence is low or the data containing "N" is removed, and the sequence-contaminated data is removed, and the sequence whose length is too short needs to be removed.
  • the high throughput sequencing is of the paired-end (PE) type, the PE sequences need to be spliced according to the intersection of the PE sequences.
  • the comparison of the sequencing result comprising the antibody sequence with the reference sequence specifically comprises: first, comparing the sequencing result with the reference sequence using local alignment software. The sequencing result is then compared with the reference sequence again to obtain the alignment result.
  • the reference sequence may be the discovered germline sequence before the V/D/J/C rearrangement, and the local comparison software used is not particularly limited, and the person skilled in the art may flexibly select according to the actual situation, according to the present invention.
  • the local alignment software employed includes, but is not limited to, BLAST, BLASTN, and LASTZ. Next, based on the alignment results, the corresponding sequences of the V gene, the D gene, the J gene, and the C gene are determined.
  • the heavy chain comprises the V, D, J and C genes
  • the primer of the common double-stranded antibody sequence is simultaneously designed and the double-stranded antibody sequence is amplified, the light chain of the double-stranded antibody includes V, J and C gene.
  • the nucleotide sequence of the antibody is determined based on the immunologically related gene sequence.
  • CDR region also referred to as a hypervariable region
  • framework region a complementarity determining region
  • various statistics of the obtained nucleotide sequence data are further performed, including the number of sequence species of the CDR3, the distribution of use of the V and J genes, and the use of VJ pairing.
  • Distribution, V, (D) insertion deletion length distribution on the J gene, base composition of the V and J genes, frequency distribution of the CDR3 sequence, and the like.
  • the expression “number of sequence types of CDR3” used herein refers to a different number of CDR3 sequences; “the distribution of use of V and J genes” refers to each V and J used in all sequences.
  • the insertion deletion length distribution on the J gene means that the VDJ gene is rearranged, and some bases are deleted at the end of the gene, and some bases are inserted between the genes. Here, the insertion or deletion of the bases is counted.
  • the length of the "base composition of the V and J genes” refers to the ratio of the base composition and the proportion of each base at each position of the statistical gene for all sequences; "the frequency distribution of the CDR3 sequences” means each CDR3 The frequency distribution of the sequence.
  • the following statistics are obtained on the translated amino acid sequence: abundance, frequency (abundance refers to the number of repetitions of one amino acid sequence; frequency refers to the content of one amino acid sequence in the population), corresponding V and J gene, and sequence information.
  • sequences with sequence abundances less than 2 are removed.
  • VHH sequence is obtained by screening, and the antibody database is constructed based on the VHH sequence.
  • the VHH sequence obtained by screening in this step includes all VHH sequences in the animal after immunization, including both the VHH sequence associated with the immunizing antigen and the VHH sequence unrelated to the immunizing antigen.
  • the indication that the amino acid sequence is the VHH sequence is one of the following:
  • the framework region has one of the following four conserved amino acids: 37F, 44E, 45R, 47G.
  • 37F means that the amino acid at position 37 is phenylalanine (F)
  • 44E means that the amino acid at position 44 is glutamic acid (E)
  • 45R means that the amino acid at position 45 is arginine ( R)
  • 47G means that the amino acid at position 47 is glycine (G);
  • the amino acid at position 37 is valine (V)
  • the amino acid at position 44 is glycine ( G)
  • the amino acid at position 45 is leucine (L)
  • W tryptophan
  • the first amino acid in the hypervariable region is zero.
  • the hinge region has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2:
  • Aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag (SEQ ID NO: 1)
  • Ggaacgaatgaagtatgcaagtgtcccaaatgtcca (SEQ ID NO: 2)
  • the average length of CDR3 is abnormal. Specifically, the length of the CDR3 is a large range of variation, whether it is a normal antibody or VHH, but in terms of the average length of the CDR3, the average length of the CDR3 of the VHH is longer, and the average length of the CDR3 of the common antibody is shorter.
  • the amino acid sequence obtained in the step (3-3) includes a nano-antibody sequence and a double-stranded antibody sequence.
  • the nano-antibody sequence and the double-chain antibody sequence can be separated, that is, the nano-antibody database and the double-chain antibody database can be simultaneously obtained, and those skilled in the art can directly select the nano-antibody database according to the needs.
  • the Nanobody sequence associated with the immunizing antigen is screened based on the frequency information of the antibody CDR3.
  • the method for screening a Nanobody of the present invention further comprises:
  • step (5) further comprises:
  • the serum of the immunized animal was subjected to IgG enrichment using Protein A/G to obtain an enriched product.
  • IgG enrichment is performed using Protein A/G, and the enrichment result is subjected to western blot or enzyme.
  • An immunosorbent assay (Elisa) assay is performed to determine that the enriched sample has a specific response to the antigenic polypeptide.
  • the antibody is affinity-purified from the enriched product using a chromatography column coupled to the antigen.
  • the known antigen polypeptide used in the foregoing is attached to a chromatography column, and the enriched product obtained in the step (5-1) is affinity-purified by the chromatography column and separately collected. Flow through the liquid and eluent. The obtained flow-through and eluate were subjected to Western blotting or enzyme-linked immunosorbent assay (ELISA), respectively, to determine that the affinity purification product specifically reacted with the known antigen polypeptide used.
  • ELISA enzyme-linked immunosorbent assay
  • the protease is pepsin, chymotrypsin, elastase, trypsin, intracellular protease Lys-C, metal endopeptidase Lys-N, endonuclease Glu-C, aspartic acid At least one of the endopeptidase Asp-N and the Clostridin Arg-C.
  • a common contaminating protein sequence can be incorporated into the antibody database to obtain a final database.
  • the contaminating protein sequence comprises a conserved region sequence of the subject species and a protein sequence from other species as interference.
  • protein mass spectrometry results can be compared with an antibody database using appropriate identification software and appropriate parameters to identify protein mass spectrometry results and identify using the Target-Decoy method.
  • the false positive rate of the results was evaluated, and the results of the low false positive rate were retained as the basis for subsequent analysis (eg, the false positive rate was 1% or less).
  • data of protein mass spectrometry analysis of a plurality of enzymatic peptides can be combined, and information such as peptide identification number, protein coverage, CDR3 region coverage, etc. of the antibody sequence can be counted according to the correspondence relationship between the antibody and the identified peptide segment, and The quantitative value of the corresponding antibody sequence is calculated. Then, based on the frequency information of the antibody sequence, the relationship between the number of peptide identification, protein coverage and antibody sequencing frequency is analyzed, and according to the relationship between them, screening is performed to obtain a nanoantibody sequence related to the immune antigen.
  • the screening conditions are as follows: the number of identified peptides is not less than 20, the coverage of multiple enzymatic integration is not less than 70% in the CDR3 region, and the coverage of the entire V-region is not Below 50%, the NGS frequency is not limited.
  • the screening conditions are as follows: the number of peptides is not less than 2, the coverage of the plurality of enzymatic integration is not limited, and the NGS frequency is ranked as the top 10.
  • the screening condition is: the number of peptides is not less than 20, the coverage of the plurality of enzymatic integration is not less than 70% in the CDR3 region, and the entire V-region coverage is not less than 35%.
  • the NGS frequency is ranked as the first 200.
  • the method for screening a Nanobody of the present invention further comprises:
  • the Nanobody is expressed by a protein expression system based on the Nanobody sequence to identify the Nanobody.
  • the method of the invention adopts the immunosense library sequencing technology to screen the nano-antibody, and can directly and directly screen and obtain the nano-antibody having no light chain and only the heavy chain, and the experimental period is only about 21 days, and the time is greatly shortened, and the preparation time of the nano-antibody is greatly shortened, and the preparation time is greatly improved.
  • the efficiency and screening of the obtained antibodies do not require random pairing of heavy and light chains for analysis, effectively eliminating heavy and light chain mismatches and causing erroneous analysis.
  • the invention provides a system 1000 for screening Nanobodies.
  • the system includes: a nucleic acid sample extraction device 100 for extracting a nucleic acid sample from tissue or peripheral blood of an antigen-immunized animal; and a sequencing device 200, a sequencing device for obtaining a sequencing result including an antibody sequence based on the nucleic acid sample; an antibody database construction device 300 for constructing an antibody database based on the sequencing result of the antibody-containing sequence; the information analysis device 400
  • the information analysis device is configured to perform information analysis based on an antibody database, and screen to obtain a nano-antibody sequence.
  • the integrated assay device is configured to integrate the information of the antibody database with the protein mass spectrometry results to obtain the Nanobody sequence.
  • the animal is a camelid.
  • the camelid is at least one of a dromedary, a bactrian camel, a guanaco, an alpaca, a llama and an alpaca.
  • the antibody sequence comprises a hypervariable region sequence and a framework region sequence.
  • the sequencing device 200 further comprises: an amplification unit for amplifying the antibody sequence in the nucleic acid sample to obtain an amplification product; a sequencing unit, the A sequencing unit is used to sequence the amplified product to obtain the sequencing result of the antibody-containing sequence.
  • the nucleic acid sample is DNA or RNA.
  • the antibody sequence in the nucleic acid sample is amplified by PCR; when the nucleic acid sample is RNA, by 5'-RACE or PCR pair The antibody sequence in the nucleic acid sample is amplified.
  • the PCR is at least one of multiplex PCR, linear amplification mediated PCR and nested PCR.
  • the sequencing unit is a high throughput sequencing platform.
  • the high throughput sequencing platform is at least selected from the group consisting of Illumina HiSeq2000, HiSeq 2500, MiSeq, MiSeqDx, NextSeq500, Hiseq X Ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, and single molecule sequencing platforms.
  • the antibody database construction device 300 further comprises: a aligning unit for aligning the sequencing result of the antibody-containing sequence with a reference sequence to determine an immune-related gene sequence a determining unit for determining a nucleotide sequence of the antibody based on the immune-related gene sequence; a translation unit for translating a nucleotide sequence of the antibody into an amino acid sequence; screening a unit for screening for a VHH sequence based on the amino acid sequence, and constructing the antibody database based on the VHH sequence.
  • the immune-related gene is at least one of a V gene, a D gene, a J gene, and a C gene.
  • the reference sequence is a pre-rearranged germline sequence of at least one of the known V gene, D gene, J gene and C gene.
  • the screening unit screens for obtaining the indication of the VHH sequence as one of the following:
  • the framework region has one of the following four conserved amino acids: 37F, 44E, 45R, 47G.
  • 37F is The amino acid at position 37 is phenylalanine (F)
  • 44E is the amino acid at position 44 is glutamic acid (E)
  • 45R is the amino acid at position 45 is arginine (R)
  • 47G is The amino acid at position 47 is glycine (G); for normal double-stranded antibodies, the amino acid at position 37 is valine (V), and the amino acid at position 44 is glycine (G), position 45.
  • the amino acid is leucine (L), and the amino acid at position 47 is tryptophan (W), wherein the first amino acid in the hypervariable region is at position 0.
  • the hinge region has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;
  • Aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag (SEQ ID NO: 1)
  • Ggaacgaatgaagtatgcaagtgtcccaaatgtcca (SEQ ID NO: 2)
  • the average length of CDR3 is abnormal. Specifically, the length of CDR3 is a large range of variation, whether it is a normal antibody or VHH, but in terms of the average length of CDR3, the average length of CDR3 of VHH is longer, and the average length of CDR3 of a normal antibody is shorter.
  • the protein spectrum analysis device 500 further includes: an enrichment unit that performs IgG enrichment on the serum of the immunized animal using Protein A/G to obtain enrichment a product; an affinity purification unit that affinity-purifies the antibody from the enriched product using a chromatography column coupled to the antigen to obtain a purified product; an enzymatic unit, the enzymatic hydrolysis a unit for denaturation and reductive alkylation of the purified product, followed by enzymatic hydrolysis with a protease to obtain an enzymatic peptide; a mass spectrometric detection unit for performing protein on the enzymatic peptide Mass spectrometry was performed to obtain a mass spectrometric detection result of the enzymatic peptide.
  • the protease is pepsin, chymotrypsin, elastase, trypsin, intracellular protease Lys-C, metal endopeptidase Lys-N, endonuclease Glu-C, aspartic acid At least one of the endopeptidase Asp-N and the Clostridin Arg-C.
  • Nano-antibody was screened by immunohistochemistry.
