Nanobody proteins, uses, expression vectors, strains and medicaments and kits

Abstract

The application belongs to the technical field of biology, and discloses an anti-H5 subtype avian influenza virus nanobody protein, and an amino acid sequence of the nanobody protein is shown as SEQ ID NO. 1. The nanobody protein is excellent in activity, affinity and specificity, has good solubility, and is beneficial to expression and purification. The application further discloses the anti-H5 subtype avian influenza virus nanobody protein, application, expression vector, strain, medicine and kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a strain of anti-H5 subtype avian influenza virus nanobody protein, use, expression vector, strain and medicine and kit. BACKGROUND

[0002] Influenza viruses cause a huge health and socio-economic burden worldwide. There are four types of influenza viruses known, designated A-D. Type A and B viruses circulate in human populations and cause epidemics (type A), pandemics and seasonal epidemics (types A and B), while types C and the recently discovered type D viruses do not cause significant disease or epidemics. Influenza A viruses are further divided into subtypes based on the antigenic properties of their two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). There are 18 known HA subtypes (H1 to H18) and 11 NA subtypes (N1 to N11). The 18 HA subtypes can be further divided into group 1 (H1, H2, H5, H6, H7, H8, H9, H11, H12, H13, H16, H17, and H18) or group 2 (H3, H4, H7, H10, H14, and H15). Of the 198 (11 x 18) possible combinations, only three (H1N1, H2N2, H3N2) are known to have caused human pandemics. The main natural host of influenza A viruses are wild aquatic birds, but poultry can also be infected and thus carry influenza A viruses. Pigs and other mammals, such as horses, dogs, seals, mink, and bats, can also be infected with influenza A viruses and can serve as a source of infection for humans. Certain subtypes found in natural hosts occasionally appear in humans, for example, H5N1, H5N6, H6N1, H7N7, H7N9, H9N2, and H10N8 viruses. Some of these zoonotic subtypes can be highly pathogenic when infecting humans and have a high mortality rate (>50% of hospitalized patients). Since 2003, H5N1 viruses have triggered disease outbreaks in poultry and wild birds in more than 60 countries across three continents, with over 800 human infections reported in 16 countries with a mortality rate close to 60%. Highly pathogenic avian influenza viruses pose a continuing threat to the poultry industry and public health.

[0003] Among all influenza virus proteins, HA has the highest rate of evolution because it is the main target of the immune response. Phylogenetic analysis suggests that different HA subtypes of influenza A virus diverged before 2000. Despite their HA proteins having as little as 40% sequence identity, they adopt the same protein fold. As a class I viral fusion protein, HA plays an important role in virus entry by binding to sialylated glycans on host receptors, respiratory epithelial cells, and promoting membrane fusion in the low pH environment of endosomal compartments after cellular entry by endocytosis. During virus replication, the uncleaved precursor of HA, HA0, is synthesized and then cleaved by cellular proteases into two subunits, HA1 and HA2, to produce the fully functional protein, in which HA1 and part of HA2 form the globular head and HA2 forms the stem of the HA protein.

[0004] Two classes of broadly neutralizing antibodies (bnAbs) against HA influenza have been discovered. Antibodies targeting the stem, such as murine monoclonal antibody (mAb) C179, human monoclonal antibody CR6261, F10, and A6, have broad heterosubtypic activity, some of which can target almost all HA strains of various subtypes and subgroups, such as CR9114, MEDI8852, these broad-spectrum neutralizing antibodies recognize the highly conserved stem region and block the virus fusion mechanism. A second class of neutralizing antibodies targeting the HA head domain has also been discovered, these head-targeting antibodies mostly recognize the receptor-binding site (RBS) and block virus attachment and entry. Most head-targeting antibodies have a limited recognition pattern in one subtype; for example, H1 -specific 5J8 and CH65, and H2-specific 8M2 antibodies. A few exceptions are C05, F045-92, and S139 / 1, which react with HA head domains from multiple HA subtypes.

