CN100551931C - TXi Baoshouti is showed - Google Patents

Abstract

The proteinaceous particle of a kind of for example phage, rrna or cell, at its surface display a kind of TXi Baoshouti (TCR).The TCR that shows preferably has the heterodimer of non-natural disulfide linkage between the constant domain residue.Such displaying particle can be used to set up multifarious TCR library to differentiate high-affinity TCR.Several high-affinity TCR are disclosed.

Description

TXi Baoshouti is showed

The present invention relates to show the proteinaceous particle of TXi Baoshouti (TCR), for example phage or ribosomal particle, and different libraries.

Background of invention

Natural TXi Baoshouti (TCR)

As for example described in the WO99/60120, TCR mediation T cell is to the identification of the main histocompatibility complex of specificity (MHC)-peptide complex body, and this is the basis of immune system cell immunity performance function.

Antibody and TCR be only two kinds and discern antigenic molecule in special mode, and TCR thereby be unique acceptor of the specific antigen peptide of offering at MHC, and the foreign matter peptide often is unusual unique sign in the cell.In when, direct physical taking place between T cell and the antigen presenting cell (APC) contact when, generation T cell recognition, and be connected startup with the pMHC mixture by antigen specific T CR.

Natural TCR is a kind of allos dimerization cell surface protein of immunoglobulin superfamily, and it links to each other with the ankyrin of the CD3 complex body that participates in the mediation signal transduction.TCR exists with α β and γ δ form, and is similar but have diverse anatomy location and perhaps different functions on both structures.I class and II class MHC part also are immunoglubulin superfaminly protein matter but have and make it can offer the multifarious binding site peptide point of the segmental height of a large amount of different APC cell surface small peptides, specially in order to angtigen presentation.

The protein of known two kinds of further types can play a role as the TCR part.(1) CD1 antigen is MHC I class associated molecule, and its gene is positioned at and is different from classical MHC I class and the antigenic karyomit(e) of II class.The CD1 molecular energy is offered peptide and non-peptide (for example fat, glycolipid) part with the form that is similar to conventional I class and II class MHC-peptide complex to the T cell.Referring to, " human leucocyte antigen general introduction " (The Leucocyte AntigenFactsbook) that show such as Barclay for example, the 2nd edition, (1997) and Bauer (1997) Eur J Immunol 27 (6) 1366-1373.(2) bacterial superantigen is can be in conjunction with the soluble toxin (Fraser (1989) Nature 339 221-223) of II class MHC molecule and a part of TCR.Many superantigens demonstrate the segmental specificity to one or both V β, and other show more random combination.In any case, superantigen can rely on it to cause the enhanced immune response with the ability that the polyclone mode activates a part of T cell.

The α β of allos dimerization and born of the same parents' outside part of gamma delta T CR comprise two polypeptide, and wherein every has the constant domain of a nearly film and the variable domains of a film far away.Each constant domain and variable domains comprise an intrachain disulfide bond.Variable domains comprises the high polymorphism ring that is similar to complementary antibody determining area (CDR).The peptide interaction that the CDR3 of α β TCR and MHC offer, and the CDR1 of α β TCR and CDR2 and peptide and MHC interaction.The polymorphism of TCR sequence is reset by the somatocyte of chain variable domains (V), diversity (D), joining region (J) and constant gene and is produced.

Functional α and γ chain polypeptide are formed by the V-J-C district of resetting, and β and δ chain polypeptide comprise the V-D-J-C district.The outer constant domain of born of the same parents has membrane-proximal region and immunoglobulin domain.There are single α and δ chain constant domain, are called TRAC and TRDC.β chain constant domain is by a kind of formation in two kinds of different beta constant domain that are called TRBC1 and TRBC2 (IMGT nomenclature).Have 4 amino acid whose changes between these β constant domain, wherein 3 are positioned at the structural domain inside that is used to produce the strand TCR that is illustrated on the phage particle of the present invention.These change the exons 1 inside that all is positioned at TRBC1 and TRBC2: N ₄K ₅-＞K ₄N ₅And F ₃₇-＞Y (IMTG numbering, TRBC1-＞TRBC2 difference), the amino acid change between last two kinds of TCR β chain is in the exon 3 of TRBC1 and TRBC2: Vi-＞E.Constant γ structural domain is made of one of TRGC1, TRGC2 (2X) or TGRC2 (3X).These two kinds of TGRC2 constant domain are just different on the coded amino acid copy number of described gene extron 2.

Some variation of the scope of every kind of TCR ectodomain.Yet, be proficient in those skilled in the art and can use such as " TXi Baoshouti general introduction " (The T Cell Receptor Facts Book, Lefranc﹠amp; Lefranc, Publ.Academic Press 2001) etc. with reference to the position of determining the structural domain border easily.

Reorganization TCR

Produce reorganization TCR and be good, because they provide the soluble T CR that is applicable to following purpose analogue,

Research TCR/ ligand interaction (for example pMHC and α β TCR)

The relevant interactional inhibitor of screening TCR

For the potential methods of treatment provides the basis

Designed multiple member at present to produce reorganization TCR.These members are divided into strand TCR and dimerization TCR two big classes, have summed up below and these two kinds of documents that member is relevant.

Strand TCR (scTCR) is the artificial member that is made of the single amino acid chain, and it is similar to natural allos dimerization TCR and combines with the MHC-peptide complex.Unfortunately, thereby make both express the also not achieving success of trial of producing functional α/β analogue scTCR in single open reading frame by connecting α and β chain simply, the chances are because the right natural unstable of alpha-beta solubility structural domain.

Correspondingly, using the technical skill of one of α chain and β chain or both various truncated sequences is essential for producing scTCR.As if these modes only can be used for very limited scTCR sequence.Soo Hoo etc. are at (1992) PNAS.89 (10): reported among the 4759-63 by use expression with the mouse TCR of single stranded form in the 2C T cell clone of a kind of α of brachymemma of 25 amino acid whose connectors connections and β chain and bacterium periplasmic expression (also can be referring to Schodin etc., (1996) Mol.Immunol.33 (9): 819-29).This design has also formed Holler etc. at (2000) PNAS.97 (10): report among the 5387-92 be derived from 2C scTCR's and with the basis of the restricted isomery epi-position of identical H2-Ld-(alloepitope) bonded m6 strand TCR.Shusta etc. have reported in (2000) NatureBiotechnology 18:754-759 and in the United States Patent (USP) 6,423,538 at the yeast that produces mutation T CR and have showed that use mouse strand 2C TCR member improves thermostability and solubility in the experiment.This report has illustrated that also the 2C TCR selective binding of these displayings expresses the ability of its similar pMHC cell.Khandekar etc. are at (1997) J.Biol.Chem.272 (51): reported the similar design at mouse D10TCR among the 32190-7, though melting to be incorporated in the bacterium kytoplasm, this scTCR and MBP express (visible Hare etc., (1999) Nat.Struct.Biol.6 (6): 574-81).Hilyard etc. are at (1994) PNAS.91 (19): reported among the 9057-61 by using V α-connector-V β design and at the specific people scTCR of a kind of common cold virus stromatin HLA-A2 of bacterium periplasmic expression.

Chung etc. have reported in (1994) PNAS.91 (26) 12654-8 and to have used V α-connector-V β-V β design and in the production of the people scTCR of mammal cell line surface expression.This report does not relate to any peptide-HLA specificity combination of scTCR.Plaksin etc. are at (1997) J.Immunol.158 (5): reported among the 2218-27 that a kind of similar V α-connector-V β-V β design is to produce at HIV gpl20-H-2D ^dThe mouse specificity scTCR of epi-position.This scTCR is with bacterium inclusion body formal representation and in external renaturation.

Many document descriptions comprise the production of the TCR heterodimer of the natural disulfide linkage that connects corresponding subunits (Nature 384 (6605): 134-41 for Garboczi etc., (1996); Garboczi etc., (1996), J Immunol157 (12): 5403-10; Chang etc., (1994), PNAS USA 91:11408-11412; Davodeau etc., (1993), J.Biol Chem.268 (21): 15455-15460; Golden etc., (1997), J.Imm.Meth.206:163-169; U.S. Patent No. 6080840).Yet TCR although it is so can be discerned by the TCR specific antibody, and they are unsettled and can not discern its native ligand, unless these parts reach relative high density.

In WO 99/60120, described a kind of correct folding soluble T CR, so it can discern its native ligand, stable in the time of one section, adding, and can be with rational quantity production.This TCR comprises by TCR α of a pair of C-terminal dimerization peptide (such as leucine zipper) and corresponding TCR β or δ chain ectodomain dimerization or γ chain ectodomain.The strategy of this production TCR is applicable to all TCR usually.

Reiter etc. are at Immunity, and 1995, describe in detail among the 2:281-287 and comprise by disulfide linkage stable TCR α and β variable domains, the structure of the shla molecule that one of them links to each other with pseudomonas (Pseudomonas) extracellular toxin (PE38).One of reason of producing this a part is to overcome strand TCR inherent instability.The position of new disulfide linkage is by (these disulfide linkage before once were imported into the antibody variable territory in this TCR variable domains, referring to (1993) such as for example Brinkmann, Proc.Natl.Acad.Sci.USA 90:7538-7542, and (1994) Biochemistry 33:5451-5459 such as Reiter) homology differentiate.Yet owing to there is not such homology between the constant domain of antibody and TCR, such technology can not be used to differentiate the appropriate site of interchain disulfide bond new between the TCR constant domain.

As mentioned above, Shusta etc. has reported in (2000) Nature Biotechnology 18:754-759 and has showed at yeast and to use strand 2C TCR member in the experiment.In the past for showing that on phage particle scTCR had carried out discussion.For example, WO 99/19129 describes the production of scTCR in detail, and has summed up the potential method of production of the phage particle of the scTCR that is used to show V α-connector-V β C beta form.Yet this application does not comprise the illustration of the production of the phage particle that described displaying TCR is described.This application is still with reference to a pending application: " described the structure of the dna vector of the dna fragmentation that comprises the coding scTCR molecule that merges with phage coat protein (gene II or gene VIII) in described unsettled U.S. Patent application 08/813,781.”

And that this application depends on that the identification of anti-TCR antibody or superantigen MHC mixture produces is soluble, the ability of the scTCR of non-phage display to be to confirm its correct conformation.Therefore, real peptide-MHC binding specificity of scTCR does not illustrate in any form definitely.At last, further study the phage display that (Onda etc., (1995) MolecularImmunology 32 (17-18) 1387-1397) discloses two mouse TCR α chains under the situation that does not have its corresponding β chain.This research explanation, show the phage particle of one of described TCR α chain (being derived from the little murine hybridoma of A1.1) preferential be fixed in the microtiter well, with by mouse I class MHC I-A ^dThe identical peptide combination of complete TCR meeting normal reaction when offering.

Screening is used

Comprise that the multiple important cell interaction of immune cynapse of TCR mediation and cell response are by cell surface receptor and are presented on touch controls between the part of other cell surfaces.These special molecule contact types are regulated significant for biochemistry correct in the human body, thereby are the research focuses.In many cases, the target of research is that design is regulated the method for cell response to prevent or to treat disease like this.

Therefore, be used to differentiate that it is important having certain specific method with human receptor or part bonded compound, because this can guide the discovery and the development of new methods for the treatment of diseases.Especially disturb the compound of some receptor-ligand binding to have immediately ability as therapeutant or carrier.

The progress of combinatorial chemistry can produce very huge library of compounds easier and effectively, and this has improved the scope of extensive compound test.Now, the most common character of the detection method that can use, suitable acceptor and the production of ligand molecular and the degree that these detection methods are suitable for high-throughput screening method of being present in of the restriction of screening procedure.

Methods of exhibiting

Often need present a kind of given peptide or polypeptide at proteinaceous particle surface.Such particle can be used as the supplementary mode (because these particles that carry peptide or polypeptide can separate with unwanted impurity by precipitation or additive method) of peptide or peptide purification.They also can be used as particulate vaccine, offer to be activated by particulate at the peptide of surface display or the immunne response of polypeptide.Yeast counter-rotating seat protein p24 and hepatitis B surface coat protein are that self-chambering is made into the proteinic example of particulate.It is the mode of offering a kind of approval of described peptide or polypeptide at the particle surface that generates that interested peptide or polypeptide and these particles form proteic fusion.

Yet the particle methods of exhibiting mainly has been used to differentiate the protein that has such as required character such as enhanced expression amount, combination and/or stabilising characteristics.These methods comprise various storehouse or " library " that is created in proteinaceous particle surface expressing protein or polypeptide.These particles have two key features, and the first, each particle is offered single protein or polypeptide, and the second, the genetic stocks of encode expressed albumen or polypeptide links to each other with described particulate genetic stocks.Described subsequently library carries out taking turns or multi-turns screen.For example, this can comprise a kind of part contacted with a kind of particle display libraries of mutant receptors and differentiate which kind of mutant receptors with high-affinity in conjunction with described part.In case screening process is finished, the acceptor with required character can separate, and its genetic stocks can increase so that described acceptor is checked order.These methods of exhibiting are divided into two big classes, show in external displaying and the body.

Methods of exhibiting depends on following steps in all bodies, wherein usually will by such as the reproducible particulate genetic nucleic acid transformant of plasmid or phage replication with expressing protein or polypeptide (Pliickthun (2001) Adv ProteinChem 55367-403).There is the multiple replicon/host system that has confirmed to be suitable for displaying in albumen or the polypeptide body.These systems comprise:

Phage/bacterial cell

Plasmid/Chinese hamster ovary celI

Carrier/yeast cell based on yeast 2 μ m plasmids

Baculovirus/insect cell

Plasmid/bacterial cell

Methods of exhibiting comprises the cell surface display method in the body, the plasmid importing host cell of the fusion rotein that is made of protein of interest matter or polypeptide and a kind of cell surface protein or polypeptide of wherein will encode.This Expression of Fusion Protein causes protein of interest or polypeptide to be illustrated in cell surface.The cell of showing these protein of interest or polypeptide can be used for as screening processes such as FACS subsequently, and the plasmid that obtains from the cell of selecting can separate and check order.Mammalian cell (Higuschi (1997) J Immunol.Methods 202193-204), yeast cell (Shusta (1999) J Mol Biol 292949-956) and bacterial cell (Sameul son (2002) J.Biotechnol 96 (2) 129-154) have been designed the cell surface display system.

A large amount of summaries of display technique in the multiple body are disclosed, for example Hudson (2002) Expert Opin BiolTher (2001) 1 (5) 845-55 and Schmitz (2000) 21 (Supp A) S106-S112.

External methods of exhibiting is based upon on the rrna basis with the various array with mRNA library translation becoming protein or polypeptide.Being connected by one of two kinds of methods between the mRNA of protein that forms or polypeptide and these molecules of coding kept.The ribosomal display of routine adopts the short catenation sequence of coding (40-100 amino acid usually) and the protein that need show or the mRNA sequence of polypeptide.Catenation sequence provides enough spaces can not produced sterically hindered by rrna with refolding for albumen or the polypeptide of showing.Described mRNA sequence lacks " termination " codon, and this guarantees that expressed proteins or polypeptide and RNA and ribosomal particle keep being connected.Relevant mRNA methods of exhibiting is based upon preparation coding protein of interest or polypeptide and carries on the mRNA basis of tetracycline DNA connector partly.In case rrna arrives the mRNA/DNA joint, translation just stops, and tetracycline and rrna form covalently bound.About the recent summary of these two kinds of relevant external methods of exhibiting referring to Amstutz (2001) Curr OpinBiotechnol 12 400-405.

Especially preferably express and the heterologous peptides of its surface protein fusion or the display technique of bacteriophage (Smith (1985) Science 217 1315-1317) of polypeptide ability based on phage particle.The process of showing for polypeptide monomer is quite general, and is to know in this area.Yet, be combined into when natural form under the situation of dimeric polypeptide, as if having only the phage display of antibody to obtain thorough research.

