CN116925213B

CN116925213B - Nanometer antibody for neutralizing botulinum toxin type A

Info

Publication number: CN116925213B
Application number: CN202311169052.7A
Authority: CN
Inventors: 胡乃静; 乔春霞; 冯健男; 彭峰浩; 罗龙龙; 王晶; 李新颖; 肖鹤; 陈国江; 刘成华; 沈倍奋
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2023-09-12
Filing date: 2023-09-12
Publication date: 2024-03-15
Anticipated expiration: 2043-09-12
Also published as: CN116925213A

Abstract

The invention discloses a nano antibody for neutralizing botulinum toxin type A, wherein the amino acid sequence of a CDR1 of the nano antibody is shown as SEQ ID NO.2, the amino acid sequence of the CDR2 is shown as SEQ ID NO.3, and the amino acid sequence of the CDR3 is GEF. Experiments prove that the nano-antibody has stronger affinity with the BoNT/A antigen, and the application of the nano-antibody can treat botulism caused by BoNT/A. Thus, the invention provides a new way for clinical diagnosis or treatment of botulinum toxin intoxication.

Description

Nanometer antibody for neutralizing botulinum toxin type A

Technical Field

The invention belongs to the field of biological medicine, and relates to a nano antibody for neutralizing botulinum toxin type A.

Background

Botulinum toxins, also known as botulinum neurotoxins, are one of the toxin proteins produced by the anaerobic gram-positive bacterium clostridium botulinum, the strongest known toxin, which can be classified into seven types (BoNT/a-BoNT/G) depending on its antigenicity, with the highest mortality of botulinum toxin type a poisoning. The half-mortalities (LD 50) of the intraperitoneal injections of Bonts were measured in mice to be 0.5-5 ng/kg, with an LD50 in humans of about 1 ng/kg. The latency period for botulinum toxin intoxication is typically hours to days, and the person with intoxication typically dies at 2-3 d. The poisoning may cause weakness, dizziness, blurred vision, paralysis of skeletal muscle, and respiratory failure in severe cases, resulting in death. In daily life, poisoning events caused by pollution of food to botulinum toxin and irregular injection of botulinum toxin in the beauty industry occur, so that the life and health of people are seriously threatened. Therefore, the method has great significance for the research of preventing and treating the botulism.

Currently, there are two main treatments for clinical botulinum toxin poisoning: specific horse serum antitoxin therapy and symptomatic therapy. The main strategy in preventing botulinum toxin is vaccination, and since the second battle, various vaccines against botulinum toxin, type AB bivalent toxoid and PBT toxoid have been developed in the United states for vaccination of high risk groups. However, toxoid vaccines still suffer from a number of drawbacks, and PBT has not been marketed by FDA approval. Therefore, the specific medicine for effectively preventing and treating the botulism is obtained, and has important significance for biosafety. The therapeutic antibody becomes the first choice and the focus of research of the team of BoNT/A specific antidote development because of high specificity, low toxic and side effects and good neutralization effect.

Nanobodies are the smallest active antigen binding proteins currently known, and a novel antibody consisting of only two heavy chains, namely heavy chain antibodies, was found in camel serum by the Hamers team in 1993, whereas the variable region fragment of heavy chain antibodies was nanobody. Compared with the conventional IgG antibody, the nano antibody has the advantages of stable structure, strong specificity, high yield and easy expression, and is widely focused as a research tool, diagnosis and treatment method. In addition, during antigen recognition, because nanobodies have long complementarity determining regions, loop ring structures are easily formed to bind to clefts and cavities in the spatial conformation of proteins, thereby recognizing hidden concave epitopes such as enzyme active sites and hidden viral epitopes. Nanobodies are therefore good candidates in the development of botulinum toxin therapeutic or diagnostic agents.

Disclosure of Invention

The invention discloses a nanometer antibody of botulinum toxin type A, which comprises CDR1, CDR2 and CDR3 of a heavy chain variable region shown in SEQ ID NO. 1.

Further, the amino acid sequence of the CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the CDR2 is shown as SEQ ID NO.3, and the amino acid sequence of the CDR3 is GEF.

