Mutant P17 oligopeptide and application thereof in preparation of nano antibody

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a mutant P17 oligopeptide and application thereof in preparation of a nano antibody. The amino acid sequence of the mutant P17 oligopeptide is KGFRDEKKRFKNTKG. The mutant P17 oligopeptide is a mutant P17 oligopeptide. The mutant P17 oligopeptide provided by the invention has good chaperone-like activity and dissolution promoting effect, has good applicability to different nano antibody strains, is fused with a nano antibody and is expressed in a prokaryotic expression system, so that the solubility and expression efficiency of the nano antibody in the prokaryotic expression system can be effectively improved, and the application prospect is broad. The soluble yield of the nano antibody is greatly improved; moreover, the mutant P17 oligopeptide is beneficial to keeping the biological activity of the antibody, the nano antibody with high activity and high expression quantity is obtained, favorable conditions are created for large-scale production of the nano antibody, and the mutant P17 oligopeptide has a wide application prospect.

Description

Technical Field

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

[0002] Nanobody (Nb) is a type of single-domain antibody containing only the variable region of the heavy chain (VHH). It exists in camels and some cartilaginous fish and is the smallest unit known to bind to the target antigen, with a molecular weight of only 15KDa. Nanobody has many advantages, such as small molecular weight, strong tissue penetration, low immunogenicity, simple humanization, high antigen binding affinity, good stability under extreme conditions, and easy recombinant expression and production. It has attracted much attention in the fields of biomedicine, food safety testing, and disease diagnosis and treatment.

[0003] The gene sequence of nano antibodies is relatively simple, involving only the coding sequence of the heavy chain variable region, which makes the large-scale fermentation production of nano antibodies based on genetic engineering technology have great advantages. At present, the Escherichia coli expression system is the most widely used nano antibody expression system. As one of the traditional model organisms, Escherichia coli is the preferred host for laboratory and industrial production of heterologous proteins because of its fast reproduction speed, thorough genetic background, and easy genetic manipulation. However, since Escherichia coli is a prokaryotic organism and lacks the post-translational modification mechanism in eukaryotic organisms, it is easy to cause low expression of antibodies, poor solubility, and the formation of inactive or low-activity inclusion bodies, which leads to the need for antibodies to undergo complex renaturation steps after purification and further easily cause the problem of degradation of the expression product. Therefore, improving the soluble expression of nano antibodies in Escherichia coli is one of the keys to achieving its wide 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), at the N-terminus or C-terminus of the target protein, the target protein can be folded correctly 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 antibodies and antigens, and the tags need to be removed after expression and purification, which may destroy the stability and biological activity of the antibody.

[0005] P17 protein is a fibrous protein in the tail of 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 P17 peptide or tag), and can deliver phage particles and small molecules, proteins, nucleic acids, and liposomes to hepatocytes. This characteristic makes the P17 tag unique in application value in antibody expression. The P17 tag has significant advantages such as improving the solubility of antibody expression, enhancing stability, and improving antigen binding ability. Although the P17 protein has shown good application potential in antibody expression, it still faces some challenges. How to further optimize the structure and function of the P17 tag to improve its applicability and soluble expression efficiency in different antibody expression systems is the top priority in current work. Summary of the invention

[0006] In view of the P17 short peptide tag of the prior art, the present invention optimizes its amino acid sequence by point mutation, and provides a novel P17 short peptide. The mutant P17 short peptide has good chaperone-like activity and solubilizing effect, and can effectively improve the solubility and expression efficiency of nano antibodies in prokaryotic expression systems, greatly improve the soluble expression yield of nano antibodies and help maintain their biological activity. Therefore, the present invention further provides the application of the mutant P17 short peptide in the preparation of nano antibodies. The present invention is specifically implemented by the following technical solutions:

[0007] The first aspect of the present invention provides a mutant P17 short peptide, 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, which comprises 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 in SEQ ID NO.5 or SEQ ID NO.6.

[0012] Furthermore, the nucleic acid molecule also 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.

[0013] The third aspect of the present invention provides an expression vector, which comprises 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, connecting 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, harvesting the prokaryotic cells after culturing for a period of time 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-solubility 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-solubility expression of the mutant P17 short peptide of the present invention and the total amount of antibodies obtained by purification are about 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, which can be directly used without removing the mutant P17 short peptide label 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 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 embodiment 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 embodiment of the present invention were transformed;

[0024] Figure 3 The electrophoresis diagram of nanobodies VHH-S-1 and VHH-S-2 obtained by purifying the expression vectors fused with different P17 tags in the embodiment of the present invention after transformation of E. coli;

[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 solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes 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 defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0028] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, 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 according to the reported significant figures and by conventional rounding methods.