For the monomer polypeptide is showed, there are two kinds of main processes:

First kind (method A) inserts carrier (phagemid) by the heterologous peptides of the DNA fusion of the phage coat protein of will encoding and encode or the DNA of polypeptide.Subsequently by with phagemid transfection bacterial cell, and use " helper phage " to infect cell transformed subsequently, show the expression of the phage particle of heterologous peptides or polypeptide.Helper phage is uncoded as described phagemid, the required proteic source of phage of the functional phage particle of generation.

Second kind (method B) is by the complete phage genome that DNA inserts and coding phage coat protein DNA merges of will encode heterologous peptides or polypeptide.Subsequently by show the expression of the phage particle of heterologous peptides or polypeptide with phage genome bacterial infection cell.This method has the advantage of " one step " process than first method.Yet, reduced the size that can successfully be packaged into the allogeneic dna sequence of final phage particle.The example that is applicable to the phage of this method has M13, T7 and lambda particles phage.

A kind of distortion of method B comprises that coding needs the DNA of the heterologous peptides of displaying to add the dna sequence dna of coding nucleotide binding domains in phage genome, and further adds corresponding nucleotide binding site to phage genome.This makes described heterologous peptides directly link to each other with phage genome.This peptide/genome complexes is packed into subsequently and is showed in the phage particle of heterologous peptides.Complete this method of having described among the WO 99/11785.

Can regain phage particle subsequently and be used to study described heterologous peptides or polypeptide in conjunction with feature.In case separate, phagemid or phage DNA can regain from the phage particle of displayed polypeptide or polypeptide, and this DNA can duplicate by PCR.The PCR product can be used for heterologous peptides or the polypeptide showed by specific phage particle are checked order.

Single-chain antibody and segmental phage display thereof have become the ordinary method of these polypeptide of research in conjunction with feature.There are multiple books that display technique of bacteriophage and phage biology (have for example been made summary, " phage display-laboratory manual " (the Phage Display-A Laboratory Manual that is shown referring to Barbas etc., 2001, Cold SpringHarbour Laboratory Press).

The third phage display method (method C) is based upon at the heterologous polypeptide that needs the position to have cysteine residues and can be expressed by phagemid or phage genome with soluble form, and causes with the improved phage surface albumen that also has cysteine residues in surperficial exposure position and be connected this fact by formation disulfide linkage between described two halfcystines.WO 01/05950 describes the use at this other method of attachment of single-chain antibody derived peptide in detail.

Summary of the invention

Natural TCR has the heterodimer of keeping as the membrane spaning domain of the required length of functional dimeric stability.As mentioned above, therefore the TCR of soluble form usefully for research and therapeutic purpose seldom adopts the displaying of insoluble natural form.On the other hand, the TCR of soluble stable form has been proved to be and has been difficult to design, and as if because most of methods of exhibiting only is described monomeric peptide and polypeptide, the display technique that is suitable for solvable dimer TCR is not also studied.And because the function of the TCR that shows depends on the correct connection of TCR dimer variable domains, the successful displaying of functional dimer TCR is not inappreciable.

WO 99/18129 comprises such statement: " according to the unsettled U. S. application No.08/813.781 that submitted on March 7th, 1997; it is open by with reference to being combined in the application's book; the DNA member of coding scTCR fusion rotein can be used for preparing phage display library ", but in this application, do not comprise the actual description of such displaying.Yet the contriver of this application has delivered one piece of paper (Weidanz (1998) J Immunol Methods 221 59-76), and two kinds of mouse scTCR displayings on phage particle have been described.

WO 01/62908 has opened the method that is used for scTCR and scTCR/Ig fusion rotein phage display altogether.Yet, the function (specificity pMHC combination) of disclosed member is not assessed.

At last, be used for the method for various TCR library at what mouse TCR had demonstrated retrovirus-mediated method at immature T cell surface display.The TCR library that is illustrated in the sudden change of prematurity T cell surface uses the pMHC tetramer to screen by flow cytometer, and this causes differentiating to similar pMHC TCR form special or its variant (Helmut etc., (2000) PNAS 97 (26) 14578-14583).

The present invention partly is based upon strand and dimer TCR can be with the formal representation that merges with proteinaceous particle surface, and can utilize on the discovery basis of the protein particulate of showing α/β analogue and gamma/delta analogue scTCR and dTCR member.The proteinaceous particle of showing TCR on this comprises the cell that the auto-polymerization particle forms albumen, phage, viral source albumen, ribosomal particle and has covalently bound cell surface protein of TCR or peptide molecule.The TCR that such protein particulate is showed is useful for purifying and screening purpose, and especially the diverse libraries of the TCR that shows as particle is used for biological elutriation and has such as the TCR that needs feature at target MHC-peptide complex high-affinity etc. with discriminating.With regard to the back this point, the scTCR that particle is showed can be used for the discriminating of required TCR, but described information may better be applied to the member of the analogue dimer TCR that finally is used for the treatment of.The present invention also comprises can be by the definite high-affinity TCR of these methods.

Detailed Description Of The Invention

One main aspect, the invention provides a kind of protein particulate, wherein at its surface display TXi Baoshouti (TCR)

(i) described protein particulate is that rrna and described TCR are that strand TCR (scTCR) polypeptide or dimer TCR (dTCR) polypeptide are right, or

(ii) described protein particulate is a phage particle, or have can with the covalently bound cell surface protein of TCR or the cell of peptide molecule, and described TCR is that people scTCR or people dTCR polypeptide are right, or

(iii) described protein particulate is a phage particle, or have can with the covalently bound cell surface protein of TCR or the cell of peptide molecule, and described TCR is that inhuman dTCR polypeptide is right, or

(iv) described protein particulate is a phage particle, or have can with the covalently bound cell surface protein of TCR or the cell of peptide molecule, and described TCR is the scTCR polypeptide, this polypeptide comprises the outer constant and corresponding TCR aminoacid sequence of variable domains sequence with the born of the same parents of natural TCR chain, connect with the corresponding variable domains sequence of variable domains sequence of a chain of natural TCR and with the catenation sequence of the corresponding constant domain sequence of constant domain sequence of another natural TCR chain, and the residue that connects the constant domain sequence, the disulfide linkage that in natural TXi Baoshouti, does not have Equivalent.

In a kind of embodiment preferred, it is a kind of right at a kind of dimer TXi Baoshouti of surface display (dTCR) polypeptide to the invention provides, or the protein particulate of a kind of single-chain T-cell receptor (scTCR) polypeptide, wherein said dTCR polypeptide to by be present in the outer constant and corresponding TCR aminoacid sequence of variable domains sequence of born of the same parents in the natural TCR chain and form, described scTCR is by forming with outer constant and corresponding aminoacid sequence of variable domains sequence of the born of the same parents of natural TCR chain and catenation sequence, described catenation sequence connect and the corresponding variable domains sequence of variable domains sequence of a chain of natural TCR and with the corresponding constant domain sequence of the constant domain sequence of another natural TCR chain; Wherein the dTCR polypeptide to or the mutual location of the variable domains sequence of scTCR polypeptide and the location basically identical among the natural TCR, and under the situation of described scTCR polypeptide, do not exist the disulfide linkage of Equivalent to connect the residue of described polypeptide among the natural TCR.Showing according to the present invention under the situation of α β scTCR or dTCR, to the mutual location of α and the segmental variable domains sequence of β and the requirement of the location basically identical among the natural α β TCR is the functional another kind of saying of TCR, and this can be by confirming described molecule and relevant TCR part (pMHC mixture, CD-1 antigenic compound, superantigen or superantigen/pMHC mixture) if combination detects-combination, and then this requirement is met.Can use Biacore 3000 with the interaction of pMHC mixture ^TMOr Biacore 2000 ^TMInstrument is measured.WO99/6120 provides and has analyzed TCR combines required method with the MHC-peptide complex detailed description.These methods can be applied to TCR/CD1 and TCR/ superantigen Study of Interaction in the same manner.For these methods are applied to the TCR/CD1 Study of Interaction, need the CD1 of soluble form, in Bauer (1997) EurJ Immunol 27 (6) 1366-1373, its production process has been described.Under the situation of gamma delta T CR of the present invention, the similar part of these molecules is unknown, thereby can use two level methods such as its conformations of checking such as antibody recognition.The special monoclonal antibody MCA991T (deriving from Serotec) of δ chain variable domains is the example that is suitable for the antibody of this task.

ScTCR of the present invention or dTCR can be illustrated on the protein particulate by for example following dual mode, for example on the phage particle, preferably on the filobactivirus particle:

(i) the right member's of dTCR polypeptide C-terminal, or the C-terminal of scTCR polypeptide, or the C-terminal of the small peptide connector that links to each other with arbitrary above-mentioned C-terminal can directly link to each other with the surperficial exposed residue of protein particulate by peptide bond.For example, described surperficial exposed residue is preferably located in the N-terminal of the gene product of phage gene III or gene VIII; And

The (ii) right member's of dTCR polypeptide C-terminal, or the C-terminal of scTCR polypeptide, or the cysteine residues of the C-terminal of the small peptide connector that links to each other with arbitrary above-mentioned C-terminal through importing links to each other by the halfcystine that disulfide linkage and protein particulate surface expose.For example, described surperficial exposed residue also is the N-terminal that is preferably located in the gene product of phage gene III or gene VIII.

Preferably above method (i).Under the situation of scTCR, the nucleic acid of coding TCR can merge as the nucleic acid that reproducible particulate particle such as phage or cell forms albumen or surface protein with coding.In addition, represent mRNA but do not have the nucleic acid of terminator codon or can translate by rrna, thereby described TCR keeps the fusion with ribosomal particle with the nucleic acid that tetracycline RNA merges.Under the situation of dTCR, the nucleic acid of a chain of coding TCR can form the nucleic acid fusion of albumen or cell surface protein with reproducible particulate particle such as coding such as phage or cell etc., and the right second chain of TCR polypeptide can link to each other with the particle of the final expression of showing article one chain.Article two, the correct functional connection of chain can be as more fully discussed in the following, and the halfcystine that can form interchain disulfide bond that exists in the constant domain by two chains is auxiliary.

The scTCR that shows

The scTCR polypeptide of showing can be the molecule that for example has following component:

First fragment of forming by the aminoacid sequence that merges with N-terminal corresponding to TCR α or δ chain variable domains sequence corresponding to the aminoacid sequence of the outer sequence of TCR α chain constant domain born of the same parents,

Second fragment of forming by the aminoacid sequence that merges with N-terminal corresponding to TCR β or γ chain variable domains sequence corresponding to the aminoacid sequence of the outer sequence of TCR β chain constant domain born of the same parents,

The catenation sequence that connects the first fragment C-terminal and the second fragment N-terminal, or vice versa, and

Article first and second, the disulfide linkage between the chain, there is not Equivalent in described disulfide linkage in natural α β or gamma delta T cells acceptor,

The length of catenation sequence and the position of disulfide linkage make the mutual location of the first and second segmental variable domains sequences and the location basically identical in natural α β or the gamma delta T cells acceptor.

In addition, the scTCR of displaying can be the molecule with following component:

By first fragment of forming corresponding to the aminoacid sequence of TCR α or δ chain variable domains,

Second fragment of forming by the aminoacid sequence that merges with N-terminal corresponding to TCR β or γ chain variable domains sequence corresponding to the aminoacid sequence of the outer sequence of TCR β chain constant domain born of the same parents, and

The catenation sequence that connects the first fragment C-terminal and the second fragment N-terminal,

Condition is that wherein said protein particulate is a phage, and described scTCR is corresponding to people TCR; Or

Molecule with following component

By first fragment of forming corresponding to the aminoacid sequence of TCR β or γ chain variable domains,

Second fragment of forming by the aminoacid sequence that merges with N-terminal corresponding to TCR α or δ chain variable domains sequence corresponding to the aminoacid sequence of the outer sequence of TCR α chain constant domain born of the same parents, and

The catenation sequence that connects the first fragment C-terminal and the second fragment N-terminal

Condition is that wherein said protein particulate is a phage, and described scTCR is corresponding to people TCR.

The dTCR that shows

The dTCR that is illustrated on the protein particulate can be a molecule composed of the following components:

Corresponding to the sequence of TCR α or δ chain variable domains sequence and corresponding to terminal first polypeptide that merges of the sequence of N of the outer sequence of TCR α chain constant domain born of the same parents, and

Corresponding to the sequence of TCR β or γ chain variable domains sequence and corresponding to terminal second polypeptide that merges of the sequence of N of the outer sequence of TCR β chain constant domain born of the same parents,

First and second polypeptide connect by the disulfide linkage that does not have Equivalent in natural α β or gamma delta T cells acceptor.

Preferably, described dTCR is illustrated on the filobactivirus and is molecule composed of the following components

Corresponding to the sequence of TCR α chain variable domains sequence and corresponding to terminal first polypeptide that merges of the sequence of N of the outer sequence of TCR α chain constant domain born of the same parents, and

Corresponding to the sequence of TCR β chain variable domains sequence and corresponding to terminal second polypeptide that merges of the sequence of N of the outer sequence of TCR β chain constant domain born of the same parents,

First and second polypeptide by replacing the TRAC*01 exons 1 Thr48 and the disulfide linkage between the cysteine residues of the Ser57 of the exons 1 of TRBCl*01 or TRBC2*01 or its non-human Equivalent connect,

Described dTCR polypeptide links to each other with the coat protein of phage by peptide bond to a member's C-terminal.

The dTCR polypeptide to the scTCR polypeptide

Being present in the outer sequence preference ground of constant domain born of the same parents among scTCR or the dTCR corresponding to these parts of people TCR, also is like this for the variable domains sequence.Yet the correspondence between such sequence does not need on the amino acid levels corresponding one by one.With respect to the people TCR sequence of N of correspondence-or C-brachymemma and/or aminoacid deletion and/or replacement allow.Especially do not participate in directly and the contacting of scTCR or dTCR bonded part because be present in the outer sequence of constant domain born of the same parents in first and second fragments, with respect to the outer constant domain sequence of the born of the same parents of natural TCR, they can be shorter, maybe can comprise to replace or disappearance.

Be present in the dTCR polypeptide to one of or scTCR polypeptide first fragment in the outer sequence of constant domain born of the same parents can comprise sequence corresponding to the outer constant Ig structural domain of born of the same parents of TCR α chain, and/or be present in the dTCR polypeptide and can comprise sequence corresponding to the outer constant Ig structural domain of born of the same parents of TCR β chain to the outer sequence of the constant domain born of the same parents in another member or scTCR polypeptide second fragment.

In one embodiment of the present invention, the member that the dTCR polypeptide is right, or first fragment of scTCR polypeptide, the TCR α chain that merges corresponding to the N-terminal with the abundant complete ectodomain of TCR α chain constant domain is complete variable domains fully; And/or the abundant complete variable domains of the TCR β chain that merges corresponding to N-terminal of right another member of polypeptide or second fragment with the abundant complete ectodomain of TCR β chain constant domain.

In another embodiment, be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in the outer sequence of constant domain born of the same parents corresponding to the constant domain of α and the β chain of the natural TCR of C-terminal brachymemma, thereby got rid of the cysteine residues that forms the natural interchain disulfide bond of TCR.In addition, these cysteine residues can be replaced such as amino-acid residues such as Serine or L-Ala by other, thereby have deleted natural disulfide linkage.In addition, natural TCR β chain comprises an azygous cysteine residues, and in the β of scTCR of the present invention sequence, this residue can be deleted or be replaced by non-cysteine residues.

In particular of the present invention, be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and β chain variable domains sequence can be jointly corresponding to the functional variable domains of first TCR, and be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequence of the constant domain born of the same parents of second TCR, first and second TCR are from identical species.Thereby, be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and β chain variable domains sequence can be corresponding to first kind of people TCR variable domains sequence, and α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequences of the constant domain born of the same parents of second kind of people TCR.For example, the outer sequence of A6Tax sTCR constant domain born of the same parents can be used as skeleton, can merge localized heterologous alpha and β variable domains on this.

