WO2018103093A1 - Primer combination constructed by variable region immune repertoire of camelid antibody and use thereof - Google Patents

Primer combination constructed by variable region immune repertoire of camelid antibody and use thereof Download PDF

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WO2018103093A1
WO2018103093A1 PCT/CN2016/109286 CN2016109286W WO2018103093A1 WO 2018103093 A1 WO2018103093 A1 WO 2018103093A1 CN 2016109286 W CN2016109286 W CN 2016109286W WO 2018103093 A1 WO2018103093 A1 WO 2018103093A1
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primer
antibody
round
gene
amplification
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PCT/CN2016/109286
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Chinese (zh)
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李新洋
杨乃波
黄谧
刘楚新
曹丽霞
丁权
马莹莹
王媚娘
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深圳华大基因研究院
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Priority to PCT/CN2016/109286 priority Critical patent/WO2018103093A1/en
Priority to CN201680089897.7A priority patent/CN110139952B/en
Publication of WO2018103093A1 publication Critical patent/WO2018103093A1/en

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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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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

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  • the present invention belongs to the field of biotechnology, and in particular, the present invention relates to a primer combination and application constructed by a camelid antibody variable region immunological library.
  • the heavy chain antibody is a heavy-chain antibody (HCAbs) that Hamers-Casterman is equivalent to a naturally-deficient light chain found in camelids in 1993, consisting only of heavy chains.
  • the antigen binding site of such antibodies is only formed by the variable region VHH single domain of the heavy chain.
  • VHH is one of the smallest molecular weight antibody fragments that have been found to have antigen-binding function to date, and has a molecular weight of 13-15 KD, which is only 1/10 of that of a conventional antibody, and is therefore also referred to as a nanobody.
  • VHH antibodies Compared with traditional antibodies, VHH antibodies have a small molecular weight and are easy to express; they exhibit higher specificity and affinity when recognizing antigenic epitopes, and can recognize antigenic epitopes with unique conformations; and have high solubility compared with traditional antibodies.
  • the characteristics of sex and stability, and the development of VHH antibodies specific for important target molecules have become a new strategy for the preparation of new antibody products in recent years.
  • the camelids of the present invention include, but are not limited to, Camelus bactrianus (Bactrian camel), dromedary (Camelus dromedarius), llama (llama, guanaco, alpaca), llama (vicuna), camel Sheep (Alpaca Suri, lamagama), llama (vigogne), llamas and camel.
  • B cell receptor is an immunoglobulin that recognizes antigen on the surface of B cells and has antigen binding specificity.
  • BCR is a tetrameric protein composed of two heavy chains and two light chains, wherein the heavy chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region, and The light chain has only the V zone and the C zone.
  • the V region consists of two domains, the heavy chain variable region (VH) and the light chain variable region (VL), which are composed of three complementarity determining regions, CDR1, CDR2, and CDR3, and the CDR arrangement order and amino acid composition are presented.
  • VH heavy chain variable region
  • VL light chain variable region
  • CDR1, CDR2, and CDR3 complementarity determining regions
  • CDR arrangement order and amino acid composition are presented.
  • the B cell immune group library was constructed by PCR technology, and the huge BCR sequence library corresponding to B cells in peripheral blood was obtained after sequencing. According to the screening criteria of certain biological information sequences, the screening of specific antibody sequences can be achieved.
  • the storage capacity and composition of the immune group library will directly affect the subsequent screening process. Therefore, those skilled in the art are working to construct a large-capacity immune library.
  • the object of the present invention is to provide a primer combination and application of a camelid antibody variable region immunological library.
  • a primer combination comprising a first upstream primer which specifically anneals to a leader region of the camelid antibody V gene I family (Clan I).
  • the first upstream primer comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 11, 2, 3, 4, 5, 6, 7, 8, 9, 10 1,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 36, 37, 38, 39, 40, 41, 42, 43, and 44; preferably, the first upstream primer sequence is set forth in SEQ ID NO.
  • the primer combination further comprises a downstream primer; preferably the downstream primer sequence is set forth in SEQ ID NO.
  • the primer combination further comprises a second upstream primer, the second upstream primer specifically annealing to the leader region of the camelid antibody V gene III family (Clan III), preferably the second The upstream primer sequence is set forth in SEQ ID NO.
  • the primer combination further comprises a third upstream primer, the third upstream primer specifically annealing to the leader region of the camelid antibody V gene II family (Clan II), preferably the second
  • the upstream primer sequence is set forth in SEQ ID NO.
  • each primer in the primer combination being a downstream primer that specifically anneals to a region of a camelid antibody J gene, and the primer combination comprises a one selected from the group consisting of One or more primer sequences: SEQ ID NO. 54, 55, 56, and 57.
  • the primer combination further comprises an upstream primer; preferably the upstream primer sequence is set forth in SEQ ID NO.
  • the primer combination comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 47, 48, 49, 50.
  • the primer combination further comprises the primer combination of the first aspect of the invention.
  • kits comprising the first aspect of the invention The primer combination, and/or the primer combination of the second aspect of the invention; and, optionally, a PCR amplification reagent.
  • the kit further comprises an upstream primer primer CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46) and/or CALL5 (5'-TGGTGGCAGGTCCCCAAGGT-3', SEQ ID NO .53).
  • CALL001 5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46
  • CALL5 5'-TGGTGGCAGGTCCCCAAGGT-3', SEQ ID NO .53.
  • a method of constructing a camelid antibody variable region immune library comprising the steps of:
  • a second round of nested PCR amplification was performed to obtain a second round of amplification product.
  • the nucleic acid in the step (1), is a cDNA reverse-transcribed into a cell-derived RNA.
  • the method further comprises the optional step of:
  • the illumina library was constructed using the second round of amplification products as a template.
  • a gene fragment of 600-1100 bp is recovered as the first round of amplification product; preferably, a gene fragment of 700-800 bp is recovered as the next round.
  • a template for amplifying the variable region of a heavy chain antibody preferably, a gene fragment of 900-1100 bp is recovered as a template for the next round of amplification of the heavy chain variable region of a conventional antibody.
  • a gene fragment of 300-600 bp is recovered as the second round of amplification product; preferably, a gene fragment of 400-500 bp is recovered as the first Two rounds of amplification products.
  • the second round of the amplified product is used as a template, and the P1 primer and the primer containing the index mark are PCR amplification of the upstream and downstream primers, and the obtained primer can be directly used for constructing the sequencing. Library on the machine.
  • a method for screening a heavy chain antibody comprising the steps of: constructing a heavy chain antibody variable region immunological library using the method of the fourth aspect of the invention; The heavy chain antibody is screened in a heavy chain antibody variable region immunological pool.
  • the method for screening heavy chain antibodies is microbial display technology and/or bioinformatics analysis methods such as phage, Escherichia coli, and the like.
  • FIG. 1 Schematic diagram of the construction of a camelid antibody variable region immunological library library by three rounds of polymerase chain reaction.
  • the leader region refers to a region upstream of the antibody gene transcribed into mRNA
  • the V, D, and J genes refer to three structural components of the antibody variable region
  • the CH1 region, the hinge region, and the CH2 region are The structure of the constant region of the antibody constitutes a gene.
  • the present inventors have obtained an upstream primer capable of efficiently annealing to the leader region of the camelid family V gene I family by extensive and intensive research; and a downstream primer capable of efficiently annealing to the camelid antibody J gene region.
  • the experimental results show that with the other primers, the primer combination provided by the present invention can be used for three rounds of amplification, and a large-capacity antibody of the camelid antibody can be constructed, and on the basis of this, the high specificity and high affinity can be screened. Chain antibody. On the basis of this, the present invention has been completed.
  • the general method for constructing a heavy chain antibody immunological library is to take peripheral blood, separate peripheral blood mononuclear cells (PBMC) with cell separation solution, extract total cellular RNA, and obtain cDNA by reverse transcription.
  • PBMC peripheral blood mononuclear cells
  • cDNA was used as a template, nested PCR was used to amplify the variable region sequences.
  • the first round was CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO.
  • CALL001 is located in the leader region of the BCR V gene III family of Camelidae
  • CALL002 is located in the highly conserved region of the CH2 domain
  • CALL001 and CALL002 can be amplified to obtain most of the variable region. Sequence of regions. The 700-800 bp fragment was used as a template to bind the upstream primer VHH-forward (5'-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3', SEQ ID NO.
  • VHH V gene III family region
  • VHH-reverse binding to the J gene region
  • 5'-GGAGACGGTGACCTGGGT-3', SEQ ID NO. 52 was used to perform a second round of PCR enrichment of the VHH gene sequence.
  • CALL refers to the abbreviation of camelid
  • VHH refers to the variable region of camelid heavy chain antibody.
  • a combination of any one or a plurality of primers comprising a camelid antibody V gene I family (Clan I) leader region is used, and the designed primers are shown in Table 1;
  • a combination comprising the camelid antibody J gene region primers was used, and the designed primers are shown in Table 2.
  • the heavy chain antibody gene and the traditional antibody gene sequence were enriched, and the gelatin was distinguished.
  • the first round of PCR product was used as a template, and the V-region primer and the J-region primer were used for two round amplification to form an antibody. Immunization group library.
