CN120865397A - C-type botulinum toxin receptor binding domain nano antibody and application thereof - Google Patents
C-type botulinum toxin receptor binding domain nano antibody and application thereofInfo
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Abstract
The invention provides a nano antibody of an anti-C-type botulinum toxin receptor binding domain and application thereof. The invention takes a C-type botulinum toxin receptor binding domain as an antibody, immunizes alpaca, extracts RNA from monocytes thereof, and screens and obtains the nanometer antibody of the C-type botulinum toxin receptor binding domain by utilizing phage display library technology. The nano antibody provided by the invention can be specifically combined with the C-type botulinum toxin receptor binding domain, has high affinity, can effectively inhibit the specific combination of the C-type botulinum toxin and a target cell membrane, and can neutralize the C-type botulinum toxin to play a protective role. The nano antibody provided by the invention is expected to be applied to preparation of C-type botulinum toxin poisoning antidote. Meanwhile, the nano antibody provided by the invention can be used as a capturing antibody and a detecting antibody to be applied to qualitative and quantitative analysis and detection of C-type botulinum toxin.
Description
Technical Field
The invention belongs to the fields of cell immunology and genetic engineering, and in particular relates to a C-type botulinum toxin nano antibody which can be used as an effective antidote.
Background
Botulinum toxin is a neurotoxin secreted by clostridium botulinum, which is highly lethal to humans and animals and is considered to be the most toxic substance among known biological and chemical toxins. Botulinum toxins are classified into 8 serotypes A-G and X depending on their antigenicity, each formed by disulfide linkage of a Light Chain (LC) of about 50kDa with a Heavy Chain (HC) of 100 kDa. Heavy chains consist of an N-terminal translocation domain (HCN) and a C-terminal receptor domain (HCR). The toxin binds to the presynaptic membrane specific receptor via HCR to form endocytic vesicles into the cell. The lower pH within the endocytic vesicle breaks the disulfide bond linking the HC to the LC, while HCN binds to the endocytic vesicle membrane, promoting release of the LC into the cytoplasm. LC is a protease domain that specifically cleaves SNARE proteins after entry into the cytoplasm, thereby blocking synaptic vesicle fusion with presynaptic membrane, inhibiting neurotransmitter release, and causing toxic reactions.
The C-type botulinum toxin (Botulinum toxin type C, boNT/C) is one of main causes of animal botulism, the whole body muscle is weak after animal food BoNT/C poisoning, serious people can have dyspnea, typical clinical botulism manifestations such as general paralysis, the morbidity is urgent, the mortality rate is high, the characteristics of mass-sending are often presented, and once the animal is poisoned, no effective detoxification medicine exists, and huge economic loss is caused for animal husbandry. If measures are not taken timely, other carnivores can be caused to have botulinum poisoning symptoms and even die due to the fact that the animal carcasses are poisoned by eating, so that clostridium botulinum is hidden and spread in the nature, and the clostridium botulinum can possibly cause greater range and serious botulinum poisoning, even threatens the food safety of human beings, and becomes a social public safety problem.
The duration of action of different types of botulinum toxins is found to be different, wherein the duration of action of BoNT/C is comparable to BoNT/a currently in wide use in the medical and medical fields, and the neuromuscular injury is less. BoNT/C is expected to become a good substitute for BoNT/A, and solves the problem of BoNT/A drug resistance. Furthermore, since BoNT/C is the only botulinum toxin protein known to date that cleaves both SNARE substrate proteins (SNAP-25 and syntaxin) at the same time, this suggests that BoNT/C is suitable for patients that do not respond naturally to BoNT/A on the one hand. On the other hand, studies have shown syntaxin over-expression in a variety of high secretion diseases, suggesting the potential for BoNT/C to be used in the preparation of drugs for such diseases. In conclusion, boNT/C has great potential application prospects in the medical field, and similarly, botulinum toxin-based substances are highly toxic substances, thereby bringing high risk of iatrogenic poisoning probability.
To date, boNT/C poisoning has not been effective antidote, and animal botulinum toxin poisoning control can only be prevented by feed control and injection of botulinum toxin type C vaccine. However, the toxin vaccine has complex process, weak immune effect and short duration of immune response induced and needs multiple times of inoculation. Horse-derived botulinum antitoxin is the only specific therapeutic preparation after BoNT/C poisoning at present, but the horse antitoxin can only act within a very short time after poisoning, and due to the difference of species, the allergic response rate is high, and serological diseases are easy to occur. In conclusion, boNT/C is the main cause of animal botulism, and the great potential of BoNT/C is widely applied to clinic, so that the risk of human iatrogenic toxin poisoning is increased. Thus, there is an urgent need in the marketplace for effective BoNT/C botulism antidotes.
Binding of botulinum toxin to receptors on the presynaptic membrane is a key step in its action into the cell. Effective inhibitors are developed aiming at the binding sites of the botulinum toxin and the receptor, and the effects of blocking the internalization of the botulinum toxin into neurons can be achieved, so that the flaccid paralysis caused by the toxin can be effectively reduced. The nanobody is derived from heavy chain antibodies produced by camels, sharks and the like, and compared with common IgG antibodies, the nanobody only contains heavy chain variable region fragments, can independently and stably exist in vitro, can independently act on antigens, and has complete antigen recognition capability. Nanobodies are 4nm long, 2.5nm in diameter, and have a molecular weight of only about 15kDa, the smallest protein currently known to have antigen binding activity. The CDR3 of the nano antibody is composed of 13-18 amino acids, is longer than that of the CDR3 of the traditional antibody, and can recognize hidden deeper antigen epitopes which are difficult to recognize by the traditional antibody. Because of unique structure of nano antibody, the nano antibody has the advantages of low production cost, weak immunogenicity, good tissue permeability and the like, and has become a hot spot for research in a plurality of medical fields.
Disclosure of Invention
Aiming at the problem that the existing C-type botulinum toxin lacks an effective antidote, the invention provides a nano antibody capable of specifically binding with a BoNT/C receptor binding domain, wherein the nano antibody is obtained by screening by utilizing a phage display library technology, can effectively inhibit the specific binding between BoNT/C and a target cell membrane thereof, can neutralize the C-type botulinum toxin, and plays a protective role in a mouse BoNT/C lethal experiment and a mouse gastrocnemius paralysis experiment. The nano antibody provided by the invention can be used as a capturing antibody and a detecting antibody in the qualitative and quantitative analysis and detection processes of C-type botulinum toxin. Meanwhile, the nano antibody provided by the invention is expected to be applied to preparation of C-type botulinum toxin antidote.
The method specifically comprises the following steps:
In a first aspect, the present invention provides a C-type botulinum toxin receptor binding domain nanobody having the sequence of the complementarity determining region CDRs of any one of (1) to (18) below:
(1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2 and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, CDR3 shown in SEQ ID NO. 6;
(3) CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, CDR3 shown in SEQ ID NO. 9;
(4) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, CDR3 shown in SEQ ID NO. 12;
(1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2 and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, CDR3 shown in SEQ ID NO. 6;
(3) CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, CDR3 shown in SEQ ID NO. 9;
(4) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, CDR3 shown in SEQ ID NO. 12;
(5) CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, CDR3 shown in SEQ ID NO. 15;
(6) CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, CDR3 shown in SEQ ID NO. 18;
(7) CDR1 shown in SEQ ID NO.19, CDR2 shown in SEQ ID NO.20, CDR3 shown in SEQ ID NO. 21;
(8) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, CDR3 shown in SEQ ID NO. 24;
(9) CDR1 shown in SEQ ID NO.25, CDR2 shown in SEQ ID NO.26, CDR3 shown in SEQ ID NO. 27;
(10) CDR1 shown in SEQ ID NO.28, CDR2 shown in SEQ ID NO.29, CDR3 shown in SEQ ID NO. 30;
(11) CDR1 shown in SEQ ID NO.31, CDR2 shown in SEQ ID NO.32, CDR3 shown in SEQ ID NO. 33;
(12) CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO.35, CDR3 shown in SEQ ID NO. 9;
(13) CDR1 shown in SEQ ID NO.36, CDR2 shown in SEQ ID NO.8, CDR3 shown in SEQ ID NO. 37;
(14) CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.8, CDR3 shown in SEQ ID NO. 9;
(15) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, CDR3 shown in SEQ ID NO. 41;
(16) CDR1 shown in SEQ ID NO.39, CDR2 shown in SEQ ID NO.40, CDR3 shown in SEQ ID NO. 42;
(17) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.43, CDR3 shown in SEQ ID NO. 44;
(18) CDR1 shown in SEQ ID NO.45, CDR2 shown in SEQ ID NO.43, and CDR3 shown in SEQ ID NO. 44.
Preferably, the sequence of the nanobody is shown in any one of SEQ ID NO. 46-65.
In a second aspect, the present invention provides a nucleic acid molecule encoding a C-type botulinum toxin receptor binding domain nanobody of the first aspect above.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect above.
In a fourth aspect, the present invention provides a host cell comprising a nucleic acid molecule as described in the second aspect above or a recombinant vector as described in the third aspect above.
In a fifth aspect, the present invention provides the use of a C-type botulinum toxin receptor binding domain nanobody as described in the first aspect above for the detection of C-type botulinum toxin for non-disease diagnostic purposes.
In a sixth aspect, the present invention provides the use of a C-type botulinum toxin receptor binding domain nanobody as described in the first aspect above for the preparation of a product for detecting C-type botulinum toxin.
In a seventh aspect, the present invention provides the use of the C-type botulinum toxin receptor binding domain nanobody of the first aspect above in the manufacture of a medicament for the treatment of botulinum toxin intoxication.