In another embodiment of the invention, be present in respectively the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR δ and γ chain variable domains sequence can be jointly corresponding to the functional variable domains of first TCR, and be present in respectively the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequence of the constant domain born of the same parents of second TCR, first and second TCR are from identical species.Thereby, be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR δ and γ chain variable domains sequence can be corresponding to first kind of people TCR variable domains sequence, and α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequences of the constant domain born of the same parents of second kind of people TCR.For example, the outer sequence of A6Tax sTCR constant domain born of the same parents can be used as skeleton, can merge allos γ and δ variable domains on this.

In a kind of particular of the present invention, be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and β or δ and γ chain variable domains sequence can be jointly corresponding to the functional variable domains of first kind of people TCR, and be present in the dTCR polypeptide to or scTCR polypeptide first and second fragments in TCR α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequence of the constant domain born of the same parents of second kind of inhuman TCR.Thereby, being present in TCR α in first and second fragments of dTCR or scTCR polypeptide and β or δ and γ chain variable domains sequence can be corresponding to first kind of people TCR variable domains sequence, and α and the outer sequence of β chain constant domain born of the same parents can be corresponding to the outer sequences of the constant domain born of the same parents of second kind of inhuman TCR.For example, the outer sequence of mouse TCR constant domain born of the same parents can be used as skeleton, can merge allogenic human α and β TCR variable domains on this.

Connector in the scTCR polypeptide

For the protein particulate of displaying scTCR of the present invention, catenation sequence connects first and second TCR fragments to form single polypeptide chain.Described catenation sequence is passable, for example, has formula-P-AA-P-, and wherein P is a proline(Pro), and AA represents wherein, and amino acid is the aminoacid sequence of glycine and Serine.

For the scTCR that shows by protein particulate of the present invention with part (being the MHC-peptide complex under the situation of α β TCR) combines for, thereby first and second fragments are matched the location of its variable domains sequence and are suitable for such combination.Therefore, catenation sequence should have sufficient length to cross between the first fragment C-terminal and the second fragment N-terminal or the distance that vice versa.On the other hand, hinder or weaken under scTCR and the target ligands bonded situation in that the catenation sequence of variable domains sequence N-terminal is terminal, preferably should avoid the excessive length of catenation sequence.

For example, be present in therein the outer sequence of constant domain born of the same parents in first and second fragments corresponding to the C-terminal brachymemma with the α of the natural TCR that removes the cysteine residues that forms natural interchain disulfide bond and the constant domain of β chain, and catenation sequence connects under the situation of the first segmental C-terminal and second sequence of N end.

Catenation sequence can comprise for example 26-41 amino acid, preferably 29,30,31 or 32 amino acid, or 33,34,35 or 36 amino acid.Specific connector has formula-PGGG-(SGGGG) ₅-P-and-PGGG-(SGGGG) ₆-P-, wherein P is a proline(Pro), G is a glycine, and S is a Serine.

Interchain disulfide bond

A preferred dTCR that shows by protein particulate of the present invention and the characteristic element of scTCR be the dTCR polypeptide between or disulfide linkage between scTCR first and second fragments.Disulfide linkage can be corresponding to the natural interchain disulfide bond that is present among the natural dimer α β TCR, or can in natural TCR, not have counterpart, be present in specific combination go into the dTCR polypeptide to or the halfcystine of the outer sequence of scTCR polypeptide first or the second segmental constant domain born of the same parents between.In some cases, natural and non-natural disulfide linkage all may need.

The position of disulfide linkage satisfy the dTCR polypeptide to or the fully mutual localized requirement in similar natural α β or the gamma delta T cells acceptor of the scTCR polypeptide first and second segmental variable domains sequences.

Disulfide linkage can sport halfcystine by non-cysteine residues on first and second fragments, and the key that causes between the sudden change residue is formed.Among the natural TCR its separately the β carbon atom separately be about

Or littler, and be preferably located in

Arrive

Interval in residue be preferred, can between the cysteine residues that imports on the position of natural residue, form disulfide linkage like this.Though can be between the residue in nearly membrane structure territory, if preferably between the residue of disulfide linkage in constant immunoglobulin domains.Can import halfcystine and be the following residue in the exons 1 of TRAC*01 and TRBCl*01 TCR β chain or TRBC2*01 of TCR α chain with the optimum position that forms disulfide linkage:

TCR α chain	TCR β chain	Natural β carbon atom is (nm) at interval
			Thr 48 Thr 45 Tyr 10 Thr 45 Ser 15	Ser 57 Ser 77 Ser 17 Asp 59 Glu 15	0.4730.5330.3590.5600.59

Following motif in the corresponding separately people TCR chain can be used to differentiate the residue (the dash area residue is the residue that is mutated into halfcystine) that suddenlys change.

α chain Thr 48:DSDVYITDKTVLDMRSMDFK (the 39-58 amino acids of the exons 1 of TRAC*01 gene) (SEQ ID 1)

α chain Thr 45:QSKDSDVYITDKTVLDMRSM (the 36-55 amino acids of the exons 1 of TRAC*01 gene) (SEQ ID 2)

α chain Tyr 10:DIQNPDPAVYQLRDSKSSDK (the 1-20 amino acids of the exons 1 of TRAC*01 gene) (SEQ ID 3)

α chain Ser 15:DPAVYQLRDSKSSDKSVCLF (the 6-25 amino acids of the exons 1 of TRAC*01 gene) (SEQ ID 4)

β chain Ser 57:NGKEVHSGVSTDPQPLKEQP (TRBCl*01﹠amp; The 48-67 amino acids of the exons 1 of TRBC2*01 gene) (SEQ ID 5)

β chain Ser 77:ALNDSRYALSSRLRVSATFW (TRBCl*01﹠amp; The 68-87 amino acids of the exons 1 of TRBC2*01 gene) (SEQ ID 6)

β chain Ser 17:PPEVAVFEPSEAEISH TQKA (TRBCl*01﹠amp; The 8-27 amino acids of the exons 1 of TRBC2*01 gene) (SEQ ID 7)

β chain Asp 59:KEVHSGVSTDPQPLKEQPAL (TRBCl*01﹠amp; The 50-69 amino acids of the exons 1 of TRBC2*01 gene) (SEQ ID 8)

β chain Glu 15:VFPPEVAVFEPSEAEISHTQ (TRBCl*01﹠amp; The 6-25 amino acids of the exons 1 of TRBC2*01 gene) (SEQ ID 9)

In other species, the TCR chain may not have the zone identical with above motif 100%.Yet, be proficient in those skilled in the art and can use above motif to differentiate the residue that is mutated into halfcystine of partly also differentiating thus of equal value of TCR α or β chain.Can use the alignment permutation technology on the one hand at this.For example, European information biology institute website ( Http:// www.ebi.ac.uk/index.html) on the ClustalW that provides can be used for specific T CR chain-ordering and above motif comparison are positioned with the TCR sequence relevant portion to the needs sudden change.

The present invention comprises α β and the γ δ analogue scTCR that protein particulate is showed in its scope, and other mammiferous α β and γ δ analogue scTCR, and these Mammalss include but not limited to mouse, rat, pig, goat and sheep.That as the above mentioned, is proficient in that those skilled in the art should be able to determine to be equivalent to above-mentioned people can import the position of cysteine residues with the position that forms interchain disulfide bond.For example, the aminoacid sequence of mouse C α and the solvable structural domain of C β has been described below, and show be equivalent to above-mentioned people can be mutated into the motif (wherein relevant residue shadow representation) of halfcystine with the mouse residue of the residue that forms the TCR interchain disulfide bond:

The solvable structural domain of mouse C α:

PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMK

AMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVP(SEQ ID 10)

The solvable structural domain of mouse C β:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGREV

HSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDK

WPEGSPKPVTQNISAEAWGRAD(SEQ ID 11)

The mouse Equivalent of people α chain Thr 48: ESGTFITDKTVLDMKAMDSK (SEQ ID 12)

The mouse Equivalent of people α chain Thr 45: KTMESGTFITDKTVLDMKAM (SEQ ID 13)

The mouse Equivalent of people α chain Tyr 10: YIQNPEPAVYQLKDPRSQDS (SEQ ID 14)

The mouse Equivalent of people α chain Ser 15: AVYQLKDPRSQDSTLCLFTD (SEQ ID 15)

The mouse Equivalent of people β chain Ser 57: NGREVHSGVSTDPQAYKESN (SEQ ID 16)

The mouse Equivalent of people β chain Ser 77: KESNYSYCLSSRLRVSATFW (SEQ ID 17)

The mouse Equivalent of people β chain Ser 17: PPKVSLFEPSKAEIANKQKA (SEQ ID 18)

The mouse Equivalent of people β chain Asp 59: REVHSGVSTDPQAYKESNYS (SEQ ID 19)

The mouse Equivalent of people β chain Glu 15: VTPPKVSLFEPSKAEIANKQ (SEQ ID 20)

As mentioned above, the outer constant domain of A6Tax scTCR born of the same parents can be used as skeleton, can merge the allos variable domains on this.Preferably, any position of allos variable domains sequence between disulfide linkage and constant domain sequence of N end is connected with the constant domain sequence.Under the situation of A6Tax TCR and β constant domain sequence, form disulfide linkage between the halfcystine that can import respectively at amino-acid residue 158 and 172 places.Therefore, if preferably allogenic α and β chain variable domains sequence link position lay respectively between 159 or 173 residues and α or the β constant domain sequence of N end.

TCR shows

Being used for biological elutriation has as being phage display at TCR methods of exhibiting in the preferred body of target peptide-required character such as MHC mixture high-affinity with discriminating.

At first, make up the DNA library of the diversity array of the scTCR of the sudden change of having encoded or dTCR.This library makes up as the DNA of the natural TCR of amplification template by using coding.Thereby at desired mutant being imported TCR DNA and finally express TCR albumen, exist multiple for being proficient in those skilled in the art known suitable method.For example fallibility PCR (EP-PCR), DNA reset technology and as the use of bacterium mutator strains such as XL-1-Red be in the TCR sequence, to import the ordinary method of suddenling change.Especially preferably, if these mutant import in the clear and definite structural domain of TCR.For example, the sudden change in the variable domains, especially complementary determining region (CDR) and/or skeleton district cause generating the various library of TCR to have the optimal position of the part of raising in conjunction with the TCR sudden change of character with generation.EP-PCR is an example that such " regiospecificity " sudden change can be imported TCR by this method.This comprises the multiple copied in the TCR DNA zone of controlled horizontal random mutation to the dna sequence dna complementary EP-PCR primer of the zone adjacency of using and needing to suddenly change to increase.By connecting or overlapping PCR, the dna sequence dna in these encoding mutant zones inserts the do not suddenly change dna sequence dna of part of coding TCR.The DNA that coding contains the regional TCR that suddenlys change can be connected the fusion rotein that is fit to displaying to produce with the DNA of coding heterologous polypeptide then.Under the situation of phage display, the expression vector of employing is phagemid or phage genome carrier, and wherein TCR DNA can be proteic with coded surface, and preferably the DNA of gIII or gVIII surface protein connects.Under the situation of scTCR, such connection is as carrying out for the phage display of any monomeric peptide or polypeptide.Under the situation of dTCR, have only a TCR chain to connect as the aforementioned.Another chain encoding with the nucleic acid of phagemid and helper phage nucleic acid coexpression in, the second chain of Biao Daing links to each other with the phage of the expression of article one chain with surface display like this.Describe in more detail as above, in both cases, correct localized halfcystine is by forming disulfide linkage by these residues in the constant domain, and it is helpful adopting in its functional location in the variable domains that causes TCR.

As for expression, comprise the nucleic acid of the chain that (a) coding dTCR polypeptide is right, and another the right chain of TCR polypeptide that (b) forms the nucleotide sequence fusion of albumen or cell surface protein with the coding particle; Or form the nucleic acid of the coding scTCR polypeptide that the nucleotide sequence of albumen or cell surface protein merges with the coding particle, dTCR is right, or comprises first carrier that is made of nucleic acid (a) and the expression vector of the composition of second carrier that is made of nucleic acid (b) contacts at the host cell that is suitable for expressing under the condition of described cell transformation with the genetic material that can cause coding.Such expression vector, expression system comprise phagemid or the phage genome carrier of coding dTCR and scTCR, and the host cell that carries them forms additional aspect of the present invention.In embodiment preferred of the present invention, phagemid or phage genome support source are from filobactivirus.

Cell transformed is hatched the expression with the protein particulate that allows the TCR displaying subsequently.These particles can be used to screen or test the TCR form that has specific enhancing characteristic with discriminating subsequently.Can separate subsequently and have the particle that strengthens characteristic in any research.The DNA of these TCR of encoding can and determine its sequence by pcr amplification subsequently.

The high expression level of known allogenic polypeptide may be deleterious for host cell.Under these circumstances, must find host's strain that allogenic polypeptide is more tolerated, or expression level must be limited in the level that can tolerate in the host cell.For example, Beekwilder etc. has reported that in (1999) Gene 228 (1-2) 23-31 the potato proteinase inhibitor (PI2) of the mutant form that only contains disappearance or amber terminator codon can successfully obtain selecting from phage display library.Under described situation, the observations explanation in the working process of reporting among the embodiment here, in more colibacillary bacterial strains, the expression level that limits the TCR of protein grain displaying of the present invention may need at least.Like this, cultivate the A6TCR that select among the embodiment 4 of back and be derived from wherein phagemid repeating several the wheel with respect to the cell of having undergone mutation of initial importing." milky white " terminator codon has been created in this sudden change in TCR β chain.This codon is " to read to lead to " integral level that causes inserting the obvious reduction that tryptophan residue and total length β chain express on this site with low frequency by the rrna of the coli strain that is adopted.

Exist multiple in the host of one or two the TCR chain expression level that may be suitable for limiting scTCR or dTCR the strategy of restriction particular expression system allogenic polypeptide expression level.For example:

Use the expression level such as the specific gene product of TCR α or β chain of weak promoter sequence-acquisitions, can customize by the promoter sequence of use varying strength.Lambda particles phage P _RMIt is the example of weak promoter.

The ribosome bind site (RBS ' s) of sudden change-suddenly change with single nucleic acid among the RBS that links to each other such as gene products such as TCR α or β chains, this can cause the expression level that reduces.For example, wild-type AGGA series jump is become AGGG.

" initiator codon " of sudden change-will suddenly change with the initiator codon of the single nucleic acid that links to each other such as gene products such as TCR α or β chains also to cause the expression level that reduces.For example, the initial son of wild-type AUG is sported GUG.

Inside, TCR β chain encoding district is inserted in missense mutation inhibiting-these sudden changes.Example comprises " milky white " terminator (UGA), the terminator of this " imprecision " cause the low frequency of tryptophane to insert and the encoding sequence rest part read lead to.

The promotor intensity adjustments of metabolite mediation-being in some promotor control expression level such as gene products such as TCR α or β chains down can be by reducing to the cell interpolation associated metabolic thing that contains this promotor.For example, can use the downward modulation of interpolation glucose to be in the expression of the gene product under the control of Lac promotor.

Its relative with for example mammalian cell of codon use-bacterial cell certain amino acid whose codon that is used to encode has " preference ".For example, the most common use of bacterial cell CGU codon coding arginine, and the most common use of eukaryotic cell AGA.The dna sequence dnas of codon that comprise a plurality of low preferences of the expression vector that is adopted by employing can reduce the expression level such as gene products such as TCR α or β chains.

Expressing relevant detailed description with above-mentioned down-regulated gene product can be referring to " gene function-intestinal bacteria and hereditary element thereof " (Gene Function-E.coli and its heritableelements, Croom Helm) and Rezinoff " operon " the 2nd edition (The Operon 2 that show in 1980 that Glass1982 showed ^NdEdit ion, Cold Spring Harbor Laboratory).

Known equally, as to provide the sugar (such as sucrose) of relative high density can improve soluble protein for bacterial cultures periplasmic expression level (referring to for example Sawyer etc., (1994) Protein Engineering 7 (11) 1401-1406).

