CN1319988C - High affinity immune globulin binding molecule and method for preparation - Google Patents
High affinity immune globulin binding molecule and method for preparation Download PDFInfo
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- CN1319988C CN1319988C CNB2004100671572A CN200410067157A CN1319988C CN 1319988 C CN1319988 C CN 1319988C CN B2004100671572 A CNB2004100671572 A CN B2004100671572A CN 200410067157 A CN200410067157 A CN 200410067157A CN 1319988 C CN1319988 C CN 1319988C
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Abstract
The present invention relates to a high affinity immune globulin combination molecule and a preparation method thereof. In the method, the structural domains of A, B, C, D and E of the staphylococcal protein A of immune globulin combination molecules from different genera, the structural domains of B1, B2, B3, B4 and B5 of peptostreptococcus magnus protein L, and the G1 and the G2 of the streptococcus protein G of C group and G group are used as structural units; the structural units are randomly connected to construct a recombined Ig combination molecule library, and the library is shown on the surface of phage; the recombined Ig combination molecule library shown on the surface of phage is sieved compatibly for three to four times with immune globulin to obtain a series of high affinity immune globulin combination molecules. The molecule displays very high immune globulin combination activity, and can be used for the detection and the purification of antibodies. The present invention can be widely used in amynologic diagnoses, such as an enzyme linked immunoadsorption assay, immunochromatographic assay detection, an immunohistochemical assay, etc.
Description
Technical field
The present invention relates to molecular biology and immunodiagnostics field, relate in particular to a kind of recombination immunoglobulin binding molecule library, its preparation and application.
Background technology
Enzyme linked immunosorbent assay (ELISA method) detects antibody and has been widely used in immunodiagnosis, blood donor's screening and the epidemiology survey that diagnosis, the especially transmissible disease of various clinical diseases such as hepatitis, HIV infect.But at present ELISA method detection sensitivity is not high, often causes omission and influences the early diagnosis of disease, affects the state of an illness adversely or causes that blood transfusion back hepatitis C and HIV infect, and people's health in serious harm.Screen whose anti-HCV and anti-HIV ELISA reagent from being used for the blood donor, still there is omission in up-to-date detection reagent (third generation reagent) at present.Wherein, these omissions be greatly in the detection system another key substance-enzyme conjugates with caused by the quantity not sufficient that combines of the antibody of antigen capture.Therefore, improving enzyme conjugates is to improve one of ELISA antibody test reagent susceptibility key factor with combining of capture antibody.
The enzyme conjugates that is used to detect at present mainly is that (Immunoglobulin, polyclonal antibody Ig) or monoclonal antibody, staphylococcus aureus protein A (protein A), C and G group streptococcus Protein G (protein G) and peptostreptococcus magnus belong to albumen L (protein L) for the anti-human normal immunoglobulin of horseradish peroxidase (HRP) mark.
Discover that some bacterium surface albumen can combine and not influence the antigenic activity of antibodies with Ig, therefore being used as enzyme conjugates is applied to immunology detection such as enzyme connection.These Ig binding molecules mainly are that staphylococcus aureus protein A (proteinA), C and G group streptococcus Protein G (protein G) and peptostreptococcus magnus belong to albumen L (protein L).Protein A mainly combines with the Fc section of Ig, with the Fc section of people and nearly all mammiferous IgG very strong combining is arranged all, but only in conjunction with wherein IgG
1, IgG
2And IgG
4Subclass can not be in conjunction with IgG
3Subclass, with other types Ig to combine activity very weak, itself and the poor effect that combines of IgM.Similar to protein A binding characteristic, protein G also can combine with the Fc section of people and nearly all mammiferous IgG, its bonding force is slightly stronger than protein A, different is, protein G can not be in conjunction with the immunoglobulin (Ig) of the other types except that IgG, therefore, not to be well suited for as immunodetection.It is another kind of that antibody binding proteins-the anerobe peptostreptococcus magnus belongs to protein L is different fully with the binding characteristic of protein A and protein G, it mainly combines with the kappa light chain variable district of Ig, can be in conjunction with the Fab fragment of dissimilar Ig, and do not influence antigen binding site, with people and nearly all mammiferous Ig all kinds of and each hypotype stronger combining arranged all, and avidity is suitable, is suitable as immunodetection.
1992, Sweden scholar Kihlberg etc. made up the fusion rotein Protein LG of protein L and protein G, found that Protein LG has that antibody binding activity can be in conjunction with Fc, Fab section and the Ig light chain of most of Ig widely.Compare with single protein L or proteinG, its antibodies characteristic is more complete, except that human Ig, also can be used for the detection of mouse endogenous antibody.1998, Sweden scholar Svensson etc. combine four structural domains in conjunction with Ig κ light chain of protein L the structural domain fusion of IgG with four of proteinA, construct a kind of new fusion protein Protein LA, bind profile is wide, can not only be in conjunction with people and some mammiferous dissimilar Ig and all subclass of IgG, the antigen binding capacity of antibody be can also not influence in conjunction with phage single chain antibody Fv (ScFv) fragment, immunodetection and all kinds of Ig of affinity chromatography purifying and Fv (ScFv) fragment can be used for.Proof Protein LA is a kind of multi-functional antibody-binding molecules.At present, soluble, the HRP mark of Protein LA or be developed to commodity by CLONTECH company in conjunction with the several formulations of agarose form.Domestic do not have analogous products to occur so far.
The protein molecule lactam enzyme by directional anagenesis in vitro is that a kind of protein that grew up is in recent years transformed New Policy, by random mutation, reorganization and directed screening to encoding gene, so as to changing, optimize the biomacromolecule characteristic, acquisition has the protein that improves function or brand-new function, the natural evolutionary process in millions of years is achieved in a short time, is the important method of transforming bioactive molecules.It is the easiest, the most effective so far in-vitro directed molecular evolution technique that DNA resets, it is with one group of nucleotide sequence random fragmentation that is closely related, be reassembled into the total length nucleotide sequence again, evolve rapidly, can obtain new protein molecular with better function by sequence.DNA resets technology and has been used widely and has achieved success in the preparation of many enzymes, antibody and important proteic transformation, novel lps molecule and protein medicaments aspect medical.
Each free a plurality of sequence height multiple structural domain of the antibodies district of protein A, protein L and protein G are formed, and the Ig land of protein A is in series by 5 (A, B, C, D, E) 58-62 amino acid residue sequence height homologous structural domains; A spot of aminoacid sequence difference is only arranged between each single structure territory, respectively have 5 amino acid different with B, B and C-structure territory as A.Also repeated to be in series by 5 (B1, B2, B3, B4, B5) 72-76 amino acid residue sequence height homologous antibodies structural domains in the Ig land of protein L, each structural domain amino acid is formed identical mostly, some site difference is only arranged, have only 4 amino acid different with the B3 structural domain as B2.The Ig of protein G is located in 2 height multiple B structural domains (G1, G2) in conjunction with activity, and each structural domain is made up of 55 amino-acid residues.Wherein the B1 of protein L, B2, B3, B4, B5 structural domain have the gene order shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 respectively; The A of protein A, B, C, D, E structural domain have the gene order shown in SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 respectively; The G1 of protein G, G2 structural domain have the gene order shown in SEQ ID NO:11, SEQ ID NO:12 respectively.Experimental results show that above-mentioned proteic one antibodies structural domain has the Ig binding ability too.The present invention utilizes DNA rearrangement technology with 12 kinds of protein L, proteinA and Protein G unijunction close the territory, and (B1, B2, B3, B4, B5, A, B, C, D, E, G1, G2 represent three kinds of albumen protein L successively, the different antibodies binding domains of protein A and Protein G, totally 12 kinds) for structure unit carries out again the random alignment combination, make up reorganization Ig binding molecule library.Utilize this library to carry out the DNA rearrangement and can form multiple different Ig binding molecule, therefrom can filter out the high affinity immune globulin stronger (Ig) binding molecule (highaffinity immunoglobulin binding molecules HAIBMs), realize protein molecule lactam enzyme by directional anagenesis in vitro of the present invention with the Ig binding ability.
Display technique of bacteriophage is as high-throughout screening method, and the in-vitro screening that makes large vol combination gene library set up the back molecules of interest becomes possibility.Display technique of bacteriophage is that a kind of of foundation in 1985 inserts the allogenic polypeptide gene in bacteriophage coat protein PIII or the PVIII gene, allogenic polypeptide is showed in the method on phage particle surface.The genotype and the phenotype of its allogenic polypeptide combine cleverly, and the foreign protein that is illustrated in phage surface directly is associated with its encoding gene, can directly carry out the activity identification and the sequential analysis of foreign protein by phage breeding amplification.Through the screening of specific aglucon, can obtain the molecules of interest of specific combination, and make the phage that contains target protein obtain up to ten thousand times and even 10 by the repetitive process of " absorption " one " wash-out " one " amplification "
8Enrichment doubly is a kind of high flux screening system of efficient molecular evolution research.The Vector for Phage Display pCANTAB5S that we use, at " structure of novel Vector for Phage Display pCANTAB5S ", Medical University Of Anhui's journal, in 2004 the 39th volume the 83rd～86 page of the 2nd phase, and has detailed description in Chinese patent literature-document number 1508254, here briefly, be in primer, to add Sac I restriction enzyme site, to be connected the fragment PCR product cloning with the IFN-α A-2b of this primer amplification in pMD-18T, again this fragment is inserted among the pCANTAB5L, cut through Sac I enzyme, reclaim linear carrier segments, be connected to form novel Vector for Phage Display pCANTAB5S again.PCANTAB5S has proofreaied and correct the frame of pCANTAB5L cloning site and has introduced new restriction site SacI.Figure 12 is seen in the structural representation of reorganization phagemid pCANTAB5S.PCANTAB5S can show reorganization Ig binding molecule library, and can carry out affine screening to phage display reorganization Ig binding molecule library by Ig, obtains serial HAIBMs.
Summary of the invention
An object of the present invention is to provide a kind of reorganization Ig binding molecule library and preparation method thereof.
Another object of the present invention provides the phage display method in a kind of Ig of reorganization binding molecule library.
Another object of the present invention provides a kind of method that obtains high-affinity Ig binding molecule phage library.
Another object of the present invention provides reorganization Ig binding molecule of the present invention library and is applied to screen high-affinity Ig binding molecule.
In a first aspect of the present invention, the present invention relates to a kind of reorganization Ig binding molecule library, this library is with the A of different Ig binding molecule staphylococcal protein A,SPAs, B, C, D, the E structural domain, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, the B5 structural domain, G group or the proteic G1 of C group streptococcus G, the single antibodies structural domain of G2 structural domain is a structural unit, resetting formation Ig binding molecule with the structural unit random dna forms, wherein the nucleotide sequence in said structure territory is respectively: SEQ ID NO:1-SEQ ID NO:12, connect with Nucleotide GAGCTC between the single structure territory, the structural unit random dna is reset the Ig binding molecule that forms and is obtained by the following method:
A) with A, B, C, D, the E structural domain of Ig binding molecule staphylococcal protein A,SPA, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, B5 structural domain, the single antibodies structural domain of G group or the proteic G1 of C group streptococcus G, G2 structural domain are introduced Sac I restriction enzyme site as tie point through pcr amplification respectively;
B) PCR is reclaimed product and cut digestion through Sac I enzyme respectively;
C) the Sac I enzyme that step (2) is obtained is cut the digestion product mixing, with the T4 ligase enzyme each single structure territory is connected reorganization at random.
In a second aspect of the present invention, the present invention relates to the preparation method in above-mentioned library, comprise the following steps:
A) with A, B, C, D, the E structural domain of Ig binding molecule staphylococcal protein A,SPA, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, B5 structural domain, the single antibodies structural domain of G group or the proteic G1 of C group streptococcus G, G2 structural domain is a structural unit, introduce Sac I restriction enzyme site as tie point through pcr amplification respectively, the nucleotides sequence of wherein said structural domain is classified as: SEQ ID NO:1-SEQ ID NO:12;
B) PCR reclaims product and cuts digestion through the SacI enzyme respectively;
C) the Sac I enzyme that step (2) is obtained is cut the digestion product mixing, with the T4 ligase enzyme each single structure territory is connected reorganization at random again.
In a third aspect of the present invention, the present invention relates to the reorganization Ig binding molecule phage library that phage display method and this method in a kind of Ig of reorganization binding molecule library are obtained, this method will be carried out the phage display of 3+3 form with this carrier by described Ig binding molecule library clone in phagemid carrier pCANTAB5S.
In a fourth aspect of the present invention, the present invention relates to a kind of method that obtains high-affinity Ig binding molecule phage library, this method Ig wrapper sheet utilizes above-mentioned reorganization Ig binding molecule phage library to take turns the affine screening of external orthogenesis IgG through 3-4 and obtains high-affinity Ig binding molecule phage library.
In a fifth aspect of the present invention, the present invention relates to the high-affinity Ig binding molecule phage library that aforesaid method obtains, by the high-affinity Ig binding molecule of phage display be that the single structure territory of single structure territory, peptostreptococcus magnus protein L with staphylococcal protein A,SPA and G group or the proteic single structure of C group streptococcus G territory are structural unit, be spaced in the Ig binding molecule that is formed by connecting any one or multiple.
More preferably, represent single structure territory, G group or the proteic single structure of the C group streptococcus G territory of the proteic single structure of peptostreptococcus magnus L territory, staphylococcal protein A,SPA respectively with ML, MA, MG, the arrangement mode of the sequence of then above-mentioned high-affinity Ig binding molecule is (ML-MA) n or (ML-MG) n or (ML-MA-MG) n or (ML-MG-MA) n or ML-(MA-ML) n or ML-(MG-ML) n, and wherein n is the multiplicity of this sequence.
More preferably, the arrangement mode of above-mentioned high-affinity Ig binding molecule sequence is B3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, any of B2-C-G2-B1-D-G1.
