Mutant P17 short peptide and its application in the preparation of nanobodies

Abstract

The present invention belongs to the field of genetic engineering technology, and in particular relates to a mutant P17 short peptide and its application in the preparation of nano antibodies. The amino acid sequence of the mutant P17 short peptide is KGFRDEKKRFKNTKG. The mutant P17 short peptide provided by the present invention has good molecular chaperone-like activity and solubility-promoting effect, and has good applicability to different nano antibody strains. It is fused with a nano antibody and expressed in a prokaryotic expression system, which can effectively improve the solubility and expression efficiency of the nano antibody in the prokaryotic expression system, and greatly improve the soluble yield of the nano antibody; and the mutant P17 short peptide of the present invention helps to maintain the biological activity of the antibody, obtains a nano antibody with high activity and high expression, creates favorable conditions for the large-scale production of nano antibodies, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a mutant P17 short peptide and application thereof in the preparation of nano antibodies. Background Art

[0002] Nanobodies (Nb) are a type of single-domain antibody consisting solely of the variable heavy chain (VHH) region. Found in camels and some cartilaginous fish, they are the smallest known unit capable of binding to a target antigen, with a molecular weight of only 15 kDa. Nanobodies offer numerous advantages, including small molecular weight, strong tissue penetration, low immunogenicity, simplified humanization, high antigen-binding affinity, excellent stability under extreme conditions, and ease of recombinant expression and production. They have garnered significant attention in fields such as biopharmaceuticals, food safety testing, and disease diagnosis and treatment.

[0003] The gene sequence of nanobodies is relatively simple, involving only the coding sequence of the heavy chain variable region, which makes the large-scale fermentation production of nanobodies based on genetic engineering technology have great advantages. At present, the E. coli expression system is the most widely used nanoantibody expression system. As one of the traditional model organisms, E. coli is the preferred host for laboratory and industrial production of heterologous proteins due to its fast reproduction speed, thorough genetic background, and easy genetic manipulation. However, since E. coli is a prokaryotic organism and lacks the post-translational modification mechanism in eukaryotic organisms, it is easy to cause low antibody expression, poor solubility, and the formation of inactive or low-activity inclusion bodies, etc. This requires the antibody to undergo a complex renaturation step after purification and is further prone to degradation of the expression product. Therefore, improving the soluble expression of nanoantibodies in E. coli is one of the keys to achieving its widespread application.

[0004] By incorporating soluble expression tags, that is, fusing one or more solubility-promoting tags, such as maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), and nitrogen utilization substance A (NusA), to the N-terminus or C-terminus of the target protein, the target protein can be properly folded to improve the soluble expression level of the expression product. However, these larger tags, such as MBP, may cause spatial interference with the interaction between the antibody and the antigen, and the tags need to be removed after expression and purification, which may damage the stability and biological activity of the antibody.

[0005] The P17 protein is a fibrous protein in the tail of the T7 bacteriophage. It specifically binds to the low-density lipoprotein receptor-related protein (LRP) on the surface of hepatocytes through a 33-amino acid polypeptide (called the P17 peptide or tag), enabling targeted delivery of phage particles and small molecules, proteins, nucleic acids, and liposomes to hepatocytes. This property makes the P17 tag uniquely valuable for application in antibody expression. The P17 tag offers significant advantages, such as improved antibody solubility, enhanced stability, and enhanced antigen-binding capacity. Although the P17 protein has shown great potential for application in antibody expression, it still faces several challenges. Further optimizing the structure and function of the P17 tag to improve its applicability and soluble expression efficiency in different antibody expression systems is a top priority in current work. Summary of the Invention

[0006] In response to the prior art P17 short peptide tag, the present invention optimizes its amino acid sequence through point mutation, providing a novel P17 short peptide. This mutant P17 short peptide has good molecular chaperone-like activity and a solubility-promoting effect, can effectively improve the solubility and expression efficiency of nanobodies in prokaryotic expression systems, greatly improve the soluble expression yield of nanobodies and help maintain their biological activity. Therefore, the present invention further provides the use of this mutant P17 short peptide in the preparation of nanobodies. The present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides a mutant P17 short peptide, wherein the amino acid sequence of the mutant P17 short peptide is KGFRDEKKRFKNTKG.

