CN108610417A - Anti-tetanus toxin neutralizing antibody, preparation method and the usage - Google Patents
Anti-tetanus toxin neutralizing antibody, preparation method and the usage Download PDFInfo
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Abstract
The present invention provides a kind of anti-tetanus toxin neutralizing antibody, preparation method and uses, and the amino acid sequence of the CDR of the light chain variable region of the antibody is as shown in SEQ ID NO.1 3；The amino acid sequence of the CDR of the heavy chain variable region of the neutralizing antibody is as shown in SEQ ID NO.4 6.The potential risks such as the neutralizing antibody non-immunogenicity of the present invention, specificity is good, affinity is high, ingredient is clear, pathogen-free domestic pollution, are widely used in various crowds, the preparation method is simple and efficient, is suitable for standardized production.
Description
Technical field
The invention belongs to cellular immunology fields, are related to a kind of full humanized anti-spasmotoxin neutralizing antibody；In addition, this hair
The bright preparation method and purposes for further relating to the neutralizing antibody.
Background technology
Lockjaw (Tetanus) is caused due to infection clostridium tetanus (clostridiumtetani)
Acute, lethal infecting both domestic animals and human the nervous system disease.Clostridium tetanus be widely present in people, animal enteron aisle and
In soil, Population carriage is up to 25%, and the wound of different type and size can become clostridium tetanus and invade
Portal.After people is infected, mass propagation is simultaneously at wound under anaerobic for clostridium tetanus in incubation period
Toxin is discharged, tetanus toxin encroaches on central nervous system by destroying human neuronal cell line, causes body spasm, four limbs strong
Directly, the symptoms such as opisthotonos finally make one because of asphyxia or death due to respiratory failure.When outbreak of war and natural calamity take place frequently
Phase, lockjaw become a severe public health problem, and tetanus infection may occur for any age groups, and the death rate is
20-30%, patient with severe symptoms and the neonatal death rate are up to 80%.
Tetanus toxin effect is rapid, and toxicity is very strong.After patient is diagnosed as infection clostridium tetani, in vivo generally
There are a large amount of bacteriums and toxin, and patient vitals cannot be saved using antibiotic.It prevents and treats tetanic the only effective
Method is that timely injection anti-tetanus toxin antibody neutralizes a toxin, and is removed toxin from internal by the mediation of antibody receptor.
Occur more and more reports chimeric, Ren Yuan and full humanized anti-spasmotoxin monoclone antibody about people-mouse in recent years.It utilizes
The genetic engineering antibody that B cell screening antibodies engineering technology is transformed full human monoclonal antibody preparation not only eliminates blood
Allergic reaction caused by albumin, and the drawbacks of overcome people source immunoglobulin production process, before there is preferable development
Scape.Using gene recombination technology, antibody molecule is transformed at the genetic level, forms chimeric antibody, i.e., it is mouse is anti-variable
Area's gene is linked with human IgG constant region；The complementary determining region of the anti-variable region of mouse is implanted into the bone of human IgG by humanized antibody
In frame area, the anti-caused immune response of mouse further reduced；Complete human antibody, such antibody are free of any alien gene,
Allergic reaction will not be caused, is one of the hot spot of current genetic engineering antibody research.Although however the antibody of these reports can
It is combined with tetanus toxin, but the potency to neutralize a toxin is not high, and the industrialization production problem of antibody not yet solves.
Therefore, there is an urgent need in the art to utilize more advanced technology, the full humanized anti-spasmotoxin of highly effective and safe is developed
Neutralizing antibody, the serum product for replacing market supply, to meet human wants.
Invention content
In view of the deficiencies of the prior art, one of the objects of the present invention is to provide a kind of suitable full humanized anti-spasmotoxin lists
Neutralizing antibody is cloned, and its preparation method and application, the antibody non-immunogenicity, specificity is good, affinity is high, at distinguishing
The potential risks such as clear, pathogen-free domestic pollution, are widely used in various crowds, and the preparation method is simple and efficient, is suitable for standardization
Production.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of antigen-binding fragment, the light chain variable region of the antigen-binding fragment
The amino acid sequence of complementary determining region CDR is as shown in SEQ ID NO.1-3；
The amino acid sequence of the complementary determining region CDR of the heavy chain variable region of the antigen-binding fragment such as SEQ ID NO.4-
Shown in 6.