[0005] In the mid-1990s, researchers found that camels possess a unique unconventional antibody, which is naturally devoid of light chains in their serum, called heavy-chain only antibodies (HCAbs). The N-terminal variable domain of HCAbs represents the smallest naturally occurring antigen-binding fragment known, called the heavy chain variable domain of the heavy chain-only antibody (VHH) or Nanobody (Nb). The molecular size of Nanobody (Nb) is 12-15 kDa, which is significantly smaller than that of a classical immunoglobulin G molecule (IgG) (~160 kDa), an antigen-binding fragment (Fab) (~50 kDa. These monomeric antibody fragments exhibit several unique advantages in terms of high affinity; ease of cloning and expression in E. coli and Saccharomyces cerevisiae; high thermal, chemical, and conformational stability; greater solubility; and ease of generating bispecific or multimeric constructs. In addition, these entities display extended complementarity determining regions (CDRs), which enable them to recognize haptens and cryptic epitopes that are less readily accessible to conventional antibodies. Good thermal, chemical, and conformational stability makes them more resistant to denaturing, degrading, or regenerating conditions in immunoassays. The single-domain nature of VHHs facilitates directed coupling on biosensor surfaces with a single chemical reactive group for labeling. All these unique advantageous properties make VHHs extremely attractive new candidates in immunoassay applications.

[0006] The applicant has previously applied for an invention patent: ZL202110714355.7, Anti-H5 subtype avian influenza virus nanobody and its application;

[0007] The main scheme is: the antibody gene is connected with the maltose binding protein (MBP) gene through a connecting peptide, an expression vector is constructed, and a fusion protein is induced to express, so as to promote the soluble expression of the anti-H5 avian influenza virus nanobody fusion protein in the expression system, thereby solving the problem that the anti-H5 avian influenza nanobody protein is insoluble in the prokaryotic system, and improving the accuracy and repeatability.

[0008] The applicant has also disclosed another prior application: ZL202110390340.X, an anti-H5 subtype avian influenza nanobody protein and its encoding gene and application;

[0009] The scheme further combines the anti-H5 subtype avian influenza virus nanobody protein in vivo and maltose binding protein tag fusion expression of E. coli, which can realize rapid detection of antigen, and be applied to the development of H5 subtype avian influenza A rapid detection kit, and the fusion protein can be used for the development of H5 subtype avian influenza A prevention and control products.

[0010] In the process of screening and preparing the anti-H5 subtype avian influenza virus nanobody protein, a common problem is that the solubility in the prokaryotic system is poor.

[0011] Meanwhile, in the long-term research, the applicant screened several hundred proteins, which cannot achieve very good affinity.

[0012] The technical problem to be solved by the scheme is how to develop a corresponding nanometer protein with strong activity, affinity, specificity and good solubility in the prokaryotic system for H5 subtype avian influenza virus. SUMMARY

[0013] The purpose of the present application is to provide an anti-H5 subtype avian influenza virus nanobody protein, which has excellent activity, affinity and specificity, and has good solubility, which is beneficial to expression and purification. The present application also discloses the anti-H5 subtype avian influenza virus nanobody protein, use, expression vector, strain, medicine and kit.

[0014] To achieve the above purpose, the present application provides the following technical scheme: an anti-H5 subtype avian influenza virus nanobody protein, which comprises three complementarity determining regions, namely CDR1, CDR2 and CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

[0015] In the above anti-H5 subtype avian influenza virus nanobody protein, it comprises a framework region, which is divided into four parts and is FR1, FR2, FR3 and FR4 in turn, and the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0016] In the above anti-H5 subtype avian influenza virus nanobody protein, the amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the anti-H5 subtype avian influenza virus nanobody protein is shown in SEQ ID NO. 9.

[0017] Meanwhile, the application also discloses the use of the anti-H5 subtype avian influenza virus nanobody protein in the preparation of the anti-H5 subtype avian influenza virus medicine.

[0018] Or,

[0019] The use of the anti-H5 subtype avian influenza virus nanobody protein in the preparation of the diagnostic kit for the H5 subtype avian influenza is also disclosed.

[0020] In addition, the application also discloses a recombinant expression vector, wherein a gene coding the anti-H5 subtype avian influenza virus nanobody protein is cloned into an expression vector to obtain the recombinant expression vector; and the nucleotide sequence of the gene coding the anti-H5 subtype avian influenza virus nanobody protein is shown in SEQ ID NO. 9.

