CN101386648A - Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof - Google Patents
Neutralizing monoclonal antibodies against B type botulinum neurotoxin, preparation method and use thereof Download PDFInfo
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Abstract
The invention discloses a neutrality monoclonal antibody against botulinum neurotoxin type B and also discloses a humanized modifier of the antibody. The antibody is prepared through the following method: firstly, the CDR region, variable region, IgG part or full gene of the antibody is prepared through molecular biological methods or other methods and is expressed in prokaryotic cells such as colibacillus, eukaryote cells such as yeast, or insect cells, vegetable cells or mammal cells such as CHO; and the antibody is obtained through purification. The antibody is characterized by strong specific neutralization activity and high affinity. In addition, the humanized antibody is characterized by low side and toxic effect. The neutrality monoclonal antibody and the humanized modifier of the antibody are suitable for the first aid treatment of botulinum neurotoxin type B intoxication and also for the detection of botulinum neurotoxin type B. Therefore, the antibody and the humanized modifier thereof have good application prospect.
Description
Technical field
The present invention relates to a kind of toxinicide monoclonal antibody, be specifically related to a kind of neutralizing monoclonal antibody of anti-botulinum neurotoxin, also relate to the preparation method and the purposes of this antibody.
Background technology
Botulinum neurotoxin (Clostridium Botulinum Neurotoxins, BN are called for short botulinus toxin) is one group of protein (comprising the A-G type) that known virulence is the strongest.When botulinus toxin from contaminated food products or after infection enters human circulation, by the release of block nerves mediator vagusstoff (excitatory synapse mediator) in neuromuscular junction, cause the sausage poisoning symptom of serious nervous disorder and carrying out property neuromuscular paralysis, cause the unable and bulbar paralysis of skeletal muscle, serious respiratory insufficiency finally occurs, heartbeat stops and causing death.What cause human poisoning mainly is A, B, E type.The case statistics shows, and botulinum toxin type B is the most common serotype of China's sausage poisoning.
Use toxinicide provide passive immunization at toxin poisoning prevention and treatment in can play effective provide protection.At present, clinical specificity treatment sausage poisoning mainly is to use meat poison multivalence toxinicide-horse serum.The polyclone toxinicide (HIG) in horse serum source has been used for 80% above sausage poisoning patient's treatment.The polyclone toxinicide can be discerned a large amount of different epi-positions, can guarantee the existence of sub-fraction protection antibody.The anti-application of Tacket CO report horse in 1984, use horse antibody also is effective before being exposed to botulinus toxin and in exposing back 24 hours.But, use horse to resist significant side effects is arranged, there is 9% case serum sickness and anaphylaxis (Black RE, Gunn RA.Hypersensitivity reactions associated with botulinal antitoxin.Am J Med 1980 to occur approximately in the Black RE report clinical treatment; 69:567-570).Strong immunological rejection that this heterology produces and serum sickness etc. have seriously limited the antitoxic clinical application of horse serum.For fear of with overcome these side effects, people prepare human normal immunoglobulin (human BIG) (Gelzleichter TR from the serum of volunteering immune blood donor, Myers MA, MentonRG, et al.Protection against botulinum toxins provided by passive immunizationwith botulinum human immune globulin J Appl Toxicol.1999Dec; 19Suppl1:S35-8), be mainly used in the treatment of baby's sausage poisoning.Yet still there are a lot of problems in the application of human normal immunoglobulin, mainly comprises propagation, goods validity and specificity difference that potential blood source catches, and the blood donor of the goods deficiency etc. of originating, and these problems have restricted its application.And the potential problems that people usually ignore are, for satisfying the volunteer that the needs of producing human normal immunoglobulin carry out the immunity of meat poison toxoid, will lose chance and right with the multiple neuroregulation disorder disease of botulinum toxin treatments.Therefore, present research emphasis has turned to novel antitoxic preparation.Hc is the receptor binding domain of botulinus toxin, contains neutralizing epitope, and this site is a high special in the different serotypes toxin.Therefore become antibody research at target.
The mouse resource monoclonal antibody is the hybridoma excretory that is formed through cytogamy by mouse B cell and rat bone marrow tumour cell, and its preparation comprises animal immune, cytogamy, selection hybridoma, detects the mass production of antibody, hybridoma cloning and monoclonal antibody.Calendar year 2001 Pless DD etc. has prepared the segmental mouse monoclonal antibody at botulinum toxin type A Hc, and it is 0.06-0.9nM that SPR analyzes antibody dissociation constant scope, and epi-position is drawn to analyze and shown that these mouse source monoclonal antibodies are attached to segmental at least two different zones of Hc.The botulinum toxin type B toxoid immunity that usefulness such as Yang GH in 2004 do not contain the toxicity composition has prepared a strain at the segmental mouse monoclonal antibody BTBH-N1 with endotoxin neutralizing activity of Hc, report 10 μ g can be effectively in and 20LD
50The Type B toxin; Bavari Sina etc. has prepared the mouse monoclonal antibody at botulinum toxin type A, can potentially be applied to the detection and the treatment of toxin, and it has been applied for patent (UnitedStates Patent Application No.20060177881); Marks, James D etc. screen the mouse source neutralizing antibody that has obtained at botulinum toxin type A by phage library, and it has been applied for patent (UnitedStates Patent Application No.20040175385).But the relevant report of research shows that the therapeutic action that the monospecific antibody clone poisons to botulinus toxin is limited, therefore impels people to seek how more efficiently monoclonal antibody, with the cocktail therapeutic strategy of development monoclonal antibody.