Preferably, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region of the antibody or its antigen-binding fragment
Row are as shown in SEQ ID NO.1 or SEQ ID NO.1 are substituted, lack or add one or several amino acid and the antibody of formation
Or its antigen-binding fragment has the amino acid sequence that same or similar activity obtains.
Preferably, the amino acid sequence of the complementary determining region CDR1 of light chain variable region amino as shown in SEQ ID NO.1
Acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain homologous
Acidic amino acid sequence, and the amino acid sequence obtained has the activity in conjunction with tetanus toxin.
Preferably, the amino acid sequence of the complementary determining region CDR2 of light chain variable region is as shown in SEQ ID NO.2 or SEQ
ID NO.2 be substituted, lack or add one or several amino acid and formation antibody or its antigen-binding fragment have it is identical
Or the amino acid sequence that shares activity obtains.
Preferably, the amino acid sequence of the complementary determining region CDR2 of light chain variable region amino as shown in SEQ ID NO.2
Acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain homologous
Acidic amino acid sequence, and the amino acid sequence obtained has the activity in conjunction with tetanus toxin.
Preferably, the benefit of light chain variable region determines the amino acid sequence of area CDR3 as shown in SEQ ID NO.3 or SEQ ID
NO.3, which is substituted, lacks or adds the antibody of one or several amino acid and formation or its antigen-binding fragment, has identical or phase
The amino acid sequence obtained like activity.
Preferably, the amino acid sequence of the complementary determining region CDR3 of light chain variable region amino as shown in SEQ ID NO.3
Acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain homologous
Acidic amino acid sequence, and the amino acid sequence obtained has the activity in conjunction with tetanus toxin.
Preferably, the amino acid sequence of the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment such as SEQ ID
Shown in NO.4 or SEQ ID NO.4 are substituted, lack or add the antibody or its antigen knot of one or several amino acid and formation
Close the amino acid sequence that there is segment same or similar activity to obtain.
Preferably, the amino acid sequence of the complementary determining region CDR1 of heavy chain variable region amino as shown in SEQ ID NO.4
Acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain homologous
Acidic amino acid sequence, and the amino acid sequence obtained has the activity in conjunction with tetanus toxin.
Preferably, the amino acid sequence of the complementary determining region CDR2 of heavy chain variable region is as shown in SEQ ID NO.5 or SEQ
ID NO.5 be substituted, lack or add one or several amino acid and formation antibody or its antigen-binding fragment have it is identical
Or the amino acid sequence that shares activity obtains.
Preferably, the amino acid sequence of the complementary determining region CDR2 of heavy chain variable region can also be shown in SEQ ID NO.5
Amino acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain
Homologous amino acid sequences, and obtain amino acid sequence have in conjunction with tetanus toxin activity.
Preferably, the amino acid sequence of the complementary determining region CDR3 of heavy chain variable region is as shown in SEQ ID NO.6 or SEQ
ID NO.6 be substituted, lack or add one or several amino acid and formation antibody or its antigen-binding fragment have it is identical
Or the amino acid sequence that shares activity obtains.
Preferably, the amino acid sequence of the complementary determining region CDR3 of heavy chain variable region can also be shown in SEQ ID NO.6
Amino acid sequence through being not higher than 20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and obtain
Homologous amino acid sequences, and obtain amino acid sequence have in conjunction with tetanus toxin activity.
Further, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment such as SEQ ID NO.7
Shown or SEQ ID NO.7 are substituted, lack or add the antibody or its antigen binding fragment of one or several amino acid and formation
Section has the amino acid sequence that same or similar activity obtains.
Preferably, the amino acid sequence of light chain variable region amino acid sequence as shown in SEQ ID NO.7 is by being not higher than
20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and the Homologous amino acid sequences that obtain, and
The amino acid sequence of acquisition has the activity in conjunction with tetanus toxin.
Further, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment such as SEQID NO.8
Shown or SEQ ID NO.8 are substituted, lack or add the antibody or its antigen binding fragment of one or several amino acid and formation
Section has the amino acid sequence that same or similar activity obtains.