Preferably, the botulinum toxin intoxication is caused by botulinum toxin type C.
In an eighth aspect, the present invention provides a reagent for qualitatively and quantitatively detecting botulinum toxin type C, the reagent comprising the C botulinum toxin receptor binding domain nanobody of the first aspect.
In a ninth aspect, the present invention provides a medicament for the treatment of botulinum toxin type C toxin mediated toxicity, the active ingredient of the medicament comprising a C botulinum toxin receptor binding domain nanobody of the first aspect described above.
In an eighth aspect, the present invention provides a reagent for qualitatively and quantitatively detecting botulinum toxin type C, the reagent comprising the nanobody of the first aspect.
In a ninth aspect, the present invention provides a medicament for the treatment of botulinum toxin intoxication, the active ingredient of which comprises the nanobody of the first aspect above.
The invention has the beneficial effects that firstly, the phage display library technology is utilized to screen and obtain the nano antibody capable of being specifically combined with the BoNT/C receptor binding domain, secondly, the nano antibody can effectively inhibit the specific combination of BoNT/C and target cell membranes thereof, can neutralize C-type botulinum toxin, plays a protective role in a mouse BoNT/C lethal experiment and a mouse gastrocnemius paralysis experiment, can be used as a capturing antibody and a detecting antibody to be applied to qualitative and quantitative analysis and detection of C-type botulinum toxin, and meanwhile, the nano antibody is hopeful to be applied to preparation of C-type botulinum toxin antidote.
Drawings
The results of the induced expression, purification and desalting of recombinant protein His6-BCHCR in FIG. 1, the induced purification of recombinant protein His6-BCHCR in lane 1, the protein molecular weight standard in lane 2, the uninduced induction of protein by 3:0.5mM IPTG, the centrifugation of the bacterial suspension in 4, the centrifugation of the supernatant of the bacterial suspension in 5, the permeation of the column chromatography by 6:Ni, the washing by 7:binding buffer, the gradient elution by 8-11:250mM imidazole, the segmented collection of 1-4;B, the desalting of recombinant protein His6-BCHCR in lane 1, the protein molecular weight standard in lane 9;3-5:20mM PB,100mM NaCl in FIG. 2:A, and the segmented collection of 1-3.
FIG. 2 shows the results of induced expression and purification of recombinant protein GST-BCHCR, lane 1, protein molecular weight standard, 2, no induction, 3, 0.5mM IPTG induction, 4, precipitation by centrifugation of the bacterial suspension, 5, supernatant by centrifugation of the bacterial suspension, 6, ni column chromatography penetration, 7, binding buffer washing, 8-12, and 1-5 of reduced glutathione elution and sectional collection.
FIG. 3 Phage-ELISA positive clone selection results, A92 total clones (numbers 1-92) and B92 total clones (numbers 93-184).
FIG. 4 BoNT/C HCR nanobody purification results, lane 1 protein molecular weight standard, lanes 2-13 nanobody A2, A3, B7, C6, D1, D12, E2, E5, E11, E12, F9, G5, respectively.
FIG. 5 shows results of targeting binding of BCHCR nanobody and cell membrane, A shows results of laser confocal detection of binding activity of nanobody to BoNT/HCR and target cell, 12 nanobody and BoNT/HCR incubated fluorescence imaging results, and BSA protein is set as control group, scale size is 5 μm, B shows statistical result of average fluorescence intensity of cell, statistical data shows mean value.+ -. Standard deviation (n=10), and P <0.01, P <0.001, ns P >0.05 compared with BoNT/HCR control group.
FIG. 6 shows the Western blot detection result of toxin uptake by the nanometer antibody A2 blocking primary culture neurons, the Western blot detection result of toxin uptake by the A2 blocking primary culture neurons, and the semi-quantitative analysis result of the inhibitory effect of A2 on the activity of BoNT/C cleavage SNAP-25, wherein the gray value is taken as a quantitative index, the ordinate is the proportion of uncleaved SNAP-25 to total SNAP-25 protein, and the data are the average value + -standard deviation (n=3). P <0.001 compared to BoNT/C group.
FIG. 7 shows the result of neutralizing and protecting BoNT/C with nanobody A2, wherein A2 is optimal, B0.1, 1,10 μg nanobody A2 is incubated with 125pg BoNT/C to neutralize BoNT/C in a dose-dependent manner to protect, and C nanobody A2 is incubated with different doses of BoNT/C at a ratio of 1:10 6 to significantly neutralize BoNT/C to protect.
FIG. 8 shows the results of the rescuing effect of nanobody on BoNT/C poisoning, A. Intraperitoneal injection of BoNT/C, caudal injection of A2, B. Intramuscular injection of BoNT/C, in situ point intramuscular injection of A2, C. Intramuscular injection of BoNT/C, caudal injection of A2.
Detailed Description
The invention will be further elucidated with reference to the following detailed description of embodiments, but the examples described hereinafter are merely illustrative of the technical solution of the invention and are not limiting thereof. Unless specifically stated otherwise, all technical means used in the following examples are conventional means known to those skilled in the art, all bioinformatics software and products used are commercially available, experimental procedures and methods are conventional methods known in the art, all materials sources, trade names and components listed as necessary are indicated at the first occurrence, and all reagents used thereafter are the same as indicated at the first occurrence unless otherwise stated.
In addition, it should be noted that the combination and application of the sites provided by the present application are accomplished by the inventor of the present application through hard creative work and optimization work.
EXAMPLE 1 purification of expression of His6-BCHCR and GST-BCHCR proteins
1. Experimental materials
Kanamycin sulfate (available from Bio-engineering Co., ltd., A506636-0025), ampicillin sodium (available from Shimadzu chemical Co., ltd., A610028-0025), IPTG (available from Bio-engineering Co., ltd., A600168-0025), imidazole (available from Bio-engineering Co., ltd., A600277-0100), tryptone (available from Simer Feishi technology Co., LP 0042B), yeast extract (available from Simer Feishi technology Co., LP 0021), sodium chloride, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, methanol, glacial acetic acid, absolute ethanol, SDS (available from Shimadzu chemical Co., ltd., A600028-0100), glycine (available from Shimadzu biological engineering Co., ltd., A610235-0005), tris (available from Bio-engineering Co., ltd., A610195-0005), coomassie blue R-250 (available from Siemen technology Co., A610037), bromophenol (available from Sijingsu Co., ltd., beijing technology Co., ltd., E.20-90), beijing technology (available from Beijing technology Co., ltd., beijing-20-90), acrylamide (available from Beijing technology Co., ltd., beijing 4-28-90); pGEX-4T (Feng Hui Biotechnology Co., ltd, YH 004), ni SepharoseTM 6: 6 Fast Flow (GE HEALTHCARE, 17-5318-01), sephadex G-25 Resin chromatography medium (Biotechnology Co., ltd., C510060-0026), GST Sepharose 4FF (GST-Tag) (Biotechnology Co., ltd., C600031), glutathione reduced GSH (available from Biotechnology Co., ltd., A600229).
2. Solution preparation
LB liquid culture medium, weighing tryptone 5.0g, yeast extract 2.5g and NaCl 5.0g, dissolving in 400ml deionized water, fixing volume to 500ml, sterilizing with 121 deg.C high pressure steam for 20min, and preserving at room temperature.
Weighing 7.5g of agar powder in 500ml of unsterilized LB liquid medium, sterilizing for 20min at 121 ℃ by high-pressure steam, adding a proper amount of antibiotics according to the experiment requirement when the temperature of the medium is cooled to 50-60 ℃, uniformly mixing, paving a flat plate, and preserving at 4 ℃.
1M IPTG 11.915g was weighed and dissolved in 40ml deionized water, then the volume was set to 50ml, filtered and sterilized by a 0.22 μm filter head, and frozen at-20 ℃.
100 Mu g/ml kanamycin sulfate, 5.0g of kanamycin sulfate is weighed and dissolved in 40ml of deionized water, the volume is fixed to 50ml, a 0.22 mu m filter head is used for filtration and sterilization, and the frozen storage is carried out at-20 ℃.
10 XPBS solution, weighing NaCl 80g,KCl 1g、Na2HPO4·12H2O 35.8g、KH2PO4·3H2O 2.7g, dissolving in 800ml deionized water, and then fixing the volume to 1l, and preserving at room temperature.
PBST, 100mL of 10 XPBS solution was measured and mixed with 900mL of deionized water, 250. Mu.L of Tween-20 was added, and the mixture was stored at room temperature.
5 XSDS-PAGE electrophoresis buffer, weighing 15.1g of Tris, 94g of glycine and 5g of SDS, dissolving in 800ml of deionized water, and then fixing the volume to 1l, and preserving at room temperature.
5 XSDS-PAGE loading buffer:1M Tris-HCl (pH 6.8) 2.5ml, SDS1g, bromophenol blue 0.05g, glycerol 2.5ml, dissolved in 8ml deionized water, and frozen at-20℃to a volume of 10 ml.
Coomassie brilliant blue R-250 staining solution, namely, weighing coomassie brilliant blue R-2501.0g, adding 250ml of isopropanol and 100ml of glacial acetic acid, and then fixing the volume to 1l, and preserving at room temperature.
The coomassie brilliant blue staining and decolorizing solution is prepared by weighing 100ml of glacial acetic acid, 50ml of absolute ethyl alcohol, and preserving at room temperature after the volume is fixed to 1 l.