More preferably, above-mentioned high-affinity Ig binding molecule has SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, the SEQ ID NO:32 sequence signature shown in arbitrary.
In a sixth aspect of the present invention, reorganization Ig binding molecule provided by the invention library can be used for screening high-affinity Ig binding molecule.The high-affinity Ig binding molecule that screening obtains can be applicable to immunology diagnosis or detection of antibodies and purifying.Especially being applied to enzyme linked immunosorbent assay or immunochromatography detects or immunohistochemical methods.
Technological line of the present invention is to adopt Protocols in Molecular Biology to make up reorganization Ig binding molecule library; This library is showed on the filobactivirus in the mode of 3+3; With Ig phage display library is carried out 3-4 and take turns affine screening, obtain serial high-affinity Ig binding molecule (HAIBMs); The dna sequence dna of the HAIBM that obtains is measured in sequential analysis, and knows its aminoacid sequence, analyzes the formation characteristics of its molecule; Prokaryotic expression, purifying HAIBM molecule are measured the binding ability of itself and Ig; Measure and show that the binding ability that HAIBM molecule phage and Ig are arranged reaches the effect in the ELISA antibody test.
Advantage of the present invention is to utilize the strategy of protein molecule lactam enzyme by directional anagenesis in vitro, screen and a series of Ig binding molecules of immunoglobulin (Ig) high-affinity bonded (HAIBMs) by making up reorganization Ig binding molecule gene library, HAIBMs can be widely used in immunology diagnosis and detection of antibodies and purifying such as enzyme linked immunosorbent assay, immunochromatography detection, immunohistochemical methods as the new Ig binding molecule with high-affinity.
Description of drawings
A, the B of Fig. 1 Protein A, the segmental pcr amplification product electrophoresis of C-structure domain gene synoptic diagram, wherein:
m：DL2000Marker
1-4:Mu-Protein A A domain gene fragment PCR amplified production
5-6:Mu-Protein A B domain gene fragment PCR amplified production
7-8:Mu-Protein A C-structure domain gene fragment PCR amplified production
C: blank
The segmental pcr amplification product electrophoresis of the D domain gene synoptic diagram of Fig. 2 Protein A, wherein:
m：DL2000Marker
1-4:Mu-Protein A D domain gene fragment PCR amplified production
C: blank
The segmental pcr amplification product electrophoresis of the E domain gene synoptic diagram of Fig. 3 Protein A, wherein:
m：DL2000Marker
1-4:Mu-Protein A E domain gene fragment PCR amplified production
C: blank
The segmental pcr amplification product electrophoresis of Fig. 4 Protein L B1-B5 domain gene synoptic diagram, wherein:
The segmental pcr amplification product of 1:Protein L B1 domain gene
The segmental pcr amplification product of 2:Protein L B2 domain gene
The segmental pcr amplification product of 3:Protein L B3 domain gene
The segmental pcr amplification product of 4:Protein L B4 domain gene
The segmental pcr amplification product of 5:Protein L B5 domain gene
C: blank
m：DL2000Marker
The segmental pcr amplification product electrophoresis of Fig. 5 Protein G G1-G2 domain gene synoptic diagram, wherein:
The segmental pcr amplification product of 1:Protein G G1 domain gene
The segmental pcr amplification product of 2:Protein G G2 domain gene
C: blank
m：DL2000Marker
Fig. 6 Protein A single structure territory+Protein L single structure territory+Protein G single structure territory ligation product electrophoresis synoptic diagram, wherein:
1: connect preceding 2: connect 30 ' 3: connect 1h
4: connect 2h 5: connect 4h 6: connect 24h
m：DL2000Marker
Fig. 7 PALGn/phage library Ig screening F1 inserts segmental pcr amplification 1-8 for mono-clonal phage in the library: mono-clonal 1-16#; C: positive control LT/pCANTAB5L; M:DL2000Marker; N: negative control pCANTAB5S
Fig. 8 PALGn/phage library Ig screening F2 inserts segmental pcr amplification 1-8 for mono-clonal phage in the library: mono-clonal 1-16#; C: positive control LT/pCANTAB5L; M:DL2000Marker; N: negative control pCANTAB5S
Fig. 9 PALGn/phage library Ig screening F3 inserts segmental pcr amplification 1-8 for mono-clonal phage in the library: mono-clonal 1-16#; C: positive control LT/pCANTAB5L; M:DL2000Marker; N: negative control pCANTAB5S
Figure 10 PALGn/phage library Ig screening F4 inserts segmental pcr amplification 1-8 for mono-clonal phage in the library: mono-clonal 1-16#; C: positive control LT/pCANTAB5L; M:DL2000Marker; N: negative control pCANTAB5S
Figure 11 respectively screens the ratio that the composition of the single structure territory (domain) in the HAIBM molecule distributes in the round IgG binding molecule library
The recombinate structural representation of phagemid pCANTAB5S of Figure 12
Embodiment
One, the structure of reorganization Ig binding molecule gene library
Reset with traditional DNA-purpose fragment DnaseI is digested the back re-assemble at random realize that the mode of lactam enzyme by directional anagenesis in vitro is different, the present invention with the single antibodies structural domain of the Ig binding molecule of different genera as the (A of protein A of structure unit, B, C, D, the E structural domain, the B1 of protein L, B2, B3, B4, the G1 of B5 structural domain and protein G, the G2 structural domain) introduces identical restriction site as Linker through pcr amplification, after enzyme is cut digestion, with the T4 ligase enzyme each single structure territory is connected reorganization at random again, constitute reorganization Ig binding molecule library.The antibodies structural domain that DNA rearrangement can formation different lengths (2-5 structural domain or more) is formed with 55-76 amino-acid residue in this library is the reorganization Ig binding molecule of least unit, and it repeats series connection at random can produce 12
nThe above different I g binding molecule of Zhonging (n is for repeating placed in-line minimum working energy unit number); Compare with Protein LA molecule, its various spread pattern can be represented with P (L/A/G) n, P is a polypeptide, (L/A/G) is any in 5 kinds of Protein L, 5 kinds of Protein A and the 2 kinds of Protein G single structure territories, and n is the number that repeats placed in-line single structure territory.Compare with Protein LA (LLL-AAAA) and the single structure formation of Protein LG, the sequence polymorphism of the diversity of the diversity of L and A/G distance, (L/A/G) repetition serial number and the generation of various L/A/G amino acid difference makes and produces the high-affinity Ig binding molecule stronger with the Ig binding ability in the library of molecules.Each single structure territory is the orderly polypeptide with a fixed structure in this molecular library, itself has the Ig binding ability, and its basic framework structure is continued to preserve in new albumen, thereby the Ig binding ability that has guaranteed combination molecule is improved and optimizes.Because of each single structure territory self has secondary structure (α spiral or βZhe Die), the space structure of recombinant molecule has higher stability than random fragment recombinant molecule, helps keeping of new albumen space conformation in addition.In order to guarantee the diversity of recombinant molecule, the contriver is by adjusting each segmental concentration, make each fragment ratio suitable, while control linkage reaction conditions, the connection product of different time is mixed, obtain the reorganization Ig binding molecule gene library of the various arrangements of different lengths, Figure of description Fig. 1-Fig. 6 has promptly shown the A of Protein A respectively, B, the segmental pcr amplification product electrophoresis of C synoptic diagram, the segmental pcr amplification product electrophoresis of the D of Protein A synoptic diagram, the segmental pcr amplification product electrophoresis of the E of Protein A synoptic diagram, the segmental pcr amplification product electrophoresis of the B1-B5 of Protein L synoptic diagram, the segmental pcr amplification product electrophoresis of the G1-G2 of Protein G synoptic diagram, ProteinA single structure territory+Protein L single structure territory+Protein G single structure territory ligation product electrophoresis synoptic diagram.
Two, the phage display in reorganization Ig binding molecule library
To recombinate Ig binding molecule library clone in phagemid carrier pCANTAB5S, be the displaying that carrier carries out the 3+3 form with the phagemid.Foreign gene inserts in the III gene of phagemid, transformed into escherichia coli TG1, and the rescue of M13K07 helper phage makes up and shows Ig binding molecule phage library.The storage capacity of being built is 3.4 * 10
7Individual bacterium colony forms, and the titre of phage is 4.1 * 10
12The unit conversion units per ml (tramsformation unit/ml, TU/ml).PCANTAB5S phagemid carrier not only keeps multiple clone site and [G4S] 3 joints of former pCANTAB5L carrier, the function that has guaranteed macromolecule (＞300 amino acid) reorganization Ig binding molecule is kept and active the displaying, also increased the SacI cloning site, can be more convenient, show the foreign gene library effectively, help enlarging storage capacity.
Three, the in-vitro directed molecular evolution screening of Ig high-affinity Ig binding molecule (HAIBMs)
The high flux screening that utilizes display technique of bacteriophage to set up screens in conjunction with the in-vitro directed molecular evolution of Ig.Use the Ig wrapper sheet, phage display Ig binding molecule library obtains HAIBMs through the affine screening of 3-4 wheel Ig.We every take turns the IgG that selected for use in the storehouse screening in conjunction with the experiment enumeration, insert the effect of index visual evaluation screenings such as fragment PCR positive colony number, the results are shown in Table 1, table 2, Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11.
Four, the molecular structure characteristics and the sequence of high-affinity ig binding molecule (HAIBMs)
The monoclonal recombinant phage in preparation screening back extracts phagemid dna, and with upstream and downstream primer pCANTAB5-S1 and its dna sequence dna of the two-way mensuration of pCANTAB5-S6 of pCANTAB5S, institute check order to be listed as and uses its sequence signature of DNASTAR software analysis.The sequential analysis of high-affinity Ig binding molecule shows the diversity that its structural domain is formed: by the A/B/C/D/E from the single antibodies structural domain-protein A of different I g binding molecule, the B1/B2/B3/B4/B5 of protein L, the G1/G2 of protein G reorganization is connected to form that (B1-B5 represents the B1-B5 structural domain of protein L, A, B, C, D, E represents the A of protein A, B, C, D, the E structural domain, G1, G2 represents the B1 of protein G, the B2 structural domain), as B1-B2-G1-A, B3-D-B3, B1-B3-B1, G1-B2-A-B1, B1-G2-B1, D-B2-B-B3, B3-D-B3-D-B3, B-G1-B2-D-B1 etc.With ML, MA, MG represents the proteic single structure of peptostreptococcus magnus L territory respectively, the single structure territory of staphylococcal protein A,SPA, G group or the proteic single structure of C group streptococcus G territory, then the molecular characterization that HAIBMs possessed is: (ML-MA) n or (ML-MG) n or (ML-MA-MG) n or (ML-MG-MA) n or ML-(MA-ML) n or ML-(MG-ML) n structure, and HAIBMs is by the single structure territory (ML) of protein L, the single structure territory (MA) of protein A or the single structure territory (MG) of protein G are spaced be formed by connecting (n is the multiplicity of this sequence).The mode of connection of representative sequence wherein is: B3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, B2-C-G2-B1-D-G1.Between the single structure territory of representing sequence protein L, protein A, proteinG, connect with GAGCTC with Nucleotide.The present invention disclosed especially have SEQ ID NO:14, SEQ IDNO:16, the high-affinity Ig binding molecule of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, the SEQ ID NO:32 sequence shown in arbitrary.
Five, the Ig of HAIBM recombinant phage is in conjunction with determination of activity
Respectively each mono-clonal HAIBM recombinant phage is carried out Ig in conjunction with determination of activity with the Ig wrapper sheet.Detect combining of recombinant phage and Ig in two ways, add e. coli tg1 in first kind of sample hole after recombinant phage and antibodies and make its infection, mono-clonal recombinant phage infectious bacteria liquid coated on the amicillin resistance plate count, with the negative contrast of helper phage, the size of how much representing phage and Ig binding ability of colony number; Add enzyme mark phage-resistance antibody (HRP/Anti-M13 conjugate) in second kind of sample hole after recombinant phage Ig combination, the OD450nm value is surveyed in the TMB colour developing, and directly detection be the results are shown in Table 3 by the quantity of the recombinant phage of the special absorption of people Ig.
Six, the function of high-affinity Ig binding molecule (HAIBMs)
HAIBMs is cloned in prokaryotic expression carrier pET32 (a), and IPTG abduction delivering HAIBMs albumen is with Ni affinity chromatography column purification HAIBMs albumen.HAIBMs with purifying wraps by 96 hole enzyme plates, establishes the SPA positive control simultaneously, and the LT negative control adds IgG HRP enzyme labelled antibody, and it is active with combining of Ig to detect HAIBMs, the results are shown in Table 4.