In a specific embodiment of the invention, the nanobody further comprises a framework region; the heavy chain variable region has the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In an alternative embodiment, the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody, and a fusion nanobody.

Monovalent nanobody: the antigen-specific nanobody is obtained by screening specific antigen from a nanobody library, can maintain a strict monomer structure due to a large number of hydrophilic residues on the surface of the nanobody, and can be combined with the antigen with high specificity and high affinity only in a monomer form.

Multivalent nanobody: multivalent antibodies are polymers of monovalent antibodies that recognize the same epitope, with higher antigen affinity than the corresponding monovalent nanobody. Multispecific antibodies are polymers of monovalent antibodies that recognize different epitopes, can bind to different targets or different epitopes of the same target, and have higher antigen recognition capabilities than monovalent antibodies. The nanobody has a simple structure, only has one structural domain, and can be polymerized together through a short connecting sequence, so that the nanobody is converted into a multivalent and multispecific form.

Fusion nanobody: the nano antibody has strict monomer characteristics and small relative molecular mass, and can be easily combined with other structures (such as BSA, igG-Fc and the like) to form new fusion molecules, such as enzymes, antibacterial peptides or developing substances and the like for prolonging half-life of the nano antibody. In the novel fusion molecule, the nanobody is bound with the target antigen thereof in a directional manner, and the part fused with the nanobody can play a corresponding function. In clinicians, they want the drug to stay in the body long enough, however, nanobody blood clearance is fast, which is not beneficial for the drug it carries to act. Therefore, the nanobody VHH and the long-life molecule are fused together by the gene technology, so that the existence time of the nanobody in blood can be prolonged, namely the half life of the nanobody can be prolonged, and a better therapeutic effect can be achieved.

In a specific embodiment of the invention, the nanobody is a monovalent nanobody.

Further, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1.

The invention also discloses a nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the nano-antibody.

In an alternative embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 8.

Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

The recombinant vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce a DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.

One type of vector is a plasmid, i.e., a circular double stranded DNA molecule, into which a DNA fragment of interest can be ligated into a plasmid loop. Another type of vector is a viral vector, which can ligate and package the DNA fragment of interest into the viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter host cells, expression of the gene of interest can be performed.

The invention also discloses a host cell comprising the nucleic acid molecule as described above or the recombinant vector as described above.

The host cell is, for example, selected from a mammalian cell selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, vero, BHK cells, CV1 cells, heLa cells, MDCK cells, hep-2 cells, and Per6 cells. Among them, 293 series cells, per6 cells and CHO cells are common mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

The invention also discloses a product for detecting, enriching or purifying BoNT/A, which comprises the nanobody described above.

The product comprises a reagent, a kit or a chip.

Further, the agent is an antibody comprising the nanobody described previously.

In some embodiments, the antibody may be any of a full length antibody, a heavy chain antibody, a chimeric antibody, a multispecific antibody (e.g., bispecific antibody, trispecific antibody, tetraspecific antibody, etc.), a murine antibody, a humanized antibody, or an antigen binding fragment. The antigen binding fragment includes any one selected from the group consisting of F (ab ') 2, fab', fab, fv, and scFv of an antibody, so long as they exhibit the desired antigen binding activity.

The "chimeric antibody" according to the present invention is an antibody in which a variable region of a non-human antibody is fused with a constant region or a framework region of a human antibody, and can reduce an immune response induced by the non-human antibody.

The antigen binding fragments, i.e., functional fragments of antibodies, generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The antigen binding fragments described above may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by automated peptide synthesizers such as those sold for example as Applied BioSystems.

Further, the agent is a nanoparticle comprising the nanobody described above or an antibody described above thereof.

The nanoparticles are selected from any one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

Further, the kit comprises the aforementioned nanobody, the aforementioned antibody, or the aforementioned nanoparticle.

In one embodiment, the nanoparticle coated with a nanobody or antibody is used as a capture antibody, and the nanobody or antibody labeled with a detectable label is used as a detection antibody.

