CN1305904C - Human anti-tetanotoxin monoclonal antibody, method for preparation and application - Google Patents

Abstract

The present invention provides a human source anti-tetanus toxin monoclonal antibody and a preparing method and the purpose thereof. The human source anti-tetanus toxin monoclonal antibody is a human source antibody which comprises an antibody variable region which comprises or doesn't comprises the constant region of the antibody; the antibody has the capability to neutralize tetanus toxin; the gene and the protein sequence of the antibody variable region are shown as the sequence 1. The human source anti-tetanus toxin monoclonal antibody has the advantages of no possibility to induce obvious anaphylactic reaction, high titer, long effect, no animal virus pollution in products and infinite production. The present invention also provides the biological functions, a measuring method, a production manufacturing method and the application of the human source anti-tetanus toxin monoclonal antibody.

Description

The preparation and the application of the anti-tetanus toxin monoclone antibody in people source

One, technical field

The present invention relates to the preparation and the application of the anti-tetanus toxin monoclone antibody in a kind of people source.

Two, background technology

The tetanus that is commonly called as is a kind of by caused common and the disease that mortality ratio is very high of tetanus bacillus infection.Tetanus bacillus is present in soil widely, dust, and iron rust etc. are located, and are easy to along with wound, and as general wound, wound the when women produces the baby or the like infects human body.After the people was by tetanus bacillus infection, in the latent period about seven days, bacterium is a large amount of breedings and release toxin in human body.Tetanus toxin causes fatal harm by destroying human neuronal cell line to the people.Mortality ratio is up to more than 90%.In a single day toxin is attached on the neurocyte, causes violent nervous symptoms immediately, and patient is agonizing, dies very soon.After patient was diagnosed as the infection tetanus bacillus, owing to have a large amount of bacteriums and toxin in the body this moment, use antibiotic had had little time to save patient's life, and unique effective methods of treatment is to use the antibody of anti-tetanus toxin.The antibody of anti-tetanus toxin is logical to carry out neutralization reaction and makes the toxin inactivation with toxin, eliminates.Though the injection tetanus vaccine can prevent above-mentioned situation to take place fully.Thereby but owing to not having vaccination or the expired situation of tetanus bacillus infection that causes of immunological effect still very general.

The antibody that present China is used for clinical anti-tetanus toxin is animal antiserum (being mainly the anti-tetanus toxin antiserum(antisera) of horse).The antiserum(antisera) of animal causes that through regular meeting the human allergy reacts as the foreign matter antigen of human body.Serious allergy sufferers is about 20%-30% of patient's sum.Even more serious is, the virus of increasing animal begins to infect human through variation, and in a single day the situation resemble the mad cattle disease occurs on the length, and its consequence is hardly imaginable.Thereby this product demands urgently eliminating, and replaced by the purified virus-free contaminating impurity antibody that waits in people source.The anti-tetanus toxin monoclone antibody that utilizes gene recombination technology to prepare the people source should be optimal selection, because this will fundamentally overcome the puzzlement and the possible harm that is caused by virus pollution of the existing sero-fast severe allergic reaction in market.

Utilize traditional cell-fusion techniques to make very difficulty of human antibody, because hybridoma technology commonly used does not still have important breakthrough in people-people's hybridoma technology.Its reason is that existing people-people's hybridoma is also very unstable because can not find ideal as the human tumor cells that merges usefulness on the one hand, the antibody of generation even do inadequately to detect and use.A lot of repressed Causative virus are arranged in the cell of the opposing party's dough figurine, in case these viruses that are activated can reactivate and duplicate, thereby the antagonist product produce to pollute, and uses very dangerous.If run into very rare antibody, the plasmacytic probability that is separated to these antibody is just very little because can form stablize hybridoma just to account for all production antibody plasmacytic seldom a part of, lose very serious.These factors are suitable as to find the cell strain of antibody to be difficult to realize with hybridoma technology.

Another method of making antibody is to utilize genetic engineering technique.The genetic engineering antibody group antibody of weighing again, it is on the basis of fully realizing IgG antibody gene structure and function, uses DNA recombinant technology antagonist molecule on gene level and splices the antibody that is assembled into.Genetic engineering antibody had both kept specificity and the biologic activity of natural antibody, more can reach the product of endless purifying by the suitability for industrialized production technology, be with a wide range of applications.

Successfully make up the several genes engineered antibody so far in the world, comprise several base types, be human mouse chimeric antibody (chimeric antibody, being about to the human IgG district is connected with the antibody variable region of mouse, thereby the antibody fragment that in same antibody molecule, contains the different genera source), reshaping antibody (reshapingantibody) also is called humanized antibody (humanized antibody, be further minimizing and merge the immune response that mouse source antibody causes in the antibody, will merge in the antibody variable region, mouse source structure and further transform); Human antibody (human antibody, the i.e. antibody of mankind itself's generation) etc.Wherein human antibody does not contain any alien gene owing to be 100% gene from the people, thereby can not cause anaphylaxis to people's application, is suitable for clinical application most.

Antibody variable region wherein has only antigen that partial sequence has antibody variable region in conjunction with feature, and Most amino-acids sequence does not wherein have antigen in conjunction with feature.Be divided in the antibody variable region sequence skeleton district (framework region, FR) and complementary determining region (complementary determining region, CDR, light-chain amino acid sequence 24-34,50-56,89-97, heavy chain 31-37,50-65,95-102).Wherein have only the CDR district to combine with antigen.Remaining is a supporting structure, and the change of the aminoacid sequence of this part still can keep antibody and original antigenic binding ability.Make up people-mouse chimeric antibody or CDR grafted antibody (CDR-grafted Ab) according to this principle people.This technology is exactly the CDR sequence of mouse source antibody is partly transplanted and to replace human antibody corresponding C DR sequence, although its aminoacid sequence of the antibody of transforming out thus changes in a large number, still keeps the function with original AI.

Utilize genetic engineering technique production people mouse to merge the technology maturation of antibody and humanized antibody (humanizedantibody), product is the marketization.Be used for the clinical mouse monoclonal antibody that mostly is so far, but it may seriously limit it in Clinical Application to the allergy that the people causes.Utilize genetically engineered preparation and produce completely that human antibody (that is all being people's gene) is world's the next item up emerging technology and industry.The key of technology is how to obtain the gene fragment of energy expressing antibodies peptide chain.A kind of way that obtains the gene fragment use of energy expressing antibodies peptide chain at present is that the bone-marrow-derived lymphocyte that will produce required antibody from blood of human body is picked out, representative technology such as antibody library method (phage antibody library).This method be with the whole antibody clonings in people's the somatic cell gene in the upstream of bacteriophage coat protein gene, make the fusion product of antibody protein fragment and coat protein be expressed in the surface of phage particle simultaneously.Thereby, only need utilize immobilized antigen, just can from antibody library, filter out the surface and have the segmental oblatio phage clone of specific antibody.But the shortcoming of this method maximum is that the antibody of its screening is not high to antigenic avidity, and direct applied clinical value is little.This is because the antibody selected from people's somatic antibody gene storehouse all is initial antibody, and the transgenation that causes through contact with antigen combines with antigen to compete etc. and screens selection process naturally.The another kind of method that obtains the human antibody gene fragment is to transform the gene of mouse, makes it produce the antibody in people source.The somebody mixes human peripheral leucocytes with the antigen of probe mark, thus the plasmocyte that will wherein produce antibody pick out, therefrom obtain antibody gene again.

The final purpose of antibody engineering is in a suitable system, makes exogenous gene high-efficient expressed.The clone that it comprises foreign gene transcribes, and translates processing, and the processes such as separation and purification of expression product.Engineered expression system can be divided into protokaryon and eukaryotic expression system.Complete antibody albumen can only be expressed with eukaryotic expression system because of being glycosylated protein.Eukaryotic expression system comprises yeast, plant, and insect and mammlian system, the wherein expression of suitable human antibody of mammlian system expression system is because this system guarantees that the glycosylation of expressed protein and people's is in full accord.The clinical application of this antagonist is very important, because some due biological functions of incorrect glycosylated antibody deficiency (as complement activation, the transformation period in the body), and can cause anaphylaxis.Recently, some new technologies are put into effect successively, and hope can make it carry out glycosylation as the Mammals by the yeast genes transformation.If the variable region part of expressing antibodies only, also available prokaryotic system is expressed such as bacterium.

Utilizing mammlian system to express can carry out at cell levels, and the myeloma cell such as personnel selection also can be undertaken by transgenic animal.The latter changes antibody gene the ovum of animal over to, makes the animal that the clones antibody of cloning by expression automatically, and from milk or other secretory product in large quantities secretion come out.This method has been avoided the industrialization investment of large scale culturing cell, has very big economic outlook.

Existing successively in recent years several pieces of reports about invention and preparation humanized anti-spasmotoxin monoclone antibody.Though but combining with tetanus toxin of having of these antibody, can not toxopexic toxicity; What have can toxopexic toxicity, but it is not high to tire.Thereby all really do not possess the humanized anti-spasmotoxin monoclone antibody that business development is worth so far.Can find and can carry out in scale operation efficient and the anti-monoclonal antibody in tetanus toxin people source is still the direction that people pursue.Antibody is toxopexic tires highly more, and the value of its clinical application is big more.Have only the toxopexic antibody of efficient also energy just have the value of the market development.The toxopexic ability of antibody is obviously by its chemical structure decision.Regrettably people do not know which type of structure can give the antibody height and tire so far.Thereby to seek efficient antibody still be the stochastic process of maying come by something with luck, but not by searching for it, consequently unpredictalbe.