[0021] In the above-mentioned recombinant expression vector, one of the expression vectors pET-3a, pET-9a, pET-28a(+), pET-22b(+), and pET-41b(+) is used.

[0022] In the above-mentioned recombinant expression vector, the gene coding the anti-H5 subtype avian influenza virus nanobody protein is inserted into the pET-28a(+) through the front end restriction site Xho I and the rear end Nco I.

[0023] Meanwhile, the application also discloses a recombinant expression strain, wherein the recombinant expression vector is transfected into a host strain to obtain the recombinant expression strain.

[0024] Finally, the application also discloses a medicine containing the anti-H5 subtype avian influenza virus nanobody protein.

[0025] In addition, the application also discloses a kit containing the anti-H5 subtype avian influenza virus nanobody protein.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] (1) The application uses the phage display technology to screen the new neutralizing nanobody protein against the H5 subtype avian influenza virus HA protein, and the nanobody is specific to the H5 subtype avian influenza virus.

[0028] (2) The nanobody against the H5 subtype avian influenza virus HA protein screened in the experiment has high solubility, high purity, high temperature resistance, strong stability, and good biological activity, and has a wide development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1is a schematic diagram of soluble anti-H5 subtype avian influenza virus HA protein new neutralizing nanobody fusion plasmid 1A2 inpET-28a(+), wherein the 1A2 gene refers to a gene encoding an anti-H5 subtype avian influenza virus HA protein new neutralizing nanobody protein.

[0030] Figure 2 is a SDS-PAGE electrophoresis detection of protein purification results, wherein 1: 180 kDa Prestained Protein Marker, 2: inducer IPTG 0.1 mmol / L, 16h induced at 16℃ soluble nanobody protein purification results.

[0031] Figure 3 is a hemagglutination inhibition (HI) chart of the screened nanobody against different standard avian influenza inactivated antigens, wherein the first row of antigens is H5 (Re-8), the second row of antigens is H7 (H7-Re1 strain), the third row of antigens is H9 (H9 subtype), the 11th column is a negative control, and the 12th column is a blank control.

[0032] Figure 4 is an ELISA reaction fold line chart of coating inactivated antigens with different concentrations of soluble anti-H5 subtype avian influenza virus nanobody protein, wherein the abscissa is the antibody concentration, and the ordinate is the OD reading at a wavelength of 450nm after adding the color developing solution. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] In the embodiments, 2x Taq Plus Master Mix was purchased from Nanjing Novozyme Bio-Pharm Co., Ltd., DNA gel recovery kit was purchased from OMEGA Corporation of the United States, Sif I endonuclease was purchased from NEB Company of the United Kingdom, primers were synthesized by Shanghai Aik Biotech Co., Ltd., HRP-M13 was purchased from Beijing Yiqi God, TMB substrate color developing solution was purchased from Yuisheng Biotech Co., Ltd., mouse anti-his monoclonal antibody and goat anti-mouse-HRP antibody were purchased from Shanghai Sangon Biotech Co., Ltd., the used competent cells were from Novozyme Bio-Pharm Co., Ltd., and SDS-PAGE reducing gel was purchased from Jinssr Biotech Co., Ltd.

[0035] Example 1 Construction of Phage Display Library

[0036] Immunization was performed by injecting young Xinjiang Bactrian camels (Gansu Lanzhou Institute of Animal Husbandry and Veterinary Medicine) with hemagglutination inhibition test antigen (Harbin Weike Biotechnology Development Co., Ltd.) of avian influenza virus H5 subtype Re-8 strain every week continuously within 42 days. Three days after the last boost, 50 mL of peripheral blood was collected for lymphocyte separation, RNA extraction, and reverse transcription to obtain cDNA;

