A nanoantibody specifically binding to FAdV-4, a fusion protein and its application

Abstract

The present invention discloses a nanobody, fusion protein and application that specifically binds to FAdV-4, and belongs to the technical field of veterinary biological products. Based on the phage nanobody display library of FAdV-4, the present invention screened specific nanoantibodies against FAdV-4 fiber protein 2 (Fiber 2), and used the self-assembly technology of ferritin to genetically engineer the nanoantibodies to express and prepare the anti-FAdV-4 polymerized nanoantibody Fenobody-75. The antibody is simple to express and purify, has stable properties and low production cost. The antiviral effect was analyzed by in vitro and in vivo tests. The results show that the Fenobody-75 of the present invention is highly safe and has a good protective effect against FAdV-4 infection. Therefore, Fenobody-75 has a good market application prospect as a biological preparation for resisting FAdV-4 infection in poultry.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a nano antibody specifically binding to FAdV-4, a fusion protein and applications thereof. Background Art

[0002] Fowl adenovirus (FAdV) belongs to the genus Avian Adenovirus in the family Adenoviridae and can infect poultry, including chickens, turkeys, geese, ducks, pheasants, and quail. FAdV is prevalent worldwide, causing significant economic losses to the poultry industry. FAdV infection can cause a variety of clinical diseases, including inclusion body hepatitis (IBH), hydropericardium hepatitis syndrome (HHS), and gizzard erosions (GE). Currently, the prevalent serotypes are primarily FAdV-4, FAdV-8, and FAdV-11, with FAdV-4 being the most prevalent.

[0003] Currently, the prevention and control of FAdV-4 infection primarily relies on vaccination. After vaccination, immunity typically takes at least seven days to develop, leaving a window period for FAdV-4 infection. Furthermore, if a FAdV-4 outbreak occurs in an unvaccinated chicken farm, the disease will spread rapidly, with chickens becoming increasingly ill or even dying within 3-10 days. During this period, emergency vaccinations may not be able to effectively protect the flock in a timely manner. In such situations, effective treatments are crucial for minimizing losses. It is well known that egg yolk antibodies have a wide market application in poultry disease prevention and control. Veterinary egg yolk antibodies are approved for use against various avian viral diseases, including duck hepatitis, gosling plague, and infectious bursal disease. Numerous studies have examined FAdV-4 yolk antibodies, all demonstrating promising preventive and therapeutic efficacy against homologous strains. However, the production cost of yolk antibodies is relatively high, and they require high-quality laying hens. Therefore, the development of other specific agents that are highly effective, easy to prepare, and low-cost are crucial for the comprehensive prevention and control of FAdV-4.

[0004] Nanobodies, as third-generation genetically engineered antibodies, are the smallest known antibodies with antigen-binding ability. Compared with traditional monoclonal antibodies, their molecular weight is only about 15kDa. In addition, this type of antibody is easy to genetically engineer and can be coupled with a variety of fragments; it is also easy to express and purify, and can be expressed using Escherichia coli expression systems, yeast expression systems, mammalian cells, or plant cells. Therefore, as a therapeutic drug, nanobodies have the advantages of good specificity and stability, simple production process, and low production cost. Therefore, the development of efficient, easy-to-prepare, and low-cost nanoantibody drugs is crucial for the comprehensive prevention and control of FAdV-4. Summary of the Invention

[0005] The purpose of the present invention is to provide a nanobody, fusion protein and application that specifically binds to FAdV-4 to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a nanobody that specifically binds to FAdV-4, whose amino acid sequence is as shown in SEQ ID Shown in NO.3.

[0008] The second technical solution of the present invention is a DNA molecule encoding the nanobody, whose nucleotide sequence is shown in SEQ ID NO.4.

[0009] The third technical solution of the present invention is a fusion protein, whose amino acid sequence is as shown in SEQ ID Shown in NO.1.

[0010] The fourth technical solution of the present invention is a DNA molecule encoding the fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] The fifth technical solution of the present invention is a recombinant expression vector, comprising the aforementioned DNA molecule.

[0012] The sixth technical solution of the present invention is the use of the nanobody, the fusion protein or the recombinant expression vector in the preparation of a drug for preventing and treating FAdV-4 infection.

[0013] The seventh technical solution of the present invention is a drug for preventing and treating FAdV-4 infection, comprising the nanobody or the fusion protein.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention is based on a phage nanobody display library of FAdV-4, screened specific nanobodies against FAdV-4 fiber protein 2 (Fiber 2), and used ferritin self-assembly technology to genetically engineer the nanobodies to express and prepare the anti-FAdV-4 multimeric nanoantibody Fenobody-75. The antibody is easy to express and purify, has stable properties, and low production cost. The antiviral effect was analyzed through in vitro and in vivo tests. The results showed that the Fenobody-75 of the present invention is highly safe and has a good protective effect against FAdV-4 infection. Therefore, Fenobody-75 has a good market application prospect as a biological agent for resisting FAdV-4 infection in poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 is the titer of anti-FAdV-4 antibodies in the serum of Bactrian camels after the fifth immunization;

[0018] Figure 2 To obtain lymphocytes from the peripheral blood of immune Bactrian camels by separating them with Ficoll lymphocyte separation medium;

[0019] Figure 3 The nanobody gene product was amplified by the first round of nested PCR for agarose gel electrophoresis analysis, with a size of 700 bp, where M is a nucleic acid marker;

[0020] Figure 4 The nanobody gene product was amplified by the second round of nested PCR for agarose gel electrophoresis analysis, and the size was approximately 400 bp;

[0021] Figure 5 The results of double enzyme digestion of nanobody and pMECS phage display vector were analyzed by agarose gel electrophoresis, where M is a nucleic acid marker, lane 1 is the enzyme-digested pMECS vector with a size of 4500 bp, and lane 2 is the nanobody fragment with a size of 400 bp;

