Recombinant fusion protein of murine single-chain antibody, DNA (deoxyribonucleic acid) molecule and library building method

Abstract

The invention discloses a recombinant fusion protein of a murine single-chain antibody, a DNA molecule and a library building method, the recombinant fusion protein of the murine single-chain antibody is the recombinant fusion protein formed by connecting the murine single-chain antibody with phage Piii protein and luciferase, the amino acid sequence of the recombinant fusion protein of the murine single-chain antibody is shown as SEQ ID NO: 5, and the amino acid sequence of the recombinant fusion protein of the murine single-chain antibody is shown as SEQ ID NO: 6. According to the invention, luciferase is coupled with a mouse source single-chain antibody, and nanoluc is small in molecular weight, so that the size of recombinant fusion protein is reduced, the electrotransfection efficiency and the electrotransfection capacity are increased, gene III fusion protein in bacteriophage is retained, namely, the capability of expression on the surface of bacteria is retained, multiple rounds of screening can be carried out to effectively enrich positive clones, and the screening efficiency is improved. The obtained library is large in capacity, the established single-chain antibody TG1 library is more than 1 * 10 < 9 > cfu / mL, and the 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 murine single-chain antibody, a DNA molecule and a library construction method. Background Art

[0002] 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 HRP-conjugated tag protein antibody is used to recognize and bind a single-chain antibody that binds to a specific ligand. 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 a phage display library, 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, thereby reducing the electroporation efficiency and library capacity.

[0004] In some literatures, for example, Michael Tesar et al. cloned Nanoluc 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 for single-chain antibodies that can bind to 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 performed 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 murine single-chain antibody, a DNA molecule and a library construction method, 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.

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

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

[0008] Optionally, the murine single-chain antibody comprises a light chain and a heavy chain: wherein, a plurality of primers are designed for the light chain and the heavy chain.

[0009] The present invention further provides a DNA molecule for encoding a recombinant fusion protein of the murine single-chain antibody as described above, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:1.

[0010] The present invention further provides a method for constructing a library of recombinant fusion proteins of murine single-chain antibodies, comprising the following steps:

[0011] S10: Using the recombinant fusion protein of the murine single-chain antibody as described in any one of the above as a template, cloning and ligating a vector to obtain a recombinant plasmid;

[0012] S20: Providing primers for the murine single-chain antibody, using the recombinant plasmid as a template, respectively amplifying the light chain and the heavy chain of the murine single-chain antibody to obtain a plurality of heavy chain fragments and light chain fragments, wherein the amino acid sequence of the murine single-chain antibody is shown in SEQ ID NO:6, and a plurality of primers are designed for the light chain and the heavy chain of the murine single-chain antibody;

[0013] S30: Mixing the plurality of heavy chain fragments and the plurality of light chain fragments, and performing PCR reactions to obtain a plurality of mixed products respectively;

[0014] S40: Respectively performing double digestion and ligation of the plurality of mixed products with the recombinant plasmid to obtain a plurality of plasmids;

[0015] S50: Mixing the plurality of plasmids, and performing electrotransformation and amplification to obtain a library of recombinant fusion proteins of murine single-chain antibodies.

[0016] Optionally, the vector in step S10 comprises an AB plasmid.

[0017] In the technical solution provided by the present invention, a new phage display single-chain antibody library is used, luciferase (nanoluc) is conjugated with the murine single-chain antibody, and the molecular weight of the nanoluc is small, so the size of the recombinant fusion protein is reduced, thus increasing the electrotransformation efficiency and library capacity, and retaining the gene III fusion protein in the phage, that is, retaining the ability to be expressed on the bacterial surface, and being able to perform multiple rounds of screening to effectively enrich positive clones. The library obtained in this way has a large capacity, and the constructed library of recombinant fusion proteins of murine single-chain antibodies is above 1x10 9 cfu / mL, and has characteristics such as high sensitivity and high signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 be obtained based on these drawings.

[0019] Figure 1 Schematic diagram of enzyme expression for the monoclonal antibody obtained in Example 1 of the present invention;

[0020] Figure 2 Schematic diagram of enzyme expression in the MH4LAB monoclonal supernatant obtained in Example 1 of the present invention.

