CN117659183A - anti-H5 subtype avian influenza nanobody protein and application thereof - Google Patents
anti-H5 subtype avian influenza nanobody protein and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
The invention belongs to the technical field of biological medicines, and discloses an anti-H5 subtype avian influenza nanobody protein and application thereof. Specifically discloses an anti-H5 subtype avian influenza virus nano antibody, which comprises 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. According to the invention, nanobody development is carried out aiming at H5 subtype avian influenza virus, and a nanobody fusion protein 5B2 which specifically binds with H5-Re14 strain with higher affinity is screened, wherein the nanobody fusion protein 5B2 is a nanobody aiming at H5 subtype avian influenza virus and having high binding force.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-H5 subtype avian influenza nanobody protein and application thereof.
Background
Influenza viruses are widely popular worldwide, a plurality of people infect the influenza viruses in winter and spring alternation of influenza outbreaks each year, in addition, the host spectrum of the influenza viruses is very wide, and the influenza viruses can be separated from various animal bodies such as bat, poultry, people, pigs, horses, dolphins and seals at present, thereby causing great threat to public health safety. Influenza viruses are members of the orthomyxoviridae, influenza virus genus, and can be classified into three types A, B, C according to the differences between viruses NP and M. Avian influenza virus (avian influenza virus, AIV) belongs to influenza a virus, and can be classified into low-pathogenic avian influenza (low pathogenic avian influenza, LPAI) and high-pathogenic avian influenza (highly pathogenic avian influenza, HPAI) according to the difference in their pathogenic abilities. Among them, highly Pathogenic Avian Influenza (HPAI) is a disease caused by H5 subtype strain or H7 subtype strain (represented by H5N1 and H7N 7), and can infect almost all poultry and wild birds, and serious clinical symptoms occur after infection, with a mortality rate of 100%. At present, the cases of the high pathogenicity avian influenza virus infection of people occur, the distribution is wide, and the virus is a new subtype which possibly evolves to generate the virus for inducing the pandemic of the human influenza through antigen drift and antigen transformation, and the potential threat is not ignored.
The structure of the avian influenza virus can be divided into a core part, a matrix protein part and an envelope part from inside to outside, genetic materials are single-strand negative strand RNA, and the avian influenza virus consists of 8 discontinuous segments, and can code at least 10 proteins (PB 1, PB2, PA, HA, NP, NA, M1, M2, NS1 and NS 2). Hemagglutinin (HA) is one of the major components constituting the membrane fibers of the avian influenza virus envelope, is present on the surface of the envelope in the form of a trimer having a molecular weight of about 75kDa and its primary structure HAs 4 domains, which are signal peptide, cytoplasmic domain, transmembrane domain and extracellular domain capable of recognizing the endoplasmic reticulum membrane, respectively. The HA trimer HAs 5 antigenic determinants, A, B, C, D, E respectively, on the head, can induce the generation of protective neutralizing antibodies, is the most important protective antigen of AIV, and can stimulate the organism to generate corresponding antibodies, thereby generating resistance. Therefore, HA is of great significance to the development and study of AIV antibodies.
According to different functions and antigen characteristics of structural proteins of AIV, various experimental methods for detecting AIV have been established at home and abroad, and serological detection such as Hemagglutination (HA), hemagglutination inhibition test (HI), enzyme-linked immunosorbent assay (ELISA), virus neutralization test (VN), agar diffusion test and the like; molecular biological assays such as Polymerase Chain Reaction (PCR), fluorescent quantitative real-time reverse transcription-polymerase chain reaction (RT-RCR), and the like. Among the above various assays for AIV, HI assay is most commonly used because it is rapid, simple and inexpensive, but because of the relatively complicated preparation of polyclonal antisera, the sensitivity and specificity are relatively poor, and the mass differences of antibodies from lot to lot are relatively large. In practical application, monoclonal antibodies still have such problems as high cost, complex process, ethical issues caused by the production of antibodies by inducing ascites of experimental animals through immunogens, and the like; polyclonal antibodies, although relatively inexpensive, suffer from the disadvantage of being prone to cross-reaction leading to erroneous subtype determination and batch-to-batch variation. The nanobody (Nb) has the characteristics of small molecular weight, good stability, high affinity, high specificity, and easy high-quality production, and can just avoid the above-mentioned disadvantages, and in recent years, development in the field of immunodetection has been rapidly advanced.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a nano antibody for resisting the H5 subtype avian influenza virus and application thereof. The nano antibody has good stability and biological activity, can be applied to diagnosis and detection of H5 subtype avian influenza virus, and has excellent detection sensitivity and accuracy.