  • the schematic diagram of the process is shown in Figure 6, as follows:
  • Primers for the C region were designed using the antibodies of the experimental animal species (single-humped camel, Bactrian camel, llama, etc.) in the IMGT database (http://www.imgt.org/) as reference sequences.
  • Figure 4 shows a schematic representation of the primer design for the heavy chain (Common VH) of VHH, a common double-stranded antibody (i.e., a conventional antibody), and the light chain of a conventional double-stranded antibody.
  • VHH primers are designed for the CH2 region
  • primers are designed for the heavy chain of the common double-stranded antibody
  • primers are designed for the CH1 region
  • primers are designed for the light chain of the common double-chain antibody.
  • primers are designed for the C region.
  • Primer design was performed using Oligo6 software, and the resulting primer sequences were delivered to a primer synthesis company for synthesis.
  • the primer sequences are as follows:
  • VHH CH2 region primer 5 ⁇ -GGTACGTGCTGTTGAACTGTTCC-3 ⁇ (SEQ ID NO: 3);
  • Heavy chain primer for common double-stranded antibody 5'-AGACCAGGCAGCCGAARG-3' (SEQ ID NO: 4);
  • Camels are immunized at a dose of 1 mg/time using known antigenic polypeptides, such as CK18, 863-17 (NDKA), immunized once every two weeks, and immunized 4 times.
  • Tissue samples of camels such as spleen, lymph nodes, intestines, oral mucosa
  • Nucleic acid samples were extracted from the obtained tissue samples and peripheral blood, respectively, and the obtained nucleic acid samples were used for subsequent experiments.
  • RNA samples were subjected to 5'-RACE and the amplification system was as follows:
  • the total volume of the above ingredients was 24 ⁇ l. After mixing, simply knead it down and bake at 42 ° C for 1 min.
  • sequence of the anchor primer is: GGCCACGCGTCGACTAGTACGGGGGGGGGG (SEQ ID NO. 8).
  • DNA samples were subjected to PCR amplification system:
  • sequences of the P1 primer and the P2 primer are as follows:
  • P2 primer TCAGAGGACGGCGGGAACAGG (SEQ ID NO. 10)
  • Pre-denaturation at 94 ° C for 3 min 35 rounds of PCR amplification were carried out according to the following reaction conditions, denaturation at 94 ° C for 0.5 min, annealing at 55 ° C for 0.5 min, extension at 72 ° C for 1-2 min, and extension at 72 ° C for 5 min.
  • a sequencing library was constructed for the amplified antibody sequences according to the instructions provided by the manufacturer of the sequencing instrument.
  • constructing a sequencing library generally includes the following steps:
  • the PCR amplification product is first purified by decapitation, and then subjected to dNTP as a substrate under the action of enzymes such as T4 DNA Polymerase, Klenow Fragment and T4 Polynucleotide Kinase. Repair, forming a DNA fragment that is phosphorylated at the end of the fill.
  • the base "A” is then added to the 3' end of the complement sequence using Klenow Frgment (3'-5'exo-) polymerase and dATP.
  • the DNA in the purified reaction system was recovered using a MiniElute PCR Purification Kit.
  • the DNA sequence at the 3' end plus the base "A” was ligated under the action of T4 DNA ligase, and the ligated product was recovered using a Mini Elute PCR Purification Kit, and quantified by Qubit et al.
  • connection product is recovered and purified.
  • the purified gel fragment DNA was recovered by agarose gel electrophoresis and a Mini Elute PCR Purification Kit.
  • a universal PCR primer was added, and a DNA high-fidelity phusion enzyme was used for PCR amplification.
  • the amplified product is purified by magnetic beads to obtain a sequencing library.
  • the resulting sequencing library was sequenced on at least one high-throughput sequencing platform such as Illumina HiSeq2000, HiSeq2500, MiSeq, MiSeqDx, NextSeq500, Hiseq X Ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, and single molecule sequencing, Thereby the sequencing results are obtained. Filtering removes low quality data, depending on the situation.
  • the principle of filtration includes: removing the data of the joint contamination, removing the portion with the lower base sequence quality value at the end of the sequence, and removing the data having a lower average base mass value or more "N". As shown in Table 1, after data filtering, the traditional antibody library left 62.87%-75.67%, and the Nanobody library left 55.14%-71.64% data volume.
  • PE sequences are spliced according to the intersection of the PE sequences.
  • Table 1 shows the preliminary results of the data. It can be seen from Table 1 that in the PE sequence splicing, the splicing success rate of all samples is above 91%.
  • the germline sequence of the discovered V/D/J/C rearrangement is used as a reference sequence (http://www.imgt.org/), using local alignment software (eg BLAST, BLASTN) Or LASTZ) to align the sequences, and then align the sequences again. Since V, D, and J have base deletion and insertion during gene rearrangement, after comparison, it is possible to more accurately determine that several bases have been specifically deleted. Based on the alignment results, the corresponding sequences of the V gene, the D gene, the J gene, and the C gene are determined.
  • the heavy chain is known to include the V, D, J, and C genes, the light chain includes the V, J, and C genes, and both the heavy and light chains include the complementarity determining region and the framework region.
  • a partial sequence can be determined by the germline sequence capable of aligning the V gene and the J gene, and the remaining sequences are determined by finding a conserved region. Thereafter, the assembled nucleotide sequence is translated into an amino acid sequence.
  • the ratio of V gene to J gene is shown in Table 2, the ratio of V gene is slightly lower, between 45% and 71%. The reason for the low ratio is that the reference sequence of the database is not complete. The ratio of J gene is very high, all of which is above 88%. Finally, there are sequences of V gene and J gene alignment, between 44% and 70%.
  • VHH sequence (including the VHH sequence associated with the immunizing antigen and the VHH sequence unrelated to the immunizing antigen) is screened from the translated amino acid sequence according to the following principles, namely:
  • the framework region has one of the following four conserved amino acids: 37F, 44E, 45R, 47G.
  • 37F means that the amino acid at position 37 is phenylalanine (F)
  • 44E means that the amino acid at position 44 is glutamic acid (E)
  • 45R means that the amino acid at position 45 is arginine ( R)
  • 47G means that the amino acid at position 47 is glycine (G);
  • the amino acid at position 37 is valine (V)
  • the amino acid at position 44 is glycine ( G)
  • the 45th amino acid is leucine (L)
  • the 47th amino acid is tryptophan (W)
  • the hinge region has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2:
  • Aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag (SEQ ID NO: 1)
  • Ggaacgaatgaagtatgcaagtgtcccaaatgtcca (SEQ ID NO: 2)
  • the average length of CDR3 is abnormal. Specifically, regardless of the normal double-stranded antibody VH or VHH, the length of CDR3 is a large range of variation, but in terms of the average length of CDR3, the average length of CDR3 of VHH is longer, and the average length of CDR3 of common antibody is longer. short.
  • step (3) 5. Perform various statistics on the nucleotide sequence data assembled in step (3), including the number of sequence types of CDR3, the distribution of use of V and J genes, the distribution of use of VJ pairing, V, (D), J gene. Insertion deletes the length distribution, the base composition of the V and J genes, the frequency distribution of the CDR3 sequences, and the like. The specific statistical results are shown in Figure 11 (conventional antibody) and Figure 12 (nanobody).
  • step (3) Perform the following statistics on the amino acid sequence translated in step (3): abundance, frequency (abundance refers to the number of repetitions of one amino acid sequence; frequency refers to the content of one amino acid sequence in the population), corresponding V and J gene, and sequence information.
  • abundance refers to the number of repetitions of one amino acid sequence; frequency refers to the content of one amino acid sequence in the population
  • V and J gene refers to the content of one amino acid sequence in the population
  • Nanobody sequence associated with the immunizing antigen is screened according to the abundance information of the antibody CDR3, and the specific screening conditions are: the abundance of the CDR3 is not less than 5, and the antibody sequence with high CDR3 abundance is preferentially selected.
  • IgG is enriched by Protein A/G to obtain enrichment. product. After obtaining the enriched product, it is determined by protein Western blotting or enzyme-linked immunosorbent assay (ELISA) that the obtained enriched product is capable of specifically binding to the known antigenic polypeptide used.
  • ELISA enzyme-linked immunosorbent assay
  • the antigen polypeptide used in Example 1 was attached to a chromatography column, and the previously obtained enriched product was affinity-purified using the column, and the flow-through and eluate were separately collected.
  • the obtained flow-through and eluate were subjected to Western blotting or enzyme-linked immunosorbent assay (ELISA), respectively, to determine that the affinity purification product specifically reacted with the known antigen polypeptide used.
  • ELISA enzyme-linked immunosorbent assay
  • Enzymatically Affinity Purified Antibody, Mass Spectrometry Detection After denaturation and reductive alkylation of the enriched antibody, it is divided into several parts, using different proteases (eg, pepsin, chymotrypsin, elastase, trypsin, The intracellular protease Lys-C, the metal endopeptidase Lys-N, the endoproteinase Glu-C, the aspartic endopeptidase Asp-N, the Clostridin Arg-C, etc. are subjected to enzymatic hydrolysis, thereby obtaining Enzymatic hydrolysis of peptides. Thereafter, the obtained enzymatic peptide was subjected to mass spectrometry to obtain a protein spectrum detection result.
  • proteases eg, pepsin, chymotrypsin, elastase, trypsin
  • Protein profiling results were identified by the software Mascot (http://www.matrixscience.com/) and used by Mascot Percolator (http://www.sanger.ac.uk/resources/software/mascotpercolator/) filters the results of the identification.
  • Mascot Percolator http://www.sanger.ac.uk/resources/software/mascotpercolator/
  • the false positive rate of the identification results obtained in the above steps was evaluated by the Target-Decoy method, and the identification result of the low false positive rate was retained as the basis for the subsequent analysis (the threshold of the false positive rate filtering was set to 1%).
  • the frequency information of the antibody sequence was analyzed to analyze the relationship between the number of peptide identification, the degree of protein coverage and the frequency of antibody sequencing, and according to their relationship, screening was performed to obtain a nanobody sequence related to the immune antigen.
  • the screening condition is one of the following:
  • the number of identified peptides is not less than 6, and the coverage of multiple enzymatic integration is not less than 50% in the CDR3 region, and the coverage of the entire V-region is not less than 40%, and the frequency of NGS is not limited;
  • the number of identified peptides is not less than 2, and the coverage of multiple enzymatic integration is not limited, and the high-throughput sequencing (NGS) frequency is ranked 10;
  • the number of identified peptides is not less than 4, the coverage of multiple enzymatic integration is not less than 50% in the CDR3 region, the coverage of the entire V-zone is not less than 35%, and the frequency of NGS is 200.
  • the number of identified peptides is not less than 6, and the coverage of multiple enzymatic integration is not less than 50% in the CDR3 region, and the coverage of the entire V-region is not less than 40%.
  • the frequency of NGS is not limited:
  • the number of identified peptides is not less than 2, and the coverage of multiple enzymatic integration is not limited.
  • the high-throughput sequencing (NGS) frequency is ranked before 100:
  • the number of identified peptides is not less than 4, the coverage of multiple enzymatic integration is not less than 50% in the CDR3 region, the coverage of the entire V-region is not less than 35%, and the order of NGS frequency is 200:
  • Example 1 The antibody gene sequence obtained by screening in Example 1 was synthesized, and the Nanobody was expressed by a protein expression system, and the Nanobody was identified as follows:
  • the bacterial pellet was dissolved on ice, and the lysate was added in an amount of 2 to 5 ml per gram, and was sufficiently resuspended.
  • the protein sample was added to an equal volume of 2 ⁇ loading buffer and incubated at 100 ° C for 5 min.
  • the results of SDS-PAGE gel electrophoresis are shown in Figure 14.
  • the antibody was identified by ELISA method.
  • the experimental steps are as follows:
  • the bacterial solution (i.e., a small amount of expressed protein) induced in the step (3) was sonicated and centrifuged at 12000 rpm for 10 minutes, and the supernatant was aspirated into a new EP tube to be used as an antibody.
  • Coat Conjugated antigen polypeptide coating concentration (10 ⁇ g/ml), 100 ⁇ l per well, overnight coating at 4 ° C, two replicates per sample.
  • Wash the plate Wash the plate more than 5 times with a plate washer and pat dry on the absorbent paper.
  • washing the plate Wash the plate more than 5 times with a plate washer and pat dry on the absorbent paper. At the same time as washing the plate, prepare the color developing solution (TMB).
  • Color development After preparing the stop solution, add 100 ⁇ l/well of the color development solution to shake the plate to accelerate the color development process. When the color is developed to a certain extent (generally, the blank and the negative color are not too high), add 50 ⁇ l/well of the stop solution, and then wait for more than 10min after adding the stop solution, so that the termination is complete and the color is uniform (the plate can be accelerated and terminated) .
  • Reading Use a microplate reader to read the plate after termination.
  • the microplate reader must be preheated for more than 30 minutes; the TMB color detection wavelength is 450/630.
  • 11-1A, 22-2B, and the like in the following table are the Nanobodies expressed by the procedure described in Example 3, such as the sequence selected in Example 2, such as >C10WBA-IgG23_26.
  • 22-2B, 12-1B, 31-3A, and the like were identified as positive antibodies.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