[0021] The realization, functional features, and advantages of the object of the present invention will be further described in conjunction with the embodiments and the drawings. Detailed implementation manners

[0022] 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. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0023] It should be noted that for those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by 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 solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or the solution where A and B are satisfied simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory 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 of 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.

[0024] Currently, the vast majority of phage display libraries use a technical method of conjugating a tag protein (such as myc, FLAG tags, etc.) after the single-chain antibody sequence. In subsequent screening and detection, an HRP-conjugated tag protein antibody is used to recognize and bind to the single-chain antibody that specifically binds to a ligand. Its disadvantages are that it requires a large number of incubation and washing steps, 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 relatively large, which greatly increases the size of the single-chain antibody-enzyme fusion protein, thereby reducing the electroporation efficiency and library capacity.

[0025] In view of this, a recombinant fusion protein of a murine single-chain antibody proposed by the present invention aims to prepare a recombinant fusion protein with high electroporation efficiency, large library capacity, reduced screening costs and steps, and high sensitivity and high signal-to-noise ratio.

[0026] To achieve the above object, the present invention provides a recombinant fusion protein of a murine single-chain antibody, wherein the recombinant fusion protein of the murine single-chain antibody is formed by connecting a murine single-chain antibody with phage Piii protein and luciferase. Among them, the amino acid sequence of the recombinant fusion protein of the murine single-chain antibody is shown in SEQ ID NO:5:

[0027] MKYLLPTAAAGLLLLAAQPAMAQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGYTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARPSSLPQFDYWGQGTTVTVSSGGGGSGGGGSGGGGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQMYHYPRTFGGGTKLEIKRTVAAAAAEYPYDVPDYAVDGGGGSGGGGSGGGGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILADYKDDDDKTRXGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

[0028] The present invention provides a recombinant fusion protein of a murine single-chain antibody of a novel phage-displayed single-chain antibody, which conjugates luciferase (nanoluc) with the murine single-chain antibody and retains the gene III fusion protein in the phage, that is, retains the ability to be expressed on the bacterial surface, and can perform multiple rounds of screening to effectively enrich positive clones. The library obtained in this way has a large 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.

[0029] Furthermore, the amino acid sequence of the murine single-chain antibody is as shown in SEQ ID NO:6:

[0030] MKYLLPTAAAGLLLLAAQPAMAQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGYTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARPSSLPQFDYWGQGTTVTVSSGGGGSGGGGSGGGGQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQMYHYPRTFGGGTKLEIKRTVAA

[0031] Furthermore, the present invention provides a DNA molecule for encoding the recombinant fusion protein of the murine single-chain antibody as described above, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:1:

[0032]

[0033] The nucleotide sequence for encoding the murine single-chain antibody as described above is shown as SEQ ID NO:2 as follows:

[0034] ATGAAATATCTGCTGCCGACGGCAGCAGCAGGTCTGCTGCTGCTGGCGGCCCAGCCGGCCATGGCCCAGGTGCAACTGCAGCAACCGGGTGCGGAGCTGGTGAAACCGGGTGCGAGCGTTAAAATGAGCTGCAAGGCGAGCGGTTACACCTTCACCAGCTATAACATGCACTGGGTTAAGCAGACCCCGGGCCGTGGTCTGGAATGGATCGGTGCGATTTACCCGGGCAACGGTTACACCAGCTATAACCAGAAGTTTAAAGGCAAGGCGACCCTGACCGCGGACAAAAGCAGCAGCACCGCGTACATGCAACTGAGCAGCCTGACCAGCGAGGACAGCGCGGTGTACTATTGCGCGCGTCCGAGCAGCCTGCCGCAGTTCGATTATTGGGGCCAAGGTACCACCGTTACCGTTAGCAGCGGTGGCGGTGGCAGCGGTGGCGGTGGCAGCGGTGGCGGTGGCCAGATCGTTCTGAGCCAAAGCCCGGCGATTCTGAGCGCGAGCCCGGGTGAAAAGGTGACCATGACCTGCCGTGCGAGCAGCAGCGTTAGCTACATGCACTGGTTCCAGCAAAAACCGGGTAGCAGCCCGAAGCCGTGGATCTATGCGACCAGCAACCTGGCGAGCGGCGTGCCGGTTCGTTTTAGCGGTAGCGGTAGCGGTACCAGCTACAGCCTGACCATTAGCCGTGTGGAGGCGGAAGATGCGGCGACCTACTATTGCCAGCAAATGTACCACTATCCGCGTACCTTTGGTGGCGGTACCAAACTGGAGATCAAGCGTACCGTTGCGGCG