The object of the first aspect of the invention is to provide an anti-H5 subtype avian influenza virus nanobody.
The object of the second aspect of the present invention is to provide a fusion protein.
The object of the third aspect of the present invention is to provide a nucleic acid molecule.
The object of the fourth aspect of the present invention is to provide a biological material related to the nucleic acid molecule of the third aspect of the present invention.
The object of the fifth aspect of the present invention is to provide the use of the nanobody of the first aspect of the invention, the fusion protein of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention or the biomaterial of the fourth aspect of the invention for the preparation of a product.
The object of the sixth aspect of the invention is to provide a product.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, an anti-H5 subtype avian influenza virus nanobody is provided, wherein the nanobody comprises 3 complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.
In some embodiments of the invention, the nanobody further comprises framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 respectively.
In some embodiments of the invention, the amino acid sequence of the nanobody is as shown in SEQ ID NO. 1.
The nanobody is a nanobody which is screened out by a phage library and aims at the H5 subtype avian influenza virus.
In a second aspect of the invention there is provided a fusion protein comprising a nanobody of the first aspect of the invention.
In some embodiments of the invention, the fusion protein further comprises a histidine-binding protein.
In some embodiments of the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 11.
In a third aspect of the invention, there is provided a nucleic acid molecule comprising the following components:
(1) A nucleic acid fragment for encoding a nanobody of the first aspect of the invention; or (b)
(2) Nucleic acid fragments for use in the fusion proteins of the second aspect of the invention.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO 9 or SEQ ID NO 10.
In a fourth aspect of the invention there is provided a biological material associated with the nucleic acid molecule of the third aspect of the invention, the biological material comprising at least one of a 1) to a 3):
a1 An expression cassette comprising the nucleic acid molecule of claim 5 or 6;
a2 A recombinant vector comprising the nucleic acid molecule of claim 5 or 6 or an expression cassette of a 1);
a3 A recombinant cell comprising the nucleic acid molecule of claim 5 or 6, the expression cassette of a 1) or the recombinant vector of a 2).
In some embodiments of the invention, the recombinant vector is a plasmid vector, a viral vector, or a cellular vector.
In some embodiments of the invention, the plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cellular vector does not include propagation material.
In some preferred embodiments of the invention, the carrier is a PET series carrier.
In some preferred embodiments of the invention, the vector is pET-9a, pET-28a (+), pET-22b (+), pET-26b (+), or pET-31b (+).
In some more preferred embodiments of the invention, the vector is pET-28a (+).
In some embodiments of the invention, the expression vector is obtained by specifically inserting the nucleic acid molecule of the third aspect of the invention into pET28a (+) via the cleavage sites XhoI and NcoI.
In some embodiments of the invention, the cell is a prokaryotic cell.
In some embodiments of the invention, the recombinant cell is a bacterium, yeast, or fungus.
In some embodiments of the invention, the recombinant cell is E.coli.
In some embodiments of the invention, the recombinant cell is E.coli BL21 (DE 3).
In a fifth aspect, the invention provides the use of a nanobody according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention or a biomaterial according to the fourth aspect of the invention in the preparation of a product.
In some embodiments of the invention, the product functions as any one of c 1) to c 5):
c1 Preparing a reagent for detecting and/or diagnosing avian influenza virus;
c2 Detecting and/or diagnosing avian influenza virus;
c3 Preparing a medicament for preventing and/or treating diseases related to avian influenza virus infection;
c4 Preventing and/or treating diseases related to avian influenza virus infection;
c5 Initiating or enhancing an immune response in the body.
In some embodiments of the invention, the product includes, but is not limited to, reagents, kits, medicaments.
In some embodiments of the invention, the medicament comprises a vaccine.
In some embodiments of the invention, the medicament may be in the form of a nasal spray, oral formulation, suppository or parenteral formulation.
In some embodiments of the invention, the avian influenza virus is an H5 subtype avian influenza virus.
In a sixth aspect, the invention provides a product comprising a nanobody of the first aspect of the invention, a fusion protein of the second aspect of the invention, a nucleic acid molecule of the third aspect of the invention or a biological material of the fourth aspect of the invention.
