Recombinant nano antibody for resisting H9 subtype avian influenza virus NP protein, recombinant nano antibody protein, engineering strain and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant nano antibody for resisting H9 subtype avian influenza virus NP protein, a recombinant nano antibody protein, an engineering strain and application thereof. The amino acid sequence of the recombinant nano antibody for resisting the H9 subtype avian influenza virus NP protein is SEQ ID NO: 2, and the nucleotide sequence of the recombinant nano antibody is SEQ ID NO: 1. The invention also provides the application of the recombinant nano-antibody for resisting the H9 subtype avian influenza virus NP protein in preparation of a nano-antibody preparation and / or a nano-antibody kit for diagnosing the H9 subtype avian influenza virus. The recombinant nano-antibody for resisting the H9 subtype avian influenza virus NP protein, provided by the invention, is screened from an established H9 subtype avian influenza virus NP protein immune library, and an immune antigen of the recombinant nano-antibody has the sequence characteristic of a virus strain (SG strain) under a current epidemic H9.4. 2.2 branch, and has higher binding suitability with an epidemic virus strain.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant nano antibody against H9 subtype avian influenza virus NP protein, a recombinant nano antibody protein, an engineering strain and applications thereof. Background Art

[0002] At present, the prevalence of H9 subtype avian influenza is relatively high in China. Compared with H5 and H7 HPAIV, H9LPAIV can be latently infected clinically. When the external environment changes drastically or is infected by secondary infection, it can cause large-scale deaths of poultry. In addition to poultry, H9 LPAIV can naturally infect mammals such as pigs, ferrets, guinea pigs and humans. Research reports have shown that when H9 and H7N9, H5N1 and H5N6 subtypes are mixed, they can provide internal genes for HPAIV, accelerate the evolution of the virus, and produce new strains with epidemic potential, which seriously endangers the health and safety of the public.

[0003] Traditional antibodies are composed of two light chains and two heavy chains, and have a large molecular weight. Unlike traditional antibodies, nanobodies only contain the variable heavy chain domain of HCAb (VHH), which is the smallest antibody structure known so far. Compared with traditional antibodies, nanobodies have a narrower CDR3 region, which can bind to sites that traditional antibodies have difficulty binding to in the past, and can withstand extreme temperatures, pH, organic solvents, and proteases. At the same time, the VHH fragment is small, with a molecular weight of about 15kDa, strong tissue permeability and high solubility, can effectively penetrate the blood-brain barrier, and can be administered through the respiratory tract. Based on the characteristics of 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 the efficiency and specificity of VHH binding are improved for different antigenic epitopes. The common tandem method can be achieved by adding a G4S hinge (G4S) between two or three VHH fragments. VHH can select the same antigen epitope or different epitopes, presenting strategies such as dual-target signal blocking and reducing viral immune escape. These advantages make nanobodies a hot spot and trend in the development of new biotherapeutic agents.

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

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

[0006] The present invention provides a recombinant nano antibody against H9 subtype avian influenza virus NP protein, a recombinant nano antibody protein, an engineered strain and applications thereof, so as to fill the gap in the research on recombinant nano antibodies against H9 subtype avian influenza virus NP protein.

[0007] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a recombinant nanobody against the NP protein of H9 subtype avian influenza virus, and its amino acid sequence is 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 nano antibody protein provided by the present invention comprises the recombinant nano antibody against the H9 subtype avian influenza virus NP protein.

[0011] The engineered 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 invention provides an application of the recombinant nanobody against NP protein of H9 subtype avian influenza virus in the preparation of a nanobody preparation for diagnosing H9 subtype avian influenza virus.

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

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

[0015] The present invention provides a recombinant nanobody against the NP protein of H9 subtype avian influenza virus, whose amino acid sequence is SEQ ID NO: 2, whose yield reaches 1.0 mg / mL, and the ELISA result of H9-NP-Nb09 against the NP protein of H9 subtype avian influenza virus is OD 450 =2.56, which has significant binding and can be used to prepare new nano-antibody preparations for pre-diagnosis of H9 subtype avian influenza virus.

[0016] The present invention provides a recombinant nano antibody against the NP protein of H9 subtype avian influenza virus, which is screened from an established H9 subtype avian influenza virus nano antibody immune library, and its immune antigen is the sequence feature of the virus strain (SG strain) under the currently popular H9.4.2.2 branch. An expression plasmid is constructed to express the NP protein, and the screened nano antibody has a higher binding compatibility with the popular virus strain. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 This is the solid phase screening ELISA test result of the H9 subtype avian influenza virus nanoantibody immune library.

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

[0020] Figure 4 This is the purification result of the recombinant nanoantibody H9-NP-Nb09. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described clearly and completely below in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0022] (I) Some sources of experimental materials are as follows:

[0023] The prokaryotic expression plasmid pET28a(+) was purchased from Beijing Solebow Technology Co., Ltd.;

[0024] Restriction endonucleases Nhe I and HindIII were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.;

[0025] The gel recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0026] T4 ligase was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.;

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

[0028] IPTG was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

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

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

[0031] The H9 subtype avian influenza virus nanoantibody immune library was purchased from Fujian Shengwei Biotechnology Co., Ltd.;

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

[0033] Restriction endonucleases EcoR I and NotI were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.;

[0034] Escherichia coli DH5α competent cells were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.;

[0035] Restriction endonuclease SalI was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.;

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

[0037] YPG medium was purchased from Beijing Solebow Technology Co., Ltd.;