Preferably, the amino acid sequence of heavy chain variable region amino acid sequence as shown in SEQ ID NO.8 is by being not higher than
20%, preferably not higher than 5% replacing, missing or adding for amino acid residue and the Homologous amino acid sequences that obtain, and
The amino acid sequence of acquisition has the activity in conjunction with tetanus toxin.
The antibody of conservative sequence variant including preferred antibody amino acid sequence is also included within the scope of the present invention.It protects
It includes not significantly changing full humanized anti-spasmotoxin neutralizing antibody binding property of the present invention to keep amino acid sequence variation
With the modification for the amino acid sequence for neutralizing property etc., the variant such as substituted derived from Similar amino acids well known in the art, amino acid
Lack, increased variant.
The antibody of the present invention further includes people source and non-human source antibodies, and with antibody identical function of the present invention or
All antibody of transformation and optimization.
Term " antibody " of the present invention refer to refer to usually by two pairs of polypeptide chains (it is each pair of have " light " (L) chain and
One " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as μ, δ,
γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.Each heavy chain is by heavy chain variable region
(VH) it is formed with heavy chain constant region (CH).Heavy chain constant region is made of 3 structural domains (CH1, CH2 and CH3).Each light chain is by light chain
Variable region (VL) and constant region of light chain (CL) composition.Constant region of light chain is made of a domain C.The areas VH and VL can be also subdivided
With denatured region (being known as complementary determining region (CDR)), to be interspersed with the more conservative area for being known as framework region (FR)
Domain.The variable region (VH and VL) of each heavy chain/light chain pair is respectively formed paratope.Term " antibody " is not by any specific
Generate the method limitation of antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody and polyclonal antibody.Antibody can
To be the antibody of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype)), IgA1, IgA2, IgD,
IgE or IgM antibody.
" antigen-binding fragment " of term antibody of the present invention refers to the polypeptide of the segment comprising full length antibody, is kept
The ability for the same antigen that specific binding full length antibody is combined, and/or the specificity of antigen is tied with full length antibody competition
It closes, also referred to as " antigen-binding portion thereof ".In a particular embodiment, antigen-binding fragment can be for example selected from the group：
Fab, Fab', F (ab') 2, Fd, Fv, dAb and complementary determining region (CDR) segment, linear antibodies, single-chain antibody (for example, scFv),
Miniantibody, double antibody and such polypeptide, it includes be enough to assign the antibody of polypeptid specificity antigen binding capacity at least
A part.
Second aspect, the present invention provides a kind of full humanized anti-spasmotoxin neutralizing antibody, the neutralizing antibody includes
Antigen-binding fragment as described in relation to the first aspect.
Preferably, the constant region of the antibody or its antigen-binding fragment includes humanized IgG 1, IgG2, IgG3 or IgG4 perseverance
Determine in area any one or at least two combination, preferably 1 constant region of humanized IgG.
The third aspect, the present invention provides a kind of DNA fragmentation, the DNA fragmentation includes encoding as described in relation to the first aspect
The nucleotide sequence of the light chain variable region and/or heavy chain variable region of antigen-binding fragment and/or the antibody as described in second aspect.
Fourth aspect, the present invention provides a kind of expression vector, the expression vector includes the DNA as described in second aspect
Segment.In a preferred embodiment, carrier of the invention is such as plasmid, clay, bacteriophage, coemid etc..
Preferably, the expression vector includes pcDNA3.3 carriers.
5th aspect, the present invention provides a kind of host cell, the host cell contains the DNA as described in second aspect
Segment and/or the expression vector as described in the third aspect.
Host cell for use in the present invention includes but not limited to microorganism, such as can be through containing antibody coding sequence
Recombinant phage dna, Plasmid DNA or cosmid DNA expression vectors conversion bacterium (Escherichia coli (E.coli), withered grass gemma
Bacillus (B.subtilis))；Saccharomycete (saccharomyces through the recombinant yeast expression vector conversion containing antibody coding sequence
(Saccharomyces), pichia (Pichia))；It is (rod-shaped through the recombinant virus expression vector containing antibody coding sequence
Virus) infection insect cell system；Through recombinant virus expression vector (cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)
(TMV)) plant cell system infecting or through recombinant plasmid expression vector (Ti-plasmids) conversion containing antibody coding sequence；
Or it carries and contains the promoter (metallothionein promoter) from mammalian cell gene group, derive from mammalian disease
The mammalian cell of the recombinant expression construct body of the promoter (adenovirus late promoter, vaccinia virus 7.5K promoters) of poison
System (COS, CHO, BHK, 293,3T3 cells).