[0022] Figure 6 The positive rate of the constructed nanobody phage display library was analyzed by agarose gel electrophoresis, and the nanobody gene inserted into the phage library was identified by PCR. Where M is a marker; 1-48 means picking 48 monoclonal clones, and the size of the positive target fragment is 500bp;

[0023] Figure 7 The reactivity of crude extracts of recombinant nanobodies with FAdV-4Fiber2 protein was detected by indirect ELISA method;

[0024] Figure 8 Agarose gel electrophoresis analysis of double-enzyme digested nanobodies and pCMV-HRP vector, where A is the nanobodies Nb12, Nb50, Nb55, Nb61, Nb66, Nb73, Nb75, and Nb91, with a size of 400 bp; B is the result of double-enzyme digestion of the recombinant vector pCMV-N1-HRP, with a size of 5000 bp;

[0025] Figure 9 After the constructed nanobody recombinant expression plasmid was transfected into HEK293T cells, the expression of Fiber2-Nbs-HRP fusion protein in HEK293T cells was analyzed by immunofluorescence, wherein Negative was the pCMV-N1-HRP empty vector;

[0026] Figure 10 ELISA was used to analyze the affinity of Fiber2-Nbs-HRP fusion protein to FAdV-4Fiber2 protein;

[0027] Figure 11 Immunofluorescence analysis was used to determine whether the Fiber2-Nbs-HRP fusion protein binds to FAdV-4 infecting LMH cells. The positive result was the serum control of FAdV-4-immunized mice.

[0028] Figure 12 This is a preliminary immunofluorescence screening of Fiber2-Nbs-HRP fusion proteins with neutralizing activity, where the negative is an irrelevant Nb-HRP control;

[0029] Figure 13 Fenobody-50, -61, -75, and -91 fragments were PCR amplified for agarose gel electrophoresis analysis, with a size of 800 bp;

[0030] Figure 14The expression of FAdV-4-Fiber 2-Fenobody-50 / 61 / 75 / 91 was identified by SDS-PAGE and Western Blot, where M is a protein marker; A is SDS-PAGE identification: lane 1 is Fenobody-50, with a molecular weight of approximately 30 kDa; lane 2 is Fenobody-61, with a molecular weight of approximately 30 kDa; lane 3 is Fenobody-75, with a molecular weight of approximately 30 kDa; lane 4 is Fenobody-91, with a molecular weight of approximately 30 kDa; B is Western Blot identification: lane 1 is Fenobody-50, with a molecular weight of approximately 30 kDa; lane 2 is Fenobody-61, with a molecular weight of approximately 30 kDa; lane 3 is Fenobody-75, with a molecular weight of approximately 30 kDa; lane 4 is Fenobody-91, with a molecular weight of approximately 30 kDa;

[0031] Figure 15 Analysis of the cytotoxicity of FAdV-4-Fiber 2 Fenobody 50 / 61 / 75 / 91 on LMH cells for CCK8. Panel A shows the cytotoxicity assay of different protein amounts of Fenobody-50, Panel B shows the cytotoxicity assay of different protein amounts of Fenobody-61, Panel C shows the cytotoxicity assay of different protein amounts of Fenobody-75, and Panel D shows the cytotoxicity assay of different protein amounts of Fenobody-95.

[0032] Figure 16 The half-maximal inhibitory concentration (IC50) of FAdV-4-Fiber2 Fenobody 50 / 61 / 75 / 91 proteins was used for immunofluorescence analysis. 50 );

[0033] Figure 17 To analyze the neutralization activity of FAdV-4 with different concentrations of FAdV-4-Fiber2 Fenobody 75 by immunofluorescence;

[0034] Figure 18 To pass TCID 50 Analyze the effects of different concentrations of Fenobody-75 on the titer of FAdV-4 progeny viruses;

[0035] Figure 19 Fluorescence quantitative PCR was used to detect the neutralization activity of Fenobody-75 at different concentrations against FAdV-4;

[0036] Figure 20 Western Blot analysis of the neutralization activity of Fenobody-75 at different concentrations against FAdV-4;

[0037] Figure 21 Survival analysis of FAdV-4 infected chickens treated with Fenobody-75;

[0038] Figure 22 Analysis of viral load in different chicken tissues after FAdV-4-infected chickens were treated with Fenobody-75. A is the liver, B is the heart, C is the spleen, D is the lung, and E is the kidney.

[0039] Figure 23 Analysis of histopathological lesions in different chickens after FAdV-4 infection was treated with Fenobody-75. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0046] The embodiment of the present invention provides a nanobody that specifically binds to FAdV-4, and its amino acid sequence is as shown in SEQ ID Shown in NO.3.

[0047] The present invention also provides a DNA molecule encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0048] The present invention also provides a fusion protein, the amino acid sequence of which is as shown in SEQ ID Shown in NO.1.

[0049] The embodiment of the present invention also provides a DNA molecule encoding the fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0050] An embodiment of the present invention also provides a recombinant expression vector comprising the aforementioned DNA molecule.

[0051] In some specific embodiments, the recombinant expression vector is pET-28a-FAdV-4-Fiber2-Fenobody75.

[0052] In some specific embodiments, the preparation method of the recombinant expression vector is: using the nucleotide sequence encoding ferritin as the framework, the nucleotide sequence encoding the nanobody is connected to the C-terminus of ferritin through a GS flexible linker, the obtained fusion sequence and the pET-28a plasmid are digested with restriction endonucleases BamHI and HindⅢ, and then connected by T4 ligase, and transformed into Escherichia coli DH5α competent cells to obtain a recombinant prokaryotic expression plasmid and named it: pET-28a-FAdV-4-Fiber2-Fenobody75.