[0029] In addition, the meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the result may be added. The term "and / or" should be regarded 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 regarded as including the following situations: (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 phage P17 protein (Uniprot: P03748) exists in the form of a trimer and is a six-fold symmetrical tail fiber protein trimer. The P17 protein is an important component of the T7 phage tail and is mainly responsible for recognizing and adsorbing receptors on the surface of host liver cells to target phage particles and other cargoes (including small molecules, proteins, siRNA, DNA polymers and liposomes) to liver cells, and then triggering a series of reactions in the phage tail, thereby injecting the phage nucleic acid into the host cell and initiating the infection process. This is mainly mediated by the association of a 33-amino acid (aa) P17 protein fragment (called P17 peptide or P17 tag) with the cell surface low-density lipoprotein receptor-related protein (LRP). Studies have found that the P17 peptide can improve the solubility of a variety of different single-chain antibodies (scFv) in Escherichia coli and enhance the thermal stability of scFv, suggesting that the P17 peptide has a molecular chaperone-like activity and has a positive role and wide application value in the field of antibody expression. The amino acid sequence of the P17 peptide is KNESSTNATNTKQWRDETKGFRDEAKRFKNTAG.

[0032] The present invention compares the unmutated P17 long peptide (P17-33aa) and the short peptide (P17-N18del) with 18aa deleted at the N-terminus in increasing the soluble expression of nano antibodies in a prokaryotic (Escherichia coli) expression system, and finds that both the unmutated P17 long and short peptides can improve the solubility of the expressed antibody, and the solubility of the P17-N18del short peptide is equivalent to that of the long peptide, and the short peptide has more advantages due to the small 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 order to increase its solubilizing activity, and then improve the expression level and soluble expression of the fusion protein. Specifically, the present invention mutates alanine (A) to lysine (K) or arginine (R), and obtains multiple mutated P17 short peptides, and the mutated P17 short peptides are connected to the N-terminus or C-terminus of the nano antibody, which can improve the expression and solubility of the prokaryotic expression antibody. The solubilizing effect of the P17-N18del short peptide introduced with A to K mutation is more obvious, and has a higher chaperone-like activity than the unmutated short peptide and the A to R mutation short peptide, which is more conducive to improving the expression efficiency and active expression yield of the nano antibody in the prokaryotic expression system.

[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 with P17-N18del.

[0035] The mutant P17 short peptide provided by the present invention has good chaperone-like activity and pro-solubility effect. It is fused with multiple nano antibodies and expressed in a prokaryotic expression system. It is 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 obtained by pro-solubility expression of the mutant P17 short peptide of the present invention and the total amount of antibodies obtained by purification are about 4-6 times that of the wild-type P17 short peptide (unmutated), and the pro-solubility effect is significant. The biological activity of the purified nano antibody is further detected by enzyme-linked immunosorbent assay. The results show that the mutant P17 short peptide is fused with the nano antibody and its antigen binding ability is comparable to the nano antibody expressed without adding the P17 short peptide. It can be seen that the addition of the mutant P17 short peptide of the present invention will 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 label 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, which creates favorable conditions for the large-scale production of nanoantibodies and has broad application prospects.

[0036] Another embodiment of the present invention provides a nucleic acid molecule, which includes 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 described in detail 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 obtained by deducing the aa sequence of the P17 short peptide through 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 appreciate that, due to the degeneracy of the genetic code, gene sequences other than those exemplified above can also encode the mutant P17 short peptide of the present invention. Therefore, the sequences exemplified above should not be used as a limitation on the protection scope 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 lytic expression of the nanobody of 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 type of tag 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 described in detail 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 a prokaryotic cell and a eukaryotic cell, which is selected according to the type of expression vector. For example, when it is a prokaryotic expression vector, the host cell is selected from a prokaryotic cell, and examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc.; when it is a eukaryotic expression vector, the host cell is selected from a eukaryotic cell, and examples of commonly used eukaryotic host cells include Saccharomyces cerevisiae, Saccharomyces cerevisiae, etc.

[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 restriction sites of BamH I and Xho I. The pET22b vector connected with the nucleic acid molecule of the present invention is used for expression 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 nanobodies preparation methods.