In another embodiment, when the kit comprises only: nanobodies or antibodies labeled with a detectable label; the nanobody or antibody at this time may be used to capture or detect the antibody.

Further, the detectable label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a colloid.

Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.

In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110 msin, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

In an alternative embodiment, the chemiluminescent reagent is selected from at least one of acridinium esters, luminol, lucigenin, crustacean fluorescein, ruthenium bipyridine, dioxane, rouge base, and peroxyoxalate.

Colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the product enriched or purified BoNT/a comprises a support and nanobodies or antibodies on the support.

In an alternative embodiment, the carrier is selected from the group consisting of magnetic beads, agarose gel microspheres, silica gel microspheres, or porous materials. For example, the activated beads are incubated with the antibody such that the beads are coated with the antibody.

Also disclosed is a method of detecting, enriching or purifying BoNT/a comprising contacting a sample comprising BoNT/a with a nanobody as described above or an antibody as described above.

The invention also discloses the application of the nano-antibody or the antibody in preparation of products for detecting, enriching or purifying BoNT/A.

The invention also discloses the application of the nano antibody or the antibody in preparation of products for diagnosing botulinum toxin type A caused by botulinum toxin.

The invention also discloses the application of the nano antibody or the antibody in preparing medicaments for treating botulinum toxin type A caused by botulinum toxin.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or auxiliary material.

By "pharmaceutically acceptable" is meant that the drug does not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a DNA fishing electrophoresis diagram of a nanobody library, wherein M represents a nucleic acid Marker, lane 1 is the variable region domain of all immunoglobulin heavy chains (VH, VHH), and lane 2 is the amplification of VHH;

FIG. 2 shows partial ELISA screening results;

FIG. 3 shows a diagram of SDS-PAGE result;

FIG. 4 shows a graph of SEC-HPLC analysis results, wherein A:218nm; b:280nm;

FIG. 5 shows a chart of Biacore analysis results;

FIG. 6 shows a graph of the results of in vivo challenge protection experiments in mice.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotidide Synthesis) (M.J. Gait, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors forMammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (CurrentProtocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example one acquisition of native camel VHH library

1. Material

Peripheral blood 100 mL of camel L9; the camel lymphocyte separation liquid is a product of the Sichuan of the ocean biology product technology Limited liability company; the RNA Easy Fast animal tissue/cell total RNA extraction kit and the FastKing cDNA first strand synthesis kit are products of Tiangen Biochemical technology Co., ltd; the PCR Primer Max enzyme is a product of TAKARA company; gene sequencing was done by su Jin Weizhi biotechnology limited; other relevant reagents are all commercially available.

2. Method and results

1. Acquisition of camel L9 peripheral blood

100 mL blood was collected from the jugular vein of a camel using EDTA-coated blood collection tubes, inverted twice to prevent clotting, and the blood samples were transferred to a laboratory.

2. Separation of lymphocytes

Using a 15 ml centrifuge tube, the blood was centrifuged at 600: 600 g for 25 min at room temperature according to the separation 5: 5 mL, blood 5: 5 mL. After centrifugation, the centrifuge tube was divided into four layers from top to bottom. The first layer is a plasma layer, the second layer is an annular milky camel lymphocyte layer, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer. The camel lymphocytes were carefully aspirated, added to a new 50 mL centrifuge tube, added with wash solution, centrifuged at 250 g for 15 min, and the supernatant discarded. The washing was repeated twice and the cells obtained by resuspension were camel lymphocytes.

3. Modulation of native VHH libraries

Taking the above lymphocyte 6×10 ⁷ Total RNA was obtained according to the procedure of RNA Easy Fast animal tissue/cell total RNA extraction kit, and cDNA library was obtained according to the procedure of FastKing cDNA first strand synthesis kit.

Amplifying the variable region domains of all immunoglobulin heavy chains (VH, VHH) from the cDNA using primers CALL001 and CALL002, and agarose gel electrophoresis, with a fragment of about 1000bp representing a conventional heavy chain antibody and a fragment of about 750bp representing a heavy chain antibody alone, i.e., a nanobody; the fragment at 750bp was recovered by gel to obtain a purified DNA fragment. Nested PCR was performed using primers VHH-Back and VHH-For, and finally a nanobody gene library was obtained (FIG. 1).