In some embodiments of the invention, the products include, but are not limited to, medicaments, including vaccines, reagents and kits.
In some embodiments of the invention, the product further comprises a pharmaceutically acceptable excipient.
In some embodiments of the invention, the adjuvant comprises a carrier, diluent, excipient, preservative, antibacterial agent, and/or immunoadjuvant.
The invention also provides a preparation method of the fusion protein of the second aspect, which comprises the steps of cloning the gene of the fusion protein of the third aspect into an expression vector to obtain a recombinant expression vector, transferring the recombinant expression vector into escherichia coli to obtain recombinant expression cells, expressing, collecting an expression product, crushing, separating and purifying.
In some embodiments of the invention, the separation and purification comprises a step of treatment with nickel column affinity chromatography.
In some embodiments of the invention, the recombinant expression cells are obtained and expressed in a manner that induces expression.
In some embodiments of the invention, the inducer that induces expression is IPTG.
In some embodiments of the invention, the final concentration of IPTG is 0.05-0.15 mmol/L.
In some embodiments of the invention, the final concentration of IPTG is 0.1mmol/L.
In some embodiments of the invention, the conditions for inducing expression are 27-29℃for 14-18 h.
In some embodiments of the invention, the recombinant expression strain is cultured to a bacterial liquid OD 600 And when the ratio is 0.6-0.8, adding an inducer for induction.
The invention adopts a prokaryotic expression system to construct and obtain recombinant bacteria for expressing the nanometer antibody of the anti-H5 subtype avian influenza virus, effectively reduces the production cost of the nanometer antibody, improves the accuracy and the repeatability, has higher purity of the produced nanometer antibody protein, and can realize the large-scale production of the nanometer antibody of the anti-H5 avian influenza virus.
The beneficial effects of the invention are as follows:
the invention develops nano antibody aiming at H5 subtype avian influenza virus, and screens nano antibody fusion protein 5B2 which specifically binds H5-Re14 strain with higher affinity. The inventors verified the ability of the virus to specifically inhibit the aggregation of erythrocytes by a hemagglutination inhibition assay, and were able to bind to the H5 subtype (H5-Re 14 strain) with a high binding force in an ELISA assay. These all indicate that the nanobody fusion protein 5B2 is a nanobody with high binding force against H5 subtype avian influenza virus.
The production method of the anti-H5 avian influenza virus nanobody protein provided by the invention is simple to operate and low in cost, and can realize the large-scale production of the anti-H5 avian influenza virus nanobody.
The H5 avian influenza virus resistant nanobody fusion protein provided by the invention has higher purity, low sensitivity in the detection process, good stability and biological activity, and provides a potential nanobody reagent for clinical prevention, treatment and detection of avian influenza virus. The method can be used for developing a novel VHH antibody immunodetection method aiming at avian influenza virus, so as to solve the problems of low antibody affinity, low purity in the production process, low sensitivity and low efficiency in the detection process in the past development process.
Drawings
FIG. 1 is a recombinant expression vector of an anti-H5 avian influenza virus nanobody fusion protein 5B2.
FIG. 2 is a diagram showing the results of PCR assay, in which lane 1 is Yeasen 2000Marker, lanes 2 to 5 are the results of amplification of a plurality of single colony PCR after transformation, and lane 6 is a negative control.
FIG. 3 is a diagram showing the result of analysis of SDS-PAGE electrophoresis of proteins, in which lane 1 shows Vazyme MP102Maker and lane 2 shows the result of expression of fusion protein 5B2.
FIG. 4 is a graph showing the results of Hemagglutination Inhibition (HI), wherein the first row of antigens is H5-Re14, the second row of antigens is H7-Re4, the third row of antigens is H9 subtype (SD 696 strain), the 11 th row is a negative control, and the 12 th row is a blank control.
FIG. 5 is a line drawing showing ELISA reaction between fusion protein 5B2 and inactivated antigen H5-Re14 strain.
