CN104892754A - Human single-chain antibody 2F-scFv having anti-botulinum toxin type B enzyme activity and application thereof - Google Patents

Abstract

The invention provides a human single-chain antibody 2F-scFv having anti-botulinum toxin type B enzyme activity. The human single-chain antibody 2F-scFv is screened out from a humanized library of antibodies having anti-botulinum toxin type B enzyme activity by utilizing artificially synthesized recombinant protein BontB and a substrate GFP-HIS6-SNAP25(62)-VAMP(57)-C used for screening the human single-chain antibody 2F-scFv having anti-botulinum toxin type B enzyme activity; and the screened human single-chain antibody 2F-scFv has an affinity constant of (1.146+/-0.525)10<6> L/mol and lays a foundation for production of specific anti-botulinum toxin type B treatment drugs and for rapid detection of botulinum toxin type B.

Description

The human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to the human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity, and the application in production for treating botulinum toxin type B poisoning by enzyme medicine, in detection botulinum toxin type B enzyme, also relate to the substrate of the human single chain variable fragments antibody 2F-scFv screening anti-botulinum toxin type B enzymic activity.

Background technology

Botulinus toxin can cause people and animal sausage poisoning, its infective dose is extremely low, and draw from primate experiment, minimum lethal dose (MLD) (LD50) is 1ng/kg, therefore, it is appointed as the biological weapon threat of the mankind being similar to anthrax by CDC of the U.S..Cause the reason of sausage poisoning to be roughly divided into four types, one, adult sausage poisoning.Two, infections in infants sausage poisoning.Three, wound infection sausage poisoning.

Botulinus toxin is that Clostridium botulinum is formed in its natural state or in the medium, is discharged in environment or nutrient solution by bacteria lysis, and the toxin of this form is called as precursor toxin, its molecular weight ranges at 300 kDa to 900 kDa.Precursor toxin exists with the form of complex body usually, by neurotoxin (Bont), can surround and protect the non-toxic albumen of toxin to form, comprise hemagglutinin (HA), non toxin non hemaglutinin (NTNH) by one or more.Said botulinus toxin is often referred to the neurotoxin part with neurotoxic effect clinically.

Botulinus toxin is according to its antigenic difference, co-exist in seven kinds of serotypes (A, B, C, D, E, F, G), although the botulinus toxin moiety of seven kinds of serotypes and its mechanism of action slightly difference, they have close molecular weight and analog structure.Ripe botulinus toxin forms by the single chain polypeptide of about 150 kDa, by the proteolytic enzyme of bacteria mediated, the cutting of the single chain polypeptide of 150kDa is cracked into two unequal polypeptide fragments during protein maturation, two fragments are connected by a disulfide linkage, form the heavy chain fragment (HC) that light chain segments (LC) that a molecular weight is 50kDa and molecular weight are 100kDa.Wherein light chain has a catalytic domain, and heavy chain comprises two functional domains that molecular weight is 50kDa, and N end is divided into membrane-spanning domain, and C end is divided into Sphingolipids,sialo binding domain.Independent light chain and the equal nontoxicity of heavy chain, only have double-strand just to have biology toxicity.

Light chain is made up of intracellular toxin albumen, has zine ion and relies on endonuclease activity, can suppress the release of neurotransmitter.(~ 20 is dark) is buried in light chain, surface negative charge in the active zone of botulinus toxin, obtains by a passage.The avtive spot of shuttle shape neurotoxin comprises a single zinc atom, and this zinc atom has always been considered to function, but does not play structural effect.By removing zinc with metal chelator, but can regain zinc by adding free zinc atom in secondary structure, the ability of toxin and Binding Capacity is almost constant, but the biologic activity of light chain greatly reduces, and is only equivalent to original 30%.Be positioned at zinc binding modules HExxH (X the is arbitrary amino acid) sequence that there is a high conservative at light chain center.The zine ion integrated by 3 Histidines containing one in HExxH sequence, and to cynapse vacuolar membrane protein, there is specific proteolysis activity, this catalytic site is arranged in the darker crack of protein surface.