3. Experimental procedure
3.1 His6-BCHCR protein induced expression
The BoNT/C HCR gene sequence was synthesized from Jin Weizhi total gene (SEQ ID NO. 66) and cloned into the NdeI and EcoRI sites of pET28a+ after codon optimization, with reference to the UniProt database (P18640, BXC _CBCP, range 866-1291) amino acid information, transformed E.coli BL21 (DE 3), frozen into glycerol bacteria. Frozen glycerol bacteria were taken from a-80℃refrigerator and 5. Mu.l inoculated into 30ml LB medium containing 80. Mu.g/ml kanamycin sulfate, and shaken at 37℃and 200rpm for 14h as a seed. 2ml of strain is inoculated in 500ml of LB culture medium containing 80 mug/ml kanamycin sulfate, shaking is carried out at 37 ℃ and 200rpm for 4-5 hours until OD 600 reaches 0.8-1.2, 1ml of strain liquid is taken as an uninduced sample, 0.5mM IPTG is added, protein expression is induced at 20 ℃ for 16 hours, and 1ml of strain liquid is taken as an induced sample. After the induction and the expression are finished, 8000g of bacterial precipitate is collected by centrifugation for 3min and is frozen and stored in a refrigerator at the temperature of minus 20 ℃. And simultaneously centrifuging the uninduced sample and the induced sample to prepare a sample of SDS-PAGE, and detecting the expression condition of the protein.
3.2 His6-BCHCR protein purification
Adding binding buffer (binding buffer: 20mM PB,pH7.4,300mM NaCl,50mM imidazole) with a gram weight of 30 times of the volume of the bacterial sediment collected by centrifugation into the bacterial sediment, carrying out homogenizing and crushing under a high-pressure homogenizer at 850bar until the bacterial sediment is clear, centrifuging at 1000rpm for 10min to obtain supernatant, loading the supernatant onto a Ni 2+ SepharoseTM Fast Flow affinity chromatography column pre-equilibrated by the binding buffer at a flow rate of 1.5ml/min, washing the chromatography column with the binding buffer at a flow rate of 1.5ml/min until an effluent OD 280 value reaches a base line, eluting a target protein with an elution buffer (20mM PB,pH7.4,300mM NaCl,250mM imidazole) at a flow rate of 1.5ml/min, and collecting the effluent. The collected effluent was then passed through a Sephadex G-25Resin desalting column, imidazole removed, stored in 20mMPB,pH7.4,100mM NaCl,5% glycerol buffer and quick frozen in liquid nitrogen and stored at-80 ℃.
3.3 Inducible expression of GST-BCHCR
The BoNT/C HCR gene sequence refers to the amino acid information of UniProt database (P18640, BXC _CBCP, range 866-1291), is synthesized by Jin Weizhi total gene (shown as SEQ ID NO. 66) after codon optimization and cloned into BamHI and EcoRI sites of pGEX 4T-1, and E.coli DH5 alpha is transformed and frozen into glycerol bacteria. The induction of the expression of the target protein was performed according to the method 3.1. 5ml of strain is inoculated in 500ml of LB culture medium containing 100 mug/ml of ampicillin sodium, shaking is carried out at 37 ℃ and 200rpm for 3 hours until OD 600 reaches 0.4-0.6, 1ml of strain liquid is taken as an uninduced sample, 0.5mM IPTG is added, after protein expression is induced for 16 hours at 20 ℃, 1ml of strain liquid is taken as an induced sample, and SDS-PAGE electrophoresis detection is adopted.
3.4 Purification of GST-BCHCR
The target protein was obtained by disrupting the bacteria with reference to 3.2, loading the supernatant onto a GST affinity chromatography column pre-equilibrated with binding buffer (20 mM PB, pH8.0, 150mM NaCl) at a flow rate of 1.5ml/min, and eluting with elution buffer (20 mM Tris-HCl, pH8.0,20mM GSH). SDS-PAGE electrophoresis was used.
4. Experimental results
And (3) passing the bacterial suspension supernatant through a nickel column affinity chromatography column by utilizing the principle that imidazole and His tag proteins are combined with nickel ions in a competitive manner. SDS-PAGE results of purified proteins are shown in FIG. 1A, and by comparing the uninduced bacterial liquid sample of lane 2 with the bacterial liquid sample of lane 3 induced by 0.5mM IPTG, a band of the target protein of induced expression can be observed at about 45kDa, and the recombinant protein His 6-BoNT/HCR with higher concentration and purity can be successfully obtained in the eluent. The target sample obtained is desalted by a desalting column, and the result is shown as B in FIG. 1, and the target protein desalting and liquid changing treatment can also be carried out by a dialysis method.
The His 6-BoNT/HCR obtained in this example can be used as an antigen for immunization of alpaca, and the GST-tagged BoNT/HCR is purchased in this example in consideration of the desire for subsequent screening of antigens to filter nanobodies against purified tags. The target protein was purified using GSH affinity chromatography columns, and the results are shown in fig. 2. Collecting eluted target protein GST-BoNT/HCR, sub-packaging, and freezing at-80 ℃ for standby.
EXAMPLE 2C construction of botulinum toxin receptor binding domain nanobody phage libraries
1. Material
Total RNA Extraction Reagent (Shanghai next, san Biotech Co., ltd., 10606ES 60); AEBSF (Beijing Soy Bao Tec., ltd., IA 0110); (NEB, R3140V); (NEB Co., R3162V), pMES.sup.4 phagemid vector (Feng Hui Biotechnology Co., ltd., ZT 642), TG1 E.coli cell line (Rui Chu Biotechnology Co., ltd., R09003), VCSM13 helper phage (apak Biotechnology Co., ltd., P007), sanPrep column PCR product purification kit (Biotechnology Co., ltd., B518141-0100), STRIPWELLTM MICROPLATE (COSTAR Co., 42592).
2. Experimental method
2.1 Extraction of RNA from Peripheral Blood Mononuclear Cells (PBMC) obtained by alpaca immunization
His6-BCHCR with the concentration of 1mg/ml and the purity of more than 90 percent is taken as an antigen to be sent to Shanxinami biotechnology development Co-Ltd for external cooperated immunization of alpaca. After 4 times of immunization, the quality detection of the alpaca antiserum is qualified, when the titer reaches 10 5, the blood of the alpaca is collected to separate peripheral blood mononuclear cells, and total RNA is extracted:
(1) Taking out TRIzol solution containing 1.2X10 8 PBMC, adding 11.0ml Total RNA Extraction Reagent to ensure that each milliliter of Total RNA Extraction Reagent contains 1×10 7 cells, repeatedly blowing and beating the mixed cells with a pipetting gun, sub-packaging into 1ml 12 tubes, and standing at room temperature for 5min to ensure complete dissociation of nucleoprotein;
(2) 200 μl of chloroform was added to each tube, and the mixture was left standing at room temperature for 3min after shaking vigorously for about 15 hours;
(3) Centrifuging at 4deg.C for 15min at 12000g, wherein three layers of materials are added in the centrifuge tube, wherein the upper layer is transparent water phase, RNA exists in water phase, the middle layer is white precipitated DNA, the lower layer is red organic phase, and the upper layer has a capacity of about 50-60% of the total amount of Total RNA ExtractionReagent;
(4) Carefully sucking the water phase into a new centrifuge tube, adding 500 μl of isopropanol, mixing, and standing at room temperature for 10min;
(5) Centrifuging at 4 ℃ for 15min at 12000g, wherein colloidal precipitation appears at the bottom of the test tube;
(6) Isopropanol was carefully discarded, 1ml of 75% ethanol in DEPC water was added to the tube, and vortexed to thoroughly wash the precipitate;
(7) Centrifuging at 4 ℃ for 5min at 7500g, and carefully discarding the supernatant;
(8) Repeating steps (6) - (7) to ensure that the precipitate is thoroughly washed;
(9) Air-drying at room temperature for 10min, adding 40 μl DEPC water to dissolve RNA, taking a small amount after complete dissolution, and performing RNA quality detection, and the rest is used for reverse transcription experiment.
2.2 Reverse transcription of RNA into cDNA
Reverse transcription into cDNA by reverse transcriptase using the extracted RNA as template, the preparation of reverse transcription reaction system (20. Mu.l system) is shown in Table 1, and the reverse transcription reaction procedure is shown in Table 2.
TABLE 1 reverse transcription reaction system
TABLE 2 reverse transcription reaction procedure
| Temperature (° C) | Time (min) |
| 25 | 5 |
| 55 | 15 |
| 85 | 5 |
2.3CDNA obtaining nanobody fragments by nested PCR
CDNA obtained by reverse transcription is subjected to nested PCR to specifically amplify nano antibody fragments. The primers required for the first-step amplification of the nested PCR are shown in Table 3, the PCR system is shown in Table 4, the PCR program is shown in Table 5, the target fragment of 700bp is obtained by amplification, the target strip is subjected to gel recovery according to the specification of a gel recovery kit, the recovered product is used as a template for the second-step nested PCR, the primers required for the second-step amplification of the nested PCR are shown in Table 6, the PCR system is shown in Table 7, the PCR program is shown in Table 8, and the gel recovery is further carried out on the nano antibody fragment of about 450bp obtained by amplification.
TABLE 3 nested PCR first-step amplification primers
| Primer(s) | Sequence(s) |
| CALL001 Primer | GTCCTGGCTGCTCTTCTACAAGG(SEQ ID NO.67) |
| CALL002 Primer | GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.68) |
TABLE 4 nested PCR first-step reaction System
TABLE 5 nested PCR first-step reaction procedure
TABLE 6 nested PCR second-step amplification primers
| Primer(s) | Sequence(s) |
| VHH-Back | GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO.69) |
| VHH-For | CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO.70) |
TABLE 7 nested PCR second-step reaction System
TABLE 8 nest PCR second step reaction procedure
2.4 Construction of phage nanobody display library
The 450bp nanobody fragment amplified by nested PCR was subjected to PstI and BstEII double digestion with pMES phagemid vector, the double digestion system being as shown in Table 9.