Can the screening of efficient antibody depend on and obtain a large amount of relevant resources.The method of all screening anti-tetanus toxin antibodies of reporting at present all can only be obtained and produce the plasmacytic wherein part of antibody, even sub-fraction, thereby it is very little to filter out high-titer antibody clone's probability.Must find better method,, filter out the probability that high-titer antibody is cloned thereby improve to obtain the plasmocyte of whole production antibody.

Three, summary of the invention

Main purpose of the present invention is to overcome the above-mentioned shortcoming that currently available products exists; and provide the preparation and the application of the anti-tetanus toxin monoclone antibody in a kind of people source; it is animal antiserum (being mainly horse anti-tetanus toxin antiserum(antisera)) based on the employed antibody that is used for clinical anti-tetanus toxin of present China; the antiserum(antisera) of animal is as the foreign matter antigen of human body; cause that through regular meeting the human allergy reacts; even more serious is; the virus of increasing animal begins to infect human through variation; thereby this product demands urgently eliminating, and replaced by the purified virus-free contaminating impurity antibody that waits in people source.

The objective of the invention is to realize by following technical scheme.

Humanized anti-spasmotoxin monoclone antibody D of the present invention, it is characterized in that, it is a human antibody, comprise antibody variable region, this antibody comprises or does not comprise the constant region of antibody, during this antibody has and the ability of tetanus toxin, the aminoacid sequence of this antibody variable region of encoding is shown in the sequence in the sequence table 11 and 3.

Aforesaid humanized anti-spasmotoxin monoclone antibody D wherein, changes, lacks or add one or several amino acid described aminoacid sequence, and still keeps in it and the ability of tetanus toxin.

The nucleotide sequence of the amino acid sequences of aforesaid humanized anti-spasmotoxin monoclone antibody D, wherein, this nucleotide sequence is shown in the sequence in the sequence table 15 and 7.

Aforesaid humanized anti-spasmotoxin monoclone antibody D, wherein, the constant region of described antibody is any in the constant region of people source heavy chain immunoglobulin or light chain; This CH is the constant region of humanized IgG, IgM, IgA, IgE or IgD, or includes the CH of hinge region or any fragment in the CH; Described constant region of light chain is the constant region of the κ or the λ of human immunoglobulin's light chain.

The preparation method of a kind of humanized anti-spasmotoxin monoclone antibody D as claimed in claim 1 of the present invention is characterized in that, it comprises that the method for the method of utilizing gene recombination technology or chemosynthesis prepares and produces; Wherein, utilize the method for gene recombination technology to prepare, comprising: the clone of A, antibody gene and sequential analysis; The structure of B, antibody gene expression vector; C, in a suitable system or host, antibody gene is expressed: D, the antibody expression host that cultivates and increase in a large number; E, the antibody separation and purification to expressing: F, to the qualitative and quantitative analysis of expressing antibodies.

The preparation method of aforesaid humanized anti-spasmotoxin monoclone antibody D, wherein, the expression vector of described antibody gene is plasmid vector or virus vector, the genetically modified carrier of animal or universal support; The expression vector of described antibody gene changes among a suitable system or the host and is expressed, and wherein system of Shi Heing or host comprise the cell of animal or plant, cell strain, bacterium or yeast; This animal is transgenic animal.

The preparation method of aforesaid humanized anti-spasmotoxin monoclone antibody D wherein, also comprises the protein sequence that detects antibody.

The application of the described humanized anti-spasmotoxin monoclone antibody D of claim 1 of the present invention in the pre-antitetanus infectation of bacteria medicine of preparation.

The application of the described humanized anti-spasmotoxin monoclone antibody D of claim 1 of the present invention in the diagnostic preparation of preparation tetanus infectation of bacteria.

The present invention also comprises following technology contents:

Humanized anti-spasmotoxin monoclone antibody of the present invention is characterized in that, it is a human antibody, comprise antibody variable region, this antibody comprises or does not comprise the constant region of antibody, and during this antibody has and the ability of tetanus toxin, the gene of antibody variable region and protein sequence are shown in sequence table 1.

Aforesaid humanized anti-spasmotoxin monoclone antibody is characterized in that, the gene of described antibody variable region and protein sequence comprise homologue shown in the sequence table 1 or modifier; Homologue be its homology of protein sequence of antibody variable region reach above-mentioned sequence 60% or above, 70% or above, 80% or more than; Modifier for the product that the amino acid or their nucleotide sequence of human antibody changed by replacement, increase or brachymemma still keep human antibody expressing antibodies albumen have in and the ability of tetanus toxin.

Aforesaid humanized anti-spasmotoxin monoclone antibody, it is characterized in that, described homologue and modifier also comprise the modification after the pirate recordings of antagonist protein is synthesized, make the antibody protein of expression have in and the ability of tetanus toxin, strengthen treatment or prophylactic effect, raising in external stability or to the defensive ability/resistance ability of body endoproteinase to reach.

Aforesaid humanized anti-spasmotoxin monoclone antibody is characterized in that, the constant region of described antibody is any in the constant region of people source heavy chain immunoglobulin or light chain; This CH is a humanized IgG, IgM, and IgA, IgE, the constant region of IgD, and include the CH of hinge region (hinge region) or any fragment in the CH; Described constant region of light chain fragment is the constant region of the κ or the λ of human immunoglobulin's light chain.

The preparation method of humanized anti-spasmotoxin monoclone antibody of the present invention is characterized in that, it comprises that the method for the method of utilizing gene recombination technology and chemosynthesis prepares and produces; Wherein, utilize the method for gene recombination technology to prepare, comprising: the clone of A, antibody gene and sequential analysis; The structure of B, antibody gene expression vector; C, hold in the justice, antibody gene is expressed: D, the antibody expression host that cultivates and increase in a large number in a suitable system or host; E, the antibody separation and purification to expressing: F, to the qualitative and quantitative analysis of expressing antibodies.

The preparation method of aforesaid humanized anti-spasmotoxin monoclone antibody is characterized in that, the expression vector of described antibody gene is plasmid vector, virus vector, the genetically modified carrier of animal or universal support.

The preparation method of aforesaid humanized anti-spasmotoxin monoclone antibody, it is characterized in that, described antibody gene carrier can change among a suitable system or the host and be expressed, and the expression system that is fit to comprises the cell of animal and plant, cell strain, bacterium or yeast; This animal also can be transgenic animal.

The method of aforesaid humanized anti-spasmotoxin monoclone antibody is characterized in that, also comprises: the sequence of A, detection antibody protein or gene nucleic acid; B, the detection antibody tetanus toxin to injection in the experimental animal body has its toxic function of inhibition.

The application of humanized anti-spasmotoxin monoclone antibody of the present invention in the pre-antitetanus infectation of bacteria medicine of preparation.

The application of humanized anti-spasmotoxin monoclone antibody of the present invention in the diagnostic preparation of preparation tetanus infectation of bacteria.

The invention has the beneficial effects as follows:

The invention provides several new humanized anti-spasmotoxin monoclone antibodies that utilize gene recombination technology to produce, its gene and protein sequence are inequality with existing report, the characteristics of this antibody have very high in the ability of tetanus toxin.By the small white mouse evidence, above-mentioned antibody can watch for animals and resist the attack of tetanus toxin.In addition, antibody compares as medicine and domestic existing antibody, also has the following advantages:

1, can not bring out tangible anaphylaxis: present domestic use be horse serum antibody, bring out severe allergic reaction easily.And this antibody is the human antibody (hundred-percent people's gene) that utilizes gene recombination technology to make, thereby can not bring out obvious anaphylaxis theoretically.

2, very high tiring arranged: these antibody have more than the much higher toxopexic ability of past report.Prove that with the small white mouse in vivo test antibody of the present invention can protect mouse to resist the attack of the tetanus toxin of lethal dose fully.

3, can produce endlessly.

4, long-acting: as to guarantee to keep in the body effective drug level.

5, can accomplish that product does not have animal virus and pollutes: the production process of this antibody can adopt the production of state-of-the-art in the world serum-free cell culture technology, can take in its Production Flow Chart not use any animal preparation, thereby product has been avoided the pollution of animal or human's virus.World-shaking mad cow disease, animal infectious diseases such as SARS are wreaked havoc the Europe and the fact all over the world, make people's acutely aware, use the biotechnological formulation of animal serum to exist huge pollution hidden trouble, even the serious threat human beings'health, and this product has advantage in this regard.

Four, accompanying drawing and subordinate list explanation

Table 1 is summed up for the plasmacytic part in people source that the present invention screens secretion anti-tetanus toxin antibody.

Table 2 is an iodine of the present invention ¹²⁵The anti-tetanus toxin antibody of mark and people source Immunoglobulin IgG1 are in rabbit intravital half life.

Table 3 is that gene recombination anti-tetanus toxin antibody of the present invention is to protection test result in the animal body.

Fig. 1 is the present invention clone's the segmental gel electrophoresis figure of some humanized anti-spasmotoxin antibody variable region cDNA.

Fig. 2 is the expression vector synoptic diagram of the humanized anti-spasmotoxin light chain of antibody of the present invention's structure.