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified. For example: ex Taq enzyme, 4X Protein SDS PAGE Loading Buffer, DL 2000Marker, DL 5000Marker, restriction endonucleases Nco I and Xho I were all purchased from TAKARA; protein Marker, 5 XCE II Buffer, exnase II, loading Buffer terminator and nucleic acid dye were purchased from Vazyme; both murine anti-His mab and High Affinity Ni-NTA medium were purchased from Kirsrui biotechnology; some commonly used biological materials, such as competent cells, vectors, helper phage, cells to be transformed, etc., are also commercially available products, e.g., E.coli competent DH 5. Alpha. From Vazyme; e.coli protein expression vector pET-28a was purchased from Solarbio; some synthetic biological materials, such as primers, sequences, etc., which require artificial synthesis, are committed to the synthesis company, e.g., the primers of the present invention are synthesized by Ai Ji biological limited.
EXAMPLE 1 construction of phage nanobody library
Immunizing a recombinant AIV inactivated vaccine (H5N 8, H5-Re 14) into a Bactrian camel, immunizing once every three weeks, immunizing four times altogether, extracting about 50mL of immunized donor peripheral lymphocyte blood, separating peripheral blood lymphocytes (PBMCs) from the blood, extracting total RNA according to the steps of the specification, performing reverse transcription to obtain cDNA, performing nested PCR amplification twice by using the cDNA as a template to obtain VHH fragments, and cutting and recovering target bands. The purified DNA is used as a template, a nested PCR is used for amplifying VHH fragments, the VHH fragments are connected with a pcantab-5E phagemid vector, and the connected products are transformed into XL-Blue bacteria to construct a nano antibody library aiming at the antigen; the phage nanobody library can be obtained by culturing the helper phage VCSM13 super-infected host bacteria XL-Blue bacteria overnight and collecting the supernatant.
EXAMPLE 2 screening of phage nanobody libraries
Coating H5 subtype avian influenza inactivating antigen (H5-Re 14 strain) on an ELISA plate with sodium carbonate-sodium bicarbonate buffer solution (0.05 mol/L, pH9.6), wherein each hole contains 100 μl of inactivating antigen 10%, and standing at 37deg.C for 2 hr; the wells were discarded, washed once with 100. Mu.L of PBS, then 300. Mu.L of 3% BSA was added and the wells were blocked at room temperature for 1h; removing the blocking solution, adding 100 mu L of phage antibody liquid, and incubating for 1h at room temperature; discarding the liquid in the holes and unbound phage, washing for 5 times with 0.05% PBST for 5 min/time, adding 100 μl of glycine-hydrochloric acid pH=2.2 eluent, collecting the eluent, adding 2M Tris base to make the phage-containing liquid neutral, and obtaining phage subjected to first round enrichment screening; amplifying the phage further, and entering the next round of screening; five rounds of "adsorption-elution-amplification" screening processes, a phage clone with greater specificity can be obtained (wherein the first round of screening uses non-specific conditions and the later rounds of screening improves specificity by changing antigen coating concentration).
Screening positive clones by ELISA method by selecting single colony: coating a single colony on an ELISA plate according to the coating method, sealing, taking a blank ELISA plate (i.e. not coated) as a blank control, adding 100 mu L/hole HRP-M13, incubating for 1h at 37 ℃, washing 5 times by 0.05% PBST, adding 100 mu L/hole TMB substrate color development solution for 5 min/time, standing at room temperature in a dark place for 20min, measuring an OD value with a wavelength of 620nm by an ELISA reader, and taking a P/N value (i.e. the ratio of positive hole OD reading to reference hole OD reading) as positive when the P/N value is more than or equal to 3. And (3) sequencing the positive clone to obtain the anti-H5 subtype avian influenza virus nano antibody sequence.
The amino acid sequence of the anti-H5 subtype avian influenza virus nano antibody is shown as SEQ ID NO.1, and comprises a Framework Region (FR) and an antibody gene Complementarity Determining Region (CDR), wherein the framework region is divided into four parts, FR1, FR2, FR3 and FR4 are defined in sequence, and the amino acid sequence of the anti-H5 subtype avian influenza virus nano antibody is 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 sequence, and the amino acid sequences of the complementarity determining region are SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 in sequence.