Table 9 double enzyme digestion System
After cleavage at 37℃for 1h, cleavage at 4℃overnight, the cleavage product was gel recovered and ligated in a molar ratio of pMES to VHH of 1:3, the ligation system being as shown in Table 10.
Table 10 connection system
| Component (A) | Volume of |
| pMES4 | 15μl |
| VHH | 15μl |
| 10×Buffer | 5μl |
| T4 DNA Ligase | 5μl |
| H2O | 10μl |
Ligation was performed overnight at 20℃by means of SanPrep column PCR product purification kit and eluted with 30. Mu.l sterile water to give ligation products, which were subjected to 20 electrotransformation using TGI competence under GeminniX2 electrometer, 1cm electrocuvette, voltage 1.8KV, 25. Mu.FD and 200 ohm. Immediately after the electrotransformation, 1ml of SOC culture solution was added, shaking at 37 ℃ and 200rpm for 1 hour, diluting 1000 times, then coating LB agar plates (containing 100 mug/ml ampicillin sodium) as a quality control plate for calculating the reservoir capacity, and coating the residual bacterial liquid on LB agar plates (containing 100 mug/ml ampicillin sodium) as reservoir establishment requirements, and culturing at 37 ℃ overnight. The number of the colonies of the quality control board is 590 clones in the next day, and the calculated library capacity is 1.18 multiplied by 10 7, so that the requirements of library establishment are met. Following the washing of colonies on agar plates, glycerol was added at a final concentration of 20% and frozen at-80 ℃. And randomly selecting 15 monoclonal antibodies to perform bacterial liquid PCR to verify the insertion rate of the nano antibody fragments, and the result shows that the insertion rate of the invention reaches 100%. Bacterial liquid PCR verification primers are shown in Table 11, PCR system is shown in Table 12, and PCR procedure is shown in Table 13.
TABLE 11 PCR verification primers for bacterial liquid
| Primer(s) | Sequence(s) |
| MP57 | TTATGCTTCCGGCTCGTATG(SEQ ID NO.71) |
| GIII | CCACAGACAGCCCTCATAG(SEQ ID NO.72) |
TABLE 12 bacterial liquid PCR verification system
TABLE 13 bacterial liquid PCR reaction procedure
3. Experimental results
To obtain nanobody gene sequences from PBMC, the present invention uses TRIzol reagent to extract total RNA from 1.2X10 7 cells and is dissolved in 40. Mu.l of sterile, enzyme-free water. The detection result of nucleic acid electrophoresis shows that three rRNA bands of 28S,18S and 5S are clearly visible, and the detection result accords with theory, so that the total RNA is complete and is not degraded. Meanwhile, the ratio of A260/A280 of the high-quality RNA is 1.8-2.1, the concentration of the RNA solution is 1,100ng/μl is measured by a nucleic acid analyzer, and the ratio of A260/A280 is 1.95, which shows that the total RNA has better purity and meets the further experimental requirements.
In order to amplify the nanobody gene sequence from the extracted total RNA, the total RNA is reverse transcribed into cDNA, and the nanobody gene fragment is amplified from the cDNA by nested PCR, thereby improving the specificity of the PCR reaction. PCR amplification is carried out by adopting different amounts of cDNA, and a 700bp target fragment containing a nano antibody gene sequence is obtained through a first round of amplification reaction. Furthermore, heavy chain fragment bands of conventional antibodies at 1,000bp are also visible. The invention uses the 1 mul cDNA as a template, 8 groups of PCR products are repeated in parallel, each group of PCR products is respectively subjected to gel recovery, the bands at 700bp positions are separated and recovered, and the concentration of the recovered products is about 20 ng/. Mu.l. Then about 20ng of recovered DNA fragment is used as a second round of amplification template, and the result of nucleic acid electrophoresis shows that the nano antibody fragment band appears at about 450bp, the band is clear, the brightness is high, and the concentration of the DNA fragment obtained by gel recovery is 26.508 ng/. Mu.l. The invention adopts nest PCR to carry out two rounds of amplification, successfully obtains the nanometer antibody gene sequence from the reverse transcription cDNA, has proper concentration, and can be used for subsequent experiments.
PMES4 is a phagemid vector, which has the functions of a filamentous phage and a plasmid vector, and is commonly used in phage display technology. In order to construct pMES-nano antibody recombinant plasmid, the invention utilizes two restriction endonucleases PstI and BstEII to respectively enzyme-cut pMES vector and nano antibody gene fragment amplified by nested PCR, so that the vector is completely linearized. The result of nucleic acid electrophoresis shows that pMES carrier has only one clean band after enzyme cutting, and the linear plasmid has slower migration rate than circular plasmid, high band position and same adhesive end as nanometer antibody gene fragment. Further, DNA having the same cohesive end was ligated with T4DNA ligase in a ratio of fragment to vector molar ratio of 1:3, and recombinant plasmid was obtained by purification of PCR product, and the concentration was found to be 14.91 ng/. Mu.l. In summary, pMES vectors and nanobody gene fragments are subjected to enzyme digestion and connection, so that recombinant plasmids are successfully constructed, and the recombinant plasmids can be used for constructing phage display libraries.
Coli TG1 is a commonly used host bacterium in phage display technology that contains an F' factor, allows phage infection and repackaging, and can suppress the amber stop codon, allowing continued expression of genes on the plasmid downstream of the stop codon, such as PIII genes required for phage display. Phage display libraries were constructed by electrotransformation pMES of the 4-nanobody recombinant plasmid into TG1 competence. In order to improve the efficiency of library construction, the electrotransformation efficiency reaches to 1.706 X10 10 cfu/. Mu.g TG1 competence, and in order to ensure the sequence diversity in the library, a plurality of groups of parallel experimental groups are arranged in the nested PCR process to reduce the preference in amplification, and meanwhile, the insertion rate of the nano antibody sequences in the library reaches 100%, the influence of empty carriers is reduced, and the library capacity is more than 10 7 colony numbers.
EXAMPLE 3C amplification, enrichment and screening of botulinum toxin receptor binding domain nanobody phage libraries
1. Experimental materials
PEG6000 (Shanghai assist Saint Biotech Co., ltd., P8250), bovine serum albumin V (Beijing Soy Bao Tec., A8020), pancreatin, AEBSF (Beijing Soy Bao Tec., IA 0110), serine inhibitors (Beijing Soy Bao Tec., IA 0110), RIPA lysate (Beijing Soy Bao Tec., R0010), PMSF protease inhibitors (Beijing Soy Bao Tec., P0100).
2. Solution preparation
2 XYT liquid culture medium, weighing tryptone 8.0g, yeast extract 5.0g, sodium chloride 2.5g, dissolving in 480ml deionized water, constant volume 500ml, sterilizing with 121 deg.C high pressure steam for 20min, and storing at room temperature.
2 XYT solid culture medium, weighing 7.5g of agar powder in 500ml of 2 XYT liquid culture medium, sterilizing with 121 deg.C high pressure steam for 20min, cooling to 50-60 deg.C, adding appropriate amount of antibiotic according to experiment requirement, mixing, and spreading to obtain flat plate.
20% (W/v) PEG6,000/2.5M NaCl solution weighing PEG6,000 200g, stirring and dissolving 146g NaCl in 800ml deionized water, then metering to 1L, sterilizing with 121 deg.C high pressure steam for 20min, and preserving at room temperature.
3. Experimental method 3.1 phage amplification
100 Μl phage pools were inoculated in 50ml of 2 XYT medium (containing 2% glucose with 100 μg/ml sodium ampicillin sodium) and incubated at 37℃at 200rpm to an OD600 of about 0.5. 20ml were placed in a new centrifuge tube, 50. Mu.l of 4X 10 10 p.f.u helper phage was added, left to stand at 37℃for 30min, after sufficient infection the pellet was collected by centrifugation, resuspended in2 XYT (containing 25. Mu.g/ml kanamycin and 100. Mu.g/ml sodium ampicillin sodium) medium, and incubated at 37℃at 200rpm overnight.
3.2 Phage enrichment
Collecting the supernatant of the overnight culture bacterial liquid at 5000g and 4 ℃ after centrifugation for 15min, adding 1/5 volume of precooled PEG6000, ice-bath for 30min,3200g and 4 ℃ after centrifugation for 10min, adding 1ml of precooled PBS, blowing and resuspension, 20000g and 4 ℃ after centrifugation for 1min, adding 250 μl of PEG6000, reversing and mixing uniformly, ice-bath for 10min,4 ℃ and 20000g after centrifugation for 15min, adding 1ml of PBS for resuspension, 4 ℃ and 20000g after centrifugation for 1min, and collecting the supernatant to obtain phage concentrate.
3.3 Phage titer assay
Fresh colonies of TG1 cells on plates were picked up and inoculated into 30ml of LB medium at 37℃and 200rpm to an OD600 of about 0.5-0.6 and placed on ice for use. Mu.l phage concentrate was diluted gradient from 10 -1 to 10 -16 with PBS and 10. Mu.l each of each dilution was transferred to 200. Mu.l EP tube containing 90. Mu.l TG1 cells, mixed well, incubated in a 37℃metal bath for 15min, and 5. Mu.l each of the infected TG1 cells were dropped onto LB (containing 100. Mu.g/ml ampicillin sodium with 2% glucose) plates while uninfected 5. Mu.l TG1 cells were set as controls and incubated overnight at 37 ℃.