Development of molecular biology in recent years, particularly the maturation of library technology has promoted the development of antibody genetic engineering greatly, have tangible technical superiority aspect the flux screening of specific monoclonal antibody, particularly from the antibody library of specific immunity, can be relatively easy to obtain high-affinity antibody.The antibody gene in immunity storehouse from immunity after individual immunoglobulin (Ig) mRNA, therefore, in immune storehouse, at certain antigenic antibody gene may be enrichment or through affinity maturation, obtain high-affinity specific antibody clone easily.Peter in 2002 etc. are the anti-BoNT/A-Hc antibody of screening from phage natural antibody library and BoNT/A phage immunity library, the result shows that antibody that immune library obtains can be discerned and in conjunction with two different epi-position I and II, ScFv in conjunction with I has endotoxin neutralizing activity (Amersdorfer P, Wong C, Smith T, et al, Genetic and immunological comparison of anti-botulinum typeA antibodies from immune and non-immune human phage libraries.Vaccine.2002Feb22; 20 (11-12): 1640-1648.).The antibody repertoire scope decision in immune antibody library can be screened the diversity and the neutralization activity of specific antibody, so the selected immunizing antigen of antibody library structure will have very strong neutralizing antibody inducibility.Early-stage Study and bibliographical information all show; the heavy chain C fragment albumen of Type B botulinum neurotoxin is protective antigen; can induce (the Yang Xiuqing etc. of immunoprotection completely of body; the preliminary study of botulinum toxin type B heavy chain C end expression of gene and provide protection; the biotechnology communication, 2005,16:147-149); be the preferred target antigen of preparation neutralizing antibody, can be applied to the screening of the structure and the monoclonal antibody gene in antibody mediated immunity library.
The botulinus toxin poisoning has sudden, uses toxinicide effective at the poisoning initial stage, do not use antibody preparation repeatedly in the time of therefore can not resembling the tumour antibody treatment, so mouse source property monoclonal antibody has the feasibility that is applied to botulinus toxin poisoning treatment.But, use mouse source antibody to treat institute's potential foreign protein transformation reactions risk in order further to reduce, provide the humanization technical tactic safer.At present, mouse monoclonal antibody humanization modified can be transplanted to the CDR sequence of mouse in people's antibody variable region framework, produces the humanized antibody that CDR transplants, perhaps according to the antibody protein structure elucidation carry out the antigenicity amino acid mutation, reinvent on the surface, and makes up mouse-people's chimeric antibody etc.
Summary of the invention
At the defective of existing botulinus toxin poisoning treatment with the toxinicide goods, the invention provides mouse source anti-Type B botulinum neurotoxin neutrality antibody MuBNbP8, the heavy chain of this antibody and the variable region of light chain have the aminoacid sequence shown in sequence in the sequence table 2 and sequence 4 respectively, and its encoding gene has the nucleotide sequence shown in sequence in the sequence table 1 and sequence 3 respectively.
Heterology problem at mouse source antibody, the present invention also provides the humanization modifier of above-mentioned antibody, be the anti-Type B botulinum neurotoxin of humanization neutrality antibody HuBNbP8, the variable region of this heavy chain of antibody and light chain has the aminoacid sequence shown in sequence in the sequence table 6 and sequence 8, and its encoding gene has the nucleotide sequence shown in sequence in the sequence table 5 and sequence 7.
Anti-Type B botulinum neurotoxin neutrality antibody of the present invention comprises that also the aminoacid sequence to above-mentioned mouse source and humanized antibody forms by interpolation, deletion, modification to amino-acid residue, has the antibody of identical function.
The anti-Type B botulinum neurotoxin of mouse of the present invention source and humanization neutrality antibody has the advantages that special neutralization is active by force, avidity is high, humanized antibody has more the characteristics of low toxic side effect, can develop into the emergency treatment preparation that the Type B botulinum neurotoxin is poisoned, not only can eliminate the anaphylaxis that the antiantitoxin horse serum easily causes, and can overcome the potential virus pollution problems that people's antitoxic immunity sphaeroprotein exists, have a good application prospect.
The present invention provides the preparation method of anti-Type B botulinum neurotoxin neutrality antibody MuBNbP8 and HuBNbP8 simultaneously.At first be preparation MuBNbP8 and HuBNbP8 encoding gene (as CDR district or variable region or IgG part or full gene), can adopt for example PCR method of chemosynthesis or genetically engineered, then protokaryon or carrier for expression of eukaryon are gone in this gene clone, as intestinal bacteria, yeast, insect cell, vegetable cell or mammalian cell expression vector etc., the preparation recombinant expression vector, wherein prokaryotic expression carrier can be pCANTAB-5E, pET series etc., Yeast expression carrier can be pPIC series, and the mammalian cell expression vector can be pEF1, pTriEx etc.Recombinant expression vector with preparation changes over to respectively in host cell such as intestinal bacteria, yeast, insect, plant or the mammalian cell etc. and expresses then, and wherein intestinal bacteria can be that GS115 etc., mammalian cell are CHO etc. for HB2151, BL21 (DE3) etc., yeast.The expression product purifying is obtained anti-Type B botulinum neurotoxin genetic engineering antibody.
Anti-Type B botulinum neurotoxin neutrality antibody MuBNbP8 gene of the present invention screens by the following method.At first reorganization prepares Type B meat poison receptor binding domain Hc albumen (BHc), preparation freund adjuvant vaccine, immunity Balb/C mouse, separation from peripheral blood lymphocyte, heavy, the chain variable region gene (VH and VK) of clonal antibody, make up phage antibody immunity library, mouse source, use the botulinum toxin type B target antigen with comparalive ease, adopt elisa plate solid phase antigen prize law, screening phage positive colony, and then obtain to have the special MuBNbP8 gene active, high-affinity that neutralizes.Implementing when of the present invention, this gene or gene fragment can be carried out chemosynthesis or are prepared by biological methods such as PCR by technology known in the art.