Fig. 3 is the expression vector synoptic diagram of the humanized anti-spasmotoxin heavy chain of antibody of the present invention's structure.

Fig. 4 is the humanized anti-spasmotoxin heavy chain of antibody and the segmental gel electrophoresis figure of light chain cdna of gene recombination of the present invention.

Fig. 5 is the determination experiment result of expressed excretory gene recombination anti-tetanus toxin antibody in the Chinese hamster ovary celI supernatant.

Fig. 6 is the provide protection figure of the single and mixed antibody of the present invention to animal.

Five, embodiment

The present invention relates to several new humanized anti-spasmotoxin monoclone antibodies, comprise by gene and protein sequence, biological function, the measuring method of these new antibodies, manufacture the method for these antibody, and utilize the medicine of these Antibody Preparation treatment tetanus bacillus infections and be used to prepare the preparation of diagnosis tetanus infectation of bacteria.

The definite definition of some terms of using among the present invention:

1, antigen: can cause the material that produces the specific immune reaction in the animal body.

2, human antibody: the antibody that human body synthesizes.Human antibody is divided into and is IgG, IgA, IgM, IgD, the big class of IgE5.Wherein IgG is divided into IgG1 again, IgG2, and IgG3, the IgG4 subclass, their heavy chain quilt is called after IgG 1 correspondingly, and IgG 2, and IgG 3, and IgG 4.Light chain then has κ or two types of λ.The gene of various dissimilar antibody is different (seeing for details down) with protein sequence.

3, the basic structure of antibody: (one) tetrapeptide chain structure: the basic structure of each antibody-like all is the symmetrical structure of four peptide chains, comprises two identical heavy chains and two identical light chains.Article two, reach between heavy chain and light chain between the heavy chain and between two light chains, borrow disulfide linkage to connect respectively.The heavy chain of antibody is made up of more than 400 amino acid.It can be fallen into 5 types according to the antigenic difference of heavy chain, the antibody of being made up of them is called IgM, IgG, and IgD, IgA, the light chain of IgE. antibody respectively contain more than 200 amino acid, and light chain has κ or two types of λ.(2) constant region and variable region: the N end (containing about 107-130 amino-acid residue) at light chain of antibody and heavy chain peptide chain is the variable region of antibody, is called the variable region of heavy chain and the variable region of light chain.The sequence of amino acid in this district changes to some extent with the antibodies specific difference, so claim variable region (being the V district).At the C of polypeptide end, the amino acid quantity in this district and put in order etc. all more stable, so be called constant region (being the C district).So the constant region sequence of different antibodies and structure are distinguished not quite each other, but the sequence of its variable region and structure difference are very big.The variable region of antibody is the position of decision antibodies specific.(3) hinge region of antibody: a rotating zone of the heavy chain of antibody (being the hinge zone).

4, homologous antibody: come from the antibody of same kind animal, if be called homologous antibody as the antibody that produces by other people such as the antibody that is applied to the people.The people does not produce tangible immune response to homologous antibody.If be used for people's antibody and be by the different genera animal produce be called not homology or heterologous antibody.Because its antigen property of antibody that the different genera animal produces is very inequality, thereby the people can produce tangible immune response to heterologous antibody.

5, the specificity of antibody: a kind of antigen and its bring out relation between the antibody of generation just as the relation of key and lock, be mutually at, special.

6, antibody and antigen bonded avidity: the avidity between antigen and antibody antigen and the antibody be meant specific antibody and its at antigen bonded tightness degree, the big more expression bonded of avidity is tight more.

7, the neutralization reaction of antibody and tetanus toxin: the different sites of tetanus toxin all can bring out generation antibody.Have only those antibody that produces at the special significant points of toxin, just can be by eliminating the function of (that is neutralizing) toxin with combining of this position.Neutralization is tired and is meant in the energy and the quantity of the required antibody of toxin of fixed amount.Required antibody amount is few more, and this antibody neutralization is tired high more.Toxopexic the tiring of antibody depended on antibody and toxin bonded tightness degree, is by the decision of the chemical structure of antibody.

8, plasmocyte: plasmocyte is an activated B cell, can produce secretory antibody.Each plasmocyte can only be produced at a kind of antigenic antibody.

9, tetanus toxin: tetanus toxin is the toxin that tetanus bacillus discharges, and is that a kind of molecular weight is 150, and the protein of 000Da size, its pathological effect are to destroy human neuronal cell line and the people is caused fatal harm.

10, albumen merges: the gene segment of two different proteins is connected, and its expression product is a single chain protein matter that is connected with peptide bond each other by two different proteins.

11, immunological rejection: animal is to importing the tissue of intravital other different genera animal, the immune response that organ or material produce, even cause this histoorgan or material necrosis.

The invention provides several new humanized anti-spasmotoxin monoclone antibodies, it is characterized in that, these antibody are human antibodies of 100%, the gene of their variable region and protein sequence are new, its structure comprises the variable region of antibody, (comprising or not comprising constant region), during this antibody has and the ability of tetanus toxin, wherein, the homologue of the gene of variable region and protein sequence such as sequence table 1 and this sequence and modifier are formed, and wherein constant region is any in the constant region of people source heavy chain immunoglobulin or light chain.

The present invention also provides the method that the overwhelming majority is produced the plasmocyte resource of antibody of obtaining, thereby improves the probability that filters out the high-titer antibody clone, and the feature of this method is to utilize the plasmocyte surface receptor to separate to obtain.

The method that the present invention also provides preparation and produced these several humanized anti-spasmotoxin monoclone antibodies is comprising the method for utilizing gene recombination technology to manufacture.The method major technique of utilizing gene recombination technology to manufacture comprises the clone and the sequential analysis of (1) antibody gene; (2) structure of antibody gene expression vector.(3) in a suitable system or host, antibody gene is expressed; (4) increase the in a large number host system of expressing antibodies; (5) the antibody separation and purification to expressing: (6) are to the qualitative and quantitative analysis of expressing antibodies.

The method that the above-mentioned method of utilizing gene recombination technology prepares and produce humanized anti-spasmotoxin monoclone antibody, wherein the expression vector of antibody gene can utilize plasmid vector, virus vector or other general carrier, also the genetically modified carrier of animal.

The method that the above-mentioned method of utilizing gene recombination technology prepares and produce humanized anti-spasmotoxin monoclone antibody, wherein the antibody gene carrier can change in the suitable system and be expressed, wherein the expression system of Shi Heing comprises cell, cell strain or yeast, the bacterium of animal and plant, and expression system also can be transgenic animal.

The testing method that the invention provides above-mentioned humanized anti-spasmotoxin monoclone antibody is, 1, detect the sequence of antibody protein or gene nucleic acid; 2, detect antibody simultaneously and in animal body tetanus toxin is had provide protection.

The invention provides above-mentioned these humanized anti-spasmotoxin monoclone antibodies are used for the treatment of the medicine of tetanus infectation of bacteria in preparation application, this antibody can be used for preparing pre-antitetanus infectation of bacteria medicine, above-mentioned antibody can be prepared various drug forms separately or with other composition, or is prepared into hybrid medicine or mixing treatment preparation with other medicines.

The invention provides of the application of above-mentioned these humanized anti-spasmotoxin monoclone antibodies at the preparation of preparation diagnosis tetanus infectation of bacteria.

The summary of technical solution of the present invention: the present invention is realized by following major technique step: what (1) filtered out the people can produce the plasmocyte that the anti-tetanus toxin antibody is tired in secretion very high; (2) from the complete variable region sequences that accesses this antibody of these cells; (3) make up the antibody gene suitable expression; (4) the antibody gene carrier is put in suitable system or the host expresses, and select the clone of stably express antibody; (5) collect also antibody purification; (6) by experiment in the external and body further filter out those can be efficiently in the antibody cloning of tetanus toxin; (7) increase the in a large number host of expressing antibodies; (8) purifying prepares antibody; (9) utilize the Antibody Preparation of producing to be used for the treatment of the medicine and the preparation of tetanus bacillus infection; (10) antibody is used for proving in the body that it is to the provide protection of animal for tetanus toxin.

Relevant detailed description of the present invention:

The invention provides several humanized anti-spasmotoxin monoclone antibodies, it is characterized in that these antibody are human antibodies, its structure comprises the variable region and the constant region of antibody, the gene of its variable region and protein sequence and existing report inequality is during this antibody has and the ability of tetanus toxin.

Above-mentioned several new humanized anti-spasmotoxin monoclone antibody, the gene of its antibody variable region and protein sequence are made up of the sequence of sequence table 1 and the homologue and the modifier of this sequence; Homologue refers to the protein sequence of antibody variable region, its homology reach above-mentioned sequence 60% or above, 70% or above, 80% or more than; Modifier refers to that this modification comprises that amino acid or their nucleotide sequence to sequence table 1 pass through to replace, increase or the brachymemma change, but the product that changes still keeps the principal character of this antibody, during promptly the antibody protein of Biao Daing has with the ability of tetanus toxin.In addition, homologue and modifier refer to that also those antagonist sequences change and the purpose of modification is to strengthen treatment or prophylactic effect in order to reach; Or in order to improve product in external stability or to the defensive ability/resistance ability of body endoproteinase; Or the modification that the patent antibody protein is done in the synthetic back of pirate recordings, as long as the product that is derived by these variations remains with the principal character of antibody of the present invention, during also the antibody protein of promptly expressing has and the ability of tetanus toxin, should belong to scope of the present invention.

