Recombinant fusion proteins of human kappa light chain single chain antibodies, dna molecules and library construction methods and applications

Abstract

The application discloses a recombinant fusion protein of human kappa light chain single-chain antibody, a DNA molecule, a library construction method and application. The recombinant fusion protein of human kappa light chain single-chain antibody is a recombinant fusion protein formed by connecting a human kappa light chain single-chain antibody with a phage PIII protein and luciferase. The amino acid sequence of the recombinin fusion protein of human kappa light chain single-chain antibody is shown as SEQ ID NO: 5. The nanoluc is coupled with the human kappa light chain single-chain antibody. The nanoluc has a small molecular weight, so that the size of the recombinant fusion protein is reduced, the electric conversion efficiency and the library capacity are increased, and the gene III fusion protein in the phage is retained, that is, the ability of expression on the bacterial surface is retained, so that multiple rounds of screening can be carried out to effectively enrich positive clones. The library capacity is large, the recombinant fusion protein library of human kappa light chain single-chain antibody constructed has a capacity of 1x10 9 cfu / mL or above, and has the characteristics of high sensitivity and high signal-to-noise ratio.

Description

TECHNICAL FIELD

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

[0002] At present, most of the phage display library adopts a technical method of coupling a tag protein (such as myc, FLAG tag, etc.) after a single-chain antibody sequence. In subsequent screening and detection, a HRP-coupled tag protein antibody is used to recognize a single-chain antibody combined with a specific ligand. The disadvantage is that a large number of incubation and washing steps are required, which is easy to introduce false positives and false negatives.

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

[0004] In some documents, such as Michael Tesar, Nanoluc is cloned into a phage display vector to replace the gene III fusion protein. The ligation product is transformed into chemically competent E. coli Top10 cells, and the bacterial culture supernatant is used to screen single-chain antibodies capable of binding to specific ligands. The disadvantage is that Nanoluc replaces the gene III fusion protein, which makes the single-chain antibody lose the ability to express on the surface of bacteria, and cannot be subjected to multiple rounds of screening to effectively enrich positive clones. The transformation of the ligation product into chemically competent cells reduces the transformation efficiency, resulting in a small library capacity. SUMMARY

[0005] The main purpose of the present application is to provide a recombinant fusion protein of human kappa light chain single-chain antibody, a DNA molecule, a library construction method and application, which aims to prepare a recombinant fusion protein with large library capacity, reduced screening cost and steps, high sensitivity and high signal-to-noise ratio.

[0006] To achieve the above-mentioned purpose, the present application provides a recombinant fusion protein of human kappa light chain single-chain antibody, which is a recombinant fusion protein connected by a human kappa light chain single-chain antibody, a phage Piii protein and luciferase, wherein the amino acid sequence of the recombinant fusion protein of human kappa light chain single-chain antibody is shown as SEQ ID NO: 5.

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

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

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

[0010] Optionally, the human kappa light chain single-chain antibody comprises a light chain and a heavy chain.

[0011] wherein a plurality of primers are designed for the light chain and the heavy chain.

[0012] The present application provides a DNA molecule for encoding the recombinant fusion protein of the human kappa light chain single-chain antibody as described above, and the nucleotide sequence of the DNA molecule is shown as SEQ ID NO: 1.

[0013] Further provided is a library construction method of the recombinant fusion protein of the human kappa light chain single-chain antibody, comprising the following steps:

[0014] S10, using the recombinant fusion protein of any one of the above as a template, cloning a connecting vector to obtain a recombinant plasmid;

[0015] S20, providing a plurality of primers of the human kappa light chain single-chain antibody, using the recombinant plasmid as a template, respectively amplifying the light chain and the heavy chain of the human kappa 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 kappa light chain single-chain antibody is shown as 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.

[0016] S30, mixing a plurality of the heavy chain fragments with each of the light chain fragments respectively, and performing PCR reaction to obtain a plurality of reaction mixture products.