6th aspect preparing antigen-binding fragment as described in relation to the first aspect and/or such as second party the present invention provides a kind of
The method of antibody described in face, the described method comprises the following steps：
(1) B cell of immune body is acquired, PCR obtains the DNA fragmentation of neutralizing antibody variable region；
(2) step (1) described DNA fragmentation is connected into expression vector, is transferred to competent cell, picking monoclonal after culture
Cell is screened；
(3) expression vector after screening is transferred to host cell, cultivates and collect supernatant, isolate and purify to obtain it is described in
And antibody.
The present invention utilizes single-cell RT-PCR and antibody screening techniques, from the healthy human B cell for having injected tetanus vaccine
In separate the variable region nucleotide sequence of anti-tetanus toxin neutralizing antibody and obtained high-purity by construction of expression vector
The full people source neutralizing antibody albumen of degree, preparation method standard is controllable, of low cost.
7th aspect, the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes as described in relation to the first aspect
Antigen-binding fragment, the antibody as described in second aspect, the DNA fragmentation as described in the third aspect, as described in fourth aspect
In expression vector or host cell as described in terms of the 5th any one or at least two combination.
Preferably, described pharmaceutical composition further includes arbitrary in pharmaceutically acceptable carrier, excipient or diluent
It is a kind of or at least two combination.
7th aspect, the present invention provides a kind of kit, the kit includes antigen knot as described in relation to the first aspect
Close segment, the antibody as described in second aspect, the DNA fragmentation as described in the third aspect, the expression vector as described in fourth aspect
In the host cell as described in terms of the 5th any one or at least two combination.
Preferably, shown kit further includes cleaning solution.
Eighth aspect, the present invention provides a kind of antibody as described in second aspect as described in relation to the first aspect, such as thirds
DNA fragmentation described in aspect, the expression vector as described in fourth aspect or the host cell as described in terms of the 5th prepare it is broken
Application in related drugs and/or the detection reagent of catching cold.
In the present invention, the lockjaw drug is for preventing and/or treating lockjaw.
In the present invention, " tetanus toxin " refers to the secretory protein that lockjaw bacillus fusiformis generates under anaerobic, is
General knowledge known in this field (discloses sequence) referring to NCBI GENEBANK databases.
In the present invention, " monoclonal antibody " refers to the antibody obtained from a kind of substantially uniform group, except minority may deposit
Natural mutation outside, in the group include identical single antibody.Monoclonal antibody is directed to single antigen with high specificity
Site is produced by cell strain, and cross contamination is not present；Modifier " monoclonal " only indicates the characteristic of antibody, refer to from
It is obtained in substantially uniform antibody population, cannot be construed to need to produce antibody with any specific process.
In the present invention, the antibody includes homologue or trim；Wherein, the homologue including the use of simple point mutation or
Multi-point combination is mutated the antibody fragment after antibody constant region or CDR partial amino-acid series are transformed and are optimized, CDR transplanting
Antibody fragment, the CDR of the single heavy chain CDR or single light chain CDR of antibody fragment, constant region and cdr amino acid sequence alterations are moved
Plant all antibody of antibody, the antibody of constant region and CDR transformations or single heavy chain and single light chain containing antibody CDR of the present invention
The scFv antibody that variable region is attached；The trim includes being carried out to the amino acid sequence or nucleotide sequence of antibody
Add, be deleted or modified after still retain neutralize tetanus toxin ability product.
In the present invention, the variable region that " variable " refers to antibody is different in sequence, forms various to specific antigen tool
There is the antibody of function of specific connecting.Changeability concentrates on the complementary determining region (CDR) or super of antibody light chain and heavy chain variable region
Become in three segments in area, includes respectively that (in variable region more conservative portion in four areas FR in the variable region of native heavy and light chain
Point), they are in generally beta sheet configuration, are connected by three CDR of formation connection ring, can form part β-pleated sheet structure.Every
CDR in chain is by the areas FR firmly against the antigen-binding site for together forming antibody together and with the CDR of another chain.It is permanent
Determine area and do not participate in the combination of antibody and antigen directly, but show different effector functions, it is anti-to such as participate in depending on for antibody
The cytotoxicity of body.