[0035] The nucleotide sequence for encoding the phage Piii protein is shown as SEQ ID NO:3 as follows:

[0036] GGCGGTTCTGGTAGCGGTGATTTCGATTACGAAAAAATGGCGAACGCCAACAAAGGTGCCATGACCGAAAATGCCGATGAAAATGCGCTGCAGAGCGATGCCAAAGGTAAACTGGATAGCGTTGCCACCGATTATGGTGCCGCCATTGATGGCTTTATTGGCGATGTTAGCGGCCTGGCGAATGGTAATGGTGCCACCGGTGATTTTGCCGGTAGCAATAGCCAGATGGCCCAGGTTGGTGATGGTGATAACAGCCCGCTGATGAACAACTTTCGTCAGTATCTGCCGAGCCTGCCGCAGAGCGTTGAATGTCGTCCGTTTGTGTTTGGCGCCGGCAAACCGTACGAATTTAGCATCGATTGTGATAAAATCAACCTGTTCCGTGGCGTTTTTGCCTTTCTGCTGTACGTGGCGACCTTTATGTATGTGTTCAGCACCTTTGCCAACATCCTGCGCAACAAAGAAAGCTAATAA

[0037] The nucleotide sequence for encoding the luciferase is shown as follows in SEQ ID NO:4:

[0038] GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAGTCTTCACGCTTGAAGACTTTGTCGGGGACTGGCGCCAAACCGCCGGTTACAATTTGGACCAAGTCCTGGAGCAGGGAGGTGTGTCTTCGTTATTCCAGAATTTGGGGGTTTCGGTGACACCGATTCAGCGCATTGTCTTATCAGGTGAGAATGGATTAAAGATTGATATTCATGTAATCATCCCCTACGAGGGCCTGTCCGGGGACCAGATGGGTCAAATCGAAAAGATTTTCAAGGTTGTGTACCCCGTGGACGACCATCATTTCAAAGTTATTTTACATTATGGTACGTTGGTGATTGACGGAGTAACACCAAACATGATCGACTACTTCGGGCGTCCTTATGAGGGCATCGCTGTTTTCGACGGCAAAAAAATCACCGTTACCGGCACGCTTTGGAATGGGAATAAAATCATCGACGAGCGCCTTATCAACCCAGATGGGTCCCTTCTGTTCCGTGTAACCATCAACGGAGTAACAGGGTGGCGTTTGTGCGAACGCATTTTGGCT

[0039] 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 to a ligand. The principle of this method relies on genetic recombination of the phage genome. A sequence of interest is inserted beside the phage coat protein pIII to form a fusion protein, and then the recombinant phage infects bacteria and its genome is replicated. Expression of the recombinant phage genome results in 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.

[0040] Nanoluc is the latest member of the luciferase system family. Compared with the commonly used luciferase and Renilla luciferase, Nanoluc emits light two orders of magnitude higher, has a stable signal, and the light emission is linearly related within a concentration range spanning 1,000,000 times, and the signal half-life ≥ 2 h. Therefore, the fluorescence signal can be detected even at low concentrations, and the detection sensitivity is greatly improved. The molecular weights of luciferase and Renilla luciferase are 62 kD and 34 kD respectively, while Nanoluc is 19 kD in size, so the size of the recombinant fusion protein is greatly reduced, which in turn increases the electroporation efficiency and library capacity, facilitating genetic engineering modification and high-throughput screening.