[0053] In some specific embodiments, the preparation method of the fusion protein is: transforming the constructed pET-28a-FAdV-4-Fiber2-Fenobody75 into Escherichia coli BL21 (DE3) to obtain recombinant bacteria of BL21 (DE3)-pET-28a-FAdV-4-Fiber2-Fenobody75, inducing expression by IPTG and extracting protein by ultrasonication to obtain the fusion protein.

[0054] The embodiments of the present invention also provide the use of the nanobody, the fusion protein or the recombinant expression vector in the preparation of a drug for preventing and treating FAdV-4 infection.

[0055] An embodiment of the present invention also provides a drug for preventing and treating FAdV-4 infection, comprising the nanobody or the fusion protein.

[0056] The fiber protein (Fiber2) is a key structural protein on the surface of FAdV-4, composed of 479 amino acids and formed by hydrolysis of the precursor protein. From its N-terminus to its C-terminus, it comprises three functional regions: the tail, the stalk, and the apical bulb. The tail region is connected to the base of the penton and contains a conserved amino acid region; the apical bulb is the functional region that allows the virus to attach to host cells. The Fiber2 protein extends outward from the capsid surface and plays a vital role in viral receptor recognition and viral transmission. Furthermore, compared with non-pathogenic strains, the Fiber2 protein is a key factor in determining the virulence of different FAdV-4 strains and is considered the primary protein for FAdV-4 immune protection. Studies have shown that the Fiber2 protein possesses subgroup-specific antigenic determinants that can induce the production of primarily type-specific neutralizing antibodies. The specific neutralizing antibodies induced by the Fiber2 protein are effective against FAdV-4 infection. Therefore, the Fiber2 protein is an ideal target for the prevention of FAdV-4 infection and the development of therapeutic drugs.

[0057] Ferritin (Fe) is a protein that is widely present in nature. From bacteria to mammals, the three-dimensional structure of ferritin is highly conserved and consists of three parts: four long helices (α-δ) consisting of 24 units, one-fifth of a short helix (ε), and a long loop composed of β and γ helices. Under certain pH conditions, ferritin can self-assemble into spherical nanostructures. Therefore, this property can be used to selectively modify the core and outer surface of ferritin in vitro. Fenobody technology inserts a nanobody sequence into the C-terminus of ferritin to replace its ε helix. Through the self-assembly properties of ferritin, the nanobody is displayed on the ferritin surface in the form of a 24-mer. The fusion protein formed by ferritin and nanobody is called Fenobody. Compared with monovalent nanobodies, Fenobody is a polymerized nanobody with higher affinity and longer half-life than monovalent nanobodies, showing great potential in the development of antibody drugs.

[0058] The present invention immunizes Bactrian camels with purified and inactivated FAdV-4 to construct a nanobody phage display library; then, using purified Fiber2 protein as a coating antigen, phage screening technology is used to obtain specific nanoantibodies against FAdV-4 Fiber2 protein. The neutralization activity of the nanoantibody is verified and analyzed through in vitro neutralization tests. Then, using Fenobody technology, based on the neutralizing active nanoantibodies, anti-FAdV-4 poly-nanoantibodies Fenobodies are expressed and prepared, and the antiviral effects of Fenobodies are analyzed through in vitro and in vivo tests. The Fenobody-75 prepared by the present invention has good preventive and therapeutic effects on FAdV-4 infection in clinical practice, providing a fast, simple, safe and effective technical means for the prevention and treatment of clinical FAdV-4, which will have good promotion and application prospects and is of great practical significance for ensuring the health and rapid development of my country's poultry industry.

[0059] In the embodiment of the present invention, FAdV-4 was isolated and preserved by the Major Animal Disease Pathogen Infection and Pathogenic Mechanism Team of the College of Veterinary Medicine of Northwest Agriculture and Forestry University; Alxa Bactrian camels were purchased from Minqin County, Wuwei City, Gansu Province.

[0060] Example 1

[0061] Screening and preparation of specific nanoantibodies against FAdV-4 Fiber2 protein

[0062] (1) Emulsification of immunogens and animal immunization

[0063] Concentrated, purified FAdV-4 was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously into the neck of four-year-old Alxa Bactrian camels (1 mL). Four subsequent immunizations were administered with 1 mL of purified FAdV-4 emulsified with an equal volume of incomplete Freund's adjuvant, every two weeks. Seven days after the final immunization, 150 mL of peripheral blood was collected from the camels.

[0064] Purified FAdV-4 (800 ng / well) was used as the coating antigen, and the anti-FAdV-4 antibody titer in camel serum after 5 immunizations was detected by indirect ELISA. The results showed that the anti-FAdV-4 antibody titer in the serum was 1:128000 ( Figure 1 ).

[0065] (2) Construction and panning of VHH phage library

[0066] 1) Isolation of peripheral blood lymphocytes

[0067] The peripheral blood lymphocytes were separated using Ficoll-Paque PLUS lymphocyte separation medium (Greinerbio-one). After centrifugation, the ring-shaped milky white substance between the plasma and the white transparent lymphocyte separation medium is the lymphocyte ( Figure 2 The lymphocytes were aspirated, washed repeatedly with PBS, centrifuged, resuspended and counted, and the number of cells was calculated as 1×10 7 The cells were divided into aliquots and stored at -80℃ for later use.

[0068] 2) Amplification of VHH gene segments in peripheral blood lymphocytes

[0069] according to The total RNA (larger than 200 bp) of isolated lymphocytes was extracted using the Plus Mini RNA Extraction Kit (QIAGEN). The first-strand cDNA was synthesized using RNA as a template using PCR-III reverse transcriptase (Life Technologies). The VHH gene was then amplified using nested PCR. First, an RNA / primer mixture was prepared, as shown in Table 1.