Compared with prior art, the present invention has the advantages that：
(1) neutralizing antibody of the invention has very strong combination activity and affinity to tetanus toxin；
(2) neutralizing antibody purity height of the invention, non-immunogenicity, ingredient are clear, and there is no pathogen contamination etc. is potential
Risk；
(3) method standard for preparing neutralizing antibody of the invention it is controllable, it is of low cost, be simple and efficient, be suitable for standard metaplasia
Production.
Description of the drawings
Fig. 1 is that the SDS-PAGE of TRN1013 antibody of the present invention schemes, wherein the 1st swimming lane is non-reducing antibody, the 2nd swimming lane
For molecular weight of albumen, the 3rd swimming lane is the antibody of reduction；
Fig. 2 is the neutralization activity testing result figure of TRN1013 antibody of the present invention and lockjaw normaltoxin；
Fig. 3 is the affine Activity determination figure of TRN1013 antibody of the present invention and lockjaw normaltoxin；
Fig. 4 is neutralization test result figure of the TRN1013 antibody of the present invention under 20 times of LD50 dosage in Mice Body.
Specific implementation mode
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art,
Or carried out according to product description, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in.Production firm is not specified in agents useful for same or instrument
Person, being can be by the commercially available conventional products of regular channel.
The term " KD " that embodiment uses refers to the Dissociation equilibrium constant of specific antibodies-antigen interactions, for describing
Binding affinity between antibody and antigen.Equilibrium dissociation constant is smaller, and antibody-antigen binding is closer, antibody and antigen it
Between affinity it is higher.In general, antibody is to be less than 10-5The Dissociation equilibrium constant (KD) of M and antigen binding, such as can be less than
10-6M、10-7M、10-8M、10-9M or 10-10M, KD are measured using surface plasma body resonant vibration art (SPR) using BIACORE instrument.
Experiment material
(1) after healthy volunteer injects 1500IU tetanus toxoid vaccines, blood was acquired respectively at the 0th, 14 and 21 day
Sample 20mL, anti-freezing；
(2) reagent：Tetanus vaccine, Ficoll lymphocyte separation mediums (Cedarlane companies), TAT-FITC, TAT-
FITC-Isotype, primer (Invitrogen), Superscript III reverse transcriptase, HotStarTaq Plus enzymes
(Invitrogen, Carlsbad, CA), agarose, Tris, LB culture mediums, ampicillin, PolyFect transfection reagents
(Qiagen, Valencia, CA), FCS, DMEM culture mediums, PBS buffer solution, BSA, HBsAg kits etc.；
(3) carrier：pcDNA3.3；
(4) bacterial strain：Bacillus coli DH 5 alpha；
(5) cell：293T.
1 mononuclearcell of embodiment detaches and thick liquid cell sorting
(1) respectively at the 0th, the 14 and 21 day blood from the healthy volunteer for having injected 1500IU tetanus toxoid vaccines
Middle acquisition 15mL blood samples are detached in the anticoagulant tube containing heparin with Ficoll, and it is outstanding to draw mononuclearcell (PBMC) layer
Liquid is washed 3 times with PBS, and supernatant is abandoned in suction；
(2) with 40 μm of membrane filtrations, target is sub-elected from PBMC after cell count using BD FACSria flow cytometers
Cell mass selects the intact individual cells of form and is placed in 96 hole PCR (Polymerase Chain Reaction, polymerase chain
Reaction) in plate (per the 20 unicellular lysates of μ L of hole), so that each hole is contained there are one B cell, -80 DEG C of refrigerators save backup.
Embodiment 2 utilizes RT-PCR separation antibody variable region genes from single B cell
(1)RT-PCR：The constant of 0.5 μM of different subtype heavy chain and light chain is added into 96 orifice plates containing single B cell
Area's primer (primer is designed using conventional method in specific site) and Superscript III reverse transcriptase, 37 DEG C of incubations 1 are small
When, carry out PCR amplification by the following conditions：95℃ 15min；95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 cycles；72
℃ 10min；4℃ 5min；The product cDNA of acquisition is in -20 DEG C of preservations；
(2)PCR：In 50 μ L systems containing 5 μ L reverse transcription products, HotStarTaq Plus enzymes, dNTPs and 0.5 μM
The specific primer (primer is designed using conventional method in specific site) of different subtype heavy chain and light chain variable region, by following item
Part carries out PCR amplification：94 DEG C of 5min of pre-degeneration；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 50s, 35 cycles；72℃ 7min；
The PCR product of acquisition is identified with 1% agarose gel electrophoresis.