In mankind's sausage poisoning, botulinum toxin type A and botulinum toxin type B occupy most important status, His 222 wherein on botulinum toxin type A light chain, Glu223 and His226 residue constitutes a part for zinc binding modules HExxH sequence jointly, and plays direct effect in the mechanism of action coordinating active zone zinc (His 222 and His226) and a water molecules (Glu223 residue).Glu261 is the 3rd part coordinating zinc.Even someone thinks that the catalytic mechanism of botulinum toxin type A and thermolysin are similar, this is because on the basis of structure based and sequence similarity, their avtive spot is also similar.Sudden change from Glu223 to Asp makes activity almost all lose, and removes His226 and makes botulinum toxin type A lose catalytic activity.By showing botulinus toxin structural analysis, the critical amino acid residues of being correlated with mechanism of action in A, botulinum toxin type B is identical.The crystalline structure of the botulinum toxin type B be combined with synaptobrevin shows, Arg369 and Tyr372 residue is positioned at close by the peptide bond of toxin cracking.With the sudden change of residue, catalytic rate constant is obviously reduced in botulinus toxin, but not affecting zinc combines and Binding Capacity, people propose, and these residues have played effect in transition state is stable.

In addition, some amino-acid residues are also had, as Phe266, Glu350Arg363 and Tyr366 in LC active centre.Their function mainly maintains and the relative position of the structure in stable light chain active centre and/or it and combined substrate, ensures the enzymolysis ability of light chain the best with this.

LC to be held with the N of HC by disulfide linkage and is connected.Under the state that disulfide linkage exists, toxin heavy chain HN structural domain forms a loop structure, by the Zn in over head and ears light chain surface crack ²⁺catalytic site covered, thus did not show enzymic activity.After double-strand toxin enters neurocyte, disulfide linkage is reduced, and the light chain discharged could show zine ion endopeptidase activity, and catalytic site can hold substrate 16 amino-acid residues.The zine ion of catalytic site is formed except coordinate bond except with the imidazole ring of 4 histidine residues, 1 water molecules being incorporated into conservative L-glutamic acid, and also form coordinate bond with the L-glutamic acid carboxyl of another molecule, 1 tryptophan modules probably also participates in the coordination of zine ion.This unique zinc ion coordination form, the space structure of toxin light chain are all different from the three-dimensional structure of other all known metalloproteases, should belong to a novel Metalloproteinase familv.

The pathogenesis of botulinus toxin

1, the identification of target cell and combination

Precursor toxin enters gi tract, and in digestive process, non-toxin component plays a protective role to neurotoxin, makes it can not be subject to the erosion of various digestive ferment and hydrochloric acid in gastric juice.Because molecular weight is excessive, lps molecule is not the mode spread, but passes through epithelium of intestinal mucosa barrier in the mode of " indent " and cellular uptake, and can not destroy gi tract.Then enter in small intestine, under the slight alkalinity environment of small intestine, the neurotoxin in front extracting toxin dissociates out, enters blood and lymphokinesis, plays its toxic action at motorius and sympathetic nerve place.Although neurotoxin can invade different tissues and organ, can not penetrate hemato encephalic barrier, its only affine target spot is peripheral cholinergic nerve tip.At cholinergic nerve endings, the light chain of toxin can suppress neurotransmitter acetylcholine in the release of neuromuscular junction, causes muscular paralysis.In this toxicity process, each functional domain of neurotoxin has participation, and concrete process can be divided into three phases.

The combination of target cell and internalization

In the identification of target cell and in conjunction with in this process, the binding domain of botulinus toxin heavy chain has played effect.Binding domain can identify cholinergic nerve endings, and with its generation specific binding.Binding domain is attached to the Sphingolipids,sialo of motor neuron and is positioned at the little protein receptor tied of terminal nerve surface of cell membrane, particularly has the GD1b of avidity, GT1b and GQ1b class Sphingolipids,sialo.Then ceramide is connected to by the Sphingolipids,sialo formed containing sialic oligosaccharides.By the research distributed to Sphingolipids,sialo and to its affine bonding force, can determine, Sphingolipids,sialo are not uniquely to promote the molecule that botulinus toxin combines, and the proposition of amboceptor model shows, before generation internalization, botulinus toxin has just been attached on protein receptor.Although there are some evidences to show, synaptotagmin may take part in the internalization of A, B and E type, and the acceptor of each member of fusobacterium family, does not also determine completely.Each toxin and its albumen co-receptor are specific cognate, and botulinum toxin type B is just similar to botulinum toxin protein part to the mixture of sialyl lactose, indicate " lock & key " principle that neurotoxin is combined with Sphingolipids,sialo.This is observed and successfully describes a viewpoint, and that is exactly that Sphingolipids,sialo combination makes botulinus toxin near protein receptor, facilitates identification and the internalization of toxin.