[0070] Table 1 RNA reverse transcription system

[0071]

[0072] After incubating the RNA / primer at 65°C for 5 minutes, the mixture was immediately placed in an ice-water bath for 1 minute. Subsequently, a cDNA synthesis mixture was prepared, the system of which is shown in Table 2.

[0073] Table 2 cDNA synthesis mixture system

[0074]

[0075] The cDNA synthesis mixture (10 μL) was added to the RNA / primer mixture, mixed well, incubated at 50° C. for 50 min, and terminated by incubation at 85° C. for 5 min.

[0076] The VHH gene was amplified by nested PCR using the reverse transcribed cDNA as a template. The primer sequences used are shown in Table 3 below.

[0077] Table 3 Primer sequences for nested PCR amplification of VHH genes

[0078]

[0079] First, primers CALL001 and CALL002 were used to perform the first round of PCR amplification, and the reaction system was shown in Table 4.

[0080] Table 4 First round PCR amplification reaction system

[0081]

[0082] The reaction program was pre-denaturation at 94°C for 5 min; 28 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and extension at 72°C for 7 min.

[0083] The PCR products were identified by 1.2% agarose gel electrophoresis, and the results showed that there was a band at the 700 bp and 900 bp positions ( Figure 3 A 700-bp PCR product was recovered using the EasyPure Quick Gel Extraction Kit according to the manufacturer's instructions. This recovered product was then used as a template for a second round of PCR amplification using the aforementioned VHH-FOR and VHH-REV primers. The reaction system is shown in Table 5.

[0084] Table 5 Second round PCR amplification reaction system

[0085]

[0086] The reaction program was as follows: pre-denaturation at 94°C for 5 min; 18 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and extension at 72°C for 5 min.

[0087] The PCR product was electrophoresed on 1.5% agarose gel, and the results showed that a 400 bp target band was obtained ( Figure 4 ). The PCR product was then recovered using the EasyPure Quick Gel Extraction Kit, which is the amplified VHH gene.

[0088] 3) Construction of VHH phage display vector

[0089] The recovered PCR products and the pMECS phage display vector were simultaneously digested with Pst I and Not I. The specific enzyme digestion system is shown in the following table (Tables 6 and 7).

[0090] Table 6 pMECS phage display vector double enzyme digestion system

[0091]

[0092] Table 7 PCR product double enzyme digestion system

[0093]

[0094] The enzyme digestion conditions were 37°C for 16 h. After digestion, the digestion products were recovered using the commercial EasyPure Quick GelExtraction Kit. The digestion results were analyzed by agarose gel electrophoresis, and the digestion fragments of about 5000 bp and 400 bp were obtained respectively ( Figure 5 ).

[0095] The recovered PCR digestion product was ligated into the digested phage display vector pMECS using T4 DNA ligase at 16°C for 16 h. The ligation system is shown in Table 8.

[0096] Table 8 VHH enzyme digestion products and enzyme digestion vector pMECS connection system

[0097]

[0098] 4) Preparation of E. coli TG1 competent cells

[0099] E. coli TG1 was cultured to OD 600nm After the pH value is 0.4-0.6, cool in an ice bath, centrifuge and wash three times with pre-cooled 10% glycerol, and finally resuspend the bacteria with 10% glycerol to prepare TG1 competent cells.

[0100] 5) Transformation of TG1 competent cells with ligation products and preparation of phage antibody library

[0101] The ligation product was added to freshly prepared E. coli TG1 competent cells, mixed thoroughly, and added to an electroporation cuvette. The competent cells were electroporated using an Eppendorf electroporator (1.8 kV, 25 μF, 200 Ω, 1 mm). Immediately after electroporation, SOC medium (2 g tryptone, 0.5 g yeast extract, 0.058 g NaCl, and 0.0186 g KCl dissolved in 96 mL ultrapure water and autoclaved at 121°C for 30 min) was added. After the medium cooled to room temperature, 2 mL of 20% glucose solution, 1 mL of 1 M MgCl2 solution, and 1 mL of 1 M MgSO4 solution were added to resuspend the cells. After shaking culture at 37°C and 120 r / min for 1 h, spread on LB / AMP-GLU large plates (10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar, dissolved in 900 mL ultrapure water, sterilized by high-pressure steam at 121°C for 30 min, and when the culture medium is cooled to about 60°C, add 100 mL 20% glucose solution and 1 mL ampicillin storage solution). After culture at 37°C for 6 to 8 h, collect the bacterial lawn with a cell scraper and add 1 / 3 volume of 50% glycerol to prepare the phage library.

[0102] 6) Determination of phage library diversity and capacity.

[0103] The electroconversion product was diluted 10-fold to 10 -5 Then, the cells were plated on LB / AMP-GLU plates and cultured at 37°C for 12 h. The number of transformants was calculated and the final storage capacity was 1.75×10 7 48 single clones were randomly selected and identified by PCR using the VHH-FOR and VHH-REV primers described in Table 3. Positive clones were identified by agarose gel electrophoresis. The target size was about 400 bp ( Figure 6 ).

[0104] (3) Screening of anti-FAdV4 Fiber2-specific nanoantibodies

[0105] 1) Rescue of phage libraries

[0106] The obtained FAdV-4-specific phage display library was inoculated into 2×YT / AMP-GLU medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl dissolved in 900 mL ultrapure water, autoclaved at 121°C for 30 min, and after the medium cooled to room temperature, 100 mL of 20% glucose solution and 1 mL of ampicillin stock solution were added). The culture was incubated at 37°C at 200 rpm until logarithmic phase. M13KO7 helper phage was then added and the culture was allowed to stand at 37°C for 30 min. The culture was then centrifuged at 2800 rpm for 10 min, the supernatant discarded, and the pelleted cells resuspended in 200 mL of 2×YT / AMP-KAN medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl dissolved in 1000 mL ultrapure water, 1 mL of kanamycin stock solution and 1 mL of ampicillin stock solution added). The culture was then incubated at 37°C at 200 rpm for 14 h. After centrifugation at 3800 rpm and 4°C for 30 min, collect the supernatant and add 1 / 5 volume of pre-chilled PEG / NaCl solution. Centrifuge at 3800 rpm and 4°C for 30 min. Add 1 mL of PBS to resuspend the phage pellet and fully dissolve it. This is the rescued phage library.