3.4 Phage selection
Mu.l of BCHCR protein coated ELISA plate at 6. Mu.g/ml was used, and irrelevant protein BSA was set as control well, coated overnight at 4 ℃. PBST was washed 3 times, blocked with 250. Mu.l 5% BSA per well, and shaken for 2h at 700 rpm. PBST was washed 5 times, 100. Mu.l of the incubated phage (50. Mu.l phage concentrate was mixed with 10. Mu.l 5% BSA, 400. Mu.l PBS and shaken at 700rpm for 0.5 h), 700rpm, 2h, PBST was washed 15 times, 100. Mu.l 2.5mg/ml pancreatin was added to each well, and samples from control and experimental wells were transferred to the prepared tubes and 5. Mu. lAEBSF (protease inhibitor) was added. The titer was determined after 10. Mu.l of samples from the experimental and control groups were diluted to 10 -8 in a gradient (method such as 3.3), while 50. Mu.l of each sample from the experimental group was added to 3ml of TG1 cells having an OD 600 value of 0.5, and allowed to stand at 37℃for 30min. The culture broth was made up to 10ml with LB and 10. Mu.l of ampicillin sodium and 2% glucose were added at a final concentration of 100. Mu.g/ml. Culturing at 37 deg.C and 200rpm overnight, adding 20% glycerol, and storing at-80 deg.C as strain obtained by first round enrichment screening.
The strains obtained by the first round of screening are repeatedly amplified, enriched and screened, the second round of phage screening strains are obtained by the screening process, and the like, the third round of phage screening and the fourth round of phage screening are completed, the titer of the experimental group is 100 times different from that of the control group after the fourth round of phage screening is finished, the specific phage is effectively enriched, and screening of phage-ELISA clones can be carried out.
4. Experimental results
To enrich phage from phage display libraries for specific binding to the target protein BoNT/HCR, the efficiency of screening is improved. In the invention, GST-BoNT/HCR protein is coated on a high-adsorption ELISA plate solid phase, through the phage screening process of four rounds of amplification-adsorption-elution, the concentrated liquid drop degree and the phage titer after screening of each round of phage are recorded, and meanwhile, a BSA coated control group is arranged to detect the condition of nonspecifically adsorbed phage. The results showed that with increasing rounds of screening, the differences in phage titers between the experimental and control groups increased gradually, indicating that specifically bound phage were effectively enriched during the screening process. Wherein in the fourth screening, the phage titer is 3×10 13 pfu/ml when enriching, the phage titer of the control group is 1.4×10 8 pfu/ml after eluting, and the phage titer of the experimental group is as high as 1.4×10 10 pfu/ml. The phage titers of the experimental group were significantly higher than the control group, 100-fold different, indicating that phage binding to the target protein were highly enriched through four rounds of panning (table 14).
TABLE 14 phage enrichment degree in four-pass screening
EXAMPLE 4 phage identification of ELISA-Positive clones
1. Experimental materials
High-efficiency RIPA tissue/cell quick lysate (Beijing Soy Bao technology Co., ltd., R0010), TMB single-component color development solution (Beijing Soy Bao technology Co., ltd., PR 1200), anti-His6 Tag Rabbit pAb (Wuhansai Weir Biotechnology Co., ltd., GB 111251), HRP X Goat Anti Rabbit IgG (H+L) (Ai Baisen Biotechnology Co., ASG 032N).
2. Experimental method 2.1 preparation of the supernatant of the Nami body
Mu.l of bacterial liquid was extracted from the fourth phage panning library, diluted to 10 -4 and 10 -5 with LB, plated on LB medium containing 100. Mu.g/ml ampicillin sodium and 2% glucose, and cultured overnight at 37 ℃. 200 individual clones were picked from the plates and incubated overnight at 37℃in a plurality of 96 wells (100. Mu.l 2 XYT, 100. Mu.g/ml sodium ampicillin, 2% glucose, 10% glycerol), 10. Mu.l of the bacterial liquid was added to 1ml 2 XYT (100. Mu.g/ml sodium ampicillin, 2% glucose) from each well, and after shaking at 37℃for 6 hours, 1. Mu.l 1M IPTG,37℃and 200rpm were added to each tube. 3200g, centrifuging at normal temperature for 10min, discarding supernatant, adding 250 μl of RIPA lysate into each tube, shaking, mixing, and shaking for 1 hr. Centrifuging at 4 ℃ and 10000rpm for 5min, and sucking 200 μl of supernatant to obtain nano-body supernatant.
2.2 Screening of Phage-ELISA Positive clones
100 Μl of 0.5 μg/ml GST-BCHCR protein coated ELISA plate was used, while irrelevant protein BSA was set as control wells and coated overnight at 4 ℃. PBST was washed 3 times, blocked by adding 250. Mu.l of 5% BSA (in PBST, w/v) per well, and shaken at 700rpm for 2h. PBST was washed 5 times, 50 μl buffer (5% bsa: pbst=1:20) was added to each well, each well of the experimental well was sequentially added with one 50 μl nanobody, while 4 control wells were added with 50 μl RIPA lysate, 700rpm, and shaken for 1h. PBST was washed 5 times, anti-6×his6 Tag rabit pAb antibody was diluted 2000-fold with buffer (5% bsa: pbst=1:20), 100 μl/well, 700rpm, and shaken for 1h. PBST was washed 5 times, hrp× Goat Anti Rabbit IgG (h+l) secondary antibody was diluted 10000-fold with buffer (5% bsa: pbst=1:20), 100 μl/well, 700rpm, and shaken for 1H. PBST was washed 10 times, TMB color development solution was added thereto, 100. Mu.l/well, and left to stand in a dark place for 30 minutes. The chromogenic reaction was stopped by adding 2M H 2SO4, 50. Mu.l/well and absorbance was measured at 450 nm.
2.3 Sequencing
And sequentially carrying out ELISA screening on 200 clones to obtain 44 monoclonal antibodies with higher OD 450 values, and carrying out nucleic acid sequencing on the clones by using an external assistant Jin Weizhi to obtain 20 nano antibody protein sequences.
3. Experimental results
In order to rapidly screen high affinity nanobody monoclonal from the sub-pool of the fourth round of phage panning, the present invention compares the affinities of the 184 nanobodies picked using ELISA methods based on specific binding of antigen-antibody. As shown in fig. 3, the OD 450 of the negative control group was 0.1062, and the OD 450 of most of the test groups met the requirement when the OD 450 of the negative control group was 2.1 times or more of the OD 450 of the negative control group. However, to select higher affinity nanobodies, gene sequencing was performed with selection of 49 monoclonal antibodies with OD 450 values exceeding 1.
