CN104861070B - The human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity and its application - Google Patents

Abstract

The present invention provides the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, it is to utilize artificial synthesized recombinant protein BontB, and the substrate GFP-HIS6-SNAP25(62 of the human single chain variable fragments antibody of the anti-botulinum toxin type B enzymatic activity of screening)-VAMP(57)-C, it is screened in humanization anti-botulinum toxin type B enzyme antibody library, affinity costant is (5.35 ± 0.903) × 10⁵L/mol, to produce, anti-botulinum toxin type B special efficacy gives treatment to drug and quickly detection botulinum toxin type B is laid a good foundation.

Description

The human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity and its application

Technical field

The invention belongs to field of biotechnology, and in particular to the human single chain variable fragments antibody 3A- of anti-botulinum toxin type B enzymatic activity ScFv, and its application, in terms of botulinum toxin type B poisoning by enzyme drug is treated in production, answering in terms of detection botulinum toxin type B enzyme With further relating to the substrate for screening the human single chain variable fragments antibody of anti-botulinum toxin type B enzymatic activity.

Background technique

Botulinum toxin can lead to people and animal botulismus, and infective dose is extremely low, obtain from primate experiment, Minimum lethal dose (MLD) (LD50) is 1ng/kg, and therefore, it has been appointed as the prestige to the mankind by U.S.'s Disease Control and Prevention Center The side of body is similar to the biological weapons of anthrax.The reason of leading to botulismus, is roughly divided into four types, one, adult botulismus.Two, Infections in infants botulismus.Three, wound infection botulismus.

Botulinum toxin is that clostridium botulinum is formed in its natural state or in the medium, is discharged by bacteria lysis In environment or culture solution, the toxin of this form is referred to as precursor toxin, and molecular weight ranges are in 300 kDa to 900 kDa.Before Body toxin exists usually in the form of complex, being capable of enclosure and protection toxin by one or more by neurotoxin (Bont) Non-toxic albumen composition, including hemagglutinin (HA), non toxin non hemaglutinin (NTNH).Clinically described botulinum toxin is usual Refer to the neurotoxin part with neurotoxic effect.

Botulinum toxin according to its antigenic difference, co-exist in seven kinds of serotypes (A, B, C, D, E, F, G), although seven kinds The botulinum toxin constituent of serotype and its mechanism of action slightly have difference, but they have similar molecular weight and similar Structure.Mature botulinum toxin is made of the single chain polypeptide of about 150 kDa, passes through the protease of bacteria mediated when protein maturation The single chain polypeptide cutting of 150kDa is cracked into two unequal polypeptide fragments, two segments are connected by a disulfide bond, shape The heavy chain fragment (HC) that the light chain segments (LC) for being 50kDa at a molecular weight and a molecular weight are 100kDa.Wherein light chain There is a catalytic domain, heavy chain includes two functional domains that molecular weight is 50kDa, and N-terminal portion is transmembrane domain, C-terminal part For gangliosides binding domain.Individual light chain and heavy chain are non-toxic, and only double-strand just has biology toxicity.

Light chain is made of intracellular toxin albumen, and there is zinc ion to rely on endonuclease activity, can inhibit releasing for neurotransmitter It puts.(~ 20 is deep) is buried in light chain in the active region of botulinum toxin, and surface negative charge can be obtained by a channel.Fusiform mind Active site through toxin includes a single zinc atom, this zinc atom is always considered functional, but does not play structure Property effect.It can be by removing zinc with metal-chelator, but by free zinc atom being added in secondary structure come again The ability of acquisition zinc, toxin and Binding Capacity is almost unchanged, but the biological activity of light chain greatly reduces, and is only equivalent to Originally 30%.There is highly conserved zinc binding modules HExxH (X is arbitrary an amino acid) sequence positioned at light chain center. The zinc ion integrated containing one by 3 histidines in HExxH sequence, and there are specific proteins to cynapse vacuolar membrane protein Hydrolysing activity, this catalytic site are located in the deeper crack of protein surface.

In mankind's botulismus, botulinum toxin type A and botulinum toxin type B occupy most important status, wherein A type meat poisoning His 222 on toxin light chain, Glu223 and His226 residue have collectively constituted one of zinc binding modules HExxH sequence Point, and risen in the mechanism of action for coordinating active region zinc (His 222 and His226) and a hydrone (Glu223 residue) Direct effect.Glu261 is the third ligand for coordinating zinc.Even it is believed that the catalytic mechanism of botulinum toxin type A with it is thermophilic Hot mycoproteinase is similar, this is because based on structure and sequence it is similar on the basis of, their active site is also similar 's.So that active almost all loses, removal His226 makes botulinum toxin type A lose catalysis for mutation from Glu223 to Asp Activity.By showing that A, key amino acid relevant to mechanism of action is residual in botulinum toxin type B to botulinum toxin structural analysis Base is identical.The crystal structure of botulinum toxin type B in conjunction with synaptobrevin shows Arg369 and Tyr372 residue position In the close peptide bond cracked by toxin.The mutation that residue is neutralized in botulinum toxin is substantially reduced catalytic rate constant, but not Zinc combination and Binding Capacity are influenced, it has been proposed that, these residues have played effect in transition state is stablized.

In addition, in the activated centre LC, there are also some amino acid residues, such as Phe266, Glu350Arg363 and Tyr366.It Function be mainly maintain and stablize light chain activated centre structure and/or it be combined the relative position of substrate, with this Guarantee the optimal enzymatic hydrolysis ability of light chain.

LC is connected by the N-terminal of disulfide bond and HC.Disulfide bond there are in the state of, toxin heavy chain HN structural domain formed one Loop structure, by the Zn in over head and ears light chain surface crack²⁺Catalytic site covered, to not show enzymatic activity.In double-strand After toxin enters nerve cell, disulfide bond is reduced, and the light chain released could show zinc ion endopeptidase activity, catalytic site 16 amino acid residues of substrate can be accommodated.The zinc ion of catalytic site removes the imidazole ring with 4 histidine residues, 1 combination It is formed outside coordinate bond in the hydrone of conservative glutamic acid, also forms coordinate bond, 1 tryptophan with the glutamic acid carboxyl of another molecule Molecule is likely to also assist in the coordination of zinc ion.This unique zinc ion coordination form of toxin light chain, space structure and other The three-dimensional structure of all known metalloproteinases is all different, and a novel Metalloproteinase familv should belong to.