  • the primers shown in Table 1 are the upstream primers of the first round of PCR, which can be annealed to the leader sequence region of the Camel antibody V gene I family BCR.
  • any one or more primers in Table 1 Amplification of the camelid antibody V gene I family gene sequence with the downstream primer CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID NO. 45), in combination with CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46) and CALL5 (5'-TGGTGGCAGGTCCCCAAGGT-3', SEQ ID NO. 53) was used to amplify the camelid antibody V gene I, II and III family gene sequences.
  • CALL001, CALL5 and CALL002 amplifies the V gene II and III family gene sequences.
  • the base substitution of any primer in Table 1 is carried out, and any one of the primers is randomly selected in the middle, and the restriction enzyme site and the protection base are added before and after, and the primer formed by any platform sequencing linker is added before and after, as long as it can amplify the corresponding Target gene sequences are all within the scope of the claims of the present application.
  • the primers shown in Table 2 are the downstream primers of the second round of PCR, which can be annealed and bound to the camelid antibody J gene family region, and the upstream primer used together: VHH-forward5'-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO. 58) .
  • the downstream primers used in the second round of PCR are based on Table 2 plus the linker sequence of the corresponding sequencing platform.
  • the sequence of the underlined part of the primers in Table 3 is the Illumina building linker sequence, the upstream primer.
  • a linker sequence such as 5'- CAGACGTGTGCTCTTCCGATCTAG ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO. 51), is also added to SEQ ID NO. 58, and the underlined portion of the sequence is the Illumina linker sequence.
  • R represents any one of A and G
  • Y represents any one of C and T
  • M represents any one of A and C
  • K represents any of G and T
  • S represents G and C.
  • W stands for any of A and T.
  • the primers in this table are substituted at any position, and any one of them is randomly selected in the middle, and the enzyme cleavage site and the protection base are added before and after, and the primers formed by any platform sequencing linker are added before and after, as long as they can amplify corresponding Target gene sequences are all within the scope of the claims of the present application.
  • the antibody can be screened by combining microbial display technology such as phage and Escherichia coli, and can also be screened by antibody analysis through high-throughput measurement technology combined with information analysis method.
  • microbial display technology such as phage and Escherichia coli
  • the method comprises the steps of: digesting the phage display vector and the immunogen bank VHH DNA fragment using a restriction endonuclease, and ligating to obtain a ligation product; and transforming the ligation product into a competent cell to construct a heavy chain antibody library.
  • the constructed phage display antibody library is added to the ELISA plate coupled with the corresponding antigen for specific enrichment, After washing off the non-specific phage, the phage which specifically binds to the corresponding antigen can be obtained, and the phage is produced and purified for the next round of screening. The same screening process is repeated for 3-4 rounds, and the enrichment is gradually obtained.
  • a primer capable of efficiently annealing to the leader region of the Camelin antibody V-initiating I family; and a primer sequence capable of efficiently annealing to the camelid antibody J gene region (Table 2) Set, use the primer set to any one or more primers).
  • a large-capacity heavy-chain antibody immunological library can be constructed, and on this basis, a highly specific and high-affinity heavy chain can be selected. antibody.
  • Three-step PCR can be directly obtained to obtain the upper library, without the need for traditional end repair, adding A and adding complicated steps of the machine joint.
  • the reference sequences on which the primers are designed are Arabian camel IGH V-REGION, Alpaca IGH V-REGION and Alpaca IGH J-REGION, etc. are from the general database of international immunology ( The international ImMunoGeneTics information Http://www.imgt.org); Primer design software is primer5.0 and Oligo7.
  • Type immune repertoire of cloning the antibody is the sequence diversity of up to 108 or more large building often rely multiplex primer polymerase chain reaction repertoire, in order to cover the tremendous diversity.
  • the structure of the antibody genes on the genome is shown in Figure 1. They exhibit high conservation in the leader and framework regions and high denaturation in the complementary binding regions.
  • Primer design process Since IMGT has not published the leader sequence of the germline V gene I family recognized by most camelids such as dromedary Bactrian camel, only the above partial sequence of alpaca (alpaca) can be referred to, which gives primers. The design creates difficulties. When designing primers, the nucleic acid sequence can only be deduced based on sequence similarity between species, degeneracy and preference of codons, and a few reported amino acid sequences of the leader regions. The primers Nos. 1, 4, 11, and 12 in Table 1 are based on the literature (Ikbel Achour, et al. Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus.
  • the primer design principle in the present invention is a. It can cover the diversity of the immune group library as much as possible; b. use as few primers as possible to reduce the competition between the primers; c. does not form a serious self-primer 2 Polymers, hairpin structures, and non-specific amplification.
  • RNA 2ug is the initial amount of reverse transcription, and reverse transcription generates cDNA; the following are the reverse transcription system and reaction conditions, and the following components are arranged, the above sample is 65 ° C, 7 min, and ice bath is 5 min.
  • Reverse transcription was carried out under the following conditions: 25 ° C, 10 min; 42 ° C, 2 h; 72 ° C, 15 min.
  • the cDNA obtained in the above step is used as a template, the primers in the leader region (such as CALL001, CALL5 and the primer No. 11 in Table 1) are used as the upstream primers, and the CALL002 is the downstream primer for the first round of ordinary PCR;
  • the gel recovered a 700 bp-800 bp region and a 900-1100 bp fragment as the next round of amplification template.
  • the PCR reaction conditions and system are as follows.
  • the DNA recovered in the above step is a template, and a band of a 700 bp-800 bp region heavy chain antibody is used as a template for amplifying a Nanobody (heavy chain antibody variable region VHH); a fragment of 900-1100 bp is recovered as an amplification conventional antibody.
  • the template of the VH region variable region (VHH-forward: 5'-CAGACGTGTGCTCTTCCGATCTAGATG-GCTSAKGTGCAGCTGGTGGAGTCTGG-3', SEQ ID NO. 51) is the upstream primer, and the J gene region primer is as shown in Table 3. ID NO.47, 48, and 50 are downstream primers.
  • the second round of ordinary PCR the PCR reaction system and conditions are as follows,
  • the PCR product in the above step is a template, and the Index_X primer containing the Hiseq library recognition marker (5'-CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO. 59) XXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO.
  • PCR reaction system and conditions are as follows:
  • the basic analysis idea is to download the immunological library sequence of the machine and the international The general immunology database database IMGT (http://www.imgt.org/vquest/refseqh.html) analyzes the germline genes of the camelid family, and statistically obtains the abundance information of the CDR3 sequence of the immune library.
  • the length distribution information, the V gene use information, the J gene use information, the VJ gene combination use information, the immune group library diversity information, and the like, and the present invention relates to the V gene use situation and the J gene use situation of the antibody immunological pool data.
  • VHH heavy chain antibody variable region
  • the antibody germline V gene in Table 4 refers to the internationally accepted immunological library obtained by analysis and statistics.
  • IMGT the species of the V gene subtype of the Camelidae V gene family
  • the number of Reads refers to the number of sequences of the detected V-germline genes that were statistically obtained after the analysis of the data of the immune group library.
  • Reads ratio (%) the proportion of sequences that match the V germline gene to all of the lower sequences. From the table, we can see that the IGHV1, IGHV2 and IGHV3 three major germline V gene use sequences are included, the total number of reads is 350,000, and the number of identified V gene subtypes is the majority of the alpaca.
  • the antibody germline J gene in the table refers to all germline gene types of the Camelidae J gene family in the IMGM database.
  • the number of Reads refers to the statistical data of the immunogen library samples. The number of sequences detected to match the corresponding V germline gene; Reads ratio (%): the ratio of the sequence matched to the V germline gene to all of the lower sequences.
  • Nanobody sequence libraries have been reported in the literature. In general, the number of clones that have been reported for nanobody libraries has typically ranged from tens or hundreds of clones with low throughput.
  • the B cells that construct the immune group are mainly derived from the active immunization of the target antigen by the body, or the memory B cells after infection in the pathogen.
  • the larger the capacity of the immune group the better the screening of high-affinity antibody sequences.
  • By constructing the relevant antibody library screening of specific antibodies can be performed, and depending on the corresponding screening equipment, high-throughput screening of antibodies can also be achieved.
  • the advantages of comprehensive VHH antibody and immune group library screening relying on the camelid primer of the present invention and the three-step method of database construction, can quickly and directly obtain a sequence database of large-capacity camelid antibodies, the database has The germline V gene subtype is comprehensive, well-structured, and covers the advantages of atypical Nano-antibodies. It provides sufficient antibody sequence library for screening high-affinity antibodies and lays a foundation for the screening of specific VHH antibodies.

Abstract

Provided are a primer combination constructed by variable region immune repertoire of camelid antibody and use thereof. The primer combination comprises a primer that can be effectively annealed and combined with a leader region of camelid antibody V gene I family, a primer that can be effectively annealed and combined with camelid antibody J gene region, and a primer combination that contain these primers. The primer combination is amplified by a third-round nested polymerase chain reaction to directly construct a large-capacity immune repertoire of camelid antibody.

Description

骆驼科抗体可变区免疫组库构建的引物组合及应用Primer combination and application of camelid antibody variable region immunization library 技术领域Technical field
本发明属于生物技术领域，具体地说，本发明涉及骆驼科抗体可变区免疫组库构建的引物组合及应用。The present invention belongs to the field of biotechnology, and in particular, the present invention relates to a primer combination and application constructed by a camelid antibody variable region immunological library.