[0041] Further, in order to establish the diversity of the library, the murine single-chain antibody described in the present invention includes a light chain and a heavy chain. Among them, multiple primers are designed for the light chain and the heavy chain. The multiple 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, and a fifth reverse primer. The nucleotide sequences of the first forward primer are respectively as shown in SEQ ID NO:7, and the nucleotide sequence of the second forward primer is as shown in SEQ ID NO:8; the nucleotide sequence of the first reverse primer is as shown in SEQ ID NO:9, 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 nucleotide sequence of the fourth reverse primer is as shown in SEQ ID NO:12, and the nucleotide sequence of the fifth reverse primer is as shown in SEQ ID NO:13.

[0042] 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;

[0043] The nucleotide sequence of the first forward primer is as shown in SEQ ID NO:7:

[0044] mL-FWD: TTCGATTATTGGGGCCAAGGTAC

[0045] The nucleotide sequence of the second forward primer is as shown in SEQ ID NO:8:

[0046] MHFWD: tggcggcccagccggccatggcc

[0047] The nucleotide sequence of the first reverse primer is as shown in SEQ ID NO:9, and its sequence is specifically:

[0048] CAGTTTGGTACCGCCACCAAAGGTMNNCGGMNNMNNMNNMNNTT GCTGGCAATAGTAGGTCGCCGCA, and the name of this sequence is mL-REV.

[0049] The nucleotide sequence of the second reverse primer is shown in SEQ ID NO:10, and its specific sequence is:

[0050] tctgcggccgcCGCCGCAACGGTACGCTTGATCTCCAGTTTGGTACCGCC ACCAAAGGT, and the name of this sequence is mLNotI-REV.

[0051] The nucleotide sequence of the third reverse primer is shown in SEQ ID NO:11, and its specific sequence is: GTACCTTGGCCCCAATAATCGAAMNNMNNMNNMNNACGCGCGCAATA GTACACCGCGCT, and the name of this sequence is MH4-REV.

[0052] The nucleotide sequence of the fourth reverse primer is shown in SEQ ID NO:12, and its specific sequence is:

[0053] GTACCTTGGCCCCAATAATCGAAMNNMNNMNNMNNMNNACGCGC GCAATAGTACACCGCGCT, and the name of this sequence is MH5-REV.

[0054] The nucleotide sequence of the fifth reverse primer is shown in SEQ ID NO:13, and its specific sequence is:

[0055] GTACCTTGGCCCCAATAATCGAAMNNMNNMNNMNNMNNMNNACG CGCGCAATAGTACACCGCGCT, and the name of this sequence is MH6-REV.

[0056] The present invention further provides a method for constructing a library of recombinant fusion proteins of murine single-chain antibodies, comprising the following steps:

[0057] S10: Using the recombinant fusion protein described in any one of the above as a template, cloning and ligating a vector to obtain a recombinant plasmid;

[0058] Further, the vector in step S10 includes plasmid AB.

[0059] In some embodiments, the specific steps include: double-digesting the murine single-chain antibody template and plasmid AB with HindIII and AgeI, cloning the template into plasmid AB to obtain plasmid ABM.

[0060] S20. Provide primers for the murine single-chain antibody. Using the recombinant plasmid as a template, amplify the light chain and heavy chain of the murine single-chain antibody respectively to obtain multiple heavy chain fragments and light chain fragments. Among them, the amino acid sequence of the murine single-chain antibody is as shown in SEQ ID NO: 6, and multiple primers are designed for the light chain and heavy chain of the murine single-chain antibody.

[0061] Specifically, in some embodiments, the operation steps include:

[0062] Reaction 1: Using the ABM plasmid as a template, with the MHFWD and MH4-REV primers, to obtain the product MH4.

[0063] Reaction 2: Using the ABM plasmid as a template, with the MHFWD and MH5-REV primers, to obtain the product MH5.

[0064] Reaction 3: Using the ABM plasmid as a template, with the MHFWD and MH6-REV primers, to obtain the product MH6.

[0065] Reaction 4: Using the ABM plasmid as a template, with the mL-FWD and mL-REV primers, to obtain the product mL.

