Recombinant Nanobody against H9 Subtype Avian Influenza Virus NP Protein, Recombinant Nanobody Protein, Engineering Strain and Their Applications

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a recombinant nanobody against the NP protein of H9 subtype avian influenza virus, a recombinant nanobody protein, an engineered strain and their applications. The recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention has an amino acid sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 1. The application of the recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention in the preparation of a nanoantibody preparation and / or a nanoantibody kit for diagnosing H9 subtype avian influenza virus. The recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention is screened from an established H9 subtype avian influenza virus nanobody immune library, and its immunizing antigen is the sequence characteristics of the virus strain (SG strain) under the current prevalent H9.4.2.2 branch, and has higher binding adaptability with the prevalent virus strain.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant nanobody against the NP protein of H9 subtype avian influenza virus, a recombinant nanobody protein, an engineering strain and their applications. Background Art

[0002] At present, the prevalence rate of H9 subtype avian influenza is relatively high in the Chinese region. Compared with H5 and H7 HPAIV, in terms of the state, H9 LPAIV can present latent infection clinically. When the external environment changes drastically or is subjected to secondary infection, it can cause large-scale death of poultry. In addition to poultry, H9 LPAIV can naturally infect mammals such as pigs, ferrets, guinea pigs and humans. Existing research reports have shown that when H9 is co-infected with subtypes such as H7N9, H5N1 and H5N6, it can provide internal genes for HPAIV, accelerate the evolution of the virus, and generate new strains with epidemic potential, seriously endangering the health and safety of the public.

[0003] Traditional antibodies are composed of two light chains and two heavy chains, with a relatively large molecular mass. Different from traditional antibodies, nanobodies only contain the variable heavy chain domain of HCAb (VHH), which is the smallest known antibody structure. Compared with traditional antibodies, nanobodies have a more elongated CDR3 region, can bind to sites that are difficult for traditional antibodies to bind to, and can tolerate extreme temperatures, pH, organic solvents, and proteases. At the same time, the VHH fragment is small, with a molecular weight of about 15 kDa, has strong tissue permeability and high solubility, can effectively penetrate the blood-brain barrier, and can be administered through the respiratory tract. Due to the small molecular weight, VHH has low immunogenicity and can also be expressed in large quantities in prokaryotic or eukaryotic biological systems for large-scale production. Through engineering transformation, different VHHs are connected by flexible peptides, and for different antigenic epitopes, the binding efficiency and specificity of VHH are improved. Common tandem methods can be achieved by adding a G4S hinge (G4S) between two or three VHH fragments. VHH can select the same antigenic epitope or different epitopes, presenting strategies such as dual-target signal blockade and reducing viral immune escape. These advantages make nanobodies a hot spot and trend in the development of new biological therapeutic agents.

[0004] At present, there are limited reports on nanobodies against H9 subtype avian influenza virus. Zhao Qin et al. disclosed a nanobody against H9N2 subtype avian influenza virus, its preparation method and application, successfully prepared a fusion protein of nanobody and HRP, and applied this fusion protein to detect antibodies against H9N2 subtype avian influenza virus (AIV) in chicken serum. It was found that the detection method constructed by this fusion protein has high sensitivity, and has the advantages of simple operation, no need to use a secondary antibody, and short time-consuming for detecting samples (Zhao Qin, Sun Yani, Wang Kun, etc. Nanobody against H9N2 subtype avian influenza virus, preparation method and application [P]. Shaanxi Province: CN111171146B, 2022-03-04.).

[0005] At present, the research on recombinant nanobodies against the NP protein of H9 subtype avian influenza virus is still in the blank stage. Summary of the Invention

[0006] The present invention provides a recombinant nanobody against the NP protein of H9 subtype avian influenza virus, a recombinant nanobody protein, an engineering strain and their applications to fill the blank in the research on recombinant nanobodies against the NP protein of H9 subtype avian influenza virus.

[0007] To solve the problems existing in the prior art, the technical solutions adopted by the present invention are as follows:

[0008] The recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention has an amino acid sequence of SEQ ID NO: 2.

[0009] The gene encoding the recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention has a nucleotide sequence of SEQ ID NO: 1.

[0010] The recombinant nanobody protein provided by the present invention contains the recombinant nanobody against the NP protein of H9 subtype avian influenza virus.

[0011] The engineering strain provided by the present invention is used to express the recombinant nanobody against the NP protein of H9 subtype avian influenza virus, and its nucleotide sequence is SEQ ID NO: 3.

[0012] The application of the recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention in the preparation of a nanoantibody preparation for diagnosing H9 subtype avian influenza virus.

[0013] The application of the recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention in the preparation of a nanoantibody kit for diagnosing H9 subtype avian influenza virus.