Seven, the application of high-affinity Ig binding molecule (HAIBMs)
HCV is antigen coated on enzyme mark row batten, after the washing, the serum that adds the infection with hepatitis C virus positive patients, the unconjugated antibody of effect back flush away, add different HAIBM mono-clonal recombinant phages (each titre transfers to same level), if phage negative control, reacted sample is divided into two groups, first group of every hole adds e. coli tg1 makes its infection, the bacterium liquid that the mono-clonal recombinant phage is infected is coated on the amicillin resistance plate and is counted, with the negative contrast of helper phage; Second group of every hole adds phage antibody HRP/Anti-M13conjugate, and OD is surveyed in the TMB colour developing
450The nm value is used to detect the quantity by the recombinant phage of the special absorption of patients serum, the results are shown in Table 4, table 5, table 6.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The structure in embodiment 1 reorganization Ig binding molecule library
1. synthetic primer
1.1 be used for protein A, protein L, the amplification of protein G single structure domain gene
(1) upstream primer from Mu-protein A/pGEM-T easy plasmid amplification Protein A-A, Protein A-B/C, Protein A-D, Protein A-E sequence is respectively:
PA-uA：5’-cacgagctcGCTGACAACAATTTCAAC-3’
PA-uB：5’-cgcgagctcGCGGATAACAAATTCAAC-3’
PA-uC：5’-cgcgagctc GCTGACAACAAATTCAAC-3’
PA-uD：5’-tccgagctcGCTGATGCGCAACAAAAT-3’
PA-uE：5’-cgcgagctcGCTCAACAAAATGCTTTT-3’
Downstream primer is respectively:
PA-dA：5’-tctgagctcTTTCGGTGCTTGAGATTC-3’
PA-dB：5’-tctgagctcTTTTGGTGCTTGTGCATC-3’
PA-dC：5’-acggagctcTTTTGGTGCTTGAGCATC-3’
PA-dD：5’-tcggagctcAAAGCCACGAACTCTAAG-3’
PA-dE：5’-agagagctcTTTTGGAGCTTGAGAGTC-3’
(2) from the B1-B5 sequence of Protein L/pGEM-T easy plasmid amplification Protein L
Upstream primer is respectively:
pLB1-u：5’-tctgagctcAAAGAAGAAACACCAGAA-3’
pLB2-u：5’-acggagctcAAAGAAAAAACCCCGGAA-3’
pLB3-u：5’-cgtagactCAAAGAAAAAACACCAGAA-3’
pLB4-u：5’-cgtagactcAAAGAAAAAACACCAGAA-3’
pLB5-u：5’-cgcagactcAAGAAAGTTGACGAAAAA-3’
Downstream primer is respectively:
pLB1-d：5’-cgcagactcTCCAGCAAATTTAATATT-3’
pLB2-d：5’-cgcagactcTCCAGCAAATTTAATATT-3’
pLB3-d：5’-tgcgagctcACCAGCGAATTTGATGTT-3’
pLB4-d：5’--cgcagactcACCTGCAAATCTAATATT-3’
pLB5-d：5’--cgtagactcTCCAGCAAATCTAATGTT-3’
(3) upstream primer from Protein G/pGEM-T easy plasmid amplification Protein G B1-B2 sequence is respectively:
pG1-u：5’-tctgagctcACTTACAAATTAATCCTT-3’
pG2-u：5’-tctgagctcACTTACAAACTTGTTATT-3’
Downstream primer is respectively:
pG1-d：5’-tatgagctcTTCAGTTACCGTAAAGGT-3’
pG2-d：5’-tctgagctcTTCAGTAACTGTAAAGGT-3’
Above-mentioned primer has all been introduced Sac I (GAGCTC-) restriction enzyme site.
Insert segmental primer sequence 1.2 be used for pcr amplification detection positive colony, for pCANTAB5S cloning site upstream and downstream sequence upstream primer is:
pCANTAB5-S1：5’-CAACGTGAAAAAATTATTATTCGC-3’
Downstream primer is:
pCANTAB5-NOT2：5’-GCCCAGCCGGCC-3’
1.3 be used for the primer sequence of positive colony dna sequencing, for the upstream and downstream sequence upstream primer of pCANTAB5E is:
pCANTAB5-S1：5’-CAACGTGAAAAAATTATTATTCGC-3’
Downstream primer is:
pCANTAB5-S6：5’-GTAAATGAATTTTCTGTATGAGG-3’
The pcr amplification of 2 each antibodies structural domain
Employed template protein A sequence is seen GenBank AB050857, and protein L sequence is seen GenBank M86697, and protein G sees GenBankY00428.Protein A/pGEM-T easy plasmid with 5 antibodies structural domains (A-E) of containing staphylococcal protein A,SPA is a template, with PA-uA/PA-d A, PA-u B/PA-d B, PA-u C/PA-d C, PA-u D/PA-dD, PA-u E/PA-d E increase respectively A, B, C, D and the E fragment of Protein A; With Protein L/pGEM-T easy plasmid is template, with pL B1-u/pL B1-d, pL B2-u/pL B2-d, pL B3-u/pL B3-d, pL B4-u/pL B4-d and pL B5-u/pL B5-d is primer, increase the respectively B1 of Protein L of PCR, B2, B3, B4 and B5 fragment; With ProteinG/pGEM-T easy plasmid is template, is primer with pG1-u/pG1-d and pG2-u/pG2-d, increase the respectively B1 of ProteinG of PCR, B2 fragment.PCR reaction volume 50 μ l add plasmid template 5ng, each 1 μ mol of upstream and downstream primer, dNTP 100 μ mol, Mg++ 2mmol, 1U Taq enzyme.Amplification condition is 94 ℃ of 30sec; 60 ℃ of 30sec; 72 ℃ of 30sec; 35 circulations.Each PCR product glue test kit purifying of Shen energy betting office, by specification carries out.Corresponding pcr amplification product electrophorogram is seen Fig. 1-Fig. 5.
3 PCR reclaim the enzyme of product and cut
Each PCR reclaims product and gets 40 μ l and make Sac I enzyme and cut reaction volume 80 μ l.Sac I4 μ l (40U), 10 * buffer8 μ l, divide 80 μ l/ to prop up, 37 ℃ of enzymes are cut and are spent the night, after 1% agarose electrophoresis, cut off the purpose band with knife blade, glue test kit with Shen energy betting office reclaims PA-A, PA-B, PA-C, PA-D, PA-E, PL B1, PL B2, PL B3, PL B4, PL B5 and PG1, PG2 endonuclease bamhi.
The structure in 4 Ig binding molecule libraries
Above-mentioned each enzyme cuts back to close fragment and gets 5 μ l (1 μ g), 10 * connection damping fluid, 8 μ l, T4DNA ligase enzyme 4 μ l (20U), reaction volume 100 μ l.Earlier dna fragmentation is added in the pipe in 65 ℃ of incubations 5 minutes with water, before ligation reagent adds, with dna solution in 0 ℃ of precooling 5 minutes, add the T4DNA ligase enzyme, connect mixture and divide 5,20 μ l/ prop up, and 16 ℃ connect 30min, 1h, 2h, 4h, 24h respectively, the connection product of different time are mixed to be used for and being connected of pCANTAB5S DNA again.Fig. 6 shows successful connection.
The phage display in embodiment 2, reorganization Ig binding molecule library
1. preparation and the linearizing of phagemid carrier pCANTAB5S DNA:
The pCANTAB5S phagemid dna can reclaim the test kit extracting by betting office's plasmid with the Shen, and by specification carries out.Agarose electrophoresis is quantitative.Sac I enzyme is cut back alkaline phosphatase (CIAP) dephosphorylation and is handled, and test kit glue from solution reclaims linearized vector, and agarose electrophoresis is quantitative.
2.Ig the binding molecule library is connected with the phagemid carrier:
A, B, C, D, the E unijunction of the protein A of 12 kinds of recovery are closed the territory endonuclease bamhi, the B1 of protein L, B2, B3, B4, B unijunction close the territory endonuclease bamhi, the mixture 80 μ l (5 μ g) that the G1 of Protein G, G2 unijunction close the territory endonuclease bamhi add T4 dna ligase 5 μ l (25U), 10 * Buffer1.5 μ l, reaction cumulative volume 100 μ l.100 μ l are divided into 20 μ l/ to be propped up, totally 5,16 ℃ of connections, merge after connecting 30min, 60min, 90min, 120min, 150min respectively, add carrier 2 μ l (2 μ g), handle 10min for 65 ℃ and be cooled to room temperature, add T4DNA ligase enzyme 5 μ l (25U) again, 10 * Buffer2 μ l, reaction cumulative volume 120 μ l merge after dividing 6 16 ℃ to connect 30min, 60min, 90min, 120min, 150min and 180min respectively.
3. the preparation of competent cell:
Original TG1 bacterium line LB flat board, overnight incubation in 37 ℃ of incubators, the dull and stereotyped mono-clonal colony inoculation of going up of picking activation is in the test tube of 2mlPsi substratum, and 37 ℃ of 250rpm shaking table shaking culture are spent the night; Culturing bacterium is transferred in the Psi substratum at 1: 50,37 ℃ of 250rpm sway and are cultured to OD value 0.4 again.Bacterium liquid 1ml/ propped up be sub-packed in the 1.5ml centrifuge tube ice-water bath 15 minutes, 4000rpm, 4 ℃ of centrifugal 10min; Remove supernatant, with 1ml 0.1M CaCl
2Carefully bacterial suspension is got up; Ice-water bath 15min, 3500rpm, 4 ℃ of centrifugal 10min; Remove supernatant, precipitate with 200 μ l 0.1M CaCl
2Bacterium is carefully multiple outstanding, and ice bath is standby.
4. the preparation of recombinant phage:
120 μ l connect product and add in the 2ml competent cell, mixing gently, ice-water bath 30min, 42 ℃ of water-bath 60s, ice-water bath 2min, add 8ml SOC substratum, 37 ℃ of 150rpm, shaking table shaking culture 1h gets 100 μ l and evenly is coated with the dull and stereotyped 37 ℃ of overnight incubation of LB acillin (100ng/ml), the counting colony number is to calculate storage capacity, and 37 ℃ of 150rpm shaking tables of all the other liquid shaking culture is spent the night.Second day, transform culture and use 100ml 2 * YT (1.6% Tryptones, 1% yeast extract paste, 0.5% sodium-chlor) activation culture 1 hour, add 10ml 4 * 10
10The M13K07 helper phage rescue of TU/ml, 37 ℃, 250rpm shaking culture 1 hour adds kantlex to concentration 50ng/ml, and 37 ℃, the 250rpm shaking culture is spent the night.Centrifugal 10 minutes of culture 5000rpm gets supernatant through 0.22 μ m membrane filtration, obtains former generation (F
0Generation) recombinant phage PALGn storehouse.Its transformation efficiency is 3.4 * 10
7(connecting contrast certainly is 0).Therefore, building the library capacity is 3.4 * 10
7Individual colony-forming unit.Molecular library is made up of 12 kinds of composed components: the G1 of the A-E domain of protein A, the B1-B5 domain of protein L and protein G, G2 domain, as calculating by the longest PAL molecule that forms 5 domain, each domain all has positive and negative dual mode to insert, and this storehouse should contain 24+24
2+ 24
3+ 24
4+ 24
5Promptly 8.3 * 10
6Plant the external source PALGn molecule of different permutation and combination methods, therefore, the PALGn-pCANTAB5S combinatorial library that we make up can comprise all PALGn of combination at random molecules fully preferably.
5. recombinant phage titer determination
With 10 times of serial dilutions of recombinant phage, respectively get the e. coli tg1 100 μ l that recombinant phage 10 μ l infect logarithmic phase, cultivate after 1 hour for 37 ℃ and be coated with LB (containing acillin 100ng/ml) flat board, put 37 ℃ of overnight incubation.Count the colony number on the different extent of dilution plates, the extension rate of growth colony number multiply by the 100 conversion unitss that are every milliliter of phage (transformation unit, TU).Detected result is showed F
0Titre for the recombinant phage in Ig binding molecule PALGn storehouse is 4.1 * 10
12TU/ml is 6.1 * 10 with identical method in the titre of the M13K07 wild type phage that contains count detection on the flat board of kalamycin resistance
12TU/ml, the titre of recombinant phage and the horizontal basically identical of the titre of wild type phage.
The in-vitro directed molecular evolution screening of embodiment 3, Ig high-affinity Ig binding molecule
1. the affine screening of the Ig in recombinant phage storehouse:
By people Ig (final concentration is 10 μ g/ml) on enzyme mark row batten 37 ℃, 2 hours, arranged batten 1 hour, 4 ℃ of preservations with the carbonate buffer solution 200 μ l of PH 9.6 bag with the sealing of 150 μ l confining liquids (PBS+10% skimmed milk).Wash the avidity screening that is used for phage library after 5 times with washing lotion (PBS+0.05%Tween20).The every hole of row's batten adds 100 μ l confining liquids and 100 μ lPALGn recombinant phages, 37 ℃ were reacted 3 hours, washing lotion (0.25%Tris+0.05% Tween20) washing 30 times, the e. coli tg1 that adds 100 μ l logarithmic phases, 37 ℃ were reacted 1 hour, get 10 μ l, 1 μ l, 0.1 μ l after the collection and be coated with the dull and stereotyped 37 ℃ of incubated overnight counting of LB (containing acillin 100ng/ml), in order to the combine situation of assessment phage library with the Ig molecule.All the other bacterium liquid add among 5ml 2 * YT, and 37 ℃, 250rpm shaking culture 1 hour adds acillin to 100ng/ml, adds 1ml4 * 10
10The M13K07 helper phage of TU/ml, 37 ℃, 250rpm shaking culture 1 hour adds kantlex to 50ng/ml, and 37 ℃, the 250rpm shaking culture is spent the night.Centrifugal 10 minutes of culture 5000rpm, supernatant be through 0.22 μ m membrane filtration, is the PALGn recombinant phage library after the affine screening of Ig.If positive control Protein A/phage two row culture IgG screen (preparation of Protein A/phage: get Protein A/pCANTAB5L plasmid 600ng and transform TG1 competent cell, the same PALGn/phage of all the other processes) synchronously.Press said process, screening that Ig is affine repeats 4 and takes turns, and carries out every titer determination of taking turns the screening storehouse simultaneously.Whenever, take turns screening and all establish negative control LT/phage two holes (the LT/phage preparation process is with Protein A/phage), blank two holes (not adding phage), make IgG in conjunction with experiment, respectively get 10 μ l, 1 μ l, 0.1 μ l and be coated with the dull and stereotyped 37 ℃ of incubated overnight counting of LB (containing Amp 100ng/ μ l).Every Ig that takes turns is in conjunction with experiment enumeration (blank is 0) and titer determination result such as table 1.Binding ability by table visible PALGn/phage library and Ig improves constantly along with the increase of screening round, has reacted screening process the binding ability of ground, library Ig is constantly evolved.