背景技术Background technique
重链抗体是Hamers-Casterman等于1993年在骆驼体内发现的一种天然缺失轻链，仅由重链所组成的抗体，所以被称为重链抗体(HCAbs)。此类抗体的抗原结合位点仅由重链的可变区VHH单结构域形成。VHH是迄今为止已发现的具有结合抗原功能的最小分子量抗体片段之一，分子量为13-15KD，仅为常规抗体的1/10，因此又被称为纳米抗体(nanobody)。与传统抗体相比，VHH抗体分子量较小，易表达；在识别抗原表位时表现为更高的特异性和亲和力，并可识别独特构象的抗原表位；同时与传统抗体相比较具有高溶解性和稳定性等特点，因而对重要靶分子特异性的VHH抗体的研发成为近年来抗体新药制备的一种新策略。本发明所述骆驼科动物，包括但不限于双峰驼(Camelus bactrianus,Bactrian camel)，单峰驼(Camelus dromedarius)，大羊驼(llama,guanaco,alpaca)，小羊驼(vicuna)，驼羊(Alpaca Suri,lamagama)，骆马(vigogne)，美洲驼和澳洲驼等。The heavy chain antibody is a heavy-chain antibody (HCAbs) that Hamers-Casterman is equivalent to a naturally-deficient light chain found in camelids in 1993, consisting only of heavy chains. The antigen binding site of such antibodies is only formed by the variable region VHH single domain of the heavy chain. VHH is one of the smallest molecular weight antibody fragments that have been found to have antigen-binding function to date, and has a molecular weight of 13-15 KD, which is only 1/10 of that of a conventional antibody, and is therefore also referred to as a nanobody. Compared with traditional antibodies, VHH antibodies have a small molecular weight and are easy to express; they exhibit higher specificity and affinity when recognizing antigenic epitopes, and can recognize antigenic epitopes with unique conformations; and have high solubility compared with traditional antibodies. The characteristics of sex and stability, and the development of VHH antibodies specific for important target molecules have become a new strategy for the preparation of new antibody products in recent years. The camelids of the present invention include, but are not limited to, Camelus bactrianus (Bactrian camel), dromedary (Camelus dromedarius), llama (llama, guanaco, alpaca), llama (vicuna), camel Sheep (Alpaca Suri, lamagama), llama (vigogne), llamas and camel.
正常机体B细胞占外周血淋巴细胞总数的10-40％，多样性B细胞主要作用是产生抗体激发体液免疫应答。B细胞抗原受体(B cell receptor，BCR)是B细胞表面识别抗原的一种免疫球蛋白，具有抗原结合特异性。BCR是由两条重链和两条轻链组成的四聚体蛋白，其中，重链可以分为可变区(V区)、恒定区(C区)、跨膜区以及胞质区，而轻链则只有V区和C区。V区由重链可变区(VH)和轻链可变区(VL)两个结构域组成，它们都由三个互补决定区CDR1，CDR2，CDR3构成，CDR的排列顺序和氨基酸组成都呈现出高度的多样性，在同一个个体内，B细胞克隆种类数可达109-1010以上，构成容量巨大的BCR库，从而使个体拥有识别各种抗原、产生各种特异性抗体的巨大潜能。利用PCR技术构建B细胞免疫组库，测序后得到外周血中B细胞相应的巨大的BCR序列库，依照一定的生物信息序列筛选标准，可实现特异性抗体序列的筛选。免疫组库的库容和组成会直接影响后续筛选过程。 因此本领域技术人员致力于构建大容量的免疫组库。Normal body B cells account for 10-40% of the total number of peripheral blood lymphocytes, and the main role of the diversity B cells is to generate antibodies to stimulate humoral immune responses. B cell receptor (BCR) is an immunoglobulin that recognizes antigen on the surface of B cells and has antigen binding specificity. BCR is a tetrameric protein composed of two heavy chains and two light chains, wherein the heavy chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region, and The light chain has only the V zone and the C zone. The V region consists of two domains, the heavy chain variable region (VH) and the light chain variable region (VL), which are composed of three complementarity determining regions, CDR1, CDR2, and CDR3, and the CDR arrangement order and amino acid composition are presented. With a high degree of diversity, the number of B cell clones in the same individual can reach 10 9 -10 10 or more, which constitutes a large capacity BCR library, so that individuals have a huge capacity to recognize various antigens and produce various specific antibodies. Potential. The B cell immune group library was constructed by PCR technology, and the huge BCR sequence library corresponding to B cells in peripheral blood was obtained after sequencing. According to the screening criteria of certain biological information sequences, the screening of specific antibody sequences can be achieved. The storage capacity and composition of the immune group library will directly affect the subsequent screening process. Therefore, those skilled in the art are working to construct a large-capacity immune library.
发明内容Summary of the invention
本发明的目的在于提供骆驼科抗体可变区免疫组库构建的引物组合及应用。The object of the present invention is to provide a primer combination and application of a camelid antibody variable region immunological library.
本发明的第一方面，提供了一种引物组合，所述引物组合中包括可以特异性退火结合到骆驼科抗体V基因I家族(Clan I)的前导区的第一上游引物。In a first aspect of the invention, there is provided a primer combination comprising a first upstream primer which specifically anneals to a leader region of the camelid antibody V gene I family (Clan I).
在另一优选例中，所述第一上游引物包含选自下组的一种或多种引物序列：SEQ ID NO.11、2、3、4、5、6、7、8、9、10、1、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、和44；优选地，所述第一上游引物序列如SEQ ID NO.11所示。In another preferred embodiment, the first upstream primer comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 11, 2, 3, 4, 5, 6, 7, 8, 9, 10 1,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 36, 37, 38, 39, 40, 41, 42, 43, and 44; preferably, the first upstream primer sequence is set forth in SEQ ID NO.
在另一优选例中，所述引物组合还包括下游引物；优选地所述下游引物序列如SEQ ID NO.45所示。In another preferred embodiment, the primer combination further comprises a downstream primer; preferably the downstream primer sequence is set forth in SEQ ID NO.
在另一优选例中，所述引物组合还包括第二上游引物，所述第二上游引物特异性退火结合到骆驼科抗体V基因III家族(Clan III)的前导区，优选地所述第二上游引物序列如SEQ ID NO.46所示。In another preferred embodiment, the primer combination further comprises a second upstream primer, the second upstream primer specifically annealing to the leader region of the camelid antibody V gene III family (Clan III), preferably the second The upstream primer sequence is set forth in SEQ ID NO.
在另一优选例中，所述引物组合还包括第三上游引物，所述第三上游引物特异性退火结合到骆驼科抗体V基因II家族(Clan II)的前导区，优选地所述第二上游引物序列如SEQ ID NO.53所示。In another preferred embodiment, the primer combination further comprises a third upstream primer, the third upstream primer specifically annealing to the leader region of the camelid antibody V gene II family (Clan II), preferably the second The upstream primer sequence is set forth in SEQ ID NO.
本发明的第二方面，提供了一种引物组合，所述引物组合中的各引物为特异性退火结合到骆驼科抗体J基因区域的下游引物，并且所述引物组合包含选自下组的一种或多种引物序列：SEQ ID NO.54、55、56、和57。In a second aspect of the invention, there is provided a primer combination, each primer in the primer combination being a downstream primer that specifically anneals to a region of a camelid antibody J gene, and the primer combination comprises a one selected from the group consisting of One or more primer sequences: SEQ ID NO. 54, 55, 56, and 57.
在另一优选例中，所述引物组合还包括上游引物；优选地所述上游引物序列如SEQ ID NO.58所示。In another preferred embodiment, the primer combination further comprises an upstream primer; preferably the upstream primer sequence is set forth in SEQ ID NO.
在一优先例中，所述引物组合包含选自下组的一种或多种引物序列：SEQ ID NO.47、48、49、50。In a preferred embodiment, the primer combination comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 47, 48, 49, 50.
在另一优选例中，所述引物组合还包括本发明第一方面所述的引物组合。In another preferred embodiment, the primer combination further comprises the primer combination of the first aspect of the invention.
本发明的第三方面，提供了一种试剂盒，所述试剂盒包含本发明第一方面 所述的引物组合，和/或本发明第二方面所述的引物组合；以及，任选地PCR扩增试剂。In a third aspect of the invention, a kit is provided, the kit comprising the first aspect of the invention The primer combination, and/or the primer combination of the second aspect of the invention; and, optionally, a PCR amplification reagent.
在另一优选例中，所述试剂盒试剂盒还包括上游引物引物CALL001(5′-GTCCTGGCTGCTCTTCTACAAGG-3′，SEQ ID NO.46)和/或CALL5(5′-TGGTGGCAGGTCCCCAAGGT-3′，SEQ ID NO.53)。In another preferred embodiment, the kit further comprises an upstream primer primer CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46) and/or CALL5 (5'-TGGTGGCAGGTCCCCAAGGT-3', SEQ ID NO .53).