[0107] Take a small amount of phage library solution and dilute it 10 times, then take a dilution of 10 -2 , 10 -4 , 10 -6 , 10 -8 , 10 -10 The sample was added to TG1 cells in the logarithmic growth phase and incubated at 37°C for 15 min. The cells were evenly spread on LB / AMP-GLU plates and cultured at 37°C for 8 h. The titer of the recombinant phage was calculated to be 9.1×10 12 PFU / mL.

[0108] 2) Selection of anti-FAdV-4Fiber2 specific recombinant phage

[0109] Purified FAdV-4Fiber2 was coated onto an ELISA plate, with PBS serving as a control. The rescued phage library was added to the ELISA plate and incubated at room temperature for 1 hour. The phage sample was discarded. Freshly prepared 0.1M triethylamine was then added to each well, and the plate was allowed to stand at room temperature for 10 minutes. The eluate was then quickly neutralized with an equal volume of 1M Tris-HCl (pH 7.4).

[0110] Take the eluate and infect 4 mL of logarithmic phase TG1 cells, let it stand at 37°C for 30 min, then add 2×YT / AMP-GLU medium and culture at 37°C at 200 rpm until OD 600nm The above steps were repeated to rescue the phage library, followed by a second and third round of panning, with phage titers measured in each round. The results showed that after three rounds of panning, the P / N value increased from 1 to 570, demonstrating effective enrichment of anti-FADV-4 Fiber 2 phage (Table 9).

[0111] Table 9 Enrichment of anti-FAdV-Fiber2 protein specific phage

[0112]

[0113] (4) Preparation of crude extracts of recombinant nanobodies

[0114] 96 single colonies were randomly selected from the plate with the third round of elution of phage titer, marked 1-96 in sequence, and inoculated into a 96-well plate. LB / AMP-GLU medium was added to each well and cultured at 37°C and 200 rpm for 8 h.

[0115] 10 μL of culture medium was aspirated from each clone and inoculated into 1 mL of TB medium. The cells were cultured in a 24-well culture plate at 37°C and 200 rpm until the OD value of the clone reached the logarithmic phase. 600nm The expression of the expressed bacteria was induced by adding IPTG to each well at a final concentration of 0.1 mM. The bacteria were centrifuged and precipitated, and the mixture was frozen and thawed three times, centrifuged at 3500 rpm and 4°C for 15 minutes, and the supernatant was collected as the crude extract of the soluble recombinant nanobody.

[0116] (5) Indirect ELISA detection of specific binding between crude extracts of recombinant nanoantibodies and FAdV-4 Fiber2 protein

[0117] Purified FAdV-4Fiber2 protein was coated onto an ELISA plate, and an irrelevant E. coli was used as a negative control. The crude extract of the soluble recombinant nanobody was diluted 1:1 with 2.5% skim milk powder and added to the ELISA plate. The plate was incubated at room temperature for 1 hour. Dissolve the plate in PBS'T (8.5 g NaCl, 3.352 g Na2HPO4·12H2O, 0.286 g NaH2PO4·2H2O in deionized water) and dilute to 1000 mL, pH 7.2-7.4. Add 0.5 mL eluent (PBS'T containing 0.05% Tween-20) to wash the plate, then add commercial mouse anti-HA-tag antibody (Beijing Quanshijin Biotechnology Co., Ltd.) and incubate at room temperature for 1 hour. Then add commercial HRP-labeled goat anti-mouse IgG (Beijing Boosun Biotechnology Co., Ltd.) and incubate at room temperature for 1 hour. Add TMB colorimetric solution (Solarbio) and color at room temperature in the dark for 15 minutes. Then terminate the reaction with 3 M concentrated sulfuric acid and read the OD value with an automatic microplate reader. 450nm The results showed that 82 of the 96 crude extracts could specifically bind to FAdV-4Fiber2 protein ( Figure 7 The positive strains were sequenced, and the results showed that 8 nanoantibodies against FAdV-4 Fiber 2 protein were successfully screened and obtained, which were named Fiber2-Nb12, Fiber2-Nb50, Fiber2-Nb55, Fiber2-Nb61, Fiber2-Nb66, Fiber2-Nb73, Fiber2-Nb75, and Fiber2-Nb91.

[0118] Example 2

[0119] Expression and Preparation of FAdV-4Fiber2 Nanobody and Horseradish Peroxidase (HRP) Fusion Protein

[0120] (1) Construction of recombinant eukaryotic expression vector of nanobody and HRP fusion protein

[0121] The eukaryotic expression vector pCMV-N1-HRP (containing His and HA tags) and the product containing the encoding nanobody obtained by PCR were double-digested using PstI and NotI endonucleases. The results of agarose gel electrophoresis showed that the target bands of about 5000bp and 400bp were successfully obtained respectively. The VHH gene obtained by enzyme digestion was then ligated to the pCMV-N1-HRP vector using T4 ligase to obtain a recombinant positive plasmid expressing the nanobody and HRP fusion protein, named pCMV-N1-Fiber2-Nbs-HRP ( Figure 8 ).

