CN108623681A - A kind of neutralizing antibody of anti-tetanus toxin and application - Google Patents
A kind of neutralizing antibody of anti-tetanus toxin and application Download PDFInfo
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- CN108623681A CN108623681A CN201810420730.5A CN201810420730A CN108623681A CN 108623681 A CN108623681 A CN 108623681A CN 201810420730 A CN201810420730 A CN 201810420730A CN 108623681 A CN108623681 A CN 108623681A
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Abstract
The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin.The neutralizing antibody of anti-tetanus toxin provided by the invention can specifically bind tetanus toxin, thus have extremely strong neutralization activity and very high affine activity, and dosage is low.The neutralizing antibody of anti-tetanus toxin provided by the invention can be used in preventing and treating the antibody of the disease of tetanus toxin infection and clostridium tetani mediation or the separation of disease condition, while can also be used to diagnose and/or monitor the antibody of tetanus toxin infection and the separation of infection due to Clostridium tetani.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention is polluted without exogenous virus, it is widely portable to various crowds.
Description
Technical field
The present invention relates to the neutralizing antibodies and application of gene engineering technology field more particularly to a kind of anti-tetanus toxin.
Background technology
Tetanus toxin is a kind of inhibition that clostridium tetani (Clostridium tetani) generates under anaerobic
The secretory protein of neurotransmitter regulator.Tetanus toxin can cause super reflex response and band muscle spasmus, be received in muscle tonue
It contracts on the basis of (myotonia hardens), the strong spasm of paroxysmal, patient with severe symptoms can be lethal, and case fatality rate may be up to 50%.
Prevention and treatment for tetanus toxin, generally use horse serum TAT (Tatanus Antitoxin, lockjaw
Antitoxin) it is treated.But the acceptance level due to the difference of human body constitution and to horse serum TAT is different, and many people can go out
The adverse reactions such as existing allergic reaction, anaphylactic shock or serum sickness.Therefore, horse serum TAT is not particularly suited for owner.With biology
The progress of medical treatment, HTIG (Human Tetanus Immunoglobulin, Homo-Tet) are progressed into
Market.HTIG can overcome adverse reaction when horse serum TAT Clinical practices, and it is horizontal to greatly improve tetanic prevention.But
Since people's blood source source is because difficult, expensive, there are the danger of exogenous virus pollution, therefore, the industrialized production of HTIG and face
Bed application is greatly limited.
In recent years, due to the development of technique for gene engineering so that prepare humanization/humanized by the method for genetic engineering
Antibody is possibly realized.Humanization/the human antibody prepared using genetic engineering, which can reduce or eliminate foreign sera albumen, to be caused
Allergic reaction, and can overcome people source immunoglobulin produce inadequate blood source and potential virus pollution possibility the problems such as, at
For the Main way studied at present.
Invention content
The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin, and to solve, existing HTIG is expensive, deposits
The problem of exogenous virus pollutes.
In a first aspect, the present invention provides a kind of neutralizing antibody of anti-tetanus toxin, including：
Including the variable heavy chain domain of VH CDR1, VH CDR2 and VH CDR3 and comprising VLCDR1, VLCDR2 and
The variable light chain domain of VLCDR3, wherein
The VH CDR1 include amino acid sequence shown in SEQ ID NO.1；
The VH CDR2 include amino acid sequence shown in SEQ ID NO.2；
The VH CDR3 include amino acid sequence shown in SEQ ID NO.3；
The VL CDR1 include amino acid sequence shown in SEQ ID NO.4；
The VL CDR2 include amino acid sequence shown in SEQ ID NO.5；
The VL CDR3 include amino acid sequence shown in SEQ ID NO.6
Preferably, further include light chain variable shown in heavy chain variable region and SEQ ID NO.8 shown in SEQ ID NO.7
Area.
Preferably, the neutralizing antibody includes：
With the VH CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.1,
And/or with the VL CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.4；With/
Or,
With the VH CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.2,
And/or with the VL CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.5；With/
Or,
With the VH CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.3,
And/or with the VL CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.6.
Preferably, the neutralizing antibody immunospecifically combines tetanus toxin, the neutralizing antibody to have at least 10-
6, the affinity to the tetanus toxin and/or the clostridium tetani of 10-7,10-8,10-9 or 10-10M.
Second aspect, the present invention provide a kind of composition, include the neutralizing antibody of the anti-tetanus toxin of first aspect.
The third aspect, the present invention provide a kind of nucleic acid molecules, the anti-tetanus poison of the nucleic acid molecule encoding first aspect
The neutralizing antibody of element.
Fourth aspect, the present invention provide a kind of expression vector, include the nucleic acid molecules of the third aspect.
5th aspect, the present invention provide a kind of recombinant cell, include the table of the nucleic acid molecules of the third aspect or fourth aspect
Up to carrier.