[0066] 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 about 400 bp.

[0067] S30. Mix the multiple heavy chain fragments and the multiple light chain fragments, and perform PCR reactions respectively to obtain multiple mixed products.

[0068] Specifically, in some embodiments, the operation steps include:

[0069] Reaction 1: Mix the first heavy chain product MH4 and the first light chain product mL at a molar ratio of 1:1. Using MH FWD and mLNotI REV as primers, perform PCR reaction to obtain the first mixed product MH4L.

[0070] Reaction 2: Mix the second heavy chain product MH5 and the second light chain product mL at a molar ratio of 1:1. Using the MH FWD and mLNotIREV primers, obtain the second mixed product MH5L.

[0071] Reaction 3: Mix the third heavy chain product MH6 and the third light chain product mL at a molar ratio of 1:1. Using the MH FWD and mLNotIREV primers, obtain the third mixed product MH6L.

[0072] 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 is further 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 800 bp.

[0073] S40. Respectively, subject the multiple mixed products and the recombinant plasmid to double digestion and ligation to obtain multiple plasmids;

[0074] Specifically, the operation steps include: digest the recombinant plasmid, and the digestion system:

[0075] Table 1 Digestion system of recombinant plasmid

[0076] Reagent Volume / Mass ABM plasmid 100 μg 10x Restriction Enzyme Buffer 50 μl Not I 15 μl Nco I 15 μl <![CDATA[ddH2O]]> fill to 500 μl

[0077] Reaction conditions: After digestion at 37°C for 7 hours, run agarose gel electrophoresis and recover the vector fragment.

[0078] The digestion systems of the multiple mixed products are shown in the following table:

[0079] Table 2 Digestion system

[0080] Reagent Volume / Mass MH4L 30 μg 10x Restriction Enzyme Buffer 30 μl Not I 10 μl Nco I 10 μl <![CDATA[ddH2O]]> fill to 300 μl

[0081] Reaction conditions: After digestion at 37°C for 7 hours, run agarose gel electrophoresis and recover the large fragment. Using the same treatment method, perform similar digestion treatments on the MH5L and MH6L fragments.

[0082] Ligate the digested vector and the digested antibody fragment into a complete vector:

[0083] Table 3 Digestion system

[0084] Reagent Volume / Mass Digested ABM 30 μg Digested MH4L 20 μg 10x Restriction Enzyme Buffer 45 μl T4 Ligase 20 μl <![CDATA[ddH2O]]> fill to 450 μl

[0085] 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, MH4LAB plasmid. Similarly, perform similar treatments on the digested MH5L and MH6L to obtain the second plasmid, MH5LAB, and the third plasmid, MH6LAB plasmid.

[0086] S50. Mix the multiple plasmids, followed by electrotransformation and amplification to obtain a recombinant fusion protein library of murine single-chain antibodies.

[0087] In summary, the method for constructing a library of recombinant fusion proteins of murine single-chain antibodies proposed in this application enables the prepared recombinant fusion proteins of murine single-chain antibodies to have a large library capacity. The established single-chain antibody TG1 library has more than 1x10 9 cfu / mL, and can be screened in multiple rounds to enrich positive clones that can bind ligands, and has the characteristics of high sensitivity, high signal-to-noise ratio, and a fluorescence signal of each monoclonal after electroporation being above 10 7 , while the fluorescence signal of untransfected cells is very low.

[0088] 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.

[0089] Example 1 Construction of the library

[0090] 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.

[0091] After collecting the bacteria, centrifuge at 3000 g for 10 minutes and discard the supernatant.

[0092] 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.

[0093] Add 5 ml of buffer (10% glycerol + 1.5% mannitol), centrifuge at 3000 g for 5 minutes, and aspirate the supernatant with a pipette tip.