Once be attached to neuron surface, parent molecule cracking, heavy chain and light chain are combined by zinc atom and arrive neuronal cell film, and are entered cell vesicle by internalization, form the acid vesicles that is wrapped in lps molecule.I.e. internalization process.This receptor-mediated endocytic processes and time, temperature and synaptic activity are all relevant.

2, transposition

The light chain of botulinus toxin must from vesica cell or endosome out, and can successfully enter cytosolic target protein by film transport under H chain N-end effect, and this process is called light chain transposition.

The same with other clostridial toxin, after botulinus toxin enters intracellular acidic vesicles, can the recurring structure restructuring because of the change of pH.Although it is apparent for entering vesicle in synaptic vesicle and born of the same parents, botulinus toxin transposition enters the characteristic of vesicle in born of the same parents and not yet absolutely determines.The reduction of pH makes botulinus toxin structure change, thus causes it to have larger hydrophobicity in the molecule, and adds the perviousness of lipid bilayer.The heavy chain of such toxin and light chain can embed in the lipid bilayer of vesicle, once embed, will form ionic channel on phospholipid bilayer and PC12 film.This passage has optionally, and the molecule that molecular weight is large cannot pass through, so generally believe that toxin light chain can be transported to neurocyte kytoplasm by ionic channel in vesicle chamber at present.There are three kinds of light chain transmembrane transport hypothesis models accordingly, i.e. pipeline model (tunnel model), fractured model (cleft model) and cracking model (lysis model).Wherein fractured model theory is thought, when low pH, the molecular construct of protein there occurs change, and now HC can form a hydrophilic crack, and wears in membrane process at light chain and surrounded by its water-wetted surface.Define wetting ability and hydrophobic interaction in this mode.The carboxyl terminal of light chain enters into kytoplasm at first because disulfide linkage is connected with heavy chain aminoterminal.After realizing transposition, because the pH of kytoplasm is higher than acidic vesicles, light chain construct is folded again, comes back to and there is catalytic activity.

At low ph values, the change on the HN territory occurred conformation of A and botulinum toxin type B, final artificial rust of crossing over forms ionic channel, and under the environment of pH in vivo, the activity of the passage of formation is maximum.It is similar that the ionic channel that C BOTULINUM TOXIN TYPE A A is formed at adipose membrane and diphtheria toxin are formed, and be also only formed at a low ph.Botulinus toxin ionic channel only allows positively charged ion to pass through, but can stop chloroquine in vitro.But for the research of small-bore ionic channel, we not yet know that the passage that botulinus toxin is formed is that toxin light chain enters cytosolic the only way which must be passed.Data presentation, the structure of light chain has a theatrical change at a low ph, may produce an optionally LC structure and be more suitable for transposition exactly.Although this is not also proved in vivo, be completely reversibility by low pH inducement structure change.

Botulinus toxin can play a role in neurotransmission, and the surface that just LC and HN joint must be exposed by proteolysis produces breach, thus makes inactive single chain molecule amount be the toxin activation of 150-kDa.The bacterium of minority and kethepsin can realize this scission reaction, and produce activated double-strand neurotoxin.Produce after otch, light chain and heavy chain still by non-covalent interaction, and are connected by a single disulfide linkage.The minimizing of the disulfide linkage (the Cys430 – Cys454 in botulinum toxin type A) of this interchain is the subordinate phase of light chain activation, and this is that light chain freely enters motor neuron cytosol and stage requisite with substrate interphase interaction.As noted, in low pH situation, light chain has a theatrical change.The change of this structure contributes to the restructuring of easy bit strip (if being near light chain really in translocation point HN territory) and causes the final activation of light chain.NGF signal transduction pathway can with botulinus toxin-LC activity maintain close ties with together with.Therefore, completing of light chain activation finally may occur in cytosol, and then transposition from the cell of low pH.