[0017] S40, performing double enzyme digestion on the plurality of reaction mixture products and the recombinant plasmid respectively, and then connecting to obtain a plurality of plasmids.

[0018] S50, mixing the plurality of plasmids, and then performing electrotransformation and amplification to obtain a library of the recombinant fusion protein of the human kappa light chain single-chain antibody.

[0019] Optionally, the vector in step S10 comprises an AB plasmid.

[0020] The present application also provides a phage library, which is prepared from a recombinant fusion protein library of human kappa light chain single chain antibodies obtained by the library construction method of the recombinant fusion protein of human kappa light chain single chain antibodies as described above.

[0021] In the technical solution provided by the present application, a new phage display single chain antibody library is used, luciferase (nanoluc) is coupled with human kappa light chain single chain antibodies, the molecular weight of the nanoluc is small, so that the size of the recombinant fusion protein is reduced, the electroporation efficiency and the library capacity are increased, and the gene III fusion protein in the phage is retained, that is, the ability to express on the surface of bacteria is retained, and multiple rounds of screening can be performed to effectively enrich positive clones, so that the library capacity is large, the recombinant fusion protein library of human kappa light chain single chain antibodies established has a capacity of 1x10 9 cfu / mL or more, and has the characteristics of high sensitivity and high signal-to-noise ratio. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other related drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0023] Figure 1 Enzyme activity diagram of the monoclonal antibody obtained in Example 1 of the present application;

[0024] Figure 2 Enzyme activity diagram in the supernatant of the overnight culture of the human kappa light chain single chain antibody library of the present application.

[0025] The implementation, functional characteristics and advantages of the present application will be further described in combination with the embodiments and drawings. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0027] It should be noted that the specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market. In addition, the meaning of "and / or" appearing throughout the text includes three parallel options. For example, "A and / or B" includes the A option, or the B option, or both A and B options. In addition, the technical solutions of various embodiments can be combined with each other, but must be based on the fact that a person skilled in the art can realize it. When the combination of technical solutions contradicts each other or cannot be realized, it should be considered that the combination of technical solutions does not exist and is not within the scope of protection required by the present application. Based on the examples in the present application, all other examples obtained by a person skilled in the art without creative labor are within the scope of protection of the present application.

[0028] At present, most of the phage display libraries use the method of coupling a tag protein (such as myc, FLAG tag, etc.) after a single-chain antibody sequence. In the subsequent screening and detection, the HRP-coupled tag protein antibody is used to recognize the single-chain antibody that binds to a specific ligand. The disadvantage is that a large number of incubation and washing steps are required, which can easily introduce false positives and false negatives. Moreover, although a sensitive signal amplification reporter system enzyme is used. However, the molecular weight of alkaline phosphatase (140kD), beta-galactosidase (115kD), horseradish peroxidase (44kD), and luciferase (62kD) and renilla luciferase (34kD) is relatively large, which greatly increases the size of the single-chain antibody-enzyme fusion protein, and further reduces the electroporation efficiency and library capacity.

[0029] In view of this, the present application provides a recombinant fusion protein of a human kappa light chain single-chain antibody, which aims to prepare a recombinant fusion protein with high electroporation efficiency, large library capacity, and reduced screening cost and steps, high sensitivity, and high signal-to-noise ratio.

[0030] To achieve the above-mentioned object, the present application provides a recombinant fusion protein of a human kappa light chain single-chain antibody, which is a recombinant fusion protein formed by connecting a human kappa light chain single-chain antibody with a phage Piii protein and luciferase. The amino acid sequence of the recombinant fusion protein of the human kappa light chain single-chain antibody is as shown in SEQ ID NO: 5:

[0031] MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKAAAEYPYDVPDYAVDGGGGSGGGGSGGGGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILADYKDDDDKTRXGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGD NSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

[0032] The present application provides a new recombinant fusion protein of phage display single-chain antibody, luciferase (nanoluc) is coupled with human kappa light chain single-chain antibody, and the ability of expressing on the surface of bacteria is retained, that is, the gene III fusion protein in phage is retained, and multiple rounds of screening can be carried out to effectively enrich positive clones, so that the library capacity obtained is large, the single-chain antibody TG1 library established is more than 1x109, and the library has the characteristics of high sensitivity, high signal-to-noise ratio, and each single clone fluorescence signal after electrotransformation is more than 107, and the fluorescence signal of untransfected cells is very low.