The primers were as follows:

CALL001：5’-GTCCTGGCTGCTCTTCTACAAGG-3’ （SEQ ID NO.9）；

CALL002：5’-GGTACGTGCTGTTGAACTGTTCC-3’ （SEQ ID NO.10）；

VHH-Back：5’- CCGTGGCCCAGGCGGCCGTCCTGGCTGCTCTTCTACA -3（SEQ ID NO.11）’；

VHH-For：5’-TGCTGGCCGGCCTGGCCTGAGGAGAYGGTGACCWGGGT -3’ （SEQ ID NO.12）

EXAMPLE two construction of native camel VHH phage library

1. Material

A gene library of natural nanobodies; the PCR Primer Max enzyme is a product of TAKARA company; t4 DNA ligase is NEB company product; gene sequencing was done by su Jin Weizhi biotechnology limited; other relevant reagents are all commercially available.

2. Method and results

1. Construction of nanobody library

a) The nano antibody library fragment is digested and connected with pComb3XTT vector:

10 mu L of nano antibody library PCR product

Shif I 1 μL

Cutstmart 5 μL

The total volume of the sterile water is 50 mu L

The above system was incubated at 37℃for 6h and recovered by a conventional DNA product purification kit.

b) The cleavage product described above was ligated to the pComb3XTT vector:

nanometer antibody library PCR product 1. Mu.L

5. Mu.L of pComb3XTT vector

T4 ligase 1. Mu.L

T4 Buffer 1 μL

The total volume of the sterile water is 10 mu L

The ligation product was electrotransformed into TG1 E.coli overnight at 16℃and spread on 2YT agar medium containing 100. Mu.g/mL ampicillin (final concentration) and 2% glucose. And meanwhile, carrying out gradient dilution to obtain the calculation reservoir capacity of the titer plate, and picking monoclonal colonies for sequencing. The obtained phage nanobody library is cultured overnight in a 2YT liquid medium containing 2% glucose of 100 mug/mL ampicillin (final concentration), and the phage nanobody library is obtained by a PEG precipitation method.

Constructing a nano antibody library onto a phage vector by using an enzyme digestion, enzyme linked and other molecular biological technology, and sequencing and identifying a colony of the phage nano antibody library to compare multiple sequences, and identifying the quality of the library; the phage library is amplified and PEG precipitated to obtain the phage nanometer antibody library for subsequent screening.

EXAMPLE phage Natural nanobody library screening of Tri-anti BoNT/A nanobodies

1. Material

Phage natural nano antibody library; boNT/a AHC antigen; PBS; tween-20; TG1; horseradish enzyme-labeled anti-M13 antibody; other reagents are commercially available products.

2. Method of

1. Panning

1.1 The BoNT/AHC antigen coated immune tubes were diluted with 10. Mu.g/mL sterile PBS, 500. Mu.L per tube, and coated overnight at 4℃with a total of 5. Mu.g antigen.

1.2 The antigen solution was discarded, 4% milk prepared with sterile PBST was added, and 1 h was blocked at room temperature. Simultaneously, 500. Mu.L of phage antibody library diluted with 4% milk was prepared 5X 10 ¹² cfu incubated at room temperature 1 h.

1.3 While blocking, 200. Mu.L of TG1 to 50 mL of 2YT liquid medium was inoculated and cultured until the OD600 was 0.8.

1.4 Milk was discarded, 500. Mu.L of blocked phage solution was added to the immune tube and incubated at room temperature for 1 h.

1.5 The PBST tube was washed 15 times and then 10 times with PBS.

1.6 Phage were eluted with 500. Mu.L of 0.1M HCL-Gly and eluted at room temperature for 10min (with a slight shaking in between for examination). 100. Mu.L of 1M Tris-HCl was added to adjust the pH of the eluate to pH 7.5.