The every Ig in Ig screening process pnagus medius library that takes turns of table 1 is in conjunction with enumeration
Annotate: F represents colony number＞300.
2. inserting segmental PCR identifies: the original storehouse of phage and each take turns the phage selection storehouse at random picking PALGn/phage Ig to the 0.5ml centrifuge tube, cultivate 5h in conjunction with 48 of mono-clonal bacterium colonies on the test panel, to cultivate bacterium liquid is template, pCANTAB5-S1 is a upstream primer, pCANTAB5-NOT1 is a downstream primer, carries out pcr amplification and inserts segmental size to detect.The pcr amplification condition is 94 ℃ of 30sec; 53 ℃ of 30sec; 72 ℃ of 45sec; 35 circulations.If positive control LT/pCANTAB5L, negative control pCANTAB5S.This experiment is in order to understand the changing conditions of the Ig binding molecule molecular weight size of being showed in the screening process pnagus medius library.Table 2 shows that with the result of Fig. 7-11 along with the continuous increase of screening round, the ratio of the HAIBM molecule of macromolecule constantly increases in the library, has reacted screening process the molecule in the library is constantly evolved to helping Ig bonded direction.
Table 2 respectively screens in the round library distribution situation that the single structure territory in 48 random choose HAIBM molecules is formed
| The single structure territory is formed | F 0 | F 1 | F 2 | F 3 | F 4 |
| 0domain 1domain 2domain 3domain＞3domain amounts to | 8 5 35 0 0 48 | 8 5 35 0 0 48 | 8 2 37 1 0 48 | 5 1 26 7 9 48 | 4 0 23 12 9 48 |
Above result shows that every IgG that takes turns the screening storehouse can react the effect of lactam enzyme by directional anagenesis in vitro rapidly intuitively in conjunction with experiment enumeration, insertion fragment PCR positive colony number.From IgG in conjunction with the condition experiment enumeration, along with the screening round increase, PALGn/phage IgG in conjunction with activity near (F
3For) and be higher than (F
4Generation) positive control IgG is in conjunction with activity; Insertion fragment PCR qualification result demonstration positive colony number increases along with the increase of screening round and tends towards stability, positive colony is formed also considerable change: along with the screening round increases, big fragment (〉=2 domain) number and positive percentage thereof increase, and small segment number and positive percentage thereof reduce.Illustrate that the short-movie section low with IgG avidity is eliminated through IgG screening, and with high the coming out of IgG avidity than long segment is screened.IgG is consistent with insertion fragment PCR qualification result in conjunction with experimental result, all conforms to theory, has embodied the powerful effect of utilizing avidity to select in the display technique of bacteriophage.
The molecular structure characteristics of embodiment 4, high-affinity Ig binding molecule (HAIBMs)
Take turns the resulting phage library of screening from the 4th, select the bigger positive colony of insertion fragment and carry out sequential analysis, the reorganization phagemid of positive colony DNA extraction agent box purifying, entrust the Shen, Shanghai betting office to carry out sequencing, sequencing primer is upstream and downstream primer pCANTAB5-S1 and the pCANTAB5-S6 of pCANTAB5E, sequencing result DNASTAR software analysis.The results are shown in Table 3-6.Sequential analysis shows the diversity that its structural domain is formed: (1) selected molecule that goes out comprises by being connected to form from single antibodies structural domain-A/B/C/D/E of protein A, the B1/B2/B3/B4/B5 of protein L of different genera Ig binding molecule and the G1/G2 reorganization of protein G; (2) in the library unijunction in the individual molecule to close the arrangement mode of structural domain various, comprising: B1-B2-G1-A, B3-D-B3, B1-B3-B1, G1-B2-A-B1, B1-G2-B1, D-B2-B-B3, B3-D-B3-D-B3, B-G1-B2-D-B1 or the like.Relatively each HAIBMs molecular structure finds that they have certain arrangement feature: (ML-MA) n or (ML-MG) n or (ML-MA-MG) n or (ML-MG-MA) n or ML-(MA-ML) n or ML-(MG-ML) n structure are spaced be formed by connecting (n is the multiplicity of this sequence) by the single structure territory (ML) of protein L, the single structure territory (MA) of protein A or the single structure territory (MG) of protein G.The arrangement mode of representative sequence wherein is: B3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, B2-C-G2-B1-D-G1.Between the single structure territory of representing sequence protein L, protein A, protein G, connect with Nucleotide GAGCTC.Nucleotide GAGCTC codified joint peptide EL (the amino acid single-letter is represented) represents the nucleotide sequence of sequence as follows:
1, represent sequence B 3-G1-B2-G2-B5 (SEQ ID NO:13):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAAATGGTA
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTACAAATTAATCCTT
AATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAATACGC
TAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAAGAGCTCAAAGAAA
AAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAACAGCAGAA
TTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAGAATATAC
AGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTACAAACTTGTTATTAATGGTA
AAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCAGAAAAAGCCTTCAAACAATACGCTAACGAC
AACGCTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTACGGTAACTGAAGAGCTCAAGAAAGTTGACGA
AAAACCAGAAGAAAAAGAACAAGTAACAATTAAAGAAAATATATATTTTGAAGATGGAACAGTACAAACTGCAACATTTA
AAGGAACATTTGCAGAAGCGACAGCAGAAGCATACAGATATGCAGATTTGTTATCAAAAGAACATGGTAAATACACAGCA
GACTTGGAAGATGGTGGATACACTATCAACATTAGATTTGCTGGA
2, represent sequence B 2-D-G1-B5-C-G1 (SEQ ID NO:15):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAG
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATGCGCAACAAAAT
AACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTT
CATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCAC
CGAAAGAGCTCACTTACAAATTAATCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCT
ACTGCAGAAAAAGTCTTCAAACAATACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGAC
CTTTACAGTTACTGAAGAGCTCAAGAAAGTTGACGAAAAACCAGAAGAAAAAGAACAAGTAACAATTAAAGAAAATATAT
ATTTTGAAGATGGAACAGTACAAACTGCAACATTTAAAGGAACATTTGCAGAAGCGACAGCAGAAGCATACAGATATGCA
GATTTGTTATCAAAAGAACATGGTAAATACACAGCAGACTTGGAAGATGGTGGATACACTATCAACATTAGATTTGCTGG
AGAGCTCGCTGACAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAG
AACAACGTAACGGCTTCATCCAAAGCCTTAAAGACGATCCTTCAGTGAGCAAAGAAATTTTAGCAGAAGCTAAAAAGCTA
AACGATGCTCAAGCACCAAAAGAGCTCACTTACAAATTAATCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGA
AGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAATACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACG
ACGATGCGACTAAGACCTTTACAGTTACTGAA
3, represent sequence B 5-A-B2-G1-B1 (SEQ ID NO:17):
AAGAAAGTTGACGAAAAACCAGAAGAAAAAGAACAAGTAACAATTAAAGAAAATATATATTTTGAAGATGGAACAGTACA
AACTGCAACATTTAAAGGAACATTTGCAGAAGCGACAGCAGAAGCATACAGATATGCAGATTTGTTATCAAAAGAACATG
GTAAATACACAGCAGACTTGGAAGATGGTGGATACACTATCAACATTAGATTTGCTGGAGAGCTCGCTGACAACAATTTC
AACAAAGAACAACAAAATGCTTTCTATGAAATCTTGAACATGCCTAACTTGAACGAAGAACAACGCAATGGTTTCATCCA
AAGCTTAAAAGATGACCCAAGTCAAAGTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCTCAAGCACCGAAAG
AGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACA
CAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAA
TGGAGAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTACAAATTAA
TCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAA
TACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAAGAGCTCAA
AGAAGAAACACCAGAAACACCAGAAACTGATTCAGAAGAAGAAGTAACAATCAAAGCTAACCTAATCTTTGCAAATGGAA
GCACACAAACTGCAGAATTCAAAGGAACATTTGAAAAAGCAACATCAGAAGCTTATGCGTATGCAGATACTTTGAAGAAA
GACAATGGAGAATATACTGTAGATGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGA
4, represent sequence B 3-D-B3 (SEQ ID NO:19):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAAATGGTA
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATGCGCAACAAAAT
AACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTT
CATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCAC
CGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGA
AAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAA
AGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGA
5, represent sequence B 3-D-B3-D-B3 (SEQ ID NO:21):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAAATGGTA
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATGCGCAACAAAAT
AACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTT
CATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCAC
CGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGA
AAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAA
AGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATG
CGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAA
CGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGA
ATCTCAAGCACCGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCT
ATGCAGATGGAAAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGAC
TTATTAGCAAAAGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGA
6, represent sequence B 2-G2-A-B3-D-G1 (SEQ ID NO:23):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAG
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTACAAACTTGTTATT
AATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCAGAAAAAGCCTTCAAACAATACGC
TAACGACAACGCTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTACGGTAACTGAAGAGCTCGCTGACA
ACAATTTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTGAACATGCCTAACTTGAACGAAGAACAACGCAATGGT
TTCATCCAAAGCTTAAAAGATGACCCAAGTCAAAGTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCTCAAGC
ACCGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATG
GAAAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCA
AAAGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGA
TGCGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGC
AACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAAC
GAATCTCAAGCACCGAAAGAGCTCACTTACAAATTAATCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGC
TGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAATACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACG
ATGCGACTAAGACCTTTACAGTTACTGAA
7, represent sequence B 1-G1-B3-G1 (SEQ ID NO:25):
AAAGAAGAAACACCAGAAACACCAGAAACTGATTCAGAAGAAGAAGTAACAATCAAAGCTAACCTAATCTTTGCAAATGG
AAGCACACAAACTGCAGAATTCAAAGGAACATTTGAAAAAGCAACATCAGAAGCTTATGCGTATGCAGATACTTTGAAGA
AAGACAATGGAGAATATACTGTAGATGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTAC
AAATTAATCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTT
CAAACAATACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAAG
AGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACA
CAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAAA
TGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCACTTACAAATTAA
TCCTTAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAA
TACGCTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAA
8, represent sequence B 2-D-B3-D-B1 (SEQ ID NO:27):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAG
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATGCGCAACAAAAT
AACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTT
CATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCAC
CGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGA
AAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAA
AGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATG
CGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAA
CGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGA
ATCTCAAGCACCGAAAGAGCTCAAAGAAGAAACACCAGAAACACCAGAAACTGATTCAGAAGAAGAAGTAACAATCAAAG
CTAACCTAATCTTTGCAAATGGAAGCACACAAACTGCAGAATTCAAAGGAACATTTGAAAAAGCAACATCAGAAGCTTAT
GCGTATGCAGATACTTTGAAGAAAGACAATGGAGAATATACTGTAGATGTTGCAGATAAAGGTTATACTTTAAATATTAA
ATTTGCTGGA
9, represent sequence B 2-C-B4-E-G1 (SEQ ID NO:29):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAG
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGACAACAAATTCAAC
AAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAGAACAACGTAACGGCTTCATCCAAAG
CCTTAAAGACGATCCTTCAGTGAGCAAAGAAATTTTAGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAAGAGC
TCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACTCAA
ACAGCAGAGTTCAAAGGAACATTTGCAGAAGCAACAGCAGAAGCATACAGATACGCTGACTTATTAGCAAAAGAAAATGG
TAAATATACAGCAGACTTAGAAGATGGTGGATACACTATTAATATTAGATTTGCAGGTGAGCTCGCTCAACAAAATGCTT
TTTATCAAGTCTTAAATATGCCTAACTTAAATGCTGATCAACGCAATGGTTTTATCCAAAGCCTTAAAGATGATCCAAGC
CAAAGTGCTAACGTTTTAGGTGAAGCTAAAAGATTAAACGAATTTCAAGCACCGAAAGAGCTCACTTACAAATTAATCCT