[0008] The second aspect of the present invention provides a nucleic acid molecule comprising a gene sequence encoding the mutant P17 short peptide as described above.

[0009] Furthermore, the gene (DNA) sequence encoding the mutant P17 short peptide is AAGGGCTTCAGAGACGAAAAAAAGAGGTTCAAGAACACGAAGGGC.

[0010] Furthermore, the nucleic acid molecule also includes a gene sequence encoding a nanobody.

[0011] Furthermore, the amino acid sequence of the nanobody is shown as SEQ ID NO.5 or SEQ ID NO.6.

[0012] Furthermore, the nucleic acid molecule further comprises a gene sequence encoding a purification tag, and the purification tag is selected from one or more of 6×His, 8×His, Trx, 3×FLAG, GST, strep(II), HA, GFP, cMyc, and mFC.

[0013] The third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0014] Furthermore, the expression vector is a pET22b vector, and the nucleic acid molecule is inserted between the BamH I and Xho I restriction sites of the pET22b vector.

[0015] The fourth aspect of the present invention provides the use of the mutant P17 short peptide, nucleic acid molecule or expression vector as described above in the preparation of nanobodies.

[0016] A fifth aspect of the present invention provides a method for preparing a Nanobody, comprising the following steps:

[0017] S1. Connect the gene sequence encoding the mutant P17 short peptide and the gene sequence encoding the nanobody to the multiple cloning site of the expression vector to obtain a recombinant expression vector;

[0018] S2. Introducing the recombinant expression vector into prokaryotic cells, culturing the prokaryotic cells for a period of time, harvesting the prokaryotic cells for cell disruption, and then collecting the cell disruption supernatant for purification to obtain the nanobody.

[0019] The advantages and positive effects of the present invention are:

[0020] The mutant P17 short peptide provided by the present invention has good molecular chaperone-like activity and pro-lytic effect, and has good applicability to different nano antibody strains. It is fused with the nano antibody and expressed in a prokaryotic expression system, which can effectively improve the solubility and expression efficiency of the nano antibody in the prokaryotic expression system. Under the same culture system and culture conditions, the yield of soluble nano antibodies obtained by pro-lytic expression of the mutant P17 short peptide of the present invention and the total amount of purified antibodies are approximately 4-6 times that of the wild-type P17 short peptide (unmutated), which greatly improves the soluble yield of the nano antibody; and the addition of the mutant P17 short peptide of the present invention helps to maintain the biological activity of the antibody, thereby obtaining a nano antibody with high activity and high expression amount. It can be directly used without removing the mutant P17 short peptide tag of the present invention, which greatly simplifies the preparation procedure of the nano antibody; the present invention creates favorable conditions for the large-scale production of nano antibodies and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1This is an electrophoresis diagram of the expression level of Nanobody VHH-S-1 in E. coli after the expression vectors fused with different P17 tags in the examples of the present invention were transformed;

[0023] Figure 2 This is an electrophoresis diagram of the expression level of Nanobody VHH-S-2 in E. coli after the expression vectors fused with different P17 tags in the examples of the present invention were transformed;

[0024] Figure 3 The electrophoretic diagrams of nanobodies VHH-S-1 and VHH-S-2 obtained by transforming E. coli with expression vectors fused with different P17 tags according to the present invention are as follows;

[0025] Figure 4 This is a graph showing the antigen binding ability test results of nanobodies fused with different P17 tags according to an embodiment of the present invention, with nanobodies VHH-S-1 and VHH-S-2 from left to right. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0028] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

[0029] In addition, the terms "including," "comprising," "containing," "having," and similar expressions are non-limiting, i.e., other steps and other components that do not affect the result may be added. The term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is considered to include the following: (i) A, (ii) B, and (iii) A and B.