Above-mentioned several new humanized anti-spasmotoxin monoclone antibody, its structure comprises the variable region of antibody or the constant region of antibody, the gene of these antibody variable regions and protein sequence are made up of the homologue and the modifier of sequence table 1 and sequence table 1, and the constant region of these antibody can be any in the constant region of people source heavy chain immunoglobulin or light chain, and promptly the variable region fragment can merge with the different human igg heavy chain constant region or the constant region of light chain.Here humanized IgG that CH refers to, IgM, IgA, IgE, IgD; This IgG wherein comprises IgG1, IgG2, IgG3, IgG4; Wherein the constant region fragment of light chain comprises the constant region of the κ or the λ of human immunoglobulin's light chain.Here CH refers to and includes hinge region (hingeregion) and CH1, CH2 and CH3 zone or any fragment wherein.

To the change and the modification of humanized anti-spasmotoxin monoclone antibody of the present invention, following various changes and modification should be included within the present invention.

Basically is if be to strengthen treatment or prophylactic effect in order to reach to the change of above-mentioned antibody and modification; Or in order to improve product at external stability (to the defensive ability/resistance ability of body endoproteinase); Or the modification that the protein of humanized anti-spasmotoxin monoclone antibody of the present invention is done in the synthetic back of pirate recordings, as change saccharification or phosphorylation composition, as long as the product that is derived by these variations remains with the principal character of this humanized anti-spasmotoxin monoclone antibody, that is the antibody protein of expressing have in and the ability of tetanus toxin, both should be included within the present invention.

This modification comprises to be passed through to replace to the amino acid of humanized anti-spasmotoxin monoclone antibody of the present invention or their nucleotide sequence, increase or brachymemma come change, but the product that changes still keeps the principal character of humanized anti-spasmotoxin monoclone antibody of the present invention, can be replaced by Isoleucine or a word used in person's names propylhomoserin such as leucine, between the acidic amino acid (or between the basic aminoacids, between the neutral amino acids, between the polare Aminosaeren or between the nonpolar amino acid, between the aromatic amino acid) or the like can replace mutually.It is generally acknowledged,, with the ability of tetanus toxin, just should belong within the scope of the invention in having simultaneously as long as product and the sequence of various antibody of the present invention, especially variable region sequences after changing have 80% or above identical.

In addition, humanized anti-spasmotoxin monoclone antibody protein of the present invention or their nucleotide sequence also can add other compound and keep the principal character of humanized anti-spasmotoxin monoclone antibody of the present invention on its pendant chemical groups, as alkali, thereby increase its solubility; Or isotropic substance, as cobalt 60, iodine 125, sulphur 35 or the like, or fluorescence, or the chimeric hybridization of their nucleotide sequence are as the toxin fragment, to reach the effect that kills and wounds target cell.These all are the methods of the reservation humanized anti-spasmotoxin monoclone antibody principal character of the present invention of widespread.

The other feature of above-mentioned humanized anti-spasmotoxin antibody of the present invention be they can be in vivo in and tetanus toxin, in this and the ability of tetanus toxin much higher more than the performance of past report, its can be in vivo with external efficiently in and tetanus toxin.The invention process case provides relevant test-results: use the small white mouse evidence, above-mentioned antibody can watch for animals and resist the attack of tetanus toxin, has very high tiring.If two kinds of different antibody are mixed use, wherein in and the dosage ratio of tetanus toxin be used alone antibody and can further obviously reduce, result of treatment significantly improves.

Humanized anti-spasmotoxin antibody of the present invention is realized by following several major technique steps: (1) filters out the plasmocyte that the people source produces secretion anti-tetanus toxin antibody; (2) from the complete variable region sequences that accesses this antibody of these cells; (3) make up the antibody gene suitable expression; (4) the antibody gene carrier is put in the suitable host expresses, and select the clone of stably express antibody; (5) by experiment in the external and body further filter out those can be efficiently in the antibody cloning of tetanus toxin.(6) measure the nucleic acid or the protein sequence of these antibody.

At first, the present invention screens the B lymph-plasma cell that to produce justacrine anti-tetanus toxin and realizes by following original creation method: at first obtain peripheral blood with the health tax blood person of tetanus vaccine immunity recently from one.Isolate main B-lymphocyte component in the peripheral blood through the method for routine, such as by the density centrifugation method.The plasmocyte of secretory antibody utilizes with the method for these albumen specific combination and these plasmocyte can be separated because the surface has some distinctive protein.Have Chemokine Receptors CXCR3 such as the plasmocyte surface.The ligand i P-10 of utilization and CXCR3 specific combination moves experimental technique by cell induction plasmocyte is isolated.With plasmocyte dilution and be put into 96 orifice plates (average every hole 1-2 cell) to each cell amplification cultivation.The plasmocyte culturing process need add some special somatomedins, such as the EL-4-B5 cell of crossing through the isotropic substance radiation exposure as vegetative cell, the lymph plain IL-2 etc. that is situated between.Through some days cultivation, the supernatant liquor of collecting cell.By detecting the antibody that has or not in (analogy detects with enzyme-linked immunoassay method) supernatant with the specific combination of tetanus toxin, filter out some plasmocyte that can secrete anti-tetanus toxin antibody clones.The present invention uses passes through the plasmocyte surface receptor and obtains the method that produces the antibody cell and compare all methods of reporting in the past and have tangible strong point, because, method of the present invention in theory can make us obtain the plasmocyte of most production antibody, thereby the probability that filters out the high-titer antibody clone is improved greatly; And other method such as the cytogamy method, all can only be obtained a plasmacytic wherein part of producing antibody, even sub-fraction, so these methods obviously reduce the probability that filters out the high-titer antibody clone.

The gene of anti-tetanus toxin antibody duplicated out from these plasmocyte clone can utilize multiple diverse ways.The method of describing in the analogy the implementation case 1, or other sensitive method extract the above-mentioned plasmacytic messenger RNA(mRNA) that filters out, and through the reverse transcription biochemical reaction whole messenger RNA(mRNA)s are transformed into cDNA; Utilize the special primer (described) of design that the heavy chain of antibody in each sample or the variable region of light chain are cloned respectively through sensitive PCR method (analogy Sparrow's nest PCR) such as embodiment 2.Certainly, also can be with the light chain and the heavy chain of whole antibody, that is comprise that the whole gene fragment clone in variable region and Heng Ding district comes out, measure the cloned genes sequence.

The invention provides the way that how to prepare above-mentioned humanized anti-spasmotoxin antibody, comprise chemical synthesis process and gene recombination method.Wherein, chemical synthesis process can be antibody protein elder generation branch to be made several fragments synthesize respectively, then, and again by before and after its sequence these fragments being connected with each other.Wherein protein synthesis can synthesize two kinds of main technique methods with solid phase or liquid phase, and the concrete grammar of these two kinds of technology is identical with general present current techique method, no longer describes at this.The method major technique of utilizing gene recombination technology to manufacture above-mentioned antibody comprises the structure of (1) antibody gene expression vector; (2) in a suitable system or host, antibody gene is expressed; (3) to expression system or host's extensive amplification; (4) separate expressing purifying antibody; (5) to the qualitative and quantitative analysis of expressing antibodies.

The present invention makes up above-mentioned anti-tetanus toxin antibody expression carrier and specifically realizes by following method: the expression vector of at first selecting to be fit to express human antibody.Because antibody is glycosylated protein, eukaryotic expression vector should be proper, and is all very suitable such as mammalian expression vector and yeast cell to express carrier.Express the carrier of anti-tetanus toxin antibody gene and can use plasmid vector, heavy chain of antibody hCg1 carrier shown in embodiment 3 and light chain of antibody hCK carrier, they all are the plasmid vectors of mammalian cell expression, respectively the expressing human source heavy chain of antibody with constant region light chain.Also virus vector, plant expression vector, Yeast expression carrier or other general carrier, or the used carrier of transgenic animal.Can have various regulatory gene fragments in the carrier, such as promotor (as the CMV promotor), and enhanser (as the EU enhanser) or the like.In order to screen conveniently, carrier also can be put into the impedance gene at different antibiotic, the gene of anti-Neomycin like this and anti-Puromycin.The gene fragment that also can add other in the carrier as long as the purpose that these genes reached is the expression of gene of relevant humanized anti-spasmotoxin monoclone antibody of the present invention, all should belong to category of the present invention.Clone's the heavy chain of antibody and the variable region gene of light chain are downcut with specific restriction enzyme, and the variable region gene of heavy chain and light chain is received respectively in the expression vector, be connected with the constant region fragment of antibody.If the clone's is the gene fragment (the whole gene fragment that comprises variable region and constant region) of whole antibody, then with in this whole gene fragment clone to a suitable expression.

