CN103374073A - Humanized monoclonal antibody that recognizes the activated form of integrin α4β7 - Google Patents
Humanized monoclonal antibody that recognizes the activated form of integrin α4β7 Download PDFInfo
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Abstract
The invention relates to a novel human derived monoclonal antibody for identifying activated integrin alpha 4 beta 7. The monoclonal antibody for specifically identifying activated integrin alpha 4 beta 7 is obtained by screening by an inventor. Based on the identification specificity, the monoclonal antibody can be used as a carrier for targeting transportation to take a medicament to positions of pathological tissues so as to fulfill an aim of treating diseases; and the monoclonal antibody also can be used as a tool for researching the activation status of the integrin alpha 4 beta 7.
Description
Technical field
The invention belongs to biological technical field.Specifically, the present invention relates to a kind of new identification activated form integrin alpha
4β
7Humanized monoclonal antibodies.
Background technology
Leukocytic locomotory movement is important step and the key link in the inflammatory reaction pathologic process, is focus and the frontier nature problem of current life science research.Integrin is the important albumen of direct mediated leucocytes locomotory movement as the important cell surface adhesion molecule of a class.The heterodimer that integrin is comprised of by non covalent bond α and two subunits of β, 18 kinds of α subunits and 8 kinds of β subunits in vertebrates, have been found, form 24 kinds of integrins, the tissue positioned of the immune response of they and organism, the motion of cell and migration, immunocyte, blood coagulation etc. are closely related.Integrin alpha
4β
7Mainly expressing at lymphocytic cell surface, is important homing receptor.Lymphocyte enters the step that inflammation tissue site performance immunization needs a series of finely regulatings by the recycle system, comprising: lymphocyte in the rolling of vascular endothelial cell, stick and pass vessel wall and arrive the inflammation tissue.In this process, there are many protein moleculars and immune factor to participate in adjusting, wherein integrin alpha
4β
7That a kind of important adhesion molecule has been brought into play important effect therein.Integrin is in close relations with a lot of human diseasess, as: inflammation, cardiovascular disorder, thrombosis, cancer etc., world-renowned pharmaceuticals is all the medicine target of integrin as new drug development.
Present studies show that the generation of inflammatory bowel and integrin alpha
4β
7Close relation.Integrin alpha
4β
7Can make by the interaction with its part MAdCAM-1 lymphocyte pass the intestines blood vessel wall and arrive inflammation part.So, with integrin alpha
4β
7Treating inflammatory bowel with its part as the pharmacy target spot becomes focus, and the medicine that occurs now all is the interaction that some inhibiting antibodies or little antagonist molecules suppress them.But these medicines all can non-specifically suppress normal immune function, cause the complication of some serious critical patient's life.
Antibody drug take monoclonal antibody as the basis has high degree of specificity to specific molecular target, as targeted drug huge potentiality is arranged in treating correlative diseases.Current, the research and development of antibody drug have become the focus in biotech drug field.At present research is found at inflammatory bowel pathological tissue place integrin alpha
4β
7Abnormal activation, and suppress α
4The interaction of integrin and its part can be played mitigation for inflammatory bowel, such as the α that has gone on the market
4Integrin antibody Tysabri-natalizumab.But this antibody recognition does not have specificity, both can identify the α of activated form
4Integrin also can be identified the α of disactivation form
4Integrin is so can cause some patients were generation progressive multifocal leukoencephalopathy in process of clinical application.By the end of at present, this area is the extraordinary integrin alpha of neither one identification specificity also
4β
7Antibody drug be used for clinical.
Summary of the invention
The object of the present invention is to provide a kind of new identification activated form integrin alpha
4β
7Humanized monoclonal antibodies.
In a first aspect of the present invention, a kind of monoclonal antibody is provided, it identifies the activated form integrin alpha specifically
4β
7
In another preference, described monoclonal antibody identification activated form integrin alpha
4β
7Two sections characteristic sequences: people source α
4Subunit 184-190 amino acids, people source β 7 subunit 331-348 amino acids; And these two sections sequences are at other source of species α
4β
7In corresponding sequence.
In another preference, described monoclonal antibody nonrecognition disactivation type integrin alpha
4β
7And other integrin.
In another preference, the aminoacid sequence of the variable region of heavy chain of described monoclonal antibody is shown in 1-116 position among the SEQ ID NO:4, and the aminoacid sequence of variable region of light chain is shown in 1-106 position among the SEQ ID NO:3.
In another preference, the CH of described monoclonal antibody and constant region of light chain are respectively CH and the constant region of light chain of immunoglobulin G while 1 (IgG1).
In another preference, the aminoacid sequence of the CH of described monoclonal antibody is shown in 119-448 position among the SEQ ID NO:4, and the aminoacid sequence of described constant region of light chain is shown in 109-213 position among the SEQ ID NO:3.
In another aspect of this invention, provide a kind of variable region of heavy chain of monoclonal antibody, its aminoacid sequence is shown in 1-116 position among the SEQID NO:4.
In another aspect of this invention, provide a kind of variable region of light chain of monoclonal antibody, its aminoacid sequence is shown in 1-106 position among the SEQID NO:3.
In another aspect of this invention, provide the purposes of described variable region of heavy chain or described variable region of light chain, for the preparation of monoclonal antibody, described monoclonal antibody specificity ground identification activated form integrin alpha
4β
7
In another aspect of this invention, provide a kind of polynucleotide, its described monoclonal antibody of encoding.
In another preference, these polynucleotide contain the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the 1-318 position among the SEQ ID NO:1, and/or the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the 1-348 position among the SEQ ID NO:2.
In another preference, these polynucleotide contain the nucleotide sequence of the described monoclonal antibody constant region of light chain of coding shown in the 325-642 position among the SEQ ID NO:1, and/or the nucleotide sequence of the described monoclonal antibody CH of coding shown in the 355-1347 position among the SEQ ID NO:2.
In another aspect of this invention, provide a kind of polynucleotide, its encode described variable region of heavy chain or described variable region of light chain.
In another preference, the sequence of the polynucleotide of the described variable region of heavy chain of encoding is shown in 1-348 position among the SEQ ID NO:2; Or the sequence of the polynucleotide of the described variable region of light chain of encoding is shown in 1-318 position among the SEQ ID NO:1.
In another aspect of this invention, provide a kind of expression vector, the expression regulation sequence that it contains described polynucleotide and links to each other with this series of operations.
In another aspect of this invention, provide a kind of host cell, it contains described expression vector, or is integrated with described polynucleotide in its genome.
In another preference, described host cell is eukaryotic cell.
In another aspect of this invention, provide a kind of method for preparing described monoclonal antibody, the method comprises:
A) provide expression vector, the expression regulation sequence that described expression vector contains described polynucleotide and links to each other with this series of operations;
B) use a) described expression vector transfection host cell of step;
C) culturing step b under the condition that the is fit to described monoclonal antibody expression) host cell of gained; With
D) separation and purification obtains described monoclonal antibody.
In another aspect of this invention, provide the purposes of described monoclonal antibody, be used for distinguishing integrin alpha
4β
7Activated form state and disactivation type state; Or for the preparation of targeted activation type integrin alpha
4β
7Medicine and system; Or be used for as the research integrin alpha
4β
7The instrument of relative disease or the relevant medicine of preparation.
In another aspect of this invention, provide a kind of targeted activation type integrin alpha
4β
7Medicine, described medicine comprises: described monoclonal antibody and derivative thereof, and be attached thereto (covalently bound, coupling, the coupling or compound) medicine.
In another aspect of this invention, provide described targeted activation type integrin alpha
4β
7The purposes of medicine, be used for the treatment of integrin alpha
4β
7The inflammatory disease that abnormal activation is relevant.
In another preference, described inflammation is inflammatory bowel (such as ulcerative colitis or crohn).
In another aspect of this invention, provide a kind of composition, it contains the described targeted activation type integrin alpha of significant quantity
4β
7Medicine, and pharmaceutically acceptable carrier.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1. integrin alpha
4β
7The structure of the expression vector of soluble proteins.
(A) α
4β
7The total length ectodomain, C-holds with the TEV point of contact, soda acid peptide, His-tag and Strep-tag.And at α
4The thigh structural domain introduced the R558A sudden change, purpose is that endogenous protease cutting site is removed.
(B) total length wild-type and mutant ' alpha '
4β
7Expression and purifying.Non-reducing represents unreduced state, i.e. α
4β
7Reducing represents the state that reduces, the α that namely separates
4And β
7
Fig. 2. Flow cytometry J19 is to integrin alpha
4β
7Evident characteristics.
(A) integrin alpha of J19 identification activation
4β
71mM Ca
2++ 1mM Mg
2+The condition integrin is in the disactivation state, 2mM Mn
2+The condition integrin is in active state.Act-1: integrin alpha
4β
7Monoclonal antibody, without identification specificity.HIgG: humanized IgG contrast.
(B) J19 identifies the integrin alpha that various conditioned stimulus are induced
4β
7Activation.Numeral is average fluorescent strength among the figure, mean+SD.
The cross immunity originality of Fig. 3 .J19.
(A) integrin alpha of J19 intersection identification Mouse and rat
4β
7(B) J19 fails to see others' source integrin alpha
4β
1And α
Eβ
7Numeral is average fluorescent strength among the figure, mean+SD.
