Recombinant fusion protein of human lambda light-chain single-chain antibody, DNA (deoxyribonucleic acid) molecule, library building method and application

Abstract

The invention discloses a recombinant fusion protein of a human lambda light-chain single-chain antibody, a DNA (deoxyribonucleic acid) molecule, a library building method and application, and the recombinant fusion protein of the human lambda light-chain single-chain antibody is the recombinant fusion protein formed by connecting the human lambda light-chain single-chain antibody, phage Piii protein and luciferase, the amino acid sequence of the recombinant fusion protein of the human lambda light-chain single-chain antibody is shown as SEQ ID NO: 5, the recombinant fusion protein library of the human lambda light-chain single-chain antibody has large capacity, the established single-chain antibody TG1 library is more than 1 * 10 < 9 > cfu / mL, positive clones capable of being combined with ligands can be screened and enriched for multiple rounds, and the recombinant fusion protein library has the characteristics of high sensitivity, high signal-to-noise ratio and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of fusion proteins, and particularly relates to a recombinant fusion protein of a human lambda light chain single-chain antibody, a DNA molecule, a library construction method and an application thereof. Background Art

[0002] At present, most of the technical methods adopted by phage display libraries are to conjugate tag proteins (such as myc, FLAG tags, etc.) after the single-chain antibody sequence. In subsequent screening and detection, HRP-conjugated tag protein antibodies are used to recognize and bind single-chain antibodies specific to certain ligands. Its disadvantages are that a large number of incubation and washing steps are required, and false positives and false negatives are easily introduced.

[0003] In phage display libraries, a reporter system is generally added, and the most sensitive signal amplification reporter system is an enzyme. However, the molecular weights of alkaline phosphatase (140 kD), β-galactosidase (115 kD), horseradish peroxidase (44 kD), luciferase (62 kD), and Renilla luciferase (34 kD) are all relatively large, which greatly increases the size of the single-chain antibody-enzyme fusion protein, and thus reduces the electrotransformation efficiency and library capacity.

[0004] In some documents, such as Michael Tesar et al., Nanoluc was cloned into a phage display vector to replace the gene III fusion protein. The ligation product was transformed into chemically competent Escherichia coli Top10 cells, and the bacterial culture supernatant was used to screen single-chain antibodies that could bind specific ligands. Its disadvantages are that after Nanoluc replaces the gene III fusion protein, the single-chain antibody loses the ability to be expressed on the bacterial surface, and multiple rounds of screening cannot be carried out to effectively enrich positive clones. The transformation of the ligation product into chemically competent cells will reduce the transformation efficiency, resulting in a small library capacity. Summary of the Invention

[0005] The main object of the present invention is to propose a recombinant fusion protein of a human lambda light chain single-chain antibody, a DNA molecule, a library construction method and an application thereof, aiming to prepare a recombinant fusion protein with a large library capacity, high sensitivity, high signal-to-noise ratio, and reduced screening cost and steps.

[0006] To achieve the above object, the present invention proposes a recombinant fusion protein of a human lambda light chain single-chain antibody, which is a recombinant fusion protein formed by connecting a human lambda light chain single-chain antibody with phage Piii protein and luciferase. Among them, the amino acid sequence of the recombinant fusion protein of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:5.

[0007] Optionally, the amino acid sequence of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:6.

[0008] Optionally, the amino acid sequence of the phage Piii protein is as shown in SEQ ID NO:7.

[0009] Optionally, the amino acid sequence of the luciferase is as shown in SEQ ID NO:8.

[0010] Optionally, the human lambda light chain single-chain antibody comprises a light chain and a heavy chain; wherein, a plurality of primers are designed for the light chain; and / or, a plurality of primers are designed for the heavy chain.

[0011] The present invention provides a DNA molecule for encoding a recombinant fusion protein of the human lambda light chain single-chain antibody as described above, and the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO:1.

[0012] The present invention further provides a method for constructing a library of a recombinant fusion protein of a human lambda light chain single-chain antibody, comprising the following steps:

[0013] S10: Using the recombinant fusion protein of the human lambda light chain single-chain antibody as described above as a template, cloning and ligating a vector to obtain a recombinant plasmid;

[0014] S20: Providing a plurality of primers for the human lambda light chain single-chain antibody, using the recombinant plasmid as a template, respectively amplifying the light chain and the heavy chain of the human lambda light chain single-chain antibody to obtain a plurality of heavy chain fragments and a plurality of light chain fragments, wherein the amino acid sequence of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:6, comprising a light chain and a heavy chain, and a plurality of primers are designed for the light chain and the heavy chain respectively;

[0015] S30: Mixing a plurality of the heavy chain fragments and a plurality of the light chain fragments, and performing a PCR reaction to obtain a plurality of reaction mixtures;

[0016] S40: Respectively performing double digestion and ligation of the plurality of reaction mixtures with the recombinant plasmid to obtain a plurality of plasmids;

[0017] S50: Mixing the plurality of plasmids, and performing electrotransformation and amplification to obtain a library of the recombinant fusion protein of the human lambda light chain single-chain antibody.