[0033] Further, the amino acid sequence of the human kappa light chain single-chain antibody is shown in SEQ ID NO: 6.

[0034] MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

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

[0036] DFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

[0037] It should be noted that a first linker protein is provided between the amino acid of the phage Piii protein and the luciferase, and the amino acid sequence of the first linker protein is shown as SEQ ID NO: 19: GGSGSG

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

[0039] VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0040] It should be noted that a second linker protein is provided between the amino acid of the luciferase and the phage Piii protein, and the amino acid sequence of the second linker protein is shown as SEQ ID NO: 20: GGGGSGGGGSGGGG

[0041] It should be noted that the nucleotide sequence of the recombinant fusion protein encoding the human kappa light chain single-chain antibody is as shown in SEQ ID NO: 1 as follows

[0042]

[0043] The nucleotide sequence encoding the human kappa light chain single chain antibody is as shown in SEQ ID NO: 2 as follows:

[0044] ATGAAATATCTGCTGCCGACGGCAGCAGCAGGTCTGCTGCTGCTGGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAACGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCTTTTCCCTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAG ACTGGTCGCATCCCGCCAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0045] The nucleotide sequence encoding the phage Piii protein is as shown in SEQ ID NO: 3 as follows:

[0046] GGCGGTTCTGGTAGCGGTGATTTCGATTACGAAAAAATGGCGAACGCCAACAAAGGTGCCATGACCGAAAATGCCGATGAAAATGCGCTGCAGAGCGATGCCAAAGGTAAACTGGATAGCGTTGCCACCGATTATGGTGCCGCCATTGATGGCTTTATTGGCGATGTTAGCGGCCTGGCGAATGGTAATGGTGCCACCGGTGATTTTGCCGGTAGCAATAGCCAGATGGCCCAGGTTGGTGATGGTGATAACAGCCCGCTGATGAACAACTTTCGTCAGTATCTGCCGAGCCTGCCGCAGAGCGTTGAATGTCGTCCGTTTGTGTTTGGCGCCGGCAAACCGTACGAATTTAGCATCGATTGTGATAAAATCAACCTGTTCCGTGGCGTTTTTGCCTTTCTGCTGTACGTGGCGACCTTTATGTATGTGTTCAGCACCTTTGCCAACATCCTGCGCAACAAAGAAAGCTAATAA

[0047] The nucleotide sequence encoding luciferase is as set forth in SEQ ID NO: 4 as follows:

[0048] GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAGTCTTCACGCTTGAAGACTTTGTCGGGGACTGGCGCCAAACCGCCGGTTACAATTTGGACCAAGTCCTGGAGCAGGGAGGTGTGTCTTCGTTATTCCAGAATTTGGGGGTTTCGGTGACACCGATTCAGCGCATTGTCTTATCAGGTGAGAATGGATTAAAGATTGATATTCATGTAATCATCCCCTACGAGGGCCTGTCCGGGGACCAGATGGGTCAAATCGAAAAGATTTTCAAGGTTGTGTACCCCGTGGACGACCATCATTTCAAAGTTATTTTACATTATGGTACGTTGGTGATTGACGGAGTAACACCAAACATGATCGACTACTTCGGGCGTCCTTATGAGGGCATCGCTGTTTTCGACGGCAAAAAAATCACCGTTACCGGCACGCTTTGGAATGGGAATAAAATCATCGACGAGCGCCTTATCAACCCAGATGGGTCCCTTCTGTTCCGTGTAACCATCAACGGAGTAACAGGGTGGCGTTTGTGCGAACGCATTTTGGCT

[0049] Phage display is a method in which antibody or polypeptide libraries are expressed on the surface of phages and specific antibody or polypeptide sequences are selected based on their ability to bind to a ligand. The principle of this method relies on the genetic recombination of the phage genome, the insertion of the sequence of interest next to the phage coat protein pill, forming a fusion protein, and the infection of bacteria by the recombinant phage, whose 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 selection step, different proteins or molecules (called ligands) are bound to the above-mentioned antibodies or polypeptides and can be enriched in subsequent selections. By sequencing, the antibody or polypeptide sequence that can bind to the ligand is obtained.