3, neurotransmitter regulator is suppressed

Neural brief summary place has synaptic vesicle to steep, and wherein comprises vagusstoff, and it is a kind of neurotransmitter can shunk through neuromuscular junction stimulated muscle.The vesicles of this vagusstoff be called by one the protein polymer of SNARE complex body combine.In order to realize the transmission of signal through neuromuscular intersection, the vagusstoff vesicles in presynaptic nerve brief summary must be released in synaptic cleft.Here, neurotransmitter is attached on special receptor on muscle plate, triggers ionic channel and opens, thus cause adjacent striate depolarize and contraction.The release of vagusstoff, needs the participation of SNARE albumen, and it can mediate the fusion of synaptic vesicle and neuronal cell film.

SNARE complex protein participates in plasma membrane fusion on synaptic vesicle, and the effect of botulinum neurotoxin light chain hinders exocytosis.More particularly, be exactly at neuromuscular junction place, neurotoxin has blocked Vesicle-Containing by cutting snare protein (the necessary material of acetylcholine molecules exocytosis) and has been discharged in extracellular environment, suppresses the release of vagusstoff, thus suppresses neurotransmission.It is confirmed that, independent snare protein cracking can not hinder SNARE complex body to be formed, but non-functional mixture but can be caused at Ca ²⁺coupling between interior stream and fusion is destroyed.Toxin, when suppressing neurotransmitter regulator, needs Ca ²⁺participation, and at cynapse tip Ca ²⁺concentration increase can affect the action effect of botulinus toxin.

Light chain causes SNARE mixture unstable as zinc endopeptidase Protein cleavage, becomes non-functional, thus suppresses vagusstoff to be discharged into synaptic cleft.The quantity that vesica is discharged into synaptic cleft is fewer, and the probability that action potential is propagated is lower, thus causes the contraction of meat fiber.Result causes the Chemodenervation of muscle self and causes flaccid paralysis.Now there are some researches show that SNARE mixture is made up of VAMP, SNAP-25 and syntaxin tri-kinds of protein, following table is depicted as target protein and the cleavage site thereof of dissimilar botulinus toxin.

The ultimate principle of display technique of bacteriophage is that foreign DNA is inserted in the gene of phage encoded coat protein pIII or pVIiI, the expression product making heterologous DNA fragments corresponding merges in the coat protein of phage, forms fusion rotein (fusion protein), is presented on phage surface.The remarkable advantage of display technique of bacteriophage is: establish direct physical link between genotype (genotype) and phenotype (phenotype), thus make screening simple and effective.By foreign protein or expression of polypeptides in phage surface, just the affinity of it and other biological or abitotic substance can be utilized to screen the phage containing goal gene according to its character.For the screening of phage antibody library, Fab (fragment antigen binding) is expressed at phage surface, with corresponding antigen for affinity ligands screens, the phage of expression specificity antibody can be screened quickly and efficiently from a large amount of clone.Therefore, prepare high-affinity antibody with display technique of bacteriophage, compared with traditional authentic monoclonal antibody technology, there is obvious advantage.

Phage antibody, at Membrane surface expression, is formed fusion rotein by Fab section or ScFv and single stranded phage coat protein and completed.Be characterized in " it both the corresponding antigen of identifiable design and with its combination, again can infection host bacterium increase again ".Utilize the characteristic that it can increase again, with target antigen by affine absorption-wash-out-amplification, the part peptide chain of target molecule can be screened.Improve affinity of antibody through sudden change and strand displacement method again, finally just obtain the specific antibody of high-affinity.The screening of phage antibody library comprises two key steps: wash in a pan sieve and qualification.Naughty sieve is jointly hatched at phage antibody library and the antigen selected, and takes turns wash-out, collect the phage combined by several.The phage-infect bacterium of acquisition is increased, then carries out the naughty sieve of next round.Through several take turns wash in a pan sieve after, just can be enriched to the polyclone bacterial strain of the phage-infect be combined with antigen-specific.Qualification process picks out mono-clonal bacterial strain from the polyclone bacterial strain of phage-infect.Be about to wash in a pan sift out phage-infect bacterium, bed board, select, high-specificity monoclonal bacterial strain can be obtained.