本发明提供了筛选纳米抗体的方法及相应的系统，所述方法采用聚合酶链式反应和cDNA 5'末端快速扩增技术，筛选获得纳米抗体，实验周期只需21天左右。

Description

筛选纳米抗体的方法及系统 技术领域
本发明涉及生物技术领域，具体涉及筛选纳米抗体的方法及系统。
背景技术
长期以来，我们认为的抗体指的是一般意义上由重链和轻链配对构成的，拥有完整CH1，CH2和CH3等完整恒定区的抗体，即所谓传统抗体，然而在1993年，Hamers-Casterman等发现了骆驼科动物的血清里存在一种只含重链不含轻链的天然重链抗体(HCAb)。克隆重链抗体的可变区得到只由一个重链可变区组成的单域抗体，称为VHH抗体(variable domain of heavy chain of heavy-chainantibody，VHH)。VHH晶体直径2.5nm，长4nm，呈圆柱状，故又称为纳米抗体(nanobody，Nb)或者单域重链抗体。
纳米抗体从发现至今，已经发展为有广泛生物应用价值和临床应用价值的高度通用分子。纳米抗体有来源于成年骆驼体内HCAbs的最小的功能性抗原结合片段，具有高度稳定性和与抗原结合的高亲合力，能与蛋白裂隙和酶活性位点的相互作用，使之作用类似于抑制剂。因此，纳米抗体可以为从肽模拟药物设计小分子酶抑制物提供新的思路。由于仅有重链，纳米抗体的制造较mAb容易。纳米抗体的独特性质，如处于极端温度和pH环境中的稳定性，可以低成本制造大产量。因此，纳米抗体在疾病的治疗和诊断中具有很大的价值，在肿瘤的抗体靶向诊断和治疗中也具有很大的发展前景。
传统方法筛选骆驼抗体采用的是噬菌体展示技术，噬菌体展示技术的主要是通过从免疫的骆驼中获取一定量的外周血或者免疫组织，分离出淋巴细胞，提取mRNA，通过RT-PCR，让CDNA插入到噬菌体外壳蛋白结构基因的适当位置，使外源基因随外壳蛋白的表达而表达，形成单克隆进行抗体库筛选。然而，噬菌体展示技术免疫对象大多是人、小鼠、兔子，极少对骆驼进行免疫，且实验周期较长，需要77天左右，筛选的是重、轻链互相配对存在的常规抗体，噬菌体等展示技术不仅费时、费工，而且技术难度大。
因而，关于筛选纳米抗体的技术仍有待改进。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个 目的在于提出一种实验周期短、效率高、不需重、轻链随机配对分析的筛选高特异性成熟纳米抗体的方法。
在本发明的一个方面，本发明提供了一种筛选纳米抗体的方法。根据本发明的实施例，该方法包括：(1)从经过免疫的动物的组织或外周血提取核酸样本；(2)基于所述核酸样本，获得包含抗体序列的测序结果；(3)基于所述包含抗体序列的测序结果，构建抗体数据库；(4)基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
本发明的方法采用免疫组库测序技术——即聚合酶链式反应(PCR)和cDNA 5’末端快速扩增(5’-RACE技术)筛选抗体，能够快速有效地直接筛选获得没有轻链只有重链的纳米抗体，实验周期只需21天左右，大大缩短了纳米抗体的筛选制备时间，大大提高了效率，且筛选纳米抗体不需要重、轻链随机配对进行分析，有效排除重轻链错配而引起错误分析，筛选获得的纳米抗体在疾病诊断治疗、药物开发以及食品检测等方面具有广阔的应用前景。
根据本发明的实施例，本发明的筛选纳米抗体的方法进一步包括：(5)对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果；(6)将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列。
根据本发明的实施例，本发明的筛选纳米抗体的方法进一步包括：(7)基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
根据本发明的实施例，所述动物为骆驼科动物。
根据本发明的实施例，所述骆驼科动物为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种。
根据本发明的实施例，所述抗体序列包括高变区序列和框架区序列。
根据本发明的实施例，步骤(2)进一步包括：(2-1)对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物；(2-2)对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果。
根据本发明的实施例，所述核酸样本为DNA或RNA。
根据本发明的实施例，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的所述抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的所述抗体序列进行扩增。
根据本发明的实施例，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种。
根据本发明的实施例，通过高通量测序装置对所述扩增产物进行测序。
根据本发明的实施例，所述高通量测序装置为选自Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche454和单分子测序装置中的至少一种。由此，测序通量较高且成本低廉。
根据本发明的实施例，步骤(3)进一步包括：(3-1)将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因序列；(3-2)基于所述免疫相关基因序列，确定所述抗体的核苷酸序列；(3-3)将所述抗体的核苷酸序列翻译成氨基酸序列；(3-4)基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库。
根据本发明的实施例，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种。
根据本发明的实施例，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系(germline)序列。
根据本发明的实施例，在步骤(3-4)中，所述氨基酸序列为所述VHH序列的指示为下列之一：
A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G。具体而言，37F是指第37位的氨基酸为苯丙氨酸(F)，44E是指第44位的氨基酸为谷氨酸(E)，45R是指第45位的氨基酸为精氨酸(R)，47G是指第47位的氨基酸为甘氨酸(G)；而对普通双链抗体来说，则为：第37位的氨基酸为缬氨酸(V)，第44位的氨基酸为甘氨酸(G)，第45位的氨基酸为亮氨酸(L)，第47位的氨基酸为色氨酸(W)，其中，高变区第一个氨基酸所在位置为0。
B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列：
aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag(SEQ ID NO:1)，
ggaacgaatgaagtatgcaagtgtcccaaatgtcca(SEQ ID NO:2)，
C.缺少CH1的至少一部分；
D.CDR3的平均长度异常。具体而言，不管是普通抗体还是VHH，CDR3的长度都是一个大的变化的范围，但是就CDR3的平均长度来说，VHH的CDR3平均长度较长，普通抗体的CDR3平均长度较短。
根据本发明的实施例，步骤(5)进一步包括：(5-1)利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物；(5-2)利用偶联所述抗原的层析柱，从所述富集产物亲和纯化所述抗体；(5-3)将步骤(5-2)中所得到的纯化产物进行变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段；(5-4)利用质谱仪对所述酶解肽 段进行蛋白质质谱检测，以便获得所述酶解肽段的质谱检测结果。
根据本发明的实施例，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
在本发明的另一方面，本发明提供了一种筛选纳米抗体的系统，根据本发明的实施例，该系统包括：核酸样本提取装置，所述核酸样本提取装置用于从经过抗原免疫的动物的组织或外周血提取核酸样本；测序装置，所述测序装置用于基于所述核酸样本，获得包含抗体序列的测序结果；抗体数据库构建装置，所述抗体数据库构建装置用于基于所述包含抗体序列的测序结果，构建抗体数据库；信息分析装置，所述信息分析装置用于基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
根据本发明的实施例，进一步包括：蛋白质谱分析装置，所述蛋白质谱分析装置用于对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果；整合分析装置，所述整合分析装置用于将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列。
根据本发明的实施例，进一步包括：鉴定装置，所述鉴定装置用于基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
根据本发明的实施例，所述动物为骆驼科动物。
根据本发明的实施例，所述骆驼科动物为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种。
根据本发明的实施例，所述抗体序列包括高变区序列和框架区序列。
根据本发明的实施例，所述测序装置进一步包括：扩增单元，所述扩增单元用于对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物；测序单元，所述测序单元用于对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果。
根据本发明的实施例，所述核酸样本为DNA或RNA。
根据本发明的实施例，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的所述抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的所述抗体序列进行扩增。
根据本发明的实施例，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种。
根据本发明的实施例，所述测序单元为高通量测序平台。
根据本发明的实施例，所述高通量测序平台为选自Illumina HiSeq2000、HiSeq2500、 MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche454和单分子测序平台中的至少一种。
根据本发明的实施例，所述抗体数据库构建装置进一步包括：比对单元，所述比对单元用于将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因序列；确定单元，所述确定单元用于基于所述免疫相关基因序列，确定抗体的核苷酸序列；翻译单元，所述翻译单元用于将所述抗体的核苷酸序列翻译成氨基酸序列；筛选单元，所述筛选单元用于基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库。
根据本发明的实施例，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种。
根据本发明的实施例，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系序列。
根据本发明的实施例，所述筛选单元筛选获得所述VHH序列的指示为下列之一：
A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G；
B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列；
C.缺少CH1的至少一部分；
D.CDR3的平均长度异常。
根据本发明的实施例，所述蛋白质谱分析装置进一步包括：富集单元，所述富集单元利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物；亲和纯化单元，所述亲和纯化单元利用偶联所述抗原的层析柱，从所述富集产物亲和纯化所述抗体，以便获得纯化产物；酶解单元，所述酶解单元用于将所述纯化产物进行变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段；质谱检测单元，所述质谱检测单元用于对所述酶解肽段进行蛋白质质谱检测，以便获得所述酶解肽段的质谱检测结果。
根据本发明的实施例，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
附图说明
图1显示了根据本发明的一个实施例，筛选纳米抗体的方法的流程示意图；
图2显示了根据本发明的另一个实施例，筛选纳米抗体的方法的流程示意图；
图3显示了根据本发明的又一个实施例，筛选纳米抗体的方法的流程示意图；
图4显示了根据本发明的实施例，引物设计的示意图；
图5显示了根据本发明的实施例，对测序结果进行分析的流程图；
图6显示了根据本发明的实施例，采用免疫组库技术筛选纳米抗体的方法的流程示意图；
图7显示了根据本发明的实施例，采用免疫组库技术结合蛋白质谱分析技术筛选纳米抗体的方法的流程示意图；
图8显示了根据本发明的一个实施例，筛选纳米抗体的系统的结构示意图；
图9显示了根据本发明的再一个实施例，筛选纳米抗体的系统的结构示意图；
图10显示了根据本发明的又一个实施例，筛选纳米抗体的系统的结构示意图；