HuBNbP8 gene of the present invention obtains by the following method.The anti-Type B botulinum neurotoxin of humanization neutrality antibody HuBNbP8 changes structure to form on the basis of the maternal antibody MuBNbP8 in mouse source, by information biology mouse source antibody MuBNbP8 being carried out the branch submodule builds, in conjunction with the homology structural analysis, the antigen property acidic amino acid of applied molecular biology technical antagonism body variable region body structure surface carries out point mutation, makes up humanized antibody clone huBNbP8.Wherein, the CDR structure all at the framework region (FWR) of antibody variable region, is not disturbed in the mutational site.The antigen-binding specificity of humanized antibody HuBNbP8 does not change, and has kept the biologic activity of maternal antibody MuBNbP8.On this basis, can further utilize humanized variable region structure to obtain the full molecular antibody of humanization or other reshaping antibodies or derivative, be more suitable for using.Implementing when of the present invention, this antibody gene or gene fragment can be carried out chemosynthesis or transgenation by technology known in the art, gene splicing equimolecular biological method is prepared.Can express in intestinal bacteria, yeast, insect, plant or mammalian cell etc., through the humanized antibody albumen of purifying acquisition homogeneous, this product immunogenicity is low and toxic side effect is little, has higher security.
The present invention provides the purposes of anti-Type B botulinum neurotoxin neutrality antibody in the detection of Type B botulinum neurotoxin and urgent prevention of sausage poisoning and treatment simultaneously.In experiment, the recombinant antibodies albumen of MuBNbP8 and HuBNbP all can combine with Type B botulinum neurotoxin antigen-specific, and does not combine with other serotype botulinus toxins such as A type, E type, F types, shows as the specific selectivity of target antigen.Antibody of the present invention is anti-at existing therapeutic anti Type B meat poison horse, has tangible emulative target antigen in conjunction with activity.HuBNbP is to the avidity height (1.34nM) of target antigen, and bonding force is strong, has very strong combination stability and target binding ability.The anti-Type B botulinum neurotoxin neutrality antibody of genetic engineering technique preparation; can effectively neutralize a toxin in vivo; suppress the caused neural toxic effect of toxin; has the passive immunization provide protection; can protect mouse to escape the attack of lethal quantity (5LD50) Type B botulinum neurotoxin, play the effect of urgent prevention and treatment.Antibody of the present invention is to the body nontoxicity, uses saferly, effective, and the mechanism of action is clear and definite.Antibody of the present invention can be used for Type B botulinum neurotoxin poisoning prevention and treatment and Type B botulinum neurotoxin and detect.
The present invention is directed to the defective that present antiserum production exists, for satisfying the demand of development humanized antibody of new generation, a kind of brand-new specificity, the anti-Type B botulinum neurotoxin of high-affinity neutrality antibody are provided, the encoding gene of this antibody also is provided, and the further design construction of using gene engineering technique based on the humanized antibody modifier of Fv (variable region), and confirm by the inside and outside biological activity research.The invention also discloses the preparation method of above-mentioned antibody.Anti-Type B botulinum neurotoxin neutralizing antibody of the present invention has the characteristics of high specificity, avidity height, good stability, and its humanized antibody has more the low characteristics of immunogenicity.Substitute as the haematogenous immunoglobulin (Ig) has many good qualities: its effective constituent is clear and definite, is anti-Type B botulinum neurotoxin neutrality antibody; The specificity height is at the receptor binding domain (BHc) of Type B botulinum neurotoxin; Reduced immunogenicity, the humanization protein structure; Production that can be quantity-unlimiting can be amplified in genetically engineered preparation.Anti-Type B botulinum neurotoxin neutrality antibody of the present invention can develop into the urgent prevention of sausage poisoning and the special detection antibody of medicine and Type B botulinum neurotoxin, has vast market prospect.
Description of drawings
Fig. 1 is for extracting the agarose electrophoresis collection of illustrative plates of the total RNA product of mouse splenic lymphocyte.
Fig. 2 is murine antibody VH and VL gene amplification collection of illustrative plates: A figure is the agarose electrophoresis of VH family gene amplified production; B figure is the agarose electrophoresis of VL family gene amplified production.Wherein M is dna molecular amount standard DL2000; The 1-6 swimming lane is the PCR product of VH family gene or VL family gene.
Fig. 3 is mouse single-chain antibody (ScFv) gene pool of assembling, the agarose electrophoresis collection of illustrative plates of heavy chain and light chain gene splicing product, and wherein M is dna molecular amount standard DL2000; The 1-6 swimming lane is the PCR product of assembling ScFv.
Fig. 4 is that the recombination to construct of mouse source (MuBNbP8) and the anti-botulinum toxin type B antibody of humanization (HuBNbP8) is identified collection of illustrative plates with expression, and wherein 1 swimming lane is after the MuBNbP8 clone induces; 2 swimming lanes are after HuBNbP8 induces; M is the low molecular weight protein (LMWP) standard; 3 swimming lanes are before MuBNbP8 induces; 4 swimming lanes are for after inducing premutation.
Fig. 5 is that the Native-PAGE electrophoresis and the Western blot of purifying preparation of the scfv fusion protein of the anti-botulinum toxin type B antibody of humanization (HuBNbP8) identifies collection of illustrative plates, and wherein 1 swimming lane is the HuBNbP8 clone's of purifying the SDS-PAGE electrophorogram of scfv fusion protein; 2 swimming lanes are the Western blot collection of illustrative plates of corresponding protein; M is the low molecular weight protein (LMWP) standard.
