Humanized rabies virus nanobody, recombinant nanobody protein and their use in preparing rabies prevention and treatment products

Abstract

The present invention relates to the field of biomedicine, and more particularly to a humanized rabies virus nanobody, a recombinant nanobody protein, and their use in the preparation of rabies prevention and treatment products. The present invention screens for a humanized rabies virus nanobody with strong binding to the rabies virus glycoprotein and the ability to cross the blood-brain barrier. EGFP and the blood-brain barrier-penetrating peptide T7 or PepH3 are then linked to the C-terminus, respectively. The antibody fusion protein gene is cloned into the prokaryotic expression vector pMal-p5x, and expressed and purified using the prokaryotic expression engineered bacterium Shuffle T7 to obtain a recombinant nanobody protein capable of intravenously entering the central nervous system for virus neutralization therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a humanized rabies virus nanobody, a recombinant nanobody protein, and applications in preparing rabies prevention and treatment products. Background Art

[0002] Rabies is an acute, severe zoonotic disease caused by the highly neurotropic rabies virus (RABV). It has a mortality rate as high as 100% and remains untreated. Rabies virus primarily targets the central nervous system (CNS). Neurotropism is a key characteristic of natural infection, and viral replication is almost exclusively within neurons. RABV first replicates in muscle cells surrounding the wound, then enters the peripheral nervous system through surrounding neuronal terminals. It then travels centripetally along the spinal cord via long-distance axonal transport and transsynaptic spread between neurons. It then undergoes retrograde transport within neuronal axons before entering the CNS and spreading to various parts of the brain. Prolonged replication in the brain ultimately causes encephalitis, a potentially fatal condition. Since the discovery of rabies, research into its treatment has been ongoing. Unfortunately, due to the unique characteristics of the rabies virus, no drug has yet been developed that can successfully cure rabies. In the early stage after viral infection, vaccination or rabies immunoglobulin (RIG) is used for preventive intervention after thoroughly washing the wound with soap and water, which can effectively prevent the development of rabies. This method is called post-exposure prophylaxis (PEP). RIG is used for patients with severe exposure as an additional measure for vaccination. It is a process of passive immunization. Currently, equine rabies immunoglobulin and human rabies immunoglobulin are mainly used. However, PEP is expensive and has limited supply. It needs to be administered quickly within a short period of time after exposure and requires multiple vaccinations, making it difficult to be widely used in underdeveloped areas (Tarantola A, et al. Rabies Vaccine and Rabies Immunoglobulin in Cambodia: Use and Obstacles to Use [J]. J Travel Med. 2015; 22(5): 348-352).

[0003] In order to better prevent and treat rabies, scientists have developed an alternative to RIG - broadly neutralizing antibodies (bnAbs) with low cost, sufficient supply, good stability and high safety. The earliest rabies monoclonal antibody is Rabishield (Gogtay NJ, et al. Comparison of a Novel HumanRabies Monoclonal Antibody to Human Rabies Immunoglobulin for PostexposureProphylaxis:A Phase2 / 3,Randomized,Single-Blind,Noninferiority,ControlledStudy[J]. Clin Infect Dis.2018;66(3):387-395) and TwinrabTM (Kansagra K, et al. APhase 3, Randomized, Open-label, Noninferiority Trial Evaluating Anti-RabiesMonoclonal Antibody Cocktail (TwinrabTM) Against Human Rabies Immunoglobulin (HRIG) [J]. Clin Infect Dis. 2021; 73(9): e2722-e2728), both passed clinical trials and were licensed and were launched in India in 2017 and 2020, respectively. Currently, other monoclonal antibody drugs are also under continuous research. RVC20 and RVC58 are two human monoclonal antibodies that bind to antigenic sites I and III of the RABV G protein, respectively. RVC20 is one of the best known spectrum-neutralizing monoclonal antibodies. It can block membrane fusion between RABV and cells, prevent RABV from entering cells and further spreading in infected hosts. It has been approved for post-exposure treatment of human rabies (de Melo GD, et al. Monoclonal antibodies against rabies: current uses in prophylaxis and intherapy [J]. Curr Opin Virol. 2022; 53: 101204).RVC58 can also recognize and lock onto the RABV-G protein in its prefusion conformation, and has been shown to more effectively neutralize extracellular viruses (Zorzan M, et al. Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy [J]. Front Immunol. 2023; 14: 1186063). After the onset of neurological symptoms in the early stages of infection, a mixture of RVC20 and RVC58 mAbs was injected into the peripheral and central nervous systems by intracerebroventricular infusion, which was able to treat rabies in mice in the early stages of the disease to a certain extent (de Melo GD, et al. A combination of two human monoclonal antibodies cures symptomatic rabies [J]. EMBO Mol Med. 2020; 12 (11): e12628).

[0004] In the early stages of rabies virus infection, the host generally does not experience any symptoms. By the time symptoms appear, the virus has already invaded and multiplied rapidly in the central nervous system. If neutralizing antibodies are to be used for treatment at this stage, how these antibodies can cross the blood-brain barrier (BBB) ​​and enter the central nervous system is crucial. The BBB is composed of brain capillary endothelial cells, pericytes, and astrocytes. The BBB and its intercellular tight junctions are the primary components of the BBB, collectively known as the barrier between plasma and brain cells, between plasma and cerebrospinal fluid, and between cerebrospinal fluid and brain cells. These barriers prevent harmful substances from entering the brain through the bloodstream while ensuring the uptake of substances necessary for brain function and the excretion of drug metabolites, maintaining a relatively stable internal environment and supporting normal neuronal function. The BBB prevents most drug molecules, including antibodies, from entering the central nervous system to exert their effects. This presents a significant obstacle to rabies treatment, and developing methods for drug delivery through the BBB is a major challenge in rabies treatment.

[0005] Nanobody (Nb) is a light chain-free antibody found in camel serum. This antibody contains only a heavy chain variable region (Variable Domain of Heavy Chain of Heavy-ChainAntibody, VHH), two constant regions CH2 and CH3, and lacks two light chains and the constant region CH1. This antibody has a diameter of 2.5nm, a length of 4nm, and a relative molecular mass of 12-14kDa. It is the smallest naturally occurring fragment that can bind to an antigen (Ji F, et al. Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in Camelids to a Wide Range of Useful Applications [J]. Methods Mol Biol. 2022; 2446: 3-17). Compared with traditional antibodies, nanoantibodies have many advantages: in addition to being able to be expressed in mammalian expression systems, they can also be expressed through prokaryotic expression systems such as Escherichia coli and yeast, with lower costs, higher expression rates, better purification effects, higher water solubility and higher stability, and are not easily inactivated at different temperatures and pH values. They can still maintain high activity after high-temperature treatment at 90°C. In addition, nanoantibodies also have lower immunogenicity and stronger antigen binding ability, which can reduce drug resistance and improve specificity (Wesolowski J, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol [J]. 2009; 198 (3): 157-174). Nanoantibodies have good tissue penetration ability due to their small size and relative molecular weight, and can penetrate the blood-brain barrier. They have important application value in drug development and basic research, especially providing a new method for the treatment of brain diseases. The use of nanomedicines to deliver to the brain through intravenous injection has become an increasingly popular field.