[0018] Optionally, the vector in step S10 comprises an AB plasmid.

[0019] The present invention also provides a phage library, which is constructed from the recombinant fusion protein of the human lambda light chain single-chain antibody as described above or from the phage library of the recombinant fusion protein of the human lambda light chain single-chain antibody obtained by the method for constructing a library of the recombinant fusion protein of the human lambda light chain single-chain antibody as described above.

[0020] In the technical solution provided by the present invention, a new phage display single-chain antibody library is adopted, and luciferase (nanoluc) is coupled with the human lambda light chain single-chain antibody. Since the molecular weight of the nanoluc is small, the size of the recombinant fusion protein is reduced, the electrotransformation efficiency and the library capacity are increased, and the gene III fusion protein in the phage is retained, that is, the ability to be expressed on the bacterial surface is retained, and multiple rounds of screening can be carried out to effectively enrich positive clones. The library obtained in this way has a large capacity, and the constructed phage library of the recombinant fusion protein of the human lambda light chain single-chain antibody is above 1x10 9 cfu / mL, and has the characteristics of high sensitivity, high signal-to-noise ratio, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a schematic diagram of the enzyme expression of the monoclonal antibody obtained in Example 1 of the present invention;

[0023] Figure 2 It is a schematic diagram of the enzyme expression in the supernatant of the overnight culture of the human lambda light chain single-chain antibody library.

[0024] The realization, functional characteristics and advantages of the object of the present invention will be further described in conjunction with the embodiments and the drawings. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0026] It should be noted that for those not specifying specific conditions in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel scenarios. Taking "A and / or B" as an example, it includes scenario A, or scenario B, or the scenario where both A and B are satisfied simultaneously. Furthermore, the technical solutions between various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] At present, the vast majority of phage display libraries adopt a technical method of conjugating a tag protein (such as myc, FLAG tag, etc.) after the single-chain antibody sequence. In subsequent screening and detection, an antibody of the tag protein conjugated with HRP is used to recognize and bind the single-chain antibody that binds to a specific ligand. Its disadvantages are that a large number of incubation and washing steps are required, which are prone to introducing false positives and false negatives. Moreover, although a sensitive signal amplification reporting system enzyme is used. However, the molecular weights of alkaline phosphatase (140 kD), β-galactosidase (115 kD), horseradish peroxidase (44 kD), luciferase (62 kD), and Renilla luciferase (34 kD) are all relatively large, greatly increasing the size of the single-chain antibody-enzyme fusion protein, thereby reducing the electroporation efficiency and library capacity.

[0028] In view of this, the present invention proposes a recombinant fusion protein of a human lambda light chain single-chain antibody, aiming to prepare a recombinant fusion protein with high electroporation efficiency, large library capacity, reduced screening cost and steps, high sensitivity, and high signal-to-noise ratio.

[0029] To achieve the above object, the present invention proposes a recombinant fusion protein of a human lambda light chain single-chain antibody, characterized in that the recombinant fusion protein of the human lambda light chain single-chain antibody is a recombinant fusion protein formed by connecting a human lambda light chain single-chain antibody with phage Piii protein and luciferase, wherein the amino acid sequence of the recombinant fusion protein of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:5.

[0030] It should be noted that the amino acid sequence of the recombinant fusion protein of the human lambda light chain single-chain antibody is as follows:

[0031] MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPTQAFDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSGARRPSVVFGGGTKLTVLGAAAEYPYDVPDYAVDGGGGSGGGGSGGGGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILADYKDDDDKTRXGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

[0032] The present invention provides a new recombinant fusion protein of a phage-displayed single-chain antibody, which conjugates luciferase (nanoluc) with a human lambda light-chain single-chain antibody and retains the gene III fusion protein in the phage, that is, retains the ability to be expressed on the surface of bacteria, and can perform multiple rounds of screening to effectively enrich positive clones. The library obtained in this way has a large library capacity. The established single-chain antibody TG1 library is above 1x10 9 cfu / mL, and has the characteristics of high sensitivity, high signal-to-noise ratio, and the fluorescence signal of each monoclonal after electroporation is above 107, while the fluorescence signal of untransfected cells is very low.