图11显示了根据本发明的一个实施例，获得传统抗体各数据统计结果图；
图12显示了根据本发明的一个实施例，获得纳米抗体各数据统计结果图；
图13显示了根据本发明的一个实施例，获得抗体肽段，抗体可变区覆盖度和CDR3区域覆盖度统计结果；以及
图14显示了根据本发明的又一个实施例，筛选纳米抗体分子量大小用SDS-PAGE胶检测结果图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明的一个方面，本发明提供了一种筛选纳米抗体的方法。根据本发明的实施例，参照图1，该方法包括以下步骤：
(1)从经过免疫的动物的组织或外周血提取核酸样本。
根据本发明的实施例，所述经过免疫的动物可以通过利用抗原对动物进行免疫获得。根据本发明的实施例，抗原的种类不受特别限制，可以是任何已知的抗原。在本发明的一些实施例中，所述抗原包括但不限于上皮细胞角蛋白18(CK18)、核苷二磷酸激酶A(NDKA863-17)。根据本发明的实施例，抗原的剂量也不受特别限制。根据本发明的一个具体示例，可以以10mg/次的剂量对动物进行免疫。
根据本发明的实施例，动物的种类不受特别限制，只要能够有效筛选获得纳米抗体，本领域技术人员可以根据实际情况灵活选择。根据本发明的一些实施例，动物为骆驼科动 物。根据本发明的一些实施例，骆驼科动物可以为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种。由此，能够有效提高获得纳米抗体的效率。根据本发明的一个具体示例，所述动物为骆驼。
(2)基于所述核酸样本，获得包含抗体序列的测序结果。
根据本发明的实施例，步骤(2)进一步包括以下步骤：
(2-1)对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物。
根据本发明的实施例，所述核酸样本为DNA或RNA。由此，有利于后续步骤进行，进而有利于提高筛选纳米抗体的效率。
根据本发明的实施例，所述抗体序列包括高变区序列和框架区序列。由此，能够有效筛选获得纳米抗体。
根据本发明的实施例，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的抗体序列进行扩增。利用PCR或者5’-RACE技术，只需在C区设计特异性引物，可以有效、全面富集抗体序列，可以统计基因家族的表达频率、V-J配对和碱基插入、缺失情况以及CDR区多样性、长度分布特征，还可全面观察到整个抗原特异性抗体库的全貌和克隆出高特异性成熟纳米抗体。
根据本发明的实施例，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种。
(2-2)对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果。
根据本发明的实施例，通过高通量测序装置对所述扩增产物进行测序。
根据本发明的另一个实施例，将所述扩增产物进行常规建库后，得到包含所述扩增产物的测序文库，通过高通量测序装置对所述测序文库进行测序。
根据本发明的一些实施例，所述高通量测序装置为选自Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion TorrentPGM、Proton、Roche454和单分子测序装置中的至少一种。由此，能够快速有效地获得核酸样本的核苷酸序列，且测序通量较高、成本低廉。
根据本发明的实施例，根据情况，可以对测序得到的测序结果进行过滤，去除低质量的数据。
(3)基于所述包含抗体序列的测序结果，构建抗体数据库。
根据本发明的实施例，步骤(3)进一步包括以下步骤：
(3-1)将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因 序列。
根据本发明的实施例，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系(germline)序列。由此，能够快速有效的确定免疫相关基因序列，且准确率较高。
根据本发明的实施例，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种。
根据本发明的实施例，根据需要，在将包含抗体序列的测序结果与参考序列进行比对之前，可以对包含抗体序列的测序结果进行过滤，去除低质量的数据和去掉接头污染的数据，如去除序列末端碱基测序质量值比较低的部分，去除序列整体碱基平均质量值低或者含“N”多的数据，去除序列有接头污染的数据，长度太短的序列也需要被去除。根据本发明的实施例，如果高通量测序是双末端(paired-end，PE)类型的，则需要根据PE序列的交集部分，将PE序列拼接起来。
根据本发明的实施例，将包含抗体序列的测序结果与参考序列进行比对具体包括：首先，利用局部比对软件将测序结果与参考序列进行比对。然后对测序结果与参考序列进行再次比对，获得比对结果。其中，所述参考序列可以为已发现的V/D/J/C重排前的germline序列，所采用的局部比对软件不受特别限制，本领域技术人员可以根据实际情况灵活选择，根据本发明的一些实施例，所采用的局部比对软件包括但不限于BLAST、BLASTN和LASTZ。接着，根据比对结果，确定V基因、D基因、J基因和C基因的相应序列。其中，重链包括V、D、J和C基因，根据需要，如果同时设计了普通双链抗体序列的引物，并扩增了双链抗体序列，则双链抗体的轻链包括V、J和C基因。
(3-2)基于所述免疫相关基因序列，确定抗体的核苷酸序列。
已知，重链和轻链均包括互补决定区(CDR区，又称高变区)和框架区。由此，可以通过能够比对上的V基因和J基因的胚系序列确定部分序列，其余的序列通过寻找保守区域来确定。
根据本发明的实施例，确定抗体的核苷酸序列之后，进一步对所得到的核苷酸序列数据进行多种统计，包括CDR3的序列种类数，V和J基因的使用分布，V-J配对的使用分布，V，(D)，J基因上的插入删除长度分布，V和J基因的碱基组成，CDR3序列的频率分布等等。
需要说明的是，本文中所使用的表达方式“CDR3的序列种类数”是指不一样的CDR3序列数目；“V和J基因的使用分布”是指在所有序列中，使用的各个V和J基因在样本占的频率；“V-J配对的使用分布”是指在所有序列中，每一种V-J基因组合占的频率，“V，(D)， J基因上的插入删除长度分布”是指VDJ基因在重排，基因的末端会有一些碱基的删除，并且基因之间会有一些碱基的插入，这里统计的即这些插入或者删除碱基的长度；“V和J基因的碱基组成”是指对所有的序列，统计基因在每个位置上碱基组成和各碱基占的比例；“CDR3序列的频率分布”是指每个CDR3序列占的频率分布。
(3-3)将所述抗体的核苷酸序列翻译成氨基酸序列。
根据本发明的实施例，对翻译得到的氨基酸序列进行下列统计：丰度、频率(丰度是指一条氨基酸序列重复的次数；频率是指一条氨基酸序列在总体中的含量)、对应的V和J基因、以及序列信息。在该步骤中，去掉序列丰度小于2的序列。
(3-4)基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库。
需要说明的是，该步骤中筛选获得的VHH序列包括免疫后动物体内的所有VHH序列，既包括与免疫抗原相关的VHH序列，也包括与免疫抗原无关的VHH序列。
根据本发明的实施例，所述氨基酸序列为所述VHH序列的指示为下列之一：
A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G。具体而言，37F是指第37位的氨基酸为苯丙氨酸(F)，44E是指第44位的氨基酸为谷氨酸(E)，45R是指第45位的氨基酸为精氨酸(R)，47G是指第47位的氨基酸为甘氨酸(G)；而对普通双链抗体来说，则为：第37位的氨基酸为缬氨酸(V)，第44位的氨基酸为甘氨酸(G)，第45位的氨基酸为亮氨酸(L)，第47位的氨基酸为色氨酸(W)。其中，高变区第一个氨基酸为零位。
B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列：
aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag(SEQ ID NO:1)，
ggaacgaatgaagtatgcaagtgtcccaaatgtcca(SEQ ID NO:2)，
C.缺少CH1的至少一部分；
D.CDR3的平均长度异常。具体而言，不管是普通抗体还是VHH，CDR3的长度都是一个大的变化的范围，但是就CDR3平均长度来说，VHH的CDR3平均长度较长，普通抗体的CDR3平均长度较短。
另外，需要说明的是，如果同时设计了普通双链抗体序列的引物，并扩增了双链抗体序列，则在步骤(3-3)中获得的氨基酸序列包括纳米抗体序列和双链抗体序列，通过筛选VHH抗体序列，可以将纳米抗体序列和双链抗体序列进行分离，即可以同时获得纳米抗体数据库和双链抗体数据库，本领域技术人员可以根据需要，直接选择纳米抗体数据库进行 后续分析，筛选获得纳米抗体；也可同时对纳米抗体数据库和双链抗体数据库进行后续分析，筛选获得纳米抗体，提高筛选的准确度。
(4)基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
根据本发明的实施例，在该步骤中，基于抗体CDR3的频率信息，筛选出与免疫抗原相关的纳米抗体序列。
根据本发明的实施例，参照图2，本发明的筛选纳米抗体的方法进一步包括：
(5)对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果。
根据本发明的实施例，步骤(5)进一步包括：
(5-1)利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物。
根据本发明的实施例，可以将免疫动物的血清除去脂肪、细胞残渣及小颗粒物质后，利用Protein A/G进行IgG富集，并对富集结果进行蛋白质免疫印迹法(western blot)或者酶联免疫吸附实验(Elisa)检测，以确定富集后的样品对抗原多肽具有特异性反应。
(5-2)利用偶联所述抗原的层析柱，从所述富集产物亲和纯化抗体。
根据本发明的实施例，将前面所采用的已知抗原多肽连接到层析柱上，并利用该层析柱对步骤(5-1)所得到的富集产物进行亲和纯化，并且分别收集流穿液和洗脱液。将所得到的流穿液和洗脱液分别进行蛋白质免疫印迹检验或者酶联免疫吸附检验(ELISA)，以确定亲和纯化产物对所使用的已知抗原多肽具有特异性反应。
(5-3)将所述富集产物变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段。
根据本发明的实施例，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
(5-4)利用质谱仪对所述酶解肽段进行蛋白质质谱分析，以便获得所述酶解肽段的质谱检测结果。
(6)将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列。
根据本发明的实施例，可以先向所述抗体数据库中，掺入常见污染蛋白序列，得到最终数据库。根据本发明的实施例，所述污染蛋白序列包括实验对象物种的保守区域序列和作为干扰的来自其他物种的蛋白序列。
根据本发明的实施例，可以采用合适的鉴定软件和合适的参数，将蛋白质质谱结果与抗体数据库进行比较，从而对蛋白质质谱结果进行鉴定，并利用Target-Decoy方法对鉴定 结果的假阳性率进行评估，保留低假阳性率的鉴定结果作为后续分析的基础(例如假阳性率为1％或5％以下)。进一步的，可以合并多种酶解肽段的蛋白质质谱结果分析的数据，根据抗体和鉴定肽段的对应关系，统计抗体序列的肽段鉴定数，蛋白覆盖度，CDR3区域覆盖度等信息，并计算对应抗体序列的定量值。然后，结合抗体序列的频数信息，分析肽段鉴定数目、蛋白覆盖度和抗体测序频率的关系，根据他们之间的关系，进行筛选，得到与免疫抗原相关的纳米抗体序列。
根据本发明的一个具体示例，筛选条件为：鉴定肽段数不低于20条，多种酶解整合的覆盖度在CDR3区域不低于70％，整个V-region(V-区)覆盖度不低于50％，NGS频数不作限制。
根据本发明的另一个具体示例，筛选条件为：鉴定肽段数不低于2条，多种酶解整合的覆盖度不作限制，NGS频数排序为前10。
根据本发明的又一个具体示例，筛选条件为：鉴定肽段数不低于20条，多种酶解整合的覆盖度在CDR3区域不低于70％，整个V-region覆盖度不低于35％，NGS频数排序为前200。
根据本发明的实施例，参考图3，本发明的筛选纳米抗体的方法进一步包括：
(7)基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
本发明的方法采用免疫组库测序技术筛选纳米抗体，能够快速有效地直接筛选获得没有轻链只有重链的纳米抗体，实验周期只需21天左右，时间大大缩短纳米抗体筛选制备时间，大大提高了效率，且筛选获得的抗体不需要重、轻链随机配对进行分析，有效排除重轻链错配而引起错误分析。
在本发明的另一方面，本发明提供了一种筛选纳米抗体的系统1000。根据本发明的实施例，参照图8，该系统包括：核酸样本提取装置100，所述核酸样本提取装置用于从经过抗原免疫的动物的组织或外周血提取核酸样本；测序装置200，所述测序装置用于基于所述核酸样本，获得包含抗体序列的测序结果；抗体数据库构建装置300，所述抗体数据库构建装置用于基于所述包含抗体序列的测序结果，构建抗体数据库；信息分析装置400，所述信息分析装置用于基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