[0006] Blood-brain barrier penetrating peptides are short peptides with the ability to cross the blood-brain barrier, generally containing 5 to 30 amino acids. They can transport molecular cargo to the brain parenchyma without affecting the integrity of the blood-brain barrier. They have a strong affinity for specific receptors and can mediate transcytosis (Zhou X, et al. Brain penetrating peptides and peptide-drug conjugates to overcome the blood-brain barrier and target CNS diseases [J]. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2021; 13(4): e1695). Transferrin receptor (TfR) is a membrane glycoprotein receptor. TfR-mediated transcytosis allows circulating iron-bound transferrin to be transported across the blood-brain barrier to the brain interstitial space. TfR expression levels are increased on the blood-brain barrier and glioma cells, making it a promising tool for brain-targeted delivery and glioma treatment. Among all TfR-targeted or non-targeted groups, the T7 peptide composed of 7 amino acids has been widely used as a ligand for constructing tumor-targeted nanodrug delivery systems because it can target TfR and easily enter cells with the help of transferrin. T7-modified liposomes (T7-LS) showed the highest blood-brain barrier penetration and brain distribution, indicating that T7-LS is a potential platform for clinically effective brain-targeted delivery (Mojarad-Jabali S, et al. Comparison of three synthetic transferrin mimetic small peptides topromote the blood-brain barrier penetration of vincristine liposomes for improved glioma targeted therapy [J]. Int J Pharm. 2022; 613: 121395). Specific domains of the dengue virus type 2 capsid protein can also be used as cross-blood-brain barrier peptide carriers, especially the PepH3 peptide segment composed of a 7-amino acid residue sequence and an α-helical structure.In vitro cell experiments revealed that the equilibrium distribution concentration across the blood-brain barrier was reached in less than 24 hours. In vivo biodistribution data for peptide derivatives labeled with radionuclides revealed that they had high brain permeability (Neves V, et al. Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism [J]. ACS Chem Biol. 2017; 12(5): 1257-1268). Fc-PepH3, obtained by coupling PepH3 with an immunoglobulin fragment, was confirmed to be non-toxic and became a successful model for crossing the blood-brain barrier (Cavaco M, et al. Conjugation of a Blood Brain Barrier Peptide Shuttle to an Fc Domain for Brain Delivery of Therapeutic Biomolecules [J]. ACS Med Chem Lett. 2021; 12(11): 1663-1668).

[0007] In summary, traditional antisera or monoclonal antibodies can effectively neutralize rabies virus in vitro, but it is very difficult for them to enter the central nervous system to neutralize and treat the virus. Summary of the Invention

[0008] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a humanized rabies virus nanobody.

[0009] Another object of the present invention is to provide a recombinant nanobody protein, which comprises the above-mentioned humanized rabies virus nanobody, and its C-terminus is connected and fused with enhanced green fluorescent protein (EGFP) and blood-brain barrier penetrating peptide (T7 or PepH3), has rabies virus neutralization ability, enhanced ability to enter the central nervous system, and can be used for the treatment of rabies and passive immunization.

[0010] Another object of the present invention is to provide the use of the above-mentioned humanized rabies virus nanobody and recombinant nanobody protein.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned recombinant nanobody protein.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A humanized rabies virus nanobody, the amino acid sequence of which is shown in SEQ ID No. 1;

[0014] The gene encoding the humanized rabies virus nanobody has a nucleotide sequence as shown in SEQ ID No. 2 or 3;

[0015] A recombinant nanobody protein comprises the humanized rabies virus nanobody, and its C-terminus is fused with EGFP and a blood-brain barrier penetrating peptide;

[0016] The blood-brain barrier penetrating peptide is at least one of the blood-brain barrier penetrating peptide T7 and the blood-brain barrier penetrating peptide PepH3;

[0017] The recombinant Nanobody protein is preferably at least one of RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3, wherein the amino acid sequence of RVG-hNb07-EGFP-T7 is shown in SEQ ID No. 4, and the amino acid sequence of RVG-hNb07-EGFP-PepH3 is shown in SEQ ID No. 5;

[0018] The gene encoding the recombinant nanobody protein, the nucleotide sequence encoding RVG-hNb07-EGFP-T7 is shown in SEQ ID No. 6, and the nucleotide sequence encoding RVG-hNb07-EGFP-PepH3 is shown in SEQ ID No. 7;

[0019] Use of the humanized rabies virus nanobody or recombinant nanobody protein in the preparation of rabies prevention and treatment products;

[0020] A recombinant vector comprising the gene encoding the humanized rabies virus nanobody or the gene encoding the recombinant nanobody protein;

[0021] The recombinant vector is obtained by connecting the gene encoding the humanized rabies virus nanobody or the gene encoding the recombinant nanobody protein to an expression vector;

[0022] The expression vector is preferably pMal-p5x;

[0023] A biomaterial expressing humanized rabies virus nanobody or recombinant nanobody protein is obtained by transferring the above-mentioned recombinant vector into an expression system;

[0024] The expression system can be bacteria or fungi;

[0025] The bacteria are preferably Escherichia coli SHuffle T7;

[0026] Application of the recombinant vector and biological material in the preparation of rabies prevention and treatment products;

[0027] The method for preparing the humanized rabies virus nanobody or recombinant nanobody protein comprises the following steps:

[0028] The biological material expressing humanized rabies virus nanobody or recombinant nanobody protein is induced to express and purified to obtain humanized rabies virus nanobody or recombinant nanobody protein.

[0029] Principle of the present invention:

[0030] After invading, the rabies virus rapidly infects the central nervous system and replicates in large quantities. However, due to the presence of the blood-brain barrier, conventional therapeutic drugs such as antibodies cannot enter the brain to neutralize the virus. Therefore, no effective drug for treating rabies has been developed. In order to obtain rabies therapeutic antibodies with high blood-brain barrier penetration ability, the present invention uses the yeast two-hybrid system to screen out humanized rabies virus nanoantibodies with strong binding to rabies virus glycoprotein and blood-brain barrier penetration ability from an artificially synthesized humanized nanoantibody library, and then connects enhanced green fluorescent protein (EGFP) and blood-brain barrier penetrating peptide (T7 or PepH3) at their C-termini, clones the obtained antibody fusion protein gene into the prokaryotic expression vector pMal-p5x, expresses and purifies it by prokaryotic expression engineering bacteria SHuffle T7, and obtains recombinant nanoantibodies that can enter the central nervous system from the vein for virus neutralization treatment. The therapeutic effect of the recombinant nanoantibody protein is comprehensively evaluated by cell immunofluorescence, virus neutralization test, post-exposure infiltration injection of infected mice and intravenous treatment.