Wherein, SEQ ID NO. 1:
MDNQVQLVESGGGSVQAGGSLRLSCLISAYDYFKAMAWFRQAPGKEREGVASI YGGNAYYADSVQGRVTISRDNAKATLYLQMNSLKPEDTAMYYCAASTRYVPTTQILH EFQYTDWGQGTQVTVSS。
SEQ ID NO:2：MDNQVQLVESGGGSVQAGGSLRLSCLIS。
SEQ ID NO:3：WFRQAPGKEREGVAS。
SEQ ID NO:4：YYADSVQGRVTISRDNAKATLYLQMNSLKPEDTAMYYC。
SEQ ID NO:5：WGQGTQVTVSS。
SEQ ID NO:6：AYDYFKAMA。
SEQ ID NO:7：IYGGNA。
SEQ ID NO:8：AASTRYVPTTQILHEFQYTD。
the nucleotide sequence of the gene for encoding the anti-H5 subtype avian influenza virus nanobody protein is shown as SEQ ID NO.9, wherein the SEQ ID NO. 9:
ATGGACAATCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTTAATCTCTGCATACGACTACTTTAAGGCAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCTTCTATCTATGGTGGTAACGCATACTATGCGGACTCCGTGCAGGGCCGCGTCACCATCTCCCGAGACAACGCCAAGGCCACGCTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCCAGTACACGCTATGTACCTACTACTCAGATCCTGCATGAATTTCAATATACCGACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
example 3 construction of nanobody fusion protein plasmid 5B2 in pET-28a (+) against H5 avian influenza Virus
(1) According to the gene sequence (SEQ ID NO. 9) of the anti-H5 avian influenza nanobody, the C terminal end of the anti-H5 avian influenza nanobody is connected with a histidine tag, the nucleotide sequence of fusion expression is shown as SEQ ID NO.10, and the amino acid sequence of the anti-H5 avian influenza virus nanobody fusion protein is shown as SEQ ID NO. 11.
Wherein SEQ ID NO.10:
ATGGACAATCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTTTAATCTCTGCATACGACTACTTTAAGGCAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCTTCTATCTATGGTGGTAACGCATACTATGCGGACTCCGTGCAGGGCCGCGTCACCATCTCCCGAGACAACGCCAAGGCCACGCTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCCAGTACACGCTATGTACCTACTACTCAGATCCTGCATGAATTTCAATATACCGACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCACATCATCATCATCATCATTAA。
SEQ ID NO.11：
MDNQVQLVESGGGSVQAGGSLRLSCLISAYDYFKAMAWFRQAPGKEREGVASI YGGNAYYADSVQGRVTISRDNAKATLYLQMNSLKPEDTAMYYCAASTRYVPTTQILH EFQYTDWGQGTQVTVSSHHHHHH。
the two ends of the fusion gene are respectively added with XhoI and NcoI cleavage sites through gene synthesis, and are connected with a pET-28a (+) vector for carrying out XhoI and NcoI double cleavage, and the vector is named as 5B2 in pET-28a (+), and the result is shown in figure 1, and the gene of the anti-H5 avian influenza nanobody and the gene with 6 histidine tags are sequentially connected. The 5B2 gene is the gene of the anti-H5 avian influenza nanobody. The resulting linker nucleotide sequence was optimized and synthesized by general biosystems (Anhui) Inc.
(2) Plasmid extraction: converting the synthetic plasmid of which the anti-H5 avian influenza nanobody fusion protein plasmid is 5B2 in pET-28a (+) obtained in the step (1) into receptor bacteria DH5 alpha, coating a LB (kanamycin (Kan) containing 30 mg/L) plate containing kanamycin with bacterial liquid for resuscitating and activating, culturing at 37 ℃ for 16 hours, selecting a plurality of single colonies for carrying out PCR (polymerase chain reaction) confirmation of the transformant, and re-transferring the single colony with a correct PCR strip size result to a coated plate; transferring a part of the transferred colonies into 15mL of liquid LB culture medium, shaking on a shaking table at 37 ℃ and 200rpm for 16 hours, and firstly, temporarily sub-packaging and placing part of bacterial liquid at 4 ℃ for preservation; a part of the bacterial liquid was collected by using a 15mL centrifuge tube, centrifuged at 8000rpm for 10min, and the supernatant was discarded to extract the plasmid.