TAATGGTAAAACATTGAAAGGCGAAACAACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAATACG
CTAACGACAACGGTGTTGACGGTGAATGGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAA
10, represent sequence B 2-C-G2-B1-D-G1 (SEQ ID NO:31):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAAC
AGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCAGATGCATTAAAGAAGGACAATGGAG
AATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGACAACAAATTCAAC
AAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAGAACAACGTAACGGCTTCATCCAAAG
CCTTAAAGACGATCCTTCAGTGAGCAAAGAAATTTTAGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAAGAGC
TCACTTACAAACTTGTTATTAATGGTAAAACATTGAAAGGCGAAACAACTACTAAAGCAGTAGACGCAGAAACTGCAGAA
AAAGCCTTCAAACAATACGCTAACGACAACGCTGTTGATGGTGTTTGGACTTATGATGATGCGACTAAGACCTTTACGGT
AACTGAAGAGCTCAAAGAAGAAACACCAGAAACACCAGAAACTGATTCAGAAGAAGAAGTAACAATCAAAGCTAACCTAA
TCTTTGCAAATGGAAGCACACAAACTGCAGAATTCAAAGGAACATTTGAAAAAGCAACATCAGAAGCTTATGCGTATGCA
GATACTTTGAAGAAAGACAATGGAGAATATACTGTAGATGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGG
AGAGCTCGCTGATGCGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACT
TAAACGAAGCGCAACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCT
AAAAAATTAAACGAATCTCAAGCACCGAAAGAGCTCACTTACAAATTAATCCTTAATGGTAAAACATTGAAAGGCGAAAC
AACTACTGAAGCTGTTGATGCTGCTACTGCAGAAAAAGTCTTCAAACAATACGCTAACGACAACGGTGTTGACGGTGAAT
GGACTTACGACGATGCGACTAAGACCTTTACAGTTACTGAA
Represent the aminoacid sequence (the amino acid single-letter is represented) of sequence as follows:
1, represent sequence B 3-G1-B2-G2-B5 (SEQ ID NO:14):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELTYKLIL
NGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEELKEKTPEEPKEEVTIKANLIYADGKTQTAE
FKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELTYKLVINGKTLKGETTTKAVDAETAEKAFKQYAND
NAVDGVWTYDDATKTFTVTEELKKVDEKPEEKEQVTIKENIYFEDGTVQTATFKCTFAEATAEAYRYADLLSKEHGKYTA
DLEDGGYTINIRFAG
2, represent sequence B 2-D-G1-B5-C-G1 (SEQ ID NO:16):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELADAQQN
NFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELTYKLILNGKTLKGETTTEAVDAA
TAEKVFKQYANDNGVDGEWTYDDATKTFTVTEELKKVDEKPEEKEQVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYA
DLLSKEHGKYTADLEDGGYTINIRFAGELADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKL
NDAQAPKELTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
3, represent sequence B 5-A-B2-G1-B1 (SEQ ID NO:18):
KKVDEKPEEKEQVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTINIRFAGELADNNF
NKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADGKT
QTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELTYKLILNGKTLKGETTTEAVDAATAEKVFKQ
YANDNGVDGEWTYDDATKTFTVTEELKEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKK
DNGEYTVDVADKGYTLNIKFAG
4, represent sequence B 3-D-B3 (SEQ ID NO:20):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADAQQN
NFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADG
KTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAG
5, represent sequence B 3-D-B3-D-B3 (SEQ ID NO:22):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADAQQN
NFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADG
KTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADAQQNNFNKDQQSAFYEILNMPNLNEAQ
RNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYAD
LLAKENGKYTVDVADKGYTLNIKFAG
6, represent sequence B 2-G2-A-B3-D-G1 (SEQ ID NO:24):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELTYKLVI
NGKTLKGETTTKAVDAETAEKAFKQYANDNAVDGVWTYDDATKTFTVTEELADNNFNKEQQNAFYEILNMPNLNEEQRNG
FIQSLKDDPSQSANLLSEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLA
KENGKYTVDVADKGYTLNIKFAGELADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLN
ESQAPKELTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
7, represent sequence B 1-G1-B3-G1 (SEQ ID NO:26):
KEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAGELTY
KLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEELKEKTPEEPKEEVTIKANLIYADGKT
QTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELTYKLILNGKTLKGETTTEAVDAATAEKVFKQ
YANDNGVDGEWTYDDATKTFTVTE
8, represent sequence B 2-D-B3-D-B1 (SEQ ID NO:28):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELADAQQN
NFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADG
KTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADAQQNNFNKDQQSAFYEILNMPNLNEAQ
RNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAY
AYADTLKKDNGEYTVDVADKGYTLNIKFAG
9, represent sequence B 2-C-B4-E-G1 (SEQ ID NO:30):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELADNKFN
KEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKELKEKTPEEPKEEVTIKANLIYADGKTQ
TAEFKGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFAGELAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPS
QSANVLGEAKRLNEFQAPKELTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
10, represent sequence B 2-C-G2-B1-D-G1 (SEQ ID NO:32):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGELADNKFN
KEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKELTYKLVINGKTLKGETTTKAVDAETAE
KAFKQYANDNAVDGVWTYDDATKTFTVTEELKEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYA
DTLKKDNGEYTVDVADKGYTLNIKFAGELADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEA
KKLNESQAPKELTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
In order to measure the HAIBM that recombinant phage shows and the binding ability of antibody, the positive monoclonal phagemid of recombinating is transformed TG1, make mono-clonal HAIBM recombinant phage (the phage preparation method is the same), each mono-clonal recombinant phage is adjusted to identical titre (1.0 * 10 with the M13K07 helper phage with 2 * YT substratum
12TU/ml), get respectively in the reacting hole that each mono-clonal phage 100 μ l adds people Ig antibody wrapper sheet, hatch 3 hours after scouring 50 times for 37 ℃.Reacted sample is divided into two groups, and every hole adds 100 μ l e. coli tg1 (OD in first group of sample hole after recombinant phage and M13K07 and antibodies
600=0.2), 37 ℃ hatch 1 hour after, getting 1 μ l mono-clonal recombinant phage infectious bacteria liquid coats on the amicillin resistance plate and counts, the M13K07 helper phage infectious bacteria liquid of contrast is coated on the amicillin resistance plate and is counted, be used to detect the quantity by the recombinant phage of the special absorption of people Ig, detected result sees Table 3.Every hole adds phage antibody HRP/Anti-M13conjugate (Amersham pharmaciabiotech company in second group of sample hole after recombinant phage and M13K07 and antibodies, working concentration is dilution in 1: 5000), hatch for 37 ℃ and add the TMB colour developing after 45 minutes, survey OD
450The nm value is used to detect the quantity by the recombinant phage of the special absorption of human IgG, and detected result sees Table 3.
The human IgG antibody of each HAIBM mono-clonal phage of table 3 is in conjunction with test
| Molecular composition in the mono-clonal phage | E. coli tg1 infects colony number | HRP phage- | |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 | SPA LB3-G1-LB2-G2-LB5 LB1-A-LB3 LB1-A-LB4-B-G1 LB2-D-G1-LB5-C-G1 LB3-B-LB3-A LB5-A-LB2-G1-LB1 LB3-D-LB3 LB3-D-LB3-D-LB3 LB2-G2-A-LB3-D-G1 LB1-G1-LB3-G1 LB4-G2-LB2 LB2-D-LB3-D-LB1 LB2-C-LB4-E-G1 LB2-C-G2-LB1-D-G1 LB1-C-LB1 M13K07 | 415 ＞500 328 453 ＞500 272 ＞500 ＞500 ＞500 ＞500 ＞500 398 ＞500 ＞500 ＞500 413 56 | 1.538 2.251 1.247 1.626 2.119 1.372 1.931 2.043 2.251 2.017 1.943 1.485 2.196 2.005 2.113 1.434 0.233 |
Annotate: each phage titre is 1.0 * 10
12TU/ml
Infect the colony number measurement result as seen from the e. coli tg1 of table 3, show that the phagocytosis physical efficiency that mono-clonal HAIBM molecule is arranged is fixing by the human IgG combination, and drop into phage titre 1.0 * 10
12The time exist the different IgG of height in conjunction with activity.And contrast wild type phage M13K07 titre is 1.0 * 10
12The time detect less than with human IgG bonded phage.Detected result and above-mentioned e. coli tg1 that colour developing is surveyed in the enzyme joint inspection infect colony number measurement result basically identical.The HAIBM molecule that this presentation of results merges at piii protein N end has Ig in conjunction with activity, show and have sequence B 3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, B2-C-G2-B1-D-G1 positive bacteriophage combine activity and are higher than the phage of showing staphylococcal protein A,SPA (SPA) with people Ig.
The function of embodiment 6 high-affinity Ig binding molecules (HAIBMs)
The design primer, upstream primer is introduced Nco I site, downstream primer is introduced Sal I site, pcr amplification positive colony sequence, after enzyme cuts back to close HAIBMs cloned among the Nco I and Sal I site in the pET32a carrier, IPTG abduction delivering HAIBMs albumen, the Ni-NTA column purification obtains purifying protein HAIBMs, ELISA detects the activity of purifying protein HAIBMs, wrapped by on row's batten by various purifying HAIBM albumen (1: 100) with the carbonate buffer solution bag, if positive control SPA (1: 100), 4 ℃ are spent the night behind 37 ℃ of 2h of negative control LT (1: 100), the human normal immunoglobulin molecule (HRP one people Ig) of horseradish peroxidase (HRP) mark that to add different dilution concentration be 1mg/ml, hatched 45 minutes for 37 ℃, PBS washing lotion washing 4 times, the OPD colour developing, enzyme connection detector reads the OD of 492nm place value.Result by table 4 shows that various purifying HAIBM albumen and people Ig have the special activity that combines, wherein encoding sequence has B3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, the molecule of the fusion gene coding of B2-C-G2-B1-D-G1 mode of connection is higher than SPA in conjunction with activity.
Table 4 HAIBM purifying protein combines activity with people Ig and detects
| Purifying HAIBM | HRP-people Ig extent of dilution (1MG/ML) | |||||||
| 1∶100 | 1∶200 | 1∶400 | 1∶800 | 1∶160 0 | 1∶320 0 | 1∶640 0 | 1∶128 00 | |
| Negative control LT | 0.289 | 0.146 | 0.076 | 0.056 | 0.027 | 0.029 | 0.010 | 0.001 |
| Positive control SPA | 2.073 | 2.055 | 1.429 | 0.820 | 0.430 | 0.224 | 0.172 | 0.090 |
| LB3-G1-LB2-G2-LB 5 | 2.255 | 2.117 | 1.956 | 1.862 | 1.342 | 0.955 | 0.515 | 0.323 |
| LB1-A-LB3 | 1.873 | 1.704 | 1.331 | 0.680 | 0.318 | 0.180 | 0.092 | 0.072 |
| LB1-A-LB4-B-G1 | 1.953 | 1.937 | 1.618 | 1.425 | 0.820 | 0.525 | 0.380 | 0.257 |
| LB2-D-G1-LB5-C-G 1 | 2.110 | 1.998 | 1.730 | 1.632 | 0.926 | 0.530 | 0.316 | 0.240 |
| LB3-B-LB3-A | 1.924 | 1.757 | 1.424 | 0.756 | 0.320 | 0.224 | 0.136 | 0.009 |
| LB5-A-LB2-G1-LB1 | 2.123 | 1.996 | 1.755 | 1.628 | 0.910 | 0.572 | 0.328 | 0.225 |
| LB3-D-LB3 | 2.080 | 2.069 | 1.613 | 1.580 | 0.877 | 0.448 | 0.346 | 0.112 |
| LB3-D-LB3-D-LB3 | 2.366 | 2.359 | 2.018 | 1.912 | 1.765 | 1.230 | 0.856 | 0.512 |
| LB2-G2-A-LB3-D-G 1 | 2.271 | 2.150 | 1.978 | 1.794 | 1.216 | 0.874 | 0.530 | 0.251 |
| LB1-G1-LB3-G1 | 2.045 | 1.923 | 1.767 | 1.673 | 1.065 | 0.654 | 0.320 | 0.203 |
| LB4-G2-LB2 | 2.083 | 2.020 | 1.413 | 0.806 | 0.410 | 0.212 | 0.214 3 | 0.088 |
| LB2-D-LB3-D-LB1 | 2.296 | 2.175 | 2.010 | 1.991 | 1.659 | 1.180 | 0.784 | 0.464 |
| LB2-C-LB4-E-G1 | 2.225 | 2.151 | 1.910 | 1.712 | 1.218 | 0.717 | 0.528 | 0.336 |
| LB2-C-G2-LB1-D-G 1 | 2.013 | 2.007 | 1.873 | 1.716 | 1.115 | 0.719 | 0.551 | 0.313 |
| LB1-C-LB1 | 1.930 | 2.073 | 1.660 | 0.911 | 0.525 | 0.340 | 0.275 | 0.118 |
The application of embodiment 7 high-affinity Ig binding molecules (HAIBMs)
Antigen coated the marking in enzyme of HCV arranged on the batten, after the washing, add 10 μ l, the third hepatopathy people's serum and 90 μ l PBS, 37 ℃ of 1h effects backs are washed 5 times with washing lotion (PBS+0.05%Tween20), remove unconjugated antibody, and adding 100 μ l titres is 1.0 * 10
1037 ℃ of 1h of various HAIBM mono-clonal recombinant phages of TU/ml establish negative control LT/Phage, positive control PA/phage.Use washing lotion (PBS+0.05%Tween20) to wash again 5 times, remove recombinant phage.Experiment is divided into two groups and detects institute's bonded recombinant phage on the enzymes mark row battens, and first group of every hole adds the e. coli tg1 of 100 μ l, infect 1h after, get mono-clonal recombinant phage infectious bacteria liquid 100 μ l and coat on the amicillin resistance plate and count; Second group of every hole adds 100 μ l phage antibody HRP/Anti-M13 conjugate, TMB colour developing behind 37 ℃ of reaction 1h, and the OD of 450nm place value detects.The antigen coated concentration of HCV is (2 μ g/ml), the results are shown in Table 5, table 6.