[0014] The beneficial effects of the present invention are:

[0015] The recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention has an amino acid sequence of SEQ ID NO: 2, and its yield reaches 1.0 mg / mL. The OD of the H9-NP-Nb09 ELISA result against the NP protein of H9 subtype avian influenza virus 450 = 2.56, showing significant binding ability, and can be used for preparing a novel nanoantibody preparation for pre-diagnosing H9 subtype avian influenza virus.

[0016] The recombinant nanobody against the NP protein of H9 subtype avian influenza virus provided by the present invention is screened from an established nanobody immune library of H9 subtype avian influenza virus. Its immunizing antigen is the sequence characteristics of the virus strain (SG strain) under the currently prevalent H9.4.2.2 branch. An expression plasmid is constructed to express the NP protein, and the screened nanobody has higher binding adaptability to the prevalent virus strain. Description of the Drawings

[0017] Figure 1 It is the purification result of the NP protein of H9 subtype avian influenza virus.

[0018] Figure 2 It is the solid-phase screening ELISA detection result of the nanobody immune library of H9 subtype avian influenza virus.

[0019] Figure 3 It is the map of the recombinant plasmid pPIC9K-H9-NP-Nb09.

[0020] Figure 4 It is the purification result of the recombinant nanobody H9-NP-Nb09. Detailed Embodiments

[0021] The technical solutions of the present invention are clearly and completely described below in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0022] (1) The sources of some experimental materials are as follows:

[0023] The prokaryotic expression plasmid pET28a(+) is purchased from Beijing Solarbio Science & Technology Co., Ltd.;

[0024] The restriction endonucleases Nhe I and HindIII are purchased from Takara Biotechnology (Beijing) Co., Ltd.;

[0025] The gel extraction kit is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0026] The T4 ligase is purchased from Takara Biotechnology (Beijing) Co., Ltd.;

[0027] The E. coli Rosetta(DE3) competent cells were purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.;

[0028] IPTG was purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.;

[0029] The Ni-NTA affinity chromatography column was purchased from Tiandi Renhe Company;

[0030] The BCA protein quantification detection kit was purchased from Thermo Fisher Scientific;

[0031] The immune library of nanobodies against H9 subtype avian influenza virus was purchased from Fujian Sunway Biotech Co., Ltd.;

[0032] The pPIC9K vector was purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0033] The restriction endonucleases EcoR I and NotI were purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.;

[0034] The E. coli DH5α competent cells were purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.;

[0035] The restriction endonuclease SalI was purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.;

[0036] The Pichia pastoris X-33 competent cells were purchased from Changsha Aikebo Biotechnology Co., Ltd.;

[0037] The YPG medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.;

[0038] Bleomycin (Zeocin) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0039] The BMMY liquid medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.;

[0040] The affinity chromatography column was purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0041] The 10% fetal bovine serum was purchased from NEWZERUM;

[0042] The anti-His-HRP tag antibody was purchased from Thermo Fisher Scientific.

[0043] (2) Prokaryotic expression and purification steps of the NP protein of H9 subtype avian influenza virus

[0044] For the NP protein of avian influenza virus subtype H9, according to the NP gene sequence information of the SG strain (NCBI database accession number OR528567.1) preserved in this experiment, a nucleic acid sequence optimized by E. coli codons was artificially synthesized (synthesized by General Biosystems (Anhui) Co., Ltd.), and Nhe I and Hind III restriction enzyme sites were introduced at both ends of its sequence. Subsequently, the artificially synthesized sequence (SEQ ID NO:4) and the prokaryotic expression plasmid pET28a(+) were digested with Nhe I and Hind III respectively. After identification by agarose gel electrophoresis, the fragments were recovered using a gel recovery kit. Under the ligation of T4 ligase, they were incubated at 16°C for 1 h and then transformed into competent E. coli Rosetta(DE3) to obtain a strain that could express the NP protein of avian influenza virus subtype H9. When the OD600nm value of this strain was cultured to about 0.5, IPTG with a final concentration of 1 mmol / L was added, and it was induced to express at 30°C for 6 h. The bacterial cells were collected and purified by Ni-NTA affinity chromatography column, and the purified recombinant protein was collected. The purification result is as Figure 1 shown. A target band appeared at 56 kDa, which was consistent with the expected size. Its concentration was determined to be 1.0 mg / mL by BCA protein quantification detection kit and stored at -70°C for later use.

[0045] (III) Resuscitation, rescue of the nanobody immune library of avian influenza virus subtype H9 and screening steps of recombinant nanobody sequences

[0046] According to the established nanobody immune library of avian influenza virus subtype H9, the immune library was resuscitated and rescued according to the experimental steps disclosed in the literature "Pardon E, Laeremans T, Triest S et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014;9(3):674-693. doi:10.1038 / nprot.2014.039".

[0047] Coat an ELISA plate with the purified H9 subtype avian influenza virus NP protein, and use the solid-phase screening technology of the antibody library (according to the method disclosed in the literature "Megan A. Schladetsch, et al. Generation of Single-Chain Variable Fragment (scFv) Libraries for Use in Phage Display [J]. Current protocols, 2021, 1(7): e182.") to screen for VHH sequences that can bind to the H9 subtype avian influenza virus NP protein, that is, the recombinant nanobody sequences against the H9 subtype avian influenza virus NP protein.