The pathogenesis of botulinum toxin

1, the identification and combination of target cell

Precursor toxin enters gastrointestinal tract, and in digestion process, non-toxin component plays a protective role to neurotoxin, makes it The erosion of various digestive ferments and gastric acid is not will receive.Since molecular weight is excessive, lps molecule not instead of in a manner of diffusion, with " indent " and the mode of cellular uptake are by epithelium of intestinal mucosa barrier, without destroying gastrointestinal tract.Subsequently into small intestine, small Under the alkalescence environment of intestines, the neurotoxin in preceding extracting toxin, which dissociates, to be come, into blood and Lymphatic Circulation, in kinesitherapy nerve and Its toxic effect is played at sympathetic nerve.Although neurotoxin can invade different tissue and organ, it is unable to penetration rate of blood brain screen Barrier, only affine target spot is peripheral cholinergic nerve tip.Mind is able to suppress in the light chain of cholinergic nerve endings, toxin Through transmitter acetylcholine in the release of neuromuscular junction, lead to muscular paralysis.In this toxicity process, Nervous toxicity Each functional domain of element has participation, and specific process can be divided into three phases.

The combination and internalization of target cell

During the identification of target cell is with this is combined, the binding domain of botulinum toxin heavy chain has played effect.Binding domain It can identify cholinergic nerve endings, and be specifically bound with it.Binding domain is integrated to the ganglioside of motor neuron Rouge and positioned at the small protein receptor tied of terminal nerve cell membrane surface, especially with the GD1b of affinity, GT1b and GQ1b class gangliosides.Then ceramide is connected to by the gangliosides that the oligosaccharides containing sialic acid forms.By right Gangliosides distribution and the research to its affine combination power, it may be determined that gangliosides are not that can uniquely promote meat poisoning malicious The molecule that element combines, and the proposition of amboceptor model shows before internalization occurs, botulinum toxin has just had been integrated into egg On polymeric immunoglobulin receptor.Show that synaptotagmin may take part in the internalization of A, B and E type in spite of some evidences, but clostridium The receptor for belonging to each member of family is determined there are no complete.Each toxin and its albumen co-receptor are specific cognates, Botulinum toxin type B is just similar to botulinum toxin protein part to the mixture of sialyl lactose, shows neurotoxin and nerve Save " lock & key " principle that glycosides rouge combines.This, which is observed, successfully illustrates a viewpoint, that is, gangliosides knot Conjunction makes botulinum toxin close to protein receptor, promotes the identification and internalization of toxin.

Once being integrated to neuron surface, parent molecule cracking, heavy chain and light chain are combined by zinc atom and reach neuron Cell membrane, and cell vesicle is entered by internalization, form the acid vesicles for being wrapped in lps molecule.That is internalization process. This receptor-mediated endocytic processes and time, temperature and synaptic activity are related.

2, transposition

The light chain of botulinum toxin must come out from vesica cell or endosome, and under H chain N- end effect The target protein of cytosol can be successfully entered by film transport, this process is referred to as light chain transposition.

As other clostridial toxins, after botulinum toxin enters intracellular acidic vesicles, it can occur because of the variation of pH Structural rearrangement.Although be into synaptic versicle and vesicle intracellular it is clear that botulinum toxin transposition enter vesicle intracellular Characteristic not yet determines absolutely.The reduction of pH makes botulinum toxin structure change, and has biggish dredge in the molecule so as to cause it It is aqueous, and increase the permeability of lipid bilayer.The heavy chain of toxin and light chain in this way can be embedded in the lipid bilayer of vesicle, once Insertion, will form ion channel on phospholipid bilayer and PC12 film.This channel is selective, big point of molecular weight Son can not pass through, so generally believing that toxin light chain can be transported to nerve cell born of the same parents from vesicle is intracavitary by ion channel at present Matter.There are three types of light chain transmembrane transport hypothesis model, i.e. pipeline model (tunnel model), fractured model (cleft accordingly Model) and model (lysis model) is cracked.Wherein fractured model theory thinks, in low pH, the molecular construct of protein Changed, HC can form a hydrophilic crack at this time, and wear in membrane process in light chain and surround its water-wetted surface. Hydrophily and hydrophobic interaction are formd in this mode.The c-terminus of light chain is because of disulfide bond and heavy chain aminoterminal phase Even cytoplasm is entered at first.After realizing transposition, since the pH of cytoplasm is higher than acidic vesicles, fold light chain construct again, weight Newly return to catalytic activity.

At low ph values, the variation on the domain the HN occurred conformation of A and botulinum toxin type B, finally across artificial membrane formed from Subchannel, and in vivo in the environment of pH, the activity in the channel of formation is the largest.C-type botulinum toxin is formed in adipose membrane Ion channel formed to diphtheria toxin it is similar, and be also only formed at a low ph.Botulinum toxin ion channel only allows sun Ion passes through, but can prevent chloroquine in vitro.However the research for small-bore ion channel, we not yet understand meat poisoning The channel that toxin is formed is the only way which must be passed that toxin light chain enters cytosol.Data show, at a low ph the knot of light chain Structure has a theatrical variation, is exactly there may be the LC structure of a selectivity and more suitable for transposition.Although this It is not proved also in vivo, but is completely reversibility by the variation of low pH inducement structure.

Botulinum toxin can play a role in neurotransmission, and LC and the joint HN must just be made to expose by proteolysis Surface generates notch, so that the toxin of inactive single chain molecule amount 150-kDa be made to activate.A small number of bacteriums and histone Enzyme can be realized this cracking reaction, and generate active double-strand neurotoxin.After generating notch, light chain and heavy chain are still logical Non-covalent interaction is crossed, and is connected by a single disulfide bond.The disulfide bond of this interchain is (in botulinum toxin type A Cys430-Cys454) reduction be light chain activation second stage, this is that light chain is freely accessible to motor neuron cytosol And it interacts between substrate the essential stage.As noted, in low pH, light chain has a drama Variation.The variation of this structure facilitates the recombination of transposition band (if being strictly close to light chain in the domain translocation point HN) and causes The final activation of light chain.NGF signal transduction pathway can be maintained close ties with together with botulinum toxin-LC activity.Therefore, The completion of light chain activation may finally occur in cytosol, the and then transposition from the cell of low pH.