6th aspect, the present invention provide a kind of kit, include the neutralizing antibody of the anti-tetanus toxin of first aspect, or
The recombination of the expression vector or the 5th aspect of the composition of second aspect or the nucleic acid molecules of the third aspect or fourth aspect is thin
Born of the same parents.
7th aspect, the present invention provide a kind of application process, including：
The neutralizing antibody of the anti-tetanus toxin of first aspect is being prepared by tetanus toxin infection and clostridium tetani
Application in the drug of infection；And/or
The neutralizing antibody of the anti-tetanus toxin of first aspect is preparing the infection of detection tetanus toxin and lockjaw shuttle
Application in the kit of bacterium infection；And/or
The composition of second aspect is in preparing treatment by the drug of tetanus toxin infection and infection due to Clostridium tetani
Application；And/or
The composition of second aspect is in preparing the kit of the infection of diagnosis tetanus toxin and infection due to Clostridium tetani
Application；
The nucleic acid molecules of the third aspect are preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application；
The expression vector of fourth aspect is preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application；
The recombinant cell of 5th aspect is preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application.
The technical solution that the embodiment of the present invention provides can include the following benefits：
The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin.Anti-tetanus toxin provided by the invention
Neutralizing antibody can specifically bind tetanus toxin, thus have extremely strong neutralization activity and very high affine activity, and use
It measures low.The neutralizing antibody of anti-tetanus toxin provided by the invention can be used in preventing and treating tetanus toxin infection and break
The disease of clostridium mediation of catching cold or the antibody of the separation of disease condition, while can also be used to diagnose and/or monitor broken wind toxin
The antibody of element infection and the separation of infection due to Clostridium tetani.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention
No exogenous virus pollution, is widely portable to various crowds.
It should be understood that above general description and following detailed description is only exemplary and explanatory, not
It can the limitation present invention.
Description of the drawings
In order to illustrate more clearly of the technical solution of the application, letter will be made to attached drawing needed in the embodiment below
Singly introduce, it should be apparent that, for those of ordinary skills, without having to pay creative labor,
Other drawings may also be obtained based on these drawings.
Fig. 1 passes through SDS-PAGE (English names to be provided in an embodiment of the present invention：Sodium dodecyl sulfate-
Polyacrylamide gelelectrophoresis, Chinese：Sodium dodecyl sulfate-polypropylene acyl ammonia gel electrophoresis)
The detection of expression result figure for the neutralizing antibody that method detects；
Fig. 2 passes through Western Blot (Chineses to be provided in an embodiment of the present invention：Immunoblotting) method detection
The detection of expression result figure of obtained neutralizing antibody；
Fig. 3 is the antigen-binding activity detection figure of neutralizing antibody provided in an embodiment of the present invention；
Fig. 4 is the affine Activity determination figure of neutralizing antibody provided in an embodiment of the present invention and tetanus toxin；
Fig. 5 is the protective effect of the tetanus toxin LD50 of 20 multiple dose of neutralizing antibody pair provided in an embodiment of the present invention
Figure；
Fig. 6 is the protective effect of the tetanus toxin LD50 of 60 multiple dose of neutralizing antibody pair provided in an embodiment of the present invention
Figure.
Specific implementation mode
In term, antibody refers to the protein molecular for the receptor acting for playing specific recognition antigen, including with specific antigen
The immunoglobulin molecules of immune response, including entire antibody, dimerization, trimerization and multimeric antibody；Bispecific antibody；Inosculating antibody
Body；Recombination and engineered antibody and its segment, as long as the antibody includes antigen binding domain.Immunoglobulin molecules have weight
Chain and light chain, each heavy chain and light chain include constant region and variable region, wherein the variable region of each heavy chain and light chain is referred to as variable
Heavy chain (variable heavy chain, VH) and variable light (variable light chain, VL).Heavy chain and light chain
Variable region includes four framework regions, is interrupted by three hypervariable regions, also referred to as complementary determining region (complementarity
Determining region, CDR), wherein the main function of CDR is the epitope for being attached to antigen.
It will also be appreciated by those of skill in the art that the amino acid sequence that the present invention covers the tetanus toxin antibody is repaiied
Decorations.For example, it may be desired to improve the binding affinity of antibody and/or other biological characteristics.Anti-tetanus toxin antibody it
Amino acid sequence variation is from introducing appropriate nucleotide variation into anti-tetanus toxin antibody nucleic acid or being prepared by peptide synthesis.It should
Equal modifications include the residue deletions and/or insertion and/or substitution that (for example) anti-tetanus toxin antibody amino acid sequence is interior.It carries out
For any combinations of missing, insertion and substitution to reach final construct, restrictive condition has by the final construct is wanted special
Sign.Amino acid variation also can be changed process after the translation of anti-tetanus toxin antibody, such as the number of change glycosylation site or
Position.