[0037] Trnol reagent was used to extract total RNA from lymphocytes, and cDNA was generated in turn as a template. In the first round of PCR, CALL001 (5'-GTC CTG GCT GCT CTT CTA CAA GG-3') and CALL002 (5'-GGT ACG TGC TGT TGA ACT GTT CC-3') primers were used to amplify the leading sequence of the conserved region within the CH2 exon of all IgG, and two bands with a size of about 900-700 bp were generated. The amplification reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, 40 cycles; in the second round of PCR, PMCF (5'-CTA GTG CGG CCG CTG AGG AGG CGG TGA CCT GGG T-3') and A6E (5'-GAT GTG CAG CTG CAG GAG TCT GGR GGA GG-3') primers were used to extract a 400 bp fragment as a template to amplify the VHH coding gene. The PCR amplification reaction conditions were as follows: 94°C for 3 min; 94°C for 150 s, 55°C for 15 s, 72°C for 15 s, 35 cycles; the obtained VHH amplicon was connected to pMECS phage through a Sif I enzyme digestion site, and the recombinant phage was electroporated into E. coli TG1 competent cells, and these cells were inoculated on 2×YT plates containing 100 μg / mL ampicillin. The bacteria were grown on agar plates containing 100 μg / mL ampicillin and 1% (w / v) glucose, and the library capacity and diversity of the bacterial library were measured by counting the number of bacteria and DNA sequencing. The amplified nanobody phage display was rescued to obtain a phage library containing an antibody library, i.e., when the obtained nanobody library bacteria were shaken to OD600 = 0.6, M13KO7 (multiplicity of infection 20:1) helper phage was added, shaken for 45 min, and then the phage particles were purified from the culture supernatant by precipitation with 20% (v / v) polyethylene glycol 8000 and 2.5 M NaCl. The obtained phage was used for ELISA screening, and all the phage amplifications were saved as phage libraries.

[0038] Example 2 Screening of positive monoclonal by ELISA method

[0039] In the first round of screening, 100 μL of inactivated antigen was coated on high binding capacity ELISA plates with sodium carbonate-bicarbonate buffer (0.05 mol / L, pH = 9.6) overnight at 4°C. After washing with phosphate buffered saline containing 0.1% Tween-20 (PBST), the wells were blocked with 3% bovine serum albumin (BSA)-PBS for 2 hours at 37°C. Then, 100 μL of the initial phage-displayed nanobody library (2 x 1011pfu) was added to the wells and incubated for 1 hour at 37°C. Unbound phage was discarded and the wells were washed 10 times with PBST. Bound phage was eluted with 100 μL of elution buffer [0.2 M glycine-HCl (pH 2.2) and 1 mg / mL BSA] for 8 minutes at 37°C with gentle shaking and immediately neutralized with 15 μL of 1 M Tris-HCl (pH 9.0). The eluted phage was subjected to a colony titering to determine the library size and amplification. In the following three rounds of screening, the number of input phage library was kept constant (2 x 1011pfu), while the volume of coated antigen was gradually reduced to 50, 25 and 10 μL, respectively. The resulting phage from the fourth round elution was diluted and plated on culture medium, and individual clones were randomly picked, expanded and identified by phage ELISA, i.e., 100 μL of diluted phage mixture was added to each well of a 96-well plate coated with 100 μL of antigen and incubated for 1 hour at room temperature. The solution was discarded and the wells were washed 5 times with buffer 6. The plate was incubated with 100 μL of anti-M13-HRP antibody diluted 1:8000 for 1 hour at room temperature in each well. After 5 washes with PBST, 100 μL of TMB was added to each well. The plate was incubated for 2 minutes at room temperature in the dark and the color development reaction was stopped with 50 μL of 1 M sulfuric acid. The reading at wavelength 450 nm was measured. Individual phage (positive) that did not wash off were streaked onto 5 mL of E. coli TG1 at OD600 = 0.5, and incubated at 37°C at 200 rpm for 2 hours; the bacteria were collected by centrifugation into 250 mL of 2 x YT medium, Kana was added to a final concentration of 50 μg / mL, and the culture was incubated at room temperature at 200 rpm for 1 hour; the culture was incubated overnight at 30°C with shaking, which was the amplified positive helper phage. The positive phage was aliquoted at -80°C with a final concentration of 25% (V / V) of glycerol, and the DNA encoding the positive phage was sent for sequencing. 11 pfu), and the volume of coated antigen was gradually reduced to 50, 25 and 10 μL, respectively. The resulting phage from the fourth round elution was diluted and plated on culture medium, and individual clones were randomly picked, expanded and identified by phage ELISA, i.e., 100 μL of diluted phage mixture was added to each well of a 96-well plate coated with 100 μL of antigen and incubated for 1 hour at room temperature. The solution was discarded and the wells were washed 5 times with buffer 6. The plate was incubated with 100 μL of anti-M13-HRP antibody diluted 1:8000 for 1 hour at room temperature in each well. After 5 washes with PBST, 100 μL of TMB was added to each well. The plate was incubated for 2 minutes at room temperature in the dark and the color development reaction was stopped with 50 μL of 1 M sulfuric acid. The reading at wavelength 450 nm was measured. Individual phage (positive) that did not wash off were streaked onto 5 mL of E. coli TG1 at OD600 = 0.5, and incubated at 37°C at 200 rpm for 2 hours; the bacteria were collected by centrifugation into 250 mL of 2 x YT medium, Kana was added to a final concentration of 50 μg / mL, and the culture was incubated at room temperature at 200 rpm for 1 hour; the culture was incubated overnight at 30°C with shaking, which was the amplified positive helper phage. The positive phage was aliquoted at -80°C with a final concentration of 25% (V / V) of glycerol, and the DNA encoding the positive phage was sent for sequencing. 11