The results show that PCR product about 400bp.
Embodiment 3 builds the expression vector of recombinant antibodies
Gel electrophoresis, which is accredited as positive and heavy chain, can match with light chain the PCR product of pairs of antibody variable gene
It is connected on pcDNA3.3 carriers using the TA methods cloned, the expression of the structure full people source neutralizing antibody of anti-tetanus toxin carries
Then expression vector is converted DH5 α competent bacterias, 37 DEG C of overnight incubations, picking on the tablet containing ampicillin by body
10 single bacterium colonies carry out PCR with specific primer, and reaction condition is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing
30s, 72 DEG C of extension 100s, 28 cycles；72 DEG C of extension 5min.5 μ L PCR products are taken to be detected with 1% agarose gel electrophoresis.
The results show that identifying the transformant containing heavy chain of antibody and light chain gene in positive transformant.
4 antibody expression of embodiment
Positive plasmid conversion DH5 α are subjected to large amplification, after rapid extraction recombinant plasmid, with transfection reagent PolyFect
293 cell of cotransfection changes a large amount of fresh cultures for 6-8 hours after transfection, and in 37 DEG C of 8%CO2It is cultivated 96 hours in incubator,
Cell conditioned medium is collected to be detected.
Embodiment 5 expresses the selective mechanisms of antibody
Using coating buffer by after 10 times of dilutions of tetanus toxoid, the every holes 100 μ L/, 4 DEG C of mistakes are added to 96 hole elisa plates
Night is coated with, and confining liquid room temperature is closed 2 hours；100 μ L are added and transiently transfect the incubation 2 hours of 37 DEG C of supernatant (primary antibody), add
HRP/anti-His-tag(1:2000 dilutions, secondary antibody) 37 DEG C be incubated 1 hour, 100 holes μ L/ of substrate developing solution are added, room temperature is kept away
After light places 5min, with 2M sulfuric acid stopped reactions, colorimetric detection is carried out under 450nm wavelength.
The expression and purification of 6 antibody of embodiment
There are the heavy chain of antibody of neutralization activity and expression vector (wherein, the light chain variable region of light chain by neutralize experimental identification
Amino acid sequence such as SEQ ID NO:Shown in 7, the amino acid sequence such as SEQ ID NO of heavy chain variable region:Shown in 8) cotransfection
293 cells change a large amount of fresh cultures for 6-8 hours after transfection, and in 37 DEG C of 8%CO2It is cultivated 96 hours in incubator；It collects and turns
It catches clearly, 4000rpm is centrifuged 1 hour, is purified using protein A-sepharose affinity chromatography；The table of antibody is examined using SDS-PAGE
Reach and purify situation.
The results are shown in Figure 1, obtains purity of protein height, can be clearly observable light chain and heavy chain after unwinding.Cotransfection table
Up to 293 cell successful expression human antibodies of plasmid vector, tetanus toxoid antigen can be effectively identified, and untransfected is expressed
293 cells and supernatants of plasmid cannot identify tetanus toxoid antigen, that is, transiently transfecting 293 successful expressions can specificity
Identify the anti-tetanus sphaceltoxin human antibody of tetanus toxoid.
7 monoclonal antibody neutralization activity of embodiment detects
The neutralization activity of the antibody of expression and purification is detected with previously mentioned identical ELISA method：Utilize packet
By liquid by after 10 times of dilutions of tetanus toxoid, 100 μ L/ are added per hole to 96 hole elisa plates, 4 DEG C coating, confining liquid are normal overnight
Temperature closing 2 hours；The antibody of 100 μ L expression and purifications is added as primary antibody, 37 DEG C of incubation 2h add HRP/anti-His-tag
(1:2000 dilutions) as 37 DEG C of secondary antibody incubation 1h, it is added 100 holes μ L/ of substrate developing solution, after room temperature avoid light place 5min, uses 2M
Sulfuric acid stopped reaction, with carrying out colorimetric detection under 450nm wavelength.