The embodiment of the present invention provides a kind of neutralizing antibody of anti-tetanus toxin, and in embodiments of the present invention, the neutralization is anti-
Body is known as antibody TRN1015.Antibody TRN1015 provided in an embodiment of the present invention includes：Including VH CDR1, VH CDR2 and VH
The variable heavy chain domain of CDR3 and variable light chain domain comprising VLCDR1, VLCDR2 and VLCDR3, wherein
VH CDR1 include amino acid residue sequence shown in SEQ ID NO.1；
VH CDR2 include amino acid residue sequence shown in SEQ ID NO.2；
VH CDR3 include amino acid residue sequence shown in SEQ ID NO.3；
VL CDR1 include amino acid sequence shown in SEQ ID NO.4；
VL CDR2 include amino acid sequence shown in SEQ ID NO.5；
VL CDR3 include amino acid sequence shown in SEQ ID NO.6.
In antibody TRN1015 provided in an embodiment of the present invention, the sequence of SEQ ID NO.1-SEQ ID NO.6 is distinguished
For：
SEQ ID NO.1：Gly Phe Gly Ile Gly Ile Tyr Thr；
SEQ ID NO.2：Ile Ser Gly Thr Ser Gln Tyr Ile；
SEQ ID NO.3：Val Ser Gly Ser Ser Leu Asp Tyr；
SEQ ID NO.4：Gln Asn Ile Gly Thr Asn；
SEQ ID NO.5：Gly Ala Ser；
SEQ ID NO.6：Tyr Gln Asn Tyr Asn Ser Pro Lys Thr.
Further, antibody TRN1015 provided in an embodiment of the present invention further includes heavy chain variable region shown in SEQ ID NO.7
With light chain variable region shown in SEQ ID NO.8, wherein the sequence of SEQ ID NO.7 is：Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Arg Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Val
Ser Gly Phe Gly Ile Gly Ile Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Thr Ser Gln Tyr Ile Tyr Tyr Ala Asn
Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Tyr Leu
Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Val Ser Gly Ser
Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser；The sequence of SEQ ID NO.8
For：Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Gly Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Gly Thr Asn Leu Asn Trp Phe Gln
Gln Lys Pro Gly Thr Ala Pro Asn Leu Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Phe Thr Leu Thr Ile
Asp Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Asn Tyr Asn Ser
Pro Lys Thr Phe Gly Gln Gly Thr Lys Val Asp Leu Lys。
In antibody TRN1015 provided in an embodiment of the present invention, antibody TRN1015 can immunospecifically combine broken
Wind toxin element, antibody have at least 10-6、10-7、10-8、10-9Or 10-10M to tetanus toxin.
Further, antibody TRN1015 provided in an embodiment of the present invention simultaneously further include with the amino acid sequence of antibody or
Person encodes sequence of any nucleotide sequence of the antibody with a degree of sequence identity or sequence homology.Specifically
Ground, antibody TRN1015 provided in an embodiment of the present invention further include having and sequence at least 80% sequence shown in SEQ ID NO.1 simultaneously
The VH CDR1 of the amino acid sequence of the row phase same sex, and/or with identical as sequence at least 80% sequence shown in SEQ ID NO.4
The VL CDR1 of the amino acid sequence of property；And/or with the ammonia with sequence at least 80% sequence identity shown in SEQ ID NO.2
The VH CDR2 of base acid sequence, and/or with the amino acid sequence with sequence at least 80% sequence identity shown in SEQ ID NO.5
The VL CDR2 of row；And/or with the VH with the sequence at least amino acid sequence of 80% sequence identity shown in SEQ ID NO.3
CDR3, and/or with the VL CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in SEQ ID NO.6.
Such as：With SEQ ID NO.1 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or higher sequence identity amino acid sequence VH CDR1；
With SEQ ID NO.2 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or higher sequence identity amino acid sequence VH CDR2；
With SEQ IDNO.3 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or higher sequence identity amino acid sequence VH CDR3；
With SEQ ID NO.5 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or higher sequence identity amino acid sequence VL CDR1；
With SEQ ID NO.6 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or higher sequence identity amino acid sequence VL CDR2；
And with SEQ ID NO.7 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or higher sequence identity amino acid sequence VL CDR3.
Antibody TRN1015 provided in an embodiment of the present invention is preferably full humanized antibody, including scFv (single
Chain antibody fragment, scFv), Fab, Fab ', F (ab ') 2, Fv, dsFv, double antibody (diabody), Fd or
Fd ' segments.But antibody TRN1015 provided in an embodiment of the present invention is not limited to above-mentioned full humanized antibody.Further, originally
The antibody TRN1015 that inventive embodiments provide further includes containing peptide linker.More preferably, peptide linker includes about 1-50 amino
Acid.