3, inhibit neurotransmitter regulator

There is synaptic vesicle bubble at neural brief summary, wherein including acetylcholine, it is to pass through neuromuscular junction to stimulate flesh A kind of neurotransmitter that meat is shunk.The vesicles of the acetylcholine are tied by the protein polymer that one is called SNARE complex It closes.In order to pass through the transmitting that neuromuscular intersection realizes signal, the acetylcholine vesicles in presynaptic nerve brief summary are necessary It is released in synaptic cleft.Herein, neurotransmitter is integrated on special receptor on muscle plate, and triggering ion channel is beaten It opens, so as to cause the depolarising and contraction of adjacent striated muscle.The release of acetylcholine needs the participation of SNARE albumen, it Merging for synaptic versicle and neuronal cell film can be mediated.

SNARE complex protein participates in plasma membrane fusion on synaptic versicle, and the effect of botulinum neurotoxin light chain is to hinder exocytosis Effect.It more specifically, is exactly at neuromuscular junction, neurotoxin by cutting snare protein, (make by acetylcholine molecules exocytosis With necessary substance) block Vesicle-Containing to be discharged into extracellular environment, inhibit the release of acetylcholine, to inhibit Neurotransmission.It is demonstrated that individually snare protein cracking will not interfere SNARE complex to be formed, but will lead to non-functional Property compound is in Ca²⁺Coupling between interior stream and fusion is destroyed.Toxin needs Ca when inhibiting neurotransmitter regulator²⁺Ginseng With, and in cynapse tip Ca²⁺Concentration increase will affect the function and effect of botulinum toxin.

Light chain causes SNARE compound unstable as zinc endopeptidase Protein cleavage, becomes non-functional, to press down Acetylcholine processed is discharged into synaptic cleft.The quantity that vesica is discharged into synaptic cleft is fewer, and the probability that action potential is propagated is got over It is low, so as to cause the contraction of meat fiber.As a result lead to the Chemodenervation of muscle itself and cause flaccid paralysis.It is existing Research shows that SNARE compound is made of tri- kinds of protein of VAMP, SNAP-25 and syntaxin, following table show different type Botulinum toxin target protein and its cleavage site.

The basic principle of display technique of bacteriophage is that exogenous DNA is inserted into phage encoded coat protein pIII or pVIiI Gene in, so that heterologous DNA fragments corresponding expression product fusion is formed fusion protein in the coat protein of bacteriophage (fusion protein), is presented on phage surface.The remarkable advantage of display technique of bacteriophage is: establishing genotype (genotype) the direct physical link between phenotype (phenotype), to make to screen simple and effective.By foreign protein or Polypeptide expression is in phage surface, so that it may utilize it with the compatibility of other biological or abitotic substance to containing mesh according to its property The bacteriophage of gene screened.By taking the screening of phage antibody library as an example, by Fab (fragment antigen Binding) expression is screened in phage surface by affinity ligands of corresponding antigen, can be quickly and efficiently from a large amount of grams The bacteriophage of grand middle screening expression specificity antibody.Therefore, high-affinity antibody and traditional is prepared with display technique of bacteriophage Authentic monoclonal antibody technology, which is compared, has apparent advantage.

Phage antibody is to be merged by Fab section or ScFv with the formation of single stranded phage coat protein in Membrane surface expression Albumen and complete.Its main feature is that " it not only can recognize corresponding antigen and in connection, but also can infect host strain and be expanded again Increase ".The ligand of target molecule can be screened with target antigen by affine absorption-elution-amplification using the characteristic that it can be expanded again Peptide chain.Mutated again and strand displacement method improves affinity of antibody, finally just obtains the specific antibody of high-affinity.Bacteriophage The screening of antibody library includes two key steps: washing in a pan sieve and identification.Naughty sieve is to be total to phage antibody library and the antigen of selection With being incubated for, is eluted by several wheels, collect the bacteriophage of combination.It by the phage-infect bacterium of acquisition and expands, then carries out next The naughty sieve of wheel.After several wheels wash in a pan sieve, the polyclonal bacterial strain of the phage-infect in conjunction with antigentic specificity can be enriched to.Identification Process is to pick out monoclonal bacterial strain from the polyclonal bacterial strain of phage-infect.Will wash in a pan the phage-infect bacterium sifted out, Bed board is selected, and high-specificity monoclonal bacterial strain can be obtained.

From there are mainly two types of the classical ways of the bacteriophage of Phage Antibody Library expression specificity antibody: (1) will On solid-phase media, such as ELISA Plate, immunotubes or affinity column, bacteriophage to be screened is then added in pure antigen coat, The bacteriophage for washing away non-compatibility or low compatibility recycles the bacteriophage of high-affinity.(2) by antigen and biotin group phase Even, it then is fixed in the paramagnetic beads for be coated with streptavidin and bacteriophage is screened.It is both needed to add in two methods Skim milk (or BSA, effect are poor) closes the site not occupied by antigen, to avoid the nonspecific combination of bacteriophage. For former approach, alkaline solution (such as triethylamine, (triethy-can be used by recycling the bacteriophage in conjunction with antigentic specificity Lamine) or acid solution (such as one hydrochloric acid of glycine, glycine_HCl) elutes, or is eluted with soluble antigen or hapten； For latter method, pass through two sulphur between destruction antigen and biotin with dithiothreitol (dithiothret01, DTT) Key elutes.The phage-infect host strain of recycling carries out next round screening after proliferation.It is general to be screened by 3-5 wheels are such It can obtain the clone of the purpose antibody of expression high-affinity.Each round screening requires to be detected, to confirm the effective of screening Property.

MRC HGMP resource center, Britain creation Human Single Fold scFv Libraries I+J ( Tomlinson I+J) it is current mature full Large human naive scFv phage library, storage capacity is more than 10⁸Full source of people phage single-chain Antibody, this research and utilization anti-botulinum toxin type B single-chain antibody of phage library elutriation specificity, and positive-single strand antibody is carried out Network analysis establishes material base to formulate botulinum toxin type B poisoning treatment antibody class drug.