[0033] Furthermore, the amino acid sequence of the human lambda light-chain single-chain antibody is as shown in SEQ ID NO:6:

[0034] MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPTQAFDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSGARRPSVVFGGGTKLTVLG

[0035] Furthermore, the amino acid sequence of the phage Piii protein is as shown in SEQ ID NO:7:

[0036] DFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

[0037] It should be noted that a first linker protein is provided between the amino acids of the phage Piii protein and the luciferase, and the amino acid sequence of the first linker protein is as shown in SEQ ID NO:21: GGSGSG

[0038] Furthermore, the amino acid sequence of the luciferase is as shown in SEQ ID NO:8:

[0039] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0040] It should be noted that a second linker protein is provided between the amino acids of the luciferase and the phage Piii protein, and the amino acid sequence of the second linker protein is as shown in SEQ ID NO:22:

[0041] GGGGSGGGGSGGGG

[0042] The present invention also provides a DNA molecule, and the nucleotide sequence encoding the recombinant fusion protein of the human lambda light chain single-chain antibody described in claim 1 is as shown in SEQ ID NO: 1.

[0043] It should be noted that the nucleotide sequence of the recombinant fusion protein encoding the human lambda light chain single-chain antibody is as follows: SEQ ID NO: 1

[0044]

[0045] It should be further noted that the nucleotide sequence encoding the humanized lambda light chain single-chain antibody is as shown in SEQ ID NO:2 as follows:

[0046] ATGAAATATCTGCTGCCGACGGCAGCAGCAGGTCTGCTGCTGCTGGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAACGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCGCCTACTCAGGCGTTTGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCTCTGGTGCGCGTCGGCCCTCGGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGC

[0047] The nucleotide sequence encoding the phage Piii protein gene is as shown in SEQ ID NO:3 as follows:

[0048] GGCGGTTCTGGTAGCGGTGATTTCGATTACGAAAAAATGGCGAACGCCAACAAAGGTGCCATGACCGAAAATGCCGATGAAAATGCGCTGCAGAGCGATGCCAAAGGTAAACTGGATAGCGTTGCCACCGATTATGGTGCCGCCATTGATGGCTTTATTGGCGATGTTAGCGGCCTGGCGAATGGTAATGGTGCCACCGGTGATTTTGCCGGTAGCAATAGCCAGATGGCCCAGGTTGGTGATGGTGATAACAGCCCGCTGATGAACAACTTTCGTCAGTATCTGCCGAGCCTGCCGCAGAGCGTTGAATGTCGTCCGTTTGTGTTTGGCGCCGGCAAACCGTACGAATTTAGCATCGATTGTGATAAAATCAACCTGTTCCGTGGCGTTTTTGCCTTTCTGCTGTACGTGGCGACCTTTATGTATGTGTTCAGCACCTTTGCCAACATCCTGCGCAACAAAGAAAGCTAATAA

[0049] The nucleotide sequence encoding the nanoluciferase (nanoluc) gene is shown as SEQ ID NO:4 as follows:

[0050] GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAGTCTTCACGCTTGAAGACTTTGTCGGGGACTGGCGCCAAACCGCCGGTTACAATTTGGACCAAGTCCTGGAGCAGGGAGGTGTGTCTTCGTTATTCCAGAATTTGGGGGTTTCGGTGACACCGATTCAGCGCATTGTCTTATCAGGTGAGAATGGATTAAAGATTGATATTCATGTAATCATCCCCTACGAGGGCCTGTCCGGGGACCAGATGGGTCAAATCGAAAAGATTTTCAAGGTTGTGTACCCCGTGGACGACCATCATTTCAAAGTTATTTTACATTATGGTACGTTGGTGATTGACGGAGTAACACCAAACATGATCGACTACTTCGGGCGTCCTTATGAGGGCATCGCTGTTTTCGACGGCAAAAAAATCACCGTTACCGGCACGCTTTGGAATGGGAATAAAATCATCGACGAGCGCCTTATCAACCCAGATGGGTCCCTTCTGTTCCGTGTAACCATCAACGGAGTAACAGGGTGGCGTTTGTGCGAACGCATTTTGGCT

[0051] The phage display method expresses an antibody or polypeptide library on the surface of a phage and screens for specific antibody or polypeptide sequences based on their ability to bind ligands. The principle of this method relies on genetic recombination of the phage genome, inserting the sequence of interest beside the phage coat protein pIII to form a fusion protein, and then the recombinant phage infects bacteria and its genome is replicated. The expression of the recombinant phage genome leads to the production of phages and the expression of the antibody or polypeptide to be screened on the surface. In the screening step, different proteins or molecules (called ligands) bind to the above-mentioned antibody or polypeptide and can be continuously enriched in subsequent screening. The antibody or polypeptide sequence that can bind to the ligand can be obtained by sequencing.