[0031] The present invention has the following advantages and effects compared to the prior art:

[0032] (1) The present invention utilizes the yeast two-hybrid system to screen out rabies virus glycoprotein-binding nanobodies from an artificially synthesized humanized nanoantibody library, and further obtains humanized rabies virus nanoantibodies with higher neutralization titers through the virus neutralization titer.

[0033] (2) The present invention modifies humanized rabies virus nanobodies with high neutralization titers, and fuses blood-brain barrier penetrating peptides T7 or PepH3 at their C-termini. By fusion expression of the blood-brain barrier penetrating peptide with the nanobody, its ability to enter the central nervous system through intravenous injection is enhanced, thereby improving the therapeutic effect on rabies. This therapeutic effect includes both the neutralization effect of infiltration injection on wound-infected viruses and the neutralization effect on viruses entering the brain, providing new ideas and methods for the prevention and treatment of rabies.

[0034] (3) The present invention also fuses enhanced green fluorescent protein EGFP to the C-terminus of the humanized rabies virus nanobody to facilitate subsequent observation and positioning of the recombinant protein.

[0035] (4) The recombinant nanobody protein containing humanized rabies virus nanobody provided by the present invention has a small molecular weight and a simple structure, and can be prepared using a prokaryotic expression system such as Escherichia coli, which greatly reduces the cost.

[0036] (5) The invention screens humanized nanoantibodies, which can be easily applied to human clinical treatment in the future, reducing immunogenicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is an analysis diagram of the results of direct immunofluorescence test of recombinant nanoantibodies on infected cells.

[0038] Figure 2 This is an analysis diagram of the Western blot detection results of recombinant nanobody protein in mouse blood, where a: serum sample of mouse injected with 200 μg RVG-hNb07-EGFP-T7 via tail vein, b: serum sample of mouse injected with 200 μg RVG-hNb07-EGFP-PepH3 via tail vein; positive control: purified recombinant nanobody protein.

[0039] Figure 3 This is an analysis diagram of the Western blot detection results of recombinant nanobody protein in mouse brain, where Nb-T7: mouse brain sample injected with RVG-hNb07-EGFP-T7, Nb-PepH3: mouse brain sample injected with RVG-hNb07-EGFP-PepH3, and Nb: mouse brain sample injected with RVG-hNb07-EGFP.

[0040] Figure 4 This is an analysis of the survival rate of mice injected intramuscularly after the recombinant nanobody protein neutralized the virus. Among them, Nb-T7: RVG-hNb07-EGFP-T7, Nb-PepH3: RVG-hNb07-EGFP-PepH3, Nb: RVG-hNb07-EGFP; the virus used for the attack was CVS-11 with a titer of 10 6 FFU / mL, the dose was 100 μL, the protein dose used in the high-dose group was 8 IU, and the protein dose used in the low-dose group was 4 IU.

[0041] Figure 5Survival analysis of mice infected with recombinant nanoantibodies after passive immunization, where Nb-T7: RVG-hNb07-EGFP-T7, Nb-PepH3: RVG-hNb07-EGFP-PepH3, Nb: RVG-hNb07-EGFP; the challenge virus was CVS-11 with a titer of 10 6 FFU / mL, the dose is 100 μL, and the protein dose for infiltration injection is 8 IU.

[0042] Figure 6 Analysis of the survival rate of infected mice treated with intravenous injection of recombinant nanobodies, where Nb-T7: RVG-hNb07-EGFP-T7, Nb-PepH3: RVG-hNb07-EGFP-PepH3, Nb: RVG-hNb07-EGFP; the challenge virus was CVS-11 with a titer of 10 6 FFU / mL, the dose is 100 μL, and the intravenous dose of recombinant protein is 8 IU. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0044] Example 1 Screening of humanized VHH fragments of rabies virus G protein nanobodies

[0045] The extracellular region of the rabies virus CVS-11 G protein gene (GenBank accession number: EU126641) was PCR amplified and then double-digested with EcoRI and BamHI. The fragment was then ligated into the pGBKT7 vector to construct a recombinant expression vector pGBKT7-RABV-JM-Get. This vector was used as bait to screen for nanoantibody VHH fragments that bind to the rabies virus G protein in a yeast two-hybrid humanized nanoantibody library (artificial synthetic library, construction method refers to Moutel S, et al. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife. 2016; 5: e16228). The specific method is as follows:

[0046] 1. Construction of recombinant expression vector

[0047] Using the rabies virus CVS-11 G protein gene (GenBank accession number: EU126641) as a template, primers were designed to amplify the extracellular region of the G protein gene (the upstream primer 5' was designed with an EcoRI restriction site, and the downstream primer 5' was designed with BamHI). PCR amplification was then performed to obtain the extracellular region fragment. The extracellular region fragment and the vector pGBKT7 (laboratory storage, commercially available) were double-digested with EcoRI and BamHI according to conventional methods, ligated, and transformed into competent cells. A single colony was picked and expanded for plasmid extraction. The recombinant plasmid was identified by PCR and sequencing to obtain the correctly connected recombinant expression vector pGBKT7-RABV-JM-Get. The nucleotide sequence of the extracellular region fragment is shown below:

[0048]

[0049] 2. Self-activation detection

[0050] pGBKT7-RABV-JM-Get and pGBKT7 (negative control) obtained in step 1.1 were transformed into AH109 yeast competent cells (Nanjing Ruiyuan Biotechnology Co., Ltd.) according to conventional methods. The transformed AH109 yeast competent cells were spread onto SD / -Trp plates and incubated at 30°C for 3-4 days. PCR was performed on six randomly selected spots using pGBKT7 vector primers to verify that all clones were correct. Three randomly selected clones were then plated onto SD / -Trp, SD / -Trp / -His, SD / -Trp / -His / -Ade, and SD / -Trp / -His / -Ade+X-α-gal plates and incubated at 30°C for 3-5 days. Yeast growth was observed to determine whether the recombinant expression vector exhibited autoactivation after transformation into yeast. The results showed that pGBKT7-RABV-JM-Get did not autoactivate.

[0051] 3. Transformation and screening of the yeast plasmid pGADT7-hVHH

[0052] AH109 yeast strain containing pGBKT7-RABV-JM-Get (bait plasmid) was used as a receptor to prepare competent cells. The humanized nanobody library plasmid pGADT7-hVHH (synthesized and constructed by Nanjing Ruiyuan Biotechnology Co., Ltd.) was transferred into it and coated with SD-TLH screening plates. The following steps were followed:

[0053] (1) Pick and identify positive cloned yeast strains carrying the recombinant expression vector pGBKT7-RABV-JM-Get from the SD-T plate, inoculate them into 50 mL of liquid SD-T medium, and culture them at 30°C and 225 rpm for 24 h.