The present inventor determines that botulinum toxin can cause people and animals by the understanding and research of the mechanism of action to botulinum toxin Illness altogether, and the intracorporal nerve synapse system of primary challenge, pathogenesis are, botulinum toxin passes through its heavy chain C-terminal structural domain It is incorporated into the presynaptic membrane of cholinergic neuron, toxoreceptor complex internalization is formed and enters cell vesicle, then in light chain zinc Peptase enzymatic activity area discharges into cytoplasm from intracellular acidic spaces.Once releasing from vesica, light chain is by cutting SNARE complex proteins are cut to prevent the release of acetylcholine, cause muscular paralysis.Therefore light chain is main pathogenic position. Currently, it is relatively comprehensive about the report of botulinum toxin type A both at home and abroad, but phase is reported about the research of botulinum toxin type B To less, while lacking botulinum toxin type B treatment method and drug, once thus causing someone suffers from Type B botulismus, It will be unable to the circumstances correctly treated in time.Antibody be treatment botulismus most efficient method, but at present research or it is limited There is many disadvantages for the heterologous serum antibody used, such as cause to be immunoreacted or infect disease, lead to not promote and answer With.Since such antibody molecule amount is larger, it is difficult to which penetrating cell film is then imitated without treatment for the toxin light chain for coming into cell Fruit, therefore the present invention is directed to clonal expression B BOTULINUM TOXIN TYPEs, screen neutrality in humanized antibody library by recombinant protein Humanized single chain antibody, to develop, anti-B BOTULINUM TOXIN TYPE special efficacy gives treatment to drug and quickly detection botulinum toxin type B lays the foundation.

Summary of the invention

The purpose of the present invention is exist and cause to be immunized to solve heterologous serum antibody in treatment botulinum toxin type B poisoning The problem of reaction or infection disease, and a kind of single-chain antibody ScFv of full source of people is provided, the source of people of anti-botulinum toxin type B enzymatic activity Single-chain antibody 3A-scFv.

The human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, base sequence such as sequence table SEQ ID Shown in NO.5.

The human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, amino acid sequence such as sequence table SEQ ID Shown in NO.6.

The human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity treats botulinum toxin type B poisoning by enzyme medicine in production The application in object space face.

Application of the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity in terms of detecting botulinum toxin type B.

Another object of the present invention is to provide a kind of bottom of human single chain variable fragments antibody for screening anti-botulinum toxin type B enzymatic activity Object.

Screen the substrate of the human single chain variable fragments antibody of anti-botulinum toxin type B enzymatic activity, base sequence such as sequence table SEQ ID Shown in NO.3.

Screen the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, amino acid sequence such as sequence table SEQ Shown in ID NO.4.

The present invention provides the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, it is using artificial synthesized Recombinant protein BontB, and the anti-botulinum toxin type B enzymatic activity of screening human single chain variable fragments antibody substrate GFP-HIS6-SNAP25 (62)-VAMP(57)-C, screen in humanization anti-botulinum toxin type B enzyme antibody library, affinity costant be (5.35 ± 0.903) × 10⁵L/mol, to produce, anti-B BOTULINUM TOXIN TYPE special efficacy gives treatment to drug and quickly detection botulinum toxin type B is established Basis.

Detailed description of the invention

Fig. 1 is double digestion pMD-19T-Bont-B plasmid electrophoresis result；1.EcoRAnd NdeDouble digestion plasmid pMD-19T- Bont-B ； 2. DNA marker DL2000；

Fig. 2 is that PCR identifies recombinant plasmid PET-28a-BontB result figure；1. DNA marker DL2000；2.3.4. For the region BontB pcr amplification product；

Fig. 3 is double digestion recombinant plasmid PET-28a-BontB qualification result；Wherein: 1.2. is I pair of enzyme of EcoR I and Nde Cut plasmid PET-28a-BontB 3.DNA marker DL2000；

Fig. 4 is that recombinant protein expression-form is identified wherein: 1. non-induction bacterium ultrasound precipitations；2. induction bacterium ultrasound precipitation；3. Non- induction bacterium ultrasound supernatant；4. induction bacterium ultrasound supernatant state 5: the whole cell of induction；6. protein Marker；

Fig. 5 is recombinant protein SDS-PAGE interpretation of result；Wherein: 1. protein marker；2. the purpose egg of preliminary purification It is white；3. the destination protein finally purified；

The double digestion of Fig. 6 recombinant plasmid PET-28a-GFP identifies (NcoI and BamHI)；Wherein 1: plasmid PET-28a The double enzyme digestion product of-GFP；M:DNA marker DL2000；

The double enzyme digestion product of Fig. 7 plasmid PET-28a-GFP-SV；M:DNA marker DL5000；

Fig. 8 3A-scFv SDS-PAGE interpretation of result after purification；1: flowing through 2:55% ammonium sulfate original sample 3: after purification scFv。

Specific embodiment

The synthesis of embodiment 1:B BOTULINUM TOXIN TYPE light chain gene

According to the botulinum toxin type B complete genome sequence that Genbank is reported, light chain gene is designed and synthesized, is inserted into simultaneously EcoRAnd NdeGene fragment clone is entered pMD-19T vector, is transformed into JM109 by restriction enzyme site.

Embodiment 2:B BOTULINUM TOXIN TYPE light chain gene is connect with PET-28a carrier

Utilize restriction endonuclease EcoRAnd NdeDouble digestion plasmid pMD-19T-Bont-B, digestion products are through agarose gel electrophoresis Analysis, as a result as shown in Figure 2, it is seen that the target gene BontB that size is about 1335 is consistent with expected size；

By EcoRAnd NdeThe carrier PET-28a large fragment and target gene BontB small fragment of double digestion recycle, and use T4 DNA ligase connection, obtains recombinant plasmid PET-28a-BontB, converts escherichia coli jm109 competent cell, resistance containing kan Agarose plate carry out preliminary screening.Picking individual colonies are cultivated in LB liquid medium；It is extracted with plasmid QIAquick Gel Extraction Kit Plasmid.

EcoR is inserted by synthesisAnd NdeThe botulinum toxin type B light chain gene sequence of restriction enzyme site is its design primer, Upstream primer is designated as P1, and downstream primer is designated as P2.

P1(23bp): 5'CATATgCCAgTTACAATAAATAA-3'

P2(23bp): 5'gAATTCTCATTTAACACTTTTAC-3'

Using recombinant plasmid PET-28a-BontB as template, P1 and P2 carry out amplification reaction for primer, obtained PCR product, Through agarose gel electrophoresis identification and analysis, (see figure 3)；

Double digestion identification is carried out with restriction enzyme and EcoR I and Nde I, and carries out the measurement of sequence.It can be seen that plasmid passes through After double digestion, after PCR amplification purpose band, there is purpose genophore (see figure 4) on 1335bp, it was demonstrated that target gene BontB Gene has been coupled on carrier, successfully constructs recombinant plasmid PET-28a-BontB.