根据本发明的实施例，进一步包括：蛋白质谱分析装置500，所述蛋白质谱分析装置用于对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果；整合分析装置600，所述整合分析装置用于将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列。
根据本发明的实施例，进一步包括：鉴定装置700，所述鉴定装置用于基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
根据本发明的实施例，所述动物为骆驼科动物。
根据本发明的实施例，所述骆驼科动物为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种。
根据本发明的实施例，所述抗体序列包括高变区序列和框架区序列。
根据本发明的实施例，所述测序装置200进一步包括：扩增单元，所述扩增单元用于对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物；测序单元，所述测序单元用于对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果。
根据本发明的实施例，所述核酸样本为DNA或RNA。
根据本发明的实施例，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的所述抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的所述抗体序列进行扩增。
根据本发明的实施例，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种。
根据本发明的实施例，所述测序单元为高通量测序平台。
根据本发明的实施例，所述高通量测序平台为选自Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche454和单分子测序平台中的至少一种。
根据本发明的实施例，所述抗体数据库构建装置300进一步包括：比对单元，所述比对单元用于将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因序列；确定单元，所述确定单元用于基于所述免疫相关基因序列，确定抗体的核苷酸序列；翻译单元，所述翻译单元用于将所述抗体的核苷酸序列翻译成氨基酸序列；筛选单元，所述筛选单元用于基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库。
根据本发明的实施例，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种。
根据本发明的实施例，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系序列。
根据本发明的实施例，所述筛选单元筛选获得所述VHH序列的指示为下列之一：
A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G。具体而言，37F是 指第37位的氨基酸为苯丙氨酸(F)，44E是指第44位的氨基酸为谷氨酸(E)，45R是指第45位的氨基酸为精氨酸(R)，47G是指第47位的氨基酸为甘氨酸(G)；而对普通双链抗体来说，则为：第37位的氨基酸为缬氨酸(V)，第44位的氨基酸为甘氨酸(G)，第45位的氨基酸为亮氨酸(L)，第47位的氨基酸为色氨酸(W)，其中，高变区第一个氨基酸所在位置为0。
B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列；
aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag(SEQ ID NO:1)，
ggaacgaatgaagtatgcaagtgtcccaaatgtcca(SEQ ID NO:2)，
C.缺少CH1的至少一部分；
D.CDR3的平均长度异常。具体而言，不管是普通抗体还是VHH，CDR3的长度都是一个大的变化的范围，但是就CDR3的平均长度来说，VHH的CDR3平均长度较长，普通抗体的CDR3平均长度较短。
根据本发明的实施例，所述蛋白质谱分析装置500进一步包括：富集单元，所述富集单元利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物；亲和纯化单元，所述亲和纯化单元利用偶联所述抗原的层析柱，从所述富集产物亲和纯化所述抗体，以便获得纯化产物；酶解单元，所述酶解单元用于将所述纯化产物进行变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段；质谱检测单元，所述质谱检测单元用于对所述酶解肽段进行蛋白质质谱检测，以便获得所述酶解肽段的质谱检测结果。
根据本发明的实施例，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
实施例1
采用免疫组库技术筛选纳米抗体，流程示意图见图6，具体如下：
一、设计用于富集纳米抗体的引物
以IMGT数据库(http://www.imgt.org/)中实验动物物种(单峰驼、双峰驼、美洲驼等)的抗体为参考序列，设计针对C区的引物。图4显示了针对VHH、普通双链抗体(即传统抗体)的重链(Common VH)以及普通双链抗体的轻链(Light Chain)进行引物设计的示意图。其中，对于VHH，是针对CH2区进行设计引物，对于普通双链抗体的重链，是针对CH1区进行设计引物，对于普通双链抗体的轻链，是针对C区进行设计引物。在本实施例中，采 用Oligo6软件进行引物设计，并将所得到的引物序列交付引物合成公司合成。引物序列如下：
VHH CH2区引物：5`-GGTACGTGCTGTTGAACTGTTCC-3`(SEQ ID NO:3)；
普通双链抗体的重链引物：5`-AGACCAGGCAGCCGAARG-3`(SEQ ID NO:4)；
普通双链抗体的轻链引物：
5`-GGAGTGGACTTGGGCTGAC-3`(SEQ ID NO:5)；
5`-TGAGGGTGTAGCCTTGGGT-3`(SEQ ID NO:6)；
5`-AARCACACGAYTGAGGCAC-3`(SEQ ID NO:7)。
二、获取免疫组织或者外周血
使用已知抗原多肽，例如CK18、863-17(NDKA)，以1mg/次的量对骆驼进行免疫，每两周免疫一次，经过4次免疫。并在每次免疫后分别获取骆驼的组织样本(例如脾脏、淋巴结、肠道、口腔黏膜)和外周血。并分别从所获取的组织样本和外周血提取核酸样本(DNA和RNA)，并将所得到核酸样本用于后续实验。
三、测序
利用前面所涉及的用于富集纳米抗体的引物，针对所得到的DNA、RNA样本进行PCR扩增，针对所得到的RNA样本进行5’-RACE(5’-端的快速扩增法)或PCR扩增，以便扩增相应的抗体序列，其中，在扩增反应体系中，核酸样本(DNA或者RNA)的起始量为1～2微克。
RNA样本进行5’-RACE，扩增体系如下：
1、混合下列样品，37℃10min；冰浴1min：
上述各引物...................................................................2.5pmol(10～25ng)
RNA.........................................................................................................1-5μg
DEPC-水................................................................................补水至15.5μl
2、依次加入以下样品：
10×PCR缓冲液........................................................................................2.5μl
25mM MgCl2...........................................................................................2.5μl
10mM dNTP混合物.......................................................................................1μl
0.1M DTT...............................................................................................2.5μl
以上成分总体积24μl。混匀后简单甩下来，42℃冰浴1min。
3、以上体系再加入1μl反转录酶II，混匀，42℃温浴50min反转录。
4、末端加C，再加入以下成分：
DEPC-水....................................................................................................6.5μl
5×末端转移缓冲液........................................................................................5.0μl
2mM dCTP...............................................................................................2.5μl
S.N.A.P.-纯化cDNA样本..............................................................10.0μl
共计体积..................................................................................................49.0μl
5、94℃温浴2-3min，冰浴1min，加入1μl末端转移酶TdT，简单离心后37℃温浴10min。然后65℃10min灭活TdT，置于冰上。
6、PCR扩增富集目标区域：
无菌蒸馏水..........................................................................31.5μl
10×PCR缓冲液[200mM Tris-HCl(pH 8.4),500mM KCl].................5.0μl
25mM MgCl2...........................................................................................3.0μl
10mM dNTP混合物.....................................................................................1.0μl
上述各引物(10μM).....................................................................................2.0μl
锚定引物(10μM).............................................................2.0μl
dC-tailed cDNA........................................................................................5.0μl
终体积.....................................................................................................49.5μl
其中，锚定引物的序列为：GGCCACGCGTCGACTAGTACGGGGGGGGGG(SEQ ID NO.8)。
再加入0.5μl的Taq DNA聚合酶，94℃预变性2min；按照如下反应条件进行35轮PCR扩增，94℃变性0.5min，55℃退火0.5-1min，72℃延伸1min；72℃最后延伸5min。
DNA样本进行PCR扩增体系：
2×KAPA预混液...........................................................25μl
P1引物(10μM).............................................................1.5μl
P2引物(10μM).............................................................1.5μl
cDNA.......................................................................................................4.0μl
DEPC-水...................................................................................................18μl
其中，P1引物和P2引物的序列分别如下所示：
P1引物：CTCTGCTCCTTCTCACCCTCCTCAC(SEQ ID NO.9)