1.7 Phage were eluted again with 500. Mu.L of 0.1M HCl-Gly and the secondary eluate was added to the mixed solution of the primary eluate and Tris for 10min at room temperature.

1.8 The phage obtained above was added in 500. Mu.L to TG 1in the 10 mL logarithmic growth phase, and cultured at 37℃for 0.5. 0.5h.

1.9 Centrifuge at 4000 rpm at 20℃for 15 min.

1.10 The supernatant was discarded, and all the pellet was plated on a 2YTAG (2YT+Amp 100. Mu.g/mL+2% glucose) plate (15 cm large plate) and incubated overnight at 37 ℃.

1.11 All TG1 strains were scraped from the plates and added to 50 mL of 2YTAG (2YT+Amp 100. Mu.g/mL+2% glucose) culture at 37℃and 220 rpm for 2.5 h to an OD of about 0.1.

1.12 strain growth reached the logarithmic phase, 10 mL strain was taken, 20-200 mu L M KO7 helper phage was added thereto, and the culture was allowed to stand at 37℃for 0.5h.

1.13 Centrifuge at 4000 rpm at 20℃for 15 min.

1.14 The supernatant was discarded, 50 mL of 2YTAK (2YT+Amp 100. Mu.g/mL+Kana 70. Mu.g/mL) medium was resuspended, and shake-cultured overnight at 28 ℃.

1.15 The phage were precipitated as described above for the next round of panning, for a total of 3 rounds of panning, and the enrichment and selection of monoclonal for ELISA validation.

2. ELISA method for analyzing monoclonal phage

2.1 Production of monoclonal phages in 96 deep well plates

2.1.1 40. Mu.L of TG1 was inoculated into 10 mL fresh 2YT medium and cultured at 37℃and 220 rpm for 3 h.

2.1.2 500. Mu.L of eluted phage was added to 10 mL of TG1 and incubated at 37℃for 0.5. 0.5h.

2.1.3 The bacterial liquid after infection was diluted by ten times (10 ^-1 ，10 ^-2 …10 ^-8 ) In 2YT medium, 50. Mu.L of each diluted sample was plated on 2YTAG (2YT+Amp 100. Mu.g/mL) solid medium at 37℃overnight.

2.1.4 mu.L of 2YTAG (2YT+Amp 100. Mu.g/mL) medium was added to each well of a 96-well deep plate, and each plate was incubated at 37℃and 220 rpm in 96 Kong Shenban with single colonies picked up and 4 h.

2.1.5 100 μl/well was aspirated and stored in 96-well plates for subsequent sequencing.

2.1.6 100. Mu.L of M13KO7 (1.2 mL M13KO7 helper phage+12 mL 2YT medium) was added to each well at 10-fold dilution, and incubated at 37℃for 0.5h.

2.1.7 Centrifugation is carried out for 15 min at 1800 rmp 4 ℃ in a 96-well deep plate.

2.1.8 The supernatant was discarded, and the plate was subjected to shaking at 28℃for 10 min.

2.1.9 1 mL fresh 2YTAK (2YT+Amp 100. Mu.g/mL+Kana 70. Mu.g/mL) medium was added to each well and incubated overnight at 28 ℃.

2.1.10 The plates were centrifuged at 1800 rmp 4℃for 15 min in 96 wells to obtain the supernatant of the monoclonal phage.

2.2 ELISA verification

2.2.1 The panels were coated with BoNT/a antigen, negative control and positive control, overnight at 4 ℃.

2.2.2 200. Mu.L of PBSM (4% skimmed milk in PBS) was added to each well and blocked at room temperature for 1 h.

2.2.3 After discarding the in-well solution, 100. Mu.L phage was added to each well and incubated at room temperature for 1 h.

2.2.4 Washing solution is prepared by 0.1% Tween-20 deionized water for 3 times.

2.2.5 mu.L of anti-M13/HRP antibody (1:5000 dilution) was added to each well and incubated for 45 min at room temperature.

2.2.6 Washing solution is prepared by 0.1% Tween-20 deionized water for 5 times.

2.2.7 mu.L TMB was added to each well for color development.