[0050] Nanoluc is the latest member of the luciferase system family. Compared with the commonly used luciferase and Renilla luciferase, Nanoluc has a luminescence of two orders of magnitude, a stable signal, a linear relationship of luminescence in a concentration range of 1000000 times, and a signal half-life of ≥2h. Therefore, Nanoluc can also detect the fluorescence signal at low concentration, and the detection sensitivity is greatly improved. The molecular weight of luciferase and Renilla luciferase is 62kD and 34kD respectively, while the size of Nanoluc is 19kD, so the size of the recombinant fusion protein is greatly reduced, and the electric conversion efficiency and library capacity are also increased, which is beneficial to the modification of genetic engineering and high-throughput screening.

[0051] Further, in order to the diversity of the library, the human kappa light chain single chain antibody in the application includes a light chain and a heavy chain, a plurality of primers are designed for the light chain and the heavy chain:

[0052] The plurality of primers designed for the light chain and the heavy chain of the human kappa light chain single chain antibody are used for amplification, wherein the plurality of primers include a first forward primer, a second forward primer, 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 and an eighth reverse primer, wherein the nucleotide sequence of the first forward primer is as shown in SEQ ID NO: 9, and the nucleotide sequence of the second forward primer is as shown in SEQ ID NO: 10; the nucleotide sequence of the first reverse primer is as shown in SEQ ID NO: 11, the second reverse primer is as shown in SEQ ID NO: 12, the third reverse primer is as shown in SEQ ID NO: 13, the fourth reverse primer is as shown in SEQ ID NO: 14, the fifth reverse primer is as shown in SEQ ID NO: 15, the sixth reverse primer is as shown in SEQ ID NO: 16, the seventh reverse primer is as shown in SEQ ID NO: 17, and the eighth reverse primer is as shown in SEQ ID NO: 18.

[0053] It should be noted that in the following sequence, M is A base or C base, and N is any base in ACGT;

[0054] The nucleotide sequence of the first forward primer is as shown in SEQ ID NO: 9, and the sequence is specifically:

[0055] TTTGACTACTGGGGCCAGGGAAC, and the sequence is named KLO FWD.

[0056] The nucleotide sequence of the second forward primer is shown as SEQ ID NO: 10, and specifically is: 5' tggcggcccagccggccatggccGAGGTGCAGCTGTTGGAGT 3', and the sequence is named HO FWD.

[0057] The nucleotide sequence of the first reverse primer light chain primer is shown as SEQ ID NO: 11, and specifically is:

[0058] CACCTTGGTCCCTTGGCCGAACGTMNNCGGMNNMNNACCMNNMNNCTGCTGACAGTAATACACTGC, and the sequence is named K1-REV.

[0059] The nucleotide sequence of the second reverse primer is shown as SEQ ID NO: 12, and specifically is:

[0060] CACCTTGGTCCCTTGGCCGAACGTMNNCGGMNNACCMNNMNNCTGCTGACAGTAATACACTGC, and the sequence is named K2-REV.

[0061] The nucleotide sequence of the third reverse primer is shown as SEQ ID NO: 13, and specifically is:

[0062] CACCTTGGTCCCTTGGCCGAACGTMNNCGGMNNMNNMNNMNNCTGCTGACAGTAATACACTGC, and the sequence is named K3-REV.

[0063] The nucleotide sequence of the fourth reverse primer is shown as SEQ ID NO: 14, and specifically is:

[0064] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNTTTCGCACAGTAATATACGGCC, and the sequence is named H4-REV.