The evaluation of Fig. 4 .J19 epitope.
(A) J19 is at β
7The evaluation of epitope on the subunit.β
1535 β
7Expression 1-535 amino acids is β
1Sequence, the 536-772 position is β
7Sequence; β
1131 β
7348 β
1Expression β
1The 131-336 position by β
7The 141-348 position replace, also be β
1The 1-130 position connect β
7The 141-348 position connect β
1The 337-778 position; Other the like.(B) J19 is at α
4The evaluation of epitope on the subunit.α
4184 α
E190 α
4Expression α
4The 184-190 amino acids by α
EUpper corresponding aminoacid sequence substitutes; Other the like.
Embodiment
The inventor utilizes the integrin alpha of activation
4β
7Come the screening antibodies storehouse as substrate, obtain a kind of specific recognition activated form integrin alpha
4β
7Monoclonal antibody (J19).Based on this identification specificity, the carrier that described monoclonal antibody can be used as the target transportation takes medicine (such as disturbing molecule) to the pathological tissue position, reaches the purpose for the treatment of disease.
The present invention relates to a kind of monoclonal antibody of restructuring, it identifies the activated form integrin alpha specifically
4β
7, nonrecognition disactivation type integrin alpha
4β
7And other integrin.Further studies show that described monoclonal antibody identification activated form integrin alpha
4β
7Middle β
7331-348 amino acids epitope, and α
4Subunit 184-190 amino acids epitope.
Term used herein " monoclonal antibody (monoclonal antibody) " refers to the antibody that obtains from the colony of the basic homogeneous of a class, and the single antibody that namely comprises in this colony is identical, the sudden change of the natural generation that may exist except minority.Monoclonal antibody is with high specificity for single antigen site.And from conventional Anti-TNF-α body preparation (normally having the different antibodies for different determinants) difference, each monoclonal antibody is for the single determinant on the antigen.Monoclonal antibody has good specificity.Modifier " mono-clonal " has represented the characteristic of antibody, is to obtain from the antibody population of basic homogeneous, and this should not be interpreted into and need to produce antibody with any special methods.
Term used herein " antibody " is to have about 150000 daltonian different four glycan albumen, and it is comprised of with two identical heavy chains (H) two identical light chains (L).Every light chain links to each other with heavy chain by a covalent disulfide bonds, and the disulfide linkage number between the heavy chain of different Immunoglobulin Isotypes is different.Every heavy chain and light chain be the intrachain disulfide bond at regular interval also.One end of every heavy chain has variable region (VH), is thereafter a plurality of constant regions.One end of every light chain has variable region (VL), and the other end has constant region; The constant region of light chain is corresponding with first constant region of heavy chain, and the variable region of light chain is corresponding with the variable region of heavy chain.
Some part of variable region is different on sequence in term used herein " variable " the expression antibody, and it has formed various specific antibodies to combination and the specificity of its specific antigen.Yet mutability is not evenly distributed in the whole antibody variable region.It concentrates in three fragments that are called in light chain and the variable region of heavy chain in complementary determining region (CDR) or the hypervariable region.Part conservative in the variable region is called framework region (FR).Each self-contained four FR district in the variable region of natural heavy chain and light chain, they are the beta sheet configuration haply, are linked to each other by three CDR that form shack, but forming section β-pleated sheet structure structure in some cases.CDR in every chain closely is close together by the FR district and has formed the antigen-binding site (referring to Kabat etc., NIH Publ.No.91-3242, volume I, 647-669 page or leaf (1991)) of antibody with the CDR of another chain.Constant region is not participated in the combination of antibody and antigen directly.
" light chain " of vertebrates antibody (immunoglobulin (Ig)) can be classified as according to the aminoacid sequence of its constant region the class in visibly different two classes (being called κ and λ).According to the aminoacid sequence of its CH, immunoglobulin (Ig) can be divided into different kinds.Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can further be divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.CH corresponding to the inhomogeneity immunoglobulin (Ig) is called α, δ, ε, γ and μ.The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling are well-known.
The inventor is with (Wedge mutant) and non-activated (WT) integrin alpha of activation
4β
7Be substrate, utilize display technique of bacteriophage screening people's source scFv (single-chain variable fragments) antibody library (Tomlinson I+J), last ELISA identifies and obtains a kind of monoclonal antibody (J19) that it can well identify the integrin of activated form.Then constant region (Fc) segment composition of the variable region sequences of the inventor J19 that the clone is obtained and people's antibody obtains people source full length antibody thereby express.About the further evaluation of J19 identification specificity, the inventor utilizes flow cytometry (flow cytometry) to detect it for the integrin alpha of cell surface expression
4β
7Identification.Experimental result shows, the integrin alpha that J19 both can specific identification ion condition induces
4β
7Activation also can identify the activator ADP of the integrin under the physiological status and the integrin alpha that PMA induces
4β
7Activation.Further experimental result shows, J19 can also identify the integrin alpha that chemokine SDF-1 α stimulates lower splenic lymphocyte surface
4β
7Activation.The integrin alpha that these activate under can specific identification physiological status near experiments of physiological condition prompting J19
4β
7, this just provides possibility for the in the future application of this antibody in disease treatment.Because J19 only identifies the integrin alpha of activation
4β
7And the nonrecognition integrin alpha
4β
1So for the research of J19 epitope, the inventor utilizes β
1And β
7Mosaic carry out the Mapping of epitope, the result shows that the recognition site of J19 is positioned at β
7The I structural domain.
As used herein, described " identification " epitope has also comprised the situation of " combination " epitope.
The aminoacid sequence of the variable region of heavy chain of the monoclonal antibody that the present invention obtains is shown in 1-116 position among the SEQ ID NO:4, and the aminoacid sequence of light chain variable is shown in 1-106 position among the SEQ ID NO:3.Preferably, the CH of described monoclonal antibody and constant region of light chain are CH and the constant region of light chain of immunoglobulin G while 1 (IgG1).More preferably, the CH of described monoclonal antibody and constant region of light chain are CH and the constant region of light chain of immunoglobulin G while 1Fc.More preferably, the aminoacid sequence of the CH of described monoclonal antibody is shown in 119-448 position among the SEQ ID NO:4, and the aminoacid sequence of described constant region of light chain is shown in 109-213 position among the SEQ ID NO:3.
The present invention also provides the polynucleotide molecule of the monoclonal antibody of the present invention of encoding.In a better example, this polynucleotide molecule contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the 1-318 position among the SEQ ID NO:1, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the 1-348 position among the SEQ ID NO:2.Be also contained among the present invention with the sequence of these nucleotide sequence degeneracys.More preferably, these polynucleotide contain the nucleotide sequence of the described monoclonal antibody constant region of light chain of coding shown in the 325-642 position among the SEQ ID NO:1, and the nucleotide sequence of the described monoclonal antibody CH of coding shown in the 355-1347 position among the SEQ ID NO:2.
Monoclonal antibody can make with the whole bag of tricks well known to those skilled in the art.For example, monoclonal antibody can use hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes) to make, or makes with recombinant DNA method (U.S. Patent No. 4,816,567).Monoclonal antibody is also available such as Clackson etc., Nature, and 352:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is separated acquisition from phage antibody library.
Behind the nucleotide sequence that obtains coding monoclonal antibody variable region of heavy chain of the present invention and variable region of light chain, usually can prepare by the following method monoclonal antibody of the present invention.
At first, provide the nucleotide sequence that contains code book invention monoclonal antibody and the expression vector of the expression regulation sequence that links to each other with this series of operations.Those of ordinary skills can understand, and the nucleotide sequence of code book invention monoclonal antibody heavy chain and light chain is inserted identical expression simultaneously be written into or insert respectively and carry out coexpression in the different expression vectors and can both obtain monoclonal antibody of the present invention.
Term used herein " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed.Expression regulation sequence comprises promotor and the termination signal that links to each other with target nucleotide sequence operability.They also comprise the suitably required sequence of translation of nucleotide sequence usually." operability links to each other " refers to that some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.For example, if promotor or enhanser have increased transcribing of encoding sequence, then it is that operability links to each other with encoding sequence.
The polynucleotide sequence of code book invention monoclonal antibody can make with conventional means well known to those skilled in the art.For example, can be according to sequence synthetic disclosed by the invention or with PCR method amplification obtain the encoding nucleotide sequence of this monoclonal antibody variable region of heavy chain and variable region of light chain.Then, these nucleotide sequences are inserted in the suitable expression vector by the suitable restriction enzyme site of selection with the whole bag of tricks well known in the art, make them respectively before expression vector entrained CH encoding sequence and constant region of light chain encoding sequence, and make them in same frame.Used expression vector is various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from Invitrogen company or Clontech company expression vector, concrete example such as pIRES2-EGFP.
Subsequently, the expression vector with above-mentioned acquisition transforms the appropriate host cell." host cell " generally comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.In the present invention, preferred eukaryotic cell.Usually be used as expressing the derive host cell of polypeptide of eukaryotic cell with mammal cell line.The breeding of mammalian cell in culture is well known in the art.See " tissue culture ", Academic Press, Kruse and Patterson edits (1973), and this article is included this paper in as a reference.Better mammalian cell is many commercially available immortal cell lines.These immortal cell lines including, but not limited to, 293T cell, Chinese hamster ovary (CHO) cell, Vero cell, HeLa cell, young hamster kidney (BHK) cell, monkey-kidney cells (COS), human liver cell cancer cells (such as Hep G2) and other many clones.They provide posttranslational modification for protein molecule, comprise that correct folding, correct disulfide linkage forms and the glycosylation in correct site.Although among the embodiment, the present invention has only enumerated with the example of 293T cell as host cell hereinafter, those skilled in the art can know that the present invention also can adopt above-mentioned these clones having read detailed description of the present invention and specific embodiment.