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

[0053] S1, connecting 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, harvesting the prokaryotic cells after culturing for a period of time 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 be optionally included. The purification method in step S2 may select a suitable affinity purification system according to the type of purification tag. 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 is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the 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 site is the site 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 lytic ability of the above-mentioned P17 tag, 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 to the C-terminal of aforementioned fusion sequence for protein purification, and the target gene fusion fragment is connected to the BamH I and Xho I restriction sites of pET22b expression plasmid by full gene synthesis, 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 inserted by the expression plasmid of the present embodiment is shown in Table 1, and the gene synthesis work is entrusted to Wuhan Jinkairui 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 bacteria transformation

[0074] The above-mentioned target gene synthesized by whole gene 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, and the positive plasmid was transferred into the prokaryotic expression host - Escherichia coli shuffle T7 (purchased from Beijing Huayueyang Biology, catalog 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 of plasmid to the divided competent cells, gently blow 3 times with a pipette to mix, and place on ice for 30min. (2) Heat shock: Preheat the water bath to 42℃ 30min 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 60s, and then quickly take it out and cool it on ice for 5min. (3) Screening of positive clones: Take out the bacterial solution from (2) and spread it lightly on solid culture medium, culture it in a constant temperature incubator at 37°C for 16 h, 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 expression reaches 0.5-0.55, 0.8mM IPTG is added to induce expression. The induction expression conditions are: 16℃, 220rpm, and culture for 16h. The induced bacteria are centrifuged at 12000rpm and 4℃ for 5min, the supernatant is discarded, and 1mL of bacterial disruption solution (including 50mM Tris-HCL (pH8.0), 250mM NaCl and 5% glycerol) is taken to suspend the bacteria. The bacterial solution is broken by ultrasonic disruptor, and then centrifuged at 12000rpm for 10min to obtain the supernatant and precipitate (inclusion body). The supernatant obtained from the broken bacteria is mixed with 6×Loading Buffer at a volume ratio of 5:1 to prepare the sample; the inclusion body is mixed with 2×Loading Buffer at a volume ratio of 1:1 to prepare the sample, and the prepared sample is 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 nano antibody 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, respectively; lane BL represents bacterial lysate without adding IPTG to induce antibody expression; lane S represents the supernatant obtained by centrifugation of bacterial lysate with addition of IPTG to induce antibody expression; lane I represents inclusion bodies obtained by centrifugation of bacterial lysate with addition of IPTG to induce antibody expression; and the box indicates the position of the nano antibody 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; while 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 the small-scale expression test showed that the addition of the mutated short peptide at the N-terminus and the C-terminus could significantly improve the solubility of the nanoantibody. Therefore, two control plasmids, four expression plasmids containing the wild-type P17 short peptide and four expression plasmids containing the P17 mutant short peptide (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 to 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 0.8mM IPTG and culture in a shaker at 16℃ and 220rpm for 16h; c) Ultrasonic disruption of bacteria: centrifuge the induced bacterial solution at 4000rpm for 10min, discard the supernatant, take 30mL of the bacterial disruption solution to suspend the bacteria; use an ultrasonic disruptor to disrupt the bacterial solution, then centrifuge at 9000rpm for 10min, and collect the supernatant.

[0081] (2) Supernatant purification: According to 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) for His tag, and the operation was as follows: a) After activating the resin with 30mL of binding buffer, the supernatant was combined with the resin under oscillation at 4°C for 45min; b) non-specific binding was removed with 30mL of washing buffer; c) the target antibody was eluted with 3mL 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 purifying Nanobodies in Example 2 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 It is nano antibody 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 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 nano antibody, and the box shows the position of the nano antibody band. The nano antibody yield in Escherichia coli transformed with each expression vector is shown in Table 3, wherein the antibody concentration is the concentration of the nano antibody solution after dialysis, the total antibody amount = antibody concentration × total volume of the nano antibody solution, and the antibody yield = total antibody amount / culture system (300 mL).

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

[0086] Plasmid name Antibody concentration (mg / mL) Total amount of antibody (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 nanoantibodies 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 the 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) taking out the titration plate coated with S protein, 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-purified nanoantibody as the primary antibody, starting with a 3-fold gradient dilution at a starting concentration of 1 μg / mL, a total of 8 gradient dilutions, adding the antibody dilution solution to the titration plate at 25 μL / well, and incubating at room temperature for 1 hour; adding the diluent Buffer to the NC control; d) removing the liquid in the well, adding Rabbit anti VHH-Biotin (purchased from GenScript, catalog number A02015) secondary antibody, and incubating at room temperature for 1 hour; e) adding TMB colorimetric solution (purchased from Thermo Fisher, catalog number 34029), and the reaction was allowed to stand at room temperature in the dark for 5 min. After color development, 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 an ELISA reader and the OD values ​​at 450nm and 630nm are measured. 450nm -OD 630nm The OD correction value is the ordinate, and the antibody concentration is the abscissa. 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 protection scope 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 also 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: Comprising 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 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, connecting 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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