[0065] The nucleotide sequence of the fifth reverse primer is shown as SEQ ID NO: 15, and specifically is:

[0066] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNTTTCGCACAGTAATATACGG, and the sequence is named H5-REV.

[0067] The nucleotide sequence of the sixth reverse primer is shown as SEQ ID NO: 16, and specifically is:

[0068] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNTTT CGCACAGTAATATA, the sequence of which is named H6-REV.

[0069] The nucleotide sequence of the seventh reverse primer is shown in SEQ ID NO: 17, and the sequence is specifically as follows:

[0070] GTTCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNMNNMNNMN NTTTCGCACAGTAATATACGGCC, the sequence of which is named H7-REV.

[0071] The nucleotide sequence of the eighth reverse primer is shown in SEQ ID NO: 18, and the sequence is specifically as follows: GGCACATCATAAGGGTATtctGCGGCCGCTTTGATTTCCACCTTGGTCCCTT GGCCGAACGT, the sequence of which is named KNotIREV.

[0072] The application further provides a library construction method of a recombinant fusion protein of a human kappa light chain single-chain antibody, comprising the following steps:

[0073] S10, the recombinant fusion protein described in any one of the above is used as a template to clone a connecting vector, so as to obtain a recombinant plasmid.

[0074] Further, the vector in step S10 comprises an AB plasmid.

[0075] In some embodiments, the specific steps comprise: the human kappa light chain single-chain antibody template and the AB plasmid are subjected to double enzyme digestion by using HindIII and AgeI, the template is cloned into the AB plasmid, so as to obtain a recombinant plasmid, which is referred to as an ABHK plasmid.

[0076] S20, a plurality of primers of a human kappa light chain single-chain antibody are provided, a light chain and a heavy chain of the human kappa light chain single-chain antibody are amplified respectively by using the recombinant plasmid as a template, so as to obtain a plurality of heavy chain fragments and a plurality of light chain fragments, wherein the amino acid sequence of the human kappa light chain single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence comprises a light chain and a heavy chain, and a plurality of primers are designed for the light chain and the heavy chain.

[0077] Specifically, in some embodiments, the operation steps comprise:

[0078] Reaction 1: the ABHK plasmid is used as a template, and HO FWD and H4-REV primers are used, so as to obtain a product H4.

[0079] Reaction 2: ABHK plasmid as template, HO FWD and H5-REV primers, product H5 was obtained.

[0080] Reaction 3: ABHK plasmid as template, HO FWD and H6-REV primers, product H6 was obtained.

[0081] Reaction 4: ABHK plasmid as template, HO FWD and H7-REV primers, product H7 was obtained.

[0082] Reaction 5: ABHK plasmid as template, KLO FWD and K1-REV primers, product K1 was obtained.

[0083] Reaction 6: ABHK plasmid as template, KLO FWD and K2-REV primers, product K2 was obtained.

[0084] Reaction 7: ABHK plasmid as template, KLO FWD and K3-REV primers, product K3 was obtained.

[0085] The above PCR reaction system and conditions are as follows: pre-denaturation at 98℃ for 30 seconds; denaturation at 98℃ for 10 seconds; annealing at 60℃ for 30 seconds; extension at 72℃ for 15 seconds, 35 cycles were performed, and then the sample was continuously extended at 72℃ for 5 min, with a total of 50 microliter system. The target fragments were recovered at about 400bp on the agarose gel electrophoresis.

[0086] S30, mixing each of the heavy chain fragments with each of the light chain fragments, and performing PCR reaction to obtain a plurality of reaction mixture products.

[0087] Specifically, in some embodiments, the operation steps include:

[0088] Reaction 1: mixing the first heavy chain product H4 and the first light chain product K1 in a molar ratio of 1:1, and performing PCR reaction with HO FWD and KNotIREV as primers to obtain the first mixed product H4K1.