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0031] The T7 bacteriophage P17 protein (Uniprot: P03748) exists as a trimer, a six-fold symmetrical tail fiber protein trimer. The P17 protein is a crucial component of the T7 phage tail, primarily responsible for recognizing and adsorbing receptors on the surface of host hepatocytes, targeting phage particles and other cargo (including small molecules, proteins, siRNAs, DNA polymers, and liposomes) to hepatocytes. This triggers a series of reactions within the phage tail, injecting the phage nucleic acid into the host cell and initiating infection. This is primarily mediated by the association of a 33-amino acid (aa) P17 protein fragment (referred to as the P17 peptide or P17 tag) with the cell surface low-density lipoprotein receptor-related protein (LRP). Studies have shown that the P17 peptide can improve the solubility of various single-chain variable fragments (scFvs) in Escherichia coli and enhance the thermal stability of scFvs, suggesting that the P17 peptide exhibits chaperone-like activity and has a promising role and broad application in the field of antibody expression. The amino acid sequence of the P17 peptide is KNESSTNATNTKQWRDETKGFRDEAKRFKNTAG.

[0032] By comparing the effects of an unmutated long P17 peptide (P17-33aa) and a short peptide with an N-terminal deletion of 18aa (P17-N18del) on increasing the soluble expression of nanobodies in a prokaryotic (Escherichia coli) expression system, the present invention found that both the unmutated long and short P17 peptides can improve the solubility of the expressed antibodies, and the solubility of the P17-N18del short peptide is comparable to that of the long peptide, with the short peptide having an advantage due to its smaller number of amino acids. The amino acid sequence of the P17-N18del short peptide is KGFRDEAKRFKNTAG.

[0033] The reason why the P17 peptide increases the solubility of the antibody is related to its hydrophilic sequence and charged residues. In order to further improve the functionality of the P17-N18del short peptide, the present invention introduces multiple cationic AA mutations in the short peptide, by increasing the positive charge density it carries, in the hope of increasing its lytic activity, and then improving the expression level and soluble expression of the fusion protein. Specifically, the present invention mutates alanine (A) to lysine (K) or arginine (R) to obtain multiple mutated P17 short peptides, and the mutated P17 short peptide is connected to the N-terminal or C-terminal of the nano antibody and can improve the expression and solubility of the prokaryotic expression antibody. The lytic effect of the P17-N18del short peptide introduced into A to K mutation is more obvious, and has higher chaperone-like activity than the short peptide of unmutated short peptide and A to R mutation, which is more conducive to improving the expression efficiency and active expression yield of nano antibodies in prokaryotic expression systems.

[0034] Based on this, an embodiment of the present invention provides a mutant P17 short peptide, whose amino acid sequence is KGFRDEKKRFKNTKG (see SEQ ID NO. 3), and the shadow indicates the mutation site compared to P17-N18del.

[0035] The mutant P17 short peptide provided by the present invention has good chaperone-like activity and pro-lytic effect. It is fused with multiple nano antibodies and expressed in a prokaryotic expression system. It was found through testing that the mutant P17 short peptide has similar biological effects regardless of whether it is added to the N-terminus or the C-terminus of the nano antibody. Under the same culture system and culture conditions, the soluble nano antibody yield and the total amount of purified antibodies obtained by pro-lytic expression of the mutant P17 short peptide of the present invention are about 4-6 times that of the wild-type P17 short peptide (unmutated), and the pro-lytic effect is significant. The biological activity of the purified nano antibody was further detected by enzyme-linked immunosorbent assay. The results showed that the mutant P17 short peptide was fused with the nano antibody and its antigen binding ability was comparable to that of the nano antibody expressed without adding the P17 short peptide. It can be seen that adding the mutant P17 short peptide of the present invention does not affect the biological function of the antibody and its binding activity to the antigen. It can be directly applied without removing the mutant P17 short peptide tag of the present invention, which is conducive to simplifying the preparation procedure of the nano antibody. In summary, the mutant P17 short peptide provided by the present invention has good applicability to different nanoantibody strains, can effectively improve the solubility of nanoantibodies in prokaryotic expression systems, greatly improve the soluble expression yield and expression efficiency of nanoantibodies, and the mutant P17 short peptide can improve the structural stability of nanoantibodies, help maintain their biological activity, and thus obtain nanoantibodies with high activity and high expression, creating favorable conditions for the large-scale production of nanoantibodies and having broad application prospects.