Method with the expression vector transfection host cell has a variety of, used Transformation Program to depend on host to be transformed.The method that heterologous polynucleotide is imported in the mammalian cell is known in the art, it comprises transfection, calcium phosphate precipitation, the Polybrene (1 of dextran mediation, 5-dimethyl-1,5-phenodiazine 11 methylene radical gather Methobromide) mediation transfection, protoplast fusion, electroporation, liposome-mediated transfection and with the direct microinjection of DNA in karyon.Better method is electroporation or liposome mediated-method etc.Come transfection such as host cells such as Chinese hamster ovary celIs such as the liposome method test kit that can adopt Invitrogen company.
Then, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained.Then use conventional immunoglobulin purification step, obtain monoclonal antibody of the present invention such as conventional separation and purification means purifying well known to those skilled in the art such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatographys.
The gained monoclonal antibody can be identified with conventional means.The binding specificity of monoclonal antibody can be measured with immunoprecipitation or external combination test (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).The binding affinity of monoclonal antibody such as available Munson etc., Anal.Biochem., the Scatchard of 107:220 (1980) analyzes to measure.
The present invention also provides the purposes of described monoclonal antibody, is used for distinguishing integrin alpha
4β
7Activated form state and disactivation type state.Distinguish integrin alpha
4β
7State help the diagnosis of clinical disease, and also be useful for scientific research, can be used for the research integrin alpha
4β
7Activate mechanism.Described monoclonal antibody can be identified the activated form integrin alpha
4β
7Middle β
7331-348 amino acids epitope, and α
4Subunit 184-190 amino acids epitope, visible integrin alpha
4β
7Change has occured in structure between activated form state or disactivation type state space-time.Under the activated form state, this epitope integrin alpha
4β
7Middle β
7The 331-348 position, and α
4Subunit 184-190 amino acids is exposed to the outside of space structure; And under disactivation type state, this epitope integrin alpha
4β
7Middle β
7The 331-348 position, α
4Subunit 184-190 amino acids is buried the inboard in space structure.
The present invention also provides the purposes of described monoclonal antibody, for the preparation of targeted activation type integrin alpha
4β
7Medicine.The design of this targeted drug and preparation be based on the specific recognition capability of described monoclonal antibody, medicine can be carried to specifically to have the activated form integrin alpha
4β
7Tissue or organ site, improve medicine useful effect ability, can also reduce in some cases medicine for the infringement of other tissue or organ.
The present invention also provides a kind of targeted activation type integrin alpha
4β
7Medicine, described medicine comprises: described monoclonal antibody and be attached thereto (covalently bound, coupling, the coupling or compound) medicine.Described medicine can be any activated form integrin alpha
4β
7The control medicine of relative disease for example is anti-inflammatory drug; It more particularly for example is the medicine for the treatment of inflammatory bowel.Described medicine for example can be a kind of disturbing molecule, such as the siRNA for specific gene.Can adopt method of attachment well known in the art that disturbing molecule is connected on the monoclonal antibody of the present invention, thereby be carried to specific position.
The present invention also provides a kind of composition, and it contains the described targeted activation type integrin alpha of significant quantity
4β
7Medicine, and pharmaceutically acceptable carrier.For example, pharmaceutical composition of the present invention can be used to treat inflammation.Term used herein " pharmaceutically acceptable " refers to when molecule body and composition suitably give the animal or human, and they can not produce disadvantageous, irritated or other untoward reaction." pharmaceutically acceptable carrier " used herein should be compatible with mutain of the present invention, can be with its blend the effect of decrease pharmaceutical composition under normal conditions not.The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is carbohydrate, such as lactose, dextrose plus saccharose; Polyvalent alcohol is such as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol; Lalgine; Emulsifying agent, as
Wetting agent is such as Sodium Lauryl Sulphate BP/USP; Tinting material; Seasonings; Tablet agent, stablizer; Antioxidant; Sanitas; Apirogen water; Deng oozing salts solution; With phosphate buffered saline buffer etc.Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly the factor such as disease condition, administering mode determine the useful dosage of patient is used.Administering mode for example can adopt injection or other therapeutic modality.
Major advantage of the present invention is:
The monoclonal antibody that inventor's screening obtains can be identified the integrin alpha of activated form specifically
4β
7, the carrier that utilizes this identification specificity to can be used as the target transportation takes medicine (such as siRNA) to pathological tissue position (as being used for the treatment of inflammatory enteritis), so just can well avoid the not next side effect of strong band of identification specificity.
On the other hand, integrin alpha
4β
7The antibody finiteness become the bottleneck of research its affinity regulation mechanism and biological function, the inventor's monoclonal antibody can overcome the inconvenience in this scientific research effectively.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Below describe based on α
4The aminoacid sequence of subunit (people) such as SEQ ID NO:33 (999aa).Below describe based on β
7The aminoacid sequence of subunit (people) such as SEQ ID NO:9 (779aa).
Introduced a N-glycosylation site between the I that has confirmed at the integrin beta subunit and Hybrid structural domain, can be with the integrin molecule activation.Based on this, make up the integrin alpha of total length
4β
7Wild-type and with the mutant soluble proteins expression vector of this sudden change, α
4Subunit has the existence of spliceosome in vivo, so in order to obtain the α of total length
4Subunit, the inventor splice site R558A (namely the 558th amino acid sports A from R) that suddenlyd change.The solubility integrin is without cross-film district and intracellular region, and (6 * His) are used for purifying with Strep-tag (WSHPQFEK), respectively at α at C-end amalgamation and expression His-tag
4Subunit and β
7The subunit back connects sour peptide (Acid) and alkali peptide (Basic) sequence, and (sour peptide sequence is: ENAQCEKELQALEKENAQLEW (SEQ ID NO:34); The alkali peptide sequence is: KNAQCKKKLQALKKKNAQLKW (SEQ ID NO:35)), make two subunits can be combined into mixture (Figure 1A).The C end has also made up respectively TEV point of contact (sequence is ENLYFQ (SEQ ID NO:36)), be used for other experiments, for example (,) electron microscopic observation integrin structure need to cut away the people with the TEV enzyme be His and the Strep label that soda acid peptide sequence and purifying need of adding up.Express alpha
4The system of subunit is inserted among the expression vector pCDNA3.1 (-) (available from Invitrogen); Express β
7The system of subunit is inserted among the expression vector pEF1-Puro (available from The Immune Disease Institute, Children ' s Hospital Boston, and Department of Pathology, Harvard Medical School).
Have a large amount of disulfide linkage and glycosylation site because integrate, can't express in protokaryon or the eukaryotic expression system such as low, the inventor has finally set up the 293T eukaryotic expression system.
Behind the protein expression, can obtain purity by Ni-NTA and Strep-Tactin two steps affinity chromatography and be higher than 95% albumen (Figure 1B).
(1) utilizes the wild-type disactivation of purifying and the α of mutant activation
4β
7Integrin albumen, by eluriating the method for (panning), screening scFv antibody library Tomlinson I+J (available from Geneservice) screens and removes and wild-type disactivation α through three-wheel
4β
7The phage that integrin albumen combines, just can obtain at last can expression specificity in conjunction with the α that activates
4β
7The phage of the scFv of integrin.Concrete screening process is exactly first solubility integrin albumen biotin labeling, single-chain antibody phage library with the people is hatched, there is the phage of specific recognition integrin just can well be combined with integrin, then mix with it with the coated magnetic bead of streptavidin and hatch, just all be fixed on biotin labeled integrin on the magnetic bead, same those phages in conjunction with integrin also have been fixed on the magnetic bead, by the separator of a magnetic magnetic bead is separated at last, wash-out obtains the phage of specific binding, increase again, do the screening of next round.The selection result behind the three-wheel, ELISA identify show obtained tens with the activated form integrin alpha
4β
7In conjunction with higher positive colony.The detailed process that ELISA identifies: first at the coated streptavidin of elisa plate, add again biotin labeled integrin, then add the phage that the mono-clonal amplification obtains, hatch the antibody that adds the phage-resistance coat protein M13 that is connected with horseradish peroxidase after for some time, add substrate and just can develop the color.In operating process, designed again positive control, namely with the specific reaction of vitamin H and streptavidin in contrast; Negative control is exactly not add biotin labeled integrin, develops the color.