The present invention also provides to expressing above-mentioned necessary system of anti-tetanus toxin antibody gene or host.The above-mentioned expression vector that builds is imported in the appropriate host by electric shocking method or other method and expresses.Antibody is glycosylated protein, so preferably adopt eukaryotic expression system to express.Such as, mammalian expression system just is well suited for the expression of human antibody, because the glycosylation of this system's assurance expressed protein and people's is in full accord.Also very suitable expression of Yeast system by genetic modification (this Yeast system carry out Mammals as glycosylation) in addition.Utilizing mammlian system to express can carry out at cell levels, such as the Chinese hamster ovary celI that has commonly used, the HEK293 cell, the COS cell, NS/O or HELA cell are gone back insect cell, as the Rf9 cell, or yeast cell, especially after genetic modification, can carry out identical glycosylated yeast of Mammals or the like.Also can be undertaken by transgenic animal.The latter specially goes into the ovum of animal with antibody gene, makes the animal that the clones antibody of cloning by expression automatically, and from milk or other secretory product in large quantities secretion come out.This method has been avoided the industrialization investment of large scale culturing cell, has very big economic outlook.The Chinese hamster ovary celI that adopts in the analogy embodiment of the invention 4 is as expressing the necessary host of above-mentioned anti-tetanus toxin antibody gene.Its strong point is: utilizing CHO to express and producing protein formulation is the most ripe and welcome in the world at present way.Big industrialized producing technology is ripe; Chinese hamster ovary celI can be grown in serum-free medium and be gone down to posterity.This makes whole process of production need not to use any animal preparation, thereby has avoided virus pollution; Produce protein technology with Chinese hamster ovary celI and obtained international endorsement.The some kinds of protein formulations of producing with this method reach clinical criterion of acceptability fully.Select can stably express secretory antibody, and in these antibody capables and the host cell of tetanus toxin.Through after the preliminary screening, further can pass through to detect the antibiotic impedance expression of gene of carrier, or detect the ability of secretory antibody.Concrete grammar is seen this paper, for example as the invention process case 4.

The invention provides and how aforesaid anti-tetanus toxin antibody gene is produced the method for expressing with the secretory protein form, add leader, expressed products is secreted in the culturing cell supernatant liquor in addition such as N-terminal at protein gene.Leader is cut out in the protein secreting process.Sequence table 1 has provided the example of some leaders.If utilize transgenic animal to express, then vector construction can be secreted into the movement of animal in the secretory protein mode with antibody, in milk.

The present invention further provides the method for how to produce above-mentioned humanized anti-spasmotoxin antibody, comprised with gene recombination, fermentation technique is the method that the means of production is produced the anti-tetanus toxin monoclone antibody.This method realizes by following technology: in principle, the above-mentioned method for preparing humanized anti-spasmotoxin antibody, as utilizing mammalian cell expression system such as Chinese hamster ovary celI, yeast host or expression system all can reach the purpose of producing antibody in batches by cultivating these hosts on a large scale.Analogy utilizes mammalian cell to produce antibody, can utilize the drum-type culturing bottle, or fiber tubular type culturing bottle, or the fermentation pot type increases the cell cultures scale.Every technology all has maturation process, and it is example that successful product is also arranged, concrete used equipment and zymotechnique as universal method, be not described further at this.Concrete grammar is for example as the invention process case 6.

The present invention also provides the way that how to improve antibody product output.The method that some kinds of different raising gene expression doses are arranged at present.Be exemplified below: change Dhfr (dihydrofolatereductase) gene over to host cell as what use in the embodiment of the invention, utilize the way that improves Dhfr enzyme substrates MTX (methotrexate) to filter out the high expressing cell strain, use for producing.Listed examples of the present invention shows that producing oozy antibody production can reach 3 milligrams/10 ⁹Cell is secreted into 1 liter of culture supernatant every day.It is more by further screening output to be improved.

Antibody gene of the present invention also can utilize animal to express production by transgenic technology.Transgenic animal can reach very high expression level, and production cost is low.

The present invention further provides the method that how will produce the separation and purification of excretory humanized anti-spasmotoxin monoclone antibody product, specifically realize by following method: many kinds of methods all can be with the antibody product separation and purification.The affinity chromatography separation method that is adopted such as embodiment 7.Affinity chromatography can utilize various can with the material of expressing antibodies specific combination, such as using a-protein among the embodiment 7, that it is coupled on carrier support.A-protein by with the antibody specific combination, reach ideal separation and purification result.Except also having some other, a-protein can make affinity chromatography with the protein or the simulated compound of antibody specific combination.Can be used to the separation antibody product except the affinity chromatography separation method also has other kind method, such as perfusion chromatography technology or the like, do not add detailed description at this, concrete grammar can be with reference to pertinent data.

The present invention also provides the evaluation and the screening method of secretion humanized anti-spasmotoxin monoclone antibody; further filter out in those energy and tetanus toxin by experiment in external and the body, and the antibody and the expressive host clone thereof that can shield to tetanus toxin in vivo.This technology can realize by following method: the anti-tetanus toxin antibody can utilize the whole bag of tricks to identify according to its chemical structure characteristic and biological function feature.Analogy can detect the chemical feature of antibody itself with integrated enzyme reaction (ELISA) method and isotopic labeling antibody competition experimental technique (RIA).Utilize immune neutralization reaction (antigen-antibody neutralization reaction) experiment with tetanus toxin, or can examine and determine the functional character of antibody the protection of animal experiment of injection tetanus toxin or the like method, and the size of the ability that neutralizes a toxin.Specifically for example, will change the cell cultures of antibody gene expression vector over to and collect culture supernatant.By coming the genetically modified cell of Analysis and Identification whether to synthesize justacrine antibody in the aforesaid method.Picking out those can secretory antibody, and this antibody can in and the cell strain of tetanus toxin.These cell strains have medical value probably.These authentication methods also can be used to the antibody quality of producing and tire and examine and determine.Above-mentioned many methods, as ELISA, RIA, antigen-antibody neutralization reaction experiment all is the conventional experimental technique of using, and need not describe in detail at this, concrete operations can be with reference to as immunological method class books and periodicals magazine or case study on implementation of the present invention 4 and 9.Tetanus toxin animal endogenous protective experiment concrete grammar is for example as embodiments of the invention 9.

The application of humanized anti-spasmotoxin antibody of the present invention: this humanized anti-spasmotoxin antibody is because in can be efficiently and tetanus toxin, can be applicable to make the various kinds of drug and the pharmaceutical composition of treatment tetanus bacillus infection, and be used to prepare various preparations or the preparation compositions of diagnosing tetanus bacillus infection, see that specifically embodiment 9 is described.Wherein be used to prepare the various kinds of drug and the pharmaceutical composition for the treatment of tetanus bacillus infection: humanized anti-spasmotoxin antibody of the present invention, because in can be efficiently and tetanus toxin, can be used to prepare medicine and pharmaceutical composition is treated the mortal injury that tetanus bacillus infection causes.Specifically see embodiments of the invention 9 for example.The present invention includes above-mentioned humanized anti-spasmotoxin antibody and can be prepared to different pharmaceutical dosage forms, separately or with other composition.Can be drug for injection such as (but being not limited to) as the formulation of medicine, oral drug or local application (comprise rectal administration, or the formulation of spray delivery such as nose spraying or mouthful inhale spraying etc.) various formulations.Analogy case study on implementation 9 is the examples as the solution intravenously administrable.The present invention comprises that also above-mentioned humanized anti-spasmotoxin antibody is prepared to combination therapy preparation or the technology with other medicines or method.Analogy (but being not limited to) antibody of the present invention can be treated infection medicine with other and be used, and reaches and not only suppresses tetanus toxin, also kills the effect that infects pathogeny.

Humanized anti-spasmotoxin antibody of the present invention has a lot of advantages as medicine, comprising: 1,, can not cause tangible anaphylaxis because this antibody is human antibody; 2, because this antibody half life in vivo is very long, can keep medicine effective concentration well; 3, since its have very high in and the ability of tetanus toxin, thereby consumption is few, result of treatment is remarkable; 4, can pass through to adopt the production technique of non-animal derived pollution, thereby lower the harm that causes by virus pollution greatly.Above advantage has obviously improved the clinical value of humanized anti-spasmotoxin antibody of the present invention as medicine widely.

Humanized anti-spasmotoxin antibody of the present invention can be used for preparing various preparations or the preparation compositions of diagnosing tetanus bacillus infection.Suppose the diagnosis that is used for tetanus bacillus infection after (but being not limited to) is with the antibody isotopic labeling.The present invention comprises that also above-mentioned humanized anti-spasmotoxin antibody is by independent or one-tenth and other medicines, associating diagnostic preparation or technology that preparation or method prepare together.

Embodiment 1: obtain and screen the people source plasmocyte that produces secretion anti-tetanus toxin antibody.