Fig. 6 is the antigen-binding specificity of mouse source (MuBNbP8) and the anti-botulinum toxin type B antibody of humanization (HuBNbP8), and wherein A type, Type B, E type, F type botulinum neurotoxin are expressed as type A respectively, type B, type E and type F.
Fig. 7 is the antigen binding kinetics curve (Biacore mensuration) of the anti-botulinum toxin type B antibody of humanization (HuBNbP8), and wherein the concentration of curve is respectively 58.8,29.4,14.7,7.35 from high to low from top to bottom, 3.6725nM.
Fig. 8 is that the anti-botulinum toxin type B antibody of humanization (HuBNbP8) combines the botulinum toxin type B curve with the anti-competition of anti-Type B botulinum neurotoxin horse.
Embodiment
The structure and the screening in anti-Type B botulinum neurotoxin phage immunity library, embodiment 1 mouse source
One, material:
1. bacterial strain: helper phage M13K07 is available from Biolab company; Host bacterium E.coli TG1 is a Pharmacia company product.
2. reagent: restriction enzymes such as NotI, SfiI are Promega company product; FicollPlus Pague and anti M13-HRP are the Pharmacia product; Total RNA extraction reagent box, RT reverse transcription test kit, pcr amplification reagent are TAKARA company product; BSA (bovine serum albumin component V) is a GIBCO company product; DEPC, Pyocianil, tsiklomitsin and sulphuric acid kanamycin are available from SIGMA company.
3. carrier: phage vector pCANTAB-5E is a Pharmacia company product.
Two, methods and results:
1.B the reorganization preparation and the animal immune of BOTULINUM TOXIN TYPE A A receptor binding domain Hc albumen (BHc)
1.1B the reorganization of BOTULINUM TOXIN TYPE A A receptor binding domain Hc albumen (BHc) preparation
With pET22b is expression vector, recombinant expressed in intestinal bacteria (BHc) albumen, expression product through Ni post affinity purification to purity is〉90%, as immunogen protein.
1.2 animal immune
With immunogen protein and after freund adjuvant is mixed with fully, the Balb/C mouse is carried out first immunisation, then every carrying out booster immunization (wherein replacing complete freund adjuvant with incomplete freund adjuvant) 2 weeks, immunity is 4 times altogether.
2. the structure in the anti-botulinum toxin type B phage in mouse source immunity library
2.1 the design of primers of phage immunity library construction, referring to bibliographical information (Essono et al., Ageneral method allowing the design of oligonucleotide primers to amplify theviariable regeions from immunoglobulin cDNA, J Immuno Meth2003,279:251-266) etc., the serial primer of design is synthetic by Shanghai Bo Ya company.
2.2 the extraction of lymphocytic separation and cell total rna
Collect anticoagulation 40ml,, wash 3 times 2000rpm, 5min, 5.8 * 10 with PBS with 20ml Ficoll centrifugation oyster white buffy coat
7Cell suspension to final volume is 1ml.Extract cell total rna 20 μ g referring to test kit operation instructions (Modified RNAgents Total RNA Isolation protocols), the results are shown in accompanying drawing 1.
2.3cDNA synthetic
Referring to TAKARA test kit (BcaBESTTM RNA PCR KIT Ver1.1) operation instructions.Total RNA with purifying is a template, and cDNA first chain is synthesized in reverse transcription, and the RT reaction conditions is: 30 ℃ 10 minutes, 42 ℃ 30 minutes, 99 ℃ 5 minutes, 5 ℃ 5 minutes.
2.4PCR amplification antibody variable region VH and VL gene
With 2 μ l cDNA is template, adds 1.6mmol/L MgCl in the PCR reactive system
2, 1mmol/L dNTP, the Tag enzyme of adding 2.5U, warm start reduces nonspecific reaction.The PCR condition is: 25 circulations of 94 ℃ of 30 seconds, 57 ℃ 50 seconds, 72 ℃ 60 seconds row, last 72 ℃ were extended 10 minutes.Amplification purpose product Vh1-6 and VL1-6 carry out purifying, and size is 300-400bp, the results are shown in accompanying drawing 2.
2.5ScFv the assembling of single-chain antibody gene (splicing overlap extension, SOE)
Measure VH chain, VL chain and the relative amount of Linker DNA respectively, wait mole to mix, assemble by PCR.At first do not have primer splicing, 94 ℃ after 5 minutes, add 0.5 μ l (2.5U) Tag polysaccharase, carry out 20 circulating reactions, 94 ℃ 1 minute, 54 ℃ 2 minutes, 72 ℃ 3 minutes, last 72 ℃ were extended 10 minutes.Primer amplification is arranged then, introduces restriction endonuclease sites Sfi I/Not I, carry out 30 and take turns the PCR circulation at 5 ' and the 3 ' end of ScFv: 94 ℃ 1 minute, 55 ℃ 2 minutes, 72 ℃ 2 minutes.Last 72 ℃ were extended 10 minutes.PCR product size is about 750bp, the results are shown in accompanying drawing 3.
2.6 the structure of recombinant phage strand ScFv antibody library
2.6.1 ScFv is cloned into phage vector
PCANTAB-5E is a Pharmacia company commercialization carrier, extracts and cmy vector DNA from the TGl bacterium that has plasmid DNA according to a conventional method.PCR product and carrier pCANTAB-5E and difference purifying with Sfi I and Not I double digestion digestion ScFv.Then ScFv strand gene is cloned into the pCANTAB-5E carrier by ligation.