[0089] Reaction 2: mixing the first heavy chain product H4 and the second light chain product K2 in a molar ratio of 1:1, and performing PCR reaction with HO FWD and KNotIREV as primers to obtain the first mixed product H4K2.

[0090] Reaction 3: mixing the first heavy chain product H4 and the third light chain product K3 in a molar ratio of 1:1, and performing PCR reaction with HO FWD and KNotIREV as primers to obtain the first mixed product H4K3.

[0091] Reaction 4: The second heavy chain product H5 and the first light chain product K1 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H5K1.

[0092] Reaction 5: The second heavy chain product H5 and the first light chain product K2 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H5K2.

[0093] Reaction 6: The second heavy chain product H5 and the first light chain product K3 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H5K3.

[0094] Reaction 7: The third heavy chain product H6 and the first light chain product K1 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H6K1.

[0095] Reaction 8: The third heavy chain product H6 and the first light chain product K2 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H6K2.

[0096] Reaction 9: The third heavy chain product H6 and the first light chain product K3 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H6K3.

[0097] Reaction 10: The fourth heavy chain product H7 and the first light chain product K1 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H7K1.

[0098] Reaction 11: The fourth heavy chain product H7 and the first light chain product K2 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H7K2.

[0099] Reaction 12: The fourth heavy chain product H7 and the first light chain product K3 were mixed in a molar ratio of 1 : 1, and PCR was performed with HO FWD and KNotI REV as primers to obtain the first mixed product H7K3.

[0100] The above PCR reaction system and conditions are as follows: pre-denaturation at 98℃ for 30 seconds; denaturation at 98℃ for 10 seconds; annealing at 60℃ for 30 seconds; extension at 72℃ for 15 seconds, 35 cycles are performed, and then the sample is continuously extended at 72℃ for 5 minutes, with a total of 50 microliters of system. Run agarose gel electrophoresis, and recover the target fragment at about 800 bp.

[0101] S40, respectively, the plurality of reaction mixture products are subjected to double enzyme digestion with the recombinant plasmid, and then a plurality of plasmids are obtained.

[0102] Specifically, the operation steps include: the recombinant plasmid is subjected to enzyme digestion, and the enzyme digestion system is as follows:

[0103] Table 1 Enzyme digestion system of recombinant plasmid

[0104]

[0105]

[0106] Reaction conditions: after enzyme digestion at 37℃ for 7 hours, run agarose gel electrophoresis, and recover the vector fragment.

[0107] The enzyme digestion system of the plurality of reaction mixture products is shown in the following table:

[0108] Table 2 Enzyme digestion system

[0109] Reagents Volume / mass H4K1 30 μg 10x enzyme buffer 30 μl Not I 10 μl Nco I 10 μl ddH2O fill to 300 μl

[0110] Reaction conditions: after enzyme digestion at 37℃ for 7 hours, run agarose gel electrophoresis, and recover the large fragment, and the same treatment method is used for H4K2, H4K3, H5K1, H5K2, H5K3, H6K1, H6K2, H6K3, H7K1, H7K2, and H7K3 fragments, which are subjected to similar enzyme digestion treatment.

[0111] The above enzyme-digested vector and the above enzyme-digested antibody fragment are connected into a complete vector:

[0112] Table 3 Connection system

[0113] Reagents Volume / mass ABHK cut 30 μg H4K1 cut 20 μg 10x enzyme buffer 45 μl T4 ligase 20 μl ddH2O fill to 450 μl

[0114] Reaction conditions: ligation at 16°C for 16-24 hours. After adding phenol to remove protein, the DNA was dissolved in ultrapure water after washing with 70% ethanol to obtain the first plasmid H4K1AB plasmid. Similarly, the enzyme-digested H4K2, H4K3, H5K1, H5K2, H5K3, H6K1, H6K2, H6K3, H7K1, H7K2, H7K3 were treated in the same way to obtain H4K2AB, H4K3AB, H5K1AB, H5K2AB, H5K3AB, H6K1AB, H6K2AB, H6K3AB, H7K1AB, H7K2AB, H7K3AB plasmids.