Antibody TRN1015 provided in an embodiment of the present invention further includes conjugate, which includes to be conjugated to antibody
The label of TRN1015 can such as be conjugated to polyethylene glycol (PEG).Further, antibody TRN1015 provided in an embodiment of the present invention is also
Including therapeutic agent or diagnosticum, e.g., diagnosticum includes but not limited to enzyme, fluorescent chemicals, electron transfer agent and radioactive label.
The embodiment of the present invention also provides a kind of composition, and the composition includes antibody TRN1015.The embodiment of the present invention provides
Composition can be diagnosis composition, or pharmaceutical composition.However, being diagnosis composition or pharmaceutical composition
Include antibody TRN1015.
When the composition of the embodiment of the present invention is diagnosis composition, which further includes accommodating antibody
The container of TRN1015.Further, which further includes conjugate, wherein conjugate includes to be conjugated to antibody
The label of TRN1015, such as detectable part/reagent.Label is directly conjugated to antibody by covalent bond or non-covalent bond
On TRN1015.In addition, label can be conjugated to antibody TRN1015 using one or more connection compounds, wherein for that will mark
It is well known to those skilled in the art that label, which are conjugated to the technology of antibody TRN1015,.Detectable part/examination as label
Agent is preferably selected from by enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emission material
One kind in the group that the paramagnetic metal ion of material and on-radiation forms.
When the composition of the embodiment of the present invention is pharmaceutical composition, which further includes pharmaceutically acceptable
Carrier, wherein pharmaceutically acceptable carrier refers to that will not cause significantly to stimulate to organism and will not eliminate applied chemical combination
The bioactivity of object and the carrier of property or diluent, e.g., physiological saline, sterile water, Ringer's solution, buffer salt solution, Portugal
The mixture of grape sugar juice, maltodextrin solution, glycerine, ethyl alcohol and two of which or more.If desired, the present invention is real
Apply example offer pharmaceutical composition can also include other conventional additives, as antioxidant, buffer, bacteriostatic agent, dispersant,
Surfactant, adhesive and lubricant, and be configured to injectable formulation, such as aqueous solution, suspension and emulsion, pill, glue
Wafer, granule and tablet.
The embodiment of the present invention also provides a kind of nucleic acid molecules, which can encode provided in an embodiment of the present invention anti-
Body TRN1015.
The embodiment of the present invention also provides a kind of expression vector, which includes above-mentioned nucleic acid molecules.The present invention is implemented
The expression vector that example provides further includes the expression regulation sequence being operatively connected with sequence of nucleic acid molecules.Wherein, the expression carries
Body be included in prokaryotic cell carry out gene expression carrier, in eukaryocyte carry out gene expression carrier and other
The carrier that gene expression is carried out in cell such as mammalian cell, plant cell, yeast cells, insect cell, imports phagocytosis
The polynucleotides of body, plasmid, clay, minichromosome, virus or retrovirus vector.
The embodiment of the present invention also provides a kind of recombinant cell, which includes above-mentioned nucleic acid molecules or expression vector.
Recombinant cell can be prokaryotic cell, eukaryocyte, and such as bacterium including Escherichia coli, streptomycete and salmonella typhimurium is thin
Those skilled in the art can be used in the art in born of the same parents, yeast cells, fungal cell, zooblast and plant cell etc.
The known any cell that can be used as mammalian host cell.
The embodiment of the present invention also provides a kind of kit, which includes above-mentioned antibody TRN1015 or composition, or
Nucleic acid molecules or expression vector or recombinant cell.Kit provided in an embodiment of the present invention can be used in detecting tetanus toxin
Infection and infection due to Clostridium tetani.Specifically, kit provided in an embodiment of the present invention can be measured by antibody TRN1015
Tetanus toxin in fluid, cell or tissue sample is horizontal.Meanwhile kit provided in an embodiment of the present invention can also be by institute
Tetanus toxin level is measured compared with control level, wherein compared with the control level of tetanus toxin, what is measured is broken
The raising of wind endotoxin level represents tetanus toxin infection.Preferably, the cell or tissue sample is obtained from human subjects, institute
It is blood, urine, saliva, lung phlegm, lavation or lymph sample to state cell or tissue sample to include.