[0040] The amino acid sequence of the anti-H5 subtype avian influenza virus nanobody protein is shown below (SEQ ID NO. 1):

[0041] ​MDNQVQLVESGGGSVQAGGSLRLSCAASADIYSSNVMGWFRQAPGKEREGVAAISPDGGNTYYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAMYYCAASTSWPDWVGGHFADLTYVAQGTQVTVSS

[0042] Wherein, the anti-H5 subtype avian influenza virus nanobody protein comprises a framework region (FR) and an antibody gene complementarity determining region (CDR), the framework region is divided into four parts, which are defined as FR1, FR2, FR3 and FR4 in turn, and the amino acid sequences are shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the complementarity determining region is divided into three parts, which can be defined as CDR1, CDR2 and CDR3 in turn, and the amino acid sequences are shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8;

[0043] SEQ ID NO. 2: MDNQVQLVESGGGSVQAGGSLRLSCAAS

[0044] SEQ ID NO. 3: WFRQAPGKEREGVAA

[0045] SEQ ID NO. 4: YYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAMYYC

[0046] SEQ ID NO. 5: VAQGTQVTVSS

[0047] SEQ ID NO. 6: ADIYSSNVMG

[0048] SEQ ID NO. 7: ISPDGGNT

[0049] SEQ ID NO. 8: AASTSWPDWVGGHFADLTY

[0050] The nucleotide sequence of the gene encoding the above-mentioned anti-H5 subtype avian influenza virus nanobody protein is shown as follows (SEQ ID NO. 9):

[0051] Atggacaatcaggtgcagctggtggagtctgggggaggctcggtgcaggctggagggtctctgagactctcctgtgcagcctctgcagacatctac agttcgaacgtcatgggctggttccgccaggctccagggaaggagcgcgagggggtcgcagctatttcgcctgatggtggtaacacatactatgccg actccgtaaagggccgattcaccatctcccgagacagcgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcggcctcaacctcctggcctgactgggtaggtggtcatttcgctgacttgacttacgtggcccaggggacccaggtcaccgtctcctca

[0052] Example 3: Construction of a novel soluble neutralizing nanobody protein particle 1A2 inpET-28a(+) against H5 subtype avian influenza virus HA protein

[0053] Based on the gene sequence encoding the anti-H5 subtype avian influenza virus nanobody protein (SEQ ID NO.9), the gene sequence of the soluble anti-H5 subtype avian influenza virus nanobody fusion protein was synthesized into the pET-28a(+) vector, named 1A2 in pET-28a(+), synthesized by Anhui General Company. After correct sequencing, a recombinant vector of 5632 bp was obtained. Figure 1 ).

[0054] Example 4: Induction of soluble anti-H5 subtype avian influenza virus nanobody fusion protein

[0055] The plasmid 1A2 in pET-28a(+) obtained in Case 3 (10 μL) was transformed into E. coli competent cells BL21(DE3). The cells were then placed on ice for 30 min, followed by heat shock in a 42℃ water bath for 90 s, and finally incubated at 37℃ on a shaker at 220 rpm for 1 h. 100 μL of the activated bacterial culture was then plated onto an LB agar plate containing 50 mg / mL kanamycin for further activation and incubation at 37℃ for 16 h. The next day, single-colony PCR was performed for confirmation (using T7 primers, where F is: TAATACGACTCACTATAGG; R is: TGCTAGTTATTGCTCAGCGG). Colonies with a target band size between 500-700 bp were transformed, and glycerol was used for preservation.