2.2.8 After addition of stop solution, the OD450 nm was measured by ELISA.

The results of a partial screening are shown in FIG. 2, where antibody number 1 binds best to the antigen, and antibody number 1 is selected for expression and designated HM.

The amino acid sequence of the HM antibody is shown as SEQ ID NO.1, the amino acid sequence of the CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the CDR2 is shown as SEQ ID NO.3, and the amino acid sequence of the CDR3 is GEF. The amino acid sequences of FR1-4 are shown as SEQ ID NO.4-7, respectively. The nucleotide sequence of the HM antibody is shown as SEQ ID NO. 8.

SEQ ID NO.1：EVQLVESGGGLVQPGGSLRLSCEASGFGSWFRFDENTVNWYRQPPGKSREFDELVARYPKSGIVTYLDSVKGRFTISRDNAKKMAFLQMNSLKPEDTAVYYCNVGEFWGQGTQVTVSS；

SEQ ID NO.2：

GFGSWFRFDENTVN

SEQ ID NO.3：

LVARYPKSGIVT

CDF3：GEF

SEQ ID NO.4：

EVQLVESGGGLVQPGGSLRLSCEAS

SEQ ID NO.5：

WYRQPPGKSREFDE

SEQ ID NO.6：

YLDSVKGRFTISRDNAKKMAFLQMNSLKPEDTAVYYCNV

SEQ ID NO.7：

WGQGTQVTVSS

SEQ ID NO.8：GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGAGGCAAGTGGGTTTGGAAGCTGGTTTAGGTTTGACGAGAACACCGTGAACTGGTATAGGCAGCCCCCTGGAAAGAGTAGGGAGTTTGACGAGCTGGTGGCAAGGTACCCTAAGAGCGGAATCGTGACCTACCTGGACTCCGTGAAGGGCAGGTTCACCATCTCTAGGGACAACGCAAAGAAGATGGCCTTCTTGCAGATGAACTCCTTGAAACCTGAGGACACAGCCGTGTACTATTGCAACGTGGGCGAATTTTGGGGACAGGGAACACAGGTGACCGTGAGCTCC

EXAMPLE expression and Property evaluation of the four anti-BoNT/A nanobody HM

1. Material

Eukaryotic expression vector pFRT-IgG1 is preserved by the room; primers were synthesized by su Jin Weizhi biotechnology limited; the transfection reagent is Invitrogen company product, sheep anti-human IgG, and horseradish enzyme labeled sheep anti-human IgG purchased from Thermo; the endonuclease is NEB company product; plasmid extraction kits were purchased from Tiangen Biochemical technologies Co., ltd; CHO cells purchased from ATCC; CD-1 mice were purchased from Vetong rituximab, and the other reagents were commercially available products.

2. Method of

1. Expression of antibodies

1.1 Construction of eukaryotic expression vectors for antibodies

pRT-IgG 1 is a highly efficient expression vector containing the human IgG1 antibody constant region gene. The nanobody gene cloning vector obtained in the second example was digested with the corresponding endonucleases (nanobody genes were digested with Nhe I and Xho I) and then sequentially ligated to the vector digested with the same endonucleases to obtain eukaryotic expression vectors. The specific implementation is as follows:

1.1.1 The HM gene of the obtained nano antibody is subjected to PCR to obtain a target fragment with an enzyme cleavage site;

1.1.2 Taking HM PCR productNhe I andXhoi digestion;

1.1.3 1 μg of pFR-IgG 1 vector was usedNhe I andXhoi digestion. Ligation of the resulting warp with DNA ligase T4Nhe I, IXhopFR-IgG 1 vector digested with I and use ofNhe I, IXhoI digested HM PCR product. The ligation product was transformed into TOP10 E.coli and spread on LB agar medium containing 100. Mu.g/mL ampicillin (final concentration). The obtained clone was cultured in LB liquid medium containing 100. Mu.g/mL ampicillin (final concentration), and the plasmid was extracted with a plasmid extraction kit (Tiangen Biochemical Co., ltd.). The extracted plasmid is subjected toNhe I andXho after digestion, 1% agarose gel electrophoresis analysis was performed to select a clone carrying the antibody HM gene.