According to ELISA result, therefrom picking positive colony, and further be connected with human antibody light chain (λ chain) constant region and heavy chain (IgG1) constant region, then expression and purification obtains antibody and carries out next step functional study.The preparation of J19 monoclonal antibody is specific as follows:
Screening scFv antibody library obtains after the J19scFv, with PCR method increase respectively variable region of light chain and variable region of heavy chain, and the primer of amplification variable region of light chain:
Forward primer (SEQ ID NO:5):
CG
GGATCCACGGACATCCAGATGACCCAG (underscore is the BamHI restriction enzyme site);
Reverse primer (SEQ ID NO:6)
CG
GAATTCCGCCCGTTTGATTTCCACCTTGGTC (underscore is the EcoRI restriction enzyme site);
The primer of amplification variable region of heavy chain:
Forward primer (SEQ ID NO:7):
GC
TCTAGACCGGCCATGGCCGAGGTG (underscore is the XbaI enzyme cutting site):
Reverse primer (SEQ ID NO:8):
CG
GGATCCGACGGTGACCAGGGTTCCCTG (underscore is the BamHI restriction enzyme site).
Be inserted into respectively two after these two variable region fragments respectively in the plasmid with CH and constant region of light chain expanding, the sequence of constant region is human IgG1's sequence, and the template of constant region amplification is to extract the cDNA that reverses behind RPMI8866 cell (ATCC) RNA.Plasmid vector with constant region of light chain is pEF1/V5-His A (Invitrogen), and the restriction enzyme site that constant region of light chain inserts is EcoR I and Xba I.With the plasmid vector of CH be pcDNA3.1 (-) (Invitrogen), the restriction enzyme site that CH inserts is BamHI and HindIII.Then the variable region of light chain utilizes BamH I and EcoR I to be inserted in the plasmid that contains constant region of light chain the full-length light chains of complete; The variable region of heavy chain utilizes Xba I and BamH I to be inserted in the plasmid that contains CH, the total length heavy chain of complete.
Recycle PCR method, amplify light chain and the heavy chain of total length.
The carrier of final the expressed Y type antibody is pIRES2-EGFP (Clontech), and at first total length sequence of heavy chain replacement EGFP sequence is inserted in this carrier, and corresponding restriction enzyme site is Nco I and Not I.Then the full-length light chains sequence is inserted in the plasmid that contains the total length heavy chain, and the corresponding restriction enzyme site of on position is Nco I and Mlu I.With the expression vector transfection 293T cell (ATCC) of above-mentioned structure, culturing cell finally can give expression to J19 antibody (being present in the culture supernatant) well.
The nucleotide sequence of J19 light chain of antibody (SEQ ID NO:1) following (underscore is the restriction enzyme site calling sequence, is the variable region of light chain encoding sequence before the underscore site, is the constant region of light chain encoding sequence afterwards):
ATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTAGTGATGGTCCTGCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCG
GAATTCCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG
The aminoacid sequence of J19 light chain of antibody (SEQ ID NO:3) following (underscore is the restriction enzyme site calling sequence, is variable region of light chain before the underscore site, is constant region of light chain afterwards):
MTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSDGPATFGQGTKVEIKRA
EFQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
The nucleotide sequence of J19 heavy chain of antibody (SEQ ID NO:2) following (underscore is the restriction enzyme site calling sequence, is the variable region of heavy chain encoding sequence before the underscore site, is the CH encoding sequence afterwards):
ATGGCCGAGGTGCAGTTGTTAGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATTCTAGTGGTTATTATACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATATTATGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC
GGATCCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
The aminoacid sequence of J19 heavy chain of antibody (SEQ ID NO:4) following (underscore is the restriction enzyme site calling sequence, is variable region of heavy chain before the underscore site, is CH afterwards):
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINSSGYYTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYYGYFDYWGQGTLVTV
GSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Embodiment 3, J19 identify the integrin alpha of activation specifically
4β
7
Purifying obtains after the J19 of total length, and in order further to verify its identification specificity, the inventor has adopted the Flow cytometry method.
The detailed process of Fig. 2 A experiment: stably express integrin alpha
4β
7The K562 cell respectively with containing 1mM Ca
2++ 1mM Mg
2+With 2mM Mn
2+The HBS damping fluid wash twice, then add J19 (10 μ g/ml), add the hIgG (available from Pierce) of equivalent in the contrast, hatched 0.5 hour for 4 ℃; Then still with containing 1mM Ca
2++ 1mM Mg
2+With 2mM Mn
2+The HBS damping fluid wash respectively twice, then add the mountain goat anti-human igg (1: 500) (available from Invitrogen) of Alexa Fluor 488 marks, hatched 0.5 hour for 4 ℃; Then with containing 1mM Ca
2++ 1mM Mg
2+With 2mM Mn
2+The HBS damping fluid wash respectively twice, flow cytometer (available from BD) detects.
The experimentation of Fig. 2 B: elementary operation is identical with Fig. 2 A, different is before adding antibody, cell first respectively with the DTT of 500 μ M and the ADP of 10 μ M (take do not add above-mentioned substance as contrast) hatched 15 minutes at 37 ℃, contrast was hatched 15 minutes at 37 ℃ equally.
Experimental result shows that shown in Fig. 2 A, J19 is at Ca
2++ Mg
2+Under the condition for integrin alpha
4β
7Identification and contrast be equal to, and at Mn
2+J19 is for integrin alpha under the condition
4β
7Recognition capability be better than contrast far away.Because studies show that in the past that integrin was at Mn
2+Activated form under the condition, so J19 is a specific recognition activated form integrin alpha
4β
7Antibody.
In addition, J19 also can identify small molecules DTT and ADP for integrin alpha
4β
7Activation (Fig. 2 B).
The evaluation of embodiment 4, J19 epitope
In order to find the epitope of J19, at first detected the cross immunity characteristic of antibody J19.The experiment detailed process is: the integrin alpha of expressing mouse, rat
4β
7And people's α
4β
1, α
Eβ
7Plasmid transient transfection 293T cell respectively, the conventional calcium phosphate transfection that transfection is adopted sees molecular cloning for details: lab guide.Integrin alpha for mouse (Mouse) and rat (Rat)
4β
7Identification, Flow cytometry finds that J19 can be good at identifying the activated form integrin alpha of Mouse and Rat
4β
7(Fig. 3 A).So the evaluation of epitope can not adopt mouse-human chimeric to carry out.In addition, both integrin alpha of nonrecognition activated form of J19
4β
1The also integrin alpha of nonrecognition activated form
Eβ
7, and only identify the integrin alpha of activated form
4β
7So its epitope is α
4Subunit and β
7On the subunit.The inventor has adopted the β in people source
1And β
7Mosaic is sought J19 at β
7Epitope on the subunit utilizes the α in people source simultaneously
4And α
EMosaic is sought J19 at α
4Subunit and β
7Epitope on the subunit.
A series of β have at first been made up
1And β
7Mosaic (mosaic be inserted into pcDNA3.1 (-) (Invitrogen) XbaI and the AflII site after transfection 293T cell).
It is as follows that mosaic makes up sequence (aminoacid sequence):
β
7Aminoacid sequence (SEQ ID NO:9):
ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
β
7Nucleotide sequence (wherein underscore is signal coding sequence) (SEQ ID NO:31):
atggtggctttgccaatggtccttgttttgctgctggtcctgagcagaggtgagagtgaattggacgccaagatcccatccacaggggatgccacagaatggcggaatcctcacctgtccatgctggggtcctgccagccagccccctcctgccagaagtgcatcctctcacaccccagctgtgcatggtgcaagcaactgaacttcaccgcgtcgggagaggcggaggcgcggcgctgcgcccgacgagaggagctgctggctcgaggctgcccgctggaggagctggaggagccccgcggccagcaggaggtgctgcaggaccagccgctcagccagggcgcccgcggagagggtgccacccagctggcgccgcagcgggtccgggtcacgctgcggcctggggagccccagcagctccaggtccgcttccttcgtgctgagggatacccggtggacctgtactaccttatggacctgagctactccatgaaggacgacctggaacgcgtgcgccagctcgggcacgctctgctggtccggctgcaggaagtcacccattctgtgcgcattggttttggttcctttgtggacaaaacggtgctgccctttgtgagcacagtaccctccaaactgcgccacccctgccccacccggctggagcgctgccagtcaccattcagctttcaccatgtgctgtccctgacgggggacgcacaagccttcgagcgggaggtggggcgccagagtgtgtccggcaatctggactcgcctgaaggtggcttcgatgccattctgcaggctgcactctgccaggagcagattggctggagaaatgtgtcccggctgctggtgttcacttcagacgacacattccatacagctggggacgggaagttgggcggcattttcatgcccagtgatgggcactgccacttggacagcaatggcctctacagtcgcagcacagagtttgactacccttctgtgggtcaggtagcccaggccctctctgcagcaaatatccagcccatctttgctgtcaccagtgccgcactgcctgtctaccaggagctgagtaaactgattcctaagtctgcagttggggagctgagtgaggactccagcaacgtggtacagctcatcatggatgcttataatagcctgtcttccaccgtgacccttgaacactcttcactccctcctggggtccacatttcttacgaatcccagtgtgagggtcctgagaagagggagggtaaggctgaggatcgaggacagtgcaaccacgtccgaatcaaccagacggtgactttctgggtttctctccaagccacccactgcctcccagagccccatctcctgaggctccgggcccttggcttctcagaggagctgattgtggagttgcacacgctgtgtgactgtaattgcagtgacacccagccccaggctccccactgcagtgatggccagggacacctacaatgtggtgtatgcagctgtgcccctggccgcctaggtcggctctgtgagtgctctgtggcagagctgtcctccccagacctggaatctgggtgccgggctcccaatggcacagggcccctgtgcagtggaaagggtcactgtcaatgtggacgctgcagctgcagtggacagagctctgggcatctgtgcgagtgtgacgatgccagctgtgagcgacatgagggcatcctctgcggaggctttggtcgctgccaatgtggagtatgtcactgtcatgccaaccgcacgggcagagcatgcgaatgcagtggggacatggacagttgcatcagtcccgagggagggctctgcagtgggcatggacgctgcaaatgcaaccgctgccagtgcttggacggctactatggtgctctatgcgaccaatgcccaggctgcaagacaccatgcgagagacaccgggactgtgcagagtgtggggccttcaggactggcccactggccaccaactgcagtacagcttgtgcccataccaatgtgaccctggccttggcccctatcttggatgatggctggtgcaaagagcggaccctggacaaccagctgttcttcttcttggtggaggatgacgccagaggcacggtcgtgctcagagtgagaccccaagaaaagggagcagaccacacgcaggccattgtgctgggctgcgtagggggcatcgtggcagtggggctggggctggtcctggcttaccggctctcggtggaaatctatgaccgccgggaatacagtcgctttgagaaggagcagcaacaactcaactggaagcaggacagtaatcctctctacaaaagtgccatcacgaccaccatcaatcctcgctttcaagaggcagacagtcccactctctga