[0054] (2) Transfer the bacterial solution from step (1) to 500 mL YPDA liquid base to make the initial OD 600 = 0.2, 30 ° C, 225 r / min shaking culture for 4-5 h, until OD 600 =0.6, then centrifuge to collect the bacteria, centrifuge at 4000 rpm for 5 min; resuspend the bacteria in 30 mL of sterile water, mix, centrifuge to collect the bacteria, and discard the supernatant; resuspend the bacteria in 20 mL of 0.1 mol / L LiAc, mix, centrifuge to collect the bacteria; resuspend the bacteria in 10 mL of 0.1 mol / L LiAc, mix, centrifuge at 4000 rpm for 5 min, and discard the supernatant;

[0055] (3) Add 9.6 mL of 50% PEG 3350, 1.44 mL of 1 mol / L LiAc, 300 μL of ssDNA (10 mg / mL), and 25 μg of the library plasmid pGADT7-hVHH to the centrifuge tube in sequence. Mix thoroughly by pipetting with a pipette tip, incubate in a 30°C water bath for 30 min, heat shock in a 42°C water bath for 25 min, and recover in a 30°C water bath for 1 h. Then, centrifuge to collect the bacteria, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0056] (4) Resuspend the cells in 6 mL of sterile water and mix gently. Take 20 μL of the culture and dilute it to spread on SD-TL plates to detect the library transformation efficiency. Spread the remaining 20 plates on SD-TLH plates. Incubate at 30°C for 3-7 days and observe the growth of the colonies. Then, pick out the single colony and transfer it to SD-TLHA+X-α-gal selection plates and continue to culture for 3-5 days.

[0057] 4. Screening results

[0058] Positive yeast clones were screened on SD-TLHA + X-α-gal screening plates, and 120 clones were selected from these plates for PCR verification. To identify the gene sequences of these positive clones screened on the SD-TLHA + X-α-gal plates, these clones were amplified from yeast cells, sequenced, and subjected to BLAST analysis. All 120 positive yeast clones were PCR amplified, sequenced, and aligned using Seqman and BLAST, ultimately yielding 14 gene sequences.

[0059] 5. Verification of positive yeast clones

[0060] Streak the positive clones corresponding to the 14 gene sequences from step 1.4 onto SD-TLHA+X-α-gal-deficient plates. Dilute the positive clones with sterile water and spot them onto SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal-deficient plates. Incubate each plate at 30°C for 3-4 days. The nine positive yeast clones screened all grew normally on SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal-deficient plates and developed a blue color on the SD-TLHA+X-α-gal plate.

[0061] 6. Determination of VHH Virus Neutralization Ability

[0062] The 9 selected VHHs were purified and expressed in yeast and then subjected to RABV virus neutralization test. The neutralizing antibody titers measured by the FAVN method (Tan A. Use of the standards of the World Organisation for Animal Health inveterinary certificates. Rev Sci Tech. 2020; 39(1): 263-271) are shown in the following table:

[0063] Table 1 VHH virus neutralization ability

[0064]

[0065]

[0066] RVG-hNb07 with the highest neutralization titer was selected for subsequent experiments, and its nucleotide sequence (SEQ ID No. 2) is as follows: GAAGTTCAATTGCAAGCTTCTGGTGGTGGTTTCGTCCAACCAGGTGGTTCCTTAAGATTGTCTTGTGCTGCCTCAGGCCGTACTTTGTCCTCCTACAGAATGGGTTGGTTCAGACAAGCTCCAGGTAAGGAAAGAGAATTTGTTTCTGCTATCTCTTGGAACGGTAGATCTACCTATTACGCTGATTCTGTCAAGGGTAGATTCACCATCTCCCGTGACAATTCCAAAAACACTGTTTACCTACAAATGAACTCTTTGAGAGCTGAAGACACTGCTACTTACTACTGTGCCGCCGCTTTGATTGGTGGTTACTACTCTGATGTTGACGCTTGGTCTTACTGGGGTCAAGGTACCCAAGTTACCGTCTCCAGT

[0067] RVG-hNb07 was used as the target for recombinant nanobody protein modification. The nucleic acid sequence (SEQ ID No. 3) optimized according to the codon preference of E. coli was as follows:

[0068] GAAGTACAGCTACAAGCTAGTGGTGGAGGGTTCGTGCAACCGGGTGGCAGCTTGCGCCTGAGCTGTGCGGCGAGCGGTCGTACCCTGTCTTCCTATCGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAGCGCGAATTTGTGAGCGCGATTTCTTGGAACGGCCGTAGCACCTACTACGCAGAC AGCGTTAAAGGTCGCTTCACCATCAGCCGTGATAATTCCAAGAACACCGTCTATCTGCAAATGAACTCGTTGAGAGCTGAGGACACCGCGACGTATTACTGCGCCGCGGCTCTGATCGGCGGCTATTACAGCGATGTTGACGCCTGGTCCTACTGGGGTCAGGGCACTCAGGTTAACGGTGTCATCC

[0069] The translated amino acid sequence (SEQ ID No. 1) is as follows:

[0070] EVQLQASGGGFVQPGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFVSAISWNGRSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAALIGGYYSDVDAWSYWGQGTQVTVSS

[0071] Example 2 Construction of recombinant plasmid and expression and purification of recombinant nanobody protein

[0072] 1. Construction of recombinant plasmid

[0073] The RV-hNb07 gene fragment was optimized by Suzhou Jinweizhi Biotechnology Co., Ltd. according to the codon preference of Escherichia coli, and a flexible peptide GGGGS×3, green fluorescent protein gene EGFP, 6×His and blood-brain barrier penetrating peptide (T7 or PepH3) were connected to its C-terminus. Three gene fragments, RVG-hNb07-EGFP, RVG-hNb07-EGFP-T7, and RVG-hNb07-EGFP-PepH3, were synthesized, and Nco I and Hind III restriction sites were introduced at both ends of the sequences. The resulting gene fragments with restriction sites and the vector pMal-p5x were double-digested with Nco I and Hind III, respectively, according to conventional methods. Ligation was performed, and competent cells were transformed. Single colonies were picked and expanded for culture, and plasmids were extracted. The recombinant plasmids were verified by PCR and sequencing and correctly inserted into pMal-p5x to obtain recombinant expression vectors pMal-RVG-hNb07-EGFP, pMal-RVG-hNb07-EGFP-T7, and pMal-RVG-hNb07-EGFP-PepH3. The recombinant expression vectors were transformed into SHuffle T7 (NEB, USA, Cat No. C3026J) competent bacteria and stored.