Embodiment 3: the SDS-PAGE interpretation of result of recombinant protein PET-28a-BontB expression product

Recombinant plasmid PET-28a-BontB is converted and expresses bacterium-e. coli bl21 (DE3), after IPTG inducing expression, SDS-PAGE is as the result is shown: recombinant protein PET-28a-BontB has an obvious expression band, size and theoretical value at the place 50KD or so It is consistent；See Fig. 5；

By recombinant protein expression bacterial lysate supernatant precipitating while SDS-PAGE electrophoresis is done, wherein the ratio of supernatant and precipitating Example is 1:10, the results show that the destination protein overwhelming majority is in supernatant, in precipitating corresponding position there is also a small amount of destination protein, Think the optimization by inductive condition, can be expressed under suitable induced environment with soluble form, see Fig. 5.

Embodiment 4: the purifying of recombinant protein PET-28a-BontB

The preliminary purification of recombinant protein: largely preparing bacterium, inducing expression recombinant protein, and soluble protein first carries out DEAE Anion exchange chromatography, and the peak Liu Chuan is collected, then carry out belonging to chelate chromatography Cu2+ column, collect the purpose of 200mM imidazoles elution Albumen.

His-Tag is removed with Thrombin digestion

Recombinant protein it is secondarily purified: the albumen after digestion carry out belong to chelate chromatography Cu2+ column, collect 250mM imidazoles elution Destination protein.The albumen of collection such as schemes through SDS-PAGE interpretation of result, can have an obvious band at the place 50KD or so, and pure Change efficiency up to 90% or more, sees Fig. 5.

Embodiment 5: the screening of the anti-Bont-B-scFv antibody of full source of people

The recombinant protein of purifying is antigen coat in 96 hole elisa Plates, and 4 DEG C overnight.Next day abandons supernatant, with 2% Milk- 37 DEG C of closing 2h of PBS, are added phage antibody library, and titre is 1.0 × 10¹³, acutely shake be incubated for 60min at room temperature, stand 60min.After discard liquid, washed 10 times with the PBS containing 0.1%Twenn-20, gently clap residual liquid in every hole after washing Dry, 50 μ L eluents (pancreatin-PBS of 5mg/mL) is added in every hole, acutely shakes 10min at room temperature, and wash-out bacteriophage collects 4 DEG C save.

With the Phage Infection E.coli TG1 under elution, and it is coated on TYE plate (containing 100 μ g/mLAmp's and 1% Glucose) 37 DEG C be incubated overnight.Phage library is expanded using helper phage KM13, bacteriophage is recycled by PEG/NaCl.Weight Multiple above procedure 3 times, totally 4 wheel screening, collects the phage library of screening.

Embodiment 6:Bomt-B Activity determination substrate-GFP-SV is gene constructed and purifies

According to SNAPE25, VAMP gene order logged in GenBank, HIS6-SNAPE62- is designed and synthesized EcoRI-VAMP57C-TAA gene, while it being inserted into III restriction enzyme site of BamHI and Hind, synthesis recombination matter respectively at gene both ends Grain pMD19-T- SV.

According to green fluorescent protein GFP gene, a pair of of specific primer is designed,

Upstream primer P1:5 '-CATGccATGGTGAGCAAGGGCG-3 '

Downstream primer P2:5 '-GCGGATCCcttgtacagCTCGTCCATG-3 '

Using P1 and P2 as primer, GFP plasmid is template, and PCR amplification GFP gene utilizes the bis- enzymes of restriction endonuclease NcoI, BamHI Carrier pET-28a and 720bp PCR recovery product is cut, pET-28a and GFP genetic fragment, connection are connected with T4DNA ligase Product transformed competence colibacillus E.coli JM109, next day picking single colonie extract plasmid.PCR, double digestion and survey are carried out to it Sequence identification obtains GFP successful clone into pET-28a, and base does not occur and loses and is mutated, success carrier construction pET-28a-GFP.

With restriction endonuclease Hind III and BamHI to the recombinant plasmid pMD19-T-SV and recombinant expression plasmid pET-28a- of synthesis 393bp small fragment and pET-28a-GFP carrier large fragment is separately recovered in GFP double digestion, with the connection of T4DNA ligase.Connection produces Object (pET-28a-GFP-SV) transformed competence colibacillus E.coli JM109, be coated with Kan(50 μ g/ml) LB plate on, 37 DEG C culture Overnight.Next day picking single colonie extracts plasmid, after double digestion and sequencing identification, show that SV gene is successfully plugged into pET- In 28a-GFP carrier, pET-28a-GFP-SV expression vector is successfully constructed.

Recombinant plasmid pET-28a-GFP-SV is converted and expresses bacterium-e. coli bl21 (DE3), IPTG inducing expression, really Determine optimal expression way: LB culture medium, 37 DEG C, 3 h.Through SDS-PAGE electrophoresis detection after ultrasonication thallus, mesh is determined The albumen overwhelming majority in supernatant, therefore destination protein is expressed with soluble form.

The purifying of recombinant protein GFP-SV, recombinant protein are expressed thallus and are cracked through ultrasound, successively hand over by DEAE anion Chromatography, metal chelate chromatography are changed, Q cation-exchange chromatography obtains sterling GFP-SV of the purity 90% or more.Pyrogen quality detection < 20EU/mg meets the needs of property of protein research.

Embodiment 7: anti-Bont-B-scFv positive strain identification

Phage Infection after screeningE.ColiHB2151 after inducing expression, is identified using ELISA, sets microplate reader measurement OD value (wavelength 490nm), each sample do diplopore measurement, take OD average value.Using 2%Milk-PBS as negative control, positive gram Grand bacterial strain determines standard are as follows: OD value is 3 times or more of negative control, obtains 200 plants of positive colony bacterial strains altogether.

The detection of positive strain bioactivity, GFP-SV is diluted with coating buffer, and every hole is coated with 30 μ g in detection plate (Pierce Maleimide Activated 96-well Plates, Black, 15153) in, 4 DEG C overnight, are washed 3 with Wash buffer It is secondary, 100 μ L supernatant, 4 μ g Bont-B is mixed after inducing expression centrifugation, be added to detection plate by 200 plants of positive strains In, 37 DEG C of effect 2h, every part of sample does diplopore measurement, then is washed 3 times with Wash buffer, detects on multi-function microplate reader Fluorescence intensity obtains 3 plants of preferable bacterial strains of activity.According to the gene sequence of the pIT-2 carrier on Tomlinson I+J kit Column synthesize two Specific PCR primers amplification scFv full genome segments.

P1 LMB3: 5’—CAG GAA ACA GCT ATG AC—3’

P2 pHEN: 5’ —CTA TGC GGC CCC ATT CA—3’

Through detecting 3 plants of positive strains, obvious 900bp band can occur, contain complete scFv.