Table 5 is used HAIBM mono-clonal phage and is detected whose anti-HCV test (infecting bacterium colony counting number method)
| Mono-clonal HAIBM | E. coli tg1 infects colony number | ||||||||
| HCV positive serum sample | HCV negative serum sample | ||||||||
| 1# | 2# | 3# | 4# | 1# | 2# | 3 # | 4 # | ||
| 1 | Negative control LT | 5 | 2 | 0 | 4 | 3 | 3 | 2 | 0 |
| 2 | Positive control SPA | 79 | 86 | 53 | 92 | 5 | 3 | 3 | 0 |
| 3 | LB3-G1-LB2-G2-LB5 | 126 | 113 | 111 | 127 | 2 | 0 | 5 | 1 |
| 4 | LB1-A-LB3 | 39 | 78 | 50 | 64 | 11 | 8 | 5 | 0 |
| 5 | LB1-A-LB4-B-G1 | 93 | 49 | 39 | 65 | 3 | 2 | 0 | 0 |
| 6 | LB2-D-G1-LB5-C-G1 | 102 | 117 | 97 | 72 | 4 | 5 | 1 | 0 |
| 7 | LB3-B-LB3-A | 74 | 98 | 50 | 65 | 0 | 13 | 0 | 18 |
| 8 | LB5-A-LB2-G1-LB1 | 93 | 76 | 134 | 128 | 9 | 6 | 14 | 3 |
| 9 | LB3-D-LB3 | 97 | 94 | 114 | 98 | 3 | 2 | 0 | 1 |
| 10 | LB3-D-LB3-D-LB3 | 171 | 144 | 128 | 167 | 1 | 2 | 3 | 0 |
| 11 | LB2-G2-A-LB3-D-G1 | 123 | 128 | 160 | 141 | 1 | 0 | 3 | 0 |
| 12 | LB1-G1-LB3-G1 | 111 | 131 | 125 | 96 | 12 | 4 | 10 | 9 |
| 13 | LB4-G2-LB2 | 64 | 32 | 56 | 73 | 3 | 2 | 0 | 0 |
| 14 | LB2-D-LB3-D-LB1 | 136 | 167 | 118 | 121 | 2 | 1 | 3 | 1 |
| 15 | LB2-C-LB4-E-G1 | 118 | 142 | 131 | 116 | 17 | 8 | 0 | 7 |
| 16 | LB2-C-G2-LB1-D-G1 | 117 | 124 | 119 | 158 | 3 | 0 | 5 | 1 |
| 17 | LB1-C-LB1 | 76 | 110 | 63 | 94 | 2 | 0 | 6 | 0 |
Annotate: each phage titre is 1.0 * 10
10U/ml
Table 6 is used HAIBM mono-clonal phage and is detected whose anti-HCV test (development process is surveyed in the enzyme joint inspection)
| Mono-clonal HAIBM | HCV positive serum sample (OD 450NM) | HCV negative serum sample (CD 450NM) | |||||||
| 1# | 2# | 3# | 4# | 1# | 2# | 3# | 4# | ||
| 1 | Negative control LT | 0.069 | 0.046 | 0.076 | 0.056 | 0.037 | 0.044 | 0.010 | 0.053 |
| 2 | Positive control SPA | 1.133 | 1.218 | 0.891 | 1.096 | 0.022 | 0.036 | 0.045 | 0.039 |
| 3 | LB3-G1-LB2-G2-LB5 | 1.855 | 1.317 | 1.356 | 1.662 | 0.042 | 0.036 | 0.055 | 0.051 |
| 4 | LB1-A-LB3 | 0.873 | 1.104 | 0.710 | 1.280 | 0.038 | 0.032 | 0.022 | 0.035 |
| 5 | LB1-A-LB4-B-G1 | 1.223 | 0.837 | 1.018 | 1.125 | 0.023 | 0.025 | 0.040 | 0.011 |
| 6 | LB2-D-G1-LB5-C-G1 | 1.710 | 1.698 | 1.430 | 1.230 | 0.031 | 0.043 | 0.036 | 0.031 |
| 7 | LB3-B-LB3-A | 0.924 | 0.457 | 0.524 | 0.756 | 0.024 | 0.022 | 0.036 | 0.019 |
| 8 | LB5-A-LB2-G1-LB1 | 1.523 | 1.185 | 1.792 | 1.328 | 0.033 | 0.072 | 0.058 | 0.046 |
| 9 | LB3-D-LB3 | 1.673 | 1.359 | 1.313 | 1.583 | 0.073 | 0.064 | 0.051 | 0.074 |
| 10 | LB3-D-LB3-D-LB3 | 1.921 | 1.759 | 1.718 | 1.812 | 0.065 | 0.047 | 0.073 | 0.047 |
| 11 | LB2-G2-A-LB3-D-G1 | 1.671 | 1.550 | 1.478 | 1.794 | 0.056 | 0.030 | 0.037 | 0.040 |
| 12 | LB1-G1-LB3-G1 | 1.545 | 1.633 | 1.567 | 1.373 | 0.045 | 0.024 | 0.022 | 0.054 |
| 13 | LB4-G2-LB2 | 1.083 | 0.420 | 0.913 | 1.006 | 0.015 | 0.022 | 0.043 | 0.038 |
| 14 | LB2-D-LB3-D-LB1 | 1.796 | 1.615 | 1.578 | 1.761 | 0.059 | 0.014 | 0.065 | 0.074 |
| 15 | LB2-C-LB4-E-G1 | 1.625 | 1.851 | 1.510 | 1.712 | 0.118 | 0.071 | 0.062 | 0.056 |
| 16 | LB2-C-G2-LB1-D-G1 | 1.813 | 1.601 | 1.573 | 1.816 | 0.115 | 0.079 | 0.062 | 0.073 |
| 17 | LB1-C-LB1 | 1.030 | 1.073 | 0.960 | 1.011 | 0.025 | 0.040 | 0.075 | 0.068 |
Annotate: each phage titre is 1.0 * 10
12U/ml
By table 5 and table 6 as seen, show phagocytosis physical efficiency and the reaction of people's whose anti-HCV positive antibody that mono-clonal HAIBM is arranged, and at the input phage titre 1.0 * 10
12The time have a different antibody binding activity of height.And contrast LT titre is 1.0 * 10
12Shi Jiben does not have antibody binding activity.Detected result and above-mentioned e. coli tg1 that colour developing is surveyed in the enzyme joint inspection infect colony number measurement result basically identical.This result has confirmed that the HAIBMs molecule has good people Ig combined function, and has the purposes in immunology diagnosiss such as enzyme linked immunosorbent assay of can be applicable to (ELISA method), immunochromatography detection, immunohistochemical methods and detection of antibodies and the purifying.
Sequence table
＜110〉Shanghai Runlong Biology Science and Technology Co., Ltd
＜120〉a kind of high affinity immune globulin binding molecule and preparation method thereof
<130>040183
<160>32
<170>PatentIn version 3.1
<210>1
<211>228
<212>DNA
<213>Peptostreptococcus magnus
<400>1
aaagaagaaa caccagaaac accagaaact gattcagaag aagaagtaac aatcaaagct 60
aacctaatct ttgcaaatgg aagcacacaa actgcagaat tcaaaggaac atttgaaaaa 120
gcaacatcag aagcttatgc gtatgcagat actttgaaga aagacaatgg agaatatact 180
gtagatgttg cagataaagg ttatacttta aatattaaat ttgctgga 228
<210>2
<211>216
<212>DNA
<213>Peptostreptococcus magnus
<400>2
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctgga 216
<210>3
<211>216
<212>DNA
<213>Peptostreptococcus magnus
<400>3
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgctgactt attagcaaaa gaaaatggta aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctgga 216
<210>4
<211>216
<212>DNA
<213>Peptostreptococcus magnus
<400>4
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaactcaaac agcagagttc aaaggaacat ttgcagaagc aacagcagaa 120
gcatacagat acgctgactt attagcaaaa gaaaatggta aatatacagc agacttagaa 180
gatggtggat acactattaa tattagattt gcaggt 216
<210>5
<211>219
<212>DNA
<213>Peptostreptococcus magnus
<400>5
aagaaagttg acgaaaaacc agaagaaaaa gaacaagtaa caattaaaga aaatatatat 60
tttgaagatg gaacagtaca aactgcaaca tttaaaggaa catttgcaga agcgacagca 120
gaagcataca gatatgcaga tttgttatca aaagaacatg gtaaatacac agcagacttg 180
gaagatggtg gatacactat caacattaga tttgctgga 219
<210>6
<211>174
<212>DNA
<213>Staphylococcus aureus
<400>6
gctgacaaca atttcaacaa agaacaacaa aatgctttct atgaaatctt gaacatgcct 60
aacttgaacg aagaacaacg caatggtttc atccaaagct taaaagatga cccaagtcaa 120
agtgctaacc tattgtcaga agctaaaaag ttaaatgaat ctcaagcacc gaaa 174
<210>7
<211>174
<212>DNA
<213>Staphylococcus aureus
<400>7
gcggataaca aattcaacaa agaacaacaa aatgctttct atgaaatctt acatttacct 60
aacttaaacg aagaacaacg caatggtttc atccaaagct taaaagatga cccaagccaa 120
agcgctaacc ttttagcaga agctaaaaag ctaaatgatg cacaagcacc aaaa 174
<210>8
<211>174
<212>DNA
<213>Staphylococcus aureus
<400>8
gctgacaaca aattcaacaa agaacaacaa aatgctttct atgaaatttt acatttacct 60
aacttaactg aagaacaacg taacggcttc atccaaagcc ttaaagacga tccttcagtg 120
agcaaagaaa ttttagcaga agctaaaaag ctaaacgatg ctcaagcacc aaaa 174
<210>9
<211>183
<212>DNA
<213>Staphylococcus aureus
<400>9
gctgatgcgc aacaaaataa cttcaacaaa gatcaacaaa gcgccttcta tgaaattttg 60
aacatgccta acttaaacga agcgcaacgc aatggtttca ttcaaagtct taaagacgat 120
ccaagccaaa gcactaacgt tttaggtgaa gctaaaaaat taaacgaatc tcaagcaccg 180
aaa 183
<210>10
<211>153
<212>DNA
<213>Staphylococcus aureus
<400>10
gctcaacaaa atgcttttta tcaagtctta aatatgccta acttaaatgc tgatcaacgc 60
aatggtttta tccaaagcct taaagatgat ccaagccaaa gtgctaacgt tttaggtgaa 120
gctaaaagat taaacgaatt tcaagcaccg aaa 153
<210>11
<211>165
<212>DNA
<213>Streptococcus sp.
<400>11
acttacaaat taatccttaa tggtaaaaca ttgaaaggcg aaacaactac tgaagctgtt 60
gatgctgcta ctgcagaaaa agtcttcaaa caatacgcta acgacaacgg tgttgacggt 120
gaatggactt acgacgatgc gactaagacc tttacagtta ctgaa 165
<210>12
<211>165
<212>DNA
<213>Streptococcus sp.
<400>12
acttacaaac ttgttattaa tggtaaaaca ttgaaaggcg aaacaactac taaagcagta 60
gacgcagaaa ctgcagaaaa agccttcaaa caatacgcta acgacaacgc tgttgatggt 120
gtttggactt atgatgatgc gactaagacc tttacggtaa ctgaa 165
<210>13
<211>1005
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>13
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgctgactt attagcaaaa gaaaatggta aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcacttacaa attaatcctt 240
aatggtaaaa cattgaaagg cgaaacaact actgaagctg ttgatgctgc tactgcagaa 300
aaagtcttca aacaatacgc taacgacaac ggtgttgacg gtgaatggac ttacgacgat 360
gcgactaaga cctttacagt tactgaagag ctcaaagaaa aaacaccaga agaaccaaaa 420
gaagaagtta ctattaaagc aaacttaatc tatgcagatg gaaaaacaca aacagcagaa 480
ttcaaaggaa catttgaaga agcaacagca gaagcataca gatatgcaga tgcattaaag 540
aaggacaatg gagaatatac agtagacgtt gcagataaag gttatacttt aaatattaaa 600
tttgctggag agctcactta caaacttgtt attaatggta aaacattgaa aggcgaaaca 660
actactaaag cagtagacgc agaaactgca gaaaaagcct tcaaacaata cgctaacgac 720
aacgctgttg atggtgtttg gacttatgat gatgcgacta agacctttac ggtaactgaa 780
gagctcaaga aagttgacga aaaaccagaa gaaaaagaac aagtaacaat taaagaaaat 840
atatattttg aagatggaac agtacaaact gcaacattta aaggaacatt tgcagaagcg 900
acagcagaag catacagata tgcagatttg ttatcaaaag aacatggtaa atacacagca 960
gacttggaag atggtggata cactatcaac attagatttg ctgga 1005
<210>14
<211>335
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>14
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu Leu
35 40 45
Ala Lys Glu Ash Gly Lys Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Thr Tyr Lys Leu Ile Leu
65 70 75 80
Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala
85 90 95
Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val
100 105 110
Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr
115 120 125
Glu Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr
130 135 140
Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu
145 150 155 160
Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala
165 170 175
Asp Ala Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp
180 185 190
Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Thr Tyr Lys
195 200 205
Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala
210 215 220
Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp
225 230 235 240
Asn Ala Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe
245 250 255
Thr Val Thr Glu Glu Leu Lys Lys Val Asp Glu Lys Pro Glu Glu Lys
260 265 270
Glu Gln Val Thr Ile Lys Glu Asn Ile Tyr Phe Glu Asp Gly Thr Val
275 280 285
Gln Thr Ala Thr Phe Lys Gly Thr Phe Ala Glu Ala Thr Ala Glu Ala
290 295 300
Tyr Arg Tyr Ala Asp Leu Leu Ser Lys Glu His Gly Lys Tyr Thr Ala
305 310 315 320
Asp Leu Glu Asp Gly Gly Tyr Thr Ile Asn Ile Arg Phe Ala Gly
325 330 335
<210>15
<211>1152
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>15
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgatgc gcaacaaaat 240
aacttcaaca aagatcaaca aagcgccttc tatgaaattt tgaacatgcc taacttaaac 300
gaagcgcaac gcaatggttt cattcaaagt cttaaagacg atccaagcca aagcactaac 360
gttttaggtg aagctaaaaa attaaacgaa tctcaagcac cgaaagagct cacttacaaa 420
ttaatcctta atggtaaaac attgaaaggc gaaacaacta ctgaagctgt tgatgctgct 480
actgcagaaa aagtcttcaa acaatacgct aacgacaacg gtgttgacgg tgaatggact 540
tacgacgatg cgactaagac ctttacagtt actgaagagc tcaagaaagt tgacgaaaaa 600
ccagaagaaa aagaacaagt aacaattaaa gaaaatatat attttgaaga tggaacagta 660
caaactgcaa catttaaagg aacatttgca gaagcgacag cagaagcata cagatatgca 720
gatttgttat caaaagaaca tggtaaatac acagcagact tggaagatgg tggatacact 780
atcaacatta gatttgctgg agagctcgct gacaacaaat tcaacaaaga acaacaaaat 840
gctttctatg aaattttaca tttacctaac ttaactgaag aacaacgtaa cggcttcatc 900
caaagcctta aagacgatcc ttcagtgagc aaagaaattt tagcagaagc taaaaagcta 960
aacgatgctc aagcaccaaa agagctcact tacaaattaa tccttaatgg taaaacattg 1020
aaaggcgaaa caactactga agctgttgat gctgctactg cagaaaaagt cttcaaacaa 1080
tacgctaacg acaacggtgt tgacggtgaa tggacttacg acgatgcgac taagaccttt 1140
acagttactg aa 1152
<210>16
<211>384
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>16
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Ala Leu
35 40 45
Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn
65 70 75 80
Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met
85 90 95
Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
100 105 110
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu
115 120 125
Asn Glu Ser Gln Ala Pro Lys Glu Leu Thr Tyr Lys Leu Ile Leu Asn
130 135 140
Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala
145 150 155 160
Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp
165 170 175
Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
180 185 190
Glu Leu Lys Lys Val Asp Glu Lys Pro Glu Glu Lys Glu Gln Val Thr
195 200 205
Ile Lys Glu Asn Ile Tyr Phe Glu Asp Gly Thr Val Gln Thr Ala Thr
210 215 220
Phe Lys Gly Thr Phe Ala Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala
225 230 235 240
Asp Leu Leu Ser Lys Glu His Gly Lys Tyr Thr Ala Asp Leu Glu Asp
245 250 255
Gly Gly Tyr Thr Ile Asn Ile Arg Phe Ala Gly Glu Leu Ala Asp Asn
260 265 270
Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu
275 280 285
Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
290 295 300
Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu
305 310 315 320
Asn Asp Ala Gln Ala Pro Lys Glu Leu Thr Tyr Lys Leu Ile Leu Asn
325 330 335
Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala
340 345 350
Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp
355 360 365
Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
370 375 380
<210>17
<211>1026
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>17
aagaaagttg acgaaaaacc agaagaaaaa gaacaagtaa caattaaaga aaatatatat 60
tttgaagatg gaacagtaca aactgcaaca tttaaaggaa catttgcaga agcgacagca 120
gaagcataca gatatgcaga tttgttatca aaagaacatg gtaaatacac agcagacttg 180
gaagatggtg gatacactat caacattaga tttgctggag agctcgctga caacaatttc 240
aacaaagaac aacaaaatgc tttctatgaa atcttgaaca tgcctaactt gaacgaagaa 300
caacgcaatg gtttcatcca aagcttaaaa gatgacccaa gtcaaagtgc taacctattg 360
tcagaagcta aaaagttaaa tgaatctcaa gcaccgaaag agctcaaaga aaaaacacca 420
gaagaaccaa aagaagaagt tactattaaa gcaaacttaa tctatgcaga tggaaaaaca 480
caaacagcag aattcaaagg aacatttgaa gaagcaacag cagaagcata cagatatgca 540