The embodiment of the present invention also provides a kind of application process, which includes：
Applications of the antibody TRN1015 in preparing by the drug of tetanus toxin infection and infection due to Clostridium tetani；With/
Or,
Antibody TRN1015 answering in preparing the kit of the infection of detection tetanus toxin and infection due to Clostridium tetani
With；And/or
Application of the composition in preparing treatment by the drug of tetanus toxin infection and infection due to Clostridium tetani；With/
Or,
Application of the composition in preparing the kit of the infection of diagnosis tetanus toxin and infection due to Clostridium tetani；With/
Or,
Nucleic acid molecules are in the drug for preparing the disease that treatment is mediated by tetanus toxin infection and clostridium tetani
Using；And/or
Expression vector is in the drug for preparing the disease that treatment is mediated by tetanus toxin infection and clostridium tetani
Using；And/or
Recombinant cell is in the drug for preparing the disease that treatment is mediated by tetanus toxin infection and clostridium tetani
Using.
The neutralizing antibody (antibody TRN1015) of anti-tetanus toxin provided by the invention can specifically bind broken wind toxin
Element, thus there is extremely strong neutralization activity and very high affine activity, and dosage is low.Anti-tetanus toxin provided by the invention
The disease or disease condition that neutralizing antibody can be used in preventing and treating tetanus toxin infection and clostridium tetani mediates
The antibody of separation, while can also be used to diagnose and/or monitor tetanus toxin infection and the separation of infection due to Clostridium tetani
Antibody.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention is polluted without exogenous virus, it is widely portable to various
Crowd.
With reference to the accompanying drawings and examples to the extraction of antibody TRN1015 provided in an embodiment of the present invention, purifying and in
It is described in further detail with performance, affine dynamic performance and protection in vivo.The reality of actual conditions is not specified in embodiment
Proved recipe method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning：Laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The screening and purifying of 1 selected by flow cytometry apoptosis B cell of embodiment and anti-tetanus toxin antibody
1, PBMC separation and thick liquid cell sorting
15ml venous blood is acquired with the volunteer of protection antibody from having injected tetanus vaccine and having produced, and is placed in
In anticoagulant tube containing heparin.15ml blood samples are isolated into PBMC (peripheral blood with Ficoll (ficoll)
Mononuclear cell, peripheral blood mononuclear cells).PBMC count after using BD FACSria flow cytometers from PBMC into
The single B cell sorting of row, 96 hole PCR (Polymerase Chain Reaction, polymerase are placed in by the intact B cell of form
Chain reaction) in plate, so that each hole is contained, there are one B cells.It is -80 DEG C of refrigerators that the 96 hole PCR plates for being equipped with B cell, which are placed in temperature,
In, it saves backup.
2, separation antibody variable region gene
By in the 96 hole PCR plates containing single B cell be added 0.5 μM each subtype heavy chain and light chain constant region primers with
And Superscript III reverse transcriptase, it is incubated 1h under the conditions of temperature is 37 DEG C.After incubation, according to 95 DEG C of 15min；
95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30cycles；72℃10min；4 DEG C of 5min conditions carry out PCR amplification, are expanded
Product cDNA.Amplified production cDNA is placed under conditions of temperature is -20 DEG C and preserves.
PCR separation antibody genes：RT (reverse transcription, reverse transcription) containing 5 μ l in 50uL systems is anti-
Answer product, HotStarTaq Plus enzymes (Invitrogen, Carlsbad, CA), each subtype heavy chain of dNTPs and 0.5uM with
The specific primer of light chain antibody, reaction condition：94 DEG C of 5min of pre-degeneration, then carry out 35 PCR cycles, and each cycle is：
94 DEG C × 30s, 55 DEG C × 30s, 72 DEG C × 50s, finally with 72 DEG C of extension 7min, obtain pcr amplification product.
3, the expression vector of recombinant antibodies is built
2 μ l pcr amplification products are taken to be detected through 1% agarose gel electrophoresis.Gel electrophoresis is accredited as the positive, and heavy chain
Pairs of antibody variable gene PCR product can be matched with light chain to be connected on pcDNA3.3 carriers using the TA methods cloned,
Connection product is converted in DH5 α competent bacterias, 37 DEG C of overnight incubations on the tablet containing ampicillin, immediately picking
10 single bacterium colonies carry out PCR, reaction condition with specific primer：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 55 DEG C are annealed
30s, 72 DEG C of extension 1min40s, 28 cycles, last 72 DEG C re-extend 5min；Take 5 μ l PCR products in 1% Ago-Gel
Upper carry out electrophoresis detection, identifies the transformant containing heavy chain of antibody or light chain gene in positive transformant.
4, antibody expression
The plasmid of the positive antibody heavy chain and light chain gene large amplification in bacillus coli DH 5 alpha will be expressed, recombination matter is carried out
Grain rapid extraction.With 293 cell of transfection reagent PolyFect cotransfections, concrete operations are referring to specification.6-8h renews after transfection
Fresh culture medium, and in 37 DEG C of 8%CO296h is cultivated in incubator, is collected cell conditioned medium and is detected.