Recently contribute the blood person with 20 years old male surname health of tetanus vaccine immunity from one and obtain 100 milliliters peripheral blood.This tax blood person does not have any chronic medical history, does not have any disease symptoms when contributing blood, accepts the tetanus vaccine immunity before 15 days, does not inject any other vaccine with the exception of this in the past year.This whole blood is handled through EDTA, and centrifugal half an hour by the density gradient of Ficoll, 3000 rev/mins.With the sucking-off of Ficoll middle layer, thereby obtain blood leucocyte.White corpuscle suspension is put into Tissue Culture Flask in 37 degree insulations 1 hour, to remove the monocyte that sticks to frosting.Anti-CD 19 antibodies link coupled magnetic bead (Dynabeads) is mixed (20 magnetic bead/1 * 10 with the not adherent white corpuscle that cleaned ⁸Cells/ml) and at 4 ℃ in turner, be incubated 30 minutes.Utilize magnetic field force that magnetic bead is separated.Bone-marrow-derived lymphocyte on the magnetic bead is separated according to the method that the Dynal product provides.All B cell suspensions in 300 microlitre nutrient solutions, are added to transwell lamina membranacea (Bacton ﹠amp; Dicson) in the last hole.Add chemokine below this pore membrane, such as IP-10.Collect the plasmocyte that moves to down the hole by last hole through being incubated 3 hours.With the dilution of the plasmocyte collected and be put into 96 orifice plates (average every hole 1-2 cell) to each cell amplification cultivation.Through 10 days cultivation, the supernatant liquor of collecting cell.Detect the antibody that asecretory anti-tetanus toxin is arranged in the supernatant by enzyme-linked immunoassay method.The enzyme linked immunoassay method is summarized as follows: spending the night is adsorbed onto on the enzyme-linked reaction plate with tetanus toxin (Beijing institute of Biological Products).Be incubated to have removed non-specific combination with the damping fluid that contains cow's milk protein.Afterwards, the cell culture supernatant of above-mentioned collection is added on the enzyme-linked reaction plate, is incubated 2 hours jointly.Add ELIAS secondary antibody (mouse-anti human IgG antibody) after the cleaning and be incubated 2 hours.Cleaning is removed unionized two and is resisted, and adds enzyme substrates the bonded ELIAS secondary antibody is developed the color.The result of enzyme mark colour developing represents the content of anti-tetanus toxin antibody in the supernatant sample.Utilizing this method to filter out some plasmocyte that can secrete anti-tetanus toxin antibody clones sums up as table 1.From 200 clones, find 23 plasma cell secretion anti-tetanus toxin antibodies altogether.The wherein antibody that produces of two clones and toxin bonded tire high especially (also being that bonded concentration is very low).

Embodiment 2: human cloning source anti-tetanus toxin antibody variable region gene fragment.

The plasmocyte clone's who at first embodiment 1 is filtered out cell is with containing NP-40, and the lysate of RNA enzyme inhibitors was 4 ℃ of dissolvings 10-30 minute.Cytolysate is mixed with the damping fluid that contains reversed transcriptive enzyme.By the reverse transcription biochemical reaction each clone's mRNA is transformed into the cDNA storehouse respectively.Specific as follows: the condition of reverse transcription reaction be 65 ℃ 3 minutes, slowly be cooled to 22 ℃, be incubated 3 minutes.Add the fresh damping fluid that contains reversed transcriptive enzyme.38 ℃ are incubated 100 minutes.Afterwards, 65 ℃ of insulations are so that the reverse transcription reaction termination.

It is as follows to design one group of primer, with the light chain and the heavy chain of clonal antibody.The VH primer comprises:

GGG AAT TCC CAT GGA CTG GAC CTG GA

GGG AAT TCA TGG ACA TAC TTT GTT CCA C

CCA AGC TTG CTC YTG GAG SAG GGY GCC AGG GGG AA

VK constant region primer comprises:

TTA GGA TCC ATG GTG TTG CAG ACC CAG GTC

TCC AGC TTC ATC AGA TGG CGG GAA GAT

The V primer comprises:

ATG GCC TGG GTC TCC TTC TAC CTA

ATG GCC TGG ATG ATG CTT CTC CTC

GAG TCA TTC TCG ACT GCT AGC CAC AGT ACG TAG GAC GGT SAS CTT GGT CC

Utilize above-mentioned primer and dNTP mixture that the heavy chain or the related leader sequence of light chain of antibody in each sample are duplicated out.5 '-end and 3 '-end connect the restriction enzyme point of contact respectively.The PCR condition is as follows: 94 ℃ 1 minute, 50 ℃ 2 minutes, 72 ℃ 2 minutes, totally 5 circulations, afterwards 72 ℃ 4 minutes; Next 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes, 35 circulations, last 72 ℃ 4 minutes.

Utilize another group primer of design to clone the variable region of light chain of antibody and heavy chain out.Fig. 1 shows is the gel electrophoresis figure of the dna fragmentation (PCR product) of several humanized anti-spasmotoxin antibody variable regions of cloning.The variable region size of duplicated heavy chain and light chain is about 440bp.Duplicated heavy chain of antibody and light chain size are about 1400bp.

The heavy chain of antibody and the gene of light chain are received respectively among pCR2.1 plasmid vector (INVITROGEN) DNA.Utilize antimycin A mpicillin to filter out the successful bacterial strain of clone.The plasmid vector for preparing this bacterial strain.Measure cloned genes sequence in this plasmid vector.

Sequence table 1 shows comprises antibody B and antibody D weight chain variabl area sequence and light chain variable region sequence.Embodiment 3: the expression vector that makes up humanized anti-spasmotoxin antibody.

Fig. 2 and Fig. 3 are heavy chain of antibody carrier hCg1 and light chain of antibody carrier hCK synoptic diagram.Our selection is a promotor with CMV in these two carriers, is enhanser with EU.The size of carrier is about 7000bp.In these two carriers, the cDNA of the constant region IgG γ 1 of IgG antibody heavy chain and the constant region k of light chain receives the downstream that support C MV is a promotor respectively.The variable region gene fragment that the foregoing description 1 is cloned the heavy chain of antibody that is attempted by the pCR2.1 plasmid vector and light chain is downcut with specific restriction enzyme, and separation and purification.The variable region gene of heavy chain and light chain is received respectively in two different expression vectors: the variable region gene of light chain is received among the light chain of antibody carrier hCK, with 5 ' terminal linking to each other of the k constant region gene fragment of its human antibody light chain that has; The variable region gene of heavy chain is received among the heavy chain of antibody carrier hCg1, the IgG of the human antibody heavy chain that has with it _γ5 of 1 constant region gene fragment ' is held and is linked to each other.The antibody that different cell clones goes out is by difference called after QA (being light chain A) and ZA (being heavy chain A); QB and ZB; QD and ZD, or the like.What Fig. 4 showed is the humanized anti-spasmotoxin heavy chain of antibody and the segmental gel electrophoresis figure of light chain cdna of gene recombination.

Embodiment 4: the expression of humanized anti-spasmotoxin antibody gene in eukaryotic cell CHO.

Concrete grammar is as follows: humanized anti-spasmotoxin heavy chain of antibody genophore and light chain of antibody genophore with connecting after the cDNA linearizing such as carrier QD and carrier ZD, change in the Chinese hamster ovary celI with electric shocking method.Because heavy chain of antibody carrier hCg1 has the gene of anti-Gentamycin, so the expression of screening heavy chain of antibody with Gentamycin (G418); Because light chain of antibody carrier hCK has the gene of anti-Puromycin, so screen the expression of light chain of antibody with Puromycin; Process G418 (1 mg/ml), and the screening in Purimycin (2.5 mcg/ml) 3-4 week filter out simultaneously to Puromycin and the insensitive cell strain of Gentamycin, freezing collection.This cell strain is the stably express cell clone.These cell clones might be produced complete antibody.For confirming this point, also need to do further detection, as implementing case 5.

Embodiment 5: the complete antibody that the antibody of identified gene reorganization and expression-secretion is made up of heavy chain and light chain.

After above-mentioned cell strain B and D cultivated for two weeks, collect cells and supernatant separately respectively.Identify by the ELISA method whether genetically modified cell synthesizes justacrine antibody.Particularly, utilize integrated enzyme reaction to do to detect test culture supernatant of collecting and the mouse-anti human IgG1 antibody for preparing and mouse-anti people chain antibody.Fig. 5 shows that the gene of clone D and B has been inserted into Chinese hamster ovary celI (hamster cell) gene and successful expression, and the synthetic antibody protein is secreted in the nutrient solution, thereby can be the dose-dependent competition that combines with anti-human body IgG antibody.In addition, confirm by gel electrophoresis and SDS-page test, the molecular weight of secretory antibody is 75kDa under reductive condition not, under the reductive condition, show two bands, its molecular weight is respectively 50kDa and 23kDa (result is for showing), has illustrated that excretory antibody is the complete body with light chain and heavy chain.

Embodiment 6: utilize serum-free medium to prepare the humanized anti-spasmotoxin antibody of producer gene reorganization.

In order to obtain high yielding cell sarain, we are with the Chinese hamster ovary celI of Dhfr gene transfer to expressing antibodies.Utilize the way that improves MTX in the nutrient solution to filter out the high expressing cell strain.Chinese hamster ovary celI is cloned D and B cultivates in containing the RPMI1640 nutrient solution of 5% calf serum.Changed a nutrient solution in per 3 days.In this process, gradually the serum-free medium of RPMI1640 nutrient solution with INVITROGEN replaced, and the concentration of calf serum is reduced to 1-2%.After this this Chinese hamster ovary celI clone D and B are cultivated 37 ℃ of difference in a large number with serum-free medium in 2 liters of drum-type culturing bottles.Constantly part is changed nutrient solution therebetween, and cell concn is maintained about 0.5-1 * 10 ⁶/ milliliter.Collect culture supernatant, it is made ultrafiltration and concentration, 4 ℃ of short-terms are preserved then; Or with its lyophilize, in-20 ℃ of prolonged preservation.

Embodiment 7: the humanized anti-spasmotoxin antibody of producing is expressed in separation and purification.