[0052] Nanoluc is the latest member of the luciferase system family. Compared with the commonly used luciferases and Renilla luciferase, Nanoluc emits light two orders of magnitude higher, has a stable signal, and the luminescence shows a linear relationship within a concentration range spanning 1,000,000-fold, and the signal half-life ≥ 2 h. Therefore, fluorescence signals can be detected even at low concentrations, greatly improving the detection sensitivity. The molecular weights of luciferase and Renilla luciferase are 62 kD and 34 kD respectively, while Nanoluc is 19 kD in size, thus greatly reducing the size of the recombinant fusion protein, and further increasing the electroporation efficiency and library capacity, which is beneficial to genetic engineering modification and high-throughput screening.

[0053] Furthermore, the human-derived lambda light chain single-chain antibody comprises a light chain and a heavy chain, and multiple primers are designed for the light chain and the heavy chain.

[0054] It should be noted that for the diversity of the library, multiple primers are designed for the light chain and the heavy chain of the human-derived lambda light chain single-chain antibody for amplification. Among them, the multiple primers include a first reverse primer, a second reverse primer, a third reverse primer, a fourth reverse primer, a fifth reverse primer, a sixth reverse primer, a seventh reverse primer, an eighth reverse primer, a ninth reverse primer, a tenth reverse primer, a first forward primer, and a second forward primer. The nucleotide sequence of the first reverse primer is as shown in SEQ ID NO:9, and the nucleotide sequence of the second reverse primer is as shown in SEQ ID NO:10; the nucleotide sequence of the third reverse primer is as shown in SEQ ID NO:11, the fourth reverse primer is as shown in SEQ ID NO:12, the fifth reverse primer is as shown in SEQ ID NO:13, the first forward primer is as shown in SEQ ID NO:14, the nucleotide sequence of the second forward primer is SEQ ID NO:15, the nucleotide sequence of the sixth reverse primer is as shown in SEQ ID NO:16, the nucleotide sequence of the seventh reverse primer is as shown in SEQ ID NO:17, the nucleotide sequence of the eighth reverse primer is as shown in SEQ ID NO:18, the nucleotide sequence of the ninth reverse primer is as shown in SEQ ID NO:19, and the nucleotide sequence of the tenth reverse primer is as shown in SEQ ID NO:20.

[0055] It should be noted that in the following sequences, M is a base A or C, and N is any one of A, C, G, and T;

[0056] The nucleotide sequence of the first reverse primer is shown in SEQ ID NO:9, and its specific sequence is: CTTGGTCCCTCCGCCGAATACCACMNNMNNMNNMNNMNNGGGAGAG GAGTTACAGTAATAGTC, and the name of this sequence is L1-REV.

[0057] The nucleotide sequence of the second reverse primer is shown in SEQ ID NO:10, and its specific sequence is:

[0058] CTTGGTCCCTCCGCCGAATACCACMNNMNNMNNMNNGGGMNNAG AGGAGTTACAGTAATAGTC, and the name of this sequence is L2-REV.

[0059] The nucleotide sequence of the third reverse primer is shown in SEQ ID NO:11, and its specific sequence is:

[0060] CTTGGTCCCTCCGCCGAATACCACMNNMNNMNNGGGMNNMNNAG AGGAGTTACAGTAATAGTC, and the name of this sequence is L3-REV.

[0061] The nucleotide sequence of the fourth reverse primer is shown in SEQ ID NO:12, and its specific sequence is:

[0062] CTTGGTCCCTCCGCCGAATACCACMNNGGGMNNMNNMNNMNNAGAGGAGTTACAGTAATAGTCTAGTC, and the name of this sequence is L4-REV.

[0063] The nucleotide sequence of the fifth reverse primer is shown in SEQ ID NO:13:

[0064] CTTGGTCCCTCCGCCGAATACCACGGGMNNMNNMNNMNNMNNAG AGGAGTTACAGTAATAGTC, and the name of this sequence is L5-REV.

[0065] The nucleotide sequence of the first forward primer is shown in SEQ ID NO:14, and its specific sequence is:

[0066] TTTGACTACTGGGGCCAGGGAAC, and the name of this sequence is KLO FWD.

[0067] The nucleotide sequence of the second forward primer is shown in SEQ ID NO:15:

[0068] 5’tggcggcccagccggccatggccGAGGTGCAGCTGTTGGAGT 3’, and the name of this sequence is HO FWD.

[0069] The nucleotide sequence of the sixth reverse primer is shown as SEQ ID NO:16:

[0070] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNTTTCGCACA GTAATATACGGCC, and the name of this sequence is H4-REV.

[0071] The nucleotide sequence of the seventh reverse primer is shown as SEQ ID NO:17:

[0072] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNTTTCGC ACAGTAATATACGG, and the name of this sequence is H5-REV.