[0056] The amplification reaction conditions refer to the product manual. The amplification system is as follows: 2 x Taq Plus Master Mix enzyme 25 μL, forward primer (10 pmol / μL) 1 μL, reverse primer (10 pmol / μL) 1 μL, gene template 2 μL, and ddH2O to 50 μL. After the PCR reaction is completed, the PCR product fragment size is verified by electrophoresis on a 1% agarose gel.

[0057] The E. coli containing the recombinant plasmid was inoculated into 5 mL of LB medium (containing a final concentration of 0.1 mg / mL kanamycin) at a ratio of 1:100, and placed in a 37°C, 220 rpm shaker for overnight culture. The seed liquid cultured overnight was transferred into a new 1 L of LB medium (containing a final concentration of 0.1 mg / mL kanamycin) at a ratio of 1:100, and cultured at 37°C, 220 rpm until the OD600 was 0.6-0.8. A final concentration of 0.1 mmol / L IPTG was added, and the culture was induced at 16°C, 220 rpm for 18 h. After induction, centrifugation was performed at 8000 rpm for 20 min, the supernatant was removed, and the bacterial cells were washed with ultrapure water. The supernatant was removed by centrifugation at 8000 rpm for 20 min, and the bacterial cells were resuspended in 20 mmol / L Tris-HCl (pH 8.0) buffer. The bacterial cells were broken by a homogenizer, and centrifuged at 12000 rpm, 4°C for 30 min. The supernatant was collected.

[0058] The protein was purified according to the following method: 1. Add 1 mL of nickel column filler of Kingsway, which is 1 column volume; 2. Add 2-5 column volumes of lysis buffer (150 mm / L NaCl, 10 mm / L Tri-HCL) to remove excess nickel ions; 3. After the deionized water flow is complete, add 2-5 column volumes of 20 mL Binding buffer (20 mm / L imidazole, 150 mm / L NaCl, 10 mm / L Tri-HCL) to balance the nickel column; 4. Gently load the supernatant after breaking, repeat loading twice to improve recovery; 5. Add 2-5 column volumes of 80 mL Wash buffer (30 mm / L imidazole, 150 mm / L NaCl, 10 mm / L Tri-HCL) to remove impurities; 6. Add 10 mL of Elution buffer (300 mm / L imidazole, 150 mm / L NaCl, 10 mm / L Tri-HCL) to elute the target protein; 7. Add 10 column volumes of 20 mL ultrapure water to wash the column;

[0059] SDS-PAGE method to verify whether the protein is soluble expression success. The supernatant and protein loading buffer mixed according to the ratio of 1:1, boiling water 10 min, sample purchased from Nanjing Kingsley SDS-PAGE gel, while adding the same amount of protein 180kDa Prestained Protein Marker, electrophoresis voltage regulation 120V gel 30 min, and then stained with fast protein staining solution 30 min; add water to the staining tank in the shaker, decolorization gel block with water 30 min can be seen washing bands. The decolorized gel is placed in the imaging system for scanning, as shown in Figure 2 The results show that the target band size is between 12KDa-15 Kda, which is consistent with the estimated protein size of about 15Kda, and the SDS-PAGE band is clear, proving that the protein is successfully expressed.

[0060] Example 5 Soluble anti-H5 subtype avian influenza virus nanobody fusion protein functional activity verification

[0061] Avian influenza virus has the phenomenon of red blood cell agglutination, and anti-H5 subtype avian influenza virus nanobody has the function of inhibiting the corresponding subtype avian influenza virus hemagglutination, i.e. hemagglutination inhibition. Therefore, we test the biological activity of the soluble anti-H5 subtype avian influenza virus nanobody fusion protein prepared in Example 4 by hemagglutination inhibition (HI) experiment, and the specific process can refer to the national standard; first, measure the virus titer by hemagglutination (HA) test, configure four units, and then perform HI experiment to verify the biological activity.