Two kinds are mainly contained: (1) is by pure antigen coated on solid-phase media from the classical way of the phage of Phage Antibody Library expression specificity antibody, as enzyme plate, immunotubes or affinity column, then phage to be screened is added, wash away the phage of non-affinity or low affinity, reclaim the phage of high-affinity.(2) antigen is connected with vitamin H group, then be fixed in be coated with streptavidin paramagnetic beads on phage is screened.All need in two kinds of methods to close not by the site that antigen occupies with skimmed milk (or BSA, its effect is poor), to avoid the nonspecific combination of phage.For last method, the phage alkali solution that recovery is combined with antigen-specific is (as triethylamine, (triethy-lamine) or acidic solution (as glycine one hydrochloric acid, glycine_HCl) wash-out, or with solvable antigen or haptens wash-out; For a rear method, carry out wash-out by DTT (dithiothret01, DTT) by the disulfide linkage destroyed between antigen and vitamin H.The phage-infect Host Strains reclaimed, carries out next round screening after propagation.Generally take turns through 3-5 the clone that such screening can obtain the object antibody of expressing high-affinity.Each takes turns screening all needs to detect, to confirm the validity of screening.

Human Single Fold scFv Libraries I+J (Tomlinson I+J) that MRC HGMP resource center of Britain creates is current ripe full Large human naive scFv phage library, and its storage capacity is more than 10 ⁸total man source phage single-chain antibody, the anti-botulinum toxin type B single-chain antibody of this phage library elutriation specificity of this research and utilization, and systems analysis is carried out to positive-single strand antibody, establish basic substance to initiative botulinum toxin type B poisoning treatment antibody class medicine.

The present inventor is by the understanding of the mechanism of action of botulinus toxin and research, determine that botulinus toxin can cause zoonosis, and the nerve synapse system in primary challenge body, its pathogenesis is, botulinus toxin is incorporated into the presynaptic membrane of cholinergic neuron by its heavy chain C terminal domains, form toxoreceptor complex internalization and enter cell vesicle, then light chain zinc endopeptidase active zone discharges into tenuigenin from intracellular acidic spaces.Once discharge from vesica, light chain stops the release of vagusstoff by cutting SNARE complex proteins, causes muscular paralysis.Therefore light chain is main pathogenic position.At present, relatively comprehensive about the report of botulinum toxin type A both at home and abroad, but it is relatively less about the research report of botulinum toxin type B, lack botulinum toxin type B methods for the treatment of and medicine simultaneously, so just cause once there be people to suffer from Type B sausage poisoning, the circumstances of correct treatment cannot be carried out in time.Antibody is the most effective means for the treatment of sausage poisoning, but the heterology serum antibody of research or limited use at present also exists many shortcomings, as caused immune response or communicate illness etc., causes applying.Because this type of antibody molecule amount is larger, be difficult to permeates cell membranes, for entering the toxin light chain of cell then without result for the treatment of, therefore the present invention is intended to clonal expression B BOTULINUM TOXIN TYPE A A, in humanized antibody storehouse, screen neutrality Humanized single chain antibody by recombinant protein, for develop anti-B BOTULINUM TOXIN TYPE A A special efficacy treatment medicine and rapid detection botulinum toxin type B lay the foundation.

Summary of the invention

The object of the invention is for solving heterology serum antibody poisoning at treatment botulinum toxin type B, there is the problem causing immune response or communicate illness, and provide a kind of single-chain antibody ScFv of total man source, the human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity.

The human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity, its base sequence is as shown in sequence table SEQ ID NO.7.

The human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity, its aminoacid sequence is as shown in sequence table SEQ ID NO.8.

The application of human single chain variable fragments antibody 2F-scFv in production for treating botulinum toxin type B poisoning by enzyme medicine of anti-botulinum toxin type B enzymic activity.

The human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity is detecting the application in botulinum toxin type B.

The invention provides the human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity, it is the recombinant protein BontB utilizing synthetic, and screen the substrate GFP-HIS6-SNAP25(62 of human single chain variable fragments antibody of anti-botulinum toxin type B enzymic activity)-VAMP(57)-C, screen in humanization anti-botulinum toxin type B enzymic activity antibody library, affinity costant is (5.35 ± 0.903) × 10 ⁵l/mol, for produce anti-B BOTULINUM TOXIN TYPE A A special efficacy treatment medicine and rapid detection botulinum toxin type B lay a good foundation.