After the expression, the correct pairing of scTCR polypeptide variable domains sequence is preferably assisted by the importing of disulfide linkage in the outer constant domain of the born of the same parents of scTCR.Bound by theory does not believe that disulfide linkage new in folding process provides extra stability for scTCR, thereby helps the first and second segmental correct pairings.

As mentioned above equally, for the dTCR phage display, the dTCR polypeptide to one of finally as the monomer polypeptide is illustrated in phage, express and right another chain coexpression in identical host cell of dTCR polypeptide.Along with the phage particle auto-polymerization, described two polypeptide oneself connection is illustrated on the phage as dimer.In addition, in this preferred embodiment on the one hand of the present invention, polypeptide is assisted by the disulfide linkage between the constant series as mentioned above to correct the folding in the connection procedure.Further details the embodiment here of phage display process with dTCR of interchain disulfide bond partly illustrates.

In addition, article one chain of the dTCR of phage display can at first be expressed, and second chain polypeptide can be in step subsequently contact with the phage of expressing, thereby is connected to functional dTCR at phage surface.

Ribosomal display is to be used for biological elutriation to have as needing the preferred external TCR methods of exhibiting of the TCR of character at target peptide-MHC mixture high specific etc. with discriminating.At first, the DNA library of the diversity array of the used above-mentioned technique construction scTCR of encoding mutant or dTCR polypeptide.The DNA library contacts with RNA polymerase subsequently to produce complementary mRNA library.Selectively, for the mRNA display technique, the mRNA sequence can be connected with the dna sequence dna that comprises the tetracycline binding site subsequently.These hereditary members contact with rrna is external under the condition that allows right first polypeptide translation of described scTCR polypeptide or dTCR subsequently.Under the situation of dTCR, the second right polypeptide of polypeptide is independently expressed and is contacted with first polypeptide of ribosomal display, for the combination of two chains, is preferably assisted by forming of disulfide linkage between the constant domain.In addition, the mRNA of two chains of coding TCR can contact with rrna is external under the condition that allows the translation of TCR chain, has formed the rrna of showing dTCR thus.These show that the rrna of scTCR or dTCR can be used to screen or test with discriminating have the TCR variant that specificity strengthens characteristic subsequently.Can separate any particle that strengthens characteristic that has subsequently.The mRNA of these TCR of encoding can use reversed transcriptive enzyme to convert the complementary dna sequence dna to subsequently.This DNA can increase and definite its sequence by PCR subsequently.

Others

The protein particulate of a kind of scTCR of displaying of the present invention or a kind of dTCR (preferably being made up of the constant and variable sequence corresponding to people's sequence) can provide with abundant purified form or with the form of purifying or isolating preparation.For example, can be to provide with the abundant isolating form of other protein.

The phage particle of multiple scTCR of displaying of the present invention or multiple dTCR can provide with the form of multivalence mixture.Thereby the present invention provides multivalent T cell receptor (TCR) mixture in one aspect, and this mixture comprises the phage particle of showing multiple scTCR or dTCR as described herein.Each of described multiple scTCR or dTCR is preferably identical.

Further aspect of the present invention provides the method that detects the TCR ligand complex, and this method comprises:

A., the protein particulate of a kind of TCR of displaying of the present invention is provided;

B. the phage that will show TCR contacts with the ligand complex of supposition; And

The protein particulate of detection displaying TCR combines with the ligand complex of supposition.

Be suitable for including but not limited to peptide-MHC mixture by the TCR part that above method is differentiated.

Separation with the TCR variant that strengthens characteristic

Further aspect of the present invention is to differentiate the method for the TCR with particular characteristics, described method comprises that the diverse libraries that is illustrated in the TCR on the protein particulate stands to select the chosen process of described characteristic, and the protein particulate of the TCR with described characteristic is showed in separation, and selectively carry out amplification procedure to increase isolating particle, and/or measure the screening process of described characteristic, differentiate those displayings have desired characteristic TCR protein particulate and separate these protein particulates, and selectively carry out amplification procedure to increase isolating particle.

The dna sequence dna of variant TCR and increase to measure sequence by PCR subsequently can obtain to encode.Can the enhanced characteristic include but not limited to ligand binding affinity and component stability.

The screening purposes

Adopt in the screening method that the protein particulate of displaying TCR of the present invention can design at the conditioning agent of the inhibitor that comprises the cellular immunization cynapse that TCR mediates for discriminating.

As be proficient in known to the those skilled in the art, exist the multiple screening that for this reason interacts between the proteinoid that the test form on suitable basis is provided.

As AlphaScreen ^TMLuminous the getting close to of enhancement type even measure system (Amplified LuminescentProximity Homogeneous Assay systems) mutually, depending on can bind receptor and the use of " donor " and " acceptor " globule of the hydrogel bag quilt of ligandin with one deck.Interaction between these acceptors and the ligand molecular furthers these globules.When these globules suffered laser, the photosensitizers in " donor " globule was transformed into the single line state that more intensifies with oxygen on every side.The diffusion of the oxygen molecule of singlet with " acceptor " globule in chemoluminescence agent reaction, this further activates the fluorophore that is included in this globule.Fluorophore is launched the light of 520-620nm subsequently, and this is that interactional signal has taken place receptor-ligand.The existence of the inhibitor of receptor-ligand binding causes this signal weakening.

Surface plasma resonance (Surface Plasmon Resonance, SPR) be a kind of critical surface optical detecting method, wherein a kind of binding partners (normally acceptor) is fixed on " chip " (sensor surface) and the detection another kind is soluble and the combination of the binding partners (normally part) of the chip surface of flowing through.The combination of part causes near the raising of the protein concentration of chip surface, and this causes the change of specific refraction in this zone.The formation of chip surface can detect the change of specific refraction by surface plasma resonance, surface plasma resonance is a kind of optical phenomena, is radiated at the light on the thin metal layer because the resonant excitation of surface charge density (surface plasma) ripple of vibration produces the reflected beam that intensity reduces with certain input angle thus.Resonance changes extremely sensitivity to the specific refraction in metallic membrane distally, and this is to be used to detect bonded signal between fixed and the soluble albumen.Allow the convenient SPR detection of interaction of molecules and the system of data analysis of using that commercial stock is arranged.Example is Iasys ^TMDevice (Fisons) and Biacore ^TMDevice.

Other critical surface optical detecting method comprises Woodbury and Venton total internal reflection fluorescent (TIRF), resonant mirror (RM) and grating coupling sensor (GCS) in greater detail in J.Chroma tog.B.725113-137 (1999).Get close to flicker detection (SPA) and be used for the interactional inhibitor (K of low-affinity between CD28 and the B7 _dBe about 4 μ M (Van der Merwe etc., J.Exp.Med.185:393-403 (1997), Jenh etc., AnalBiochem 165 (2) 287-93 (1998)) and the SCREENED COMPOUND storehouse.SPA is a kind of radioactivity measurement method that is used to from some radioisotopic β ray energy is transferred to the scintillator that is fixed on indicator surface.The short transmission of beta-particle just can be glimmered apart from having guaranteed to have only when beta-particle is transmitted to scintillator in extremely approaching scope in the solution.When being used for interacting between the protein, a kind of interacting partner labelled with radioisotope, and another kind of or combine with the globule that comprises scintillator, or be coated on the surface jointly with scintillator.If this assay method can optimum be provided with, radio isotope should be enough approaching with scintillator so that have only that photo emissions just is activated when combining between two kinds of protein.

A further aspect of the present invention is to differentiate the method for interactional inhibitor between the protein particulate of displaying TCR of the present invention and the TCR binding partner, this method is included under testing compound existence or the non-existent condition will show that the protein particulate of TCR contacts with the TCR binding partner, and whether the existence of definite testing compound reduces and shows that the protein particulate of TCR and combining of TCR binding partner, such reduction are thought and differentiated a kind of inhibitor.

A further aspect of the present invention is to differentiate the method for interactional potential inhibitor between the protein particulate of displaying TCR of the present invention and the TCR binding partner (for example MHC-peptide complex); this method comprises that protein particulate or the TCR binding partner companion that will show TCR contact with testing compound; and whether definite testing compound in conjunction with protein particulate and/or the TCR binding partner of showing TCR, and such combination is thought and differentiated a kind of potential inhibitor.Of the present invention this on the one hand can be such as using Biacore ^TMObtain specific application in those critical surface optical detecting methods that system carries out.

High-affinity TCR

The present invention also provides to have at specific T CR part than the wild-type TCR TCR of the sudden change of high-affinity more.Estimate that these high-affinities TCR has special purpose for the diagnosis and the treatment of disease.

Term as used herein " high-affinity TCR " be meant with concrete TCR ligand interaction and as having of measuring by surface plasma resonance at as described in the K of TCR part _dValue less than the Kd value of corresponding natural TCR or as having of measuring by surface plasma resonance at as described in wear rate (off-rate, the K of TCR part _Off) less than the scTCR and the dTCR of the sudden change of the wear rate of corresponding natural TCR.

High-affinity scTCR of the present invention or dTCR preferably undergo mutation at least one complementary determining region and/or skeleton district with respect to natural TCR.

In one aspect of the invention, it is had specific TCR part is peptide-MHC mixture (pMHC) to a kind of specific high-affinity TCR.

Another aspect of the present invention, it is the MHC classification that a kind of specific high-affinity TCR has specific TCR part to it.

A further aspect of the present invention, it is HLA-A2tax peptide (LLFGYPVYV) (SEQ ID 21) mixture that a kind of specific high-affinity TCR has specific TCR part to it.

A further aspect of the present invention, it is HLA-A2NY-ESO peptide (SLLMITQC) (SEQ ID 22) mixture that a kind of specific high-affinity TCR has specific TCR part to it.

High-affinity scTCR or high-affinity dTCR chain one or two chains can be with radiography compound (for example being suitable for the marker of diagnostic purpose) marks.The high-affinity TCR through mark like this is useful in the method that is used for detecting the TCR part that is selected from CD-1 antigenic compound, bacterial superantigen and MHC-peptide/superantigen mixture, and this method comprises that the high-affinity TCR that TCR part and this part is special (or many heights affinity TCR mixture) contacts; And the combining of detection and described TCR part.In four poly-high-affinity TCR mixtures, (for example use biotinylated heterodimer to form) and can use fluorescence streptavidin (commercial stock) that detectable mark is provided.The fluorescently-labeled tetramer is suitable for using in facs analysis, for example carries high-affinity TCR in order to detection it is had the antigen presenting cell of specific peptide.

The another kind of mode that can detect solubility high-affinity TCR of the present invention is by the TCR specific antibody, the especially use of monoclonal antibody.The anti-TCR antibody that exists multiple commerce to provide, as α F1 and β F1, they discern the constant domain of α and β chain respectively.

In addition or in addition, high-affinity TCR of the present invention (or its multivalence mixture) can with a kind of therapeutic substance (for example, the toxicity part that is used for killer cell, or as the immuno-stimulating material of interleukin or cytokine) link to each other (for example covalency or other modes connect).Multivalence high-affinity TCR mixture of the present invention is compared with the wild-type of non-many bodies or the TCR heterodimer of high-affinity, can have enhanced and TCR part bonded ability.Thereby, according to multivalence high-affinity TCR mixture of the present invention in the body of offering the specific antigen cell or external tracking or target particularly useful, and can be used as the intermediate that generation further has the multivalence high-affinity TCR mixture of such purposes.Therefore, high-affinity TCR or multivalence high-affinity TCR mixture can be used as the prescription that allows in the pharmacy of using in the body provides.

The present invention also provides a kind of method to target cell delivery of therapeutic material, this method is included in and allows under described high-affinity TCR or multivalence high-affinity TCR mixture and the described target cell bonded condition, with the potential target cell with contact according to high-affinity TCR of the present invention or multivalence high-affinity TCR mixture, described high-affinity TCR or multivalence high-affinity TCR mixture are that the TCR part is special and have a coupled therapeutic substance.

Especially, soluble high-affinity TCR or multivalence high-affinity TCR mixture can be used for to the location delivery therapeutic substance of offering the specific antigen cell.This when antitumor, may be useful especially under multiple situation.Therapeutic substance can be sent like this so that its part is exercised its effect rather than only acted on its bonded cell.Like this, imagined a kind of special strategy of the antibumor molecules that links to each other with tumour-specific high-affinity TCR or multivalence high-affinity TCR mixture.

Many therapeutic substances can be used for this purposes, for example radioactive compound, enzyme (for example pore-forming protein) or chemotherapy material (for example cis-platinum).For guaranteeing that toxin can be positioned at the liposome interior that links to each other with streptavidin at desired location performance toxic action, this compound is slowly discharged like this.This can prevent the destruction in the interior transportation of body and guarantee that toxin has maximum effect after TCR combines with the related antigen presenting cells.

Other suitable therapeutic substances comprise:

Small molecules cell toxicant material, promptly molecular weight kills and wounds the compound of mammalian cell ability less than having of 700Da.Such compound also can comprise the toxic metal with cytotoxicity.In addition, need to understand, these small molecules cell toxicant materials also comprise prodrug, promptly decompose under physiological condition or transform to discharge the compound of cell toxicant material.Such examples of substances comprises cis-platinum, the maytenin derivative, but thunder mycin (rachelmycin), calicheamycin (calicheamicin), docetaxel, Etoposide, gemcitabine, ifosfamide, Rinotecan, melphalan, mitoxantrone, the non-nurse sodium of porphin phytochrome II (porfimer sodium photofrinII), Temozolomide (temozolmide), Hycamtin, trimetrexate glucuronic acid fat (trimetrexate glucuronate), auristatin E vincristine(VCR) (auristatin E vincristine) and Zorubicin;

The cytotoxin peptide promptly has protein or its fragment of killing and wounding the mammalian cell ability.Example comprises Ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme and RNA enzyme;

Radionuclide, i.e. the unstable isotope of one or more α or beta-particle or gamma-ray element is also launched in decay simultaneously.Example comprises iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; Sequestrant can be used to promote these radionuclides and high-affinity TCR or its polymeric combination;

Prodrug is as the enzyme prodrug of antibody guiding;

Immunostimulant, the i.e. part of immune response stimulating.Example comprises as IL-2, as the cytokine of IL-8, platelet factor 4, the proteic chemokine of melanoma growth-stimulating or the like, antibody or its fragment, the complement activation factor, foreign protein structural domain, the heterologous protein structural domain, virus/bacterioprotein structural domain and virus/bacterial peptide.

Solvable high-affinity TCR of the present invention or multivalence high-affinity TCR mixture can be connected with the enzyme that prodrug can be changed into medicine.This makes that this prodrug is medicine (promptly by sTCR target) in the position transition of needs only.

The treatment of numerous diseases can be by being strengthened through the specific medicine localization of solvable high-affinity TCR potentially.For example, estimate that the special A6TCR of high-affinity HLA-A2-tax disclosed herein (LLFGYPVYV) (SEQ ID 21) can use in the diagnosis of HTLV-1 and methods of treatment, and the special NY-ESO TCR of high-affinity HLA-A2-NY-ESO disclosed herein (SLLMITQC) (SEQ ID 22) can use in the diagnosis of cancer and methods of treatment.

Exist the virus disease (for example HIV, SIV, EBV, CMV) of medicine may have benefited near cells infected, discharging or the activated medicine.For cancer, near the location tumour or the metastases can strengthen the effect of toxin or immunostimulant.In autoimmune disease, immunosuppressive drug can slowly discharge, and has on longer time span the effect of locality more like this and minimum to the whole immunological competence influence of main body.Preventing that under the situation of transplant rejection, the interaction energy of immunosuppressive drug is optimized with the same manner.For vaccine delivery, vaccine antigen can be confined near the antigen presenting cell, thus the effect of enhancement antigen.This method also can be applied to the radiography purpose.

Solvable high-affinity TCR of the present invention can thereby the suppressor T cell activation be used to regulate the T cell activation by combine also with the specific TCR part.The autoimmune disease (for example type i diabetes) that relates to cell-mediated inflammation of T and/or tissue injury may be handled according to this method.For this application, the knowledge of the special peptide epitopes that need offer by relevant pMHC.