β
1Aminoacid sequence (SEQ ID NO:10):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK
β
1Nucleotide sequence (wherein underscore is signal coding sequence) (SEQ ID NO:32):
atgaatttacaaccaattttctggattggactgatcagttcagtttgctgtgtgtttgctcaaacagatgaaaatagatgtttaaaagcaaatgccaaatcatgtggagaatgtatacaagcagggccaaattgtgggtggtgcacaaattcaacatttttacaggaaggaatgcctacttctgcacgatgtgatgatttagaagccttaaaaaagaagggttgccctccagatgacatagaaaatcccagaggctccaaagatataaagaaaaataaaaatgtaaccaaccgtagcaaaggaacagcagagaagctcaagccagaggatattactcagatccaaccacagcagttggttttgcgattaagatcaggggagccacagacatttacattaaaattcaagagagctgaagactatcccattgacctctactaccttatggacctgtcttactcaatgaaagacgatttggagaatgtaaaaagtcttggaacagatctgatgaatgaaatgaggaggattacttcggacttcagaattggatttggctcatttgtggaaaagactgtgatgccttacattagcacaacaccagctaagctcaggaacccttgcacaagtgaacagaactgcaccagcccatttagctacaaaaatgtgctcagtcttactaataaaggagaagtatttaatgaacttgttggaaaacagcgcatatctggaaatttggattctccagaaggtggtttcgatgccatcatgcaagttgcagtttgtggatcactgattggctggaggaatgttacacggctgctggtgttttccacagatgccgggtttcactttgctggagatgggaaacttggtggcattgttttaccaaatgatggacaatgtcacctggaaaataatatgtacacaatgagccattattatgattatccttctattgctcaccttgtccagaaactgagtgaaaataatattcagacaatttttgcagttactgaagaatttcagcctgtttacaaggagctgaaaaacttgatccctaagtcagcagtaggaacattatctgcaaattctagcaatgtaattcagttgatcattgatgcatacaattccctttcctcagaagtcattttggaaaacggcaaattgtcagaaggcgtaacaataagttacaaatcttactgcaagaacggggtgaatggaacaggggaaaatggaagaaaatgttccaatatttccattggagatgaggttcaatttgaaattagcataacttcaaataagtgtccaaaaaaggattctgacagctttaaaattaggcctctgggctttacggaggaagtagaggttattcttcagtacatctgtgaatgtgaatgccaaagcgaaggcatccctgaaagtcccaagtgtcatgaaggaaatgggacatttgagtgtggcgcgtgcaggtgcaatgaagggcgtgttggtagacattgtgaatgcagcacagatgaagttaacagtgaagacatggatgcttactgcaggaaagaaaacagttcagaaatctgcagtaacaatggagagtgcgtctgcggacagtgtgtttgtaggaagagggataatacaaatgaaatttattctggcaaattctgcgagtgtgataatttcaactgtgatagatccaatggcttaatttgtggaggaaatggtgtttgcaagtgtcgtgtgtgtgagtgcaaccccaactacactggcagtgcatgtgactgttctttggatactagtacttgtgaagccagcaacggacagatctgcaatggccggggcatctgcgagtgtggtgtctgtaagtgtacagatccgaagtttcaagggcaaacgtgtgagatgtgtcagacctgccttggtgtctgtgctgagcataaagaatgtgttcagtgcagagccttcaataaaggagaaaagaaagacacatgcacacaggaatgttcctattttaacattaccaaggtagaaagtcgggacaaattaccccagccggtccaacctgatcctgtgtcccattgtaaggagaaggatgttgacgactgttggttctattttacgtattcagtgaatgggaacaacgaggtcatggttcatgttgtggagaatccagagtgtcccactggtccagacatcattccaattgtagctggtgtggttgctggaattgttcttattggccttgcattactgctgatatggaagcttttaatgataattcatgacagaagggagtttgctaaatttgaaaaggagaaaatgaatgccaaatgggacacgggtgaaaatcctatttataagagtgccgtaacaactgtggtcaatccgaagtatgagggaaaatga
β
1535 β
7(β
1The 1-535 position connect the 543-779 position of β 7) aminoacid sequence (SEQ ID NO:11):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCEC
DDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALC DQCPGCKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGAD HTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
β
7542 β
1(be β
7The 1-542 position connect β
1The 536-778 position) aminoacid sequence (SEQ ID NO:12):
ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCEC
DNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTD PKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNG NNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYE GK
β
1336 β
7(β
1The 1-336 position connect β
7The 349-779 position) aminoacid sequence (SEQ ID NO:37):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSA
VGELSEDSSNVV QLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEEL IVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQ SSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPG CKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAI VLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
β <sub TranNum="479"> 7 </ sub> 348 β <sub TranNum="480"> 1 </ sub> (be β <sub TranNum="481"> 7 </ sub> The 1-348 position connect β <sub TranNum="482"> 1 </ sub> the first position 337-778) amino acid sequence (SEQ ID NO: 38)：ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSA<u TranNum="483">VGTLSANSSNVIQLI IDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFE ISITSNKCPKKDSDSF</u><u TranNum="484">KIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRH CECSTDEVNSEDMDAYCRKENSSEICSNNGE</u><u TranNum="485">CVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYT GSACDCSLDTSTCEASNGQICNGRGICECGV</u><u TranNum="486">CKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKV ESRDKLPQPVQPDPVSHCKEKDVDDCWFYFT</u><u TranNum="487">YSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDR REFAKFEKEKMNAKWDTGENPIYKSAVTTVV</u><u TranNum="488">NPKYEGK</u>
β
1131 β
7348 β
1(β
1The 131-336 position by β
7The 141-348 position replace, also be β
1The 1-130 position connect β
7The 141-348 position connect β
1The 337-778 position) aminoacid sequence (SEQ ID NO:13):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMD
LSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLP FVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDD TFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDI?IPIVAGVVAGIVLIGLALLLIWKLLMI?IHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK
β
7141 β
1336 β
7(be β
7The 141-348 position by β
1The 131-336 position replace, also be β
7The 1-140 position connect β
1The 131-336 position connect β
7The 349-779 position) aminoacid sequence (SEQ ID NO:14):
ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMD
LSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGF GSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVT RLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
β
1255 β
7348 β
1(be β
1The 255-336 position by β
7The 266-348 position replace, also be β
1The 1-254 position connect β
7The 266-348 position connect β
1The 337-778 position) aminoacid sequence (SEQ ID NO:39):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLV
VFTSDD TFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDI?IPIVAGVVAGIVLIGLALLLIWKLLMI?IHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK
β
7266 β
1336 β
7(be β
7The 266-348 position by β
1The 255-336 position replace, also be β
7The 1-265 position connect β
1The 255-336 position connect β
7The 349-779 position) aminoacid sequence (SEQ ID NO:16):
ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLV
VFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
β
1319 β
7348 β
1(be β
1The 319-336 position by β
7The 331-348 position replace, also be β
1The 1-318 position connect β
7The 331-348 position connect β
1The 337-778 position) aminoacid sequence (SEQ ID NO:15):
QTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVT
SAALPVYQELSKLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK
β
7331 β
1336 β
7(be β
7The 331-348 position by β
1The 319-336 position replace, also be β
7The 1-330 position connect β
1The 319-336 position connect β
7The 349-779 position) aminoacid sequence (SEQ ID NO:40):
ELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVT
EEFQPVYKELKNLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL
Mosaic plasmid construction method:
β
1535 β
7, β
7542 β
1, β
1336 β
7, β
7348 β
1, β
1131 β
7348 β
1, β
7141 β
1336 β
7, β
1255 β
7348 β
1, β
7266 β
1336 β
7, β
1319 β
7348 β
1And β
7331 β
1336 β
7What the structure of these several plasmids adopted is a kind of method, is exactly overlapping extension PCR (overlapping extension PCR).Concrete construction process is with β
7542 β
1Be example, sequence alignment β
1And β
7Dna sequence dna, find at β
7The dna encoding sequence of the 542nd aa near and β
1Dna sequence dna herein is identical, designs the primer of a pair of reverse complemental with this identical sequence, then respectively in conjunction with β
75 ' end primer PCR of dna sequence dna expands and coding β
7The sequence of 1-542aa, in conjunction with β
13 ' end primer PCR of dna sequence dna expands and coding β
1543 sequences to C end.Because it is complementary sequence that these two sections sequences have respectively an end, so take turns PCR and just expand and whole chimeric encoding sequence carrying out second.Chimeric 5 ' the end primer of each of last total length has an XbaI enzyme cutting site, and 3 ' end primer has an AflII restriction enzyme site, is inserted at last pcDNA3.1 (-) (Invitrogen) in the carrier.β in like manner
7141 β
1348 β
7Structure also be the same method, just first round PCR is three sections, then second takes turns three sections one and does template and expand and total length mosaic encoding sequence.