本发明的第四方面，提供了一种构建骆驼科抗体可变区免疫组库的方法，所述方法包括步骤：In a fourth aspect of the invention, a method of constructing a camelid antibody variable region immune library is provided, the method comprising the steps of:
(1)第一轮扩增(1) First round of amplification
提供骆驼科动物免疫细胞的核酸作为模板，使用本发明第一方面所述的引物组合，进行第一轮PCR扩增，获得第一轮扩增产物；Providing a nucleic acid of a camelid immune cell as a template, using the primer combination of the first aspect of the invention, performing a first round of PCR amplification to obtain a first round of amplification product;
(2)第二轮扩增(2) Second round of amplification
以第一轮扩增产物为模板，使用本发明第二方面所述的引物组合，进行第二轮巢式PCR扩增，获得第二轮扩增产物。Using the first round of amplification product as a template, using the primer combination described in the second aspect of the present invention, a second round of nested PCR amplification was performed to obtain a second round of amplification product.
在另一优选例中，步骤(1)中，所述的核酸为细胞来源的RNA反转录成的cDNA。In another preferred embodiment, in the step (1), the nucleic acid is a cDNA reverse-transcribed into a cell-derived RNA.
在另一优选例中，所述方法还包括任选地步骤：In another preferred embodiment, the method further comprises the optional step of:
(3)以所述第二轮扩增产物为模板进行illumina文库构建。(3) The illumina library was constructed using the second round of amplification products as a template.
在另一优选例中，所述步骤(1)中，扩增完成后，回收600-1100bp的基因片段作为所述第一轮扩增产物；优选地回收700-800bp的基因片段作为下一轮扩增重链抗体可变区的模板；优选地回收900-1100bp的基因片段作为下一轮扩增传统抗体重链可变区的模板。In another preferred embodiment, in the step (1), after the amplification is completed, a gene fragment of 600-1100 bp is recovered as the first round of amplification product; preferably, a gene fragment of 700-800 bp is recovered as the next round. A template for amplifying the variable region of a heavy chain antibody; preferably, a gene fragment of 900-1100 bp is recovered as a template for the next round of amplification of the heavy chain variable region of a conventional antibody.
在另一优选例中，所述步骤(2)中，扩增完成后，回收300-600bp的基因片段作为所述第二轮扩增产物；优选地回收400-500bp的基因片段作为所述第二轮扩增产物。In another preferred embodiment, in the step (2), after the amplification is completed, a gene fragment of 300-600 bp is recovered as the second round of amplification product; preferably, a gene fragment of 400-500 bp is recovered as the first Two rounds of amplification products.
在另一优选例中，所述步骤(3)中，分别以第二轮扩增产物为模板，P1引物和含有index标记的引物为上下游引物进行PCR扩增，获得可直接用于构建测序上机的文库。In another preferred embodiment, in the step (3), the second round of the amplified product is used as a template, and the P1 primer and the primer containing the index mark are PCR amplification of the upstream and downstream primers, and the obtained primer can be directly used for constructing the sequencing. Library on the machine.
本发明的第五方面，提供了一种筛选重链抗体的方法，所述方法包括步骤：使用本发明第四方面所述的方法构建重链抗体可变区免疫组库；以及，在所述的重链抗体可变区免疫组库中筛选所述重链抗体。 According to a fifth aspect of the invention, a method for screening a heavy chain antibody, the method comprising the steps of: constructing a heavy chain antibody variable region immunological library using the method of the fourth aspect of the invention; The heavy chain antibody is screened in a heavy chain antibody variable region immunological pool.
在另一优选例中，筛选重链抗体的方法为噬菌体、大肠杆菌等微生物展示技术和/或生物信息学分析方法。In another preferred embodiment, the method for screening heavy chain antibodies is microbial display technology and/or bioinformatics analysis methods such as phage, Escherichia coli, and the like.
在另一优选例中，所述的生物信息学分析方法为N＝突变率*0.4+CDR3丰度比例*0.3+CDR2丰度比例*0.2+CDR1丰度比例*0.1)*100，当N≥80时，确定为目标抗体序列。In another preferred embodiment, the bioinformatics analysis method is N=mutation rate*0.4+CDR3 abundance ratio*0.3+CDR2 abundance ratio*0.2+CDR1 abundance ratio*0.1)*100, when N≥ At 80 o'clock, it was determined to be the target antibody sequence.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.
附图说明DRAWINGS
图1：三轮聚合酶链式反应构建骆驼科抗体可变区免疫组库文库的示意图。前导区是指抗体基因转录成mRNA后在位于上游的一段区域，V、D和J基因指的是抗体可变区的三种结构组成基因，CH1区，铰链区，和CH2区等指的是抗体恒定区的结构组成基因。Figure 1: Schematic diagram of the construction of a camelid antibody variable region immunological library library by three rounds of polymerase chain reaction. The leader region refers to a region upstream of the antibody gene transcribed into mRNA, and the V, D, and J genes refer to three structural components of the antibody variable region, and the CH1 region, the hinge region, and the CH2 region are The structure of the constant region of the antibody constitutes a gene.
具体实施方式detailed description
本发明人通过广泛而深入的研究，获得了能够有效退火结合至骆驼科抗体V基因I家族的前导区的上游引物；以及能够有效退火结合至骆驼科抗体J基因区域的下游引物。实验结果表明，配合其他引物使用本发明提供的引物组合进行三轮扩增，能够构建大容量的骆驼科抗体免疫组库，在此基础上可以筛选出高特异性、和高亲和性的重链抗体。在此基础上，完成了本发明。The present inventors have obtained an upstream primer capable of efficiently annealing to the leader region of the camelid family V gene I family by extensive and intensive research; and a downstream primer capable of efficiently annealing to the camelid antibody J gene region. The experimental results show that with the other primers, the primer combination provided by the present invention can be used for three rounds of amplification, and a large-capacity antibody of the camelid antibody can be constructed, and on the basis of this, the high specificity and high affinity can be screened. Chain antibody. On the basis of this, the present invention has been completed.
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。Before the present invention is described, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiments, and is not intended to
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。 All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. As used herein, when used in reference to a particular recited value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein.
免疫组库构建Immunization library construction
构建重链抗体免疫组库的一般方法为，取外周血，用细胞分离液分离外周血单个核细胞(PBMC)，提取细胞总RNA，经逆转录得到cDNA。以cDNA为模板，运用巢式PCR进行可变区序列的扩增，第一轮以CALL001(5′-GTCCTGGCTGCTCTTCTACAAGG-3′，SEQ ID NO.46)和CALL002(5′-GGTACGTGCTGTTGAACTGTTCC-3′，SEQ ID NO.45)作为引物扩增可变区至CH2区，CALL001位于驼科BCR V基因III家族的前导序列区域，CALL002位于CH2结构域高度保守区，CALL001和CALL002可扩增获得大多数可变区序列。胶回收其中700-800bp片段做模板，以结合V基因III家族区域的上游引物VHH-forward(5’-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3’，SEQ ID NO.51)和结合J基因区域的下游引物VHH-reverse(5’-GGAGACGGTGACCTGGGT-3’，SEQ ID NO.52)来进行第二轮PCR富集VHH基因序列。其中CALL指的是骆驼科动物(camelid)的缩写，VHH指的是骆驼科重链抗体可变区。The general method for constructing a heavy chain antibody immunological library is to take peripheral blood, separate peripheral blood mononuclear cells (PBMC) with cell separation solution, extract total cellular RNA, and obtain cDNA by reverse transcription. Using cDNA as a template, nested PCR was used to amplify the variable region sequences. The first round was CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46) and CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID NO.45) as a primer to amplify the variable region to the CH2 region, CALL001 is located in the leader region of the BCR V gene III family of Camelidae, CALL002 is located in the highly conserved region of the CH2 domain, and CALL001 and CALL002 can be amplified to obtain most of the variable region. Sequence of regions. The 700-800 bp fragment was used as a template to bind the upstream primer VHH-forward (5'-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3', SEQ ID NO. 51) of the V gene III family region and the downstream primer VHH-reverse (binding to the J gene region). 5'-GGAGACGGTGACCTGGGT-3', SEQ ID NO. 52) was used to perform a second round of PCR enrichment of the VHH gene sequence. Wherein CALL refers to the abbreviation of camelid, and VHH refers to the variable region of camelid heavy chain antibody.
其中，第一轮PCR中，由于CALL001引物的局限性，仅能覆盖骆驼科V基因III家族部分可变区序列，在第一轮PCR之后，V基因I，II家族序列已经丢失。第二轮PCR，仅用一对引物进行VHH基于序列的富集，所构建的免疫组库不能包含所有重链可变区。并且现有文献和专利大多都只关注V基因III家族，对V基因I，II家族序列都不甚关心，但是很多高亲和力抗体也可以源自于骆驼科V基因I家族和II家族(Clan I和II家族)。Among them, in the first round of PCR, due to the limitation of the CALL001 primer, only the partial variable region sequence of the camelid V gene III family can be covered. After the first round of PCR, the V gene I, II family sequence has been lost. In the second round of PCR, VHH sequence-based enrichment was performed using only one pair of primers, and the constructed immune pool could not contain all heavy chain variable regions. Most of the existing literatures and patents only focus on the V gene III family, and are not very concerned about the V gene I, II family sequences, but many high affinity antibodies can also be derived from the camelid V gene I family and the II family (Clan I And the II family).