[0122] (2) Expression and preparation of nanobody and HRP fusion protein

[0123] The successfully constructed pCMV-N1-Fiber2-Nbs-HRP positive plasmid was After the medium and PEI transfection reagent were mixed evenly, they were added to HEK293T cells and cultured at 37°C, 5% CO2 for 48 hours, and the supernatant was collected. The transfected HEK293T cells were fixed with 4% paraformaldehyde and incubated at room temperature with a commercial His monoclonal antibody (Quanshijin Biotechnology Co., Ltd.), followed by incubation with FITC-labeled goat anti-mouse IgG. Immunofluorescence was used to detect whether the recombinant fusion protein was expressed in the HEK293T cells; the results showed that the HEK293T cells transfected with the positive plasmid emitted green fluorescence, indicating that the fusion protein was successfully expressed ( Figure 9 ).

[0124] Example 3

[0125] Preliminary screening of FAdV-4 neutralizing nanoantibodies

[0126] (1) ELISA verification of the specific binding of nanoantibodies, HRP fusion protein and FAdV-4Fiber2 protein

[0127] HEK293T cells were transfected with the pCMV-N1-Fiber2-Nbs-HRP positive plasmid and cultured at 37°C, 5% CO2 for 48 hours. The supernatant was collected. The collected supernatant was diluted proportionally (1:10, 1:50, 1:100, 1:500, 1:1000, 1:2000) and directly added to the ELISA plate coated with FAdV-4Fiber 2 protein (400 ng / well). After TMB color development, the results showed that except for Nb55, which did not bind to Fiber2 protein, Nb-12, -50, -61, -66, -73, -75 and -91 all bound to FAdV-4Fiber2 protein with high affinity ( Figure 10 ).

[0128] (2) IFA identification of the binding of nanoantibodies to FAdV-4

[0129] LMH cells were seeded in 24-well plates (6×10 4cells / well) and cultured in a 37°C, 5% CO2 incubator. When the cell density reached approximately 75%, FAdV-4 was inoculated at a 0.1 MOI and cultured in a 37°C, 5% CO2 incubator for another 48 hours. The culture medium was discarded, and 400 μL of 70% ice-cold ethanol was added to each well and fixed at 4°C for 30 minutes. The fixative was discarded, and 400 μL of 1% BSA was added to each well and blocked at 37°C for 1 hour. 200 μL of FAdV-4-Fiber2-Nbs was added, and FAdV-4-positive mouse serum was used as a control (1:200 dilution) and PBS was used as a negative control. The cells were incubated overnight at 4°C, and commercial His monoclonal antibody (Quanshijin Biotechnology Co., Ltd.) was incubated at room temperature. Then, FITC-labeled goat anti-mouse IgG was incubated and observed under a fluorescence microscope to determine whether FAdV-4-Fiber 2-Nbs bound to FAdV-4. IFA results showed that FAdV-4-Fiber 2-Nb12, -50, -61, -66, -73, -75 and -91 all bound to FAdV-4 ( Figure 11 ).

[0130] (3) IFA preliminary screening of nanoantibodies with the ability to neutralize FAdV-4

[0131] LMH cells were seeded in 24-well plates (6×10 4 cells / well) and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 75%, the FAdV-4-Fiber2-Nbs screened above were incubated with 0.1 MOI of FAdV-4 at 4°C overnight and then inoculated into LMH cells. 24 hours after infection, the culture medium was discarded, 400 μL of 70% ice ethanol was added to each well, and fixed at 4°C for 30 minutes. FAdV-4-positive mouse serum (1:200 dilution) was used as the primary antibody and incubated at room temperature for 1 hour. FITC-Goat@MouseIgG (1:400 dilution) was used as the secondary antibody and incubated at room temperature in the dark for 1 hour. Finally, 200 μL of DAPI (1:600 ​​dilution) was added to each well and incubated at room temperature in the dark for 10 minutes. Under an inverted fluorescence microscope, the fewer cells showing green fluorescence, the stronger the neutralizing activity of the nanoantibody. The results showed that the four nanoantibodies FAdV-4-Fiber2-Nb50, -61, -75 and -91 had strong in vitro virus neutralization effects ( Figure 12 ).

[0132] Example 4

[0133] Preparation of anti-FAdV-4 fenobody and analysis of its in vitro antiviral activity

[0134] (1) Construction of recombinant prokaryotic expression vector of nanobody and ferritin fusion protein

[0135] Primers were designed based on the gene sequences encoding nanobodies and ferritin. The primer sequences are shown in Table 10. The gene sequences of the four nanobodies that neutralize FAdV-4 were connected to the ferritin sequence using GS flexible linker. The obtained fusion gene sequences encoding nanobodies and ferritin were then digested with the pET-28a vector using HindIII and BamH I. The digestion system is shown in Table 11. The gene sequence obtained by digestion was then connected to the vector sequence using T4 ligase. The successful construction of the vector was confirmed by bacterial liquid PCR ( Figure 13 ), which were named pET-28a-FAdV-4-Fiber 2-Fenobody-50, -61, -75 and -91.

[0136] Table 10 Amplification Fenobody Primer Sequences

[0137]

[0138] Table 11 pET-28a-FAdV-4-Fiber2-Fenobodies connection system

[0139]

[0140] (2) Expression and purification of Fenobody50, -61, -75, and -91

[0141] The pET-28a-FAdV-4-Fiber2-Fenobody50, -61, -75 and -91 positive plasmids were transformed into Escherichia coli BL21 (DE3) to obtain recombinant expression bacteria of different Fenobody. The culture was carried out at 37℃ and 200 rpm until the logarithmic phase (OD 600nm 0.6-0.8), add IPTG with a final concentration of 0.1mM to induce expression, continue to culture at 37℃ for 8h, collect the bacterial solution, and use Ni column to purify after ultrasonic disruption. After SDS-PAGE and Western Blot analysis, the expected 30kDa target band ( Figure 14 ).