gatgcattaa agaaggacaa tggagaatat acagtagacg ttgcagataa aggttatact 600
ttaaatatta aatttgctgg agagctcact tacaaattaa tccttaatgg taaaacattg 660
aaaggcgaaa caactactga agctgttgat gctgctactg cagaaaaagt cttcaaacaa 720
tacgctaacg acaacggtgt tgacggtgaa tggacttacg acgatgcgac taagaccttt 780
acagttactg aagagctcaa agaagaaaca ccagaaacac cagaaactga ttcagaagaa 840
gaagtaacaa tcaaagctaa cctaatcttt gcaaatggaa gcacacaaac tgcagaattc 900
aaaggaacat ttgaaaaagc aacatcagaa gcttatgcgt atgcagatac tttgaagaaa 960
gacaatggag aatatactgt agatgttgca gataaaggtt atactttaaa tattaaattt 1020
gctgga 1026
<210>18
<211>342
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>18
Lys Lys Val Asp Glu Lys Pro Glu Glu Lys Glu Gln Val Thr Ile Lys
1 5 10 15
Glu Asn Ile Tyr Phe Glu Asp Gly Thr Val Gln Thr Ala Thr Phe Lys
20 25 30
Gly Thr Phe Ala Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu
35 40 45
Leu Ser Lys Glu His Gly Lys Tyr Thr Ala Asp Leu Glu Asp Gly Gly
50 55 60
Tyr Thr Ile Asn Ile Arg Phe Ala Gly Glu Leu Ala Asp Asn Asn Phe
65 70 75 80
Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn
85 90 95
Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp
100 105 110
Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu
115 120 125
Ser Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys
130 135 140
Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr
145 150 155 160
Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala
165 170 175
Tyr Arg Tyr Ala Asp Ala Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val
180 185 190
Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Glu
195 200 205
Leu Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
210 215 220
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln
225 230 235 240
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala
245 250 255
Thr Lys Thr Phe Thr Val Thr Glu Glu Leu Lys Glu Glu Thr Pro Glu
260 265 270
Thr Pro Glu Thr Asp Ser Glu Glu Glu Val Thr Ile Lys Ala Asn Leu
275 280 285
Ile Phe Ala Asn Gly Ser Thr Gln Thr Ala Glu Phe Lys Gly Thr Phe
290 295 300
Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp Thr Leu Lys Lys
305 310 315 320
Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu
325 330 335
Asn Ile Lys Phe Ala Gly
340
<210>19
<211>627
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>19
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgctgactt attagcaaaa gaaaatggta aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgatgc gcaacaaaat 240
aacttcaaca aagatcaaca aagcgccttc tatgaaattt tgaacatgcc taacttaaac 300
gaagcgcaac gcaatggttt cattcaaagt cttaaagacg atccaagcca aagcactaac 360
gttttaggtg aagctaaaaa attaaacgaa tctcaagcac cgaaagagct caaagaaaaa 420
acaccagaag aaccaaaaga agaagttact attaaagcaa acttaatcta tgcagatgga 480
aaaacacaaa cagcagaatt caaaggaaca tttgaagaag caacagcaga agcatacaga 540
tatgctgact tattagcaaa agaaaatggt aaatatacag tagacgttgc agataaaggt 600
tatactttaa atattaaatt tgctgga 627
<210>20
<211>209
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>20
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu Leu
35 40 45
Ala Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn
65 70 75 80
Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met
85 90 95
Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
100 105 110
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu
115 120 125
Asn Glu Ser Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu
130 135 140
Pro Lys Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly
145 150 155 160
Lys Thr Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala
165 170 175
Glu Ala Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr
180 185 190
Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala
195 200 205
Gly
<210>21
<211>1038
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>21
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgctgactt attagcaaaa gaaaatggta aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgatgc gcaacaaaat 240
aacttcaaca aagatcaaca aagcgccttc tatgaaattt tgaacatgcc taacttaaac 300
gaagcgcaac gcaatggttt cattcaaagt cttaaagacg atccaagcca aagcactaac 360
gttttaggtg aagctaaaaa attaaacgaa tctcaagcac cgaaagagct caaagaaaaa 420
acaccagaag aaccaaaaga agaagttact attaaagcaa acttaatcta tgcagatgga 480
aaaacacaaa cagcagaatt caaaggaaca tttgaagaag caacagcaga agcatacaga 540
tatgctgact tattagcaaa agaaaatggt aaatatacag tagacgttgc agataaaggt 600
tatactttaa atattaaatt tgctggagag ctcgctgatg cgcaacaaaa taacttcaac 660
aaagatcaac aaagcgcctt ctatgaaatt ttgaacatgc ctaacttaaa cgaagcgcaa 720
cgcaatggtt tcattcaaag tcttaaagac gatccaagcc aaagcactaa cgttttaggt 780
gaagctaaaa aattaaacga atctcaagca ccgaaagagc tcaaagaaaa aacaccagaa 840
gaaccaaaag aagaagttac tattaaagca aacttaatct atgcagatgg aaaaacacaa 900
acagcagaat tcaaaggaac atttgaagaa gcaacagcag aagcatacag atatgctgac 960
ttattagcaa aagaaaatgg taaatataca gtagacgttg cagataaagg ttatacttta 1020
aatattaaat ttgctgga 1038
<210>22
<211>346
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>22
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu Leu
35 40 45
Ala Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn
65 70 75 80
Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met
85 90 95
Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
100 105 110
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu
115 120 125
Asn Glu Ser Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu
130 135 140
Pro Lys Glu Glu Val Thr Ile Lys Ala Ash Leu Ile Tyr Ala Asp Gly
145 150 155 160
Lys Thr Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala
165 170 175
Glu Ala Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr
180 185 190
Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala
195 200 205
Gly Glu Leu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln
210 215 220
Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln
225 230 235 240
Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr
245 250 255
Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
260 265 270
Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile
275 280 285
Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe
290 295 300
Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp
305 310 315 320
Leu Leu Ala Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys
325 330 335
Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly
340 345
<210>23
<211>1149
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>23
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcacttacaa acttgttatt 240
aatggtaaaa cattgaaagg cgaaacaact actaaagcag tagacgcaga aactgcagaa 300
aaagccttca aacaatacgc taacgacaac gctgttgatg gtgtttggac ttatgatgat 360
gcgactaaga cctttacggt aactgaagag ctcgctgaca acaatttcaa caaagaacaa 420
caaaatgctt tctatgaaat cttgaacatg cctaacttga acgaagaaca acgcaatggt 480
ttcatccaaa gcttaaaaga tgacccaagt caaagtgcta acctattgtc agaagctaaa 540
aagttaaatg aatctcaagc accgaaagag ctcaaagaaa aaacaccaga agaaccaaaa 600
gaagaagtta ctattaaagc aaacttaatc tatgcagatg gaaaaacaca aacagcagaa 660
ttcaaaggaa catttgaaga agcaacagca gaagcataca gatatgctga cttattagca 720
aaagaaaatg gtaaatatac agtagacgtt gcagataaag gttatacttt aaatattaaa 780
tttgctggag agctcgctga tgcgcaacaa aataacttca acaaagatca acaaagcgcc 840
ttctatgaaa ttttgaacat gcctaactta aacgaagcgc aacgcaatgg tttcattcaa 900
agtcttaaag acgatccaag ccaaagcact aacgttttag gtgaagctaa aaaattaaac 960
gaatctcaag caccgaaaga gctcacttac aaattaatcc ttaatggtaa aacattgaaa 1020
ggcgaaacaa ctactgaagc tgttgatgct gctactgcag aaaaagtctt caaacaatac 1080
gctaacgaca acggtgttga cggtgaatgg acttacgacg atgcgactaa gacctttaca 1140
gttactgaa 1149
<210>24
<211>383
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>24
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Ala Leu
35 40 45
Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Thr Tyr Lys Leu Val Ile
65 70 75 80
Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala
85 90 95
Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Ala Val
100 105 110
Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr
115 120 125
Glu Glu Leu Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe
130 135 140
Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly
145 150 155 160
Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu
165 170 175
Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Glu Leu Lys
180 185 190
Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala Asn
195 200 205
Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly Thr
210 215 220
Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu Leu Ala
225 230 235 240
Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr Thr
245 250 255
Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn Asn
260 265 270
Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro
275 280 285
Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp
290 295 300
Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn
305 310 315 320
Glu Ser Gln Ala Pro Lys Glu Leu Thr Tyr Lys Leu Ile Leu Asn Gly
325 330 335
Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr
340 345 350
Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly
355 360 365
Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
370 375 380
<210>25
<211>792
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>25
aaagaagaaa caccagaaac accagaaact gattcagaag aagaagtaac aatcaaagct 60
aacctaatct ttgcaaatgg aagcacacaa actgcagaat tcaaaggaac atttgaaaaa 120
gcaacatcag aagcttatgc gtatgcagat actttgaaga aagacaatgg agaatatact 180
gtagatgttg cagataaagg ttatacttta aatattaaat ttgctggaga gctcacttac 240
aaattaatcc ttaatggtaa aacattgaaa ggcgaaacaa ctactgaagc tgttgatgct 300
gctactgcag aaaaagtctt caaacaatac gctaacgaca acggtgttga cggtgaatgg 360
acttacgacg atgcgactaa gacctttaca gttactgaag agctcaaaga aaaaacacca 420
gaagaaccaa aagaagaagt tactattaaa gcaaacttaa tctatgcaga tggaaaaaca 480
caaacagcag aattcaaagg aacatttgaa gaagcaacag cagaagcata cagatatgct 540
gacttattag caaaagaaaa tggtaaatat acagtagacg ttgcagataa aggttatact 600
ttaaatatta aatttgctgg agagctcact tacaaattaa tccttaatgg taaaacattg 660
aaaggcgaaa caactactga agctgttgat gctgctactg cagaaaaagt cttcaaacaa 720
tacgctaacg acaacggtgt tgacggtgaa tggacttacg acgatgcgac taagaccttt 780
acagttactg aa 792
<210>26
<211>264
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>26
Lys Glu Glu Thr Pro Glu Thr Pro Glu Thr Asp Ser Glu Glu Glu Val
1 5 10 15
Thr Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gln Thr Ala
20 25 30
Glu Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr
35 40 45
Ala Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala
50 55 60
Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Thr Tyr
65 70 75 80
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu
85 90 95
Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn
100 105 110
Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr
115 120 125
Phe Thr Val Thr Glu Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys
130 135 140
Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr
145 150 155 160
Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala
165 170 175
Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr Thr Val
180 185 190
Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Glu
195 200 205
Leu Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr
210 215 220
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln
225 230 235 240
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala
245 250 255
Thr Lys Thr Phe Thr Val Thr Glu
260
<210>27
<211>1050
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>27
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgatgc gcaacaaaat 240
aacttcaaca aagatcaaca aagcgccttc tatgaaattt tgaacatgcc taacttaaac 300
gaagcgcaac gcaatggttt cattcaaagt cttaaagacg atccaagcca aagcactaac 360
gttttaggtg aagctaaaaa attaaacgaa tctcaagcac cgaaagagct caaagaaaaa 420
acaccagaag aaccaaaaga agaagttact attaaagcaa acttaatcta tgcagatgga 480
aaaacacaaa cagcagaatt caaaggaaca tttgaagaag caacagcaga agcatacaga 540
tatgctgact tattagcaaa agaaaatggt aaatatacag tagacgttgc agataaaggt 600
tatactttaa atattaaatt tgctggagag ctcgctgatg cgcaacaaaa taacttcaac 660
aaagatcaac aaagcgcctt ctatgaaatt ttgaacatgc ctaacttaaa cgaagcgcaa 720
cgcaatggtt tcattcaaag tcttaaagac gatccaagcc aaagcactaa cgttttaggt 780
gaagctaaaa aattaaacga atctcaagca ccgaaagagc tcaaagaaga aacaccagaa 840
acaccagaaa ctgattcaga agaagaagta acaatcaaag ctaacctaat ctttgcaaat 900
ggaagcacac aaactgcaga attcaaagga acatttgaaa aagcaacatc agaagcttat 960
gcgtatgcag atactttgaa gaaagacaat ggagaatata ctgtagatgt tgcagataaa 1020
ggttatactt taaatattaa atttgctgga 1050
<210>28
<211>350
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>28
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Ala Leu
35 40 45
Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn
65 70 75 80
Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met
85 90 95
Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
100 105 110
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu
115 120 125
Asn Glu Ser Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu
130 135 140
Pro Lys Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly
145 150 155 160
Lys Thr Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala
165 170 175
Glu Ala Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr
180 185 190
Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala
195 200 205
Gly Glu Leu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln
210 215 220
Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln
225 230 235 240
Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr
245 250 255
Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