Sequencing is identified its sequence and is obtained through Blast database analysis: showing that 3 plants of positive strains are that source of people is single-stranded anti- Body is analyzed as follows:

3A-scFv:

ATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCG GCC

I M K Y L L P T A A A G L L L L A A

CAG CCG GCC ATG GCC GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA

Q P A M A E V Q L L E S G G G L V

CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT

Q P G G S L R L S C A A S G F T F

CDR-H1

AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG

S S Y A M S W V R Q A P G K G L E

CDR-H2

TGG GTC TCA AAT ATT TCT TCT AAT GGT AAT GCT ACA GCT TAC GCA GAC TCC GTG

W V S N I S S N G N A T A Y A D S V

AAG GGC CGG TTC ACC ATC TCC AGA AAC AAT TCC AAG AAC ACG CTG TAT CTG

K G R F T I S R N N S K N T L Y L

CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA

Q M N S L R A E D T A V Y Y C A K

CDR-H3

TAT ACT TAT GCT TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCG

Y T Y A F D Y W G Q G T L V T V S

Linker

AGC GGT GGA GGC GGT TCA GGC GGA GGT GGC AGC GGC GGT GGC GGG TCG ACG

S G G G G S G G G G S G G G G S T

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC

D I Q M T Q S P S S L S A S V G D

AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA AAT

CDR-L1

R V T I T C R A S Q S I S S Y L N

TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT TAT GCA

W Y Q Q K P G K A P K L L I Y Y A

CDR-L2

TCC AAT TTG CAA AGC GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT GGG

S N L Q S G V P S R F S G S G S G

ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC

T D F T L T I S S L Q P E D F A T Y

CDR-L3

TAC TGT CAA CAG GAT TCT TAT ACT CCT TCT ACG TTC GGC CAA GGG ACC AAG

Y C Q Q D S Y T P S T F G Q G T K

GTG GAA ATC AAA CGG GCG GCC GCA CAT CAT CAT CAC CAT CAC GGG GCC GCA

V E I K R A A A H H H H H H G A A

GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG

E Q K L I S E E D L N G A A

2F-scFv:

ATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCG

I M K Y L L P T A A A G L L L L A

GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG

A Q P A M A E V Q L L E S G G G L

CDR-H1

GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC

V Q P G G S L R L S C A A S G F T

TTT AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG

F S S Y A M S W V R Q A P G K G L

CDR-H2

GAG TGG GTC TCA TCT ATT GCT TCT ACT GGT TCT AAT ACA GCT TAC GCA GAC TCC

E W V S S I A S T G S N T A Y A D S

GTG AAG GGC CGG TTC ACC ATC TCC AGA AAC AAT TCC AAG AAC ACG CTG TAT

V K G R F T I S R N N S K N T L Y

CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG

L Q M N S L R A E D T A V Y Y C A

CDR-H3

AAA GGT ACT GGT ACT TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC

K G T G T F D Y W G Q G T L V T V

Linker

TCG AGC GGT GGA GGC GGT TCA GGC GGA GGT GGC AGC GGC GGT GGC GGG TCG

S S G G G G S G G G G S G G G G S

ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA

T D I Q M T Q S P S S L S A S V G

CDR-L1

GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA

D R V T I T C R A S Q S I S S Y L

AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTA ATC TAT ACT

N W Y Q Q K P G K A P K L L I Y T

CDR-L2

GCA TCC TAC TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT

A S Y L Q S G V P S R F S G S G S

GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA

G T D F T L T I S S L Q P E D F A

CDR-L3

ACT TAC TAC TGT CAA CAG GCT ACT ACT AGT CCT TAT ACG TTC GGC CAA GGG

T Y Y C Q Q A T T S P Y T F G Q G

ACC AAG GTG GAA ATC AAA CGG GCG GCC GCA CAT CAT CAT CAC CAT CAC GGG

TKVEIKRAAAHHHHHHG

GCC GCA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG

A A E Q K L I S E E D L N G A A

4C-scFv:

ATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCG GCC

I M K Y L L P T A A A G L L L L A A

CAG CCG GCC ATG GCT GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA

Q P A M A E V Q L L E S G G G L V

CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT

Q P G G S L R L S C A A S G F T F

CDR-H1

AGC AGC TAT GCC ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG

S S Y A M S W V R Q A P G K G L E

CDR-H2

TGG GTC TCA GGT ATT TCT CCT ATG GGT ACT CGT ACA TCG TAC GCA GAC TCC

W V S G I S P M G T R T S Y A D S

GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT

V K G R F T I S R D N S K N T L Y

CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG

L Q M N S L R A E D T A V Y Y C A

CDR-H3

AAA AAT GCT AAT GGG TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC

K N A N G F D Y W G Q G T L V T V

Linker

TCG AGC GGT GGA GGC GGT TCA GGC GGA GGT GGC AGC GGC GGT GGC GGG TCG

S S G G G G S G G G G S G G G G S

ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA

T D I Q M T Q S P S S L S A S V G

CDR-L1

GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA

D R V T I T C R A S Q S I S S Y L

AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT AGG

N W Y Q Q K P G K A P K L L I Y R

CDR-L2

GCA TCC GTT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT

A S V L Q S G V P S R F S G S G S

GAG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA

E T D F T L T I S S L Q P E D F A

CDR-L3

ACT TAC TAC TGT CAA CAG CGG AGT CGG CCG CCT ATT ACG TTC GGC CAA GGG

T Y Y C Q Q R S R P P I T F G Q G

ACC AAG GTG GAA ATC AAA CGG GCG GCC GCA CAT CAT CAT CAC CAT CAC GGG

T K V E I K R A A A H H H H H H G

GCC GCA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG

A A E Q K L I S E E D L N G A A

Embodiment 8: anti-Bont-B-scFv purifying and the measurement of antibody affinity costant KD value

3 plants of strong positive clones bacterial strains are cultivated at 37 DEG C, until OD600 to 0.9, is added 30 DEG C of overnight incubations (16h) of inducer, 4 DEG C of overnight culture, 3500 × g is centrifuged 30min, and supernatant is the scFv of expression, size 31000.Induction supernatant successively passes through After 55% saturation degree ammonium sulfate precipitation and rProtein-A affinity chromatography, sample of the purity 90% or more is obtained.