High-affinity TCR according to therapeutic of the present invention or radiography provides as the aseptic part of pharmaceutical compositions that should comprise the carrier that allows in the pharmacy usually usually.This pharmaceutical composition can be (the depending on the needed method of patient's administration) of any suitable form.It can provide with the form (should provide in sealed vessel usually) of unitary dose with as the part of test kit.Such test kit can (though not necessarily) comprise instruction manual usually.It can comprise a plurality of described unit dosage forms.

Described pharmaceutical composition can be adapted to pass through any suitable administration, for example injection, transdermal or by sucking, preferably injection (comprising subcutaneous injection, intramuscular injection or most preferably intravenous injection) approach.Such composition can be prepared by known method in any pharmaceutics field, for example passes through under aseptic condition active ingredient and carrier or mixed with excipients.

The age of the individuality that depend on the disease or the disorder that need treatment, needs treatment and healthy state or the like factor, the dosage of material of the present invention can change in wide scope, and the suitable dosage that should final decision uses of doctor.

The present invention also provides the method that obtains high-affinity TCR chain, and this method is included in and causes and hatch such host cell and isolated polypeptide subsequently under the condition that high-affinity TCR chain expresses.

The preferable feature of each aspect of the present invention is as necessary modification is made in each other aspect.Here the technical literature of mentioning formerly is combined in the lawful most complete scope.

Embodiment

Further described the present invention in following examples, these embodiment do not limit the scope of the invention by any way.

Below accompanying drawing is illustrated, wherein:

Fig. 1 a and 1b represent respectively to suddenly change with the soluble A6TCR α chain of introducing halfcystine codon and the nucleotide sequence of β chain.The halfcystine codon that shadow representation is introduced.

Fig. 2 a represents to comprise the T that is used to produce new interchain disulfide bond ₄₈The outer aminoacid sequence of the A6TCR α chain born of the same parents of → C sudden change (underlined), Fig. 2 b represents to comprise the S that is used to produce new interchain disulfide bond ₅₇The outer aminoacid sequence of the A6 TCR β chain born of the same parents of → C sudden change (underlined).

Fig. 3 has described TCR α and β chain has been cloned into phagemid carrier.Chart has been described Vector for Phage Display.RSB is a ribosome bind site.S1 or S2 are used for the signal peptide of protein secreting to colibacillus periplasm.Asterisk * represents translation stop codon.Any chain of TCR α chain or β chain can merge with the phage coat protein, though have only TCR β chain and phage coat protein to merge in the described chart.

Fig. 4 has described the dna sequence dna of phagemid pEX746:A6 in detail.

The expression of phage particle fusion rotein in intestinal bacteria of Fig. 5 bacterium coat protein and allos dimerization A6TCR.The fusion rotein of allos dimerization A6TCR:gIII uses immunoblotting to detect.Phage particle concentrates by intestinal bacteria XL-1-Blue preparation and with PEG/NaCl.Sample is carried in reduction or the non-reduced sample loading buffer.Swimming lane 1 is the sample that contains correct sequence clone 7, and swimming lane 2 is the samples that contain the clone 14 of disappearance in α chain encoding gene.Allos dimerization A6TCR:gIII fusion rotein is examined at the 125kDa place.

Fig. 6 has illustrated that the pMHC peptide complex of the allos dimerization A6TCR that is illustrated on the phage detects in conjunction with active ELISA.Clone 7 specificitys in conjunction with HLA A2-Tax mixture.Clone 14 can not be in conjunction with any pMHC, because there is not TCR to combine with this phage particle.

The schematic illustrations of Fig. 7 a strand A6TCR-C-κ DNA ribosomal display member.

Fig. 7 b and 7c have described complete the dna encoding chain and the aminoacid sequence of the strand A6TCR-C-κ DNA ribosomal display member of encoding among the pUC19 respectively in detail.

Fig. 8 has described the dna sequence dna of pUC19-T7 in detail.

Fig. 9 has described the dna sequence dna of the strand A6TCR-C-κ DNA ribosomal display member of being cloned among the pUC19-T7 in detail.

Figure 10 shows the immunoblotting that detects by the in vitro translated strand A6TCR-C-κ that uses Ambion rabbit skein cell lysate.

Figure 11 derives from the RT-PCR of the strand A6TCR-C-κ mRNA on the globule of ribosomal display reaction.

Figure 12 a has described the dna sequence dna of the β chain of A6TCR clone 9 sudden changes in detail; The nucleic acid of sudden change is represented with runic.

Figure 12 b has described the aminoacid sequence of the β chain of A6TCR clone 9 sudden changes in detail, represents with asterisk * corresponding to the position of the opal temination codon of introducing.

Figure 13 has described the dna sequence dna of the β chain of A6TCR clone 49 sudden changes in detail; The nucleic acid of sudden change is represented with runic.Because this is a kind of " silence " sudden change, does not introduce variation by this sudden change in the final aminoacid sequence.

Figure 14 a has described the dna sequence dna of the A6TCR β chain of A6TCR clone 134 sudden changes in detail; The nucleic acid of sudden change is represented with runic.

Figure 14 b has described the aminoacid sequence of the A6TCR β chain of A6TCR clone 134 sudden changes that detect by the BIAcore assay method in detail; The amino acid of sudden change is represented with runic.

Figure 14 c has described the amino acid of the A6TCR β chain of A6TCR clone 134 sudden changes that detect by phage E LISA assay method in detail; The amino acid of sudden change is represented with runic.

Figure 15 A6TCR clone 134 and HLA-A2Tax and HLA-A2NY-ESO bonded BIAcore data.

Figure 16 is used to measure A6TCR clone 134 and HLA-A2Tax bonded T _OffThe BIAcore data.

Figure 17 a and 17b represent the α of sudden change of NY-ESO TCR and the dna sequence dna of β chain respectively.

Figure 18 a and 18b represent the α of sudden change of NY-ESO TCR and the aminoacid sequence of β chain respectively.

Figure 19 a and 19b have described the DNA and the aminoacid sequence of the NY-ESO TCR β chain that is combined in the pEX746:NY-ESO phagemid respectively in detail.

Figure 20 is illustrated in the phage particle of displaying NY-ESO TCR in the phage E LISA assay method and the specific combination of HLA-A2-NY-ESO.

Figure 21 represents to incorporate into the dna sequence dna of DR1 α chain that coding and DRA0101 sequence 3 ' are held the codon of the Fos dimerization peptide that links to each other.Shadow representation Fos codon and biotinylation tag pin are represented with runic.

Figure 22 represents to be used in the Sf9 insect cell dna sequence dna of the pAcAB3 bicistronic mRNA carrier that II class HLA-peptide complex expresses.The BamH I restriction site (GGATCC) that is used to insert the Bgl II restriction site (AGATCT) of HLA α chain and is used to insert HLA β chain is used shadow representation.

The codon that Figure 23 represents to incorporate into the Jun dimerization peptide that coding links to each other with DRB0401 sequence 3 ' end and coding and DRB0401 sequence 5 ' are held the dna sequence dna of the DR1 β chain of the HLA-lotus peptide codon that links to each other.Shadow representation Jun codon, the Flu HA peptide codon of HLA-load is represented with underscore.

Figure 24 represent high-affinity A6TCR clone 134 with as the flow cell bonded BIAcore spike of following bag quilt:

Flow cell 1 (FC1)-blank

Flow cell 2 (FC2)-HLA-A2 (LLGRNSFEV) (SEQ ID 23)

Flow cell 3 (FC3)-HLA-A2 (KLVALGINAV) (SEQ ID 24)

Flow cell 4 (FC4)-HLA-A2 (LLGDLFGV) (SEQ ID 25)

Figure 25 represent high-affinity A6TCR clone 134 with as the flow cell bonded BIAcore spike of following bag quilt:

Flow cell 1 (FC1)-blank

Flow cell 2 (FC2)-HLA-B8 (FLRGRAYGL) (SEQ ID 26)

Flow cell 3 (FC3)-HLA-B27 (HRCQAIRKK) (SEQ ID 27)

Flow cell 4 (FC4)-HLA-Cw6 (YRSGIIAVV) (SEQ ID 28)

Figure 26 represent high-affinity A6TCR clone 134 with as the flow cell bonded BIAcore spike of following bag quilt:

Flow cell 1 (FC1)-blank

Flow cell 2 (FC2)-HLA-A24 (VYGFVRACL) (SEQ ID 29)

Flow cell 3 (FC3)-HLA-A2 (ILAKFLHWL) (SEQ ID 30)

Flow cell 4 (FC4)-HLA-A2 (LTLGEFLKL) (SEQ ID 31)

Figure 27 represent high-affinity A6TCR clone 134 with as the flow cell bonded BIAcore spike of following bag quilt:

Flow cell 1 (FC1)-blank

Flow cell 2 (FC2)-HLA-DR1 (PKYVKQNTLKLA) (SEQ ID 32)

Flow cell 3 (FC3)-HLA-A2 (GILGFVFTL) (SEQ ID 33)

Flow cell 4 (FC4)-HLA-A2 (SLYNTVATL) (SEQ ID 34)

Figure 28 represent high-affinity A6TCR clone 134 with as the flow cell bonded BIAcore spike of following bag quilt:

Flow cell 1 (FC1)-blank

Flow cell 4 (FC4)-HLA-A2 (LLFGYPVYV) (SEQ ID 21)

Figure 29 a and 29b represent interactional Biacore figure between solvable high-affinity NY-ESO TCR and the HLA-A2NY-ESO.

Figure 30 a and 30b represent interactional Biacore figure between solvable " wild-type " NY-ESO TCR and the HLA-A2NY-ESO.

Figure 31 a and 31b represent interactional Biacore figure between solvable A6TCR of mutant (clone 1) and the HLA-A2Tax.

Figure 32 a and 32b represent interactional Biacore figure between solvable A6TCR of mutant (clone 111) and the HLA-A2Tax.

Figure 33 a and 33b represent interactional Biacore figure between solvable A6TCR of mutant (clone 89) and the HLA-A2Tax.

Figure 34 represents interactional Biacore figure between solvable A6TCR of mutant (comprising clone 71 and clone's 134 sudden changes) and the HLA-A2Tax.

Figure 35 represents interactional Biacore figure between solvable A6TCR of mutant (comprising clone 1 and β G102 → A sudden change) and the HLA-A2Tax.

Figure 36 a represents interactional Biacore figure between solvable A6TCR of mutant (comprising clone 89 and clone's 134 sudden changes) and the HLA-A2Tax to 36c.

Figure 37 a and 37b represent interactional Biacore figure between solvable A6TCR of mutant (comprising clone 71 and clone's 89 sudden changes) and the HLA-A2Tax.

Figure 38 has described following A6TCR clone's β chain varied texture domain amino acid sequence in detail:

The 38a-wild-type, 38b-clone 134,38c-clone 89, the residue of 38d-clone 1 and 38e-clone 111 sudden changes represents that with runic the residue that is equal to is the other residue that may reside in specific position.

Figure 39 A and 39BI

The design of primers of embodiment 1-A6 Tax TCR α and β chain and mutagenesis form the new required cysteine residues of interchain disulfide bond to introduce

For the A6Tax Threonine 48 with exons 1 among the TRAC*01 is mutated into halfcystine, designed following primer (sudden change is represented with lowercase):

5’-C ACA GAC AAA tgT GTG CTA GAC AT(SEQ ID 35)

5’-AT GTC TAG CAC Aca TTT GTC TGT G(SEQ ID 36)

For the A6Tax Serine 57 with exons 1 among TRBCl*01 and the TRBC2*01 is mutated into halfcystine, designed following primer (sudden change is represented with lowercase):

5’-C AGT GGG GTC tGC ACA GAC CC(SEQ ID 37)

5’-GG GTC TGT Gca GAC CCC ACT G(SEQ ID 38)

PCR mutagenesis

Use α strand primer or β strand primer respectively, as described below the expression plasmid that contains A6Tax TCR α or β chain gene is suddenlyd change.

100 nanogram plasmids are mixed with 5 microlitre 10mM dNTP, 25 microlitre 10xPfu damping fluids (Stratagene), 10 Pfu of unit polysaccharases (Stratagene) and use H ₂O is adjusted to 240 microlitres with final volume.With adding the final concentration dilution in this mixture of 48 microlitres is that the primer of 0.2 μ M makes the end reaction volume reach 50 microlitres.After initial 95 ℃ of denaturing steps of 30 seconds, reaction mixture carries out 15 sex change (95 ℃, 30 seconds), annealing (55 ℃, 60 seconds) and extends (73 ℃, 8 minutes) circulation on Hybaid PCR express PCR instrument.Digested 5 hours at 37 ℃ with 10 DpnI of unit Restriction Enzymes (New England Biolabs) with after product.The reactant transformed competence colibacillus XL-1-Blue bacterial strain of 10 microlitres digestion and 37 ℃ of growths 18 hours.Picking list bacterium colony and at 5 milliliters of TYP+ penbritins (16 grams per liter Tryptoness, 16 grams per liter yeast extracts, 5 grams per liter NaCl, 2.5 grams per liter K ₂HPO ₄, 100 mg/litre penbritins) and middle grow overnight.According to the specification sheets of giving birth to manufacturer on Qiagen mini-prep post plasmid DNA purification and on the sequencing equipment of Oxford University department of biochemistry by its sequence of automatization order-checking check.Fig. 1 a and 2a represent the mutant nucleic acid and the aminoacid sequence of α chain respectively, and Fig. 1 b and 2b represent the mutant nucleic acid and the aminoacid sequence of β chain respectively.

Embodiment 2-Construction of Vector for Phage Display and A6 TCR α and β chain cloned into this phagemid carrier

In order on the filobactivirus particle, to show the allos dimerization A6TCR that contains the non-natural interchain disulfide bond, made up the phagemid carrier to express the fusion rotein that constitutes by allos dimerization A6TCR that contains the non-natural interchain disulfide bond and phage coat protein.These carriers comprise a pUC19 starting point, M13 starting point, a bla (amicillin resistance) gene, Lac promotor/operon and a CAP binding site.Summarized the design of these carriers among Fig. 3, this figure has described the carrier of coding A6TCR β chain-g3 or A6TCR β chain-g8 fusion rotein except soluble A6TCR α chain.A6TCR α preparation and that incorporate the required additional cysteine residues sudden change of the new interchain disulfide bond of formation containing as shown in Fig. 1 a and 1b into and β chain are as the A6TCR α of the phagemid that produces this TCR of coding and the source of β chain among the embodiment 1.The global DNA sequence of having represented the phagemid structure (pEX746) that adopted among Fig. 4.

The molecular cloning method of carrier construction has been described in " molecular cloning: laboratory manual " that J.Sambrook and D.W.Russell showed (Molecularcloning:A laboratory manual).

The primer of enumerating in the table 1 is used to the structure of described carrier.An example of PCR program is 94 ℃ of 1 circulations in 2 minutes, 94 ℃ 5 seconds, 53 ℃ 5 seconds and 72 ℃ of 25 circulations in 90 seconds subsequently, 72 ℃ of 1 circulations in 10 minutes, and 4 ℃ of insulations subsequently subsequently.Expand High Fidelity Taq archaeal dna polymerase is available from Roche.