The invention sequences 49 nanobodies altogether, 20 nanobodies with unique amino acid sequences are identified from which are named A2, A3, A10, A11, B1, B2, B5, B7, B8, C6, C12, D1, D12, E2, E5, E11, E12, F9, G5 and G11 respectively, and the FR regions are highly homologous. The specific sequences are respectively as follows:
A2：LQESGGGLVQAGGSLRLSCAASGSTFRIDVMGWYRQAPGKRRELVAVLGSGGRTDYADSVKGRFSILGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPEHEYWGQGTQVT(SEQ ID NO.46 Shown in SEQ ID NO. 1), CDR1: GSTFRIDVM (shown in SEQ ID NO. 1), CDR2: VLGSGGRTDYA (shown in SEQ ID NO. 2), CDR3: NAEEPEHEY (shown in SEQ ID NO. 3);
A3：LQESGGGLVQAGGSLRLACAASGSTFGIDVMGWYRQAPGKQRELVAVIGSGGRTDYGDSVKGRFSISGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPEYEYWGQGTQVT(SEQ ID NO.47 Shown in SEQ ID NO. 4), CDR1: GSTFGIDVM, CDR2: VIGSGGRTDYG (shown in SEQ ID NO. 5), CDR3: NAEEPEYEY (shown in SEQ ID NO. 6);
A10：LQESGGGLVQAGGSLRLSCAASGTTFNIDVMGWYRQAPGKQRDMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT(SEQ ID NO.48 Shown in SEQ ID NO. 7), CDR1: GTTFNIDVM, CDR2: VMNTGGRTDYA (shown in SEQ ID NO. 8), CDR3: NADGPSYDY (shown in SEQ ID NO. 9);
A11：LQESGGGLVQAGGSLRLSCAASEDTFSITMGWYRQAPGKQRELVATINIQNNTNYAESLKGRVTINRGNAKNTVYLQISSLKPEDTAVYYCYQRTMVSTYWGQGTQVT(SEQ ID NO.49 Shown in SEQ ID NO. 10), CDR1: EDTFSITM, CDR2: TINIQNNTNYA (shown in SEQ ID NO. 11), CDR3: YQRTMVSTY (shown in SEQ ID NO. 12);
B1：LQESGGGLVQPGGSLGLSCAASGSIYSINAMGWYRQAPGKQRELVATISGPERTSWTNYANSVKGRFTISRDYGRYTVYLQMNSLNPEDTAVYYCYADVVVSDPHGDGDYWGQGTQVT(SEQ ID NO.50 Shown in SEQ ID NO. 13), CDR1: GSIYSINAM, CDR2: TISGPERTSWTNYA (shown in SEQ ID NO. 14), CDR3: YADVVVSDPHGDGDY (shown in SEQ ID NO. 15);
B2：LQESGGGLVQPGGSLRLSCAASGRIFSINAMGWYRQAPGNQREFVAGISRGGNIVYADSVQGRSTISIDNAKNTMYLQMNSLKPEDTAVYWCNAGGWTDSGRYVMGNYWGQGTQVT(SEQ ID NO.51 Represented by SEQ ID No. 16), CDR1: GRIFSINAM, CDR2: GISRGGNIVYA (represented by SEQ ID No. 17), CDR3: NAGGWTDSGRYVMGNY (represented by SEQ ID No. 18);
B5：LQESGGGLVQAGGSLRLSCAASGSTLIIDVMGWYRQAPGKEREMVAVMNTGGRTDFADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGPTYDYWGQGTQVT(SEQ ID NO.52 Shown in SEQ ID NO. 19), CDR1: GSTLIIDVM, CDR2: VMNTGGRTDFA (shown in SEQ ID NO. 20), CDR3: NADGPTYDY (shown in SEQ ID NO. 21);
B7：LQESGGGLVQAGGSLRLSCAASGTTFNIDVMGWYRQAPGKQRDMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNNLKPEDTAVYYCNADGPSYDYWGQGTQVT(SEQ ID NO.53 Shown in SEQ ID NO. 7), CDR1: GTTFNIDVM, CDR2: VMNTGGRTDYA (shown in SEQ ID NO. 8), CDR3: NADGPSYDY (shown in SEQ ID NO. 9);
B8：LQESGGGLVQAGGSLRLSCAASGSTSSRNPMGWYRQAPGKQRELVATISTQGTITNYADSVKGRFTISRDNTQNTVYLQMSSLKPEDTAVYYCNAINPPSGSWGQGTQVT(SEQ ID NO.54 Represented by SEQ ID No. 22), CDR1: GSTSSRNPM, CDR2: TISTQGTITNYA (represented by SEQ ID No. 23), CDR3: NAINPPSGS (represented by SEQ ID No. 24);
C6：LQESGGGLVQAGGSLRLSCAASGDTLSISRMGWYRQAPGKLRELVAQIMMGGSAIYGDSVKGRFTISKDSANNIVYLQMNSLKPEDTAVYYCNARGAWSHQNYWGQGTQVT(SEQ ID NO.55 Shown in SEQ ID NO. 25), CDR1: GDTLSISRM, CDR2: QIMMGGSAIYG (shown in SEQ ID NO. 26), CDR3: NARGAWSHQNY (shown in SEQ ID NO. 27);
C12：LQESGGGLVQAGGSLTLSCAASGFAFSNAPMGWYRQAPGKRRELVATVSSLGGTTNYADSVKGRFTISRDNAKNTVYMQMNNLKPEDTAVYYCNTINGYLVGRNYWGQGTQVT(SEQ ID NO.56 Shown in SEQ ID No. 28), CDR1: GFAFSNAPM (shown in SEQ ID No. 28), CDR2: TVSSLGGTTNYA (shown in SEQ ID No. 29), CDR3: NTINGYLVGRNY (shown in SEQ ID No. 30);
D1：LQESGGGLVQAGGSLRLSCAASGITFSIDVLGWYRQAPGKQRELVAVLNSGGTTDYADSVKGGFTISTDNANNTVYLQMNSLKPEDAAVYYCNAHGPSYDYWGQGTQVT(SEQ ID NO.57 Represented by SEQ ID NO. 31), CDR1: GITFSIDVL, CDR2: VLNSGGTTDYA (represented by SEQ ID NO. 32), CDR3: NAHGPSYDY (represented by SEQ ID NO. 33);
D12：LQESGGGLVQAGGSLRLSCTASGIVDTIDTMGWYRQAPGKQREMVAVRSISGNTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT(SEQ ID NO.58 Shown in SEQ ID NO. 34), CDR1: GIVDTIDTM, CDR2: VRSISGNTDYA (shown in SEQ ID NO. 35), CDR3: NADGPSYDY (shown in SEQ ID NO. 9);
E2：LQESGGGLVQAGGSLRLSCAASGITFDIDVMGWYRQAPGKQREMVAVMNTGGRTDYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNADGLSYDYWGQGTQVT(SEQ ID NO.59 Represented by SEQ ID NO. 36), CDR1: GITFDIDVM, CDR2: VMNTGGRTDYA (represented by SEQ ID NO. 8), CDR3: NADGLSYDY (represented by SEQ ID NO. 37);
E5：LQESGGGLVQAGGSLRLSCTASGSTFSIDVMGWYRQAPGKQRELVAVMNTGGRTDYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNADGPSYDYWGQGTQVT(SEQ ID NO.60 Represented by SEQ ID NO. 38), CDR1: GSTFSIDVM, CDR2: VMNTGGRTDYA, represented by SEQ ID NO.8, CDR3: NADGPSYDY, represented by SEQ ID NO. 9;
E11：LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRELVAVISSSGNTDFADFVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETPTSEYWGQGTQVT(SEQ ID NO.61 Shown in SEQ ID NO. 39), CDR1: GTTLGIDVM, CDR2: VISSSGNTDFA (shown in SEQ ID NO. 40), CDR3: NAETPTSEY (shown in SEQ ID NO. 41);
E12：LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRDLVAVISSSGNTDFADSVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETVTSEYWGQGTQVT(SEQ ID NO.62 Shown in SEQ ID NO. 39), CDR1: GTTLGIDVM, CDR2: VISSSGNTDFA (shown in SEQ ID NO. 40), CDR3: NAETVTSEY (shown in SEQ ID NO. 42);
F9：LQESGGGLVQPGGSLRLSCAASGSTFRIDVMGWYRQAPGKQREMVAVIGSGGRTDYADSVKGRFSISGDNAKNTVYLQMNSLKPEDTAVYYCNAEEPESEYWGQGTQVT(SEQ ID NO.63 Shown in SEQ ID NO. 1), CDR1: GSTFRIDVM, CDR2: VIGSGGRTDYA (shown in SEQ ID NO. 43), CDR3: NAEEPESEY (shown in SEQ ID NO. 44);
G5：LQESGGGLVQAGGSLRLSCAASGSIFRIDVMGWYRQAPGKQREMVAVIGSGGRTDYADSVKGRFSISGDNAKNIVYLQMNSLKPEDTAVYYCNAEEPESEYWGQGTQVT(SEQ ID NO.64 Shown in SEQ ID NO. 45), CDR1: GSIFRIDVM, CDR2: VIGSGGRTDYA (shown in SEQ ID NO. 43), CDR3: NAEEPESEY (shown in SEQ ID NO. 44);
G11：LQESGGGLVQAGGSLRLSCAASGTTLGIDVMGWYRQAPGKQRELVAVISSSGNTDFADSVKGRFTIDGDFAKNTVHLQMNSLKPEDTAVYYCNAETPTSEYWGQGTQVT(SEQ ID NO.65 Shown in SEQ ID No. 39), CDR1: GTTLGIDVM, CDR2: VISSSGNTDFA (shown in SEQ ID No. 40) and CDR3: NAETPTSEY (shown in SEQ ID No. 41).
EXAMPLE 5 expression purification of nanobodies
1. Experimental materials
As in example 1.
2. Experimental method
2.1 Induction of expression of nanobodies
From the 20 kinds of protein sequence different nanometer antibody bacterial liquid obtained after the sending and testing, respectively taking 5 mu l to inoculate in 30ml LB medium containing 100 mu g/ml ampicillin sodium, shaking at 37 ℃ and 200rpm for 14h as bacterial strain. Then, 5ml of each strain is inoculated into 500ml of LB culture medium containing 100 mug/ml ampicillin sodium, shaking is carried out at 37 ℃ and 200rpm for 3 hours until OD 600 reaches 0.4-0.8, 1ml of bacterial liquid is taken as an uninduced sample, 0.5mM IPTG is added, and 1ml of bacterial liquid is taken as an induced sample after protein expression is induced for 16 hours at 20 ℃. After the induction and the expression are finished, 8000g of bacterial precipitate is collected by centrifugation for 3min and is frozen and stored in a refrigerator at the temperature of minus 20 ℃. And simultaneously centrifuging the uninduced sample and the induced sample to prepare a sample of SDS-PAGE, and detecting the expression condition of the protein.
2.2 Purification of nanobodies
Adding binding buffer (20mM PB,pH 7.4,20mM NaCl,100mM imidazole) with the gram weight of 20 times of the volume into the centrifugally collected bacterial precipitate, carrying out homogenizing and crushing under a high-pressure homogenizer at 850bar until bacterial is clear, centrifuging at 1000rpm for 10min to obtain supernatant, loading the supernatant onto a Ni 2+ SepharoseTM 6 Fast Flow affinity chromatography column pre-equilibrated by the binding buffer at the flow rate of 1.5ml/min, washing the chromatography column until the effluent OD 280 value reaches a base line by the binding buffer at the flow rate of 1.5ml/min, eluting the target protein by an elution buffer (20mM PB,pH 7.4,20mM NaCl,500mM imidazole) at the flow rate of 1.5ml/min, collecting the effluent, and analyzing the purified nano antibody by SDS-PAGE.
3. Experimental results
The invention adopts nickel column chromatography to purify nanobody protein, the purification result is shown in figure 4, 12 nanobody protein solutions which are A2, A3, B7, C6, D1, D12, E2, E5, E11, E12, F9 and G5 are obtained by purifying the nanobody protein with 20 unique sequences, and the rest 8 can not be effectively induced to be expressed, and the positions of the bands are slightly different due to different sequences but are all about 15 kDa.