[0073] The nucleotide sequence of the eighth reverse primer is shown as SEQ ID NO:18:

[0074] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNTTT CGCACAGTAATATA, and the name of this sequence is H6-REV.

[0075] The nucleotide sequence of the ninth reverse primer is shown as SEQ ID NO:19:

[0076] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNMN NTTTCGCACAGTAATATACGGCC, and the name of this sequence is H7-REV.

[0077] The nucleotide sequence of the tenth reverse primer is shown as SEQ ID NO:20:

[0078] CATAAGGGTATTCTGCGGCCGCGCCTAGGACGGTCAGCTTGGTCCC TCCGCCGAA, and the name of this sequence is LNotI REV.

[0079] The present invention further provides a method for constructing a library of recombinant fusion proteins of human lambda light chain single-chain antibodies, comprising the following steps:

[0080] S10. Using the above-mentioned recombinant fusion protein as a template, cloning and ligating a vector to obtain a recombinant plasmid;

[0081] Further, the vector in step S10 includes an AB plasmid.

[0082] In some embodiments, the specific steps include: double-digesting the human lambda light chain single-chain antibody template and the AB plasmid with HindIII and AgeI, cloning the template into the AB plasmid to obtain the ABHL plasmid.

[0083] S20. Provide a plurality of primers for the human lambda light chain single-chain antibody, and use the recombinant plasmid as a template to amplify the light chain and heavy chain of the human lambda light chain single-chain antibody respectively to obtain a plurality of heavy chain fragments and a plurality of light chain fragments. Among them, the amino acid sequence of the human lambda light chain single-chain antibody is as shown in SEQ ID NO: 2, including a light chain and a heavy chain, and a plurality of primers are designed for the light chain and the heavy chain respectively.

[0084] Specifically, in some embodiments, the operation steps include:

[0085] Reaction 1: Using the ABHL plasmid as a template, with the HO FWD and H4-REV primers, to obtain the product H4.

[0086] Reaction 2: Using the ABHL plasmid as a template, with the HO FWD and H5-REV primers, to obtain the product H5.

[0087] Reaction 3: Using the ABHL plasmid as a template, with the HO FWD and H6-REV primers, to obtain the product H6.

[0088] Reaction 4: Using the ABHL plasmid as a template, with the HO FWD and H7-REV primers, to obtain the product H7.

[0089] Reaction 5: Using the ABHL plasmid as a template, with the KLO FWD and L1-REV primers, to obtain the product L1.

[0090] Reaction 6: Using the ABHL plasmid as a template, with the KLO FWD and L2-REV primers, to obtain the product L2.

[0091] Reaction 7: Using the ABHL plasmid as a template, with the KLO FWD and L3-REV primers, to obtain the product L3.

[0092] Reaction 8: Using the ABHL plasmid as a template, with the KLO FWD and L4-REV primers, to obtain the product L4.

[0093] Reaction 9: Using the ABHL plasmid as a template, with the KLO FWD and L5-REV primers, to obtain the product L5.

[0094] The above PCR reaction system and conditions are as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds; annealing at 60°C for 30 seconds; extension at 72°C for 15 seconds. After 35 cycles, the sample continues to be extended at 72°C for 5 minutes, with a total system volume of 50 μL. Run agarose gel electrophoresis and recover the target fragment at approximately 400 bp.

[0095] S30. Mix multiple of the heavy chain fragments with multiple of the light chain fragments and perform a PCR reaction to obtain multiple reaction mixtures.

[0096] Specifically, in some embodiments, the operation steps include:

[0097] Reaction 1: Mix the first heavy chain product H4 and the first light chain product L1 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H4L1.

[0098] Reaction 2: Mix the first heavy chain product H4 and the second light chain product L2 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H4L2.

[0099] Reaction 3: Mix the first heavy chain product H4 and the third light chain product L3 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H4L3.

[0100] Reaction 4: Mix the first heavy chain product H4 and the first light chain product L4 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H4L4.

[0101] Reaction 5: Mix the first heavy chain product H4 and the first light chain product L5 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H4L5.

[0102] Reaction 6: Mix the second heavy chain product H5 and the first light chain product L1 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H5L1.

[0103] Reaction 7: Mix the second heavy chain product H5 and the second light chain product L2 at a molar ratio of 1:1, and use HO FWD and LNotIREV as primers to perform a PCR reaction to obtain the first mixture H5L2.

[0104] Reaction 8: Mix the second heavy chain product H5 and the third light chain product L3 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotIREV as primers to obtain the first mixed product H5L3.

[0105] Reaction 9: Mix the second heavy chain product H5 and the fourth light chain product L4 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotIREV as primers to obtain the first mixed product H5L4.