[0074] RVG-hNb07-EGFP nucleotide sequence:

[0075]

[0076] Amino acid sequence of RVG-hNb07-EGFP:

[0077] EVQLQASGGGFVQPGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFVSAISWNGRSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAALIGGYYSDVDAWSYWGQGTQVTVSSGSAAAGGGGSGGGGSGGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH.

[0078] Nucleotide sequence of RVG-hNb07-EGFP-T7 (SEQ ID No. 6):

[0079]

[0080] Amino acid sequence of RVG-hNb07-EGFP-T7 (SEQ ID No. 4):

[0081] EVQLQASGGGFVQPGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFVSAISWNGRSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAALIGGYYSDVDAWSYWGQGTQVTVSSGSAAAGGGGSGGGGSGGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHHAIYPRH.

[0082] Nucleotide sequence of RVG-hNb07-EGFP-PepH3 (SEQ ID No. 7):

[0083]

[0084] RVG-hNb07-EGFP-PepH3 amino acid sequence (SEQ ID No. 5):

[0085] EVQLQASGGGFVQPGGSLRLSCAASGRTLSSYRMGWFRQAPGKEREFVSAISWNGRSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAALIGGYYSDVDAWSYWGQGTQVTVSSGSAAAGGGGSGGGGSGGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP VPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHAGILKRW.

[0086] 2 Expression and purification of recombinant nanobody protein

[0087] 2.1 Expression of recombinant nanobodies

[0088] (1) Take out the positive SHuffle T7 expression bacteria stored in step 1 from the refrigerator, inoculate it into a 10mL EP tube and add 5mL Amp + LB medium was placed in a constant temperature shaker at 30°C and 200 r / min and cultured for 12 to 16 hours as the primary seed solution.

[0089] (2) Take 100 μL of the first-stage seed solution and inoculate it into 5 mL of Amp + LB medium was added and cultured in a constant temperature shaker at 30°C and 200 rpm for 12 to 16 h.

[0090] (4) Add Amp to a final concentration of 1-5% (mass fraction) in 1L TB medium, add 10mL of cultured secondary seed solution, and culture at a constant temperature shaker at 30°C and 200r / min for 3-4h until the bacterial solution OD 600 When the concentration reached about 0.5, 10 mL of 0.1 mol / L IPTG was added and the expression was induced for 24 h.

[0091] (5) After 1 day, remove the bacterial suspension and pour it into 6 50 mL centrifuge tubes. Centrifuge at 4°C and 5000 rpm for 25 min. Discard the supernatant and continue pouring the bacterial suspension into the tubes. Repeat the process until all the bacterial suspension is collected.

[0092] (6) Resuspend the enriched cells in 20 mL of PBS or Buffer A (20 mM potassium phosphate buffer, pH 7.4, 40 mM imidazole, 500 mM sodium chloride) to prepare 20 mL of suspension per tube, for a total of 3 tubes.

[0093] (7) Place the ice-water mixture in an ice box or a 1L beaker, insert the bacterial suspension into the ice, and place it in an ultrasonic disruptor. Place the ultrasonic probe 1-3 cm away from the bottom of the tube and sonicate at 300W for 3 seconds, with a 5-second pause, for a total of 30 minutes. During the sonication process, pay attention to the distance between the probe and the bottom of the tube to avoid contact with the tube wall.

[0094] (8) After ultrasonic disruption, observe whether the suspension becomes transparent and clear. Centrifuge it at 4°C and 5000 rpm for 30 min. Filter the supernatant with a 0.22 μm filter to prepare the stock solution.

[0095] 2.2 Purification of recombinant nanobodies

[0096] (1) Place the filtered stock solution in ice and place the HisTrap TM Connect the HP (5 mL) affinity chromatography column to the GE AKTApure chromatograph or NGC Quest 10Plus chromatograph (the system needs to be flushed in advance). Check whether the sample loading hole is connected correctly and whether there is gas in the liquid inlet tube.

[0097] (2) Pa≤0.3, 3 mL / min, pass 30 mL of 20% ethanol solution to clean the column, and continue to pass 30 mL of ultrapure water to wash away the ethanol.

[0098] (3) Pa≤0.3, 3 mL / min, pass 30 mL of Buffer A (20 mM potassium phosphate buffer pH 7.4, 40 mM imidazole, 500 mM sodium chloride) into the equilibrated column, observe the UV absorption peak value, and return to zero after the UV absorption peak value tends to be flat.

[0099] (4) Pa≤0.3, 2 mL / min, pump A to load the sample until all the sample is aspirated.

[0100] (5) Pa≤0.3, 3 mL / min, pass 30 mL of Buffer A to equilibrate the column again to wash away impurities and weakly bound proteins.

[0101] (6) Pa ≤ 0.3, 2 mL / min, place the inlet of pump A in Buffer A, and the inlet of pump B in Buffer B (20 mM phosphate buffer, 500 mM imidazole, 500 mM sodium chloride). Set a 0% to 100% Buffer B, 50 mL linear elution program. Place the collection tubes in the collection wells (NGC Fraction Collector) in order.

[0102] (7) Identify the collected protein solution based on the elution peak of the linear elution profile.

[0103] (8) After the linear elution process is completed, continue to introduce 30 mL of 100% Buffer B, Pa≤0.3, 3 mL / min.

[0104] (9) Pa≤0.3, 3 mL / min, 30 mL of ultrapure water was introduced.

[0105] (10) Pa≤0.3, 3 mL / min, pass 20% volume fraction ethanol into the sealed chromatography column, remove the chromatography column and store it at 4°C.

[0106] (11) Boil the dialysis bag (10 kDa) in ultrapure water for 10 min. Na2CO3 or NaHCO3 can be added.

[0107] (12) The protein solution collected from the elution peak was added to the dialysis bag, clamped at both ends, and 900 mL of PBS was added to a 1 L beaker. The bag was placed in a biological refrigerator at 4 °C and a magnetic stirrer was used at 300 r / min for 6 h. After 6 h, new PBS was replaced and the dialysis was continued for another 6 h.

[0108] (13) Pour out the dialysis solution in the beaker, add 900 mL of PBS and continue dialysis for 6 h. Repeat the operation once.

[0109] (14) The purified protein after dialysis was filtered through a 0.22 μm filter, and 0.5 μL / mL of Factor Xa protease was added and digested at 23°C for 12–16 h.

[0110] (15) Place the protein solution after enzyme digestion in a centrifuge and centrifuge at 4°C and 5000 rpm for 10 min.

[0111] (16) Fill the gravity column with Amylose resin to a volume of 3 mL and introduce 6 times the column volume of ultrapure water.