[0036] Another embodiment of the present invention provides a nucleic acid molecule comprising a gene sequence encoding the mutant P17 short peptide as described above.

[0037] The advantages of the nucleic acid molecule over the prior art are the same as those of the mutant P17 short peptide over the prior art as described above, and will not be repeated here.

[0038] Nucleic acid molecules include DNA molecules (eg, genomic DNA or cDNA) and / or RNA molecules (eg, mRNA), and the molecules can be single-stranded or double-stranded.

[0039] The sequence of the nucleic acid molecule can be derived from the aa sequence of the P17 short peptide by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0040] Illustratively, the gene (DNA) sequence encoding the mutant P17 short peptide described above is AAGGGCTTCAGAGACGAAAAAAAGAGGTTCAAGAACACGAAGGGC (see SEQ ID NO. 7).

[0041] Those skilled in the art will understand that due to the degeneracy of the genetic code, gene sequences other than the above examples can also encode the mutant P17 short peptide of the present invention. Therefore, the above examples should not be used as a limitation on the scope of protection of the present invention.

[0042] Optionally, the nucleic acid molecule further comprises a gene sequence encoding a nanobody. The sequence of the nanobody has little effect on the solubility-promoting ability of the mutant P17 short peptide of the present invention, and therefore, the nanobody can be any nanobody.

[0043] It should be noted that the connection order of the nanobody and the mutant P17 short peptide is not particularly limited. The mutant P17 short peptide can be located at the N-terminus or C-terminus of the nanobody. In other words, the gene sequence encoding the mutant P17 short peptide can be located at the 5' end or 3' end of the gene sequence encoding the nanobody.

[0044] In a typical embodiment, the amino acid sequence of the nanobody is shown in SEQ ID NO. 5 or SEQ ID NO. 6. The present invention achieves the soluble expression of the nanobody with the amino acid sequence shown in SEQ ID NO. 5-6, and the obtained nanobody has a high soluble expression level and good biological activity, but it should be further emphasized that the nanobody that can be used for the mutant P17 short peptide of the present invention is not limited to this one antibody or protein.

[0045] Optionally, the nucleic acid molecule further includes a gene sequence encoding a purification tag. The purification tag is used to facilitate the separation and purification of the nanobody, and the purification tag can be selected from one or more of 6×His, 8×His, Trx, 3×FLAG, GST, strep(II), HA, GFP, cMyc, and mFC. Those skilled in the art can specifically set the tag type according to the purification requirements to adapt to different purification systems and equipment, and the present invention is not limited to this.

[0046] In a typical embodiment, the purification tag is 6×His.

[0047] Yet another embodiment of the present invention provides an expression vector, which includes the nucleic acid molecule described above.

[0048] The advantages of the expression vector over the prior art are the same as those of the mutant P17 short peptide over the prior art as described above, and will not be repeated here.

[0049] Optionally, the expression vector includes a prokaryotic expression vector (such as an Escherichia coli expression vector, a Bacillus subtilis expression vector), a eukaryotic expression vector (such as a yeast expression vector), or a viral expression vector (such as a lentivirus, adenovirus). Correspondingly, the host cell includes prokaryotic cells and eukaryotic cells, and is selected according to the type of expression vector. For example, when it is a prokaryotic expression vector, the host cell is selected from prokaryotic cells, and examples of commonly used prokaryotic host cells include Escherichia coli and Bacillus subtilis; when it is a eukaryotic expression vector, the host cell is selected from eukaryotic cells, and examples of commonly used eukaryotic host cells include Saccharomyces cerevisiae and Saccharomyces cerevisiae.

[0050] In a preferred embodiment, the expression vector is a pET22b vector, and the nucleic acid molecule is inserted between the multiple cloning sites of the pET22b vector, specifically, between the BamH I and Xho I restriction sites. The pET22b vector of the present invention connected with the above-mentioned nucleic acid molecule is expressed in a prokaryotic host cell (such as Escherichia coli). After obtaining the host cell transformed with the expression vector, the cell is cultured under suitable conditions to obtain soluble expressed nanobodies in the cell culture medium, and then separated by conventional means to obtain high-purity, high-concentration, high-yield, and high-activity nanobodies.