5, the selective mechanisms of antibody are expressed
Using tetanus vaccine as antigen, it is used in combination coating buffer that will be coated with 96 hole elisa plates after 0 times of dilution of antigen 1, per hole
4 DEG C of 100uL is coated with overnight, and 2 h are closed with confining liquid room temperature.Using the transient transfection supernatant of 100uL as 37 DEG C of incubations of primary antibody
2h, with HRP/anti-His-tag (1:2000 dilutions) as 37 DEG C of incubation 1h of secondary antibody, 100 holes μ l/ of substrate developing solution are added, often
After warm avoid light place 5min, with 2M sulfuric acid stopped reactions, analysis is detected with 450nm wavelength.
6, antibody great expression and purifying
There are the heavy chain of antibody of neutralization activity and 293 cell of expression vector cotransfection of light chain by neutralize experimental identification, turns
6-8h changes fresh culture after dye, and in 37 DEG C of 8%CO296h is cultivated in incubator.Collect transfection supernatant, 4000rpm centrifugations
1h is purified using albumen (Protein) A affinity chromatographies.Antibody is examined using SDS-PAGE and Western Blot
Expression and purifying situation.
7, result
As a result as shown in Figure 1, 2, confirm that obtaining purer purpose resists by SDS-PAGE methods and Western Blot methods
Body can be clearly observable antibody light chain and heavy chain after unwinding.
The neutralization activity of embodiment 2ELISA detection antibody TRN1015
1, experimentation
Using lockjaw normaltoxin as antigen, it is used in combination coating buffer that will be coated with 96 hole elisa plates after 0 times of dilution of antigen 1, per hole
In containing the overnight coating that volume is 100 μ, temperature is 4 DEG C, close 2h with confining liquid room temperature.By the antibody of expression and purification
TRN1015 is used as 37 DEG C of incubation 2h of primary antibody after being serially diluted, with HRP/anti-His-tag (1:2000 dilutions) it is used as two
Anti- 37 DEG C of incubations 1h, are added room temperature avoid light place 5min after substrate developing solution, with 2M sulfuric acid stopped reactions, with 450nm wavelength into
Row detection and analysis.
2, result
The results are shown in Figure 3, and (antibody concentration is about after the antibody of expression and purification carries out 10000 dilutions：0.0002μg/
Ml), antibody TRN1015 remains able to neutralize lockjaw normaltoxin, therefore, antibody TRN1015 provided in an embodiment of the present invention
With extremely strong neutralization activity.
The affine activity dynamics of 3 antibody TRN1015 of embodiment are analyzed
1, prize law is tested
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 1ug/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 MgCl2 are used after completing 1 cycle
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.
4, result
As a result as shown in Figure 4 and Table 1, neutralizing antibody TRN1015 to the affinity of tetanus toxin up to 10-9The quantity of mol
Grade shows that antibody TRN1015 provided in an embodiment of the present invention has very high affine activity；Moreover, the antibody and between
In conjunction with rate (ka) be 3.88E+04Ms-1, dissociation rate (kd) reach 9.40E-05s-1, show the antibody combination/compound
Object is with good stability；Also, the antibody concentration has carried out repetitive cycling twice when being 4ug/mL, and analysis result is very
Close, repetition shows the stability of experiment condition and instrument state, it was demonstrated that the reliability of testing result.
Table 1：The affine Activity determination result of antibody TRN1015 and tetanus toxin
The protection test in vivo of 4 antibody TRN1015 of embodiment
1, the measurement (LD of median lethal dose50)
Prepared tetanus toxin is diluted 10 successively with dilution2, 103, 104, 105, 106, 107, each dilution
At least dilute 2ml, take 0.2ml inject mouse, every group 4.Observation 5 days.LD is calculated according to experimental result50, experimental group difference
Use 20 times and 60 times of LD50Dosage.
2, TRN1015 detects the protection of small white mouse
Experiment is divided into 3 groups, every group of 4 mouse, every mouse injects 0.4ml, wherein negative control group includes 0.2ml
Toxin+0.2ml borate buffered salines；Positive controls include 0.2ml toxin+0.2ml antitoxins；Experimental group includes 0.2ml
Toxin+0.2ml monoclonal antibodies.
The normal antitoxin diluted is quantitatively drawn in mixing and the monoclonal antibody to be checked of different dilutions is respectively charged into small test tube,
Often the dilution test toxin of equivalent is added in pipe, is uniformly mixed, jumps a queue, and 37 DEG C of incubation 1h carry out subcutaneous abdomen to small white mouse immediately
Injection.When mixing, drawing the suction pipe of normal antitoxin, monoclonal antibody to be checked and toxin must not use with.