EXAMPLE 6 nanobody inhibits BCHCR specific binding to target cell membrane
1. Experimental materials
Neuro-2a (CL 0243, feng Hui organism), sephadex G25 medium (C510060, shanghai Co., ltd.), SF-680 fluorescent dye (1035, beijing Fubaike Biotechnology Co., ltd.).
2. Experimental method
2.1 SF-680 fluorescent dye marks BCHCR protein:
1) BCHCR protein treatment the solution of BCHCR protein was exchanged for 0.1M NaHCO 3 using a 10kDa ultrafiltration tube.
2) Dye dissolution-dye solid powder was dissolved to 1mg/ml using DMSO.
3) Labeling by adding 15. Mu.l of dye to 0.8mL of BCHCR mg/mL protein solution, shaking in a shaker at 25℃and 150rpm for 1h at 4℃and standing overnight at 4℃in the absence of light.
4) And (3) desalting and purifying, namely preparing a dextran Sephadex G25 medium gravity desalting column in advance, washing the filler with deionized water with the volume of 5 times of the column volume, balancing with PBS buffer solution with the volume of 5 times of the column volume, adding the marked protein, adding PBS for desalting after the solution completely enters the filler, and collecting a fluorescent protein sample according to the indication of an ultraviolet detector. 2.2 nanobody inhibition BCHCR targeting binding to cell membranes
1) Cell resuscitation, namely, cell freezing and preserving pipes are taken out from a liquid nitrogen tank, quickly transferred into a water bath kettle preheated at 37 ℃ for thawing, 1ml of cell culture medium is added after thawing and transferred into a 5ml centrifuge tube, 1000g of the cell culture medium is centrifuged for 3min, the supernatant is discarded, 1ml of culture medium is added for gently blowing off cells, and the cells are inoculated into a culture dish and placed into a 37 ℃ CCO 2 incubator for culturing.
2) And (3) when the cell density is up to 90%, sucking and discarding the culture medium, adding a small amount of PBS along the side surface of the cell culture dish to clean the cells, sucking and discarding the PBS, adding 1ml of pancreatin, placing the cells in an incubator to digest for 1min, adding 1ml of culture medium to stop digestion, lightly blowing the bottle bottom and the cells to fully suspend and suck the cells into a centrifuge tube, centrifuging 1000g for 3min to remove the supernatant, adding 1ml of culture medium again to mix uniformly, transferring 25% of cell suspension into the culture dish containing 8ml of culture medium, shaking gently before and after, placing the culture dish into a 37-DEG CCO 2 incubator, and continuing to culture.
3) Cell slide was washed with 0.2m hcl followed by 75% ethanol and placed in a 12 well cell culture plate. Approximately 1.5X10 5 cell suspensions per well were added and 1mL of medium was added and mixed well. Culturing at 37deg.C in a 5% CO 2 incubator.
4) And (3) cell freezing, namely when the density of the Neuro-2a cells is about 90%, adding 2mL of cell freezing solution into the cells after digestion and centrifugation, fully and uniformly mixing, split charging into two freezing pipes, placing the two freezing pipes into a program cooling box, placing the two freezing pipes in a refrigerator at-80 ℃ for overnight, and transferring the two freezing pipes into a liquid nitrogen tank for long-term storage.
5) Recombinant protein BoNT/C HCR cell targeting verification, namely adding BoNT/C HCR protein marked by Super-Flour 680 fluorescent dye with the final concentration of 1 mu M into a 12-hole cell culture plate containing a Neuro-2a cell slide, incubating for 2 hours, and setting fluorescent dye marked BSA as a control group. The culture medium containing BoNT/HCR was discarded, 500. Mu.l of 4% histiocyte fixative was added, left standing for 15min, the fixative was discarded, and PBS was added for washing. The cell slide was carefully removed and placed on a slide containing an anti-fluorescent decay envelope and dried in the dark. The binding of the BoNT/C HCR protein to the Neuro-2a cell membrane was observed by confocal laser imaging microscopy.
3. Experimental results
Neuro-2a cells were used as subjects to examine whether nanobodies could inhibit the ability of BoNT/C HCR to bind to cell membranes. Mixing the nanobody and the BoNT/HCR protein marked by fluorescent molecules according to the molar ratio of 10:1, and then adding the mixture into a cell culture solution for incubation for 2 hours. Mean fluorescence intensity of cells was counted by confocal laser imaging observation and Image J, and the numerical differences of the treated group and the control group of BoNT/C HCR were compared using one-way analysis of variance (ANOVA) and Dunnett's test, respectively, with statistical significance for differences in P < 0.05. The experimental results are shown in fig. 5, wherein the average fluorescence intensity of the cells of the experimental group incubated by the nanobody A2 and the BoNT/C HCR is 40.654 +/-1.77, which is significantly lower than that of the control group only by the BoNT/C HCR (the average fluorescence intensity of the cells is 50.36+/-2.71, and P < 0.001). The BSA protein did not affect the binding of BoNT/HCR to cell membranes (average fluorescence intensity of cells 51.85.+ -. 2.77, nsP > 0.05). Thus, A2 was selected for subsequent activity verification.
Example 7 Western blot-based detection of the Effect of nanobodies on BoNT/C target cell uptake
1. Experimental materials
SNAP-25 rabbit polyclonal antibody (GB 11678-100, wohan Sieve Biotechnology Co., ltd.), anti-beta actin mouse mAb (GB 12001, wohan Sieve Biotechnology Co., ltd. );Goat anti mouse IgG(H+L)(HRP)(RS0001,Immunoway);Goat anti Rabbit IgG(H+L)(HRP)(RS0002,Immunoway);Syntaxin 1 antibody (HPC-1) (sc-12736,Santa Cruz Biotechnology), HEPES (1112 GR025, guangzhou Siro Biotechnology Co., ltd.), ECL chemiluminescent kit (36208 ES60, shanghai Santa Biotechnology Co., ltd.), full length BoNT/C prepared by a literature method using E.coli as a recipient (Zanetti G et al., PLoS Pathog.2017;13 (8): E1006567.Doi: 10.1371/journ. Ppat. 1006567) SD pregnant for about 18 days was purchased from the laboratory animal center in Lanzhou university, license number SCXK (sweet) 2023-0003.
2. Solution preparation
Neuronal cell Medium to 100ml of Neurobasal TM Plus medium was added 2ml of B-27 TM additive (50X) and 1ml of L-glutamine, mixed well and stored at 4 ℃.
10 XD-Hanks 8.0g of NaCl, 0.4g of KCl, 0.35 g of Na 2HPO4·12H2O 0.126g,NaHCO3, g g and 0.06g of KH 2PO4 g are weighed and dissolved in 8ml of deionized water, the pH is adjusted to 7.0, the volume is fixed to 10ml, and the mixture is preserved at 4 ℃.
1M HEPES 11.92g HEPES was weighed and dissolved in 40ml deionized water, and the volume was then fixed to 50ml, and the solution was sterilized by filtration using a 0.22 μm filter head and stored at-20 ℃.
The dissecting solution is prepared by weighing 10ml of 2M glucose 1ml,1M HEPES 1ml,10 XD-Hanks, adding 88ml deionized water, mixing, and preserving at 4 ℃.
10 Xtransfer buffer solution, weighing 30.3g of Tris, 151.5g of glycine, dissolving in 800ml of deionized water, and then keeping the volume to 1L at room temperature.
The Western blot membrane transfer buffer solution is 10 times 100ml of membrane transfer solution, 200ml of methanol, and the volume is fixed to 1L and the solution is stored at room temperature.
10 XTBS solution, weighing Tris 24g, sodium chloride 88g dissolved in 800ml deionized water, adjusting pH to 7.6 with hydrochloric acid, then constant volume to 1L, and preserving at room temperature.
TBST 100ml of 10 XTBS solution was measured and mixed with 900ml of deionized water, and 500. Mu.l of Tween-20 was added thereto for storage at room temperature.
3. Experimental method
3.1 Extraction of hippocampal neurons
1) To each well of a 24-well cell culture plate, 200. Mu.l of 0.1mg/ml polylysine was added and coated overnight at 4 ℃. Sucking out polylysine, and cleaning with sterile water for three times.
2) Pregnant mice were sacrificed for about 18 days during pregnancy using high concentration CO 2, the pregnant mice were quickly removed from their body and their heads were cut off and placed in pre-chilled dissected solution.
3) To a 15ml centrifuge tube, 12ml of dissection solution was added and placed on ice. The fetal mouse brain was segmented by microscopic observation in an ultra clean bench, the adhered cortical tissue and blood vessels were carefully removed, and the hippocampal tissue was removed and placed in a centrifuge tube.
4) The dissections were carefully discarded, 1ml of pre-heated pancreatin digest was added to the centrifuge tube, incubated at 37 ℃ for digestion, and the centrifuge tube was shaken every 10min for complete digestion.
5) After digestion for about 30-40min, the pancreatin digestion solution is discarded, 10ml of the dissecting solution is sucked up and added into the centrifuge tube, and the mixture is inverted and evenly mixed to clean the pancreatin digestion solution remained in the tube. Repeating the steps three times until the pancreatin is completely removed.
6) The in-tube cells were transferred to a new 1.5ml centrifuge tube and 2ml neuronal cell culture medium was added to it, and the cells were resuspended well until no apparent cell clumps were apparent. An appropriate amount of cell suspension was added to the polylysine-treated 24-well cell culture plate, and 500. Mu.l of medium was added thereto, followed by gentle mixing. Culturing in a 37 ℃ and 5% CO 2 incubator for 12-14 days until the neuron cells are mature.