2.6.2 the structure of recombinant phage strand ScFv antibody library
To connect the quick bacterium of TGl electricity transmission that the product electricity transforms 200 μ l prepared beforehand, the electric commentaries on classics is set to: 2500V, shocked by electricity 30 seconds.Electricity changes the back and adds 5-10ml 2 * YT-G (containing 2% glucose), 37 ℃ of shaking culture 1 hour.Recombinant conversion bacterium 10 μ l are coated with the 2 * YT-ammonia benzyl plate that contains 2% glucose, 30 ℃ of overnight incubation.By calculating bacterium colony detection storage capacity is 1.6 * 10
8
3. the screening of anti-Type B botulinum neurotoxin phage positive colony
3.1 the enrichment of single-chain phage antibody library screening
After the phage library process M13KO7 superingection, the reorganization phagemid of generation can be released in the microbial culture supernatant, is used for the screening of phage positive colony.Micro-orifice plate screening method is selected in the enrichment in phage single-chain antibody storehouse screening, with antigen coated 96 orifice plates of toxin, adsorbs then-the four-wheel screening process of wash-out-enrichment, and the enrichment multiple reaches 110 times.
3.2 the sequential analysis of phage positive colony
The phage positive colony MuBNbP8 that selects is checked order, obtain the nucleotide sequence of its encoding gene.Compare with known sequence in the Kabat database, determine the family location; Deriving obtains the antibody gene amino acid sequence coded, and the result shows, the long 363bp of VH encoding gene of anti-Type B botulinum neurotoxin antibody cloning MuBNbP8, shown in sequence in the sequence table 1,121 amino acid of encoding shown in sequence in the sequence table 2, belong to mouse heavy chain IgG VH family; The long 336bp of VL encoding gene, shown in sequence in the sequence table 3,112 amino acid of encoding shown in sequence in the sequence table 4, belong to K chain family.Kabat and Genbank database retrieval analytical results show that they are the immune globulin variable region gene of finding first, are the newfound anti-Type B botulinum neurotoxin antibody genes of a strain.Correctly be connected by the DNA Linker of coding (Gly4Ser) 3 between VL gene and the VH gene and constitute the ScFv structure.The VH of antibody and VL carry out textural association and reconstruct by different modes, can make up and obtain a series of function antibody allosteric bodies, as single-chain antibody (ScFv), disulfide linkage stable form antibody (dsFv), strand disulfide linkage stable form antibody (ScdsFv), binary (diabody), Fab antibody fragment, Multidomain antibody or full molecular antibody or the like.
The structure of the anti-Type B botulinum neurotoxin of embodiment 2 humanizations antibody cloning
One, material:
1. bacterial strain: host bacterium E.coli DH5a is a Novagen company product.
2. reagent: pcr amplification reagent is TAKARA company product.
3. carrier: the pMD18-T carrier is a TaKaRa company product,
Two, methods and results:
1.MuBNbP8 transgenation
Utilize Blast searching database input MuBNbP8 antibody sequence and human antibody among the GenBank to do the homology comparison, determine conserved residues.Utilize computer aided design (CAD) to determine that mouse source antibody carries out humanization modified site simultaneously, its principle is the surface that the residue of transformation is positioned at antibody molecule, and the residue replacement does not influence antibody and position, antigen bonded space.Determine that according to homology comparison and computer aided design (CAD) the mutational site is: the K4 → Q4 of heavy chain, Q6 → V6, L12 → V12, the D17 → E17 of S77 → A77 and light chain.Fv gene with MuBNbP8 is a template, adopts the mutant primer design, the amplification mutant gene, and connect T carrier sequencing analysis.
2.HuBNbP8 Gene Sequence Analysis
The mutant gene analysis revealed, introduced sudden change in the fixed point of the framework region success of heavy chain and light chain respectively, mutating acid comprises the AAA → CAA of heavy chain, CAG → GTG, CTG → GTG, TCC → GCC, shown in sequence in the sequence table 5, the GAT → GAA of light chain is shown in sequence in the sequence table 7, other gene order remains unchanged, and the encoding sequence of the humanized antibody clone's heavy chain that makes up by transgenation and the variable region of light chain is shown in sequence in the sequence table 6 and sequence 8.
The reorganization preparation of embodiment 3 mouse sources and the anti-botulinum toxin type B genetic engineering antibody of humanization
One, material:
1. bacterial strain: host bacterium E.coli ER2566, TB1, DH5a are New England Biolab (NEB) company product; E.coli BL21 (DE3) is a Novagen company product.
2. reagent: Amylose affinity column, anti-MBP enzyme mark monoclonal antibody are available from NEB company; Pcr amplification reagent is TAKARA company product; Plasmid extracts test kit in a small amount available from sky root company, and T4 ligase enzyme, restriction enzyme are NEB company product; DL2000, dna molecular amount standard and middle low molecular weight protein (LMWP) standard are a day root company product; Pyocianil is available from SIGMA company.
3. carrier: recombinant expression vector pMAL-C2X is a NEB company product.
Two, methods and results:
1.MuBNbP8 and the reorganization of HuBNbP8 preparation
1.1 the structure of recombinant expression plasmid
Extract plasmid pMAL-C2X, with EcoR I and HindIII double digestion and reclaim carrier segments.Fv gene with MuBNbP8 and HuBNbP8 clone is a template respectively, obtain to introduce the goal gene Fv product of EcoR I and HindIII restriction enzyme site by design of primers and pcr amplification, connect into the carrier segments of having handled, sequence verification successful construction expression plasmid pMAL-MuFv and pMAL-HuFv after the double digestion digestion.