P2引物：TCAGAGGACGGCGGGAACAGG(SEQ ID NO.10)
94℃预变性3min；按照如下反应条件进行35轮PCR扩增，94℃变性0.5min,55℃退火0.5min，72℃延伸1-2min；72℃最后延伸5min。
按照测序仪器制造商所提供的操作说明，针对所扩增的抗体序列，构建测序文库。
如以illumina Hiseq测序平台为例，根据操作说明，构建测序文库大致包括以下步骤：
1.末端修复、3’末端加碱基“A”
PCR扩增产物先经过切胶回收纯化，然后在T4DNA聚合酶(T4DNA Polymerase)、Klenow片段(Klenow Fragment)和T4多核苷酸激酶(T4Polynucleotide Kinase)等酶的作用下，以dNTP为底物进行末端修复，形成补平的末端磷酸化的DNA片段。然后利用Klenow Frgment(3’-5’exo-)聚合酶及dATP在补平序列的3’末端加上碱基“A”。用MiniElute PCR Purification Kit回收纯化反应体系中的DNA。
2.连接接头
3’末端加上碱基“A”的DNA序列在T4DNA连接酶的作用下进行接头连接，用Mini Elute PCR Purification Kit回收连接产物，采用Qubit等方法对其进行定量。
3.连接产物切胶回收纯化
利用琼脂糖凝胶电泳以及Mini Elute PCR Purification Kit回收纯化凝胶片段DNA。
4.PCR扩增
以步骤(3)得到的DNA为模板，加入通用PCR引物，DNA高保真phusion酶进行PCR扩增。扩增产物用磁珠进行纯化后即可得到测序文库。
将所得到的测序文库在Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche 454和单分子测序等至少一种高通量测序平台上进行测序，从而得到测序结果。根据情况，进行过滤除去低质量的数据。
四、分析测序结果：
图5中显示了对测序结果进行分析的流程。具体包括：
1.对测序数据进行过滤。过滤的原则包括：去掉接头污染的数据，去除序列末端碱基测序质量值比较低的部分，去除序列整体碱基平均质量值低或者含“N”多的数据。如表1所示，数据过滤后，传统抗体文库剩下62.87％-75.67％，纳米抗体文库剩下55.14％-71.64％的数据量。
2.如果高通量测序是双末端(paired-end，PE)类型的，则根据PE序列的交集部分，将PE序列拼接起来。
表1是数据初步处理结果，从表1中可看出，在PE序列拼接中，所有样本的拼接成功率都在91％以上。
表1：数据初步处理
Figure PCTCN2015086253-appb-000001
3.将已发现的V/D/J/C重排前的胚系(germline)序列作为为参考序列(http://www.imgt.org/)，用局部比对软件(如BLAST、BLASTN或LASTZ)对序列进行比对，之后对序列进行再次比对。由于V、D、J在基因重排时会有碱基的删除和插入，经过再次对比，能够更精确的确定出具体删除了几个碱基。根据比对结果，确定V基因、D基因、J基因和C基因的相应序列。已知重链包括V、D、J和C基因，轻链包括V、J和C基因，重链和轻链均包括互补决定区和框架区。由此，可以通过能够比对上的V基因和J基因的胚系序列确定部分序列，其余的序列通过寻找保守区域来确定。之后，将所组装的核苷酸序列翻译成氨基酸序列。V基因和J基因的比对率如表2数据所示，V基因的比对率稍低，在45％-71％之间，比对率低的原因可能是数据库的参考序列还不完全；J基因的比对率很高，全部在88％以上；最后同时有V基因和J基因比对上的序列，在44％-70％之间。
表2：V基因和J基因的比对率
Figure PCTCN2015086253-appb-000002
4.筛选出VHH序列。
按照下列原则从所翻译得到的氨基酸序列中筛选出VHH序列(包括与免疫抗原相关的VHH序列以及与免疫抗原无关的VHH序列)，即：
A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G。具体而言，37F是指第37位的氨基酸为苯丙氨酸(F)，44E是指第44位的氨基酸为谷氨酸(E)，45R是指第45位的氨基酸为精氨酸(R)，47G是指第47位的氨基酸为甘氨酸(G)；而对普通双链抗体来说，则为：第37位的氨基酸为缬氨酸(V)，第44位的氨基酸为甘氨酸(G)，第45位的氨基酸为亮氨酸(L)，第47位的氨基酸为色氨酸(W)
B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列：
aacccaagataccccaaccacaaccaaaaccacaaccacaaccacaaccacaaccaaaaccacaaccaaaacctgaaccagaatgcacgtgtcccaaatgtccag(SEQ ID NO:1)，
ggaacgaatgaagtatgcaagtgtcccaaatgtcca(SEQ ID NO:2)，
C.缺少CH1的至少一部分；
D.CDR3的平均长度异常。具体而言，不管是普通双链抗体VH还是VHH，CDR3的长度都是一个大的变化的范围，但是就CDR3的平均长度来说，VHH的CDR3平均长度较长，普通抗体的CDR3平均长度较短。
以上原则，至少选择一个。本实施例中，优选同时使用四个原则。
5.将步骤(3)中所组装的核苷酸序列数据进行多种统计，包括CDR3的序列种类数，V和J基因的使用分布，V-J配对的使用分布，V，(D)，J基因上的插入删除长度分布，V和J基因的碱基组成，CDR3序列的频率分布等等。具体统计结果如图11(传统抗体)和图12(纳米抗体)所示。
6.将步骤(3)中翻译得到的氨基酸序列进行下列统计：丰度、频率(丰度是指一条氨基酸序列重复的次数；频率是指一条氨基酸序列在总体中的含量)、对应的V和J基因、以及序列信息。将所得到的统计结果构建成抗体数据库，包括双链抗体数据库和单链抗体数据库。在该步骤中，序列的丰度小于2的序列会从数据库中去掉。所构建的抗体数据库部分展示如表3。
表3:抗体数据库部分展示
Figure PCTCN2015086253-appb-000003
Figure PCTCN2015086253-appb-000004
Figure PCTCN2015086253-appb-000005
7.根据抗体CDR3的丰度信息，筛选出与免疫抗原相关的纳米抗体序列，具体筛选条件为：CDR3的丰度不低于5，并且优先选择CDR3丰度高的抗体序列。
实施例2
在本实施例中，在实施例1的基础上，进一步结合蛋白质谱分析技术，筛选纳米抗体，采用免疫组库技术结合蛋白质谱分析技术筛选纳米抗体的流程示意图见图7，具体如下：
一、蛋白质谱实验技术方法
1.亲和纯化抗体
(1)IgG富集
将经过抗原(例如CK18、863-17(NDKA))免疫后的骆驼的血清(即外周血)除去脂肪、细胞残渣及小颗粒物质后，利用Protein A/G进行IgG富集，从而得到富集产物。在得到富集产物之后，通过蛋白质免疫印迹检验或者酶联免疫吸附检验(ELISA)，确定所得到的富集产物能够与所使用的已知抗原多肽发生特异性结合。
(2)亲和纯化
将实施例1中所采用的抗原多肽连接到层析柱上，并利用该层析柱对前面所得到的富集产物进行亲和纯化，并且分别收集流穿液和洗脱液。将所得到的流穿液和洗脱液分别进行蛋白质免疫印迹检验或者酶联免疫吸附检验(ELISA)，以确定亲和纯化产物对所使用的已知抗原多肽具有特异性反应。
2.酶解亲和纯化抗体、质谱检测将经过富集的抗体变性并还原烷基化之后，等分成若干份，分别用不同种蛋白酶(例如：胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C等)进行酶解，从而得到酶解肽段。之后，将所得到的酶解肽段进行质谱检测，得到蛋白质谱检测结果。
二、蛋白质谱的信息分析方法
1.数据库构建
向实施例1中测序分析得到的抗体数据库中，掺入常见污染蛋白序列，得到最终数据库。这里所使用的污染蛋白序列包括实验对象物种的保守区域序列和作为干扰的来自其他物种的蛋白序列。最终数据库的组成如表4所示：
表4最终数据库及其序列数目情况
数据来源 序列数目
4号骆驼第二次免疫传统抗体重链测序(频数大于1) 1732
4号骆驼第二次免疫纳米抗体测序(频数大于1) 1479
4号骆驼第四次免疫传统抗体重链测序(频数大于1) 1424
4号骆驼第四次免疫纳米抗体测序(频数大于1) 2246
10号骆驼第二次免疫传统抗体重链测序(频数大于1) 989
10号骆驼第二次免疫纳米抗体测序(频数大于1) 1360
10号骆驼第四次免疫传统抗体重链测序(频数大于1) 1580
10号骆驼第四次免疫纳米抗体测序(频数大于1) 3104
10号骆驼传统抗体重链测序(频数大于1) 876
10号骆驼纳米抗体测序(频数大于1) 1697
7号骆驼传统抗体重链测序(频数大于1) 601
7号骆驼纳米抗体测序(频数大于1) 1132
常见污染蛋白 115
酵母蛋白 6718
总计 25053
2.数据库鉴定
通过软件Mascot(http://www.matrixscience.com/)对蛋白质谱结果进行鉴定，并用Mascot  Percolator(http://www.sanger.ac.uk/resources/software/mascotpercolator/)对鉴定结果进行过滤。所使用的软件参数如下表：
搜索引擎 Mascot Percolator
酶解方式 胰蛋白酶/糜蛋白酶
固定修饰 半胱氨酸碘乙酰胺化-Carbamidomethyl(C)
可变修饰 甲硫氨酸氧化-Oxidation(M)、去酰胺基deamidate(NQ)
最大漏切数 1
母离子质量偏差 20ppm
碎片离子质量偏差 0.05Da
假阳性率阈值 肽段水平1％
蛋白推断标准 每个鉴定蛋白要求至少含有一条或一条以上的独有肽段
3.假阳性率控制和鉴定肽段筛选
利用Target-Decoy方法对上述步骤得到的鉴定结果的假阳性率进行评估，保留低假阳性率的鉴定结果作为后续分析的基础(设定假阳性率过滤阈值为1％)。
4.覆盖度，丰度统计与相关性分析
合并多种酶解的数据，根据抗体和鉴定肽段的对应关系，统计抗体序列的肽段鉴定数，抗体可变区覆盖度，CDR3区域覆盖度等信息，并计算对应抗体序列的定量值。其中，覆盖度是指所有鉴定肽段覆盖的长度占总长度的比例；肽段鉴定数目是指所有高于假阳性率过滤阈值的鉴定肽段数目。所得的抗体肽段，抗体可变区覆盖度和CDR3区域覆盖度结果统计如图13所示。
结合抗体序列的频数信息分析肽段鉴定数目，蛋白覆盖度和抗体测序频率的关系，根据他们的关系，进行筛选，得到与免疫抗原相关的纳米抗体序列。
具体的，筛选条件为下列之一：
(1)鉴定肽段数不低于6条，多种酶解整合的覆盖度在CDR3区域不低于50％，整个V-region覆盖度不低于40％，NGS频数不作限制；
(2)鉴定肽段数不低于2条，多种酶解整合的覆盖度不作限制，高通量测序(NGS)频数排序前10；
(3)鉴定肽段数不低于4条，多种酶解整合的覆盖度在CDR3区域不低于50％，整个V-区覆盖度不低于35％，NGS频数排序前200。
依照上述筛选条件，分别获得以下序列：
(1)鉴定肽段数不低于6条，多种酶解整合的覆盖度在CDR3区域不低于50％，整个V-region覆盖度不低于40％，NGS频数不作限制：
Figure PCTCN2015086253-appb-000006
(2)鉴定肽段数不低于2条，多种酶解整合的覆盖度不作限制，高通量测序(NGS)频数排序前100：
Figure PCTCN2015086253-appb-000007
Figure PCTCN2015086253-appb-000008
(3)鉴定肽段数不低于4条，多种酶解整合的覆盖度在CDR3区域不低于50％，整个V-区覆盖度不低于35％，NGS频数排序前200：
Figure PCTCN2015086253-appb-000009
具体的抗体序列示例如下所示：
>C10WBA-IgG23_26
MAHVQLVESGGGSVQAGGSLKLSCAVSPYIFTRCGLGWYRQAPGNVRELVSSMDSDGTTIYADSVKGRFTISQDNEKNMLYLQMNSLKTEDTAMYYCAADHQDPSRGAYFDHCNYMGQGTQVTVS
>C10WBA-IgG23_1064
MADVQLVESGGGLVQPGGSLRLSCVGSGFGPKVYCMGWFRQAPGKEREGVAAVDSEGNTSYVESVKGRFTISQDKDKNTVYLEMNNLKPEDTAMYYCAAELQIPLNRQVAGRSWHCPLLAPVSAGGRHSGVWGQGTQVTVS
>C10WBA-IgG23_666
MADVQLVESGGGSVQAGGSLRLSCSISPAISHRNCMAWFRQVPGNEREGVAAIYLTAGATHYIDSVKGRFTISEDSAENTMYLQMENLKSEDTGMYYCAADFFPFKGGAASLAPMCLNGLEYRHRGQGTQVTVS
实施例3
将实施例1中筛选获得的抗体基因序列合成，通过蛋白表达系统表达纳米抗体，对所述纳米抗体进行鉴定，具体如下：
一、构建表达载体
1.酶切
20μl质粒pET28a DNA，1μl EcoRI，1μl BamHI，5μl 10×K缓冲液，23μl ddH2O，37℃5h。使用琼脂糖凝胶DNA回收试剂盒，切胶回收目的片段(即纳米抗体基因序列)和载体。
2.连接、转化
(1)连接：
1μl载体，3μl目的片段，1μl T4连接酶，5μl 2×缓冲液，混匀，室温反应30min以上。
(2)转化
1)取出零下80℃保存的感受态细胞(BL21)，放在冰上缓慢解冻。
2)将感受态细胞加入连接产物中或质粒DNA中(1μl)混匀，冰上放置30min。
3)42℃热激90s。