3.2 Nanobody and BoNT/C Co-incubation with neuronal cells
Nanobody A2 was mixed with BoNT/C at 1nM, 45nM,150nM, and the nanobody A2 was allowed to bind well to BoNT/C by shaking at 700rpm in a 37℃metal bath for 45 min. And the mixture was filtered and added to neuronal cell culture medium and incubated with cells for 48h. A buffer control group was also set.
3.3 RIPA lysate extraction of cell total protein
The medium was aspirated and an appropriate amount of PBS buffer was added to the wells to clean the cell surface of residual medium. PBS was discarded, and 100. Mu.l of RIPA lysate (containing 1. Mu.l of PMSF protease inhibitor) was added to each well, and the cells were gently scraped with a gun head and lysed on ice for 30min. The cell suspension was transferred to a 1.5ml centrifuge tube. Centrifuge at 10,000rpm at 4℃for 10min. The supernatant was pipetted into a new 1.5ml centrifuge tube and the supernatant was added with the appropriate amount of 5 XSDS-PAGE loading buffer and denatured for 10min with a 100℃metal bath.
3.4 Western blot detection method
1) SDS-PAGE electrophoresis separation of proteins of different molecular weights by SDS-PAGE.
2) Transferring, namely cutting out gel region containing target proteins beta-actin, syntaxin and SNAP-25, cutting the PDVF film according to the size of gel block, and placing in methanol to activate for 30s. The cellulose pad, the filter paper, the gel, the PVDF film, the filter paper and the cellulose pad are assembled in sequence, and all layers are closely attached. Meanwhile, when the gel is placed in a film transfer groove, the gel faces to the negative electrode, the PVDF film faces to the positive electrode, and 1l of film transfer liquid is added. In an ice-water bath, 300mA is transferred to a membrane for 2h.
3) Blocking, adding 5% skimmed milk powder (dissolved in TBST, w/v), soaking PVDF membrane at room temperature for 2h.
4) Primary antibody was incubated by diluting 5,000-fold the rabbit polyclonal antibody to β -actin, syntaxin and SNAP-25, respectively, with TBST solution. And further cutting PVDF membrane according to the molecular weight difference of beta-actin, syntaxin and SNAP-25 protein, putting the cut PVDF membrane into corresponding antibody diluent, and incubating for 2h at room temperature.
5) Washing the membrane, namely washing the membrane for 3 times at room temperature by using TBST solution for 10min each time.
6) Incubation of secondary antibody two antibodies Goat anti mouse IgG (H+L) (HRP) and Goat anti rabbit IgG (H+L) (HRP) were diluted 1:5,000, respectively, and a PVDF membrane containing β -actin, syntaxin was placed in Goat anti mouse IgG (H+L) (HRP) antibody dilution, while a PVDF membrane containing SNAP-25 was placed in Goat anti rabbit IgG (H+L) (HRP) antibody dilution, and incubated for 1H at room temperature.
7) And (5) washing the membrane, wherein the step (5) is the same as the step (2).
8) And imaging, namely dripping a proper amount of ECL chemiluminescent liquid on the PVDF film for development, and placing the film in a gel imager for photographing and recording chemiluminescent signals.
4. Experimental results
This example evaluates whether nanobodies have significant blocking effect on the cleavage activity of the substrate protein Syntaxin and SNAP-25 by neurone ingestion of full-length BoNT/C by BoNT/C. The result of Westernblot detection is shown in FIG. 6, wherein serial dilutions of nanobody A2 and BoNT/C are mixed and then added into neuronal cells for co-incubation, and nanobody A2 dose-dependently inhibits cleavage of substrate proteins Syntaxin and SNAP-25 by BoNT/C. When the molar ratio of the nanobody A2 to the BoNT/C is 45:1, the nanobody A2 can completely block the uptake of toxin by neurons.
Example 8 in vivo neutralization Activity and efficacy detection of nanobodies and evaluation of BoNT/C poisoning rescue Effect
1. Experimental materials
Kunming female mice (18-22 g) were purchased from the university of Lanzhou laboratory animal center under license number SCXK (Gan) 2023-0003.
2. Experimental method
The neutralizing activity of the nano-antibody to BoNT/C and the delaying effect of the nano-antibody to BoNT/C caused paralysis are detected by a mouse intraperitoneal injection euthanasia method and a mouse toe abduction DAS scoring method respectively so as to evaluate the in vivo biological activity and the drug effect of the nano-antibody.
2.1 Evaluation of the protective Effect of nanobodies on blocking BoNT/C toxicity by intraperitoneal injection euthanasia in mice
Mice were randomly divided into 4 groups of 4 mice each. Nanobodies were serially diluted to 0.005mg/kg,0.05mg/kg and 0.5mg/kg and mixed with a lethal dose of 125 pg/BoNT/C only. The metal bath was shaken at 700rpm for 45min at 37℃to ensure adequate binding of nanobodies to BoNT/C. Mice were intraperitoneally injected with 100 μl of the mixture, while BoNT/C control groups were set. Mice survival was observed every 12 h.
2.2 DAS evaluation method for evaluating efficacy of nanobody in delaying toe paralysis of BoNT/C mice
Mice were randomly divided into 8 groups of 4 mice each. BoNT/C was diluted 5pg/μl,7.5pg/μl,10pg/μl and 12.5pg/μl, and mixed with nanobody at a molar ratio of 1:10 6, respectively, so that the concentration of nanobody was 0.5 μg/μl,0.75 μg/μl,1 μg/μl and
1.25. Mu.g/. Mu.l. The metal bath was shaken at 700rpm for 45min at 37℃to allow sufficient binding of nanobodies to BoNT/C. Mice were injected intramuscularly with 10 μl of the mixture to the left hind limb gastrocnemius, and mice were observed for gastrocnemius paralysis levels every 24h and recorded.
To further verify whether nanobody can rescue mice with systemic poisoning and regional muscle paralysis, detection was performed by mice intraperitoneal injection mortem and mice toe abduction DAS scoring, respectively.
2.3 Evaluation of the rescue effect of nanobodies on BoNT/C poisoning in mice by intraperitoneal injection euthanasia in mice
Mice were randomly divided into 4 groups of 4 mice each. Mice were given intraperitoneal injections of 125 pg/lethal dose of BoNT/C, followed by 5mg/kg of nanobody by tail vein at 1h,3h,6h, respectively, and survival was observed at intervals.
2.4 The DAS scoring method evaluates the rescue effect of nanobody on BoNT/C poisoning of mice:
Mice were randomly divided into 8 groups of 8 mice each. Mice were given 10 μl of left hind limb gastrocnemius intramuscular injection, 100 pg/BoNT/C, followed by intramuscular injection of 0.5mg/kg of nanobody or 5mg/kg of nanobody at the same gastrocnemius site at 1h,3h,6h, respectively, and mice were observed for gastrocnemius paralysis levels at intervals.
3. Experimental results
And selecting a nano antibody capable of blocking the binding of BoNT/HCR and a cell membrane, and verifying the in-vivo neutralization effect of the nano antibody on the activity of complete toxin. After co-incubation of 10pg nanobodies with 125pg of recombinant full length BoNT/C, mice were injected intraperitoneally or gastrocnemially. The results showed that nanobodies B7 and E12 failed to neutralize BoNT/C toxicity, but A2 could completely neutralize the toxin, resulting in a 100% survival rate in mice (shown as A in FIG. 7). Furthermore, nanobody A2 had a dose-dependent effect on neutralization of full length BoNT/C by intraperitoneal and gastrocnemius injections (shown as B and C in fig. 7).
Further, mice were given different doses of BoNT/C either intraperitoneally or gastrocnemially, and nanobody A2 was injected 1h,3h, and 6h after injection, respectively, for post-toxin rescue. As a result, it was found that the intramuscular injection or intravenous injection of nanobody A2 had a remarkable rescue effect, and that the rescue effect was the best for 1h of poisoning time (shown in FIG. 8).
Among the nano antibodies obtained by screening, the nano antibody A2 has the most remarkable neutralizing effect and post-poisoning rescuing effect on BoNT/C, and has great potential to be applied to preparing BoNT/C poisoning antidotes. In addition, 20 nanobodies obtained by screening of the present invention have significant specific binding to BoNT/HCR on a molecular level (fig. 3). At the cellular level, A2, A3, B7, C6, D1, E2, E5, E11, E12 and G5 significantly inhibited BoNT/C HCR binding to target cells (fig. 5). Therefore, the nano-antibody provided by the invention can be used as a capturing antibody and a detecting antibody in qualitative and quantitative analysis and detection of C-type botulinum toxin, and can be used as the capturing antibody or the detecting antibody in the kit for detecting the BoNT/C content and the activity, which can be understood by the person skilled in the art.
The treatment effect of the BoNT/A widely applied in clinic is usually weakened due to repeated injection of patients, and the treatment effect and the duration of action of the BoNT/C are very similar to those of the BoNT/A, so that the BoNT/A can treat dystonia and blepharospasm, and has no drug resistance and adverse reaction, and is hopefully a substitute of the BoNT/A. Therefore, it is imperative to develop detoxification chaperone products against the risk of human iatrogenic toxin poisoning. BoNT/C, on the other hand, is one of the major causes of botulism in animals. However, there is no effective therapeutic agent for BoNT/C poisoning, multivalent antitoxins are prone to cause allergic reactions, and have high production costs and limited supply. Therefore, the invention screens nano antibodies aiming at BoNT/C based on phage display technology so as to block the combination of BoNT/C and receptor, so that the BoNT/C cannot enter cells to exert toxic effect, and provides a new treatment method for BoNT/C poisoning.
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