Table 1 is used to make up the primer of A6TCR Vector for Phage Display

The primer title	Sequence 5 ' to 3 '
		YOL1	TAATAATACGTATAATAATATTCTATTTCAAG GAGACAGTC(SEQ ID 39)
ZOL2	CAATCCAGCGGCTGCCGTAGGCAATAGGTATT TCATTATGACTGTCTCCTTGAAATAG (SEQ ID 40)
		YOL3	CtaCGGCAGCCGCTGGATTGTTATTACTCGCG GCCCAGCCGGCCATGGCccag (SEQ ID 41)
TOL4	GTTCTGCTCCACTTCCTTCTGGGCCATGGCCG GCTGGGCCG(SEQ ID 42)

TOL5	CAGAAGGAAGTGGAGCAGAAC (SEQ ID 43)
		TOL6	CTTCTTAAAGAATTCTTAATTAACCTAGGTTA TTAGGAACTTTCTGGGCTGGGGAAG(SEQ ID 44)
YOL7	GTTAATTAAGAATTCTTTAAGAAGGAGATATA CATATGAAAAAATTATTATTCGCAATTC (SEQ ID 45)
		TOL8	CGCGCTGTGAGAATAGAAAGGAACAACTAAAG GAATTGCGAATAATAATTTTTTCATATG (SEQ ID 46)
TOL9	CTTTCTATTCTCACAGCGCGCAGGCTGGTGTC ACTCAGAC(SEQ ID 47)
		YOL10	ATGATGTCTAGATGCGGCCGCGTCTGCTCTAC cccaggcctc(SEQ ID 48)
YOL11	GCATCTAGACATCATCACCATCATCACTAGAC TGTTGAAAGTTGTTTAGCAAAAC(SEQ ID 49)
		TOL12	CTAGAGGGTACCTTATTAAGACTCCTTATTAC GCAGTATG(SEQ ID 50)

The expression of fusion rotein in intestinal bacteria of embodiment 3-bacterium coat protein and allos dimerization A6TCR

In order to verify the member that produces among the embodiment 2, use the previous described phage (Li etc. that show antibody scFv that are used to produce, 2000, Journal of Immunological Methods 236:133-146) and follow-up improvement preparation show the A6TCR phage particle that contains the non-natural interchain disulfide bond.The intestinal bacteria XL-1-Blue cell that contains pEX746:A6 phagemid (promptly encode solvable A6TCR α chain and by phagemid that produce described in the embodiment 2 and the A6TCR β chain that phage gIII albumen merges) is used to inoculate 5 milliliters of Lbatg and (contains 100 mcg/ml penbritins, 12.5 37 ℃ of shaking culture of culture spend the night (16 hours) the subsequently Lennox L substratum of mcg/ml tsiklomitsin and 2% glucose).50 microlitre overnight culture are used to inoculate 5 milliliters of TYPatg, and (TYP is 16 grams per liter peptones, 16 grams per liter yeast extracts, 5 grams per liter NaCl and 2.5 grams per liter K ₂HPO ₄), 37 ℃ of shaking culture of culture are up to OD subsequently _600nm=0.8.Add helper phage M13K07 to final concentration 5 * 10 to culture ⁹Cfu/ml.Then culture leave standstill for 37 ℃ hatch 30 minutes and subsequently 200rpm vibrated other 30 minutes.Substratum with above-mentioned culture changes TYPak (TYP that contains 100 mcg/ml penbritins and 25 mcg/ml kantlex) into subsequently, and 25 ℃ of 250rpm shaking culture of culture are 36 to 48 hours subsequently.4 ℃ of 4000rpm of culture centrifugal 30 minutes subsequently.Supernatant filters also through 0.45 micron syringe filter, and 4 ℃ of preservations are used for further concentrating or analyzing.

Thread coat protein detects by immunoblotting with the fusion rotein that contains the allos dimerization A6TCR of non-natural interchain disulfide bond in the supernatant liquor.The reduction and non-reduced sample loading buffer in about 10 ¹¹The cfu phage particle loads on each swimming lane of SDS-PAGE gel.Isolating albumen carries out an anti-mark with anti-M13 gIII monoclonal antibody, uses subsequently and horseradish peroxidase (HRP) link coupled two anti-marks.Opti-4CN substrate reagent box with Bio-Rad detects HRP activity (Fig. 5) subsequently.These data show that A6TCR clone that disulfide linkage connects 1 merges with filobactivirus coat protein (gIII albumen).

Contain the detection of the functional allos dimerization A6 TCR of non-natural interchain disulfide bond on the embodiment 4-filobactivirus particle

Use phage E LISA method to detect the existence that is illustrated in functional (HLA-A2-tax bonded) A6TCR on the phage particle.

TCR-phage E LISA

The combining to use subsequently with anti-rabbit monoclonal antibodies link coupled alkaline phosphatase (AP) with the anti-fd antiserum(antisera) of elementary rabbit (Sigma) of the phage particle of showing A6TCR among the ELISA and fixed peptide-MHC (Sigma) detected.Non-differential protein binding site can seal with 2%MPBS or 3%BSA-PBS on the flat board.

Material and reagent

1. bag is cushioned liquid, PBS

2.PBS：138mM NaCl，2.7mM KCl，10mM Na ₂HPO ₄，2mM KH ₂PO ₄

3.MPBS，3％marvel-PBS

4.PBS-Tween：PBS，0.1％Tween-20

5. substrate solution, Sigma FAST pNPP, catalog number (Cat.No.) N2770

Method

1.NeutrAvidin the reaction tank of avidin bag quilt cleans twice with PBS.

2. add vitamin H-HLA-A2Tax or vitamin H-HLA-A2NYESO that 25 microlitre concentration are 10 microlitre/milliliters, and at room temperature hatched 30 to 60 minutes.

3. reaction tank cleans twice with PBS.

4. add 300 microlitre 3%MPBS, and incubated at room 1 hour.TCR-phage suspension is mixed with isopyknic 3%MPBS and incubated at room.

5. reaction tank cleans twice with PBS.

6. add 25 microlitres phage-A6TCR/MPBS mixture, ice bath 1 hour.

7. reaction tank cleans 3 times with ice-cold PBS-Tween, and cleans 3 times with ice-cold PBS.

8. add the anti-fd antibody of rabbit that 25 microlitres were diluted in MPBS in ice-cold 1: 1000, and ice bath 1 hour.

9. reaction tank cleans 3 times with ice-cold PBS-Tween, and cleans 3 times with ice-cold PBS.

10. added 25 microlitres ice-cold 1: 50,000 is diluted in anti-rabbit monoclonal antibodies-alkaline phosphatase conjugates of MPBS, and ice bath 1 hour.

11. reaction tank cleans 3 times with ice-cold PBS-Tween, and cleans 3 times with ice-cold PBS.

12. add 150 microlitre alkaline phosphatases Huang and read signal in 405 nanometers to each reaction tank.

The presentation of results of showing among Fig. 6, clone 1 has produced and has showed the phage particle of energy specificity in conjunction with the A6TCR of its similar pMHC (HLA-A2Tax).

Dna sequence analysis to the A6TCR of this displaying is presented at non-existent " milky white " terminator codon in the expression vector member sequence that exists in respective embodiments 2 in the TCR β chain.This codon can " be read to lead to " with low frequency by the rrna of the coli strain that is adopted, and causes inserting on this position the remarkable reduction of tryptophan residue and total length β chain expression level.Infer to have only the cell of the A6TCR sequence of expressing this sudden change in the cultivation circulation of embodiment 3, to be survived by this phenomenon, and estimate that therefore the high level by the A6TCR of original expression vector expression is deleterious to host cell.

Embodiment 5-strand TCR (scTCR) ribosomal display

Structure in order to the ribosomal display scTCR carrier that produces the ribosomal display pcr template

For generation does not have wrong and stable DNA pcr template to carry out ribosomal display experiment subsequently thus, the ribosomal display member is cloned among the DNA plasmid pUC19 that is easy to obtain.Vector construction carries out in two steps to avoid using big Oligonucleolide primers (and relative Problem-Error).Shown among Fig. 7 a among the final A6scTCR-C-κ DNA ribosomal display member of schematic form and Fig. 7 b and shown its DNA and protein sequence.This member can be used as PstI/EcoRI double digestion digestion product and cuts off from pUC19.

J.Sambrook and D.W.Russell show the molecular cloning method of having described carrier construction in " molecular cloning: laboratory manual ".

The primer of enumerating in the table 2 is used for the structure of described carrier.The PCR program that is adopted is as follows: 94 ℃ of 1 circulations in 2 minutes, 94 ℃ 30 seconds, 55 ℃ 20 seconds and 72 ℃ of 25 circulations in 120 seconds subsequently, 72 ℃ of 1 circulations in 5 minutes, and 4 ℃ of insulations subsequently subsequently.The Pfu archaeal dna polymerase is available from Strategene.The Oligonucleolide primers that uses has been described in the table 2.

Make up pUC19-T7-step 1

The structure of pUC19-T7 is described below, and this causes forming the pUC19 carrier that contains a T7 promoter region of following short interval region and the suitableeest eucaryon Kozak sequence.Because this is essential for any transcripting starting that adheres to sequence in the rabbit skein cell solute, this becomes the basic component of ribosomal display member.The sequence (as A6scTCR-C-κ) that is used for ribosomal display can be connected between the NcoI and EcoR I restriction site of pUC19-T7 carrier.

By reactant being heated to 94 ℃ of 10 minutes and slow cool to room temperature, the reverse connection (Rev-Link) of equimolar amount being connected (For-Link) annealing with forward.This causes forming double-stranded DNA mixture as follows.

5’AGCTGCAGCTAATACGACTCACTATAGGAACAGGCCACCATGG

CGTCGATTATGCTGAGTGATATCCTTGTCCGGTGGTACCCTAG 3’

(SEQ ID 51)

5 ' district comprises outstanding sticking terminal with Hind III restriction site complementary, and 3 ' district comprises and the BamHI complementary is sticking terminal.

The annealed oligonucleotide is connected among the pUC19 of Hind III/BamHI double digestion, this plasmid by the agarose gel electrophoresis purifying, cut off and extract test kit and be further purified with Qiagen glue.Connect product transformed into escherichia coli XL-1-Blue.Independent pUC19-T7 clone checks order to confirm existing of correct sequence.Shown this sequence among Fig. 8.

Make up A6scTCR-C-κ carrier-step 2

The structure of strand A6scTCR-C-κ dna sequence dna requires to produce and must be combined into segmental 3 the PCR fragments of A6scTCR-C-κ subsequently.These fragments comprise (a.) 5 ' district and the A6TCR α chain variable domains of NcoI site adjacency and the glycine Serine connector part of 3 ' district and BamHI restriction site adjacency.This product produces with the conventional PCR of primer 45 and 50 (seeing Table 2) by carrier pEX202.The A6TCR β variable domains and the constant domain of fragment (b.) 5 ' district and BamHI restriction site adjacency are being followed glycine Serine connector part thereafter.This product produces with the conventional PCR of primer 72 and 73 (seeing Table 2) by carrier pEX207.The part in the people C-κ district that fragment (c.) produces with the conventional PCR of primer 61-60 (seeing Table 2) by the p147 carrier.All PCR products separate on the 1.6%TBE sepharose, cut off the DNA band of correct size and use Qiagen gel extraction kit purifying.

Fragment (b.) and (c.) merge by the overlapping PCR of the complementarity in its primer sequence 73 and 61 (seeing Table 2) by routine.PCR is undertaken by primer 72 and 60 (seeing Table 2).The PCR product separates on the 1.6%TBE sepharose, cuts off the DNA band of correct size and uses Qiagen gel extraction kit purifying.This fragment called after (d.).

Fragment (a.) NcoI and BamHI double digestion, and fragment (d.) BamHI and EcoRI double digestion.PUC19-T7 NcoI and EcoRI double digestion.The DNA product that all enzymes are cut separates on the 1.2%TBE sepharose, cuts off the DNA band of correct size and uses Qiagen gel extraction kit purifying.The pUC19-T7 that ligase enzyme is cut, fragment (a.) and (d.) and transformed into escherichia coli XL-1-Blue.The transformant order-checking is to confirm correct sequence.Shown the sequence of being cloned into the A6scTCR-C-κ ribosomal display member among the pUC19 among Fig. 9 with PstI and EcoRI site adjacency.

The oligonucleotide (available from MWG) that table 2 uses

Rev-Link	5’GATCCCATGGTGGCCTGTTCCTATAGTGAGTCGTATTAGCTGC(SEQ ID 52)
		For-Link	5’AGCTGCAGCTAATACGACTCACTATAGGAACAGGCCACCATGG(SEQ ID 53)
45-A6	5’CCACCATGGGCCAGAAGGAAGTGGAGCAGAACTC(SEQ ID 54)
		7A6-Beta(RT-PCR)(a)	5’CGAGAGCCCGTAGAACTGGACTTG(SEQ ID 55)
49-A6-BamHI-F	5’GTGGATCCGGCGGTGGCGGGTCGAACGCTGGTGTCACTCAGACCCC (SEQ ID 56)
		50-A6-BamHI-R	5’CCGGATCCACCTCCGCCTGAACCGCCTCCACCGGTGACCACAAC CTGGGTCCCTG(SEQ ID 57)
60-Kappa-rev-EcoRI	5’CTGAGAATTCTTATGACTCTCCGCGGTTGAAGCTC(SEQ ID 58)
		61-Betac-Kappa-forl	5’TGACGAATTCTGACTCTCCGCGGTTGAAGCTC(SEQ ID 59)
71 T7-primers	5’AGCTGCAGCTAATACGACTCACTATAGG(SEQ ID 60)
		72 A6-beta	5’GGCCACCATGGGCAACGCTGGTGTCACTCAGACCCC(SEQ ID 61)
73-A6-cons-rev	5’TGAACCGCCTCCACCGTCTGCTCTACCCCAGGCCTCGGCG(SEQ ID 62)
		75 Kappa-rev	5’TGACTCTCCGCGGTTGAAGCTC(SEQ ID 63)

The explanation that the sc A6 TCR-C-κ that translates by in-vitro transcription produces

Be used for the preparation of the sc A6 TCR-C-κ PCR product of in-vitro transcription translation

Here we described under the condition that biotinylated Methionin exists the synthetic of sc A6TCR-C-κ by the in-vitro transcription translation and subsequently to it by immunoblotting with the detection of the streptavidin detection of alkali phosphatase enzyme mark.

Use prepares sc A6TCR-C-κ PCR product as sc A6TCR-C-κ carrier and primer 71 and 60 of template in conventional PCR reaction.Primer 60 comprises terminator codon so that scTCR discharges from rrna.Pfu polysaccharase (Strategene) is used for improving the fidelity of PCR building-up process.The PCR product separates on the 1.6%TBE sepharose, cuts off the DNA band of correct size and uses Qiagen gel extraction kit purifying.Use transcription and translation test kit that is connected with Ambion PROTEINscript II and the above-mentioned PCR product of 300 nanograms of catalog number (Cat.No.) 1280-1287 to carry out the transcription and translation reaction.Carry out 3 transcription and translation reactions according to the scheme of manufacturers.The change of being done is to add from Transcend ^TMThe biotinylated Methionin of on-radiation translation detection system.

Reaction 1: have under the situation of the biotinylated Methionin of 2 microlitres the reaction of 300 nanogram sc A6TCR-C-κ

Reaction 2: do not have under the situation of the biotinylated Methionin of 2 microlitres the reaction of 300 nanogram sc A6TCR-C-κ

Reaction 3: have under the situation of the biotinylated Methionin of 2 microlitres the control reaction of no DNA

Every kind of reactant of 2 microlitres is gone up at the Novex of 4-20% gradient SDS-PAGE gel (Invitrogen) and is separated.Also separated multiple dilution contrast biotinylation TCR in addition.With gel transfer printing mark and with streptavidin alkaline phosphatase detection albumen and according to Transcend ^TMThe described Western that uses at alkaline phosphatase of on-radiation translation detection system scheme

The stable form substrate carries out the colorimetry colour developing subsequently.Shown the immunoblotting result among Figure 10.

In the reactant of the A6scTCR-C-κ of no DNA contrast and lifeless matter elementization Methionin, do not have as expected to find near correct big or small band, and in the reactant of the A6scTCR-C-κ that has biotinylation Methionin, can observe the band of approximate correct size.This illustrates synthesizing by the sc A6TCR-C-κ TCR of in-vitro transcription translation.