[0112] (17) Add 5 column volumes of Column Buffer A (20 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA) to rinse the column.

[0113] (18) The supernatant after centrifugation in step (15) is added to a gravity column and the flow-through (containing the target protein) is collected until the supernatant has completely flowed through.

[0114] (19) Add 5 column volumes of column buffer B (20 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, 40 mM maltose) and collect the eluate.

[0115] (20) Three column volumes of ultrapure water, three column volumes of 0.1% SDS, one column volume of ultrapure water, and five column volumes of 20% ethanol were added in sequence, and the filler was poured out and stored at 4 °C.

[0116] (21) The purified recombinant nanobody protein (RVG-hNb07-EGFP, RVG-hNb07-EGFP-T7, or RVG-hNb07-EGFP-PepH3) was quantified using a BCA protein quantification kit.

[0117] Example 3

[0118] 1. Recombinant Nanobody Protein for Direct Fluorescent Antibody Detection

[0119] (1) Prepare NA cells (mouse brain neuroma cells) (preserved by the Veterinary Microbiology Laboratory, College of Veterinary Medicine, South China Agricultural University), culture them in a culture dish until they become a monolayer, discard the supernatant and wash them once with PBS, add 1 mL of trypsin to digest for 45-50 seconds until the cells are loose, discard the trypsin, add 3 mL of complete culture medium and pipette until they are dispersed, calculate the required cell dilution amount according to the number of plates, add culture medium to dilute, take a 96-well plate, add the prepared cell solution to the plate with a spray gun, and add 100 μL of NA cell solution (10 5 After plating, observe the cells under a microscope for uniformity and appropriate density. Place the cells in a 37°C incubator overnight. Add 100 μL of the diluted CVS-11 virus solution (preserved by the Veterinary Microbiology Laboratory, College of Veterinary Medicine, South China Agricultural University) to each well using a dispenser at an MOI of 0.1. Incubate the cells in a 37°C incubator for 48 hours.

[0120] (2) After NA cells were infected with CVS-11 virus for 48 h, they were fixed with 80% acetone and subjected to direct immunofluorescence experiments using commercial RABV antibodies (FITC Anti-Rabies Monoclonal Globulin, purchased from Fujirebio Diagnostics, USA) and recombinant nanobody proteins (RVG-hNb07-EGFP, RVG-hNb07-EGFP-T7, or RVG-hNb07-EGFP-PepH3). Both commercial RABV antibodies and recombinant nanobody proteins were diluted to 5 μg / mL for use.

[0121] The results showed that the two recombinant nanobody proteins (RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3) detected the fixed samples of CVS-11 infected cells, and both showed bright and specific fluorescent spots, indicating that they can specifically bind to the glycoprotein of RABV. Compared with commercial fluorescent antibodies, the fluorescent spots of recombinant nanobodies are larger and ring-shaped, making them easier to identify ( Figure 1 ).

[0122] 2. Degradation of recombinant nanobody protein in mouse blood

[0123] Five-week-old SPF Kunming female mice (purchased from the Experimental Animal Center of Southern Medical University) were injected with 200 μg / mouse of the recombinant nanobody proteins RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3 via the tail vein. Blood was collected from the orbital cavity using an orbital blood collection needle 1, 2, 4, and 8 hours later. The collected blood was placed in a 37°C incubator at an angle for 1 hour, then placed in a 4°C refrigerator at an angle overnight, and then centrifuged at low speed for 5 minutes. The supernatant was aspirated into a clean centrifuge tube to complete serum extraction. After serum was diluted, reducing protein loading buffer was added and the cells were treated in a metal bath at 100°C for 10 min. Western blot analysis was performed using a 6×His-tagged monoclonal antibody (Proteintech Group, Cat No. 66005-1-Ig) and a beta-Actin polyclonal antibody (Proteintech Group, Cat No. 20536-1-AP).

[0124] Depend on Figure 2 As shown in Figures a and b, protein bands with molecular weights consistent with the recombinant Nanobody protein were detected in serum samples at 1, 2, 4, and 8 hours, with no significant changes in band thickness or depth. Therefore, it can be inferred that both recombinant Nanobody proteins remained stable in the blood within 8 hours of tail vein injection into mice, with no rapid degradation, at 1, 2, 4, and 8 hours.

[0125] 3. Detection of recombinant nanobody protein crossing the blood-brain barrier and entering the brain

[0126] Five-week-old SPF Kunming female mice were injected via the tail vein with 200 μg / mouse of the recombinant nanobody proteins RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3. A control group containing the nanobody protein RVG-hNb07-EGFP without the blood-brain barrier-penetrating peptide was used. Eight mice were included in each group. Four hours later, the mice were anesthetized by intraperitoneal injection of 80 μL of 10% chloral. After the mice lost their reflexes, they were placed supine on an operating table and secured with tacks around their limbs and mouth. After disinfecting with alcohol cotton, the abdominal and thoracic cavities were cut open to expose the heart. The right atrial appendage was cut open, and the injection needle was then inserted into the left ventricle. PBS was slowly perfused. When dark blood flowed out of the atrial appendage, the right atrium was successfully punctured. When the outflowing fluid became clear, the liver changed from dark red to grayish white, and the limbs and tail changed from light red to white, the perfusion was sufficient. After perfusion, the mouse was placed prone on the operating table, the forelimbs were fixed with nails, and after disinfecting with alcohol cotton, the head was fixed with forceps in the left hand. With the right hand, a transverse incision was made across the neck, cutting forward along the foramen magnum to the nasal cavity, bisecting the skull. The left and right skull bones were separated with forceps to expose the brain. The brain was removed with forceps and placed in a 1.5mL centrifuge tube. The mouse brain tissue was ground with a grinding rod on ice. Protein lysis buffer containing 1% protein inhibitor was added, mixed, and stored on ice. Centrifuge at 10,000 rpm for 5 minutes at 4°C. Pipette the supernatant into clean centrifuge tubes and store at -80°C. Dilute the brain tissue serum with PBS buffer, add reducing protein loading buffer, mix well, and incubate in a 100°C metal bath for 10 minutes. Then, perform Western blot analysis using the 6× His tag antibody and beta-Actin polyclonal antibody from step 2, respectively.

[0127] turn out( Figure 3 ), the specific bands shown by the samples injected with RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3 were darker, while the bands of the control group RVG-hNb07-EGFP were lighter. This shows that after the recombinant nanobody proteins RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3 were injected through the tail vein, the protein content entering the mouse brain tissue was higher than that of the recombinant nanobody proteins without blood-brain barrier penetrating peptides, and the brain penetration effect was better, indicating that the addition of T7 and PepH3 peptides can significantly enhance the recombinant nanobody proteins to cross the blood-brain barrier and enter the central nervous system.