[0094] Resuspend the precipitate in 1.5 ml of buffer (10% glycerol + 1.5% mannitol), add the constructed plasmid MH4LAB and mix well, then place on ice for 10 minutes. Aliquot 50 μl into pre-cooled electroporation cuvettes and perform electroporation at 1200 V for 5 ms. Add 110 μl of 2-TY glucose medium, place the electroporation cuvette in a 37°C incubator for 1 hour, collect all the transformants and evenly spread them on 40 150-mm 2-TY agar plates (2% glucose, 100 μg / ml ampicillin), and culture overnight at 30°C. Prepare MH5LAB and MH6LAB in the same way.

[0095] After the colonies grew out, 3 ml of cryopreservation solution (25% glycerol, 100 μg / ml ampicillin, 1% glucose) was added to each plate. The colonies on the plate were scraped off with a sterile glass rod and the cell suspension was collected. This was the constructed TG1 library. 50 μl was reserved, and the reserved bacterial solution was serially diluted 10-fold for standby.

[0096] The library capacity was calculated, and the capacity of the murine single-chain antibody library was 5.74x10 9 .

[0097] Example 2 Quality Detection of the Library

[0098] (1) Randomly select 23 MH monoclonal clones 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 nanoluc substrate, and detect the fluorescence signal. The results are shown in the appendix Figure 1 as follows: The fluorescence signal in untransfected TG1 cells was very low (<104), while the fluorescence signal of each monoclonal clone after electroporation was above 107.

[0099] (2) Spot 5 μl of the MH4LAB monoclonal culture solution on a nitrocellulose membrane, dry it at room temperature, and block it with 5% BSA. Then add nanoluc substrate. As shown in the appendix Figure 2 [[ID=I8]]as follows: It was found that there was no signal in blank and untransfected TG1, and a strong signal appeared in MH4LAB. The above results indicate that the construction of the library was successful.

[0100] 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 the murine single-chain antibody, and the gene III fusion protein in the phage is retained. The recombinant fusion protein of the murine single-chain antibody is screened out. The recombinant fusion protein of the murine single-chain antibody has a large library capacity. The established single-chain antibody TG1 library is above 1x10 9 cfu / mL, and can be screened multiple rounds to enrich positive clones that can bind ligands, and has the characteristics of high sensitivity, high signal-to-noise ratio, and the fluorescence signal of each monoclonal clone after electroporation is above 10 7 and the fluorescence signal of untransfected is very low.

[0101] 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 present invention's specification 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 murine single-chain antibody, characterized in that, The recombinant fusion protein of the murine single-chain antibody is formed by linking the murine single-chain antibody with phage Piii protein and luciferase. Among them, the amino acid sequence of the recombinant fusion protein of the murine single-chain antibody is as shown in SEQ ID NO:

5.

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

6.

3. The recombinant fusion protein of the murine single-chain antibody as described in claim 1, characterized in that, The murine single-chain antibody includes a light chain and a heavy chain: Among them, multiple primers are designed for the light chain and the heavy chain.

4. A DNA molecule, characterized in that, For encoding the recombinant fusion protein of the murine single-chain antibody as described in claims 1 to 4, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO:

1.

5. A method for constructing a library of recombinant fusion proteins of murine single-chain antibodies, characterized in that, Comprising the following steps: S10: Using the murine single-chain antibody described in any one of claims 1 to 3 as a template, cloning and ligating a vector to obtain a recombinant plasmid; S20: Providing primers for the murine single-chain antibody, using the recombinant plasmid as a template, respectively amplifying the light chain and the heavy chain of the murine single-chain antibody to obtain multiple heavy chain fragments and light chain fragments. Among them, the amino acid sequence of the murine single-chain antibody is as shown in SEQ ID NO:6, and multiple primers are designed for the light chain and the heavy chain of the murine single-chain antibody; S30: Mixing the multiple heavy chain fragments and the multiple light chain fragments, and performing PCR reactions to obtain multiple mixed products respectively; S40: Respectively double-digesting and ligating the multiple mixed products with the recombinant plasmid to obtain multiple plasmids; S50: After mixing the multiple plasmids, electrotransforming and amplifying to obtain a recombinant fusion protein library of the murine single-chain antibody.

6. The method for constructing a library of the recombinant fusion protein of the murine single-chain antibody as claimed in claim 5, wherein, The vector in step S10 includes plasmid AB.

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