[0051] The embodiments of the present invention also provide the use of the mutant P17 short peptide, nucleic acid molecule or expression vector as described above in the preparation of nanobodies, especially in the production of nanobodies using a prokaryotic expression system, and provide related nanobody preparation methods.

[0052] Specifically, a method for preparing a nanobody comprises the following steps:

[0053] S1. Connect the gene sequence encoding the mutant P17 short peptide and the gene sequence encoding the nanobody to the multiple cloning site of the expression vector to obtain a recombinant expression vector;

[0054] S2. Introducing the recombinant expression vector into prokaryotic cells, culturing the prokaryotic cells for a period of time, harvesting the prokaryotic cells for cell disruption, and then collecting the cell disruption supernatant for purification to obtain the nanobody.

[0055] In step S1, a gene sequence encoding a purification tag may optionally be included. The purification method in step S2 may be adapted to the type of purification tag and the appropriate affinity purification system. For example, a histidine tag (6His) may be affinity purified using a Ni column.

[0056] In step S2, the prokaryotic cells are preferably Escherichia coli cells, specifically shuffle T7 cells.

[0057] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0058] 1. Construction of Nanobody Expression Vector

[0059] The present invention designs unmutated P17 long (P17-33aa), short peptide (P17-N18del) and mutated P17 short peptide, and increases the soluble expression level of nano antibodies in Escherichia coli by comparing the aforementioned different forms of P17 tags. It is found that the unmutated P17 long and short peptides and the mutated P17 short peptides connected to the N-terminus or C-terminus of the nano antibody can improve the antibody solubility, and the soluble ability of the short peptide after mutation is more advantageous than that of the long and short peptide before mutation. The amino acid sequence of the P17 tag in this embodiment is as follows:

[0060] P17-33aa: KNESSTNATNTKQWRDETKGFRDEAKRFKNTAG (see SEQ ID NO.1);

[0061] P17-N18del:KGFRDE A KRFKNT A G (see SEQ ID NO. 2), the underlined sites are to be mutated;

[0062] P17-N18del-mutation 1: KGFRDEKKRFKNTKG (see SEQ ID NO. 3), the shadow indicates the mutation site;

[0063] P17-N18del-mutation 2: KGFRDERKRFKNTRG (see SEQ ID NO. 4), the shadow indicates the mutation site.

[0064] Two nanoantibodies targeting the surface spike protein (S protein) of the new coronavirus variant "Omicron" were selected as verification objects for the above-mentioned P17 tag's solubility-promoting ability, and were named VHH-S-1 and VHH-S-2 respectively.

[0065] The amino acid sequence of VHH-S-1 is shown below:

[0066] QVQLVESGGGVVQPGRSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAGISSGSSTY YADSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAADFTYLRYGVSYSPARYDYWGQ GTMVTVSS (see SEQ ID NO.5);

[0067] The amino acid sequence of VHH-S-2 is shown below:

[0068] EVQLVESGGGVVQPGRSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAGISSGSSTY YADSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAADFTYLRYGVSYSPARYDYWGQ GTQVTVSS (see SEQ ID NO. 6).

[0069] Select pET22b expression plasmid and Escherichia coli as host bacteria to build prokaryotic expression system.P17 tag is connected to the N-terminal and C-terminal of nano antibody, and histidine (6His) tag is connected at the C-terminal of aforementioned fusion sequence for protein purification, by full gene synthesis, target gene fusion fragment is connected between BamH I and Xho I restriction enzyme site of pET22b expression plasmid, and the protein obtained by target gene expression includes from upstream to downstream: (N) P17 tag-nano antibody-6His (C), or (N) nano antibody-P17 tag-6His (C).The target gene of the expression plasmid insertion of the present embodiment is as shown in Table 1, and gene synthesis work entrusts Wuhan Jin Kairui Bioengineering Co., Ltd. to complete.