1.1MuBNbP8 and the soluble-expression of HuBNbP8 in intestinal bacteria
With pMal-C2X is recombinant expression plasmid pMAL-MuFv, pMAL-HuFv and the empty carrier difference transformed into escherichia coli ER2566 of vector construction, and PCR identifies and selects recombinant strain.Carry out abduction delivering: the activatory engineering bacteria that spends the night is transferred in LB+ glucose-Amp substratum, 37 ℃ are cultured to OD600=0.4-0.6, add IPTG and induce the plateau that can reach expression level in 4 hours, centrifugal subsequently (4 ℃ to final concentration 0.3mmol/L, 6000rpm/min 6min) collects thalline.The expression product molecular weight that SDS-PAGE analyzes Fv is about 66KDa, and accompanying drawing 4 shows consistent with expected results.The thin layer scanning analysis shows that recombinant expressed level is about 15%.The expressing protein positioning analysis shows that expressing protein is present in the born of the same parents with soluble form.
SDS-PAGE analyzes: the bacterium liquid after getting 1ml and inducing, the centrifugal 2min of 12000rpm abandons supernatant, and thalline adds 100 μ l sample-loading buffer (10% sucrose, 2%SDS, 50mmol/L Tris-HCl, pH6.8,10% mercaptoethanol, 0.002% tetrabromophenol sulfonphthalein) hanged precipitation, boiled in the boiling water 5 minutes, 12000rpm is centrifugal, gets supernatant 10 μ l electrophoresis.The protein band of contrast empty carrier and recombinant plasmid transformed engineering bacteria, observation has or not exogenous gene expression.
1.2 the purifying of Fv antibody fusion protein
Utilize the MBP-tag of expression product N-terminal, carry out affinitive layer purification (purification media is available from NEB company), the eluted protein of collecting is carried out NATIVE-PAGE and Western blot analysis, see accompanying drawing 5, and carry out protein quantification.
Concrete purification step is:
The preparation of cell crude extract: the thalline adding 10ml of centrifugal collection is crossed post damping fluid suspension thalline.Ultrasonication cell on ice bath (pulse is 15 seconds), 4 ℃ of 12000rpm/min centrifugal 30 minutes then, collect supernatant.
Merge the affinity purification of antibody:
1 prepacked column washes with the distilled water of 8-12 times of column volume.
2 prepacked columns are crossed post damping fluid balance with 9 times of column volumes.
3 join ultrasonic supernatant liquor in the post bed, control flow velocity 0.5-1ml/min.
The elution buffer drip washing of 12 times of column volumes of 4 usefulness is not conjugated protein, each 6ml, totally 4 times.
The elution buffer wash-out of 4 times of column volumes of 5 usefulness and the MBP of Amylose resin-bonded merge antibody protein, the about 0.5-1ml/min of control flow velocity.Collect 3-5 pipe sample eluent, collected volume is the 1ml/ pipe.
6 SDS-PAGE detect elution samples.
Wherein, the Amylose affinity column is crossed the post damping fluid: 20mM Tris-HCl, pH7.4,200mM NaCl, 1mM EDTA (additive: 1mM sodium azide; 10mM β-mercaotoethanol or 1mMDTT); Elution buffer: cross post damping fluid+10mM maltose
The biologic activity analysis of embodiment 4 anti-botulinum toxin type B genetic engineering antibodies
One, material:
1. reagent: anti His-HRP is a Pierce company product; BSA (bovine serum albumin component V) is a GIBCO company product; Ammonium thiocyanate is available from Beijing biochemical reagents company; Anti-Type B botulinum neurotoxin horse is anti-identifies institute available from Chinese pharmaceutical biological product; Avidity is measured with reagent such as HBS solution, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N hydroxyl amber imines (NHS), thanomin and is Pharmacia company product.
2. material: the CM5 chip is a Pharmacia company product; The Balb/C mouse is available from Military Medical Science Institute's Experimental Animal Center.
3. instrument: Biacore3000 is a GE company product.
Three, methods and results:
1. the antigen-binding activity of anti-Type B botulinum neurotoxin genetic engineering antibody
1.1 the ELISA of antigen-binding activity detects
With the ELISA detection validation MuBNbP8 and HuBNbP8 recombinant antibodies only combine with Type B toxin antigen-specific, and with other serotype botulinus toxin antigen such as A type, E type, the debond of F type toxin antigen, show as accompanying drawing 6.The somatotype that utilizes the type specificity of high-affinity MuBNbP8 and HuBNbP8 can be applied to the Type B botulinum neurotoxin detects.
1.2 the Biacore of binding affinity detects
The principle of work of Biacore (Biomolecular Interaction Analysis) is based on the surface plasma resonance sensing, and (Surface Plasmon Resonance, SPR) technology is come the interaction between the real-time tracing biomolecules.Earlier antigen is fixed on sensor chip surface during experiment, again antibody protein to be analyzed is expelled to sensor chip surface, detectable antibody combines with antigen and dissociated whole process, accompanying drawing 7 has just shown the dynamic binding curve of the dna recombinant expression product (3.675nmol/L-58.8nmol/L) of HuBNbP8, but the bonding strength of the avidity value reaginic antibody of measuring is an important indicator of estimating the antibody applicability.Analytical results shows that the avidity of HuBNbP8 is 1.34 * 10
-9M belongs to high-affinity antibody.
The high-affinity of anti-Type B botulinum neurotoxin antibody and susceptibility are more suitable for detecting in the special hypersensitivity of Type B botulinum neurotoxin in conjunction with characteristics.