[0115] S50, after mixing the first plasmid, the second plasmid, the third plasmid, the fourth plasmid, the fifth plasmid, the sixth plasmid, the seventh plasmid, the eighth plasmid, the ninth plasmid, the tenth plasmid, the eleventh plasmid and the twelfth plasmid, the recombinant fusion protein library of human kappa light chain single chain antibody is obtained by electrotransformation and amplification.

[0116] In summary, the library construction method of the recombinant fusion protein of human kappa light chain single chain antibody provided in the present application makes the recombinant fusion protein of human kappa light chain single chain antibody prepared have the advantages of large library capacity, the TG1 library of single chain antibody established has a capacity of 1x10 9 The above, and can be screened for multiple rounds, and the positive clones capable of binding to ligands are enriched, and have the characteristics of high sensitivity and high signal-to-noise ratio.

[0117] The technical solutions of the present application are further described in detail in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application, and are not used to limit the present application.

[0118] Example 1: Construction of library

[0119] 1) The electrocompetent cells (TG1) were inoculated into 1000 ml of 2-TY medium (containing 0.01% F-68) and cultured at 18°C to OD 0.6.

[0120] 2) After collecting the bacteria, centrifugation was performed at 3000g for 10 minutes, and the supernatant was removed.

[0121] 3) The precipitates were washed with 1000 ml, 500 ml, and 200 ml of wash buffer (10% glycerol, 1.5% mannitol, 1 mM Hepes), respectively, and the residual liquid was removed as much as possible.

[0122] 4) 5 ml of buffer (10% glycerol + 1.5% mannitol) was added, and centrifugation was performed at 3000g for 5 minutes, and the supernatant was removed with a gun head.

[0123] 5) Precipitate resuspended in 1.5ml buffer (10% glycerol + 1.5% mannitol), add the constructed plasmid H4K1AB respectively, mix well and put on ice for 10 minutes. 50μl aliquot into pre-cooled electrotransformation cup, 1200V electric shock for 5ms. Add 110μl 2-TY glucose medium, the electrotransformation cup is placed in 37 degree incubator for 1 hour, after all the transformants are collected, average coating in 40 150mm 2-TY agar plates (2% glucose, 100μg / ml ampicillin), 30 degrees overnight, the same method to prepare H4K2AB, H4K3AB, H5K1AB, H5K2AB, H5K3AB, H6K1AB, H6K2AB, H6K3AB, H7K1AB, H7K2AB, H7K3AB plasmid.

[0124] 6) After the colonies grow, add 3ml freezing solution (25% glycerol, 100μg / ml ampicillin, 1% glucose) to each plate, use a sterile glass rod to scrape all the colonies on the plate and collect the bacterial suspension, which is the constructed TG1 library, take 50μl, and take 10-fold gradient dilution for standby.

[0125] Calculate the library capacity, the human kappa light chain single chain antibody library capacity is 4.21x10 9 cfu / mL.

[0126] Example 2 Quality detection of the library

[0127] (1) Randomly pick 42 HKAB monoclonal colonies on the plate, culture overnight in 2-TY medium. The next day, centrifuge 30μl supernatant into a white opaque plate, add nanoluc substrate, and detect the fluorescence signal. The results are shown in Figure 1. Figure 1 As shown in Figure 1, the fluorescence signal in untransfected TG1 cells is very low (<104), and the fluorescence signal of each monoclonal after electrotransformation is above 107.

[0128] (2) Culture untransfected TG1 and HK library overnight in 2-TY medium, with or without IPTG, harvest the supernatant the next day, add nanoluc substrate after WB gel transfer and membrane. As shown in Figure 2: Figure 2 The results show that untransfected TG1 has no band, and nanoluc-coupled single chain antibody can be detected in HK culture supernatant. The above results show that the library construction is successful.