[0070] Table 1 Expression plasmids constructed in the examples of the present invention

[0071]

[0072] 2. Nanobody-induced expression

[0073] 2.1 Host Bacterial Transformation

[0074] The above-mentioned target gene synthesized by whole gene synthesis was inserted into the BamH I and Xho I restriction sites of the prokaryotic expression vector pET22b. The plasmid construction was successfully verified by sequencing. The positive plasmid was transferred into the prokaryotic expression host - Escherichia coli shuffle T7 (purchased from Beijing Huayueyang Biological, product number GX1009-100s) by heat shock method. The specific steps are as follows: (1) Plasmid addition: In the ultra-clean workbench, the thawed shuffle T7 competent cells were divided into sterilized pre-cooled 1.5mL EP tubes, 20μL per tube. Add 1μL plasmid to the divided competent cells, gently pipette 3 times to mix, and place on ice for 30 minutes. (2) Heat shock: Preheat the water bath to 42℃ 30 minutes in advance, place the EP tube containing the mixture in (1) in the water bath and press the timer to start the countdown. Heat shock for 60 seconds, then quickly take it out and cool it on ice for 5 minutes. (3) Screening of positive clones: Take out the bacterial solution from (2) and spread it lightly on the solid culture medium, culture it in a constant temperature incubator at 37°C for 16 hours, take a single colony for sequencing to verify whether the positive plasmid is successfully transformed, and screen to obtain the positive clone strain that has successfully transformed the target gene.

[0075] 2.2 Small-scale expression test

[0076] Pick a single colony of the positive clone strain and inoculate it into 3 mL LB liquid medium containing antibiotics (50 μg / mL ampicillin and 34 μg / mL chloramphenicol), and culture it in a shaking incubator at 220 rpm and 37°C until the bacterial solution OD reaches 0. 600 When the protein reaches 0.5-0.55, 0.8 mM IPTG was added to induce expression. Induction conditions were: 16°C, 220 rpm, 16 h. Centrifuge the induced cells at 12,000 rpm, 4°C for 5 min, discard the supernatant, and resuspend the cells in 1 mL of lysis buffer (50 mM Tris-HCl (pH 8.0), 250 mM NaCl, and 5% glycerol). Disrupt the cells using an ultrasonic disruptor, then centrifuge at 12,000 rpm for 10 min to obtain the supernatant and precipitate (inclusion bodies). Mix the supernatant with 6× Loading Buffer at a 5:1 volume ratio for sample preparation; mix the inclusion bodies with 2× Loading Buffer at a 1:1 volume ratio for sample preparation. The prepared samples were subjected to SDS-PAGE gel electrophoresis.

[0077] Figure 1-2 The electrophoresis diagrams showing the expression levels of nanobodies in E. coli transformed with different expression vectors are shown; wherein, Figure 1 It is the nanobody VHH-S-1, Figure 2It is nanobody VHH-S-2; in the figure, lane MK represents a molecular marker, and the molecular weights from top to bottom are 116, 66, 45, 35, 25, 18, and 14 kDa, lane BL represents bacterial lysate without the addition of IPTG to induce antibody expression, lane S represents the supernatant obtained by centrifugation of bacterial lysate with the addition of IPTG to induce antibody expression, lane I represents the inclusion bodies obtained by centrifugation of bacterial lysate with the addition of IPTG to induce antibody expression, and the box indicates the position of the nanobody band. The results showed that there was almost no expression in the supernatant of the bacterial cell disruption liquid without the addition of P17 peptide. Under the same expression conditions, the addition of long peptides and short peptides could increase the solubility rate of the nanoantibody, and the P17 tag placed at the N-terminus or C-terminus of the nanoantibody had a solubility-promoting effect; the solubility-promoting effect of the short peptide was better than that of the long peptide, and the nanoantibody fused with the K-mutated P17 short peptide showed the highest solubility; the solubility of the nanoantibody fused with the R-mutated short peptide was not significantly increased compared with the wild-type P17 short peptide, and its solubility-promoting effect was weaker than that of the K-mutated short peptide, so it was not subsequently expressed and purified in large quantities.