在本发明一个优选的实施方式中，在第一轮PCR中，使用了包含骆驼科抗体V基因I家族(Clan I)前导区的任意一条或者多条引物的组合，设计的引物见表1；在第二轮PCR中，使用了包含骆驼科抗体J基因区域引物的组合，设计的引物见表2。第一轮PCR对重链抗体基因和传统抗体基因序列进行富集，以及切胶区分，再以第一轮PCR产物为模板，用V区引物和J区引物进行二轮扩增，形成了抗体免疫组库。In a preferred embodiment of the present invention, in the first round of PCR, a combination of any one or a plurality of primers comprising a camelid antibody V gene I family (Clan I) leader region is used, and the designed primers are shown in Table 1; In the second round of PCR, a combination comprising the camelid antibody J gene region primers was used, and the designed primers are shown in Table 2. In the first round of PCR, the heavy chain antibody gene and the traditional antibody gene sequence were enriched, and the gelatin was distinguished. Then, the first round of PCR product was used as a template, and the V-region primer and the J-region primer were used for two round amplification to form an antibody. Immunization group library.
表1：骆驼科抗体V基因I家族(Clan I)的前导区引物序列集合Table 1: Primer Primer Sequence Collection of Camelid Antibody V Gene I Family (Clan I)
Figure PCTCN2016109286-appb-000001
Figure PCTCN2016109286-appb-000001
Figure PCTCN2016109286-appb-000002
Figure PCTCN2016109286-appb-000002
Figure PCTCN2016109286-appb-000003
Figure PCTCN2016109286-appb-000003
说明：表1所示引物为第一轮PCR的上游引物，能退火结合到驼科抗体V基因I家族BCR的前导序列区域，构建驼科免疫组库时，表1中任意一条或者多条引物与下游引物CALL002(5′-GGTACGTGCTGTTGAACTGTTCC-3′，SEQ ID NO.45)扩增驼科抗体V基因I家族基因序列，联合CALL001(5′-GTCCTGGCTGCTCTTCTACAAGG-3′，SEQ ID NO.46)和CALL5(5′-TGGTGGCAGGTCCCCAAGGT-3′，SEQ ID NO.53)使用，用以扩增驼科抗体V基因I、II和III家族基因序列。CALL001、CALL5与CALL002组合可扩增V基因II和III家族基因序列。表1所述引物任何位点碱基替换，中间随机截取任意一段，前后加上酶切位点和保护碱基，以及前后加上任何平台测序接头形成的引物，只要其能够扩增出相应的靶基因序列，都属于本申请权利要求的保护范围之内。Note: The primers shown in Table 1 are the upstream primers of the first round of PCR, which can be annealed to the leader sequence region of the Camel antibody V gene I family BCR. When constructing the camelid immune pool, any one or more primers in Table 1 Amplification of the camelid antibody V gene I family gene sequence with the downstream primer CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID NO. 45), in combination with CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO. 46) and CALL5 (5'-TGGTGGCAGGTCCCCAAGGT-3', SEQ ID NO. 53) was used to amplify the camelid antibody V gene I, II and III family gene sequences. The combination of CALL001, CALL5 and CALL002 amplifies the V gene II and III family gene sequences. The base substitution of any primer in Table 1 is carried out, and any one of the primers is randomly selected in the middle, and the restriction enzyme site and the protection base are added before and after, and the primer formed by any platform sequencing linker is added before and after, as long as it can amplify the corresponding Target gene sequences are all within the scope of the claims of the present application.
表2：骆驼科抗体J基因区域引物序列集合Table 2: Camel antibody J gene region primer sequence collection
序号Serial number 引物序列(5’-3’)Primer sequence (5'-3') SEQ ID NO.SEQ ID NO.
11 CTGMRGAGACGGTGACCWGCTGMRGAGACGGTGACCWG 5454
22 GGAGACGGTGACCTGGGTGGAGACGGTGACCTGGGT 5555
33 CTGMRGAGACGGTGACCWGGGTSCCYTKGCCCCACTGMRGAGACGGTGACCWGGGTSCCYTKGCCCCA 5656
44 KGAGACAGTGACCAGGGTKGAGACAGTGACCAGGGT 5757
表3：适用于Illumina平台的骆驼科抗体J基因区域引物序列集合Table 3: Camel antibody J gene region primer sequence set for Illumina platform
Figure PCTCN2016109286-appb-000004
Figure PCTCN2016109286-appb-000004
说明：表2所示引物为第二轮PCR的下游引物，能退火结合到驼科抗体J基因家族区域，配合使用的上游引物：VHH-forward5’-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3’(SEQ ID NO.58)。对于不同的测序平台而言，第二轮PCR所用的下游引物在表2的基础上加上相应测序平台的接头序列，如表3引物中下划线部分的序列即为Illumina建库接头序列，上游引物同样在SEQ ID NO.58的基础上加入接头序列，如5’-CAGACGTGTGCTCTTCCGATCTAGATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3’(SEQ ID NO.51)，下划线部分的序列即为Illumina建库接头序列。Note: The primers shown in Table 2 are the downstream primers of the second round of PCR, which can be annealed and bound to the camelid antibody J gene family region, and the upstream primer used together: VHH-forward5'-ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO. 58) . For different sequencing platforms, the downstream primers used in the second round of PCR are based on Table 2 plus the linker sequence of the corresponding sequencing platform. The sequence of the underlined part of the primers in Table 3 is the Illumina building linker sequence, the upstream primer. A linker sequence, such as 5'- CAGACGTGTGCTCTTCCGATCTAG ATGGCTSAKGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO. 51), is also added to SEQ ID NO. 58, and the underlined portion of the sequence is the Illumina linker sequence.
表2和表3中所示R代表A、G任意一种；Y代表C、T任意一种；M代表A、C任意一种；K代表G、T任意一种；S代表G、C任意一种；W代表A、T任意一种。此表所述引物任何位点碱基替换，中间随机截取任意一段，前后加上酶切位点和保护碱基，以及前后加上任何平台测序接头形成的引物，只要其能够扩增出相应的靶基因序列，都属于本申请权利要求的保护范围之内。In Table 2 and Table 3, R represents any one of A and G; Y represents any one of C and T; M represents any one of A and C; K represents any of G and T; and S represents G and C. One; W stands for any of A and T. The primers in this table are substituted at any position, and any one of them is randomly selected in the middle, and the enzyme cleavage site and the protection base are added before and after, and the primers formed by any platform sequencing linker are added before and after, as long as they can amplify corresponding Target gene sequences are all within the scope of the claims of the present application.
经过两轮扩增的PCR产物，可结合噬菌体、大肠杆菌等微生物展示技术筛选抗体，也可通过高通量测技术结合信息分析方法经行抗体分析筛选。After two rounds of amplification of the PCR product, the antibody can be screened by combining microbial display technology such as phage and Escherichia coli, and can also be screened by antibody analysis through high-throughput measurement technology combined with information analysis method.
重链抗体筛选Heavy chain antibody screening
常规是采用噬菌体展示***对获得的免疫组库进行抗体筛选的。通常包括步骤：使用限制性内切酶酶切噬菌体展示载体及免疫组库VHH DNA片段，并连接，获得连接产物；将连接产物转化至感受态细胞中，构建重链抗体文库。将构建的噬菌体展示抗体文库加入偶联了相应抗原的酶标板，进行特异性富集， 洗去非特异性噬菌体后，即可获得特异性结合相应抗原的噬菌体，产生并纯化噬菌体用于下一轮的筛选，相同筛选过程重复3-4轮，逐步得到富集。It is conventional to perform antibody screening on the obtained immunogen bank using a phage display system. Generally, the method comprises the steps of: digesting the phage display vector and the immunogen bank VHH DNA fragment using a restriction endonuclease, and ligating to obtain a ligation product; and transforming the ligation product into a competent cell to construct a heavy chain antibody library. The constructed phage display antibody library is added to the ELISA plate coupled with the corresponding antigen for specific enrichment, After washing off the non-specific phage, the phage which specifically binds to the corresponding antigen can be obtained, and the phage is produced and purified for the next round of screening. The same screening process is repeated for 3-4 rounds, and the enrichment is gradually obtained.
经3-4轮筛选后含有噬菌体的细胞培养皿中，挑选1000个单个菌落接种培养。培养完成后粗提抗体，并将抗体转移到经相应抗原包被的ELISA板中，进行阳性克隆筛选。培养阳性克隆菌，提取质粒并进行测序，即可筛选获得特异性的重链抗体。After 3-4 rounds of screening in a cell culture dish containing phage, 1000 individual colonies were selected for inoculation culture. After the completion of the culture, the antibody was crudely extracted, and the antibody was transferred to an ELISA plate coated with the corresponding antigen to perform positive clone screening. Positive clones are cultured, plasmids are extracted and sequenced, and specific heavy chain antibodies can be screened.
本发明的主要优点在于：The main advantages of the invention are:
(1)提供了能够有效退火结合到驼科抗体V因I家族的前导区的引物(SEQ ID NO.11)；以及能够有效退火结合到驼科抗体J基因区域(表2所示的引物序列集合，使用该引物集合任意一条或多条引物)。(1) A primer (SEQ ID NO. 11) capable of efficiently annealing to the leader region of the Camelin antibody V-initiating I family; and a primer sequence capable of efficiently annealing to the camelid antibody J gene region (Table 2) Set, use the primer set to any one or more primers).