Accompanying drawing explanation

Fig. 1 is double digestion pMD-19T-Bont-B plasmid electrophoresis result; 1.EcoR and Nde double 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. BontB region pcr amplification product is.

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

Fig. 4 is that recombinant protein expression-form is identified wherein: 1. do not induce bacterium ultrasound precipitation; 2. induce bacterium ultrasound precipitation; 3. do not induce the ultrasonic supernatant of bacterium; 4. induce the ultrasonic supernatant state 5 of bacterium: the whole cell of induction; 6. protein Marker;

Fig. 5 is recombinant protein SDS-PAGE interpretation of result; Wherein: 1. protein marker; 2. the target protein of preliminary purification; 3. the target protein of final purifying;

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

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

2F-scFv SDS-PAGE interpretation of result after Fig. 8 purifying; 1: stream wears 2:55% ammonium sulfate former state 3: scFv after purifying.

Embodiment

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

According to the botulinum toxin type B complete genome sequence of Genbank report, design and synthesize light chain gene, insert EcoR simultaneously and Nde restriction enzyme site, enters pMD-19T vector, is transformed in JM109 by gene fragment clone.

embodiment 2:B BOTULINUM TOXIN TYPE A A light chain gene is connected with PET-28a carrier

Utilize restriction endonuclease EcoR and Nde double digestion plasmid pMD-19T-Bont-B, digestion products, through agarose gel electrophoresis analysis, the results are shown in Figure shown in 2, can see the goal gene BontB that size is about 1335, conforms to the size of expection;

By EcoR and Nde carrier PET-28a large fragment and the goal gene BontB small segment of double digestion reclaim, and connect with T4 DNA ligase, obtain recombinant plasmid PET-28a-BontB, transformation of E. coli JM109 competent cell, the agarose plate containing kan resistance carries out preliminary screening.Picking individual colonies is cultivated in LB liquid nutrient medium; Reclaim test kit with plasmid and extract plasmid.

EcoR is inserted by synthesis and Nde the botulinum toxin type B light chain gene sequence of restriction enzyme site, for it designs primer, upstream primer is designated as P1, and downstream primer is designated as P2.

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

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

With recombinant plasmid PET-28a-BontB for template, P1 and P2 is that primer carries out amplified reaction, the PCR primer obtained, through agarose gel electrophoresis identification and analysis, and (see figure 3);

Double digestion qualification is carried out, the mensuration of line order of going forward side by side row with restriction enzyme and EcoR I and Nde I.Visible plasmid, after double digestion, after pcr amplification object band, 1335bp all has goal gene band (see figure 4), proves that goal gene BontB gene has been connected 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

Transformed by recombinant plasmid PET-28a-BontB and express bacterium-e. coli bl21 (DE3), after IPTG abduction delivering, SDS-PAGE result shows: recombinant protein PET-28a-BontB has one obviously to express band at about 50KD place, and size conforms to theoretical value; See Fig. 5;

Cleer and peaceful precipitation on expression of recombinant proteins bacterium lysate is done SDS-PAGE electrophoresis simultaneously, wherein the ratio of supernatant and precipitation is 1:10, result shows, target protein is most in supernatant, in precipitation also there is a small amount of target protein in corresponding position, think by the optimization of inductive condition, can express with soluble form under suitable induced environment, see Fig. 5.

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

The preliminary purification of recombinant protein: prepare bacterium in a large number, abduction delivering recombinant protein, soluble proteins first carries out DEAE anion-exchange chromatography post, and collects Liu Chuan peak, then carries out genus chelate chromatography Cu2+ post, collects the target protein of 200mM imidazoles wash-out.