Making up the primer sequence is:
β
7-F：gctctagaatggtggctttgccaatggtcc(SEQ?ID?NO：17)；
β
7-R：cccttaagtcagagagtgggactgtctgcctc(SEQ?ID?NO：18)；
β
1-F：gctctagaatgaatttacaaccaattttc(SEQ?ID?NO：19)；
β
1-R：cccttaagtcattttccctcatacttcg(SEQ?ID?NO：20)；
β
7-542-F：tgcgagtgtga(SEQ?ID?NO：21)；
β
7-542-R：tcacactcgca(SEQ?ID?NO：22)；
β
7-348-F：cctaagtctgcagt(SEQ?ID?NO：25)；
β
7-348-R：actgcagacttagg(SEQ?ID?NO：26)；
β
7-141-F：tactaccttatggacctg(SEQ?ID?NO：23)；
β
7-141-R：caggtccataaggtagta(SEQ?ID?NO：24)；
β
7-266-F：cggctgctggtgtt(SEQ?ID?NO：41)；
β
7-266-R：aacaccagcagccg(SEQ?ID?NO：42)。
β
1319 β
7348 β
1And β
7331 β
1336 β
7Structure be the ordinary method that adopts rite-directed mutagenesis, such as β
7331 β
1345 β
7Just expression is β
7331-348 amino acids series jump be β
1Upper corresponding 319-336 amino acids sequence, vice versa.
Primer sequence is:
β
7331β
1336β
7-F：aaggagctgaaaaacttgatccctaagtctgcagttg(SEQ?ID?NO：27)；
β
7331β
1336β
7-R：gtaaacaggctgaaattcttcggtgacagcaaagatg(SEQ?ID?NO：28)；
β
1319β
7348β
1-F：caggagctgagtaaactgattcctaagtcagcagtaggaacatta(SEQ?ID?NO：29)；
β
1319β
7348β
1-R：gtagacaggcagtgcggcactagtaactgcaaaaattgtctgaat(SEQ?ID?NO：30)。
Then the constructed plasmid wink that can express chimera protein is turned the 293T cell and carries out Flow cytometry, obtain at last the epitope of J19 identification at β
7The 331-348 amino acids of subunit (Fig. 3 B, sequence is SAALPVYQELSKLIPKSA).Each mosaic can normal expression, and its expression efficiency passes through α
4Antibody 9F10 (by available from Developmental Studies Hybridoma Bank, the hybridoma purifying of University of Iowa) measure, then according to wild-type α
4β
7Expression efficiency and the identification of J19 carry out stdn.
Next has made up a series of α
4And α
EMosaic (mosaic be inserted into pcDNA3.1 (-) (Invitrogen) XbaI and the AflII site after turn the 293T cell).
It is as follows that mosaic makes up sequence (aminoacid sequence):
α
4Aminoacid sequence (SEQ ID NO:33):
YNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVIIIISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD
α
4Nucleotide sequence (wherein underscore is signal coding sequence) (SEQ ID NO:43):
atggcttgggaagcgaggcgcgaacccggcccccgaagggccgccgtccgggagacggtgatgctgttgctgtgcctgggg gtcccgaccggccgcccctacaacgtggacactgagagcgcgctgctttaccagggcccccacaacacgctgttcggctactcggtcgtgctgcacagccacggggcgaaccgatggctcctagtgggtgcgcccactgccaactggctcgccaacgcttcagtgatcaatcccggggcgatttacagatgcaggatcggaaagaatcccggccagacgtgcgaacagctccagctgggtagccctaatggagaaccttgtggaaagacttgtttggaagagagagacaatcagtggttgggggtcacactttccagacagccaggagaaaatggatccatcgtgacttgtgggcatagatggaaaaatatattttacataaagaatgaaaataagctccccactggtggttgctatggagtgccccctgatttacgaacagaactgagtaaaagaatagctccgtgttatcaagattatgtgaaaaaatttggagaaaattttgcatcatgtcaagctggaatatccagtttttacacaaaggatttaattgtgatgggggccccaggatcatcttactggactggctctctttttgtctacaatataactacaaataaatacaaggcttttttagacaaacaaaatcaagtaaaatttggaagttatttaggatattcagtcggagctggtcattttcggagccagcatactaccgaagtagtcggaggagctcctcaacatgagcagattggtaaggcatatatattcagcattgatgaaaaagaactaaatatcttacatgaaatgaaaggtaaaaagcttggatcgtactttggagcttctgtctgtgctgtggacctcaatgcagatggcttctcagatctgctcgtgggagcacccatgcagagcaccatcagagaggaaggaagagtgtttgtgtacatcaactctggctcgggagcagtaatgaatgcaatggaaacaaacctcgttggaagtgacaaatatgctgcaagatttggggaatctatagttaatcttggcgacattgacaatgatggctttgaagatgttgctatcggagctccacaagaagatgacttgcaaggtgctatttatatttacaatggccgtgcagatgggatctcgtcaaccttctcacagagaattgaaggacttcagatcagcaaatcgttaagtatgtttggacagtctatatcaggacaaattgatgcagataataatggctatgtagatgtagcagttggtgcttttcggtctgattctgctgtcttgctaaggacaagacctgtagtaattgttgacgcttctttaagccaccctgagtcagtaaatagaacgaaatttgactgtgttgaaaatggatggccttctgtgtgcatagatctaacactttgtttctcatataagggcaaggaagttccaggttacattgttttgttttataacatgagtttggatgtgaacagaaaggcagagtctccaccaagattctatttctcttctaatggaacttctgacgtgattacaggaagcatacaggtgtccagcagagaagctaactgtagaacacatcaagcatttatgcggaaagatgtgcgggacatcctcaccccaattcagattgaagctgcttaccaccttggtcctcatgtcatcagtaaacgaagtacagaggaattcccaccacttcagccaattcttcagcagaagaaagaaaaagacataatgaaaaaaacaataaactttgcaaggttttgtgcccatgaaaattgttctgctgatttacaggtttctgcaaagattgggtttttgaagccccatgaaaataaaacatatcttgctgttgggagtatgaagacattgatgttgaatgtgtccttgtttaatgctggagatgatgcatatgaaacgactctacatgtcaaactacccgtgggtctttatttcattaagattttagagctggaagagaagcaaataaactgtgaagtcacagataactctggcgtggtacaacttgactgcagtattggctatatatatgtagatcatctctcaaggatagatattagctttctcctggatgtgagctcactcagcagagcggaagaggacctcagtatcacagtgcatgctacctgtgaaaatgaagaggaaatggacaatctaaagcacagcagagtgactgtagcaatacctttaaaatatgaggttaagctgactgttcatgggtttgtaaacccaacttcatttgtgtatggatcaaatgatgaaaatgagcctgaaacgtgcatggtggagaaaatgaacttaactttccatgttatcaacactggcaatagtatggctcccaatgttagtgtggaaataatggtaccaaattcttttagcccccaaactgataagctgttcaacattttggatgtccagactactactggagaatgccactttgaaaattatcaaagagtgtgtgcattagagcagcaaaagagtgcaatgcagaccttgaaaggcatagtccggttcttgtccaagactgataagaggctattgtactgcataaaagctgatccacattgtttaaatttcttgtgtaattttgggaaaatggaaagtggaaaagaagccagtgttcatatccaactggaaggccggccatccattttagaaatggatgagacttcagcactcaagtttgaaataagagcaacaggttttccagagccaaatccaagagtaattgaactaaacaaggatgagaatgttgcgcatgttctactggaaggactacatcatcaaagacccaaacgttatttcaccatagtgattatttcaagtagcttgctacttggacttattgtacttctgttgatctcatatgttatgtggaaggctggcttctttaaaagacaatacaaatctatcctacaagaagaaaacagaagagacagttggagttatatcaacagtaaaagcaatgatgattaa
α
EAminoacid sequence (SEQ ID NO:44):
FNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTPGPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSSELTGTCSLLGPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEEEDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECNFALVQYGGVIQTEFDLRDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMVVLTDGGIFEDPLNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLIASDPDETHAFKVTNYMALDGLLSKLRYNIISMEGTVGDALHYQLAQIGFSAQILDERQVLLGAVGAFDWSGGALLYDTRSRRGRFLNQTAAAAADAEAAQYSYLGYAVAVLHKTCSLSYIAGAPRYKHHGAVFELQKEGREASFLPVLEGEQMGSYFGSELCPVDIDMDGSTDFLLVAAPFYHVHGEEGRVYVYRLSEQDGSFSLARILSGHPGFTNARFGFAMAAMGDLSQDKLTDVAIGAPLEGFGADDGASFGSVYIYNGHWDGLSASPSQRIRASTVAPGLQYFGMSMAGGFDISGDGLADITVGTLGQAVVFRSRPVVRLKVSMAFTPSALPIGFNGVVNVRLCFEISSVTTASESGLREALLNFTLDVDVGKQRRRLQCSDVRSCLGCLREWSSGSQLCEDLLLMPTEGELCEEDCFSNASVKVSYQLQTPEGQTDHPQPILDRYTEPFAIFQLPYEKACKNKLFCVAELQLATTVSQQELVVGLTKELTLNINLTNSGEDSYMTSMALNYPRNLQLKRMQKPPSPNIQCDDPQPVASVLIMNCRIGHPVLKRSSAHVSVVWQLEENAFPNRTADITVTVTNSNERRSLANETHTLQFRHGFVAVLSKPSIMYVNTGQGLSHHKEFLFHVHGENLFGAEYQLQICVPTKLRGLQVVAVKKLTRTQASTVCTWSQERACAYSSVQHVEEWHSVSCVIASDKENVTVAAEISWDHSEELLKDVTELQILGEISFNKSLYEGLNAENHRTKITVVFLKDEKYHSLPIIIKGSVGGLLVLIVILVILFKCGFFKRKYQQLNLESIRKAQLKSENLLEEEN