4)冰浴2min后，加入800μl无抗性的LB培养基。
5)37℃培养45min。
6)5000rpm离心3min，弃大部分上清，留约100-150μl，重悬菌体，卡那抗性的LB平板，涂板。
7)晾干，于37℃培养箱中倒置培养过夜。
3，挑单克隆及培养
(1)从转化的平板挑单克隆到1.5ml LB液体培养基中，于37℃，200rpm下培养至OD＝0.6-0.8。
(2)将1.5ml LB培养的种子液转接入1LLB扩大培养液中，至对数期OD＝0.6-0.8时加入IPTG(0.5mM)诱导，再于37℃，200rpm下培养2h。
(3)收集菌液，4℃，13200r/min离心15分钟，去上清液，加0.01mol/L PBS清洗沉淀，并再次离心，将沉淀保存于-80℃。
4、蛋白样品收集及镍柱纯化
(1)冰上溶解细菌沉淀，以每克2-5ml的量加入裂解液，充分重悬。
(2)加入溶菌酶至终浓度1mg/ml，混匀，冰上放置30分钟。
(3)冰上超声菌体，200-300W，每次10秒，共6次，每次间隔10秒。
(4)加入RNaseA(10ug/ml)和DNase(5ug/ml)，冰上放置10-15分钟。
(5)4℃，10000r/min离心20-30分钟，收集上清液，0.45mm滤膜过滤。
(6)取2ml Ni-NTA凝胶装柱。
(7)在柱上缓慢加入10倍柱体积的平衡液，以充分平衡Ni-NTA凝胶，流速为1ml/min。
(8)取过滤后的细菌裂解液，匀速加入凝胶，直至样品完全进入凝胶后，用10倍柱体积的平衡液继续洗涤凝胶，保存流速为1ml/min。
(9)用分别含50mmol/L、100mmol/L，200mmol/L咪唑的洗脱液各5ml进行阶段洗脱，流速为2ml/min，在下端用试管接住流出液体，每管1ml，试管标记顺序。
5、SDS-PAG电泳检测
将蛋白样品加入等体积的2×加样缓冲液，100℃孵育5min，SDS-PAGE胶电泳检测结果如图14所示。
二、抗体鉴定
抗体鉴定采用ELISA方法，实验步骤如下：
将步骤(3)诱导的菌液(即小量表达蛋白)超声后12000rpm离心10min，吸上清到新EP管中，作为抗体使用。
1.包被：偶联抗原多肽包被浓度(10μg/ml)，每孔100μl，4℃包被过夜，每个样品两个重复。
2.封闭：包被完的板子，用洗板机洗板3次，于吸水纸上拍干，然后用1％BSA封闭，200μl/孔，4℃封闭过夜或37℃封闭2h。
3.加一抗：封闭完的板子，用洗板机洗板3次，于吸水纸上拍干，根据不同实验，取好相应板子，加入相应一抗(一抗为小量表达蛋白上清及纯化抗体)，37℃孵育2h。
4.洗板：用洗板机洗板5次以上，于吸水纸上拍干。
5.加二抗：小量表达蛋白上清的孔加His抗体(1:2000)作为二抗，其余孔加PBS，37℃孵育1h。
6.洗板：用洗板机洗板5次以上，于吸水纸上拍干。洗板同时，配制好显色液(TMB)。
7.显色：准备好终止液后，加入显色液100μl/孔，可以晃动板子加速显色过程。显色到一定程度时(一般不能让空白和阴性显色太高)，加入50μl/孔终止液，加入终止液后需要静置10min以上，使终止彻底、颜色均一(可以晃动板子加速终止过程)。
8.读数：使用酶标仪，对终止后的板子进行读数，其中，酶标仪必须预热30min以上；TMB显色检测波长为：450/630。
在一定的稀释条件(如1：10)下，阳性结果与阴性对照(PBS对照)之间的OD值的比值达到1.5或以上(在此例中为PBS对照：0.117×1.5＝0.175，即>0.175即为阳性)，认为相应纳米抗体有活性，结果如下表所示。下表中11-1A、22-2B等为根据实施例2中筛选出来的序列，如>C10WBA-IgG23_26等通过实施例3中描述的步骤表达出来的纳米抗体。在此例中，22-2B，12-1B，31-3A等鉴定为阳性抗体。
Figure PCTCN2015086253-appb-000010
Figure PCTCN2015086253-appb-000011
Figure PCTCN2015086253-appb-000012
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种筛选纳米抗体的方法，其特征在于，包括：
    (1)从经过免疫的动物的组织或外周血提取核酸样本；
    (2)基于所述核酸样本，获得包含抗体序列的测序结果；
    (3)基于所述包含抗体序列的测序结果，构建抗体数据库；
    (4)基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
  2. 根据权利要求1所述的方法，其特征在于，进一步包括:
    (5)对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果；
    (6)将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列，
    任选地，进一步包括：
    (7)基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
  3. 根据权利要求1或2所述的方法，其特征在于，所述动物为骆驼科动物，
    任选地，所述骆驼科动物为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种，
    任选地，所述抗体序列包括高变区序列和框架区序列。
  4. 根据权利要求1或2所述的方法，其特征在于，步骤(2)进一步包括：
    (2-1)对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物；
    (2-2)对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果，
    任选地，所述核酸样本为DNA或RNA，
    任选地，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的所述抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的所述抗体序列进行扩增，
    任选地，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种，
    任选地，通过高通量测序装置对所述扩增产物进行测序，
    任选地，所述高通量测序装置为选自Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx、NextSeq500、HiseqXten、Life SOLiD、Ion Torrent PGM、Proton、Roche 454和单分子测序装置中的至少一种，
    任选地，步骤(3)进一步包括：
    (3-1)将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因序列；
    (3-2)基于所述免疫相关基因序列，确定抗体的核苷酸序列；
    (3-3)将所述抗体的核苷酸序列翻译成氨基酸序列；
    (3-4)基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库，
    任选地，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种，
    任选地，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系序列，
    任选地，在步骤(3-4)中，所述氨基酸序列为所述VHH序列的指示为下列之一：
    A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G；
    B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列；
    C.缺少CH1的至少一部分；
    D.CDR3的平均长度异常。
  5. 根据权利要求2所述的方法，其特征在于，步骤(5)进一步包括：
    (5-1)利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物；
    (5-2)利用偶联所述抗原的层析柱，从所述富集产物亲和纯化所述抗体；
    (5-3)将步骤(5-2)中所得到的纯化产物进行变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段；
    (5-4)利用质谱仪对所述酶解肽段进行蛋白质质谱检测，以便获得所述酶解肽段的质谱检测结果，
    任选地，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
  6. 一种筛选纳米抗体的系统，其特征在于，包括：
    核酸样本提取装置，所述核酸样本提取装置用于从经过抗原免疫的动物的组织或外周血提取核酸样本；
    测序装置，所述测序装置用于基于所述核酸样本，获得包含抗体序列的测序结果；
    抗体数据库构建装置，所述抗体数据库构建装置用于基于所述包含抗体序列的测序结果，构建抗体数据库；
    信息分析装置，所述信息分析装置用于基于抗体数据库进行信息分析，筛选获得纳米抗体序列。
  7. 根据权利要求6所述的系统，其特征在于，进一步包括:
    蛋白质谱分析装置，所述蛋白质谱分析装置用于对所述经过免疫的动物的血清进行蛋白质质谱分析，以便获得蛋白质质谱结果；
    整合分析装置，所述整合分析装置用于将所述抗体数据库的信息与所述蛋白质质谱结果进行整合分析，以便获得所述纳米抗体序列，
    任选地，进一步包括：
    鉴定装置，所述鉴定装置用于基于所述纳米抗体序列，通过蛋白表达系统表达所述纳米抗体，以便对所述纳米抗体进行鉴定。
  8. 根据权利要求6或7所述的系统，其特征在于，所述动物为骆驼科动物，
    任选地，所述骆驼科动物为单峰驼、双峰驼、原驼、家羊驼、小羊驼和羊驼的至少一种，
    任选地，所述抗体序列包括高变区序列和框架区序列。
  9. 根据权利要求6或7所述的系统，其特征在于，所述测序装置进一步包括：
    扩增单元，所述扩增单元用于对所述核酸样本中的抗体序列进行扩增，以便获得扩增产物；
    测序单元，所述测序单元用于对所述扩增产物进行测序，以便获得所述包含抗体序列的测序结果，
    任选地，所述核酸样本为DNA或RNA，
    任选地，当所述核酸样本为DNA时，通过PCR对所述核酸样本中的所述抗体序列进行扩增；当所述核酸样本为RNA时，通过5’-RACE或PCR对所述核酸样本中的所述抗体序列进行扩增，
    任选地，所述PCR为多重PCR、线性扩增介导的PCR和巢式PCR的至少一种，
    任选地，所述测序单元为高通量测序平台，
    任选地，所述高通量测序平台为选自Illumina HiSeq2000、HiSeq2500、MiSeq、MiSeqDx NextSeq500、Hiseq X ten、Life SOLiD、Ion Torrent PGM、Proton、Roche 454和单分子测序平台中的至少一种，
    任选地，所述抗体数据库构建装置进一步包括：
    比对单元，所述比对单元用于将所述包含抗体序列的测序结果与参考序列进行比对，以便确定免疫相关基因序列；
    确定单元，所述确定单元用于基于所述免疫相关基因序列，确定抗体的核苷酸序列；
    翻译单元，所述翻译单元用于将所述抗体的核苷酸序列翻译成氨基酸序列；
    筛选单元，所述筛选单元用于基于所述氨基酸序列，筛选获得VHH序列，并基于所述VHH序列构建所述抗体数据库，
    任选地，所述免疫相关基因为V基因、D基因、J基因和C基因中的至少一种，
    任选地，所述参考序列为已知的V基因、D基因、J基因和C基因中至少一种的重排前的胚系序列，
    任选地，所述筛选单元筛选获得所述VHH序列的指示为下列之一：
    A.框架区存在下列4个保守氨基酸之一：37F，44E，45R，47G；
    B.铰链区具有SEQ ID NO:1和SEQ ID NO:2所示的序列；
    C.缺少CH1的至少一部分；
    D.CDR3的平均长度异常。
  10. 根据权利要求7所述的系统，其特征在于，所述蛋白质谱分析装置进一步包括：
    富集单元，所述富集单元利用Protein A/G，对所述经过免疫的动物的血清进行IgG富集，从而得到富集产物；
    亲和纯化单元，所述亲和纯化单元利用偶联所述抗原的层析柱，从所述富集产物亲和纯化所述抗体，以便获得纯化产物；
    酶解单元，所述酶解单元用于将所述纯化产物进行变性并还原烷基化后，用蛋白酶进行酶解，以便获得酶解肽段；
    质谱检测单元，所述质谱检测单元用于对所述酶解肽段进行蛋白质质谱检测，以便获得所述酶解肽段的质谱检测结果，
    任选地，所述蛋白酶为胃蛋白酶、糜蛋白酶、弹性蛋白酶、胰蛋白酶、胞内蛋白酶Lys-C、金属内肽酶Lys-N、蛋白内切酶Glu-C、天冬氨酸肽链内切酶Asp-N、梭菌蛋白酶Arg-C中的至少一种。
PCT/CN2015/086253 2014-08-07 2015-08-06 筛选纳米抗体的方法及系统 Ceased WO2016019888A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/502,438 US10954550B2 (en) 2014-08-07 2015-08-06 Method and system for screening nanobody
CN201580042224.1A CN106661629B (zh) 2014-08-07 2015-08-06 筛选纳米抗体的方法及系统