[0142] (3) Cytotoxicity analysis of Fenobody50, -61, -75 and -91

[0143] LMH cells were seeded in 96-well plates (1.5×10 4Cells / well) were placed in a 37°C, 5% CO2 incubator. Fenobody 50, -61, -75, and -91 purified in Example 4 (2) were diluted in F12 medium to give final concentrations of 1 mg / mL, 10 mg / mL, 20 mg / mL, and 30 mg / mL, respectively. When the cell density reached 90%, the cells were washed with PBS (K + ) After washing the cells three times, add diluted Fenobody 50, -61, -75, and -91. Set up four replicate wells for each dilution, and set up four blank controls. After incubation at 37°C in the dark for 1 hour, discard the Fenobodies, add 100 μL of 2% F12 medium to each well, and continue incubation for 36 hours. Discard the medium, add 10 μL of CCK-8 solution to each well, and incubate at 37°C in the dark for 1 hour. Read the OD value on a microplate reader. 450nm The cytotoxicity of Fenobody50, -61, -75 and -91 was analyzed. The results showed that when pET28a-FAdV-4-Fiber2-Fenobody50, -61, -75 and -91 were added in the range of 0-30 μg, there was no significant effect on the activity of LMH cells ( Figure 15 ).

[0144] (4) Fenobody 50, -61, -75 and -91 neutralize FAdV-4IC 50 Determination of

[0145] Fenobody 50, -61, -75 and -91 purified in Example 4 (2) were serially diluted from 20 mg / mL using F12 medium (2 -1 -2 -10 ) were incubated with 0.1 MOI of FAdV-4 at 4°C overnight and then added to LMH cells. After 24 h, the IC values ​​of Fenobody50, -61, -75 and -91 proteins were detected. 50 The results showed that the IC of Fenobody75 50 was 0.6166 μg / mL, and Fenobody50, -61 and -91 had no neutralization effect on FAdV-4 ( Figure 16 ).

[0146] (5) Analysis of the antiviral activity of Fenobody75 protein in vitro

[0147] 0.1 μg, 1 μg and 10 μg of Fenobody75 were incubated with 0.1 MOI of FAdV-4 at 4°C overnight to infect LMH cells. After 24 h, the supernatant was collected and the TCID 50The results of IFA showed that the number of infected cells decreased with the increase of Fenobody75 dose, and the neutralization activity of Fenobody75 in vitro was compared with the negative group (NDV-VII-Fenobody71 incubation group). Figure 17 );TCID 50 The results showed that as the dose of Fenobody75 increased, the FAdV-4 virus titer gradually decreased in a dose-dependent manner ( Figure 18 ); The expression of FAdV-4Hexon protein was detected by Western Blot, and the FAdV-4Hexon band was scanned in grayscale. The results showed that with the increase of Fenobody75 dose, the expression of FAdV-4Hexon protein gradually decreased in a dose-dependent manner ( Figure 19 The above results indicate that Fenobody75 protein has significant in vitro neutralization activity against FAdV-4.

[0148] The amino acid sequence of Fenobody75 is shown in SEQ ID NO.1:

[0149] SEQ ID NO.1:MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEE IGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDGGGGSGGGGSGGGGSESGGGS VQTGGSLRLSCVVSGYSTMPYYMTWFRQSPGKEREGVSNIISHDADTFYAAPVKGRFTISHDMTENTLYLQMNDLRPEDTGLYYCKAVVSQCWNWLSQEAYEYWGQGTQVTVSSAA.

[0150] The nucleotide sequence encoding Fenobody75 is shown in SEQ ID NO.2:

[0151] SEQ ID NO.2: ATGCTGAGCGAACGCATGCTGAAGGCTTTAAACGACCAGCTGAATCGTG AACTGTACAGCGCCTATTTATACTTCGCCATGGCCGCCTATTTTGAAGATCTGGGTTTAGAGGGTTTCGCCAATTGGATGAAAGCCCAAGCTGAAGAAGAGATCGGTCATGCTTTACGCTTCTACAACTATATCTATGATCGCAACGGCCGCGTGGAACTGGACGAAATTCCGAAACCGCCGAAAGAGTGGGAAAGCCCGCTGAAAGCCTTTGAGGCCGCCTACGAGCACGAAAAATTCATCAGCAAAAGCATTTATGAACTGGCCGCTTTAGCCGAGGAAGAAAAAGACTATAGCACCCGCGCCTTTCTGGAGTGGTTTATCAACGAGCAAGTTGAAGAAGAAGCCAGCGTGAAGAAAATTTTAGACAAACTGAAATTTGCCAAGGACGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGAGTCTGGGGGAGGCTCGGTGCAAACTGGAGGGTCTCTGAGACTGTCCTGTGTAGTATCTGGTTATAGCACAATGCCATACTACATGACATGGTTCCGCCAAAGTCCAGGCAAGGAGCGCGAGGGAGTCTCAAATATTATCAGTCATGATGCGGACACATTCTATGCCGCGCCGGTGAAGGGCCGATTCACCATTTCCCATGACATGACCGAGAACACCCTGTATCTCCAAATGAACGACCTGAGACCTGAAGACACTGGCTTGTACTACTGTAAAGCAGTTGTTTCACAGTGTTGGAACTGGCTGTCCCAAGAGGCGTATGAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC。

[0152] The amino acid sequence of the Fiber2-Nb75 antibody is shown in SEQ ID NO.3:

[0153] SEQ ID NO.3: QVQLQESGGGSVQTGGSLRLSCVVSGYSTMPYYMTWFRQSPGKEREGVSN IISHDADTFYAAPVKGRFTISHDMTENTLYLQMNDLRPEDTGLYYCKAVVSQCWNWLSQEAYEY WGQGTQVTVSSAA.