α
ENucleotide sequence (wherein underscore is signal coding sequence) (SEQ ID NO:45):
atgtggctcttccacactctgctctgcatagccagcctggccctgctggccgctttcaatgtggatgtggcccggccctggctcacgcccaagggaggtgcccctttcgtgctcagctcccttctgcaccaagaccccagcaccaaccagacctggctcctggtcaccagccccagaaccaagaggacaccagggcccctccatcgatgttcccttgtccaggatgaaatcctttgccatcctgtagagcatgtccccatccccaaggggaggcaccggggagtgaccgttgtccggagccaccacggtgttttgatatgcattcaagtgctggtccggcggcctcacagcctcagctcagaactcacaggcacctgtagcctcctgggccctgacctccgtccccaggctcaggccaacttcttcgaccttgaaaatctcctggatccagatgcacgtgtggacactggagactgctacagcaacaaagaaggcggtggagaagacgatgtgaacacagccaggcagcgccgggctctggagaaggaggaggaggaagacaaggaggaggaggaagacgaggaggaggaggaagctggcaccgagattgccatcatcctggatggctcaggaagcattgatcccccagactttcagagagccaaagacttcatctccaacatgatgaggaacttctatgaaaagtgttttgagtgcaactttgccttggtgcagtatggaggagtgatccagactgagtttgaccttcgggacagccaggatgtgatggcctccctcgccagagtccagaacatcactcaagtggggagtgtcaccaagactgcctcagccatgcaacacgtcttagacagcatcttcacctcaagccacggctccaggagaaaggcatccaaggtcatggtggtgctcaccgatggtggcatattcgaggaccccctcaaccttacgacagtcatcaactcccccaaaatgcagggtgttgagcgctttgccattggggtgggagaagaatttaagagtgctaggactgcgagggaactgaacctgatcgcctcagacccggatgagacccatgctttcaaggtgaccaactacatggcgctggatgggctgctgagcaaactgcggtacaacatcatcagcatggaaggcacggttggagacgcccttcactaccagctggcacagattggcttcagtgctcagatcctggatgagcggcaggtgctgctcggcgccgtcggggcctttgactggtccggaggggcgttgctctacgacacacgcagccgccggggccgcttcctgaaccagacagcggcggcggcggcagacgcggaggctgcgcagtacagctacctgggttacgctgtggccgtgctgcacaagacctgcagcctctcctacatcgcgggggctccacggtacaaacatcatggggccgtgtttgagctccagaaggagggcagagaggccagcttcctgccagtgctggagggagagcagatggggtcctattttggctctgagctgtgccctgtggacattgacatggatggaagcacggacttcttgctggtggctgctccattttaccacgttcatggagaagaaggcagagtctacgtgtaccgtctcagcgagcaggatggttctttctccttggcacgcatactgagtgggcaccccgggttcaccaatgcccgctttggctttgccatggcggctatgggggatctcagtcaggataagctcacagatgtggccatcggggcccccctggaaggttttggggcagatgatggtgccagcttcggcagtgtgtatatctacaatggacactgggacggcctctccgccagcccctcgcagcggatcagagcctccacggtggccccaggactccagtacttcggcatgtccatggctggtggctttgatattagtggcgacggccttgccgacatcaccgtgggcactctgggccaggcggttgtgttccgctcccggcctgtggttcgcctgaaggtctccatggccttcacccccagcgcactgcccatcggcttcaacggcgtcgtgaatgtccgtttatgttttgaaatcagctctgtaaccacagcctctgagtcaggcctccgcgaggcacttctcaacttcacgctggatgtggatgtggggaagcagaggagacggctgcagtgttcagacgtaagaagctgtctgggctgcctgagggagtggagcagcggatcccagctttgtgaggacctcctgctcatgcccacagagggagagctctgtgaggaggactgcttctccaatgccagtgtcaaagtcagctaccagctccagacccctgagggacagacggaccatccccagcccatcctggaccgctacactgagccctttgccatcttccagctgccctatgagaaggcctgcaagaataagctgttttgtgtcgcagaattacagttggccaccaccgtctctcagcaggagttggtggtgggtctcacaaaggagctgaccctgaacattaacctaactaactccggggaagattcctacatgacaagcatggccttgaattaccccagaaacctgcagttgaagaggatgcaaaagcctccctctccaaacattcagtgtgatgaccctcagccggttgcttctgtcctgatcatgaactgcaggattggtcaccccgtcctcaagaggtcatctgctcatgtttcagtcgtttggcagctagaggagaatgcctttccaaacaggacagcagacatcactgtgactgtcaccaattccaatgaaagacggtctttggccaacgagacccacacccttcaattcaggcatggcttcgttgcagttctgtccaaaccatccataatgtacgtgaacacaggccaggggctttctcaccacaaagaattcctcttccatgtacatggggagaacctctttggagcagaataccagttgcaaatttgcgtcccaaccaaattacgaggtctccaggttgtagcagtgaagaagctgacgaggactcaggcctccacggtgtgcacctggagtcaggagcgcgcttgtgcgtacagttcggttcagcatgtggaagaatggcattcagtgagctgtgtcatcgcttcagataaagaaaatgtcaccgtggctgcagagatctcctgggatcactctgaggagttactaaaagatgtaactgaactgcagatccttggtgaaatatctttcaacaaatctctatatgagggactgaatgcagagaaccacagaactaagatcactgtcgtcttcctgaaagatgagaagtaccattctttgcctatcatcattaaaggcagcgttggtggacttctggtgttgatcgtgattctggtcatcctgttcaagtgtggcttttttaaaagaaaatatcaacaactgaacttggagagcatcaggaaggcccagctgaaatcagagaatctgctcgaagaagagaattag
α
4184 α
E190 α
4(α
4The 184-190 position by α
EThe 404-410 position replace, also be α
4The 1-183 position connect α
EThe 404-410 position connect α
4The 191-999 position) aminoacid sequence (SEQ ID NO:46):
YNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIVMGAP
GAFDWSGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD
α
E404 α
4410 α
E(α
EThe 404-410 position by α
4The 184-190 position replace, also be α
EThe 1-403 position connect α
4The 184-190 position connect α
EThe 411-1161 position) aminoacid sequence (SEQ ID NO:47):
FNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTPGPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSSELTGTCSLLGPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEEEDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECNFALVQYGGVIQTEFDLRDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMVVLTDGGIFEDPLNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLIASDPDETHAFKVTNYMALDGLLSKLRYNIISMEGTVGDALHYQLAQIGFSAQILDERQVLLGAV
GSSYWTGGALLYDTRSRRGRFLNQTAAAAADAEAAQYSYLGYAVAVLHKTCSLSYIAGAPRYKHHGAVFELQKEGREASFLPVLEGEQMGSYFGSELCPVDIDMDGSTDFLLVAAPFYHVHGEEGRVYVYRLSEQDGSFSLARILSGHPGFTNARFGFAMAAMGDLSQDKLTDVAIGAPLEGFGADDGASFGSVYIYNGHWDGLSASPSQRIRASTVAPGLQYFGMSMAGGFDISGDGLADITVGTLGQAVVFRSRPVVRLKVSMAFTPSALPIGFNGVVNVRLCFEISSVTTASESGLREALLNFTLDVDVGKQRRRLQCSDVRSCLGCLREWSSGSQLCEDLLLMPTEGELCEEDCFSNASVKVSYQLQTPEGQTDHPQPILDRYTEPFAIFQLPYEKACKNKLFCVAELQLATTVSQQELVVGLTKELTLNINLTNSGEDSYMTSMALNYPRNLQLKRMQKPPSPNIQCDDPQPVASVLIMNCRIGHPVLKRSSAHVSVVWQLEENAFPNRTADITVTVTNSNERRSLANETHTLQFRHGFVAVLSKPSIMYVNTGQGLSHHKEFLFHVHGENLFGAEYQLQICVPTKLRGLQVVAVKKLTRTQASTVCTWSQERACAYSSVQHVEEWHSVSCVIASDKENVTVAAEISWDHSEELLKDVTELQILGEISFNKSLYEGLNAENHRTKITVVFLKDEKYHSLPIIIKGSVGGLLVLIVILVILFKCGFFKRKYQQLNLESIRKAQLKSENLLEEEN
α
4211 α
E216 α
4(α
4The 211-216 position by α
EThe 436-441 position replace, also be α
4The 1-210 position connect α
EThe 436-441 position connect α
4The 217-999 position) aminoacid sequence (SEQ ID NO:48):
YNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQN
EAAQYSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD
α
E436 α
4441 α
E(α
EThe 436-441 position by α
4The 211-216 position replace, also be α
EThe 1-435 position connect α
4The 211-216 position connect α
EThe 442-1161 position) aminoacid sequence (SEQ ID NO:49):
FNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTPGPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSSELTGTCSLLGPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEEEDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECNFALVQYGGVIQTEFDLRDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMVVLTDGGIFEDPLNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLIASDPDETHAFKVTNYMALDGLLSKLRYNIISMEGTVGDALHYQLAQIGFSAQILDERQVLLGAVGAFDWSGGALLYDTRSRRGRFLNQTAAAAADA
QVKFGSYLGYAVAVLHKTCSLSYIAGAPRYKHHGAVFELQKEGREASFLPVLEGEQMGSYFGSELCPVDIDMDGSTDFLLVAAPFYHVHGEEGRVYVYRLSEQDGSFSLARILSGHPGFTNARFGFAMAAMGDLSQDKLTDVAIGAPLEGFGADDGASFGSVYIYNGHWDGLSASPSQRIRASTVAPGLQYFGMSMAGGFDISGDGLADITVGTLGQAVVFRSRPVVRLKVSMAFTPSALPIGFNGVVNVRLCFEISSVTTASESGLREALLNFTLDVDVGKQRRRLQCSDVRSCLGCLREWSSGSQLCEDLLLMPTEGELCEEDCFSNASVKVSYQLQTPEGQTDHPQPILDRYTEPFAIFQLPYEKACKNKLFCVAELQLATTVSQQELVVGLTKELTLNINLTNSGEDSYMTSMALNYPRNLQLKRMQKPPSPNIQCDDPQPVASVLIMNCRIGHPVLKRSSAHVSVVWQLEENAFPNRTADITVTVTNSNERRSLANETHTLQFRHGFVAVLSKPSIMYVNTGQGLSHHKEFLFHVHGENLFGAEYQLQICVPTKLRGLQVVAVKKLTRTQASTVCTWSQERACAYSSVQHVEEWHSVSCVIASDKENVTVAAEISWDHSEELLKDVTELQILGEISFNKSLYEGLNAENHRTKITVVFLKDEKYHSLPIIIKGSVGGLLVLIVILVILFKCGFFKRKYQQLNLESIRKAQLKSENLLEEEN
α
4240 α
E246 α
4(α
4The 240-246 position by α
EThe 463-469 position replace, also be α
4The 1-239 position connect α
EThe 463-469 position connect α
4The 247-999 position) aminoacid sequence (SEQ ID NO:50):
YNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGA
PQYKHHGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD
α
E463 α
4469 α
E(α
EThe 463-469 position by α
4The 240-246 position replace, also be α
EThe 1-462 position connect α
4The 240-246 position connect α
EThe 470-1161 position) aminoacid sequence (SEQ ID NO:51):
FNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTPGPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSSELTGTCSLLGPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEEEDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECNFALVQYGGVIQTEFDLRDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMVVLTDGGIFEDPLNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLIASDPDETHAFKVTNYMALDGLLSKLRYNIISMEGTVGDALHYQLAQIGFSAQILDERQVLLGAVGAFDWSGGALLYDTRSRRGRFLNQTAAAAADAEAAQYSYLGYAVAVLHKTCSLSYIAGA
PQHEQIGAVFELQKEGREASFLPVLEGEQMGSYFGSELCPVDIDMDGSTDFLLVAAPFYHVHGEEGRVYVYRLSEQDGSFSLARILSGHPGFTNARFGFAMAAMGDLSQDKLTDVAIGAPLEGFGADDGASFGSVYIYNGHWDGLSASPSQRIRASTVAPGLQYFGMSMAGGFDISGDGLADITVGTLGQAVVFRSRPVVRLKVSMAFTPSALPIGFNGVVNVRLCFEISSVTTASESGLREALLNFTLDVDVGKQRRRLQCSDVRSCLGCLREWSSGSQLCEDLLLMPTEGELCEEDCFSNASVKVSYQLQTPEGQTDHPQPILDRYTEPFAIFQLPYEKACKNKLFCVAELQLATTVSQQELVVGLTKELTLNINLTNSGEDSYMTSMALNYPRNLQLKRMQKPPSPNIQCDDPQPVASVLIMNCRIGHPVLKRSSAHVSVVWQLEENAFPNRTADITVTVTNSNERRSLANETHTLQFRHGFVAVLSKPSIMYVNTGQGLSHHKEFLFHVHGENLFGAEYQLQICVPTKLRGLQVVAVKKLTRTQASTVCTWSQERACAYSSVQHVEEWHSVSCVIASDKENVTVAAEISWDHSEELLKDVTELQILGEISFNKSLYEGLNAENHRTKITVVFLKDEKYHSLPIIIKGSVGGLLVLIVILVILFKCGFFKRKYQQLNLES?I?RKAQLKSENLLEEEN
Mosaic plasmid construction method:
α
4And α
EThe mosaic construction process be the ordinary method that adopts rite-directed mutagenesis, such as α
4184 α
E190 α
4Just expression is α
4184-190 amino acids series jump be α
EUpper corresponding 404-410 amino acids sequence, vice versa.
Primer sequence is:
α
4184α
E190α
4-F：gactggtccggatctctttttgtctacaatat(SEQ?ID?NO：52)；
α
4184α
E190α
4-R：aaaggcccctggggcccccatcacaatta(SEQ?ID?NO：53)；
α
E404α
4410α
E-F：tactggactggcggggcgttgctctacgacac(SEQ?ID?NO：54)；
α
E404α
4410α
E-R：agatgatccgacggcgccgagcagcacctg(SEQ?ID?NO：55)；
α
4211α
E216α
4-F：cagtacagctatttaggatattcagtcggagc(SEQ?ID?NO：56)；
α
4211α
E216α
4-R：cgcagcctcattttgtttgtctaaaaaagcc(SEQ?ID?NO：57)；
α
E436α
4441α
E-F：tttggaagttacctgggttacgctgtggccgt(SEQ?ID?NO：58)；
α
E436α
4441α
E-R：ttttacttgcgcgtctgccgccgccgctgtctg(SEQ?ID?NO：59)；
α
4240α
E246α
4-F：aacatcatgggaaggcatatatattcagcattg(SEQ?ID?NO：60)；
α
4240α
E246α
4-R：tgtaccgtggagctcctccgactacttcggtag(SEQ?ID?NO：61)；
α
E463α
4469α
E-F：gcagattggtgccgtgtttgagctccagaag(SEQ?ID?NO：62)；
α
E463α
4469α
E-R：tcatgttgaggagcccccgcgatgtaggaga(SEQ?ID?NO：63)。
Then the constructed plasmid wink that can express chimera protein is turned the 293T cell and carries out Flow cytometry, obtain at last the epitope of J19 identification at α
4The 184-190 amino acids of subunit (Fig. 3 B, sequence is GSSYWTG).Each mosaic can normal expression, and its expression efficiency passes through β
7Antibody FIB27 (available from BD) measure, then according to wild-type α
4β
7Expression efficiency and the identification of J19 carry out stdn.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (17)
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| CN114907468A (en) * | 2022-06-10 | 2022-08-16 | 东莞理工学院 | A kind of Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017026331A1 (en) * | 2015-08-11 | 2017-02-16 | 国立大学法人大阪大学 | Antibody |
| CN107922938A (en) * | 2015-08-11 | 2018-04-17 | 国立大学法人大阪大学 | Antibody |
| JPWO2017026331A1 (en) * | 2015-08-11 | 2018-05-31 | 国立大学法人大阪大学 | antibody |
| JP2019201641A (en) * | 2015-08-11 | 2019-11-28 | 国立大学法人大阪大学 | antibody |
| US10654931B2 (en) | 2015-08-11 | 2020-05-19 | Osaka University | Antibody |
| US10988540B2 (en) | 2015-08-11 | 2021-04-27 | Osaka University | Antibody |
| CN107922938B (en) * | 2015-08-11 | 2021-09-17 | 国立大学法人大阪大学 | Antibodies |
| CN113577281A (en) * | 2020-04-30 | 2021-11-02 | 中国科学院分子细胞科学卓越创新中心 | Agents and methods for modulating integrin beta subunit |
| WO2021218594A1 (en) * | 2020-04-30 | 2021-11-04 | 中国科学院分子细胞科学卓越创新中心 | REAGENT AND METHOD FOR REGULATING INTEGRIN β SUBUNIT |
| CN114907468A (en) * | 2022-06-10 | 2022-08-16 | 东莞理工学院 | A kind of Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof |
| CN114907468B (en) * | 2022-06-10 | 2023-05-23 | 东莞理工学院 | Apostichopus japonicus integrin polyclonal antibody, antigen protein and preparation method thereof |
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