(2)分别使用本发明提供的几种引物组合进行三轮扩增，能够构建大容量的重链抗体免疫组库，在此基础上可以筛选出高特异性、和高亲和性的重链抗体。(2) Using three primer combinations provided by the present invention to perform three rounds of amplification, a large-capacity heavy-chain antibody immunological library can be constructed, and on this basis, a highly specific and high-affinity heavy chain can be selected. antibody.
(3)分别使用本发明提供的引物组合进行三轮扩增，能够扩增出包含非典型纳米抗体等的更全面的抗体免疫组库。已经报道存在一类非典型的重链抗体可变区(Non-classical VHH)，即含有传统抗体四位特征氨基酸的纳米抗体，这四位已报道的特征氨基酸分别是指第42，49，50和52位氨基酸：缬氨酸，甘氨酸，亮氨酸和色氨酸。(3) Three rounds of amplification using the primer combinations provided by the present invention, respectively, can amplify a more comprehensive antibody immunological pool containing atypical Nanobodies and the like. It has been reported that there is a class of atypical heavy chain antibody variable regions (Non-classical VHH), that is, Nanobodies containing four characteristic amino acids of conventional antibodies, the four reported characteristic amino acids refer to 42, 42, 50, respectively. And 52 amino acids: valine, glycine, leucine and tryptophan.
(4)可以实现三步PCR直接得到上机文库，不需要传统的末端修复，加A和加上机接头的复杂步骤。(4) Three-step PCR can be directly obtained to obtain the upper library, without the need for traditional end repair, adding A and adding complicated steps of the machine joint.
(5)对抗体免疫组库进行高通量测序之后，得到数据量庞大的抗体序列库，依照基于样本间的表达差异，序列突变率和丰度等因素而制定的筛选标准，可直接从序列库中筛出针对靶点抗原的有潜在高亲和力的抗体序列。(5) After high-throughput sequencing of the antibody immunological pool, a database of antibody sequences with a large amount of data is obtained, and the screening criteria based on the differences in expression between the samples, the mutation rate and the abundance of the sequences can be directly obtained from the sequence. Potentially high affinity antibody sequences directed against the target antigen are screened in the library.
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。 The present invention will be further described in detail below with reference to specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples, which do not specify the detailed conditions, are generally subjected to the conditions described in the "Molecular Cloning Laboratory Guide" (translation by Huang Peitang et al., Beijing: Science Press, 2002) in accordance with conventional conditions such as Sambrook.J. , or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are available from commercially available sources unless otherwise specified.
实施例1Example 1
1.引物设计方面：1. Primer design:
引物设计所依据的参考序列Arabian camel IGH V-REGION，Alpaca IGH V-REGION和Alpaca IGH J-REGION等是来自国际免疫学方面通用数据库(
Figure PCTCN2016109286-appb-000005
the international ImMunoGeneTics information
Figure PCTCN2016109286-appb-000006
http://www.imgt.org)；引物设计软件是primer5.0和Oligo7等。免疫组库中克隆的种类，也就是抗体序列多样性庞大可达108以上，组库的构建往往依赖多重引物的聚合酶链式反应，以期覆盖巨大的多样性。抗体基因在基因组上的结构如图1，它们在前导区和框架区展现出较高的保守性，在互补结合区展现出高变性。The reference sequences on which the primers are designed are Arabian camel IGH V-REGION, Alpaca IGH V-REGION and Alpaca IGH J-REGION, etc. are from the general database of international immunology (
Figure PCTCN2016109286-appb-000005
The international ImMunoGeneTics information
Figure PCTCN2016109286-appb-000006
Http://www.imgt.org); Primer design software is primer5.0 and Oligo7. Type immune repertoire of cloning, the antibody is the sequence diversity of up to 108 or more large building often rely multiplex primer polymerase chain reaction repertoire, in order to cover the tremendous diversity. The structure of the antibody genes on the genome is shown in Figure 1. They exhibit high conservation in the leader and framework regions and high denaturation in the complementary binding regions.
引物设计过程：因目前IMGT没有公布单峰驼双峰驼等大多数骆驼科动物公认的胚系V基因I家族的前导区序列，只有羊驼(alpaca)的上述部分序列可以参考，这给引物的设计造成困难。设计引物时，只能根据物种间的序列相似性，密码子的简并性和使用偏好性，以及少数已经报道的前导区氨基酸序列反向推导其核酸序列。如表1中第1,4,11,和12号引物等就是根据文献(Ikbel Achour,et al.Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus.doi:10.4049/jimmunol.181.3.2001.The Journal of Immunology.2008Aug 1；181(3):2001-9.)中氨基酸序列MDWSWGALFLVAVAAGVHS反推并考虑物种相似性、密码子简并性和引物设计原则而最终设计确定的。本发明中引物设计原则是a.尽可能地能覆盖免疫组库序列多样性；b.尽可能地使用较少的引物条数，减少引物之间的竞争；c.不形成严重的自身引物二聚体，发卡结构，和非特异扩增等。Primer design process: Since IMGT has not published the leader sequence of the germline V gene I family recognized by most camelids such as dromedary Bactrian camel, only the above partial sequence of alpaca (alpaca) can be referred to, which gives primers. The design creates difficulties. When designing primers, the nucleic acid sequence can only be deduced based on sequence similarity between species, degeneracy and preference of codons, and a few reported amino acid sequences of the leader regions. The primers Nos. 1, 4, 11, and 12 in Table 1 are based on the literature (Ikbel Achour, et al. Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus. doi: 10.4049/jimmunol.181.3.2001. The Journal The immunological sequence of MDNSWGALFLVAVAAGVHS in the Immunology. 2008 Aug 1;181(3):2001-9.) was reversed and considered based on species similarity, codon degeneracy and primer design principles. The primer design principle in the present invention is a. It can cover the diversity of the immune group library as much as possible; b. use as few primers as possible to reduce the competition between the primers; c. does not form a serious self-primer 2 Polymers, hairpin structures, and non-specific amplification.
2.抗体免疫组库构建2. Antibody immunization library construction
(1)抽取免疫骆驼科动物的外周血10mL，分离出外周血单个核细胞，提取其RNA；(1) 10 mL of peripheral blood of immune camelids was isolated, and peripheral blood mononuclear cells were isolated and RNA was extracted;
(2)以上步骤中RNA 2ug为反转录起始量，反转录生成cDNA；以下为反转录的体系及反应条件，配置以下成分,以上样品65℃，7min，冰浴5min。(2) In the above step, RNA 2ug is the initial amount of reverse transcription, and reverse transcription generates cDNA; the following are the reverse transcription system and reaction conditions, and the following components are arranged, the above sample is 65 ° C, 7 min, and ice bath is 5 min.
Figure PCTCN2016109286-appb-000007
Figure PCTCN2016109286-appb-000007
Figure PCTCN2016109286-appb-000008
Figure PCTCN2016109286-appb-000008
接着加入以下成分反转录，每孔加入4μL。按如下条件进行反转录：25℃，10min；42℃，2h；72℃，15min。Next, the following components were added for reverse transcription, and 4 μL was added to each well. Reverse transcription was carried out under the following conditions: 25 ° C, 10 min; 42 ° C, 2 h; 72 ° C, 15 min.
Figure PCTCN2016109286-appb-000009
Figure PCTCN2016109286-appb-000009
(3)以上步骤中得到的cDNA为模板，以前导区引物(如CALL001、CALL5和表1中的第11号引物)作为上游引物，以CALL002为下游引物，进行第一轮普通PCR；后切胶回收700bp-800bp区域和900-1100bp的片段作为下一轮扩增模板。PCR反应条件和体系如下，(3) The cDNA obtained in the above step is used as a template, the primers in the leader region (such as CALL001, CALL5 and the primer No. 11 in Table 1) are used as the upstream primers, and the CALL002 is the downstream primer for the first round of ordinary PCR; The gel recovered a 700 bp-800 bp region and a 900-1100 bp fragment as the next round of amplification template. The PCR reaction conditions and system are as follows.
Figure PCTCN2016109286-appb-000010
Figure PCTCN2016109286-appb-000010
Figure PCTCN2016109286-appb-000011
Figure PCTCN2016109286-appb-000011
(4)以上步骤中回收的DNA为模板，700bp-800bp区域重链抗体的条带作为扩增纳米抗体(重链抗体可变区VHH)的模板；回收900-1100bp的片段作为扩增传统抗体重链可变区VH的模板，以V基因区域引物(VHH-forward:5’-CAGACGTGTGCTCTTCCGATCTAGATG-GCTSAKGTGCAGCTGGTGGAGTCTGG-3’，SEQ ID NO.51)等为上游引物，以J基因区域引物如表3中SEQ ID NO.47、48、50为下游引物，进行 (4) The DNA recovered in the above step is a template, and a band of a 700 bp-800 bp region heavy chain antibody is used as a template for amplifying a Nanobody (heavy chain antibody variable region VHH); a fragment of 900-1100 bp is recovered as an amplification conventional antibody. The template of the VH region variable region (VHH-forward: 5'-CAGACGTGTGCTCTTCCGATCTAGATG-GCTSAKGTGCAGCTGGTGGAGTCTGG-3', SEQ ID NO. 51) is the upstream primer, and the J gene region primer is as shown in Table 3. ID NO.47, 48, and 50 are downstream primers.