[0078] 2.3 Large-scale expression and purification

[0079] The results of small-scale expression tests showed that adding the mutated short peptide to both the N-terminus and the C-terminus could significantly improve the solubility of the nanoantibody. Therefore, two control plasmids, four expression plasmids containing wild-type P17 short peptides, and four expression plasmids containing P17 mutant short peptides (P17-N18del-mutation 1) were selected for expanded expression and purification.

[0080] (1) Large-scale expression, the operation is as follows: a) Inoculation and activation of positive strains: take the bacterial solution with correct expression in the small test, inoculate it into 2 mL LB liquid medium, and culture it in a shaker at 37°C and 220 rpm overnight; b) Expansion culture and induction: transfer all the activated bacterial solution into 300 mL LB liquid medium, and culture it in a shaker at 37°C and 220 rpm. When the OD 600 When the value reaches 0.5-0.55, add 0.8 mM IPTG and incubate in a shaker at 16°C and 220 rpm for 16 h. c) Ultrasonic disruption: Centrifuge the induced bacterial solution at 4000 rpm for 10 min, discard the supernatant, and take 30 mL of the disruption solution to suspend the bacteria; disrupt the bacterial solution using an ultrasonic disruptor, then centrifuge at 9000 rpm for 10 min, and collect the supernatant.

[0081] (2) Supernatant purification: Based on the 6His tag carried by the plasmid, the harvested supernatant was purified using a Ni-NTA affinity purification matrix pre-packed column (purchased from Lanxiao, product number A4023205) with the following steps: a) After activating the resin with 30 mL of binding buffer, the supernatant was allowed to bind to the resin with shaking at 4°C for 45 min; b) non-specific binding was removed with 30 mL of washing buffer; c) the target antibody was eluted with 3 mL of elution buffer and the eluate was collected; d) the eluate was dialyzed into 1× PBS to obtain the purified nanobody. The formula of the buffer used for purification is shown in Table 2.

[0082] Table 2 Buffer formula used for purification of Nanobodies in Examples of the present invention

[0083] Buffer name Tris (mM) NaCl (mM) Imidazole (mM) pH BindingBuffer 50 250 0 pH8.0 Washing Buffer 50 250 50 pH8.0 ElutionBuffer 50 250 500 pH8.0

[0084] The purified nanobody was mixed with 2× Loading Buffer at a volume ratio of 1:1 to prepare a sample, and SDS-PAGE gel electrophoresis was performed to determine the amount of protein in the eluate. The results are shown in Figure 3 ,in, Figure 3 It is the nanobody VHH-S-1, Figure 4 The nanobody VHH-S-2; lane MK in the figure represents a molecular marker, and the molecular weights from top to bottom are 116, 66, 45, 35, 25, 18, and 14 kDa, lane BSA represents 0.4 mg / mL bovine serum albumin (BSA), which is used as a control, lane MK represents a molecular marker, lane Nb represents the purified nanobody, and the box shows the position of the nanobody band. The nanobody yield in E. coli transformed with each expression vector is shown in Table 3, where the antibody concentration is the concentration of the nanobody solution after dialysis, the total amount of antibody = antibody concentration × total volume of the nanobody solution, and the antibody yield = total amount of antibody / culture system (300 mL).

[0085] Table 3 Effect of P17 tag on soluble expression level of Nanobodies in Example 3 of the present invention

[0086] Plasmid name Antibody concentration (mg / mL) Total antibody amount (mg) Antibody production (mg / L) purity Plasmid S-1 0.2 0.5 1.67 95％ Plasmid S-2 0.2 0.6 2 95％ Plasmid 2 0.5 1.5 5 95％ Plasmid 3 1 2.64 8.8 95％ Plasmid 6 0.6 1.8 6 95％ Plasmid 7 1 2.77 9.23 95％ Plasmid 10 0.75 2.25 7.5 95％ Plasmid 11 1.14 3.43 11.43 95％ Plasmid 14 0.7 2.1 7 95％ Plasmid 15 1.07 3.2 10.67 95％

[0087] from Figure 3 As can be seen from Table 3, the expression level of the nanobody without the addition of the P17 tag after large-scale expression is low, while the soluble expression level of the antibody with the addition of the mutant short peptide of the present invention is significantly improved, and the yield of soluble nanobody is about 4-6 times that of the control group. The solubility-promoting effect of the P17 tag peptide added at the N-terminus and C-terminus is similar.