[0062] (1) HA experiment:

[0063] ① In 96-well V-shaped plate 1-11 holes, add 25 μL PBS, and in the 12th hole, add 50 μL PBS;

[0064] ② Add 25 μL of hemagglutination inhibition experiment antigen purchased from Harbin Weike Biotechnology Development Company H5 subtype Re-8 strain to the first hole, mix well and dilute by 2 times to the 11th hole, discard 25 μL in the 11th hole, and do not add in the 12th hole;

[0065] ③ Add 25 μL of diluted PBS to each of holes 1-11.

[0066] ④ Gently shake the mixed 1% chicken red blood cell suspension, and then add 25 μL to each of holes 1-12. After gentle shaking, stand at room temperature (24-25℃) for 40 min, and observe the results.

[0067] The 1% chicken red blood cell suspension is prepared as follows: the blood of SPF chicken is drawn into an anticoagulant tube, centrifuged at 400g for 5 minutes, and washed with PBS; the white blood cells on the surface of the red blood cells are removed by centrifugation and suction, and the process is repeated for 3-5 times, and then the PBS: red blood cells are mixed at 99:1 to prepare the 1% chicken red blood cell suspension. The results are determined as follows: 100% agglutination, film-shaped, agglutination particles are uniformly distributed on the entire bottom of the well; 75% agglutination, red blood cells in the form of drops on the bottom of the well, and a film is formed around; 50% agglutination, blood drops surrounded by agglutination particles, not in the form of a film; 25% agglutination, only a small amount of agglutination particles around the blood drops; 0% agglutination, round drops are precipitated on the bottom of the well, and there are no agglutination particles. The complete hemagglutination is used as the titer of the virus.

[0068] Four-unit configuration: after the HA titer is measured, the four-unit antigen (4HAU) is configured by diluting the stock solution by 2 n-2 times, and after the four-unit antigen is configured, the four-unit verification is performed according to the steps of testing HA, and when complete hemagglutination occurs in the current two wells, it is the four-unit antigen configured.

[0069] (2) HI experiment:

[0070] ① 25 μL PBS is added to wells 1-11 of a 96V plate, and 50 μL PBS is added to well 12;

[0071] ② 25 μL of soluble anti-H5 subtype avian influenza virus nanobody fusion protein (supernatant prepared in Example 5) is added to well 1, and then sequentially diluted by 2 times to well 10, and then 25 μL PBS is discarded, and wells 11 and 12 are not added;

[0072] ③ 25 μL of AIV H5 subtype Re-8 strain 4-unit antigen suspension is added to wells 1-11, and is allowed to stand at room temperature (24-25°C) for at least 30 minutes, and well 12 is not added;

[0073] ④ 25 μL of 1% red blood cells is added to wells 1-12; gently shake and mix, and allow to stand at room temperature (24-25°C) for 40 minutes, and then observe the results.

[0074] The H7 and H9 hemagglutination inhibition experiments are control experiments of the antibody, and the steps are the same as those of the hemagglutination inhibition experiment of the H5 subtype Re-8 strain. The H5, H7 and H9 avian influenza hemagglutination inhibition test antigens are Re-8 strain, H7-Re1 strain and H9 subtype, respectively, which are purchased from Harbin Weike Biotechnology Co., Ltd.

[0075] The HI results show that the four-unit antigens of the first, second and third lines are H5, H7 and H9 avian influenza hemagglutination inhibition test antigens, respectively, wherein the soluble anti-H5 subtype avian influenza virus nanobody fusion protein has a hemagglutination inhibition titer of 8 log2 for the H5 antigen, and no hemagglutination inhibition for H7 and H9, which is a specific binding anti-H5 avian influenza nanobody fusion protein antibody. Figure 3

[0076] Example 6 Verification of the binding ability of soluble anti-H5 avian influenza virus nanobody protein to antigen