第二轮普通PCR；PCR反应体系和条件如下，The second round of ordinary PCR; the PCR reaction system and conditions are as follows,
Figure PCTCN2016109286-appb-000012
Figure PCTCN2016109286-appb-000012
Figure PCTCN2016109286-appb-000013
Figure PCTCN2016109286-appb-000013
(5)以上步骤中PCR产物为模板，以含有Hiseq文库识别标记的Index_X引物(5’-CAAGCAGAAGACGGCATACGAGAT(SEQ ID NO.59)XXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO.60)-3’，其中XXXXXX为区别文库的index)为上游引物，含有Hiseq文库接头的P1引物(5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’，SEQ ID NO.61)为下游引物,进行Hiseq文库构建的第三轮PCR，加上上机接头，测序产生数据。其中PCR反应体系和条件如下：(5) The PCR product in the above step is a template, and the Index_X primer containing the Hiseq library recognition marker (5'-CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO. 59) XXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO. 60)-3', wherein XXXXXX is a differential library Index) is the upstream primer, the P1 primer containing the Hiseq library linker (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', SEQ ID NO.61) is the downstream primer, the third round of PCR for the construction of the Hiseq library, plus the upper connector, sequencing Generate data. The PCR reaction system and conditions are as follows:
Figure PCTCN2016109286-appb-000014
Figure PCTCN2016109286-appb-000014
Figure PCTCN2016109286-appb-000015
Figure PCTCN2016109286-appb-000015
Figure PCTCN2016109286-appb-000016
Figure PCTCN2016109286-appb-000016
3.抗体免疫组库序列分析3. Antibody immunization library sequence analysis
由上面所述引物构建出来的羊驼抗体免疫组库序列经过二代高通量测序以后，经过运行Imonitor软件的生物信息学分析，基本的分析思路是，将下机的免疫组库序列与国际通用的免疫组库数据库IMGT(http://www.imgt.org/vquest/refseqh.html)中驼科家族胚系基因进行分析比对，统计得出该免疫组库的CDR3序列区域丰度信息，长度分布信息，V基因使用信息J基因使用信息VJ基因组合使用信息，该免疫组库多样性信息等，与本发明相关即是抗体免疫组库数据的V基因使用情况和J基因使用情况。After the second-generation high-throughput sequencing of the alpaca antibody immunological library sequence constructed by the above primers, after the bioinformatics analysis of the Imonitor software, the basic analysis idea is to download the immunological library sequence of the machine and the international The general immunology database database IMGT (http://www.imgt.org/vquest/refseqh.html) analyzes the germline genes of the camelid family, and statistically obtains the abundance information of the CDR3 sequence of the immune library. The length distribution information, the V gene use information, the J gene use information, the VJ gene combination use information, the immune group library diversity information, and the like, and the present invention relates to the V gene use situation and the J gene use situation of the antibody immunological pool data.
4.V基因使用情况和J基因使用情况4.V gene usage and J gene usage
仅以重链抗体可变区(VHH)免疫组库的分析情况为例来说明，如表4所示，IGHV1即代表驼科V基因I家族的V基因，类似地IGHV2代表II家族，IGHV3代表III家族。可以看出，使用我们本发明中表1和表3中提到的引物后三种家族基因都能够有效扩增出来；如表4所示，可以扩增得出全部的J区域基因种类。Only the analysis of the heavy chain antibody variable region (VHH) immune group library is taken as an example. As shown in Table 4, IGHV1 represents the V gene of the Camelidae V gene I family, similarly IGHV2 represents the II family, and IGHV3 represents III family. It can be seen that the three family genes can be efficiently amplified using the primers mentioned in Tables 1 and 3 of the present invention; as shown in Table 4, all of the J region gene types can be amplified.
表4：羊驼抗体VHH免疫组库V基因使用情况Table 4: V gene use of alpaca antibody VHH immune group
Figure PCTCN2016109286-appb-000017
Figure PCTCN2016109286-appb-000017
Figure PCTCN2016109286-appb-000018
Figure PCTCN2016109286-appb-000018
Figure PCTCN2016109286-appb-000019
Figure PCTCN2016109286-appb-000019
说明：表4中的抗体胚系V基因指的是分析统计得到的国际通用免疫组库 数据库IMGT中驼科V基因家族胚系V基因亚型种类；Reads条数指的是免疫组库样本下机数据分析后统计得出的被测到的匹配到相应V胚系基因的序列条数；Reads比例(％)：匹配到该条V胚系基因下的序列占所有下机序列的比例。从表中我们可以看到，IGHV1，IGHV2和IGHV3三大胚系V基因使用序列均有包含，总的reads条数达35万条，鉴定到的V基因亚型的使用种类数占羊驼所有已经公开的V基因亚型种类数的80％(67/84，IMGT中羊驼的胚系V基因亚型有84条)。自然地，抗体序列库(免疫组库)越大，含有高亲和力高特异性的抗体序列可能性就越大。较少有文献报道如此高通量地纳米抗体序列库的二代测序。一般而言，已报道的纳米抗体文库经过测序的克隆数量通常在几十条或者几百条不等，通量较低；且IGHV1，IGHV2和IGHV3三大胚系V基因亚型寥寥几种，覆盖较少。Description: The antibody germline V gene in Table 4 refers to the internationally accepted immunological library obtained by analysis and statistics. In the database IMGT, the species of the V gene subtype of the Camelidae V gene family; the number of Reads refers to the number of sequences of the detected V-germline genes that were statistically obtained after the analysis of the data of the immune group library. ; Reads ratio (%): the proportion of sequences that match the V germline gene to all of the lower sequences. From the table, we can see that the IGHV1, IGHV2 and IGHV3 three major germline V gene use sequences are included, the total number of reads is 350,000, and the number of identified V gene subtypes is the majority of the alpaca. 80% of the number of V gene subtypes have been disclosed (67/84, there are 84 germline V gene subtypes of alpaca in IMGT). Naturally, the larger the antibody sequence library (immunization pool), the greater the possibility of antibody sequences containing high affinity and high specificity. Fewer second-generation sequencing of such high-throughput Nanobody sequence libraries has been reported in the literature. In general, the number of clones of the reported Nanobody libraries after sequencing is usually in the order of tens or hundreds, and the flux is low; and the IGHV1, IGHV2 and IGHV3 three major germline V gene subtypes are several, Less coverage.
表4：羊驼抗体免疫组库J基因使用情况Table 4: Usage of J gene in alpaca antibody immunization group
Figure PCTCN2016109286-appb-000020
Figure PCTCN2016109286-appb-000020
说明：表中的抗体胚系J基因指的是国际通用免疫组库数据库IMGT中驼科J基因家族所有胚系基因种类；Reads条数指的是免疫组库样本下机数据分析后统计得出的被测到的匹配到相应V胚系基因的序列条数；Reads比例(％)：匹配到该条V胚系基因下的序列占所有下机序列的比例。Description: The antibody germline J gene in the table refers to all germline gene types of the Camelidae J gene family in the IMGM database. The number of Reads refers to the statistical data of the immunogen library samples. The number of sequences detected to match the corresponding V germline gene; Reads ratio (%): the ratio of the sequence matched to the V germline gene to all of the lower sequences.
从表中我们可以看到，总的reads条数达35万条，能鉴定到全部的J基因亚型的使用(除了IGHJ1为假基因，其余全部可以分析鉴定到)。较少有文献报道如此高通量地纳米抗体序列库的二代测序。一般而言，已报道的纳米抗体文库经过测序的克隆数量通常在几十条或者几百条不等，通量较低。From the table we can see that the total number of reads is 350,000, which can identify the use of all J gene subtypes (except IGHJ1 is a pseudogene, all others can be analyzed and identified). Fewer second-generation sequencing of such high-throughput Nanobody sequence libraries has been reported in the literature. In general, the number of clones that have been reported for nanobody libraries has typically ranged from tens or hundreds of clones with low throughput.
5.潜在高亲和力抗体序列的生物信息筛选5. Bioinformatic screening of potential high affinity antibody sequences
重链抗体可变区VHH免疫组库测序出来，数量巨大，为此我们制定了多套 严格合理的序列筛选的标准，对所有序列进行筛选优先级得分排序。如上所述此例中序列库数量达350 000多条，这里仅以一套筛选标准(打分公式)为例，总分＝(突变率*0.4+CDR3丰度比例*0.3+CDR2丰度比例*0.2+CDR1丰度比例*0.1)*100。突变率测序得到的抗体序列与IMGT胚系参考基因匹配时不一致的碱基数比上全部匹配序列长度。依照此公式计算出来的分数对序列进行排序，我们会高通量地筛出得分高于80分的抗体序列，我们认为利用这些序列表达出来的抗体有潜在的高亲和力。The heavy chain antibody variable region VHH immune group library was sequenced, and the number was huge. For this reason, we have developed multiple sets. Strict and reasonable sequence screening criteria, screening priority rankings for all sequences. As mentioned above, the number of sequence libraries in this example is more than 350 000. Here, only one set of screening criteria (scoring formula) is taken as an example. The total score = (mutation rate * 0.4 + CDR3 abundance ratio * 0.3 + CDR 2 abundance ratio * 0.2+ CDR1 abundance ratio *0.1)*100. The number of bases in which the antibody sequence obtained by the mutation rate sequencing was inconsistent with the IMGT germline reference gene was matched to the length of all matching sequences. By sorting the sequences according to the scores calculated by this formula, we will screen high-throughput antibody sequences with scores above 80, and we believe that antibodies expressed using these sequences have potentially high affinity.