Using the affinity costant of non-competing enzyme immunoassay measurement 2F, 4C and 3A single-chain antibody, respectively with 4 μ g/mL, 2 μ g/ ML, 1 μ g/mL and 0.5 μ g/mL are coated with Bont-B, and single-chain antibody concentration is adjusted to 10^-6Mol/L, doubling dilution 1:2 ~ 1: 512, use 1:5000 times of diluted Protein A-HRP antibody as secondary antibody, OPD colour developing measures OD490nm light absorption value, each Sample does diplopore measurement, takes OD average value.According to the sigmoid curve figure of antigen-antibody binding reaction, can solve anti-in difference The antibody concentration of writing on the blackboard light absorption value under original content is brought into formula KA=(n-1)/2 (nAb '-Ab) and calculates affinity costant, wherein Ab ' and Ab indicates to generate the antibody concentration (mol/L) of half light absorption value, n=Ag/Ag ', as n=2 when antigen is Ag ' and Ag Available 3 KA values, when n=4, can obtain 2 KA values, and when n=8 obtains 1 KA value, six KA values are averaged exactly final Affinity costant numerical value.

The affinity costant that 4C single-chain antibody is found out from table is (4.38 ± 0.445) × 10⁵The affinity costant of L/mol, 3A is (5.35 ± 0.903) × 10⁵L/mol, 2F are (1.146 ± 0.525) × 10⁶L/mol。

<110>MILITARY VETERINARY INST ACADE

<120>B BOTULINUM TOXIN TYPE light chain gene base sequence

<160> 10

<210> 1

<211> 1335

<212> DNA

<213>artificial

<400> 1

catatgccag ttacaataaa taattttaat tataatgatc ctattgataa taataatatt 60

attatgatgg agcctccatt tgcgagaggt acggggagat attataaagc ttttaaaatc 120

acagatcgta tttggataat accggaaaga tatacttttg gatataaacc tgaggatttt 180

aataaaagtt ccggtatttt taatagagat gtttgtgaat attatgatcc agattactta 240

aatactaatg ataaaaagaa tatattttta caaacaatga tcaagttatt taatagaatc 300

aaatcaaaac cattgggtga aaagttatta gagatgatta taaatggtat accttatctt 360

ggagatagac gtgttccact cgaagagttt aacacaaaca ttgctagtgt aactgttaat 420

aaattaatca gtaatccagg agaagtggag cgaaaaaaag gtattttcgc aaatttaata 480

atatttggac ctgggccagt tttaaatgaa aatgagacta tagatatagg tatacaaaat 540

cattttgcat caagggaagg cttcgggggt ataatgcaaa tgaagttttg cccagaatat 600

gtaagcgtat ttaataatgt tcaagaaaac aaaggcgcaa gtatatttaa tagacgtgga 660

tatttttcag atccagcctt gatattaatg catgaactta tacatgtttt acatggatta 720

tatggcatta aagtagatga tttaccaatt gtaccaaatg aaaaaaaatt ttttatgcaa 780

tctacagatg ctatacaggc agaagaacta tatacatttg gaggacaaga tcccagcatc 840

ataactcctt ctacggataa aagtatctat gataaagttt tgcaaaattt tagagggata 900

gttgatagac ttaacaaggt tttagtttgc atatcagatc ctaacattaa tattaatata 960

tataaaaata aatttaaaga taaatataaa ttcgttgaag attctgaggg aaaatatagt 1020

atagatgtag aaagttttga taaattatat aaaagcttaa tgtttggttt tacagaaact 1080

aatatagcag aaaattataa aataaaaact agagcttctt attttagtga ttccttacca 1140

ccagtaaaaa taaaaaattt attagataat gaaatctata ctatagagga agggtttaat 1200

atatctgata aagatatgga aaaagaatat agaggtcaga ataaagctat aaataaacaa 1260

gcttatgaag aaattagcaa ggagcatttg gctgtatata agatacaaat gtgtaaaagt 1320

gttaaatgag aattc 1335

<210> 2

<211> 441

<212> PRT

<213>artificial

<400> 2

Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn

5 10 15

Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Met Gly Arg

20 25 30

Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu

35 40 45

Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly

50 55 60

Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn

65 70 75 80

Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe

85 90 95

Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile

100 105 110

Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu

115 120 125

Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn

130 135 140

Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile

145 150 155 160

Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly

165 170 175

Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln

180 185 190

Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu

195 200 205

Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro

210 215 220

Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr

225 230 235 240

Gly Ile Lys Val Asn Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe

245 250 255

Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe

260 265 270

Gly Gly Gln Asp Pro Ser Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile

275 280 285

Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn

290 295 300

Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr

305 310 315 320

Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly

325 330 335

Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu

340 345 350

Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys

355 360 365

Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys

370 375 380

Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asp Ile

385 390 395 400

Ser Asp Lys Asn Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile

405 410 415

Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr

420 425 430

Lys Ile Gln Met Cys Lys Ser Val Lys

435 440

<210> 3

<211> 1112

<212> DNA

<213>artificial

<400> 3

ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 60

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 120

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 180

ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 240

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 300

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 360

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 420

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 480

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 540

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 600

actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 660

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg 720

gatcccatca tcatcatcat catatggatg aaaacctaga gcaggtgagc ggcatcatcg 780

ggaacctccg tcacatggcc ctggatatgg gcaatgagat cgatacacag aatcgccaga 840

tcgacaggat catggagaag gctgattcca acaaaaccag aattgatgag gccaaccaac 900

gtgcaacaaa gatgctggga agtggtgaat tcgcccaggt ggatgaggtg gtggacatca 960

tgagggtgaa cgtggacaag gtcctggagc gagaccagaa gctgtcggag ctggacgacc 1020

gtgcagatgc actccaggcg ggggcctccc agtttgaaac aagcgcagcc aagctcaagc 1080

gcaaatactg gtggaaaaac tgctaaaagc tt 1112

<210> 4

<211> 367

<212> PRT

<213>artificial

<400> 4

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly

225 230 235 240

Ser His His His His His His Met Asp Glu Asn Leu Glu Gln Val Ser

245 250 255

Gly Ile Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu

260 265 270

Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp

275 280 285

Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met

290 295 300

Leu Gly Ser Gly Glu Phe Ala Gln Val Asp Glu Val Val Asp Ile Met

305 310 315 320

Arg Val Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu

325 330 335

Leu Asp Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu

340 345 350

Thr Ser Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Cys

355 360 365 367

<210> 5

<211> 870

<212> DNA

<213>artificial

<400> 5

ataatgaaat acctattgcc tacggcagcc gctggattgt tattactcgc ggcccagccg 60

gccatggccg aggtgcagct gttggagtct gggggaggct tggtacagcc tggggggtcc 120

ctgagactct cctgtgcagc ctctggattc acctttagca gctatgccat gagctgggtc 180

cgccaggctc cagggaaggg gctggagtgg gtctcaaata tttcttctaa tggtaatgct 240

acagcttacg cagactccgt gaagggccgg ttcaccatct ccagaaacaa ttccaagaac 300

acgctgtatc tgcaaatgaa cagcctgaga gccgaggaca cggccgtata ttactgtgcg 360

aaatatactt atgcttttga ctactggggc cagggaaccc tggtcaccgt ctcgagcggt 420

ggaggcggtt caggcggagg tggcagcggc ggtggcgggt cgacggacat ccagatgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcagagca ttagcagcta tttaaattgg tatcagcaga aaccagggaa agcccctaag 600