2. the competition of the anti-Type B botulinum neurotoxin genetic engineering antibody of humanization is in conjunction with activity
Antigen coated 96 holes of ELISA:B type botulinum neurotoxin (contain 0.02%NaN with 3%BSA-PBS
3) sealing, add after respectively the HuBNbP8 antibody (MBP fusion rotein) of gradient concentration being mixed with the anti-Type B botulinum neurotoxin horse of amount is anti-together, with MBP albumen is contrast, 37 ℃ of incubations 1 hour, 0.05%Tween20-PBS (PBST) and PBS wash plate respectively three times, add the anti-MBP antibody of HRP mark, behind 37 ℃ of incubation 1h, to tmb substrate colour developing (5mg TMB, 0.05mol/L NaHCO
3, 0.5mmol/L MgCl
2), read A on the microplate reader
450
The result shows that recombinant humanized antibody HuBNbP8 albumen has good specific combination activity external, but the anti-Type B botulinum neurotoxin of effective competition horse is anti-, with Type B botulinum neurotoxin generation specific combination effect, sees accompanying drawing 8.
3. the neutralization activity of the anti-Type B botulinum neurotoxin genetic engineering antibody of humanization (protection of animal experiment)
Attack 10 of poison groups, with the mouse that the Type B botulinum neurotoxin of 5LD50 dosage is injected, all dead in 48 hours; 10 of antibody drug treatment groups, Type B botulinum neurotoxin and 200 μ g antibody fusion proteins with 5LD50 dosage are incubated overnight for 4 ℃, the abdominal injection animal, the mouse survival is 9 in 48 hours, survival is 6 in 96 hours, and the mean time to death of dead mouse also is longer than and is attacked malicious treated animal more than 5 times, show anti-Type B botulinum neurotoxin monoclonal antibody can be effectively in and botulinum neurotoxin, obviously postpone the neurovirulent generation of toxin, mouse is played the passive immunization provide protection; Control group MBP albumen does not have endotoxin neutralizing activity, and mouse is all dead, the death time on attack poison separately and organize no significant difference; 5 of antibody drug control groups, every injection 1mg antibody protein, animal is survival and no abnormal all, and heavy dose of recombinant antibodies albumen nontoxicity is described, the results are shown in subordinate list 1.
Table 1.HuBNbP8 clone's recombination expression product is active to neutralization in the body of BoNTb (5LD50)
＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
＜120〉anti-Type B botulinum neurotoxin neutralizing monoclonal antibody, Preparation Method And The Use
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Ala?Gln?Val?Lys?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly
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Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser
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Tyr?Tyr?Ile?Tyr?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Gly?Trp
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Ile?Gly?Glu?Ile?Tyr?Pro?Ser?Asn?Gly?Gly?Thr?Asn?Phe?Asn?Glu?Lys
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aagttcagtg?gcagtggatc?agggacagat?ttcagtctca?gtatcaacag?tctggagact 240
gaagattttg?gagtgttttt?ctgtcaacag?agtgacacct?ggccgtacac?gttcggaggg 300
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Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Thr?Pro?Gly
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Asp?Ser?Val?Ser?Leu?Ser?Cys?Gly?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Ser?His?Glu?Ser?Pro?Arg?Leu?Leu?Ile
35 40 45
Lys?Phe?Ala?Ser?Gln?Ser?Ile?Ser?Gly?Ile?Pro?Ser?Lys?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Ser?Ile?Asn?Ser?Leu?Glu?Thr
65 70 75 80
Glu?Asp?Phe?Gly?Val?Phe?Phe?Cys?Gln?Gln?Ser?Asp?Thr?Trp?Pro?Tyr
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aggcctggac?aaggccttgg?gtggattgga?gagatttatc?ctagcaatgg?tggcacaaat 180
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Ala?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Val?Lys?Pro?Gly
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Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser
20 25 30
Tyr?Tyr?Ile?Tyr?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Gly?Trp
35 40 45
Ile?Gly?Glu?Ile?Tyr?Pro?Ser?Asn?Gly?Gly?Thr?Asn?Phe?Asn?Glu?Lys
50 55 60
Phe?Lys?Thr?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ala?Ser?Thr?Ala
65 70 75 80
Tyr?Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Thr?Val?Tyr?Tyr
85 90 95
Cys?Ala?Arg?Met?Gly?Asn?Tyr?Gly?Gly?Ser?Tyr?Phe?Asp?Tyr?Trp?Gly
100 105 110
Gln?Gly?Thr?Thr?Leu?Thr?Val?Ser?Ser
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aagttcagtg?gcagtggatc?agggacagat?ttcagtctca?gtatcaacag?tctggagact 240
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<210> 8
<211> 112
<212> PRT
<213>
<400> 8
Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val Thr?Pro?Gly
1 5 10 15
Glu?Ser?Val?Ser?Leu?Ser?Cys?Gly?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Ser?His?Glu?Ser?Pro?Arg?Leu?Leu?Ile
35 40 45
Lys?Phe?Ala?Ser?Gln?Ser?Ile?Ser?Gly?Ile?Pro?Ser?Lys?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Ser?Ile?Asn?Ser?Leu?Glu?Thr
65 70 75 80
Glu?Asp?Phe?Gly?Val?Phe?Phe?Cys?Gln?Gln?Ser?Asp?Thr?Trp?Pro?Tyr
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Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Ala?Ala?Ala?Glu
100 105 110
Claims (10)
1. anti-Type B botulinum neurotoxin neutralizing monoclonal antibody, the variable region that it is characterized in that heavy chain and light chain has the aminoacid sequence shown in sequence in the sequence table 2 and sequence 4 respectively.