[0106] Reaction 10: Mix the second heavy chain product H5 and the fifth light chain product L5 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H5L5.

[0107] Reaction 11: Mix the third heavy chain product H6 and the first light chain product L1 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H6L1.

[0108] Reaction 12: Mix the third heavy chain product H6 and the first light chain product L2 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H6L2.

[0109] Reaction 13: Mix the third heavy chain product H6 and the first light chain product L3 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H6L3.

[0110] Reaction 14: Mix the third heavy chain product H6 and the first light chain product L4 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H6L4.

[0111] Reaction 15: Mix the third heavy chain product H6 and the first light chain product L5 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H6L5.

[0112] Reaction 16: Mix the fourth heavy chain product H7 and the first light chain product L1 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H7L1.

[0113] Reaction 17: Mix the fourth heavy chain product H7 and the first light chain product L2 in a molar ratio of 1:1, and perform a PCR reaction using HO FWD and LNotI REV as primers to obtain the first mixed product H7L2.

[0114] Reaction 18: The fourth heavy chain product H7 and the first light chain product L3 were mixed at a molar ratio of 1:1, and PCR was performed using HO FWD and LNotI REV as primers to obtain the first mixed product H7L3.

[0115] Reaction 19: The fourth heavy chain product H7 and the first light chain product L2 were mixed at a molar ratio of 1:1, and PCR was performed using HO FWD and LNotI REV as primers to obtain the first mixed product H7L4.

[0116] Reaction 20: The fourth heavy chain product H7 and the first light chain product L2 were mixed at a molar ratio of 1:1, and PCR was performed using HO FWD and LNotI REV as primers to obtain the first mixed product H7L5.

[0117] The above PCR reaction system and conditions were as follows: pre-denaturation at 98 °C for 30 seconds; denaturation at 98 °C for 10 seconds; annealing at 60 °C for 30 seconds; extension at 72 °C for 15 seconds. After 35 cycles, the sample was further extended at 72 °C for 5 minutes in a total system of 50 μL. Run agarose gel electrophoresis and recover the target fragment at approximately 800 bp.

[0118] S40: The multiple reaction mixed products were respectively double-digested with the recombinant plasmid and then ligated to obtain multiple plasmids.

[0119] Specifically, the operation steps included: digesting the recombinant plasmid, and the digestion system:

[0120] Table 1 Digestion System of Recombinant Plasmid

[0121] Reagent Volume / Mass ABHL plasmid 100 μg 10x Restriction Enzyme Buffer 50 μl Not I 15 μl Nco I 15 μl <![CDATA[ddH2O]]> fill to 500 μl

[0122] Reaction conditions: After digesting at 37 °C for 7 hours, run agarose gel electrophoresis and recover the vector fragment.

[0123] The digestion systems of the first mixed product, the second mixed product, the third mixed product, the fourth mixed product, the fifth mixed product, the sixth mixed product, the seventh mixed product, the eighth mixed product, the ninth mixed product, the tenth mixed product, the eleventh mixed product, and the twelfth mixed product are shown in the following table:

[0124] Table 2 Digestion System

[0125] Reagent Volume / Mass H4L1 30 μg 10x Restriction Enzyme Buffer 30 μl Not I 10 μl Nco I 10 μl <![CDATA[ddH2O]]> fill to 300 μl

[0126] Reaction conditions: After digestion at 37°C for 7 hours, perform agarose gel electrophoresis, recover the large fragment, and perform similar digestion treatments on the H4L2, H4L3, H5L1, H5L2, H5L3, H6L1, H6L2, H6L3, H7L1, H7L2, and H7L3 fragments using the same treatment method.

[0127] Ligate the digested vector and the digested antibody fragment to form a complete vector:

[0128] Table 3 Ligation system

[0129] Reagent Volume / Mass Digested ABHL 30 μg Digested H4L1 20 μg 10x Restriction Enzyme Buffer 45 μl T4 Ligase 20 μl <![CDATA[ddH2O]]> fill to 450 μl

[0130] Reaction conditions: Ligate at 16°C for 16 - 24 hours. Add phenol to remove proteins, wash with 70% ethanol, and dissolve the DNA in ultrapure water to obtain the first plasmid, H4L1AB plasmid. Similarly, perform similar treatments on H4L2, H4L4, H4L5, H5L1, H5L2, H5L3, H5L4, H5L5, H6L1, H6L2, H6L3, H6L4, H6L5, H7L1, H7L2, H7L3, H7L4, and H7L5 after digestion to obtain H4L2AB, H4L3AB, H4L4AB, H4L5AB, H5L1AB, H5L2AB, H5L3AB, H5L4AB, H5L5AB, H6L1AB, H6L2AB, H6L3AB, H6L4AB, H6L5AB, H7L1AB, H7L2AB, H7L3AB, H7L4AB, and H7L5AB plasmids.