[0088] 3. Functional verification of nanoantibodies

[0089] Plasmid S-1 and plasmid S-2 were used as controls, and the purified nanobody was tested by ELISA using Omicron S protein (purchased from Sinobiological, catalog number 40592-V08H140) to verify the activity of the antibody binding to the antigen. The ELISA test includes the following steps: a) coating 1 μg / mL Omicron S protein on the titration plate at 25 μL / well and incubating at 4°C overnight; b) removing the S protein-coated titration plate, removing the liquid in the well, adding blocking solution (5% BSA), and incubating at room temperature for 1 hour; c) removing the blocking solution, using the above-mentioned purified nanobody as the primary antibody, starting with a 3-fold gradient dilution starting from a starting concentration of 1 μg / mL, a total of 8 dilution gradients, and adding the antibody dilution solution to the titration plate at 25 μL / well, and incubating at room temperature for 1 hour; adding dilution buffer to the NC control; d) removing the liquid in the well, adding Rabbit anti VHH-Biotin (purchased from GenScript, product number A02015) secondary antibody, and incubating at room temperature for 1 hour; e) adding TMB color development solution (purchased from Thermo Fisher Scientific) The cells were plated with a 4% PBS (Fisher, catalog number 34029) and allowed to react at room temperature in the dark for 5 min. After color development, a stop solution (700 mM oxalic acid solution) was added to each well to terminate the reaction.

[0090] After the reaction is terminated, the ELISA titration plate is placed in a microplate reader and the OD values ​​at 450nm and 630nm are measured. 450nm -OD 630nm The OD correction value is the vertical axis, and the antibody concentration is the horizontal axis. Draw the antigen-antibody binding curve, as shown in Figure 4 It can be seen that the activity data of the nanobody with the mutant P17 short peptide tag of the present invention is basically consistent with that of the control antibody, and has good antigen binding activity, indicating that the addition of the P17 short peptide will not affect the biological function of the antibody and its ability to bind to the antigen.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A mutant P17 short peptide, characterized in that: Its amino acid sequence is KGFRDEKKRFKNTKG.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a gene sequence encoding the mutant P17 short peptide as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The gene sequence encoding the mutant P17 short peptide is AAGGGCTTCAGAGACGAAAAAAAGAGGTTCAAGAACACGAAGGGC.

4. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule also includes a gene sequence encoding a nanobody.

5. The nucleic acid molecule according to claim 4, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO.5 or SEQ ID NO.

6.

6. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule further includes a gene sequence encoding a purification tag, and the purification tag is selected from one or more of 6×His, 8×His, Trx, 3×FLAG, GST, strep(II), HA, GFP, cMyc, and mFC.

7. An expression vector, characterized in that The method comprises the nucleic acid molecule according to any one of claims 2 to 6.

8. The expression vector according to claim 7, characterized in that The expression vector is a pET22b vector, and the nucleic acid molecule is inserted between the BamH I and Xho I restriction enzyme cutting sites of the pET22b vector.

9. Use of the mutant P17 short peptide according to claim 1, the nucleic acid molecule according to any one of claims 2 to 6, or the expression vector according to any one of claims 7 to 8 in the preparation of nanobodies.

10. A method for preparing a nanobody, characterized in that: The following steps are involved: S1. Connect the gene sequence encoding the mutant P17 short peptide and the gene sequence encoding the nanobody to the multiple cloning site of the expression vector to obtain a recombinant expression vector; wherein the amino acid sequence of the mutant P17 short peptide is KGFRDEKKRFKNTKG; S2. Introducing the recombinant expression vector into prokaryotic cells, culturing the prokaryotic cells for a period of time, harvesting the prokaryotic cells for cell disruption, and then collecting the cell disruption supernatant for purification to obtain the nanobody.

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