讨论：discuss:
构建免疫组库的B细胞主要来源于机体经靶点抗原的主动免疫，或者是病原体体内感染后的记忆B细胞，免疫组库的容量越大，越有利于筛选到高亲和力的抗体序列，一旦构建完成相关的抗体库，那么就可以进行特异性抗体的筛选，同时依赖于相应的筛选设备，还可以实现抗体的高通量筛选。为此，综合VHH抗体和免疫组库筛选的优势，依靠本发明的驼科引物，以及三步法建库方案，测序后可以快速且直接的得到大容量的驼科抗体的序列数据库，数据库具有胚系V基因亚型全面，结构合理，涵盖非典型纳米抗体等优点，为筛出高亲和力抗体提供足够的抗体序列库，为特异性VHH抗体的筛选奠定基础。The B cells that construct the immune group are mainly derived from the active immunization of the target antigen by the body, or the memory B cells after infection in the pathogen. The larger the capacity of the immune group, the better the screening of high-affinity antibody sequences. By constructing the relevant antibody library, screening of specific antibodies can be performed, and depending on the corresponding screening equipment, high-throughput screening of antibodies can also be achieved. To this end, the advantages of comprehensive VHH antibody and immune group library screening, relying on the camelid primer of the present invention and the three-step method of database construction, can quickly and directly obtain a sequence database of large-capacity camelid antibodies, the database has The germline V gene subtype is comprehensive, well-structured, and covers the advantages of atypical Nano-antibodies. It provides sufficient antibody sequence library for screening high-affinity antibodies and lays a foundation for the screening of specific VHH antibodies.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (15)

  1. 一种引物组合，其特征在于，所述引物组合中包括可以特异性退火结合到骆驼科抗体V基因I家族(Clan I)的前导区的第一上游引物。A primer combination, characterized in that the primer combination comprises a first upstream primer which can specifically anneal to a leader region of the camelid antibody V gene I family (Clan I).
  2. 如权利要求1所述的引物组合，其特征在于，所述第一上游引物包含选自下组的一种或多种引物序列：SEQ ID NO.11、2、3、4、5、6、7、8、9、10、1、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、和44；优选地，所述第一上游引物序列如SEQ ID NO.11所示。The primer set according to claim 1, wherein said first upstream primer comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 11, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44; preferably, the first upstream primer sequence is set forth in SEQ ID NO.
  3. 如权利要求1所述的引物组合，其特征在于，所述引物组合还包括下游引物；优选地所述下游引物序列如SEQ ID NO.45所示。The primer set of claim 1 wherein said primer combination further comprises a downstream primer; preferably said downstream primer sequence is set forth in SEQ ID NO.
  4. 如权利要求1所述的引物组合，其特征在于，所述引物组合还包括第二上游引物，所述第二上游引物特异性退火结合到骆驼科抗体V基因III家族(Clan III)的前导区，优选地所述第二上游引物序列如SEQ ID NO.46所示。The primer set according to claim 1, wherein said primer combination further comprises a second upstream primer, said second upstream primer specifically annealing to bind to a leader region of Camelid antibody V gene III family (Clan III) Preferably, said second upstream primer sequence is set forth in SEQ ID NO.
  5. 如权利要求1所述的引物组合，其特征在于，所述引物组合还包括第三上游引物，所述第三上游引物特异性退火结合到骆驼科抗体V基因II家族(Clan II)的前导区，优选地所述第二上游引物序列如SEQ ID NO.53所示。The primer set according to claim 1, wherein said primer combination further comprises a third upstream primer, said third upstream primer specifically annealing to bind to a leader region of Camelid antibody V gene II family (Clan II) Preferably, said second upstream primer sequence is set forth in SEQ ID NO.
  6. 一种引物组合，其特征在于，所述引物组合中的各引物为特异性退火结合到骆驼科抗体J基因区域的下游引物，并且所述引物组合包含选自下组的一种或多种引物序列：SEQ ID NO.54、55、56、和57。A primer combination, characterized in that each primer in the primer combination is a downstream primer that specifically anneals to a region of the camelid antibody J gene, and the primer combination comprises one or more primers selected from the group consisting of Sequence: SEQ ID NO. 54, 55, 56, and 57.
  7. 如权利要求2所述的引物组合，其特征在于，所述引物组合还包括上游引物；优选地所述上游引物序列如SEQ ID NO.58所示。The primer set of claim 2, wherein the primer combination further comprises an upstream primer; preferably the upstream primer sequence is set forth in SEQ ID NO.
  8. 如权利要求2所述的引物组合，其特征在于，所述引物组合包含选自下组的一种或多种引物序列：SEQ ID NO.47、48、49、50。The primer set of claim 2, wherein the primer combination comprises one or more primer sequences selected from the group consisting of SEQ ID NO. 47, 48, 49, 50.
  9. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1所述的引物组合，和/或权利要求6所述的引物组合；以及，任选地PCR扩增试剂。A kit, comprising the primer combination of claim 1, and/or the primer combination of claim 6; and, optionally, a PCR amplification reagent.
  10. 一种构建骆驼科抗体可变区免疫组库的方法，其特征在于，所述方法包括步骤：A method for constructing a camelid antibody variable region immune group library, the method comprising the steps of:
    (1)第一轮扩增(1) First round of amplification
    提供骆驼科动物免疫细胞的核酸作为模板，使用权利要求1所述的引物组合，进行第一轮PCR扩增，获得第一轮扩增产物； Providing a nucleic acid of a camelid immune cell as a template, using the primer combination of claim 1, performing a first round of PCR amplification to obtain a first round of amplification product;
    (2)第二轮扩增(2) Second round of amplification
    以第一轮扩增产物为模板，以权利要求2中所述的引物组合，进行第二轮巢式PCR扩增，获得第二轮扩增产物。The second round of nested PCR amplification was carried out by using the first round of amplification product as a template and the primer combination described in claim 2 to obtain a second round of amplification product.
  11. 如权利要求10所述的方法，其特征在于，所述方法还包括任选地步骤：The method of claim 10 wherein said method further comprises the optional step of:
    (3)以所述第二轮扩增产物为模板进行illumina文库构建。(3) The illumina library was constructed using the second round of amplification products as a template.
  12. 如权利要求10所述的方法，其特征在于，所述步骤(1)中，扩增完成后，回收600-1100bp的基因片段作为所述第一轮扩增产物；优选地回收700-800bp的基因片段作为下一轮扩增重链抗体可变区的模板；优选地回收900-1100bp的基因片段作为下一轮扩增传统抗体重链可变区的模板。The method according to claim 10, wherein in the step (1), after the amplification is completed, a gene fragment of 600-1100 bp is recovered as the first round of amplification product; preferably, 700-800 bp is recovered. The gene fragment serves as a template for the next round of amplification of the variable region of the heavy chain antibody; a 900-1100 bp gene fragment is preferably recovered as a template for the next round of amplification of the heavy chain variable region of a conventional antibody.
  13. 如权利要求10所述的方法，其特征在于，所述步骤(2)中，扩增完成后，回收300-600bp的基因片段作为所述第二轮扩增产物；优选地回收400-500bp的基因片段作为所述第二轮扩增产物。The method according to claim 10, wherein in the step (2), after the amplification is completed, a gene fragment of 300-600 bp is recovered as the second round of amplification product; preferably, 400-500 bp is recovered. The gene fragment serves as the second round of amplification product.
  14. 一种筛选重链抗体的方法，其特征在于，所述方法包括步骤：使用权利要求10所述的方法构建重链抗体可变区免疫组库；以及，在所述的重链抗体可变区免疫组库中筛选所述重链抗体。A method for screening a heavy chain antibody, the method comprising the steps of: constructing a heavy chain antibody variable region immunological library using the method of claim 10; and, in said heavy chain antibody variable region The heavy chain antibody is screened in an immunological pool.
  15. 如权利要求14所述的方法，其特征在于，筛选重链抗体的方法为噬菌体、大肠杆菌等微生物展示技术和/或生物信息学分析方法；优选地，所述的生物信息学分析方法为；设定N＝(突变率*0.4+CDR3丰度比例*0.3+CDR2丰度比例*0.2+CDR1丰度比例*0.1)*100，当N≥80时，确定为目标抗体序列。 The method according to claim 14, wherein the method for screening the heavy chain antibody is a phage, Escherichia coli or the like microbial display technology and/or a bioinformatics analysis method; preferably, the bioinformatics analysis method is; N=(mutation rate*0.4+CDR3 abundance ratio*0.3+CDR2 abundance ratio*0.2+CDR1 abundance ratio*0.1)*100 was set, and when N≥80, it was determined as the target antibody sequence.
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