[0048] Among them, the ELISA test results of the solid-phase screening of the antibody library are shown in Figure 2 As shown, a total of 18 VHH sequences were screened. Among them, the Nb09 had the highest OD value. Its nucleotide sequence was SEQ ID NO:1, and the amino acid sequence encoded by it was SEQ ID NO:2.

[0049] (IV) Construction and identification steps of the recombinant plasmid

[0050] After digesting the above-screened recombinant nanobody sequence (number Nb09) and the pPIC9K vector with restriction enzymes EcoR I and NotI, recovering them by 1% agarose gel electrophoresis, ligating the obtained target fragments with T4 ligase, and simultaneously transforming them into competent Escherichia coli DH5α cells. After overnight culture on the plate, pick single colonies for sequencing and identification the next day. Name the recombinant plasmid with correct sequencing and identification as pPIC9K-H9-NP-Nb09. Its map is shown in Figure 3 , and store it at -20 °C for later use.

[0051] (V) Construction steps of the engineering strain

[0052] Use the restriction enzyme SalI to linearize the recombinant plasmid pPIC9K-H9-NP-Nb09, then add it to the competent cells of Pichia pastoris X-33, mix gently and evenly, transfer it to a pre-cooled electroporation cup for ice bath for 5 minutes, transfer it to an electroporator for electroporation treatment. Immediately add 1 mL of pre-cooled sorbitol (1 M) after electroporation. After pipetting, transfer it to a 1.5 mL centrifuge tube, place it in a 30 °C incubator, statically culture for 1 h, centrifuge at 4000 r / min at room temperature for 4 min, collect the thalli, resuspend them with 100 μL of YPG medium, and after treatment, coat them on the YPG solid medium containing bleomycin (100 μg / mL). Pick single colonies for PCR identification after culturing at 37 °C for 3 days. The identification results are shown in Figure 2As shown, the correctly identified single colony is the engineered strain obtained in the present invention for expressing recombinant nanobody, and its nucleotide sequence is SEQ ID NO:3.

[0053] (VI) Preparation steps of recombinant nanobody H9-NP-Nb09

[0054] Inoculate the above-mentioned engineered strain into 20 mL of YPG culture medium for rejuvenation. The next day, pick 5 mL and inoculate it into a 1 L flask containing 250 mL of YPG culture medium, and culture it overnight at 28 °C and 200 r / min. After centrifuging to collect the thalli, resuspend them with an equal volume of BMMY liquid medium, and induce for 120 hours at 28 °C and 200 r / min. Methanol (final concentration 0.5%) needs to be supplemented every 24 hours. Collect the supernatant, purify it according to the affinity chromatography column instruction manual, and obtain the recombinant protein. The obtained recombinant protein is analyzed for molecular weight and protein purity using SDS-PAGE and Western blot methods.

[0055] Among them, the Western blot identification results are shown in Figure 4 As shown, for the prepared recombinant protein, its relative molecular mass is 14.6 kDa, which is consistent with the expected value, its concentration is 1 mg / mL, and the purity reaches 90%. Thus, the recombinant nanobody is obtained and named H9-NP-Nb09.

[0056] (VII) Detection steps for the binding activity of recombinant nanobody H9-NP-Nb09

[0057] Verify the binding activity of recombinant nanobody H9-NP-Nb09 by ELISA detection.

[0058] First, use the BCA method to determine the concentration of the purified NP protein, dilute it with 0.05 mol / L, pH 9.6 carbonate buffer, coat it at 1 μg / well, use 10% fetal bovine serum as the blocking solution, use recombinant nanobody H9-NP-Nb09 as the primary antibody, use anti-His-HRP tag antibody as the secondary antibody, add TMB for color development, terminate the reaction with 3M H2SO4, and measure the OD 450 absorbance value. The results show that the OD 450 of recombinant nanobody H9-NP-Nb09 = 2.56, which indicates that the recombinant nanobody H9-NP-Nb09 prepared in the present invention has good binding activity.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Recombinant nanobody against H9 subtype avian influenza virus NP protein, characterized in that, Its amino acid sequence is SEQ ID NO:

2.

2. The gene encoding the recombinant nanobody against the NP protein of avian influenza virus subtype H9 as claimed in claim 1, characterized in that, Its nucleotide sequence is SEQ ID NO:

1.

3. Use of the recombinant nanobody against the NP protein of H9 subtype avian influenza virus as claimed in claim 1 in the preparation of a nano-antibody preparation for diagnosing H9 subtype avian influenza virus.

4. Use of the recombinant nanobody against the NP protein of H9 subtype avian influenza virus as claimed in claim 1 in the preparation of a nano-antibody kit for diagnosing H9 subtype avian influenza virus.

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