260 265 270
Glu Leu Lys Glu Glu Thr Pro Glu Thr Pro Glu Thr Asp Ser Glu Glu
275 280 285
Glu Val Thr Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gln
290 295 300
Thr Ala Glu Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr
305 310 315 320
Ala Tyr Ala Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp
325 330 335
Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly
340 345 350
<210>29
<211>948
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>29
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgacaa caaattcaac 240
aaagaacaac aaaatgcttt ctatgaaatt ttacatttac ctaacttaac tgaagaacaa 300
cgtaacggct tcatccaaag ccttaaagac gatccttcag tgagcaaaga aattttagca 360
gaagctaaaa agctaaacga tgctcaagca ccaaaagagc tcaaagaaaa aacaccagaa 420
gaaccaaaag aagaagttac tattaaagca aacttaatct atgcagatgg aaaaactcaa 480
acagcagagt tcaaaggaac atttgcagaa gcaacagcag aagcatacag atacgctgac 540
ttattagcaa aagaaaatgg taaatataca gcagacttag aagatggtgg atacactatt 600
aatattagat ttgcaggtga gctcgctcaa caaaatgctt tttatcaagt cttaaatatg 660
cctaacttaa atgctgatca acgcaatggt tttatccaaa gccttaaaga tgatccaagc 720
caaagtgcta acgttttagg tgaagctaaa agattaaacg aatttcaagc accgaaagag 780
ctcacttaca aattaatcct taatggtaaa acattgaaag gcgaaacaac tactgaagct 840
gttgatgctg ctactgcaga aaaagtcttc aaacaatacg ctaacgacaa cggtgttgac 900
ggtgaatgga cttacgacga tgcgactaag acctttacag ttactgaa 948
<210>30
<211>316
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>30
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Ala Leu
35 40 45
Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Asn Lys Phe Asn
65 70 75 80
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
85 90 95
Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro
100 105 110
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
115 120 125
Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu
130 135 140
Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln
145 150 155 160
Thr Ala Glu Phe Lys Gly Thr Phe Ala Glu Ala Thr Ala Glu Ala Tyr
165 170 175
Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr Thr Ala Asp
180 185 190
Leu Glu Asp Gly Gly Tyr Thr Ile Asn Ile Arg Phe Ala Gly Glu Leu
195 200 205
Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Asn
210 215 220
Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser
225 230 235 240
Gln Ser Ala Asn Val Leu Gly Glu Ala Lys Arg Leu Asn Glu Phe Gln
245 250 255
Ala Pro Lys Glu Leu Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu
260 265 270
Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys
275 280 285
Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr
290 295 300
Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
305 310 315
<210>31
<211>1161
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉its proteins encoded be can with immunoglobulin (Ig) high-affinity bonded fusion gene
<400>31
aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgcagatgc attaaagaag gacaatggag aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgacaa caaattcaac 240
aaagaacaac aaaatgcttt ctatgaaatt ttacatttac ctaacttaac tgaagaacaa 300
cgtaacggct tcatccaaag ccttaaagac gatccttcag tgagcaaaga aattttagca 360
gaagctaaaa agctaaacga tgctcaagca ccaaaagagc tcacttacaa acttgttatt 420
aatggtaaaa cattgaaagg cgaaacaact actaaagcag tagacgcaga aactgcagaa 480
aaagccttca aacaatacgc taacgacaac gctgttgatg gtgtttggac ttatgatgat 540
gcgactaaga cctttacggt aactgaagag ctcaaagaag aaacaccaga aacaccagaa 600
actgattcag aagaagaagt aacaatcaaa gctaacctaa tctttgcaaa tggaagcaca 660
caaactgcag aattcaaagg aacatttgaa aaagcaacat cagaagctta tgcgtatgca 720
gatactttga agaaagacaa tggagaatat actgtagatg ttgcagataa aggttatact 780
ttaaatatta aatttgctgg agagctcgct gatgcgcaac aaaataactt caacaaagat 840
caacaaagcg ccttctatga aattttgaac atgcctaact taaacgaagc gcaacgcaat 900
ggtttcattc aaagtcttaa agacgatcca agccaaagca ctaacgtttt aggtgaagct 960
aaaaaattaa acgaatctca agcaccgaaa gagctcactt acaaattaat ccttaatggt 1020
aaaacattga aaggcgaaac aactactgaa gctgttgatg ctgctactgc agaaaaagtc 1080
ttcaaacaat acgctaacga caacggtgtt gacggtgaat ggacttacga cgatgcgact 1140
aagaccttta cagttactga a 1161
<210>32
<211>387
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉can with immunoglobulin (Ig) high-affinity bonded fusion rotein
<400>32
Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Ala Leu
35 40 45
Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Asn Lys Phe Asn
65 70 75 80
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu
85 90 95
Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro
100 105 110
Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala
115 120 125
Gln Ala Pro Lys Glu Leu Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr
130 135 140
Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu
145 150 155 160
Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Ala Val Asp Gly Val Trp
165 170 175
Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Glu Leu Lys
180 185 190
Glu Glu Thr Pro Glu Thr Pro Glu Thr Asp Ser Glu Glu Glu Val Thr
195 200 205
Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gln Thr Ala Glu
210 215 220
Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala
225 230 235 240
Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp
245 250 255
Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala
260 265 270
Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile
275 280 285
Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln
290 295 300
Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala
305 310 315 320
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Glu Leu Thr Tyr Lys Leu
325 330 335
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val
340 345 350
Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn
355 360 365
Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr
370 375 380
Val Thr Glu
385
Claims (11)
1. reorganization Ig binding molecule library, it is characterized in that: described library is by the A with different Ig binding molecule staphylococcal protein A,SPAs, B, C, D, the E structural domain, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, the B5 structural domain, G group or the proteic G1 of C group streptococcus G, the single antibodies structural domain of G2 structural domain is a structural unit, resetting the Ig binding molecule that forms with the structural unit random dna forms, wherein the nucleotide sequence in said structure territory is respectively: SEQ ID NO:1-SEQ ID NO:12, connect with Nucleotide GAGCTC between the single structure territory, the structural unit random dna is reset the Ig binding molecule that forms and is obtained by the following method:
(1) with A, B, C, D, the E structural domain of Ig binding molecule staphylococcal protein A,SPA, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, B5 structural domain, the single antibodies structural domain of G group or the proteic G1 of C group streptococcus G, G2 structural domain are introduced Sac I restriction enzyme site as tie point through pcr amplification respectively;
(2) PCR is reclaimed product and cut digestion through Sac I enzyme respectively;
(3) the Sac I enzyme that step (2) is obtained is cut the digestion product mixing, with the T4 ligase enzyme each single structure territory is connected reorganization at random.
2. the preparation method in library according to claim 1 comprises the following steps:
(1) with A, B, C, D, the E structural domain of Ig binding molecule staphylococcal protein A,SPA, the proteic B1 of peptostreptococcus magnus L, B2, B3, B4, B5 structural domain, the single antibodies structural domain of G group or the proteic G1 of C group streptococcus G, G2 structural domain is a structural unit, introduce Sac I restriction enzyme site as tie point through pcr amplification respectively, the nucleotides sequence of wherein said structural domain is classified as: SEQ ID NO:1-SEQ ID NO:12;
(2) PCR reclaims product and cuts digestion through Sac I enzyme respectively;
(3) the Sac I enzyme that step (2) is obtained is cut the digestion product mixing, with the T4 ligase enzyme each single structure territory is connected reorganization at random again.
3. method as claimed in claim 2 is characterized in that, comprises also after the step (3) that the connection product with different time mixes acquisition reorganization Ig binding molecule library.
4. the phage display method in Ig binding molecule library of recombinating is characterized in that the described Ig binding molecule of claim 1 library clone carrying out the phage display of 3+3 form with this carrier in phage vector pCANTAB5S.
5. adopt the reorganization Ig binding molecule phage library that method obtained of claim 4.
6. method that obtains high-affinity Ig binding molecule phage library, it is characterized in that: use the Ig wrapper sheet, utilize the described reorganization of claim 5 Ig binding molecule phage library to take turns the affine screening of external orthogenesis IgG and obtain high-affinity Ig binding molecule phage library through 3-4.
7. with the high-affinity Ig binding molecule phage library that method obtained of claim 6.
8. high-affinity Ig binding molecule phage library as claimed in claim 7, it is characterized in that: by the high-affinity Ig binding molecule of phage display is that the single structure territory of single structure territory, peptostreptococcus magnus protein L with staphylococcal protein A,SPA and G group or the proteic single structure of C group streptococcus G territory are structural unit, be spaced in the Ig binding molecule that is formed by connecting any one or multiple.
9. high-affinity Ig binding molecule phage library as claimed in claim 8, represent single structure territory, G group or the proteic single structure of the C group streptococcus G territory of the proteic single structure of peptostreptococcus magnus L territory, staphylococcal protein A,SPA respectively with ML, MA, MG, it is characterized in that: described structural unit is spaced mode of connection and is (ML-MA) n or (ML-MG) n or (ML-MA-MG) n or (ML-MG-MA) n or ML-(MA-ML) n or ML-(MG-ML) n, and wherein n is the multiplicity of this sequence.
10. high-affinity Ig binding molecule phage library as claimed in claim 9, it is characterized in that: the series arrangement mode by the high-affinity Ig binding molecule of phage display is B3-G1-B2-G2-B5, B2-D-G1-B5-C-G1, B5-A-B2-G1-B1, B3-D-B3, B3-D-B3-D-B3, B2-G2-A-B3-D-G1, B1-G1-B3-G1, B2-D-B3-D-B1, B2-C-B4-E-G1, any one in the Ig binding molecule of B2-C-G2-B1-D-G1 or multiple.
11. the application in reorganization Ig binding molecule library is characterized in that: with the described library screening high-affinity of arbitrary claim Ig binding molecule in the claim 1 or 5 or 7 to 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100671572A CN1319988C (en) | 2004-10-14 | 2004-10-14 | High affinity immune globulin binding molecule and method for preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100671572A CN1319988C (en) | 2004-10-14 | 2004-10-14 | High affinity immune globulin binding molecule and method for preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1634990A CN1634990A (en) | 2005-07-06 |
| CN1319988C true CN1319988C (en) | 2007-06-06 |
Family
ID=34846632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| CN102443061A (en) * | 2005-10-14 | 2012-05-09 | 英国希尔思生物分子有限责任公司 | High-functional antibody biological synthesis |
| CN103270044B (en) * | 2010-12-21 | 2016-03-09 | Jsr株式会社 | The method of affinity chromatography carrier and separating immune globulin |
| WO2013018880A1 (en) * | 2011-08-04 | 2013-02-07 | 独立行政法人産業技術総合研究所 | Novel modified protein comprising tandem-type multimer of mutant extracellular domain of protein g |
| CN102304173A (en) * | 2011-09-05 | 2012-01-04 | 中国科学院苏州纳米技术与纳米仿生研究所 | Recombinant protein G and preparation method and use thereof |
| CN103014880B (en) | 2012-12-20 | 2015-06-24 | 天津大学 | Novel affinity ligand polypeptide library of immunoglobulin G constructed based on protein A affinity model and application of design method |
| JP2016079153A (en) * | 2014-10-21 | 2016-05-16 | 株式会社プロテイン・エクスプレス | Protein g mutant |
| CN108064286A (en) | 2015-01-26 | 2018-05-22 | 株式会社钟化 | Saltant type immunoglobulin kappa chain variable region binding peptide |
| JPWO2016121701A1 (en) * | 2015-01-26 | 2017-11-09 | 株式会社カネカ | Affinity separation matrix for protein purification containing immunoglobulin κ chain variable region |
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| WO2017191747A1 (en) * | 2016-05-06 | 2017-11-09 | 株式会社カネカ | Method for producing protein including κ chain variable region |
| WO2017195638A1 (en) | 2016-05-09 | 2017-11-16 | 株式会社カネカ | Method for refining antibody or antibody fragment containing κ-chain variable region |
| CN106279419A (en) * | 2016-08-08 | 2017-01-04 | 吴江近岸蛋白质科技有限公司 | A kind of antibody binding proteins Protein L and preparation method thereof |
| CN106970219A (en) * | 2017-04-28 | 2017-07-21 | 北京金豪制药股份有限公司 | One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent |
| CN111057153B (en) * | 2019-12-06 | 2021-09-07 | 广州康盛生物科技股份有限公司 | Immunoglobulin binding protein and preparation method and application thereof |
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| WO2003093306A2 (en) * | 2002-05-02 | 2003-11-13 | Chir0N Srl | Nucleic acids and proteins from streptococcus groups a & b |
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