[0128] 4. Neutralization titer determination of recombinant nanobody protein

[0129] BHK-21 cells (baby hamster kidney cells) (preserved by the Veterinary Microbiology Laboratory, College of Veterinary Medicine, South China Agricultural University) were thawed in advance and, after stabilization, plated onto 10 mm culture dishes and grown into monolayers for subsequent use. Two 96-well cell culture plates were prepared: one for sample testing, with 4 rows × 8 columns forming a sample area, and three sections per plate; the other for standard plates: the standard plate consisted of a standard serum (0.5 IU) control area (4 rows × 8 columns), a 1 IU (positive control) area (4 rows × 8 columns), a virus titer test area (4 rows × 6 columns), a negative control (PBS) area (4 rows × 1 column), and a cell control area (4 rows × 1 column). After clearly marking the culture dish with a marker, the prescribed amount of DMEM medium was added to each well (150 μL to the virus test area and 100 μL to the remaining wells).

[0130] Thaw one tube of 30 IU / mL human rabies immunoglobulin (BRP) standard serum (purchased from Shanghai Bio Biotech Co., Ltd.) at -80°C on ice. Pipette 4 μL into a centrifuge tube and add 240 μL of DMEM medium to dilute to a neutralizing titer of 0.5 IU / mL. Dilute the same method to obtain a 1 IU / mL human rabies immunoglobulin (BRP) standard serum as a positive control. 50 μL of each of the 0.5 IU / mL standard reference serum and the recombinant nanobody protein to be tested were added to the four wells in the first column of the standard serum control area and each sample reference area, respectively. After thoroughly mixing the contents of each well in the first column of each area (150 μL) with a multichannel pipette, 50 μL was transferred to each well in the second column. After changing the pipette tip, the contents of each well in the second column were thoroughly mixed (150 μL), and 50 μL was transferred to each well in the third column, and so on... to the eighth column (the sixth column for the standard serum reference area). After thoroughly mixing the contents of each well, 50 μL was aspirated and discarded. At this point, the contents of each well were all 100 μL, and the serum dilution factors in columns 1 to 8 were 3, 9, 27, 81, 243, 729... The number of repeats was 4 rows.

[0131] Take out rabies virus CVS-11 from -80℃, thaw on ice, and dilute it in DMEM medium in a sterilized plate to make the final titer of the virus 100TCID 50 / 50μL. After thorough mixing, use a multichannel pipette to add 50μL of virus diluent to each well in the sample test area, each well in the standard serum control area, and each well in the first column of the virus titer test area. Use a multichannel pipette to thoroughly mix the contents of the wells in the second column of the virus titer re-test area, then aspirate 50μL and transfer it to each well in the third column. After changing the pipette tip, thoroughly mix the contents of the wells in the third column (200μL), aspirate 50μL and transfer it to the wells in the fourth column, and so on... to the eighth column. After thoroughly mixing the contents of each well, aspirate 50μL and discard. At this point, the contents of each well are all 150μL. The virus dilution multiples in columns 1 to 6 are 1, 4, 16, 64, 256, and 1024, respectively, and the number of repetitions is 4 rows. After completing the above process, transfer to a 37°C incubator and incubate for 60 minutes.

[0132] BHK-21 cells grown into a monolayer were obtained, digested with trypsin, and suspended in DMEM medium containing 20% ​​serum. 50 μL (about 2×10 4 cells). Place in a 37°C, 5% CO2 incubator and culture for approximately 48 hours.

[0133] Remove the cell culture plate, discard the viral solution, and use a multichannel pipette to add 100 μL of pre-chilled 80% acetone solution to each well of the cell culture plate. Quickly discard the solution, then add 100 μL of pre-chilled 80% acetone solution. Continue fixing at -20°C for 30 minutes. Wash the plate three times with PBS for 1 minute each, vigorously discarding the final wash solution. Dilute FITC-labeled anti-rabies virus nucleoprotein fluorescent antibody (purchased from Fujirebio Diagnostics, USA) with PBS to approximately 0.004 mg / mL working solution of each antibody. After mixing thoroughly, add 50 μL to each well using a multichannel pipette and incubate overnight at 4°C.

[0134] Discard the fluorescent antibody solution and wash the plate three times with PBS for 1 minute each time. Drain the plate vigorously after the final wash. Observe under a fluorescence microscope and prepare recording paper according to the plate design. Any well with at least one fluorescent cell is marked as "+"; otherwise, it is marked as "-."

[0135] According to the recorded table, enter the two parameters of the virus titer retest in the corresponding position of the FAVN automatic calculation software on the computer: the maximum dilution factor of the entire column "+" and the number of "+"s that appear after the factor. The software automatically calculates the TCID of each 50μL of the virus working solution. 50 Enter two parameters for the standard reference serum and the test serum: the maximum dilution factor with a column of "-" and the number of "-"s following that factor. The software automatically calculates the neutralization titer of the test serum.

[0136] When the neutralization titer is lower than 0.5IU / mL, it means that the neutralizing antibodies in the protein are not sufficient to protect animals from a strong rabies attack; when the neutralization titer is greater than or equal to 0.5IU / mL, it means that the neutralizing antibodies in the protein can provide effective protection when animals are attacked by a strong rabies virus.

[0137] When the virus working solution is between 30 and 300 TCID 50 When the test result of TCID / 50μL is close to 1IU, and the BHK-21 cells and DMEM culture medium used are free of virus contamination, the neutralization titer of the tested serum can be considered valid, otherwise the test should be repeated. 50 The calculation formula is: lgTCID 50 =L+D(S-0.5), where L is the logarithm of the lowest serum dilution, D is the dilution factor, and S is the sum of the protection ratios.

[0138] To evaluate the effectiveness of recombinant nanobody proteins in neutralizing viruses, neutralization titers were determined using the FAVN assay. Four replicates were performed at each dilution, and the neutralization titers of the recombinant nanobody proteins were calculated using direct immunofluorescence (Table 2). The virus neutralization titer of RVG-hNb07-EGFP-T7 was slightly higher than that of RVG-hNb07-EGFP-PepH3 and RVG-hNb07-EGFP.

[0139] Table 2 Neutralization titer determination of recombinant nanobody protein

[0140]

[0141] 5. In vitro neutralization ability of recombinant nanobody protein against rabies virus CVS-11

[0142] Five-week-old SPF Kunming female mice were used and divided into 7 groups: PBS control group, RVG-hNb07-EGFP (high-dose group and low-dose group), RVG-hNb07-EGFP-T7 (high-dose group and low-dose group), and RVG-hNb07-EGFP-PepH3 (high-dose group and low-dose group), with 8 mice in each group. The PBS group was treated with 100 μL 10 6 FFU / mL of CVS-11 was mixed with 20 μL of sterilized PBS buffer. The high-dose group used 100 μL of 10 6 FFU / mL of CVS-11 was mixed with 8 IU of recombinant nanobody protein. The low-dose group used 100 μL of 10 6 FFU / mL of CVS-11 was mixed with 4 IU of recombinant Nanobody protein. The mixture was allowed to stand on ice for 30 minutes before intramuscular injection into mice. The mice were observed and recorded for disease progression daily.