ctcctgatct attatgcatc caatttgcaa agcggggtcc catcaaggtt cagtggcagt 660

ggatctggga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 720

tactactgtc aacaggattc ttatactcct tctacgttcg gccaagggac caaggtggaa 780

atcaaacggg cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcatc 840

tcagaagagg atctgaatgg ggccgcatag 870

<210> 6

<211> 289

<212> PRT

<213>artificial

<400> 6

Ile Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu

5 10 15

Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly

20 25 30

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

35 40 45

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

50 55 60

Gly Lys Gly Leu Glu Trp Val Ser Asn Ile Ser Ser Asn Gly Asn Ala

65 70 75 80

Thr Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asn

85 90 95

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Thr Tyr Ala Phe Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn

195 200 205

Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Asp Ser Tyr Thr Pro Ser Thr Phe Gly Gln Gly

245 250 255

Thr Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His

260 265 270

Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

275 280 285

Ala

289

<210> 7

<211> 870

<212> DNA

<213>artificial

<400> 7

ataatgaaat acctattgcc tacggcagcc gctggattgt tattactcgc ggcccagccg 60

gccatggccg aggtgcagct gttggagtct gggggaggct tggtacagcc tggggggtcc 120

ctgagactct cctgtgcagc ctctggattc acctttagca gctatgccat gagctgggtc 180

cgccaggctc cagggaaggg gctggagtgg gtctcatcta ttgcttctac tggttctaat 240

acagcttacg cagactccgt gaagggccgg ttcaccatct ccagaaacaa ttccaagaac 300

acgctgtatc tgcaaatgaa cagcctgaga gccgaggaca cggccgtata ttactgtgcg 360

aaaggtactg gtacttttga ctactggggc cagggaaccc tggtcaccgt ctcgagcggt 420

ggaggcggtt caggcggagg tggcagcggc ggtggcgggt cgacggacat ccagatgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcagagca ttagcagcta tttaaattgg tatcagcaga aaccagggaa agcccctaag 600

ctcctaatct atactgcatc ctacttgcaa agtggggtcc catcaaggtt cagtggcagt 660

ggatctggga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 720

tactactgtc aacaggctac tactagtcct tatacgttcg gccaagggac caaggtggaa 780

atcaaacggg cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcatc 840

tcagaagagg atctgaatgg ggccgcatag 870

<210> 8

<211> 289

<212> PRT

<213>artificial

<400> 8

Ile Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu

5 10 15

Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly

20 25 30

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

35 40 45

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

50 55 60

Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ala Ser Thr Gly Ser Asn

65 70 75 80

Thr Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asn

85 90 95

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Thr Gly Thr Phe Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr Ala Ser Tyr

195 200 205

Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Ala Thr Thr Ser Pro Tyr Thr Phe Gly Gln Gly

245 250 255

Thr Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His

260 265 270

Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

275 280 285

Ala

289

<210> 9

<211> 870

<212> DNA

<213>artificial

<400> 9

ataatgaaat acctattgcc tacggcagcc gctggattgt tattactcgc ggcccagccg 60

gccatggctg aggtgcagct gttggagtct gggggaggct tggtacagcc tggggggtcc 120

ctgagactct cctgtgcagc ctctggattc acctttagca gctatgccat gagctgggtc 180

cgccaggctc cagggaaggg gctggagtgg gtctcaggta tttctcctat gggtactcgt 240

acatcgtacg cagactccgt gaagggccgg ttcaccatct ccagagacaa ttccaagaac 300

acgctgtatc tgcaaatgaa cagcctgaga gccgaggaca cggccgtata ttactgtgcg 360

aaaaatgcta atgggtttga ctactggggc cagggaaccc tggtcaccgt ctcgagcggt 420

ggaggcggtt caggcggagg tggcagcggc ggtggcgggt cgacggacat ccagatgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcagagca ttagcagcta tttaaattgg tatcagcaga aaccagggaa agcccctaag 600

ctcctgatct atagggcatc cgttttgcaa agtggggtcc catcaaggtt cagtggcagt 660

ggatctgaga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 720

tactactgtc aacagcggag tcggccgcct attacgttcg gccaagggac caaggtggaa 780

atcaaacggg cggccgcaca tcatcatcac catcacgggg ccgcagaaca aaaactcatc 840

tcagaagagg atctgaatgg ggccgcatag 870

<210> 10

<211> 289

<212> PRT

<213>artificial

<400> 10

Ile Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu

5 10 15

Ala Ala Glu Pro Ala Met Ala Gly Val Gln Leu Leu Glu Ser Glu Glu

20 25 30

Glu Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

35 40 45

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro

50 55 60

Gly lys Gly Leu Glu Trp Val Ser Gly Ile Ser Pro Met Gly Thr Arg

65 70 75 80

Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

85 90 95

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Thr Gly Thr Phe Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Glu Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu leu Ile Tyr Arg Ser Val leu

195 200 205

Gln Ser Gly Val Pro Pro Ser Arg Phe Ser Gly Ser Gly Ser Glu Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Arg Ser Arg Pro Pro Ile Thr Phe Gly Gln Gly

245 250 255

Thr Lys Val Glu Ile Lys Arg Ala Ala Ala His His His His His His

260 265 270

Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

275 280 285

Ala

289

Claims (4)

1. the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, base sequence such as sequence table SEQ ID NO.5 It is shown.

2. the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity, amino acid sequence such as sequence table SEQ ID Shown in NO.6.

3. the human single chain variable fragments antibody 3A-scFv of anti-botulinum toxin type B enzymatic activity described in claim 1 treats Type B meat in production Application in terms of toxin poisoning by enzyme drug.

4. detecting the kit of botulinum toxin type B, it includes the source of people of anti-botulinum toxin type B enzymatic activity described in claim 1 Single-chain antibody 3A-scFv.