2. anti-Type B botulinum neurotoxin neutralizing monoclonal antibody according to claim 1, the variable region encoding gene that it is characterized in that heavy chain and light chain has the nucleotide sequence shown in sequence in the sequence table 1 and sequence 3 respectively.
3. the humanization modifier of claim 1 or 2 described anti-Type B botulinum neurotoxin neutralizing monoclonal antibodies, the variable region that it is characterized in that heavy chain and light chain has the aminoacid sequence shown in sequence in the sequence table 6 and sequence 8 respectively.
4. humanization modifier according to claim 3, the variable region encoding gene that it is characterized in that heavy chain and light chain has the nucleotide sequence shown in sequence in the sequence table 5 and sequence 7 respectively.
5. the preparation method of the arbitrary described anti-Type B botulinum neurotoxin neutralizing monoclonal antibody of claim 1-4 or its humanization modifier comprises the steps:
(1) the anti-Type B botulinum neurotoxin neutralizing monoclonal antibody of preparation or its humanization modifier encoding gene comprise CDR district or variable region or IgG part or full gene;
(2) protokaryon or carrier for expression of eukaryon are gone in this gene clone, the preparation recombinant expression plasmid;
(3) recombinant expression plasmid with preparation changes over to respectively in prokaryotic cell prokaryocyte or the eukaryotic cell and expresses.
6. preparation method according to claim 5 wherein prepares anti-Type B botulinum neurotoxin neutralizing monoclonal antibody or its humanization modifier encoding gene and adopts chemosynthesis or gene engineering method.
7. preparation method according to claim 5, wherein carrier for expression of eukaryon is yeast, insect, plant or mammalian cell expression vector; Prokaryotic cell prokaryocyte is intestinal bacteria, and eukaryotic cell is yeast, insect cell, vegetable cell or mammalian cell.
8. according to claim 5 or 7 described preparation methods, wherein prokaryotic expression carrier is pCANTAB-5E or pET series, and carrier for expression of eukaryon is pPIC series, pEF1 or pTriEx; Intestinal bacteria are HB2151 or BL21 (DE3), and eukaryotic cell is GS115 or CHO.
9. arbitrary described preparation anti-Type B botulinum neurotoxin neutralizing monoclonal antibody of claim 1-4 or the humanization modifier purposes in preparation Type B botulinum neurotoxin poisoning prevention or healing potion.
10. arbitrary described preparation anti-Type B botulinum neurotoxin neutralizing monoclonal antibody of claim 1-4 or the humanization modifier purposes in preparation Type B botulinum neurotoxin detection reagent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102414564A (en) * | 2009-04-27 | 2012-04-11 | 莫茨制药有限及两合公司 | Means and methods for the determination of the amount of neurotoxin polypeptide and of its catalytic and proteolytic activities |
| CN104736166A (en) * | 2012-05-30 | 2015-06-24 | 哈佛大学校长及研究员协会 | Engineered botulinum neurotoxin |
| KR101964990B1 (en) * | 2017-10-30 | 2019-04-02 | 국방과학연구소 | Neutralizing monoclonal humanized antibody for botulinum toxin type b or type e |
| CN112135840A (en) * | 2018-03-13 | 2020-12-25 | 斯米维特公司 | Single domain antibodies that bind to tetanus neurotoxin |
| CN114671948A (en) * | 2022-05-30 | 2022-06-28 | 北京弘进久安生物科技有限公司 | Antibodies against botulinum toxin type A and uses thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE19925739A1 (en) * | 1999-06-07 | 2000-12-21 | Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh | Therapeutic with a botulinum neurotoxin |
| US7838008B2 (en) * | 1999-12-07 | 2010-11-23 | Allergan, Inc. | Methods for treating diverse cancers |
| GB2416122A (en) * | 2004-07-12 | 2006-01-18 | Ipsen Ltd | Botulinum neurotoxin composition |
| CN100575363C (en) * | 2007-03-07 | 2009-12-30 | 中国人民解放军军事医学科学院微生物流行病研究所 | Human source anti-A botulinum neurotoxin genetic engineering antibody and preparation method thereof and purposes |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102414564A (en) * | 2009-04-27 | 2012-04-11 | 莫茨制药有限及两合公司 | Means and methods for the determination of the amount of neurotoxin polypeptide and of its catalytic and proteolytic activities |
| CN102414564B (en) * | 2009-04-27 | 2015-01-21 | 莫茨制药有限及两合公司 | Means and methods for the determination of the amount of neurotoxin polypeptide and of its catalytic and proteolytic activities |
| CN104736166A (en) * | 2012-05-30 | 2015-06-24 | 哈佛大学校长及研究员协会 | Engineered botulinum neurotoxin |
| KR101964990B1 (en) * | 2017-10-30 | 2019-04-02 | 국방과학연구소 | Neutralizing monoclonal humanized antibody for botulinum toxin type b or type e |
| CN112135840A (en) * | 2018-03-13 | 2020-12-25 | 斯米维特公司 | Single domain antibodies that bind to tetanus neurotoxin |
| CN112135840B (en) * | 2018-03-13 | 2024-01-23 | 斯米维特公司 | Single domain antibodies that bind to tetanus neurotoxin |
| CN114671948A (en) * | 2022-05-30 | 2022-06-28 | 北京弘进久安生物科技有限公司 | Antibodies against botulinum toxin type A and uses thereof |
| CN114671948B (en) * | 2022-05-30 | 2022-08-19 | 北京弘进久安生物科技有限公司 | Antibodies against botulinum toxin type A and uses thereof |
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