[0154] The nucleotide sequence encoding the Fiber2-Nb75 antibody is shown in SEQ ID NO.4:

[0155] SEQ ID NO.4: CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAAACTGGAGGG TCTCTGAGACTGTCCTGTGTAGTATCTGGTTATAGCACAATGCCATACTACATGACATGGTTCCGCCAAAGTCCAGGCAAGGAGCGCGAGGGAGTCTCAAATATTATCAGTCATGATGCGGACACATTCTATGCCGCGCCGGTGAAGGGCCGATTCACCATTTCCCAT GACATGACCGAGAACACCCTGTATCTCCAAATGAACGACCTGAGACCTGAAGACACTGGCTTGTACTACTGTAAAGCAGTTGTTTCACAGTGTTGGAACTGGCTGTCCCAAGAGGCGTATGAATACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC.

[0156] The amino acid sequence of ferritin is shown in SEQ ID NO.5:

[0157] SEQ ID NO.5: MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEE IGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKD.

[0158] The nucleotide sequence encoding ferritin is shown in SEQ ID NO.6:

[0159] SEQ ID NO.6: ATGCTGAGCGAACGCATGCTGAAGGCTTTAAACGACCAGCTGAATCGTG AACTGTACAGCGCCTATTTATACTTCGCCATGGCCGCCTATTTTGAAGATCTGGGTTTAGAGGGTTTCGCCAATTGGATGAAAGCCCAAGCTGAAGAAGAGATCGGTCATGCTTTACGCTTCTACAACTATATCTATGATCGCAACGGCCGCGTGGAACTGGACGAAATTCCGAAACCGCCGAAAGAGTGGGGAAA GCCCGCTGAAAGCCTTTGAGGCCGCCTACGAGCACGAAAAATTCATCAGCAAAAGCATTTATGAACTGGCCGCTTTAGCCGAGGAAGAAAAAGACTATAGCACCCGCGCCTTTCTGGAGTGGTTTATCAACGAGCAAGTTGAAGAAGAAGCCAGCGTGAAGAAAATTTTAGACAAACTGAAATTTGCCAAGGAC.

[0160] Effect Examples

[0161] Analysis of the effect of Fenobody75 protein in preventing and treating FAdV-4 virus infection

[0162] Animal challenge therapy experiments were conducted based on the purified Fenobody75 protein obtained in Example 4(2). The experimental groups were as follows:

[0163] Thirty-six SPF chickens were randomly divided into six groups: PBS control group, challenge group only, Fenobody injection group only, prevention group, challenge 1 hour treatment group, and challenge 24 hours treatment group. The chickens in each group were housed in isolation. 5.16 TCID 50Chickens in the FAdV-4 and PBS control groups received an intramuscular injection of 2 mL of PBS. The preventive group received an intramuscular injection of 10 mg / kg of Fenobody75 1 hour before challenge. The 1-hour treatment group received an intramuscular injection of 10 mg / kg of Fenobody75 1 hour, 12 hours, 24 hours, and 48 hours after challenge. The 24-hour treatment group received an intramuscular injection of 10 mg / kg of Fenobody75 24 hours, 36 hours, 48 ​​hours, and 72 hours after challenge. The Fenobody-injected control group and the 1-hour treatment group received an intramuscular injection of 10 mg / kg of Fenobody75 simultaneously to assess the toxicity of Fenobody to the chickens. The animal groups are shown in Table 12, and the in vivo Fenobody75 testing is shown in Table 13.

[0164] Table 12 Grouping of Fenobody75 in vivo challenge protection test

[0165]

[0166]

[0167] Neutralization protection efficiency of Fenobody75. The experimental results showed that all the experimental chickens in the challenge group died 6 days after the challenge, with a mortality rate of 100%, indicating that the challenge was successful. In the 1h challenge group, one chicken died on the 4th and 5th days after the challenge, respectively, and the survival rate of the chickens was 66%. In the 24h challenge group, one chicken died on the 3rd and 4th days after the challenge, respectively, and the survival rate of the chickens was 66%; no chickens died in the PBS group, the Fenobody control group, and the Fenobody prevention group, and the survival rate was 100% ( Figure 20 After 7 days of feeding, the chickens were uniformly killed and two samples of heart, spleen, liver, lung and kidney were collected for viral load detection and pathological histological analysis. The results of tissue and organ virus quantification showed that FAdV-4 could be detected in the heart, spleen, liver, lung and kidney of the chickens in the challenge group; the virus was only detected in the liver in each treatment group; FAdV-4 was only detected in the kidney in the prevention group, and the load was significantly lower than that in the challenge group ( Figure 21 HE section staining results showed that the heart, lungs, liver, spleen and kidneys of the chickens in the challenge group showed obvious pathological damage. The histopathological damage of the chickens in the Fenobody75 treatment groups and the prevention group was greatly alleviated, among which the prevention group and the group treated 1 hour after the challenge showed the mildest pathological damage ( Figure 22 ).

[0168] In summary, the Fenobody75 prepared by the present invention has a significant FAdV-4 neutralizing effect in in vitro experiments. The results of in vivo challenge treatment experiments show that injection of Fenobody75 can effectively improve the survival rate of FAdV-4-infected chickens and reduce the viral load and pathological damage in various tissues and organs. Therefore, the Fenobody75 provided by the present invention can effectively prevent and treat FAdV-4 infection, reduce the economic losses caused by FAdV-4 to the poultry industry, and has significant economic value and application market.

[0169] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A nanobody that specifically binds to FAdV-4, characterized in that Its amino acid sequence is shown in SEQ ID NO.

3.

2. A DNA molecule encoding a Nanobody according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A fusion protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

4. A DNA molecule encoding the fusion protein according to claim 3, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that: Comprising the DNA molecule as described in claim 4.

6. Use of the nanobody according to claim 1, the fusion protein according to claim 3 or the recombinant expression vector according to claim 5 in the preparation of a drug for preventing and treating FAdV-4 infection.

7. A drug for preventing and treating FAdV-4 infection, characterized in that Comprising the nanobody according to claim 1 or the fusion protein according to claim 3.

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