Example 4 Induction of expression of anti-H5 avian influenza Virus nanobody fusion proteins
(1) The plasmid expressing the anti-H5 avian influenza virus nanoantibody fusion protein stored in the above example was transformed onto recipient E.coli BL21 (DE 3), placed on ice for 30min, heat-shocked in a water bath at 42℃for 90s, 1mL of LB broth was added, resuscitated and activated on a shaker at 220rpm at 37℃for 1min, 6000rpm was centrifuged for 1min, 90% of the supernatant was discarded, the bacterial solution was smeared on LB (30 mg/L kanamycin) plates containing kanamycin for resuscitated and activated, after 16H incubation at 37℃a plurality of single colonies were picked up and confirmed by transformant PCR using T7 primers (T7-F: 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 12); T7-R:5'-TGCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 13)), and the results are shown in FIG. 2. Re-transferring the clone colony of the target strip with the size of about 600bp to a coated plate, and storing; the specific operation steps are as follows:
amplification system: PCR Maste25. Mu.L of r Mix enzyme, 1. Mu.L of forward primer (10 pmol/. Mu.L), 1. Mu.L of reverse primer (10 pmol/. Mu.L), 3. Mu.L of gene template, and ddH 2 O was made up to 50. Mu.L.
Amplification reaction conditions: 98 ℃ for 3min;98 ℃ for 10s, 58 ℃ for 20s, 72 ℃ for 30s,35 cycles; and at 72℃for 5min.
After completion of the PCR reaction, 1% agarose gel electrophoresis was used. Gel electrophoresis shows a band of interest with a size of about 600 bp.
(2) Transferring a part of the transferred colonies into 100mL of liquid LB culture medium, and shaking on a shaking table at 37 ℃ and 200 rpm; waiting bacteria liquid OD 600 At a value of 0.6, induction was performed with IPTG at a final concentration of 0.1mM; after 16h of induction, the bacterial liquid was collected using a 50mL centrifuge tube, centrifuged at 8000rpm for 10min, and the supernatant was discarded.
(3) By using protein lysate Lysis buffer (H) 2 PO 4 ·H 2 6.9g of O (MW137.99 g/mol), namely 0.05M/L, 17.54g of NaCl (MW58.44 g/mol), namely 0.3M/L, 0.68g of imidozole (MW68.08 g/mol), namely 0.01mM/L, adding about 900mL of deionized water, stirring and dissolving, adding NaOH to adjust the pH value of the solution to 8.0, adding deionized water to fix the volume to 1000 mL), taking about 30mL of the solution to resuspend the preserved bacterial liquid, crushing the bacterial liquid by using an ultrasonic crusher, wherein the ultrasonic program is crushing for 5s, and the ultrasonic crushing is carried out for 30min at intervals of 5 s.
(4) Centrifuging the ultrasonically crushed product at 8000rpm for 20min, collecting supernatant, performing nickel column affinity chromatography, concentrating and purifying the supernatant to obtain purified anti-H5 avian influenza virus nanobody protein. The specific operation method is as follows: 0.5mL HisTrap affinity columns from GE Healthcare was taken at Poly-Prep Chromatography Columns (from BIO-RAD), 7mL of the above protein lysate Lysis buffer was added three times each time, and a total of 21mL was added to equilibrate the column, then the supernatant after cleavage was slowly added, and either was slowly dropped into a waste jar, finally an eluent Elutation buffer (H) 2 PO 4 ·H 2 6.9g of O (MW137.99 g/mol), namely 0.05M/L, 17.54g of NaCl (MW58.44 g/mol), namely 0.3M/L, 0.68g of imidozole (MW 68.08 g/mol), namely 0.25mM/L, adding about 900mL of deionized water, stirring and dissolving, adding NaOH to adjust the pH value of the solution to 8.0, adding deionized water to a volume of 1000 mL) of 1mL,collecting eluent, dialyzing with PBS at 4deg.C overnight, collecting dialyzate to obtain purified protein, determining target peak by SDS-PAGE, and obtaining purified nanobody fusion protein 5B2 (i.e. anti-H5 avian influenza virus nanobody fusion protein) as shown in figure 3; adding 20% by volume of glycerol, and storing in a refrigerator at-20deg.C. Example 5 functional verification of anti-H5 avian influenza Virus nanobody fusion protein (nanobody fusion protein 5B 2)