[0077] To determine the binding characteristics of the antibody, an ELISA plate (Nunc, USA) was coated with 20 μL / well of inactivated antigen at 4°C overnight. Then the plate was blocked with 5% milk in PBS Tween 20 (PBST). After washing with PBST, 10 μL of purified nanobody was added to the wells in serial 2-fold dilutions and incubated at 37°C for 2 hours, then 200 μL / well of blocking solution was added and incubated at 37°C for 2 hours; after blocking, the plate was washed 3 times with PBST for 5 minutes each time, then 100 μL of mouse-derived His (1:5000 dilution of monoclonal antibody with PBS) was added and incubated at 37°C for 1 hour, after which the plate was washed 3 times with PBST for 5 minutes each time, then 100 μL of goat anti-mouse horseradish peroxidase (HRP, 1:10000 dilution with PBS) was added and incubated at 37°C for 1 hour. After washing and color development, the absorbance was measured at 450 nm.

[0078] The ELISA results show that as the concentration of soluble anti-H5 avian influenza virus nanobody decreases (within a certain range), the OD 450 value also decreases, and the EC50 is calculated by graph prism to be 167.7 ng( Figure 4 ), indicating good binding ability.

[0079] During the screening of the antibody proteins of the present application, we used the screening principles of Example 1 and Example 2 above, and a total of more than 500 antibody proteins with potential application prospects were screened;

[0080] Some of the proteins showed soluble expression during the induction expression of Example 4, but their binding ability and specificity did not meet the minimum application standards;

[0081] Some proteins could not be expressed in large quantities during induction expression, indicating poor solubility, so subsequent verification was stopped and solubility improvement was carried out instead.

[0082] Due to space limitations, we cannot provide all the screened proteins, and the sequences of some of the proteins are listed in Table 1 below to illustrate the difficulty of screening the target proteins of the present application, as follows: ​

[0083] Table 1. Partial proteins screened

[0084]

[0085] Therefore, compared with the prior art, the present application has the following advantages:

[0086] 1. The antibody protein with excellent soluble expression is selected;

[0087] 2. The affinity is more excellent.

[0088] Based on the above two advantages, compared with the existing products, the development prospect is wider.

[0089] It is apparent for those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, and the present application can be realized in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all aspects as illustrative and not restrictive, and the scope of the present application is defined by the appended claims rather than the foregoing description, and it is intended to embrace all changes falling within the meaning and range of equivalents of the elements of the claims. Any reference signs in the claims should not be considered as limiting the claims involved.

Claims

1. A nanobody protein against H5 subtype avian influenza virus, characterized in that, It contains three complementarity-determining regions, namely CDR1, CDR2, and CDR3; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

2. The anti-H5 subtype avian influenza virus nanobody protein according to claim 1, characterized in that, It includes a frame region, which is divided into four parts, namely FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

3. The anti-H5 subtype avian influenza virus nanobody protein according to claim 1 or 2, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding the nanobody protein against H5 subtype avian influenza virus is shown in SEQ ID NO.

9.

4. Use of the anti-H5 subtype avian influenza virus nanobody protein as described in any one of claims 1 to 3 to prepare a drug for the preparation of an anti-H5 subtype avian influenza virus; or, The use of the anti-H5 subtype avian influenza virus nanobody protein as described in claim 1 to prepare a diagnostic kit for H5 subtype avian influenza.

5. A recombinant expression vector, characterized in that, The recombinant expression vector is obtained by cloning the gene encoding the anti-H5 subtype avian influenza virus nanobody protein into the expression vector; the nucleotide sequence of the gene encoding the anti-H5 subtype avian influenza virus nanobody protein is shown in SEQ ID NO.

9.

6. The recombinant expression vector according to claim 5, characterized in that, One of the expression vectors pET-3a, pET-9a, pET-28a(+), pET-22b(+), and pET-41b(+).

7. The recombinant expression vector according to claim 5, characterized in that, The gene for the anti-H5 subtype avian influenza virus nanobody protein was inserted into pET-28a(+) via the front-end restriction enzyme site Xho I and the back-end Nco I.

8. A recombinant expression strain, characterized in that, The host strain was obtained by transfecting the recombinant expression vector as described in any one of claims 5 to 7.

9. A drug, characterized in that, It contains the anti-H5 subtype avian influenza virus nanobody protein as described in any one of claims 1 to 3.

10. A reagent kit, characterized in that, It contains the anti-H5 subtype avian influenza virus nanobody protein as described in any one of claims 1 to 3.

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