3, result
As a result as shown in Figure 5,6, when the attack of the tetanus toxin of the LD50 agent at 20 times, negative control group mouse for 24 hours-
48h is all dead, positive controls mouse and each experimental group (0.62ug/ml, 1.85ug/ml, 5.56ug/ml, 16.67ug/ml
With 50ug/ml dosage) mouse all survives in 7 days；Under the tetanus toxin attack of the LD50 agent at 60 times, negative control group is small
For mouse in all death interior for 24 hours, dosage is that the experimental mice of 0.62ug/ml observes whole death, dosage in 120h
The experimental mice of 1.85ug/ml is then all dead in 144h, experimental group (5.56ug/ml, 16.67ug/ of middle and high dosage
Ml and 50ug/ml dosage) mouse all survives in 7 days.Show that antibody TRN1015 provided in an embodiment of the present invention is anti-with standard
The potency (10IU/ml) of toxin quite, can be effectively protected the attack of animal defense lethal dose tetanus toxin, anti-with standard
The protectiveness of toxin is almost the same.Meanwhile the actual amount of antibody TRN1015 provided in an embodiment of the present invention is anti-far below standard
Toxin, this shows its effect more preferably than normal antitoxin.Thereby, it is possible to illustrate antibody TRN1015 provided in an embodiment of the present invention
The vivotoxin of mouse can be effectively neutralized, mouse is protected.
Those skilled in the art will readily occur to its of the present invention after considering specification and putting into practice the disclosure invented here
Its embodiment.This application is intended to cover the present invention any variations, uses, or adaptations, these modifications, purposes or
Person's adaptive change follows the general principle of the present invention and includes undocumented common knowledge in the art of the invention
Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are by following
Claim is pointed out.
It should be understood that the relational terms of such as " first " and " second " or the like be used merely to an entity or
Operation is distinguished with another entity or operation, and without necessarily requiring or implying between these entities or operation, there are any
This actual relationship or sequence.The invention is not limited in the precision architecture for being described above and being shown in the accompanying drawings,
And various modifications and changes may be made without departing from the scope thereof.The scope of the present invention is only limited by the attached claims
System.
Sequence table
<110>Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd
Guangzhou Tylenol enlightening bio tech ltd
<120>A kind of neutralizing antibody of anti-tetanus toxin and application
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Ile Ser Gly Thr Ser Gln Tyr Ile
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<213>Artificial sequence (Artificial Sequence)
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35 40 45
Ser Ser Ile Ser Gly Thr Ser Gln Tyr Ile Tyr Tyr Ala Asn Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Tyr
65 70 75 80
Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
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Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Thr Phe Thr Leu Thr Ile Asp Ser Leu Gln Pro
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100 105
Claims (10)
1. a kind of neutralizing antibody of anti-tetanus toxin, which is characterized in that including：
Including the variable heavy chain domain of VH CDR1, VH CDR2 and VH CDR3 and including VLCDR1, VLCDR2 and VLCDR3
Variable light chain domain, wherein
The VH CDR1 include amino acid sequence shown in SEQ ID NO.1；
The VH CDR2 include amino acid sequence shown in SEQ ID NO.2；
The VH CDR3 include amino acid sequence shown in SEQ ID NO.3；
The VL CDR1 include amino acid sequence shown in SEQ ID NO.4；
The VL CDR2 include amino acid sequence shown in SEQ ID NO.5；
The VL CDR3 include amino acid sequence shown in SEQ ID NO.6.
2. neutralizing antibody according to claim 1, which is characterized in that further include weight chain variable shown in SEQ ID NO.7
Light chain variable region shown in area and SEQ ID NO.8.
3. neutralizing antibody according to claim 1, which is characterized in that the neutralizing antibody includes：
With the VH CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.1, and/
Or with the VL CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.4；And/or
With the VH CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.2, and/
Or with the VL CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.5；And/or
With the VH CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.3, and/
Or with the VL CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.6.
4. neutralizing antibody according to claim 1, which is characterized in that the neutralizing antibody immunospecifically combines broken
Wind toxin element A segments and/or C segments and/or neutralization tetanus toxin, the neutralizing antibody have at least 10-6、10-7、10-8、10-9
Or 10-10The affinity to the tetanus toxin and/or the clostridium tetani of M.
5. a kind of composition, which is characterized in that the neutralization including the anti-tetanus toxin described in any one of claim 1-4 is anti-
Body.
6. a kind of nucleic acid molecules, which is characterized in that anti-broken described in any one of described nucleic acid molecule encoding claim 1-4
The neutralizing antibody of wind toxin element.
7. a kind of expression vector, which is characterized in that including the nucleic acid molecules described in claim 6.
8. a kind of recombinant cell, which is characterized in that including the nucleic acid molecules described in claim 6 or the table described in claim 7
Up to carrier.
9. a kind of kit, which is characterized in that the neutralization including the anti-tetanus toxin described in any one of claim 1-4 is anti-
The nucleic acid molecules described in composition or claim 6 described in body or claim 5 or the expression described in claim 7 carry
Body or recombinant cell according to any one of claims 8.