The preparation of sc A6 TCR-C-κ ribosomal display PCR product

Use has prepared sc A6TCR-C-κ PCR product as the A6scTCR-C-κ carrier and the PCR primer 71 and 75 (seeing Table 2) of template in conventional PCR reaction.Primer 75 does not comprise terminator codon.Pfu polysaccharase (Strategene) is used for improving the fidelity of PCR building-up process.The PCR product separates on the 1.6%TBE sepharose, cuts off the DNA band of correct size and uses Qiagen gel extraction kit purifying.

The ribosomal display process

Sc A6TCR-C-κ transcribing and translating

Use the transcription and translation test kit (catalog number (Cat.No.) 1280-1287) that is connected with Ambion PROTEINscript II to transcribe/translation reaction.

The responsive transcription thing

Following responsive transcription thing is placed in 0.5 milliliter the Ambion non-sticky test tube (catalog number (Cat.No.) 12350).

Content test tube 1 (normal A6) test tube 2 (contrast)

Water	4.53 microlitre	5.7 microlitre
			Template (PCR product)	Sc A6TCR-C-κ PCR product 1.17 microlitres (300 nanogram)	No DNA
5 * transcribe mixture	2 microlitres	2 microlitres
			Enzyme mixture
	2 microlitres	2 microlitres
			Superasin (Ambion RNA enzyme inhibitors)	0.3 microlitre	0.3 microlitre
Final volume	10 microlitres	10 microlitres

Described test tube on the PCR module that Re Gai closes 30 ℃ hatched 60 minutes.

The translation reaction thing

Following translation reaction thing is placed in 0.5 milliliter the Ambion non-sticky test tube.

Content test tube 1 (normal A6) test tube 2 (contrast)

The skein cell solute	105 microlitres	105 microlitres
			The 25mM magnesium acetate	3 microlitres	3 microlitres
The translation mixture	7.5 microlitre	7.5 microlitre
			Methionine(Met)	7.5 microlitre	7.5 microlitre
Water
		18 microlitres	18 microlitres
Superasin (Ambion RNA enzyme inhibitors)				3 microlitres	3 microlitres
	The responsive transcription thing	Above-mentioned test tube 1 product of 6 microlitres	Above-mentioned test tube 2 products of 6 microlitres

Each test tube comprises the volume that enough 3 times 50 microlitres are selected.Test tube mix the back on the PCR module that Re Gai closes 30 ℃ hatched 60 minutes.Add RQ1DNA enzyme (Promega) that 3 units do not contain the RNA enzyme after 30 minutes with initial dna profiling in the degraded test tube 1, and in test tube 2, add the RQ1DNA enzyme (Promega) that 3 units do not contain the RNA enzyme.Add 18 microlitre heparin solutions and add 18 microlitre heparin solutions to translation reaction thing 2 after 60 minutes to translation reaction thing 1.Ice bath is preserved sample so that select at the globule of HLA bag quilt.

The bag quilt of magnetic bead

The resuspended streptavidin magnesphere (Roche catalog number (Cat.No.) 1641778) of 20 microlitres is transferred in aseptic 1.5 milliliters of eppendorf test tubes that do not contain the RNA enzyme.Globule is fixing and removal supernatant liquor with magnetic-particle separator (Roche catalog number (Cat.No.) 1641794).Globule does not contain 1 * PBS (10 * PBS Ambion catalog number (Cat.No.) 9624, the Ambion H of RNA enzyme subsequently with 100 microlitres ₂O catalog number (Cat.No.) 9930) cleans.Having carried out 3 PBS altogether cleans.

Globule is resuspended and divide them equally two test tubes (each 10 microlitre) in 20 microlitre PBS.A test tube will be used to produce the globule of contrast sealing, and another test tube is used to produce the globule of HLA-A2-Tax bag quilt.

Add 80 microlitre BSA/ biotin solution and mixing to contrast globule test tube.The BSA/ biotin solution is formulated as follows.The PBS that contains 0.1%BSA (Ambion is ultrapure, catalog number (Cat.No.) 2616) to 990 microlitres adds 10 microlitre 0.2M Tris alkali 0.1M biotin solution.20 microlitre heparin solutions (containing 138mg/ml heparin (Sigma) among 1 * PBS) and mixing solutions have also been added.Globule intermittently mixed room temperature was hatched 1 hour.3 usefulness 100 microlitre PBS clean and contain among the PBS of 0.1%BSA resuspended at 10 microlitres subsequently.

The globule of HLA-TAX bag quilt is prepared as follows.Add 40 microlitre HLA-A2-Tax (1.15mg/ml that described in WO99/60120, prepares) and mixing to 10 microlitre globules.Globule incubated at room 15 minutes is also added 20 microlitre BSA (50mg/ml, Ambion catalog number (Cat.No.) 2616) and 20 microlitre heparin solutions (seeing above-mentioned) and mixing subsequently.Globule was hatched other 45 minutes, added 20 microlitre BSA/ biotin solution subsequently.3 usefulness of globule, 100 microlitre PBS clean and contain among the PBS of 0.1%BSA resuspended at 10 microlitres subsequently.

Use the elutriation of magnetic bead

Sample size and every part of sample size of sc A6TCR translation reaction thing being divided into 3 part of 50 microlitre volume obtain any following globule of 2 microlitres:

Contrast (no HLA)

HLA-A2-Tax

HLA-A2-Tax adds the soluble scA6TCR of 10 micrograms

Sample size and every part of sample size that contrast translation reaction thing also is divided into 3 part of 50 microlitre volume obtain any following globule of 2 microlitres:

Contrast (no HLA)

HLA-A2-Tax

HLA-A2-Tax adds the soluble scA6TCR of 10 micrograms

This has produced 6 test tubes altogether.Test tube follows mixing intermittently to hatch 60 minutes for 5 ℃ on the PCR module.

3 ice-cold damping fluids (PBS, 5mM magnesium acetate and 0.2%Tween (Sigma does not contain the RNA enzyme)) of usefulness 100 microlitres clean globule subsequently.Every part of sample size globule contains in 1 * RQ1DNA enzymic digestion damping fluid of 1 microlitre (40 unit) Superasin and 1 microlitre (1 unit) RQ1DNA enzyme resuspended at 50 microlitres subsequently.Globule on the PCR module 30 ℃ hatched 30 minutes.

Globule cleans 3 times also with ice-cold H with the ice-cold damping fluid of 100 microlitres (PBS, 5mM magnesium acetate and 0.2%Tween) subsequently ₂O cleans 1 time.Globule is resuspended in the H that 10 microlitres do not contain the RNA enzyme ₂O.Globule refrigerates subsequently to prepare to carry out RT-PCR.

The RT-PCR of scA6TCR-C-κ mRNA on the magnetic bead that obtains in the ribosomal display reaction

Described in manufacturers's scheme, the RT-PCR that uses the single test tube RT-PCR test kit of Titan (catalog number (Cat.No.) 1855476) to carry out on the magnetic bead reacts.Together with primer 45 and 7 and 0.3 microlitre Superasin RNA enzyme inhibitors, add 2 microlitre magnetic beads in each RT-PCR reaction.

For each RT-PCR reaction is provided with second single PCR reaction, the difference of this reaction is not contain reversed transcriptive enzyme except Roche high-fidelity polysaccharase.Described second reaction is as the contrast that DNA is polluted.Use the Ing of sc A6TCR-C-κ carrier to be provided with the positive control of RT-PCR in addition.

The reactant circulation is as follows.Hatched 30 minutes by 50 ℃ in sample, subsequently by on the PCR module 94 ℃ hatch and carried out the RT-PCR step with the deactivation reversed transcriptive enzyme in 3 minutes.Reactant is following to carry out 38 PCR circulations altogether:

94 ℃ 30 seconds

55 ℃ 20 seconds

68 ℃ 130 seconds

The PCR reaction is hatched end in 4 minutes for 72 ℃.

Exactissima diligentia is polluted to avoid the RNA enzyme in all ribosomal display step process.RT-PCR separates on 1.6% TBE sepharose with the PCR reactant, and this can be referring to Figure 11.Analysis to gel shows, does not exist DNA pollution and all PCR products to be derived from mRNA.The DNA band of correct size illustrates in the swimming lane 2, has selected the sc A6TCR-C-κ of ribosomal display by the globule of HLA-A2-Tax bag quilt.Swimming lane 3 shows that we can be by adding the specificity selection that soluble sc A6TCR suppresses the sc A6TCR-C-κ of this ribosomal display.With respect to the sample that does not suppress in the swimming lane 2, the obvious reduction of band brightness has illustrated this point in the swimming lane 3.For the globule of the no HLA bag quilt that contrasts, do not find the combination of the sc A6TCR-C-κ of ribosomal display.

Embodiment 6

Be illustrated in the sequential analysis of the A6TCR on the phage particle and improve the method for showing characteristic

Made up after the carrier of on phage, showing A6TCR by PCR and molecular cloning, the bacterial clone that can produce the phage of showing A6TCR has been screened by phage E LISA as described in embodiment 4.3 clones different in the ELISA binding assay, have been identified with the HLA-A2-tax bonded.These clones all comprised " wild-type " A6TCR DNA's or with regulate the sudden change that sequence links, these sudden changes have been described in the following table:

Derive from functional clone to the TCR A6 screening of phage display

Title	Feature
			Clone
7	Be positioned at v β gene the 3rd ribosome bind site before from A AGGAGA sports A AGGG GA
			Clone
9	In v β CDR3, introduce an opal codon	Global DNA among Figure 12 a and the 12b and aminoacid sequence
			Clone 49	In v β FR1, introduce an amber codon." silence " sudden change that does not influence final aminoacid sequence has been introduced in this sudden change	Global DNA sequence among Figure 13 a

As if these clones comprised the sudden change that causes that A6TCR β chain expression level reduces.We infer that as the Cytotoxic result who is caused by the TCR high expression level, it is selected that low cloning by expression is better than high-expression clone.

The mutagenesis in embodiment 7-A6TCR CDR3 zone

The CDR3 zone of A6TCR is to introduce sudden change produces the possibility of high-affinity mutant with research target.

Used overlapping PCR that the sequence of α and β CDR3 is improved to use oligonucleotide YOL54,5 ' CAGCTGGGGGAAGCTTCAGTTTGGAGCAG3 ' (SEQ ID 64) and YOL55,5 ' CTGCTCCAAACTGAAGCTTCCCCCAGCTG3 ' (SEQ ID 65) introduces unique restriction site Hind III to the α chain, and with YOL565 ' GTACTTCTGTGCCTCGAGGCCGGGACTAG3 ' (SEQID 66) and YOL575 ' CTAGTCCCGGCCTCGAGGCACAGAAGTAC3 ' (SEQ ID 67) the β chain is introduced unique restriction site Xho I.

Used PCR to introduce the sudden change of affinity maturation.A6TCR clone 9 (opal codons of incorporating an introducing in β chain CDR3 sequence into) are as a source of template DNA, and the TCR chain increases with mutant primer (describing in detail in following table) and YOL225 ' CATTTTCAGGGATAGCAAGC3 ' (SEQ ID 68) (β chain) or YOL135 ' TCACACAGGAAACAGCTATG3 ' (SEQ ID 69) (α chain).

Be used for introducing the primer of sudden change at the CDR3 of A6 α and β chain

The primer title	5 ' → 3 ' sequence
		YOL59	TGTGCCTCGAGGIMKNNKNNKNNKNNKN NKCGACCAGAGCAGTACTTCG (SEQ ID 70)
YOL60	TGTGCCTCGAGGCCGNNKNNKNNKNNKN NKNNKCCAGAGCAGTACTTCGGGC (SEQ ID 71)
		YOL61	TGTGCCTCGAGGCCGNNKNNKNNKNWKN NKNNKCGACCAGAGCAGTACTTCG (SEQ ID 72)
YOL62	TGTGCCTCGAGGCCGNNKNNKNNKNWKN NKNNKGGAGGGCGACCAGAGCAG (SEQ ID 73)
		YOL63	TGTGCCTCGAGGCCGGGANNKNNKNNKN NKNNKNNKGGGCGACCAGAGCAGTAC (SEQ ID 74)
YOL68	TGTGCCTCGAGGIMKNNKNNKNNKNNKN NKCCAGAGCAGTACTTCGggc (SEQ ID 75)
		YOL69	TGTGCCTCGAGGNNKNNKNNKNNKNNKN NKGAGCAGTACTTCGggccg (SEQ ID 76)
YOL70	TGTGCCTCGAGGNNKNNKNNKNNKNNKN NKCAGTACTTCGggccgggc (SEQ ID 77)
		YOL71	TGTGCCTCGAGGccgNNKNKNNKNNKg ggCGACCAGAGCAGTACTTCG (SEQ ID 78)
YOL58	AAACTGAAGCTTMNNMNNMNNMNNMNNT GTAACGGCACAGAGGTAG (SEQ ID 79)
		YOL72	AAACTGAAGCTTMNNMNNgctgtcMNNT GTAACGGCACAGAGGTAG (SEQ ID 80)
YOL73	AAACTGAAGCTTMNNMNNMNNgctgtcM NNTGTAACGGCACAGAGGTAG (SEQ ID 81)
		YOL74	AAACTGAAGCTTMNNMNNgc tgtcMNNA ACGGCACAGAGGTAG (SEQ ID 82)

α chain fragment is with NcoI and HindIII digestion and with Qiagen test kit purifying again, and carrier is prepared with Qiagen test kit gel-purified subsequently by cloning 9 with NcoI and HindIII digestion.β chain fragment is with XhoI and NotI digestion and with Qiagen test kit purifying again, and carrier is prepared with Qiagen test kit gel-purified subsequently by cloning 9 with XhoI and NotI digestion.The insertion fragment of purifying and carrier mix with 3: 1 mol ratio and T4 ligase enzyme damping fluid, T4 ligase enzyme and the water that do not contain ribozyme.16 ℃ of water-baths of ligation are spent the night.For each sudden change library, according to the scheme that manufacturers provides, the connection product of the purifying of 0.5 to 1 microlitre is with the ratio electricity transformed into escherichia coli TG1 of per 40 microlitres electricity transformed competence colibacillus cell (Stratagen), 0.2 micrograms of DNA altogether.After electricity transformed, cell was resuspended and be tiled in and contain the YTE that replenished 100 mcg/ml penbritins and the 2% glucose 244mm * 244mm tissue culture ware of (contain 15 gram agar, 18 gram NaCl, 10 gram Tryptoness and 5 in 1 liter and restrain yeast extracts) with 37 ℃ of 960 microlitre SOC substratum immediately.30 ℃ of overnight incubation of plate.Subsequently, scrape from plate and get cell with 5 milliliters of DYT that replenish 15% glycerine (containing 16 gram Tryptoness, 10 gram yeast extracts and 5 gram NaCl in 1 liter, 125 ℃ of autoclavings 15 minutes).

In order to prepare the phage particle of showing A6TCR, the library storage liquid of 500 to 1000 microlitres is inoculated in 500 milliliters of DYTag (DYT that contains 100 mcg/ml penbritins and 2% glucose).Culture grows into OD (600nm) and reaches 0.5.100 milliliters of cultures infect also 37 ℃ of water-baths with helper phage (Ml 3K07 (Invitrogen) or HYPERPHAGE (Progen Biotechnik, GmbH 69123Heidelberg)) and hatched 30 minutes.Replace substratum with 100 milliliters of DYTak (DYT that contains 100 mcg/ml penbritins and 25 micrograms/kantlex).25 ℃ of vibrations of culture (300rpm) were subsequently hatched 20 to 36 hours.

Embodiment 8

The separation of high-affinity A6TCR mutant

Use two kinds of diverse ways to carry out the separation of high-affinity A6TCR mutant.