With Thrombin enzyme excision His-Tag

Recombinant protein secondarily purified: enzyme cut after albumen carry out genus chelate chromatography Cu2+ post, collect the target protein of 250mM imidazoles wash-out.The albumen collected, through SDS-PAGE interpretation of result, as figure, can have an obvious band, and purification efficiency can reach more than 90%, sees Fig. 5 at about 50KD place.

embodiment 5: the screening of the anti-Bont-B-scFv antibody in total man source

The recombinant protein of purifying is antigen coated on 96 hole enzyme plates, and 4 DEG C are spent the night.Next day abandons supernatant, and the 37 DEG C of closed 2h of the Milk-PBS with 2%, add phage antibody library, and titre is 1.0 × 10 ¹³, acutely rock under room temperature and hatch 60min, leave standstill 60min.After discard liquid, wash 10 times with containing the PBS of 0.1%Twenn-20, pat dry by liquid light residual in every hole after washing, every hole adds 50 μ L elutriants (pancreatin-PBS of 5mg/mL), acutely rocks 10min, wash-out bacteriophage under room temperature, collects 4 DEG C of preservations.

With the Phage Infection E.coli TG1 under wash-out, and coat (glucose containing 100 μ g/mLAmp and 1%) 37 DEG C of incubated overnight on TYE flat board.Utilize helper phage KM13 amplification phage library, reclaim phage by PEG/NaCl.Repeat above process 3 times, totally 4 take turns screening, collect the phage library of screening.

gene constructed and the purifying of embodiment 6:Bomt-B Activity determination substrate-GFP-SV

According to SNAPE25, VAMP gene order logged in GenBank, design and synthesize HIS6-SNAPE62-EcoRI-VAMP57C-TAA gene, insert BamHI and Hind III restriction enzyme site respectively at gene two ends simultaneously, synthesis recombinant plasmid pMD19-T-SV.

According to green fluorescent protein GFP gene, design a pair Auele Specific Primer,

Upstream primer P1:5 '-CATGccATGGTGAGCAAGGGCG-3 '

Downstream primer P2:5 '-GCGGATCCcttgtacagCTCGTCCATG-3 '

With P1 and P2 for primer, GFP plasmid is template, pcr amplification GFP gene, restriction endonuclease NcoI, BamHI double digestion carrier pET-28a and 720bp PCR is utilized to reclaim product,, connect pET-28a and GFP gene fragment with T4DNA ligase enzyme, connect product conversion competence E.coli JM109, next day, picking list bacterium colony, extracted plasmid.PCR, double digestion and order-checking qualification are carried out to it, show that GFP successful clone is in pET-28a, base does not occur and loses and sudden change, successful carrier construction pET-28a-GFP.

With restriction endonuclease Hind III and BamHI to the recombinant plasmid pMD19-T-SV of synthesis and recombinant expression plasmid pET-28a-GFP double digestion, reclaim 393bp small segment and pET-28a-GFP carrier large fragment respectively, connect with T4DNA ligase enzyme.Connect product (pET-28a-GFP-SV) transformed competence colibacillus E.coli JM109, coating Kan(50 μ g/ml) LB flat board on, 37 DEG C of overnight incubation.Next day, picking list bacterium colony, extracted plasmid, after double digestion and order-checking qualification, showed that SV gene is successfully inserted in pET-28a-GFP carrier, successfully built pET-28a-GFP-SV expression vector.

Recombinant plasmid pET-28a-GFP-SV is transformed and expresses bacterium-e. coli bl21 (DE3), IPTG abduction delivering, determine best phraseology: LB substratum, 37 DEG C, 3 h.Through SDS-PAGE electrophoresis detection after ultrasonication thalline, determine that target protein is most in supernatant, therefore target protein is expressed with soluble form.

The purifying of recombinant protein GFP-SV, expression of recombinant proteins thalline is through ultrasonic degradation, and successively through DEAE anion-exchange chromatography, metal chelate chromatography, Q cation-exchange chromatography, obtains the sterling GFP-SV of purity more than 90%.Pyrogen detects <20EU/mg, meets the needs of property of protein research.

embodiment 7: anti-Bont-B-scFv positive strain qualification

Phage Infection after screening e.ColihB2151, after abduction delivering, utilizes ELISA to identify, put microplate reader and measure OD value (wavelength is 490nm), each sample does diplopore and measures, and gets OD mean value.Take 2%Milk-PBS as negative control, positive colony bacterial strain settles the standard and is: OD value is more than 3 times of negative control, obtains 200 strain positive colony bacterial strains altogether.