The results are shown in Figure 2, and (antibody concentration is about 0.0005 μ g/ to TRN1013 antibody of the diluted concentration more than 50,000 times
ML it) can still be neutralized with antigen, there is extremely strong neutralization activity.
8 monoclonal antibody of embodiment is affine Activity determination
Affinity of antibody test uses prize law, buffer solution HBS-EP, CM5 chip to be first coupled with proteinA.
Then dilution capture antibody makes its a concentration of 1 μ g/mL, binding time 50s.By analyte lockjaw element element to gradually increase
Concentration flows successively through chip, respectively obtains signal curve.Each concentration is recycled as 1, and 3M MgCl are used after completing 1 cycle2
Regeneration chip is to be returned to the original state for not capturing antibody, recovery time 30s.With BiaCore X-100System softwares
Obtained signal curve is analyzed, the affine activity of neutralizing antibody and tetanus toxin provided in an embodiment of the present invention is obtained
Detection figure.
As a result as shown in Figure 3 and Table 1, the rate (ka) of the combination of full humanized anti-spasmotoxin neutralizing antibody is 5.51E+
04Ms-1, dissociation rate (kd) reach 6.07E-05s-1, the Dissociation equilibrium constant (KD) of the antibody reaches 1.10E-9M, shows this
Antibody has very high affinity to tetanus toxin；.
The affinity measurement result tetanus toxoid (antigen) of 1 full humanized anti-spasmotoxin neutralizing antibody of table
| Antibody Designation | ka(1/Ms) | kd(1/s) | KD(M) |
| TRN1013 | 5.51E+04 | 6.07E-05 | 1.10E-0.9 |
9 neutralization test,in vivo of embodiment
(1) measurement of median lethal dose (LD50)
According to 2015 editions《Products in China regulation》Normal antitoxin and toxin dilution are prepared, prepared toxin is used
Dilution carries out gradient dilution (10,20,40,80,160,320,640,1280,2560,5120 and 10240 times), and 0.2mL is taken to note
Mouse is penetrated, every group 4, is observed 5 days, LD50 is calculated according to experimental result, experimental group uses 20 times × LD50 amounts.
(2) monoclonal antibody titration
Monoclonal antibody to be checked is diluted to 100 μ g/mL (monoclonal antibody concentration ＞ 1mg/ml) with dilution, i.e., with after toxin mixed in equal amounts
A concentration of 50 μ g/mL of monoclonal antibody in per 0.4ml injection volumes.
Quantitative diluted normal antitoxin and the monoclonal antibody to be checked drawn of mixing is respectively charged into small test tube, and often equivalent is added in pipe
Dilution test toxin, be uniformly mixed, jump a queue, 37 DEG C be incubated 1 hour, inject immediately.
It is the healthy laboratory mouse of 18-22g to take 28 weight, every group 4, is divided into 7 groups.By mixture (blank control
Group includes 0.2mL toxin (Toxin20LD50)；Negative control group includes 0.2mL toxin+0.2mL borate buffered salines
(20LD50+PAb) and negative antibody control group (20LD50+TRN006)；Positive controls include 0.2mL toxin+0.2mL antitoxin
Element；Experimental group includes 0.2mL toxin+0.2mL monoclonal antibodies (20LD50+TRN1013)) small white mouse abdomen, every injection is subcutaneously injected
0.4mL.The daily upper and lower noon, respectively observation was primary, continuous one week, the morbidity of record small white mouse and death condition.