In order to examine whether the anti-H5 avian influenza virus nanobody fusion protein has the function of inhibiting hemagglutination, the present example examined the antibody by the Hemagglutination Inhibition (HI) experiment, the procedure was as follows: the inactivated virus titer was measured by a Hemagglutination (HA) assay, four units were prepared, and then the HI assay was performed. The operation of the Hemagglutination (HA) test of this example was conducted according to the GB/T14926.53-2001 standard, and the hemagglutination inhibition test was conducted according to the GB/T14926.54-2001 standard. The method comprises the following steps:
(1) HA experiments:
(1) 25. Mu.L of PBS was added to wells 1-11 of the 96-well plate, and 50. Mu.L of PBS was added to well 12;
(2) adding 25 mu L of inactivated H5-Re14 strain HI test antigen (purchased from Harbin Biotechnology development Co.) into the 1 st hole, uniformly mixing, sucking into the 2 nd hole, sequentially diluting to the 11 th hole in multiple ratio, uniformly mixing, discarding 25 mu L, and adding no additive into the 12 th hole;
(3) from 1 to 11 wells, diluted 25 μl PBS was added per empty;
(4) mixing 1% of red blood cells by gentle shaking, and adding 25 mu L of red blood cells into each of 1-12 holes; vibrating, standing at room temperature (24-25 ℃) for 40min, and observing a result; wherein, 1% chicken erythrocyte suspension is prepared: drawing anticoagulated chicken blood, centrifuging at 700rpm for 5 min; washed with PBS. And centrifuging again, sucking the white blood cells on the surface of the precipitated red blood cells, and continuously cleaning until the white blood cells are not on the surface of the red blood cells, wherein the PBS lotion is transparent and colorless. The mixture was gently mixed in a ratio of PBS: red blood cells=99:1 to prepare a 1% chicken red blood cell suspension.
Four units are prepared: after the HA titer is measured, four units of antigen (4 HAU) are prepared according to a method of diluting stock solution by 2n-2 times, after the preparation, the HA is subjected to four-unit verification, and when the prepared four units are verified, the hemagglutination phenomenon occurs in the first two holes, namely the four units are qualified in preparation.
(2) HI experiment:
(1) 25. Mu.L of PBS was added to wells 1-11 of the 96-well plate, and 50. Mu.L of PBS was added to well 12;
(2) adding 25 mu L of anti-H5 avian influenza virus nanobody protein into the 1 st hole, uniformly mixing and sucking the mixture to the 2 nd hole, sequentially diluting the mixture to the 10 th hole in a multiple ratio, uniformly mixing and discarding the mixture, wherein the 11 th hole and the 12 th hole are not added;
(3) adding diluted 25 mu L of AIV H5 subtype Re-14 strain 4 unit antigen suspension into 1-11 holes, standing at room temperature (24-25 ℃) for at least 30min, and adding no AIV H5 subtype Re-14 strain 12;
(4) mixing 1% of red blood cells by gentle shaking, and adding 25 mu L of red blood cells into each of 1-12 holes; shaking, standing at room temperature (24-25 ℃) for 40min, and observing the result.
The procedure of the H7 and H9 hemagglutination inhibition experiments was the same as that of the H5-Re14 strain. The H5, H7 and H9 avian influenza hemagglutination inhibition test antigens are H5-Re14 strain, H7-Re4 strain and H9 subtype (SD 696 strain) respectively, and are all purchased from Hasteven biotechnology Co.
HI results show that the titer of the anti-H5 avian influenza nanobody fusion protein antibody to H5 antigen is 6log2, the IC100 is 3.13 mug/mL, and the IC100 refers to the concentration of the nanobody fusion protein 5B2 which completely inhibits 4HAU unit antigen from agglutinating erythrocytes at the highest dilution; and the nanobody fusion protein 5B2 has no hemagglutination inhibition on H7 and H9 subtypes, and is a specifically-combined nanoprotein antibody for resisting H5 avian influenza virus (figure 4).
Example 6 application of anti-H5 avian influenza Virus nanobody fusion protein to antigen specific detection
The anti-H5 avian influenza virus nanobody fusion protein is a specific antibody aiming at H5 avian influenza, and can be used for screening expressed nanobodies to detect coated antigens.