(filtering molecular size is 10,000Da) behind ultrafiltration and concentration with the CHOB that collects among the embodiment 6 and CHOD cell culture supernatant.This antibody concentrated solution is carried out purifying by the affinity column layer absorbing method that a-protein prepares.At first CL-4B-Protein A (Pharmacia) is slowly put into chromatography column, and with phosphoric acid (Tris) the damping fluid balance of PH7.0.This antibody concentrated solution slowly is added in the chromatography column, is incubated 10 minutes so that antibody combination with it under the room temperature.With 0.1M glycine (Glycine) damping fluid of pH3.0 antibody elution is got off (elution speed 100-150 centimetre/hour).The 0.1M Glycine damping fluid of using pH2.0 afterwards again is with the further wash-out of antibody.ELISA method by anti-human body IgG is measured, and obtains 100 milligrams of human antibodies from 300 milliliters of supernatants.Measure through separation and purification, the output of CHO B and CHO D cells produce secretory antibody is per 1 * 10 ⁶Cell is secreted 20 micrograms every day.Because this clone cell is all insensitive to Puromycin and Neomycin, the complete antibody that expressed proteins is made up of heavy chain and light chain chimeric protein is described.

Embodiment 8: anti-tetanus toxin antibody medicine in animal body is for dynamic experiment.

50 microgram purifying are also used iodine ¹²⁵The antibody of the anti-tetanus toxin of mark, or people source Immunoglobulin IgG1 (Sigma) is squeezed into respectively in the rabbit body of 2 kilograms of body weight by left ear vein.Got 5 milliliters of blood every 1 hour from the auris dextra vein of rabbit.Since second day, change into taking out from the latter and get sample one time every 1-2 days.By measuring the content of people's heavy chain immunoglobulin in the isotope detection blood.Shown in following table 2, I ¹²⁵The antibody protein of the anti-tetanus toxin of mark is identical with the half life of the Immunoglobulin IgG1 in people source, all is approximately 110 hours.Because known person source Immunoglobulin IgG1 is 21 days the intravital transformation period the people, thereby the transformation period should be similar to people source Immunoglobulin IgG1 in its body of the antibody protein of anti-tetanus toxin of the present invention, should be that the sero-fast transformation period of at present clinical used horse is doubly a lot.

Embodiment 9: humanized anti-spasmotoxin antibody is to the provide protection test of animal.

The antibody B of a-protein affinity chromatography separation and purification and the thick prepared product of D are carried out the test of provide protection to animal, and compare with the curative effect of the medicine anti-tetanus bovine serum preparation of clinical unique use at present.Be exemplified below: anti-(medicine of present clinical use is the horse serum preparation of anti-tetanus toxin) 4.5IU/ml of tetanus toxin mark (by the calibrating of Beijing institute of Biological Products) and tetanus toxin (Wuhan Biological Products Inst.) are dissolved in borate buffer solution respectively.It is some to get the test small white mouse, and three every group, totally 5 groups.Not purified above-mentioned cell D excretory supernatant liquor lyophilize thing is dissolved in aquae destillata.Cell B that contains humanized anti-spasmotoxin antibody and D secretion supernatant with different concns (0.1mg/kg-10mg/kg, protein/body weight) mix with the tetanus toxin of fixed amount.In contrast, the tetanus toxin of 0.5IU/ml being marked anti-D mixes with the tetanus toxin of same dosage.With each mixture at 37 ℃ in conjunction with after 1 hour, respectively to mouse muscle injection (0.4ml/ only).Every morning, afternoon, each was observed once, record mouse invasion and death condition.Result's (table 3) shows that the medicine of present clinical use is that horse serum preparation (the tetanus toxin mark is anti-) treatment group animal after 69 hours of anti-tetanus toxin is all dead.And be 0.4mg/kg body weight or all normal when above with the animal of humanized anti-spasmotoxin Antybody therapy of the present invention at therapeutic dose, do not fall ill.Absolutely the watch for animals attack of tetanus toxin of defence lethal quantity of humanized anti-spasmotoxin antibody of the present invention.Revision test obtains identical result.Preliminary experiment also shows if with antibody B and D hybrid injection, uses a kind of antibody required dosage to reduce 3-10 times with isodose tetanus toxin required dosage than simple in it, and repeated experiments obtains identical result.

Preliminary experiment shows that also tiring of the humanized anti-spasmotoxin antibody of gene recombination of the present invention is quite high, and experimental result shows that toxopexic the tiring of antibody raw product is equivalent to 31, the thick prepared product of 000IU/ gram protein.The antibody that this test is used is to utilize to contain the production of bovine serum nutrient solution, and only through containing a large amount of non-specially immunoglobulin (Ig)s in the thick prepared product of protein.So it should be 31 that the real neutralization of humanized anti-spasmotoxin antibody of the present invention is tired, the several times at least of 000IU/ gram, it is quite high to tire.

Caption

Table 1: the plasmacytic part in people source of screening secretion anti-tetanus toxin antibody is summed up

Clone's title	With the heavy chain of antibody reaction result	With tetanus toxin antigen-reactive result	The clone of gene recombinant antibody is in choosing
				1A3	+	+	+
1C8	+	+	+
				1H5	+	--	--
2B3	+	+	+
				2D6	+	+	+
2E3	+	--	--
				2G5	+	+	--

Table 2: iodine ¹²⁵The anti-tetanus toxin antibody of mark and people source Immunoglobulin IgG1 are in rabbit intravital half life

Injection protein title

Half life in rabbit plasma (hour)

		Humanized anti-spasmotoxin antibody	110
People source Immunoglobulin IgG1	110
		People source chemokine protein matter	0.25

Table 3: gene recombination anti-tetanus toxin antibody is to protection test result in the animal body

The tetanus mark is anti-: 4.5IU/ milliliter, Beijing biological products assay institute

Tetanus toxin: June calendar year 2001, Wuhan Biological Products Inst.

Experimental animal: about 16 grams of common mouse/only

(test one) morbidity and death condition

-: normal

+++: occurring degree by gently to heavily be expressed as+, ++, +++, ++ ++

: death

*: the time after the injection

The antibody injected dose	21 hours *	27 hours *	45 hours *	51 hours *	69 hours *	75 hours *
							0.1mg/kg	---	---	ΦΦΦΦ
1mg/kg	---	---	---	---	---	---
							5mg/kg	---	---	---	---	---	---
10mg/kg	---	---	---	---	---	---
							Mark anti-0.5mg/kg	---	---	---	---	++++ ++++ ΦΦ	ΦΦΦΦ

(test two) morbidity and death condition

-: normal

++ ++: occurring degree by gently to heavily be expressed as+, ++, +++, ++ ++

Φ: death

*: the time after the injection

The antibody injected dose	21 hours *	27 hours *	45 hours *	51 hours *	69 hours *	75 hours *
							0.2mg/kg	---	---	ΦΦΦΦ
0.4mg/kg	---	---	---	---	---	---
							0.6mg/kg	---	---	---	---	---	---
0.8mg/kg	---	---	---	---	---	---
							Mark anti-0.5mg/kg	---	---	---	---	+++ +++ +++	ΦΦΦΦ ++++

Fig. 1 is the present invention clone's the segmental gel electrophoresis figure of some humanized anti-spasmotoxin antibody variable region cDNA.What show is the gel electrophoresis figure of the dna fragmentation (PCR product) of several humanized anti-spasmotoxin antibody variable regions of cloning.The variable region size of duplicated heavy chain and light chain is about 440bp.From left to right be variable region of light chain QA according to this in arrow indication position among the figure, QB, Qc, variable region of heavy chain ZB.

Fig. 2 is the expression vector synoptic diagram of the humanized anti-spasmotoxin light chain of antibody of the present invention's structure.CMV is a promotor among the figure, and EU is an enhanser.The size of carrier is about the variable region that 7000bp.VK is a light chain, and CK is the constant region of light chain, and Nhe I and part that xho I marks are the point of contact of this restriction enzyme, and Ampicillin and Puromycin are this antibiotic impedance gene.

Fig. 3 is the expression vector synoptic diagram of the humanized anti-spasmotoxin heavy chain of antibody of the present invention's structure.CMV is a promotor among the figure, and EU is an enhanser.The size of carrier is about the variable region that 7000bp.VH is a heavy chain, and CH is the constant region of heavy chain, and Nhe I and part that xho I marks are the point of contact of this restriction enzyme, and Ampicillin and Neomycin are this antibiotic impedance gene.

Fig. 4 is the humanized anti-spasmotoxin heavy chain of antibody and the segmental gel electrophoresis figure of light chain cdna of gene recombination of the present invention.The gel electrophoresis figure of two the people source anti-tetanus toxin antibody light chains that are that show and the cDNA fragment (PCR product) of light chain.Duplicated heavy chain and light chain size are respectively 1.4kb and 0.7kb.From left to right be (1) 100bp molecular criteria thing according to this shown in the figure, (2) B heavy chain of antibody cDNA fragment, (3) D heavy chain of antibody cDNA fragment, (4) B light chain of antibody cDNA fragment, (5) D light chain of antibody cDNA fragment, (6) plasmid vector.

Fig. 5 is the determination experiment result of expressed excretory gene recombination anti-tetanus toxin antibody in CHOB of the present invention and the CHOD cell conditioned medium.The cell CHOB and the CHOD culture supernatant of collecting are added on the enzyme-linked reaction plate, combine test with mouse-anti human IgG 1 antibody for preparing.The result of enzyme mark colour developing represents the content of human antibody in the supernatant sample.What show is that the expressed human antibody albumen of Chinese hamster ovary celI (hamster cell) of clonal antibody D (circle) and B (trilateral) gene transfer is secreted in the nutrient solution, thereby can be the dose-dependent competition that combines with anti-human body IgG antibody.