The results are shown in Figure 4, and the small white mouse of negative control group and negative antibody control group is all dead within 48 hours,
The experimental mice of a concentration of+50 μ g/mL dosage of 20 times of median lethal doses (LD50) of monoclonal antibody shows in whole survivals as the present invention
Monoclonal antibody in 50 μ g/mL dosage, quite with the potency (10IU/ml) of normal antitoxin, can effective protection animal defense it is lethal
The attack of dosage tetanus toxin, it is almost the same with the protectiveness of normal antitoxin.Meanwhile the actual amount of monoclonal antibody of the present invention is remote
Far below normal antitoxin, show that the full humanized anti-spasmotoxin neutralizing antibody of the present invention has extremely strong potency effect.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Sequence table
<110>Guangzhou Tylenol enlightening bio tech ltd of Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd of Ji'nan University
<120>Anti-tetanus toxin neutralizing antibody, preparation method and the usage
<130> 20180427
<141> 2018-04-28
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<213>Artificial synthesized ()
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<213>Artificial synthesized ()
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35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
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115
Claims (10)
1. a kind of antigen-binding fragment, which is characterized in that the complementary determining region CDR of the light chain variable region of the antigen-binding fragment
Amino acid sequence as shown in SEQ ID NO.1-3；
The amino acid sequence of the complementary determining region CDR of the heavy chain variable region of the antigen-binding fragment such as SEQ ID NO.4-6 institutes
Show；
Further, light chain and/or the complementary determining region of heavy chain variable region are by replacing, missing or adding one or several amino
The amino acid that acid obtains, and the antigen-binding fragment formed has same or similar activity.
2. antigen-binding fragment according to claim 1, which is characterized in that the light chain variable region of the antigen-binding fragment
Amino acid sequence is as shown in SEQ ID NO.7 or SEQ ID NO.7 are substituted, lack or add one or several amino acid
And the antigen-binding fragment formed has the amino acid sequence that same or similar activity obtains；
The amino acid sequence of the heavy chain variable region of the antigen-binding fragment is as shown in SEQ ID NO.8 or SEQ ID NO.8 are passed through
Replace, miss or add the ammonia that there is the antigen-binding fragment of one or several amino acid and formation same or similar activity to obtain
Base acid sequence.
3. a kind of full humanized anti-spasmotoxin neutralizing antibody, which is characterized in that the neutralizing antibody include such as claim 1 or
Antigen-binding fragment described in 2；
Preferably, the constant region of the antibody include in humanized IgG 1, IgG2, IgG3 or IgG4 constant region any one or extremely
Few two kinds of combination, preferably 1 constant region of humanized IgG.
4. a kind of DNA fragmentation, which is characterized in that the DNA fragmentation includes encoding antigen binding as claimed in claim 1 or 2
The nucleotide sequence of the light chain variable region and/or heavy chain variable region of segment and/or antibody as claimed in claim 3.
5. a kind of expression vector, which is characterized in that the expression vector includes DNA fragmentation as claimed in claim 4；
Preferably, the expression vector includes pcDNA3.3 carriers.
6. a kind of host cell, which is characterized in that the host cell contains DNA fragmentation as claimed in claim 4 and/or such as
Expression vector described in claim 5；
Preferably, the host cell includes Chinese hamster ovary celI or 293 cells.
7. a kind of method preparing antigen-binding fragment as claimed in claim 1 or 2 and/or antibody as claimed in claim 3,
It is characterized in that, the described method comprises the following steps：
(1) B cell of immune body is acquired, PCR obtains the DNA fragmentation of antibody variable region；
(2) step (1) described DNA fragmentation is connected into expression vector, is transferred to competent cell, picking monoclonal cell after culture
It is screened；
(3) expression vector after screening is transferred to host cell, cultivates and collect supernatant, isolate and purify to obtain it is described neutralize it is anti-
Body.
8. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes antigen binding as claimed in claim 1 or 2
Segment, antibody as claimed in claim 3, DNA fragmentation as claimed in claim 4, expression vector as claimed in claim 5 or
In host cell as claimed in claim 6 any one or at least two combination；
Preferably, described pharmaceutical composition further includes any one in pharmaceutically acceptable carrier, excipient or diluent
Or at least two combination.
9. a kind of kit, which is characterized in that shown kit includes antigen-binding fragment as claimed in claim 1 or 2, such as weighs
Profit requires 3 antibody, DNA fragmentation as claimed in claim 4, expression vector as claimed in claim 5 or as right is wanted
Ask in the host cell described in 6 any one or at least two combination；
Preferably, shown kit further includes cleaning solution.
10. a kind of antigen-binding fragment as claimed in claim 1 or 2, antibody as claimed in claim 3, as claimed in claim 4
DNA fragmentation, expression vector as claimed in claim 5 or host cell as claimed in claim 6 preparing broken expelling wind drug
Application in object and/or detection reagent.
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| CN112225813A (en) * | 2020-10-21 | 2021-01-15 | 北京智仁美博生物科技有限公司 | Antibodies against tetanus toxin and uses thereof |
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