The embodiment provides a method for detecting H5 avian influenza virus, which comprises the following steps:
(1) Antigen coating: respectively taking 30 mu L of H5 avian influenza hemagglutination inhibition test antigen (H5-Re 14 strain), diluting the antigen according to the ratio of antigen to carbonate coating buffer solution=3:7, adding 100 mu L of AIV H5-Re14 strain antigen suspension diluted from 1-12 holes, incubating at 37 ℃ for 2H or incubating at 4 ℃ overnight, and discarding liquid in a plate;
(2) Closing: blocking for 1h with 300. Mu.L of 3% BSA;
(3) An antibody: the anti-H5 avian influenza virus nanobody fusion protein 5B2 (prepared in example 4) is the primary antibody, the nanobody 5B2 is diluted to 11 th holes in a 2-fold ratio, each gradient is 3 repeated holes, and each hole is 100 mu L; the twelfth well was only added with an equal amount of PBS as a blank;
(3) Washing: washing 3-5 times by using PBS washing liquid containing 0.05% Tween-20, wherein each washing and soaking time is 5min for 5 times;
(4) And (2) secondary antibody: diluting the mouse anti-his polyclonal antibody of gold Style company with PBS according to the ratio of 1:3000, adding 100 mu L of diluted antibody into each hole, and incubating for 1h;
(5) Washing: washing 3-5 times by using PBS washing liquid containing 0.05% Tween-20, wherein each washing and soaking time is 5min, and washing 5 times;
(6) Three antibodies: the goat anti-mouse-HRP antibody of Shanghai engineering company is diluted with PBS according to the proportion of 1:5000, 100 mu L of diluted antibody is added to each hole, and the mixture is incubated for 1h;
(7) Washing: washing 3-5 times by using PBS washing liquid containing 0.05% Tween-20, wherein each washing and soaking time is 5min, and washing 5 times;
(8) Color development: adding HRP substrate chromogenic solution to carry out chromogenic for 20min,
(9) And (3) terminating: 100. Mu.L of stop solution was added to each well and the reading was performed with a microplate reader at a wavelength of 650 nm.
ELISA results showed: as the gradient of the nano fusion antibody 5B2 decreases, the P/N ratio also decreases, wherein the average blank control value is 0.04, and the maximum P/N ratio is greater than 3; half maximal Effector Concentration (EC) of the nanofusion antibody against H5 avian influenza hemagglutination inhibition assay antigen (H5-Re 14 strain) was calculated using GraphPad Prism 7.0 50 ) Is 187.9ng/mL. The prokaryotic expression of the nanometer fusion antibody against the H5 subtype avian influenza virus is proved to have good biological activity (figure 5).
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The nanometer antibody against the H5 subtype avian influenza virus is characterized by comprising 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.
2. Nanobody according to claim 1, characterized in that it further comprises framework regions FR1, FR2, FR3, FR4, wherein the amino acid sequences of FR1, FR2, FR3, FR4 are shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, respectively.
3. Nanobody according to claim 1 or 2, characterized in that the amino acid sequence of the nanobody is shown in the sequence SEQ ID No. 1.
4. A fusion protein comprising the nanobody of any one of claims 1-3; preferably, the fusion protein further comprises a histidine-binding protein; and/or the amino acid sequence of the fusion protein is shown as SEQ ID NO. 11.
5. A nucleic acid molecule comprising the following components:
(1) A nucleic acid fragment for encoding the nanobody of any of claims 1 to 3; or (b)
(2) A nucleic acid fragment encoding the fusion protein of claim 4.
6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO 9 or SEQ ID NO 10.
7. A biological material associated with the nucleic acid molecule of claim 5 or 6, said biological material comprising at least one of a 1) to a 3):
a1 An expression cassette comprising the nucleic acid molecule of claim 5 or 6;
a2 A recombinant vector comprising the nucleic acid molecule of claim 5 or 6 or an expression cassette of a 1);
a3 A recombinant cell comprising the nucleic acid molecule of claim 5 or 6, the expression cassette of a 1) or the recombinant vector of a 2).
8. Use of a nanobody according to any one of claims 1 to 3, a fusion protein according to claim 4, a nucleic acid molecule according to claim 5 or 6 or a biological material according to claim 7 for the preparation of a product.
9. The use according to claim 8, wherein the product functions as any one of c 1) to c 5): c1 Preparing a reagent for detecting and/or diagnosing avian influenza virus;
c2 Detecting and/or diagnosing avian influenza virus;
c3 Preparing a medicament for preventing and/or treating diseases related to avian influenza virus infection;
c4 Preventing and/or treating diseases related to avian influenza virus infection;
c5 Initiating or enhancing an immune response in the body.
10. A product comprising the nanobody of any one of claims 1 to 3, the fusion protein of claim 4, the nucleic acid molecule of claim 5 or 6, or the biological material of claim 7.
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