[0131] S50. After mixing the multiple plasmids, perform electrotransformation and amplification to obtain a recombinant fusion protein library of human - derived lambda light - chain single - chain antibodies.

[0132] The present invention also provides a phage library, which is prepared from the recombinant fusion protein library of human - derived lambda light - chain single - chain antibodies obtained by the method for constructing a library of recombinant fusion proteins of human - derived lambda light - chain single - chain antibodies as described above.

[0133] In summary, the method for constructing a library of recombinant fusion proteins of human - derived lambda light - chain single - chain antibodies proposed in this application enables the prepared recombinant fusion proteins of human - derived lambda light - chain single - chain antibodies to have a large library capacity. The established single - chain antibody TG1 library has more than 1x10 9 above, and can be screened in multiple rounds to enrich positive clones that can bind ligands, and has characteristics such as high sensitivity and high signal - to - noise ratio.

[0134] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0135] Construction of the Library in Example 1

[0136] 1) Inoculate electrocompetent cells (TG1) into 1000 ml of 2-TY medium (containing 0.01% F-68) and culture at 18 °C until the OD reaches 0.6.

[0137] 2) After collecting the bacteria, centrifuge at 3000 g for 10 minutes and discard the supernatant.

[0138] 3) Wash the precipitate with 1000 ml, 500 ml, and 200 ml of wash buffer (10% glycerol, 1.5% mannitol, 1 mM Hepes) respectively to remove the residual liquid as much as possible.

[0139] 4) Add 5 ml of buffer (10% glycerol + 1.5% mannitol), centrifuge at 3000 g for 5 min, and aspirate the supernatant with a pipette tip.

[0140] 5) Resuspend the precipitate in 1.5 ml of buffer (10% glycerol + 1.5% mannitol), add the constructed plasmid H4L1AB respectively, mix well and place on ice for 10 minutes. Aliquot 50 μl into pre-chilled electroporation cuvettes and electroporate at 1200 V for 5 ms. Add 110 μl of 2-TY glucose medium, place the electroporation cuvette in an incubator at 37 °C for 1 hour. After pooling all the transformants, spread them evenly on 40 150-mm 2-TY agar plates (2% glucose, 100 μg / ml ampicillin) and incubate overnight at 30 °C. Prepare plasmids H4L2AB, H4L3AB, H5L1AB, H5L2AB, H5L3AB, H6L1AB, H6L2AB, H6L3AB, H7L1AB, H7L2AB, and H7L3AB in the same way.

[0141] 6) After the colonies grow, add 3 ml of cryopreservation solution (25% glycerol, 100 μg / ml ampicillin, 1% glucose) to each plate, scrape all the colonies on the plate with a sterile glass rod and collect the cell suspension. This is the constructed TG1 library. Take 50 μl and make 10-fold serial dilutions of the reserved bacterial solution for standby.

[0142] Calculate the library titer, and the titer of the human-derived lambda light chain single-chain antibody library is 6.58x10 9 cfu / mL.

[0143] Quality Detection of the Library in Example 2

[0144] (1) Randomly select 42 HKAB monoclonal antibodies on the plate and culture them overnight in 2-TY medium. The next day, centrifuge and transfer 30 μl of the supernatant to a white opaque plate, add the nanoluc substrate, and detect the fluorescence signal. The results are shown in the appendix Figure 1 As shown, the fluorescence signal in untransfected TG1 cells is very low (<104), while the fluorescence signal of each monoclonal antibody after electroporation is above 107.

[0145] (2) Culture untransfected TG1 and HL libraries overnight in 2-TY medium, with or without IPTG. Harvest the supernatant the next day, run SDS-PAGE, transfer the membrane, and then add the nanoluc substrate. As shown in the appendix Figure 2 As shown: It was found that there was no band in untransfected TG1, and the single-chain antibody conjugated with nanoluc could be detected in the supernatant of the HL medium. The above results indicate that the construction of the library was successful.

[0146] Example 3 Construction of a human lambda phage display library

[0147] 1) Add 200 ml of 2TY medium to an Erlenmeyer flask and inoculate the HK library bacteria

[0148] 2) Dilute to OD600 = 0.1, add 100 μg / ml ampicillin and 1% glucose. Culture at 37 °C until OD600 = 0.5. After a 5-minute water bath at 37 °C, add 20:1 M13KO7 and incubate in a water bath for 40 minutes.