[0129] Example 3 Human kappa single chain antibody phage display library

[0130] 1) Add 200ml 2TY medium to a triangular flask, inoculate with HK library bacteria

[0131] 2) Dilute to OD600 = 0.1, add 100μg / ml ampicillin, 1% glucose. Incubate at 37°C to OD600 = 0.5, add 20:1 M13KO7, water bath for 40 minutes

[0132] 3) 3000g for 20 minutes, remove supernatant, resuspend pellet in 100μg / ml ampicillin, 50μg / ml kanamycin

[0133] 4) Incubate at 30°C, 220rpm overnight

[0134] 5) 3000g for 20 minutes

[0135] 6) Transfer supernatant to a new container, add 1 / 5 volume of 20% (w / v) PEG 6000 / 2.5M NaCl (PEG / NaCl), mix well, place on ice for 2 hours

[0136] 7) 10800g for 20 minutes

[0137] 8) Resuspend pellet in 40ml PBS

[0138] 9) Add 1 / 5 volume of PEG / NaCl, mix well, place on ice for at least 20 minutes

[0139] 10) 10800g for 15 minutes, remove supernatant

[0140] 11) Short spin, remove residual PEG / NaCl

[0141] 12) Add 20ml 10% glycerol in PBS, aliquot 500μl per tube

[0142] 13) Determine phage titer by 10-fold dilution. 109fold dilution has 10 clones, 10x10 9 x200 = 2x10 12 / ml, each aliquot of 500μl should contain >10 12 pfu

[0143] 14) Store at -20

[0144] To sum up, the technical scheme provided by the application adopts a new phage display single-chain antibody library, couples luciferase (nanoluc) with human kappa light chain single-chain antibody, and retains the gene III fusion protein in the phage, so that the human kappa light chain single-chain antibody recombinant fusion protein is screened out, the library established by the human kappa light chain single-chain antibody recombinant fusion protein has a large capacity, the single-chain antibody TG1 library established has a capacity of 1x10 9 cfu / mL or more, and can be screened for multiple rounds, so that positive clones capable of binding to ligands are enriched, and has the characteristics of high sensitivity and high signal-to-noise ratio.

[0145] The above only describes the preferred embodiments of the application, and does not limit the patent scope of the application, and any equivalent structural transformation made by using the content of the application specification, or direct / indirect application in other related technical fields is included in the patent protection scope of the application.

Claims

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

5. The recombinant fusion protein of the human kappa light chain single-chain antibody is obtained by translating the DNA molecule shown in SEQ ID NO:1 into the TG1 strain.

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

6.

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

7.

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

8.

5. A method for constructing a library of a recombinant fusion protein of a human kappa light chain single-chain antibody, characterized in that, Includes the following steps: S10. Using the recombinant fusion protein of the human kappa light chain single-chain antibody as described in any one of claims 1 to 4 as a template, a cloning ligation vector is used to obtain a recombinant plasmid. S20. Provide a human kappa light chain single-chain antibody. Using the recombinant plasmid as a template, amplify the light chain and heavy chain of the human kappa light chain single-chain antibody to obtain multiple heavy chain fragments and multiple light chain fragments. The amino acid sequence of the human kappa light chain single-chain antibody is shown in SEQ ID NO:6, which includes a light chain and a heavy chain. Design multiple primers for the light chain and the heavy chain. S30. Mix each of the heavy chain fragments with each of the light chain fragments respectively, and perform PCR reaction to obtain multiple reaction mixture products; S40. The multiple reaction mixtures are respectively double-digested with the recombinant plasmid and ligated to obtain multiple plasmids; S50. After mixing the multiple plasmids, electroporate them into the recombinant fusion protein library of TG1 strain and amplified human Kappa light chain single-chain antibody.

6. The method for constructing a library of the recombinant fusion protein of human kappa light chain single-chain antibody as described in claim 5, characterized in that, The vector in step S10 includes the AB plasmid.

7. A phage library, characterized in that, The phage library is constructed from the recombinant fusion protein library of human kappa light chain single-chain antibody obtained by the library construction method of the recombinant fusion protein of human kappa light chain single-chain antibody as described in claim 5 or 6.

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