[0143] The results show that ( Figure 4 ), all the mice in the PBS control group died, and among the experimental groups, except for one mouse in the low-dose RVG-hNb07-EGFP-PepH3 group that died on the 14th day of infection, the survival rate was 87.5%. The mice in the other experimental groups survived, gradually gained weight, were in good mental state, did not develop the disease, and had a survival rate of 100%, indicating that the three recombinant nanoantibodies have a good in vitro neutralizing effect on rabies virus CVS-11.

[0144] 6. Passive immunotherapy effect of infiltration injection of recombinant nanobody protein on challenged mice

[0145] Five-week-old SPF Kunming female mice were used and divided into 5 groups: PBS control group, RVG-hNb07-EGFP-T7 group, RVG-hNb07-EGFP-PepH3 group, RVG-hNb07-EGFP group, and human rabies immunoglobulin (Hualan Biotechnology Co., Ltd.) group, with 10 mice in each group. All mice were anesthetized by intraperitoneal injection of 80 μL of 10% trichloraldehyde. The back of the mouse was shaved with an electric shaver, and 4×4 small holes were pierced on the back of the mouse with a 10 mL needle to form a wound. The size and range of the wound of each mouse were kept consistent as much as possible. Then, 100 μL of 10 6 Apply CVS-11 at a concentration of 500 FFU / mL to the wound. Two hours later, the wounds of the experimental group mice were infiltrated with 8 IU of the protein. The control group received no infiltration injection. The mice were observed and their disease progression was recorded daily.

[0146] like Figure 5 As shown, all mice in the PBS challenge control group died, while the survival rate of the RVG-hNb07-EGFP-T7 group and the RVG-hNb07-EGFP-PepH3 group was 80%, and the mice died on the 14th day after the challenge, which was later than the 11th day in the control group. The number of mice in the RVG-hNb07-EGFP group died was relatively large, with a survival rate of only 20%. The human rabies immunoglobulin test group had the highest survival rate of 90%. The above results indicate that the recombinant nanobody protein has a certain effect on the treatment of challenged mice by infiltration injection, among which the treatment effect of the RVG-hNb07-EGFP-T7 group and the RVG-hNb07-EGFP-PepH3 group with blood-brain barrier penetrating peptide was significantly better than that of RVG-hNb07-EGFP.

[0147] 7. Therapeutic effect of tail vein injection of recombinant nanobodies on challenged mice

[0148] Five-week-old SPF Kunming female mice were used and divided into 4 groups: PBS control group, RVG-hNb07-EGFP-T7 group, RVG-hNb07-EGFP-PepH3 group, and RVG-hNb07-EGFP group, with 10 mice in each group. All mice were injected intramuscularly with 100 μL 10 6 FFU / mL of CVS-11 was injected via the tail vein with 8 IU of recombinant Nanobody protein on days 4 and 6 (our previous research has shown that the virus has entered the brain at this time (Zhang D, He F, Bi S, et al. Genome-Wide Transcriptional Profiling Reveals Two Distinct Outcomes in Central Nervous System Infections of Rabies Virus. Front Microbiol. 2016;7:751)). The control group was injected with PBS. The mice were observed and recorded for disease progression daily.

[0149] Depend on Figure 6 As can be seen, all mice in the PBS control group died. The RVG-hNb07-EGFP-T7 group experienced mouse deaths on days 15, 16, and 17 after challenge, with only five mice surviving, for a survival rate of 50%. The RVG-hNb07-EGFP-PepH3 group also experienced mouse deaths on days 15, 16, and 17, with only six mice surviving, for a survival rate of 60%. The RVG-hNb07-EGFP group experienced mouse deaths on days 13, 14, 15, 16, and 17, with only three mice surviving, for a survival rate of 30%. Compared with the control group, the onset of mouse death in all three experimental groups was delayed, and survival rates were improved to a certain extent, with the RVG-hNb07-EGFP-PepH3 group having the highest survival rate. These results indicate that all three recombinant nanobody proteins were effective in treating rabies-challenged mice, with RVG-hNb07-EGFP-PepH3 showing the best therapeutic effect and RVG-hNb07-EGFP showing the worst. These results suggest that the fusion of the blood-brain barrier-penetrating peptide T7 and PepH3 significantly enhances the nanobody's therapeutic efficacy against rabies.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A humanized rabies virus nanobody, characterized in that Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding the humanized rabies virus nanobody according to claim 1, characterized in that Its nucleotide sequence is shown as SEQ ID No. 2 or 3.

3. A recombinant nanobody protein, characterized in that The invention comprises the humanized rabies virus nanobody according to claim 1, and its C-terminus is connected to be fused with EGFP and a blood-brain barrier penetrating peptide.

4. The recombinant nanobody protein according to claim 3, characterized in that: The blood-brain barrier penetrating peptide is at least one of the blood-brain barrier penetrating peptide T7 and the blood-brain barrier penetrating peptide PepH3.

5. The recombinant nanobody protein according to claim 3 or 4, characterized in that: The recombinant nanobody protein is at least one of RVG-hNb07-EGFP-T7 and RVG-hNb07-EGFP-PepH3, wherein the amino acid sequence of RVG-hNb07-EGFP-T7 is shown in SEQ ID No.4, and the amino acid sequence of RVG-hNb07-EGFP-PepH3 is shown in SEQ ID No.

5.

6. The recombinant Nanobody protein according to claim 3 or 4, characterized in that: The nucleotide sequence of the gene encoding the recombinant nanobody protein is shown as SEQ ID No.6 or as SEQ ID No.

7.

7. Use of the humanized rabies virus nanobody according to claim 1 or the recombinant nanobody protein according to any one of claims 3 to 6 in the preparation of a product for preventing and treating rabies.

8. A recombinant vector comprising a gene encoding a humanized rabies virus nanobody or a gene encoding a recombinant nanobody protein; The nucleotide sequence of the gene encoding the humanized rabies virus nanobody is shown in SEQ ID No. 2 or 3; The nucleotide sequence of the gene encoding the recombinant nanobody protein is shown as SEQ ID No.6 or as SEQ ID No.

7.

9. A biomaterial expressing humanized rabies virus nanobody or recombinant nanobody protein, which is obtained by transferring the recombinant vector according to claim 8 into an expression system.

10. Use of the recombinant vector according to claim 8 or the biomaterial according to claim 9 in the preparation of a product for preventing and treating rabies.

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