Fig. 6 is for comparing the provide protection figure of monospecific antibody to animal.Give the tetanus toxin of every group of mouse (2 every group) same lethal dose of intramuscular injection, give the mixture (1: 1 weight ratio) of the antibody D of mouse peritoneal injection of antibodies D (125ng/ only) or various dose after overnight respectively.What show among the figure is the used antibody dosage that do not cause death that can watch for animals.

The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Sequence table 1: inventor source anti-tetanus toxin antibody heavy chain and variable region of light chain nucleic acid and aminoacid sequence

＜110〉Gong Xiaodi

＜120〉humanized anti-spasmotoxin monoclone antibody

<160>8

<210>1

<211>143

<212>PRT

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the proteinic aminoacid sequence of the variable region of light chain of humanized anti-spasmotoxin monoclone antibody D (PS.QD)

<400>1

Met Asp Arg Gly Ala Leu Ala His Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Tht Cys Arg Ala Ser

35 40 45

Gln Ile Ile Gly Ser Tyr Leu Asn Trp Tyr Gln Gln lys pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile His Thr Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Arg Glu Asp Ile Ahr Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser Tyr Ser Gly Leu Arg Trp Thr phe Gly Gln Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140 143

<210>2

<211>142

<212>PRT

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the proteinic aminoacid sequence of the variable region of light chain of humanized anti-spasmotoxin monoclone antibody B (PS.QB)

<400>2

Met Asp Met Glu Phe Leu Val Gln Leu Leu Phe Val Leu Leu Leu Trp

1 5 10 15

Leu Pro Asp Ile Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr

20 25 30

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

35 40 45

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

50 55 60

Thr Pro Arg Leu Leu Ile Phe Gly Ala Ser Thr Arg Ala Asn Gly Ile

65 70 754 80

Pro Ala Arg Phe Ser Gly Ser Gly Tyr Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

Tyr Asn Asn Gly Ser Gly Ala Phe Gly Gln Gly Thr Ser Trp Arg Ser

115 120 125

Asn Glu Leu Trp Leu His His Leu Ser Ser Ser Ser Arg His

130 135 140 142

<210>2

<211>147

<212>PRT

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the proteinic aminoacid sequence of the variable region of heavy chain of humanized anti-spasmotoxin monoclone antibody D (PS.ZD)

<400>3

Met Glu Leu Gly Leu Ser Trp Val Phe Arg Ala Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

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

35 40 45

Ser Arg Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Thr Ser Tyr Asp Gly Gly Asn Lys Tyr Tyr Ala

65 70 75 80

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

85 90 95

Thr Leu Phe Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Gly Ser Met Val Arg Gly Asp Gly Arg Asp

115 120 125

Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

130 135 140

Lys Gly Pro

147

<210>4

<211>148

<212>PRT

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the proteinic aminoacid sequence of the variable region of heavy chain of humanized anti-spasmotoxin monoclone antibody B (PS.ZB)

<400>4

Met Asp Trp Thr Trp Arg Val Phe Phe Val Val Ala Ala Ala Thr Gly

1 5 10 15

Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ser Ser Val Lys Val Ser Cys Asn Leu Ser Gly Gly Thr Phe

35 40 45

Asn Ile Tyr Ala Ile Ser Asp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Gly Val Ile Pro Ile His Gly Ala Val His Tyr Ala

65 70 75 80

Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Met

100 105 110

Tyr Tyr Cys Gly Leu Leu Thr Thr Lys Asn Tyr Tyr Tyr Tyr Tyr Thr

115 120 125

Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro

148

<210>5

<211>430

<212>CDS

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the variable region of light chain nucleotide sequence (NS.QD) of humanized anti-spasmotoxin monoclone antibody D

<400>5

Atggacaggg gggccctggc gcaccttctg ggtctcctgc tactctggct ccgaggtgcc 60

agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgccgggc aagtcagatc attggcagtt atttaaattg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc cataccgcat ccagtttgca aagtggggtc 240

ccaccaaggt tcagtggcag tggatctggg acagatttca ctcttaccat cagcagtctg 300

caacgtgaag atattgcaac ttactactgt caacagagtt acagtggcct caggtggacg 360

ttcggccaag ggaccaagct ggagatcaaa cgaactgtgg ctgcaccatc tgtcttcatc 420

ttcccgccat ---- 430

<210>7

<211>442

<212>CDS

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the variable region of heavy chain nucleotide sequence (NS.ZD) of humanized anti-spasmotoxin monoclone antibody D

<400>7

Atggagcttg ggctgagctg ggtttttcgc gctgctcttt taagaggtgt ccagtgtcag 60

gtgcagctgg tggagtctgg gggaggtgtg gtccagcctg ggaggtccct gagactctcc 120

tgtgttggct ctggattcac cttcagtcgt tatgctatgc attgggtccg ccaggctccg 180

ggcaaggggc tggagtgggt ggcatcaaca tcatatgatg gaggcaataa atactacgca 240

gactccgtga agggccgatt caccatttcc agagacaatt ccaagaacac cttatttgtg 300

caaatgagca gcctgagagc agaggacacg gctgtttatt actgtgcgag agatgggagt 360

atggttcgcg gagacggaag ggatgacttc tggggccagg gaaccatggt caccgtctcc 420

tcagctagca ccaagggccc at 442

---cg gtc ttc ccc ctg gca ccc tcc tcc gca agc ttt ata aag ggc gaa ttc

<210>8

<211>445

<212>CDS

＜213〉people (human)

<220>

<221>V-region

<222>

＜223〉the variable region of heavy chain nucleotide sequence (NS.ZB) of humanized anti-spasmotoxin monoclone antibody B

<400>8

Atggactgga cctggagggt cttcttcgtg gtggcagcgg ctacaggtgt ccagtcccag 60

gtacagctgg tgcagtcagg ggccgaggtg aagaagcctg ggtcctcggt gaaggtctcc 120

tgcaaccttt ctggaggcac cttcaacatc tatgctatca gctgggtgcg acaggcccct 180

ggacaagggc ttgagtggat gggaggggtc atcccgatcc atggtgcagt gcactacgcc 240

cagaagttcc aggatagagt caccattacc gcggacgagt ccacgagcac agcctacatg 300

gagctgagca gcctgagatc tgacgacacg gccatgtatt attgtggcct tctgactacg 360

aagaattact actactacta caccatggac gtctggggcc aagggaccat ggtcaccgtc 420

tcctcagcta gcaccaaggg cccat 445

Claims (9)

1, a kind of humanized anti-spasmotoxin monoclone antibody D, it is characterized in that, it is a human antibody, comprise antibody variable region, this antibody comprises or does not comprise the constant region of antibody, during this antibody has and the ability of tetanus toxin, the aminoacid sequence of this antibody variable region of encoding is shown in the sequence in the sequence table 11 and 3.

2, humanized anti-spasmotoxin monoclone antibody D according to claim 1 is characterized in that, described aminoacid sequence is changed, lacks or adds one or several amino acid, and still keep in it and the ability of tetanus toxin.

3, the nucleotide sequence of the amino acid sequences of the described humanized anti-spasmotoxin monoclone antibody D of coding claim 1 is characterized in that this nucleotide sequence is shown in the sequence in the sequence table 15 and 7.

4, humanized anti-spasmotoxin monoclone antibody D according to claim 1 is characterized in that, the constant region of described antibody is any in the constant region of people source heavy chain immunoglobulin or light chain; This CH is the constant region of humanized IgG, IgM, IgA, IgE or IgD, or includes the CH of hinge region or any fragment in the CH; Described constant region of light chain is the constant region of the κ or the λ of human immunoglobulin's light chain.

5, the preparation method of a kind of humanized anti-spasmotoxin monoclone antibody D as claimed in claim 1 is characterized in that, it comprises that the method for the method of utilizing gene recombination technology or chemosynthesis prepares and produces; Wherein, utilize the method for gene recombination technology to prepare, comprising: the clone of A, antibody gene and sequential analysis; The structure of B, antibody gene expression vector; C, in a suitable system or host, antibody gene is expressed: D, the antibody expression host that cultivates and increase in a large number; E, the antibody separation and purification to expressing: F, to the qualitative and quantitative analysis of expressing antibodies.

6, the preparation method of humanized anti-spasmotoxin monoclone antibody D according to claim 5 is characterized in that, the expression vector of described antibody gene is plasmid vector or virus vector, the genetically modified carrier of animal or universal support; The expression vector of described antibody gene changes among a suitable system or the host and is expressed, and wherein system of Shi Heing or host comprise the cell of animal or plant, cell strain, bacterium or yeast; This animal is transgenic animal.

7, the preparation method of humanized anti-spasmotoxin monoclone antibody D according to claim 5 is characterized in that, also comprises the protein sequence that detects antibody.

8, the application of the described humanized anti-spasmotoxin monoclone antibody D of claim 1 in the pre-antitetanus infectation of bacteria medicine of preparation.

9, the application of the described humanized anti-spasmotoxin monoclone antibody D of claim 1 in the diagnostic preparation of preparation tetanus infectation of bacteria.

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