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410386936.2 2014-08-07
CN201410386936 2014-08-07

Publications (1)

Publication Number Publication Date
WO2016019888A1 true WO2016019888A1 (zh) 2016-02-11

Family

ID=55263170

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/086253 Ceased WO2016019888A1 (zh) 2014-08-07 2015-08-06 筛选纳米抗体的方法及系统

Country Status (3)

Country Link
US (1) US10954550B2 (zh)
CN (1) CN106661629B (zh)
WO (1) WO2016019888A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116457368A (zh) * 2020-05-01 2023-07-18 匹兹堡大学联邦高等教育系统 用于鉴定纳米抗体和纳米抗体亲和力的组合物和方法
CN114116857B (zh) * 2022-01-26 2022-06-17 北京晶泰科技有限公司 抗体数据库的生成、更新和查询方法、装置及存储介质
CN115636879B (zh) * 2022-09-30 2024-01-05 南京集思慧远生物科技有限公司 一种基于二代测序和蛋白质组学的纳米抗体生产及其应用方法
CN116381067A (zh) * 2022-10-19 2023-07-04 沈阳农业大学 羊驼和骆驼血液中纳米抗体的提取鉴定和开发应用
CN120322678A (zh) * 2022-11-02 2025-07-15 浙江纳米抗体技术中心有限公司 使用序列特征筛选纳米抗体
CN118599003B (zh) * 2024-06-24 2025-07-11 君研生物科技(山西)有限公司 一种与胰蛋白酶类似物TrypLE特异性结合的羊驼源纳米抗体及其应用
CN121006619A (zh) * 2025-01-02 2025-11-25 广州国家实验室 构建纳米抗体编码dna文库的方法及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103866401A (zh) * 2014-03-28 2014-06-18 中国农业科学院油料作物研究所 一种黄曲霉毒素纳米抗体基因库、构建方法、用途及黄曲霉毒素b1纳米抗体2014afb-g15

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7329353B2 (en) * 2004-01-23 2008-02-12 Amgen Inc. LC/MS method of analyzing high molecular weight proteins
AU2005325801A1 (en) * 2005-01-31 2006-08-03 Ablynx N.V. Method for generating variable domain sequences of heavy chain antibodies
WO2011008349A2 (en) * 2009-05-26 2011-01-20 Duke University Methods of identifying infectious disease and assays for identifying infectious disease
CN102219853B (zh) 2010-04-15 2013-10-30 上海人类基因组研究中心 抗h5n1型禽流感病毒的小羊驼vhh重链抗体及其制备方法和用途
EP3524258B1 (en) * 2011-06-22 2025-10-01 Apellis Pharmaceuticals, Inc. Methods of treating chronic disorders with complement inhibitors
JP6338235B2 (ja) * 2013-11-22 2018-06-06 国立研究開発法人産業技術総合研究所 低分子化抗体のスクリーニング方法及び製造方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103866401A (zh) * 2014-03-28 2014-06-18 中国农业科学院油料作物研究所 一种黄曲霉毒素纳米抗体基因库、构建方法、用途及黄曲霉毒素b1纳米抗体2014afb-g15

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TILLIB, S.V . ET AL.: "Fingerprint-like Analysis of 'Nanoantibody' Selection by Phage Display Using Two Helper Phage Variants", ACTA NATURAE, vol. 2, no. 3, 30 June 2010 (2010-06-30) *

Also Published As

Publication number Publication date
US10954550B2 (en) 2021-03-23
US20170226566A1 (en) 2017-08-10
CN106661629B (zh) 2021-03-16
CN106661629A (zh) 2017-05-10

Similar Documents

Publication Publication Date Title
US10175249B2 (en) Proteomic identification of antibodies
EP2572203B1 (en) Determination of antigen-specific antibody sequences in blood circulation
US11866785B2 (en) Tumor specific antibodies and T-cell receptors and methods of identifying the same
WO2016019888A1 (zh) 筛选纳米抗体的方法及系统
US11773510B2 (en) Method of discovering specific functional antibodies
Tu et al. Landscape of variable domain of heavy‐chain‐only antibody repertoire from alpaca
Erasmus et al. A single donor is sufficient to produce a highly functional in vitro antibody library
Bonissone et al. Serum proteomics expands on identifying more high-affinity antibodies in immunized rabbits than deep b-cell repertoire sequencing alone
CN106065031A (zh) 基于杂交瘤技术和高通量测序的抗体发现方法
CA3236602A1 (en) Methods and compositions for protein detection
US20250012808A1 (en) Parallel antibody engineering compositions and methods
Wang et al. Development of functional anti‐Gn nanobodies specific for SFTSV based on next‐generation sequencing and proteomics
EP3072976B1 (en) Method for determining and system for determining polypeptide bonding to target molecule
Ehling et al. Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2
US20240117526A1 (en) Methods and compositions for protein detection
JP2025506683A (ja) 抗体の開発可能性特性が最大化された単一ドメイン抗体ライブラリ
Le Bihan et al. de Novo Sequencing of Antibodies for Identification of Neutralizing Antibodies in Human Plasma Post SARS-CoV-2 Vaccination
HK1234447B (zh) 筛选纳米抗体的方法及系统
CN110139952B (zh) 骆驼科抗体可变区免疫组库构建的引物组合及应用
Marano Optimization of a pipeline for the development of recombinant monoclonal antibodies for diagnostics
Cheng et al. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes
WO2026055339A1 (en) Methods for identifying cognate immunoglobulin pairs from individual b cells
HK1234447A1 (zh) 筛选纳米抗体的方法及系统
CN121108322A (zh) 一种抗牛赤羽病毒的鼠源单克隆抗体的制备及其应用
Wang High-throughput analysis of native antibody repertoires for therapeutics discovery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15830205

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15830205

Country of ref document: EP

Kind code of ref document: A1