As a result of the above procedure, a plasmid pFNT-IgG 1-HM carrying the HM gene of the anti-BoNT/A nanosource antibody was obtained.

1.2 Expression of nanobody HM

The eukaryotic expression vector is transiently transfected into CHO cells by a liposome-mediated method, the supernatant is harvested after 8 days, and the content of antibodies in the supernatant is detected by a double-sandwich ELISA method by using goat anti-human IgG and horseradish enzyme-labeled goat anti-human IgG. Experimental results show that the target protein in the supernatant can be recognized by goat anti-human IgG antibodies, contains constant regions of the human IgG antibodies and has the characteristics of the human IgG antibodies. The antibody expression level was 770.60mg/L. Antibodies were purified using a MabSelectSure affinity column. The purified antibodies were detected by SDS-PAGE, and the results are shown in FIG. 3, indicating successful antibody expression. Remarks: in the figure, M represents a protein Marker, R represents an HM reduction band, and N-R represents an HM non-reduction band.

2. Characterization of nanobody HM

2.1 SEC-HPLC analysis

2.1.1 step

The mobile phase is 50 mmol/mL sodium phosphate and 200 mmol/mL sodium chloride, the flow rate is 1 mL/min, the sample injection volume is 7 mu L, the detection wavelength is 218nm and 280nm, and the chromatogram is integrated and related data are calculated by using a Labsolutions system after acquisition.

2.1.2 results

The results are shown in FIG. 4, where the HM main peak retention time was about 7.9min, the peak area was 99%, indicating that the HM purity was not less than 99%, and the statistics are shown in Table 1.

TABLE 1 Peak Chart statistical Chart

2.2 Biacore detection of nanobody affinity

2.2.1 step

a) Starting up

Biacore T200 was turned on according to the instruction manual.

b) Sample dilution

10XHbS-EP+ (pH 7.4) was diluted 10-fold with deionized water to prepare 200mL. The antibody HM was diluted to 0.3. Mu.g/mL with running buffer solution and the sample dilution concentration was 10nM,5nM,2.5 nM,1.25 nM,0.625 nM,0.3125nM.

c) Sample detection and analysis

And running a kinetic/Affinity program to measure and analyze the result.

2.2.2 results

The results are shown in FIG. 5 and Table 2.

TABLE 2 nanobody affinity results

3. Functional detection of nanobody HM

Research on therapeutic function of nano antibody HM by using in-vivo toxicity attack protection experiment of mice

3.1 step

18-20g of CD-1 female mice were selected, 2 times LD50 botulinum toxin was set in Control group, antibodies of different concentrations were mixed with 2 times LD50 botulinum toxin, incubated for 30min at room temperature, and then 200. Mu.L was intraperitoneally injected/administered. Mice were observed 2-6 times daily for 3-5 days, mice were observed for status and mortality was recorded.

3.2 results

The results are shown in FIG. 6, where after 60h observation, the antibody HM was able to protect mice from 100% survival at a minimum concentration of 0.25mg/kg, and Control group survived only 20%, indicating that HM was able to effectively protect mice from botulinum toxin type A poisoning.

The above embodiments are only described for the understanding of the method of the present invention and the core idea thereof, and several improvements and modifications may be made to the present invention, which also fall within the scope of the present invention as defined in the appended claims.

Claims

1. A nano antibody for neutralizing botulinum toxin type A is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1.

2. A nucleic acid molecule encoding the nanobody of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid molecule of claim 2 or the recombinant vector of claim 3.

5. A product for detecting BoNT/a, comprising the nanobody of claim 1.

6. Use of the nanobody of claim 1 for the preparation of a product for detecting, enriching or purifying BoNT/a.

7. Use of the nanobody of claim 1 for the preparation of a product for diagnosing botulinum toxin type a-induced botulinum toxin poisoning.

8. Use of the nanobody of claim 1 for the preparation of a medicament for the treatment of botulinum toxin type a-induced botulinum toxin poisoning.