10. a kind of application process, which is characterized in that including：
The neutralizing antibody of anti-tetanus toxin described in any one of claim 1-4 prepare by tetanus toxin infection and
Application in the drug of infection due to Clostridium tetani；And/or
The neutralizing antibody of anti-tetanus toxin described in any one of claim 1-4 prepare detection tetanus toxin infection with
And the application in the kit of infection due to Clostridium tetani；And/or
Composition described in claim 5 is in preparing treatment by the drug of tetanus toxin infection and infection due to Clostridium tetani
Application；And/or
Composition described in claim 5 is in preparing the kit of the infection of diagnosis tetanus toxin and infection due to Clostridium tetani
Application；And/or
Nucleic acid molecules described in claim 6 are preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application；And/or
Expression vector described in claim 7 is preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application；And/or
Recombinant cell according to any one of claims 8 is preparing disease of the treatment by tetanus toxin infection and clostridium tetani mediation
Drug in application.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810420730.5A CN108623681B (en) | 2018-05-04 | 2018-05-04 | A kind of neutralizing antibody of anti-tetanus toxin and application |
| PCT/CN2018/090488 WO2019210552A1 (en) | 2018-05-04 | 2018-06-08 | Neutralizing antibody against tetanus toxin and use thereof |
| BR112020013094-0A BR112020013094A2 (en) | 2017-12-29 | 2018-12-28 | TOTALLY HUMAN NATURAL NEUTRALIZING MONOCLONAL ANTIBODY AGAINST TETANIC TOXIN AND ITS APPLICATIONS |
| AU2018395100A AU2018395100A1 (en) | 2017-12-29 | 2018-12-28 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| PCT/CN2018/124958 WO2019129214A1 (en) | 2017-12-29 | 2018-12-28 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| EP18896977.8A EP3733699A4 (en) | 2017-12-29 | 2018-12-28 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| US16/958,213 US11725046B2 (en) | 2017-12-29 | 2018-12-28 | Human neutralizing anti-tetanus toxin monoclonal antibody and its applications |
| JP2020555284A JP7368670B2 (en) | 2017-12-29 | 2018-12-28 | Completely natural human neutralizing monoclonal antibody against tetanus toxin and its application |
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| CN201810420730.5A CN108623681B (en) | 2018-05-04 | 2018-05-04 | A kind of neutralizing antibody of anti-tetanus toxin and application |
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Cited By (2)
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|---|---|---|---|---|
| WO2019129214A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| 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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| CN1634991A (en) * | 2003-12-30 | 2005-07-06 | 龚小迪 | Human anti-tetanotoxin monoclonal antibody, method for preparation and application |
| CN101220096A (en) * | 2003-12-30 | 2008-07-16 | 龚小迪 | Preparation and application of humanized anti-spasmotoxin monoclone antibody |
| CN102206275A (en) * | 2011-04-27 | 2011-10-05 | 上海生物制品研究所 | Anti-tetanotoxin monoclonal neutral antibody, and composition and application thereof |
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| ATE414720T1 (en) * | 2000-12-05 | 2008-12-15 | Alexion Pharma Inc | RATIONALLY DESIGNED ANTIBODIES |
| CN100391975C (en) * | 2003-07-21 | 2008-06-04 | 北京明新高科技发展有限公司 | Human source anti- tetanus exotoxin antibody and preparation method and use thereof |
| CN102875674B (en) * | 2011-10-27 | 2015-02-04 | 成都蓉生药业有限责任公司 | Anti-tetanotoxin antibody, and preparation method and application thereof |
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2018
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- 2018-06-08 WO PCT/CN2018/090488 patent/WO2019210552A1/en active Application Filing
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|---|---|---|---|---|
| CN1634991A (en) * | 2003-12-30 | 2005-07-06 | 龚小迪 | Human anti-tetanotoxin monoclonal antibody, method for preparation and application |
| CN101220096A (en) * | 2003-12-30 | 2008-07-16 | 龚小迪 | Preparation and application of humanized anti-spasmotoxin monoclone antibody |
| CN102206275A (en) * | 2011-04-27 | 2011-10-05 | 上海生物制品研究所 | Anti-tetanotoxin monoclonal neutral antibody, and composition and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019129214A1 (en) * | 2017-12-29 | 2019-07-04 | 珠海泰诺麦博生物技术有限公司 | Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof |
| 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 |
| CN113416249A (en) * | 2020-10-21 | 2021-09-21 | 北京智仁美博生物科技有限公司 | Compositions against tetanus toxin |
| CN112225813B (en) * | 2020-10-21 | 2021-12-21 | 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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| WO2019210552A1 (en) | 2019-11-07 |
| CN108623681B (en) | 2019-05-24 |
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