Positive strain biological activity assay, GFP-SV coating buffer is diluted, every hole is wrapped by 30 μ g in check-out console (Pierce Maleimide Activated 96-well Plates, Black, 15153) in, 4 DEG C are spent the night, and wash 3 times with Wash buffer, by 200 strain positive strains, after abduction delivering is centrifugal, 4 μ g Bont-B cleer and peaceful on 100 μ L are mixed, joins in check-out console, 37 DEG C of effect 2h, every increment product do diplopore and measure, wash 3 times with Wash buffer again, fluorescence intensity in multi-functional microplate reader, draw the active bacterial strain preferably of 3 strains.According to the gene order of the pIT-2 carrier on Tomlinson I+J test kit, synthesize two Specific PCR primers amplification scFv full genome fragments.

P1 LMB3: 5’—CAG GAA ACA GCT ATG AC—3’

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

, all can there is obvious 900bp band, containing complete scFv in 3 strain positive strains after testing.

Order-checking its sequence of qualification also draws through Blast database analysis: show that 3 strain positive strains are human single chain variable fragments antibody, be 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 WV 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 YA 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 WV 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 GQ 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 antibody affinity costant KD pH-value determination pH

3 strain strong positive clones bacterial strains, 37 DEG C of cultivations, to OD600 to 0.9, add inductor 30 DEG C of overnight incubation (16h), overnight culture 4 DEG C, the centrifugal 30min of 3500 × g, and supernatant is the scFv expressed, and size is 31000.Induction supernatant, successively after 55% saturation ratio ammonium sulfate precipitation and rProtein-A affinity chromatography, obtains the sample of purity more than 90%.

Utilize non-competing enzyme immunoassay to measure the affinity costant of 2F, 4C and 3A single-chain antibody, wrap by Bont-B with 4 μ g/mL, 2 μ g/mL, 1 μ g/mL and 0.5 μ g/mL respectively, single-chain antibody concentration is adjusted to 10 ^-6mol/L, doubling dilution 1:2 ~ 1:512, the Protein A-HRP antibody doubly diluted with 1:5000 resists as two, and OPD develops the color, and measures OD490nm light absorption value, and each sample does diplopore and measures, and gets OD mean value.According to the sigmoid curve figure of antigen-antibody binding reaction, the antibody concentration of writing on the blackboard light absorption value under different antigen concentration can be solved, be brought into formula KA=(n-1)/2 (nAb '-Ab) calculate affinity costant, wherein Ab ' and Ab represents when antigen is Ag ' and Ag, produce the antibody concentration (mol/L) of half light absorption value, n=Ag/Ag ', 3 KA values can be obtained as n=2,2 KA values can be obtained during n=4, obtain 1 KA value during n=8, it is exactly final affinity costant numerical value that six KA values are taken the mean.

Find out that the affinity costant of 4C single-chain antibody is for (4.38 ± 0.445) × 10 from table ⁵the affinity costant of L/mol, 3A is (5.35 ± 0.903) × 10 ⁵l/mol, 2F are (1.146 ± 0.525) × 10 ⁶l/mol.

Sequence table

<110> MILITARY VETERINARY INST ACADE

The human single chain variable fragments antibody 2F-scFv of the anti-botulinum toxin type B enzymic activity of <120> and application thereof

 

<160> 10

 

<210> 1

<211> 1335

<212> DNA

<213> is 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> is 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> is 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> is 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 The 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> is 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> DNA

<213> is 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> is 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> is 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 Aal 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> is 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> is artificial

 

<400> 10

 

Ile met lys tyr leu leu pro thr ala ala ala gly leu leu leu leu

5 10 15

Ala Ala Gla 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 Thy 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 2F-scFv of anti-botulinum toxin type B enzymic activity, its base sequence is as shown in sequence table SEQ ID NO.7.

2. the human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity, its aminoacid sequence is as shown in sequence table SEQ ID NO.8.

3. the application of human single chain variable fragments antibody 2F-scFv in production for treating botulinum toxin type B poisoning by enzyme medicine of anti-botulinum toxin type B enzymic activity according to claim 1.

4. the human single chain variable fragments antibody 2F-scFv of anti-botulinum toxin type B enzymic activity according to claim 1 is detecting the application in botulinum toxin type B.