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

[0039] BMMY liquid culture medium was purchased from Beijing Solebow Technology Co., Ltd.;

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

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

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

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

[0044] According to the NP gene sequence information in the SG strain (NCBI database accession number OR528567.1) preserved in this experiment, the NP protein of H9 subtype avian influenza virus was artificially synthesized with an E. coli codon-optimized nucleic acid sequence (synthesized by General Biosystems (Anhui) Co., Ltd.), and Nhe I and Hind III restriction sites were introduced at both ends of the 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, and after identification by agarose gel electrophoresis, the fragments were recovered using a gel recovery kit, incubated at 16°C for 1h under the ligation action of T4 ligase, and transformed into E. coli Rosetta (DE3) competent cells to obtain a strain that can express the NP protein of H9 subtype avian influenza virus. The strain was cultured until the OD600nm value was about 0.5, IPTG with a final concentration of 1 mmol / L was added, and the expression was induced at 30°C for 6 h. The bacteria were collected and purified by Ni-NTA affinity chromatography column to collect the purified recombinant protein. The purification results are as follows: Figure 1 As shown, the target band appeared at 56 kDa, which was consistent with the expected value. The concentration was determined to be 1.0 mg / mL by BCA protein quantitative detection kit and stored at -70°C for future use.

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

[0046] According to the established H9 subtype avian influenza virus nanoantibody immune library, the immune library was revived and rescued according to the experimental steps disclosed in the document "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] The purified H9 subtype avian influenza virus NP protein was coated on an ELISA plate, and the antibody library solid phase screening technology (according to the method disclosed in the document "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.") was used to screen the VHH sequence that can bind to the H9 subtype avian influenza virus NP protein, that is, the recombinant nanoantibody sequence against the H9 subtype avian influenza virus NP protein.

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

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

[0050] The above-screened recombinant nanobody sequence (numbered Nb09) and pPIC9K vector were digested with restriction endonucleases EcoR I and NotI, and then recovered by 1% agarose gel electrophoresis. The obtained target fragments were ligated with T4 ligase and transformed into Escherichia coli DH5α competent cells. After overnight culture on plates, single colonies were picked for sequencing identification the next day, and the recombinant plasmid identified correctly by sequencing was named pPIC9K-H9-NP-Nb09, and its map is shown in Figure 3 , stored at -20℃ for future use.

[0051] (V) Steps for constructing engineered strains

[0052] The recombinant plasmid pPIC9K-H9-NP-Nb09 was linearized using restriction endonuclease SalI, then added to Pichia pastoris X-33 competent cells, gently mixed, transferred to a pre-cooled electroporation cup for ice bath for 5 minutes, transferred to an electroporator for electrotransformation, and immediately added 1 mL of pre-cooled sorbitol (1 M) after electroporation. After pipetting, the tube was transferred to a 1.5 mL centrifuge tube and placed in a 30°C incubator for static culture for 1 h. Centrifuged at 4000 r / min for 4 min at room temperature, the cells were collected, and resuspended in 100 μL of YPG medium. After treatment, the cells were coated on YPG solid medium containing bleomycin (100 μg / mL), and cultured at 37°C for 3 days. Single colonies were picked for PCR identification. The identification results are shown in Figure 2As shown, the correctly identified single colony is the engineered strain for expressing recombinant nanobodies obtained by the present invention, and its nucleotide sequence is SEQ ID NO: 3.

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

[0054] The above-mentioned engineered strain was inoculated into 20mL YPG culture medium for rejuvenation, and 5mL was picked up the next day and inoculated into a 1L shaking flask containing 250mL YPG culture medium, and cultured overnight at 28°C and 200r / min. After centrifugation, the bacteria were resuspended in an equal volume of BMMY liquid culture medium, and induced for 120 hours at 28°C and 200r / min, and methanol (final concentration 0.5%) was added every 24 hours. The supernatant was collected, purified according to the affinity chromatography column instructions, and the recombinant protein was obtained. The obtained recombinant protein was 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, the prepared recombinant protein has a relative molecular mass of 14.6 kDa, which is consistent with the expected value, a concentration of 1 mg / mL, and a purity of 90%. Thus, a recombinant nanobody was obtained, named H9-NP-Nb09.

[0056] (VII) Binding activity detection steps of recombinant nanobody H9-NP-Nb09

[0057] The binding activity of the recombinant nanoantibody H9-NP-Nb09 was verified by ELISA detection.

[0058] First, the BCA method was used to determine the concentration of purified NP protein. The protein was diluted with 0.05 mol / L, pH 9.6 carbonate buffer and coated at 1 μg / well. The blocking solution was 10% fetal bovine serum. The primary antibody was recombinant nanoantibody H9-NP-Nb09. The secondary antibody was anti-His-HRP tag antibody. TMB was added for color development. The reaction was terminated with 3 M H2SO4. The OD was measured. 450 Absorbance value. The results show that the recombinant nanobody H9-NP-Nb09 OD 450 =2.56, which indicates that the recombinant nanobody H9-NP-Nb09 prepared by 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein, 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. A recombinant nanobody against the NP protein of H9 subtype avian influenza virus, characterized in that: Its amino acid sequence is SEQ ID NO:

2.

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

1.

3. A recombinant nanobody protein, characterized in that: A recombinant nanobody comprising the anti-H9 subtype avian influenza virus NP protein according to claim 1.

4. An engineered strain, characterized in that: The recombinant nanobody used to express the anti-H9 subtype avian influenza virus NP protein according to claim 1 has a nucleotide sequence of SEQ ID NO:

3.

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

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

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