[0149] 3) Centrifuge at 3000 g for 20 minutes, discard the supernatant, and resuspend the pellet in 100 μg / ml ampicillin and 50 μg / ml kanamycin

[0150] 4) Culture overnight at 220 rpm at 30 °C

[0151] 5) Centrifuge at 3000 g for 20 minutes

[0152] 6) Transfer the supernatant to a new container, add 1 / 5 volume of 20% (w / v) PEG 6000 / 2.5 M NaCl (PEG / NaCl), mix well, and place on ice for 2 hours

[0153] 7) Centrifuge at 10800 g for 20 min

[0154] 8) Resuspend the pellet in 40 ml of PBS

[0155] 9) Add 1 / 5 volume of PEG / NaCl, mix well, and place on ice for at least 20 minutes

[0156] 10) Centrifuge at 10800 g for 15 minutes and discard the supernatant

[0157] 11) Centrifuge briefly to remove the residual PEG / NaCl

[0158] 12) Add 20 ml of 10% glycerol in PBS and aliquot into 500-μl tubes.

[0159] 13) Dilute the phage titer by 10-fold serial dilution. The titer of the HL library is 3x10 12 / ml and aliquot into 500-μl tubes

[0160] 14) Store at -20

[0161] In summary, in the technical solution provided by the present invention, a new phage display single-chain antibody library is used, luciferase (nanoluc) is conjugated with a human lambda light chain single-chain antibody, and the gene III fusion protein in the phage is retained. A recombinant fusion protein of the human lambda light chain single-chain antibody is screened out. The library established by this recombinant fusion protein of the human lambda light chain single-chain antibody has a large capacity. The established single-chain antibody TG1 library has a titer of 1x10 9 cfu / mL or higher, and can be screened in multiple rounds to enrich positive clones that can bind ligands, and has characteristics such as high sensitivity and high signal-to-noise ratio.

[0162] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made using the content of the specification of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A recombinant fusion protein of a human lambda light chain single-chain antibody, characterized in that, The recombinant fusion protein of the human lambda light chain single-chain antibody is a recombinant fusion protein formed by linking the human lambda light chain single-chain antibody with phage Piii protein and luciferase. Among them, the amino acid sequence of the recombinant fusion protein of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:

5.

2. The recombinant fusion protein of the human lambda light chain single-chain antibody as described in claim 1, characterized in that, The amino acid sequence of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:

6.

3. The recombinant fusion protein of the human lambda light chain single-chain antibody according to claim 1, characterized in that, The amino acid sequence of the phage Piii protein is as shown in SEQ ID NO:

7.

4. The recombinant fusion protein of the human lambda light chain single-chain antibody according to claim 1, characterized in that, The amino acid sequence of the luciferase is as shown in SEQ ID NO:

8.

5. The recombinant fusion protein of the human lambda light chain single-chain antibody according to claim 1, characterized in that, The human lambda light chain single-chain antibody includes a light chain and a heavy chain: Among them, multiple primers are designed for the light chain; and / or multiple primers are designed for the heavy chain.

6. A DNA molecule, characterized in that, For encoding the recombinant fusion protein of the human lambda light chain single-chain antibody described in claim 1, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO:

1.

7. A method for constructing a library of recombinant fusion proteins of human lambda light chain single-chain antibodies, characterized in that, Comprising the following steps: S10. Using the recombinant fusion protein of the human lambda light chain single-chain antibody described in any one of claims 1 to 5 as a template, cloning and ligating a vector to obtain a recombinant plasmid; S20. Providing multiple primers for the human lambda light chain single-chain antibody, using the recombinant plasmid as a template, respectively amplifying the light chain and heavy chain of the human lambda light chain single-chain antibody to obtain multiple heavy chain fragments and multiple light chain fragments. Among them, the amino acid sequence of the human lambda light chain single-chain antibody is as shown in SEQ ID NO:6, including a light chain and a heavy chain, and multiple primers are designed for the light chain and the heavy chain respectively; S30. Mixing multiple of the heavy chain fragments and multiple of the light chain fragments, and performing a PCR reaction to obtain multiple reaction mixtures; S40. Respectively double-digesting and ligating the multiple reaction mixtures with the recombinant plasmid to obtain multiple plasmids; S50. After mixing the multiple plasmids, performing electrotransformation and amplification to obtain a recombinant fusion protein library of the human lambda light chain single-chain antibody.

8. The method for constructing a library of the recombinant fusion protein of the human-derived lambda light chain single-chain antibody according to claim 7, characterized in that, The vector in step S10 includes plasmid AB.

9. A phage library, characterized in that, The phage library is constructed from the recombinant fusion protein of the human lambda light chain single-chain antibody described in any one of claims 1 to 5 or from the recombinant fusion protein library of the human lambda light chain single-chain antibody obtained by the method for constructing a library of the recombinant fusion protein of the human lambda light chain single-chain antibody described in claim 7 or 8.

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