First method comprises the phage particle that uses Maxisorp immunity test tube (Invitrogen) to select to show with HLA-A2Tax mixture bonded sudden change A6TCR.The immunity test tube wraps quilt by 1 to 2 milliliter of PBS ambient temperature overnight that contains 10 mcg/ml streptavidins.Test tube cleans twice with PBS, and adds 1 milliliter of PBS that contains 5 mcg/ml biotinylation HLA-A2Tax mixtures and incubated at room subsequently 30 minutes.Except following change, the residue scheme that the high-affinity binding substances is selected as discussed previously (Li etc. (2000) Journal ofImmunological Methods 236:133-146).Carrying out 3 takes turns or 4 takes turns selection.The concentration of the biotinylation HLA-A2Tax mixture of selecting for the first round is 5 mcg/ml, is 0.5 mcg/ml for second concentration of taking turns, and the concentration of third round is 0.05 mcg/ml, and four-wheel is 0.005 mcg/ml.For selection, take turns in the first round and second and to have used the M13K07 helper phage in the selection, in selection subsequently, used super phage.

The second method that adopts is to select to show with HLA-A2Tax mixture bonded the phage particle of sudden change A6TCR in solution.According to the scheme prewashing of manufacturers the paramagnetic globule (Dynal M280) of streptavidin bag quilt.For the 1st taking turns, the 2nd take turns, the 3rd taking turns and the 4th take turns selection, with 10 ¹²To 10 ¹³The concentration of cfu shows that the phage particle of sudden change A6TCR is 2 * 10 with concentration respectively ^-8M, 2 * 10 ^-9M, 2 * 10 ^-10M and 2 * 10 ^-11The biotinylation HLA-A2Tax mixture premix of M.Show that the phage particle of A6TCR and the mixture of HLA-A2Tax mixture rotated incubated at room 1 hour gently, with the M280 magnetic capture of 200 microlitres (the 1st takes turns) or 50 microlitres (the 2nd, 3,4 take turns) streptavidin bag quilt and the phage particle of biotinylation HLA-A2Tax mixture bonded displaying A6TCR.After phage particle is caught, use Dynal magnetic particle thickener frequently to wash magnetic bead (use PBS Tween20 3 times, 2 usefulness contain the PBS Tween20 of 2% alipoidic milk power, use PBS 2 times, and 2 usefulness contain the PBS of 2% alipoidic milk power, and 2 times are used PBS).After the last cleaning, magnetic bead is resuspended and be accompanied by and rotated incubated at room gently 5 to 10 minutes in the 100mM of 1 milliliter of freshly prepd pH11.5 second triamine.The phage particle of wash-out is immediately with the 1M Tris-HCl neutralization of 300 microlitre pH7.0 from the magnetic bead.According to previously described method, half elutriant is used to infect the phage particle (Li etc., (2000) Journal of Immunological Methods 236:133-146) that the e. coli tg1 of 10 milliliters of freshly prepd OD (600nm) value 0.5 is selected with amplification.

The 3rd take turns take turns screening with the 4th after, 95 single bacterium colonies of picking and be used for being inoculated in 100 microlitre DYTag on 96 orifice plates from plate.37 ℃ of vibrations of culture overnight incubation.Subinoculate in 100 microlitre DYTag with the overnight culture of 2 to 5 microlitres subsequently, and 37 ℃ of vibrations were hatched 2 to 3 hours or become muddy until culture.In order to use the helper phage cells infected, contain 5 * 10 with 25 microlitres ⁹The DYTag of cfu helper phage infects culture and 37 ℃ and hatched 30 minutes.Replace substratum with DYTak.Dull and stereotyped under the 300rpm oscillating condition 25 ℃ hatched 20 to 36 hours.By centrifugal 10 minutes sedimentation cells of 4 ℃ of 3000g.Supernatant liquor is used to screen high-affinity A6TCR mutant by competitive phage E LISA as described below.

The Nunc-Immuno Maxisorp reaction tank of streptavidin bag quilt cleans twice with PBS.Each reaction tank added the biotinylated HLA-A2-Tax mixture of 25 microlitres, 5 mcg/ml and incubated at room 30 to 60 minutes, cleaned twice with PBS subsequently.By add 300 microlitres contain 3% alipoidic milk power PBS and subsequently incubated at room sealed the nonspecific proteins binding site in the reaction tank in 2 hours.In order to prepare the phage particle of showing allos dimerization A6TCR, phage particle with contain 0,20 and the PBS that contains 3% alipoidic milk power of 200nM HLA-A6-Tax mixed also incubated at room 1 hour.Added this phage and incubated at room 1 hour to the reaction tank of HLA-A2-Tax bag quilt, then clean 3 times and clean 3 times with PBS subsequently with the PBS that contains 0.1%Tween20.Bonded shows that phage particle such as the anti-fd antibody of embodiment 4 described usefulness (Sigma) of TCR detect.

Differentiate the high-affinity A6TCR mutant of a plurality of supposition, and in following two tables, enumerated its CDR3 sequence and corresponding wild type sequence.In all β chain mutant and a α chain mutant, found amber terminator codon (X).

A6TCR β chain mutant

The clone	The CDR3 sequence
		Wild-type	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGAGCAGTAG (SEQ ID 83) A S R P G L A G G R P E Q Y (SEQ ID 84)
134	GCCTCGAGGCCGGGGCTGATGAGTGCGTAGCCAGAGCAGTAC (SEQ ID 85) A S R P G L M S A X P E Q Y (SEQ ID 86)
		86	GCCTCGAGGCCGGGGCTGAGGTCGGCGTAGCCAGAGCAGTAC (SEQ ID 87) A S R P G L R S A X P E Q Y (SEQ ID 88)
87	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGAGGCGTAG (SEQ ID 89) A S R P G L A G G R P E A X (SEQ ID 90)
		89	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGAGGATTAG (SEQ ID 91) A S R P G L A G G R P E D X (SEQ ID 92)
85	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGATCAGTAG (SEQ ID 93) A S R P G L A G G R P D Q X (SEQ ID 94)
		83	GCCTCGAGGCCGGGTCTGTAGGCTGGGCGACCAGAGCAGTAC (SEQ ID 95) A S R P G L X A G R P E Q Y (SEQ ID 96)
1	GCCTCGAGGCCGGGGCTGGTTCCGGGGCGACCAGAGCAGTAG (SEQ ID 97) A S R P G L V P G R P E Q X (SEQ ID 98)
		2	GCCTCGAGGCCGGGGCTTGTGTCTGCTTAGCCAGAGCAGTAC (SEQ ID 99) A S R P G L V S A X P E Q Y (SEQ ID 100)
111	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCACATCCGTAG (SEQ ID 101) A S R P G L A G G R P H P X (SEQ ID 102)
		125	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGATGCGTAG (SEQ ID 103) ASRPGLAGGRPDAX (SEQ ID 104)
133	GCCTCGAGGCCGGGTCTGATTAGTGCTTAGCCAGAGCAGTAC (SEQ ID 105) A S R P G L I S A X P E Q Y (SEQ ID 106)

A6TCR α chain mutant

The clone	CDR3
		Wild-type	GCCGTTACAACTGACAGCTGGGGGAAGCTTCAG (SEQ ID 107) A V T T D S W G K L Q (SEQ ID 108)
149	GCCGTTACAACTGACAGCTGGGGGCCGCTTCAG (SEQ ID 109) A V T T D S W G P L Q (SEQ ID 110)
		65	GCCGTTACAACTGACAGCTGGGGGAAGATGCAG (SEQ ID 111) A V T T D S W G K M Q (SEQ ID 112)
66	GCCGTTACAACTGACAGCTGGGGGAAGTTGCAT (SEQ ID 113) A V T T D S W G K L H (SEQ ID 114)
		153	GCCGTTACAACTGACAGCTGGGGGTAGCTTCAT (SEQ ID 115) A V T T D S W G X L H (SEQ ID 116)
71	GCCGTTACAACTGACAGCTGGGGGGAGCTTCAT (SEQ ID 117) A V T T D S W G E L H (SEQ ID 118)
		70	GCCGTTACAACTGACAGCTGGGGGAGGCTGCAT (SEQ ID 119) A V T T D S W G R L H (SEQ ID 120)
121	GCCGTTACAACTGACAGCTGGGGGCAGCTTCAT (SEQ ID 121) A V T T D S W G Q L H (SEQ ID 122)
		117	GCCGTTACAACTGACAGCTGGGGGAAGGTTCAT (SEQ ID 123) A V T T D S W G K V H (SEQ ID 124)
72	GCCGTTACAACTGACAGCTGGGGGAAGGTGAAT (SEQ ID 125) A V T T D S W G K V N (SEQ ID 126)
		150	GC C GTTACAACTGACAGCTGGGGGAAGCTTCTG (SEQ ID 127) A V T T D S W G K L L (SEQ ID 128)

Embodiment 9

Contain the generation with solvable allos dimerization A6TCR of non-natural disulfide linkage between the constant domain of CDR3 sudden change

(Quiagen UK) has separated the coding high-affinity A6TCR mutant of being differentiated among the embodiment 8 from relevant Bacillus coli cells to use the Mini-Prep test kit.

Use phagemid dna as template and use the pcr amplification of following primer be used to increase solvable TCR α and β chain DNA sequence.

A6TCR α chain forward primer

ggaattc atcgatg cagaaggaagtggagcag(SEQ ID 129)

(the ClaI restriction site is represented with underscore)

General TCR α chain reverse primer

gtaca cggccgggtcagggttctggatatac(SEQ ID 130)

(the EagI restriction site is represented with underscore)

A6 β chain forward primer

Tctctc attaatgaatgctggtgtcactcagacccc(SEQ ID 131)

(the AseI restriction site is represented with underscore)

General β chain reverse primer

Tagaa accggtggccaggcacaccagtgtggc(SEQ ID 132)

(the AgeI restriction site is represented with underscore)

Under the situation of TCR β chain, carried out further PCR splicing with the amber terminator codon in the codon replacement CDR3 district that uses coding L-glutamic acid.When the amber terminator codon was suppressed in the intestinal bacteria, the glutamine residue was normally introduced rather than translation termination.Therefore, when containing the TCR of amber codon at phage display, this position comprises the glutamine residue.Yet when this TCR β chain gene was transferred to expression plasmid, glutaminic acid residue was selected as the another kind of glutamine.The primer that is used for this PCR splicing is as follows.

YOL124 CTGCTCTGGTTCCGCACTC

(SEQ ID 133)

YOL125 GAGTGCGGAACCAGAGCAG

(SEQ ID 134)

Verified the dna sequence dna (opinion) of the solvable A6TCR β chain of sudden change at Figure 14 a of the A6TCR β chain DNA sequence of sudden change with at Figure 14 b of the A6TCR β chain amino acid sequence of sudden change by automatic sequencing.Figure 14 c shown and do not contained the aminoacid sequence of glutamine to the A6TCR β chain of L-glutamic acid alternate sudden change, promptly as by the sequence among the isolating clone 134 of phage E LISA.

These A6TCR α and β chain DNA sequence are subsequently in order to produce the solvable A6TCR described in WO 03/020763.In brief, these two chains are expressed in different culture of Escherichia coli with the form of inclusion body.In-vitro separation inclusion body, sex change and common renaturation subsequently.

Embodiment 10

BIAcore surface plasma resonance feature description with HLA-A2Tax bonded high-affinity A6 TCR

(BIAcore 3000 to have used the surface plasma resonance biological inductor ^TM) analyze combining of high-affinity clone 134A6TCR (see respectively at the global DNA of the TCR β chain of sudden change and Figure 15 a and the 15b of aminoacid sequence) and HLA-A2Tax part.The pMHC mixture (seeing following description) that produces helps this process, this mixture is fixed on bag by on the mating surface of streptavidin in the semi-directional mode, and this allows effectively to detect simultaneously solvable TCR and 4 kinds of combinations with interior different pMHC (being fixed in the different flow cells).The manual HLA of injection mixture allows the accurate level of fixed l quasi-molecule is carried out simple operations.

From the protein subunit matter that contains composition of bacterial expression and the external renaturation of inclusion body of synthetic peptide, the vitamin H that carries out purifying and vitro enzyme subsequently changes into biotinylated I class HLA-A2Tax mixture (O ' Callaghan etc., (1999) Anal.Biochem.266:9-15).Expression contains with appropriate configuration and replaces the HLA heavy chain that albumen is striden the C-terminal biotinylation label in film and cytoplasmic structure territory.Obtained the bacterial cultures of about 75 mg/litre inclusion body expression levels.HLA light chain or β2Wei Qiudanbai also with the inclusion body form from suitable member at expression in escherichia coli, expression level is about 500 mg/litre bacterial culturess.

The dissolving Bacillus coli cells also is purified to inclusion body about 80% purity.The sex change in 6M Guanidinium hydrochloride, 50mM Tris (pH 8.1), 100mM NaCl, 10mM DTT and 10mM EDTA of inclusion body protein, and, be 30 mg/litre heavy chains, 30 mg/litre β 2m refolding in 0.4M L-arginine-HCl, 100mM Tris (pH 8.1), 3.7mM cystamine, mM mercaptoethylamine, 4 mg/ml peptides (for example tax 11-19) to concentration by with single pulse denatured protein being added the mode of refolding damping fluid below 5 ℃.Refolding was finished in the time of 4 ℃ at least in 1 hour.

By dialysis exchange buffering liquid in the Tris of 10 times of volume pH 8.1 damping fluid.Need change twice damping fluid ionic strength with abundant reduction solution.Protein solution filters and loads on POROS 50HQ anion-exchange column (8 milliliters of bed volumes) by 1.5 microns acetate cellulose filters subsequently.0-500mM NaCl gradient elution protein with linearity.The HLA-A2-peptide complex is at about 250mM NaCl place wash-out, collects the component at peak value place, adds proteinase inhibitor mixture (Calbiochem) and refrigerates component on ice.

Use equilibrated Pharmacia quick desalination post in same buffer changes the damping fluid of the HLA-A2 mixture of biotinylation mark into 10mM Tris pH 8.1,5mM NaCl.Behind the wash-out, proteinaceous component is immediately in cooled on ice and add proteinase inhibitor mixture (Calbiochem).Add biotinylation reagent subsequently: 1mM vitamin H, 5mM ATP (pH is buffered to 8), 7.5mM MgCl ₂With 5 mcg/ml BirA enzymes (according to O ' Callaghan etc., the described method purifying of (1999) Anal.Biochem.266:9-15).The mixture incubated at room is spent the night.

Use the HLA-A2 mixture of gel permeation chromatography purifying biological elementization.Pharmacia Superdex 75HR10/30 chromatography column is with filtering PBS pre-equilibration and load 1 milliliter of biotinylation reaction mixture and carry out chromatography with the PBS of 0.5 ml/min flow velocity.Biotinylated HLA-A2 mixture in about 15 milliliters of forms of sentencing single peak by wash-out.Merge proteinaceous component, in cooled on ice and add the proteinase inhibitor mixture.Use coomassie binding assay (PerBio) to determine protein concn and at the biotinylation HLA-A2 of-20 ℃ of frozen sample sizes mixture.Amine coupling process by routine is streptavidin fixedly.

Contain interaction between the high-affinity A6Tax TCR of new interchain key and HLA-A2Tax or the irrelevant HLA-A2NY-ESO combination (it produces as mentioned above) at BIAcore 3000 ^TMAnalyze on surface plasma resonance (SPR) biological inductor.SPR measures the variation with near the specific refraction small flow pond interoceptor of reacton (RU) expression, and this is to be used to the principle that detects receptor-ligand interaction and analyze its avidity and kinetic parameter.By through with the crosslinked vitamin H of β 2m and with the streptavidin of flow cell activating surface chemically crosslinked between combine independent HLA-A2 peptide complex be fixed in the different flow cells, prepared the probe flow cell.Detect with the flow through surface of different flow cells of constant flow rate by sTCR subsequently, measured the SPR reaction with this.At first, interactional specificity is verified with the constant flow rate of 5 mul/min 4 different surfaces of flowing through by solvable A6TCR: the HLA-A2Tax mixture bag quilt with about 1000RU, the 2nd HLA-A2NY-ESO mixture bag quilt, and 2 blank flow cells (seeing Figure 15) with streptavidin bag quilt with about 1000RU.

The avidity of the raising of the A6TCR of sudden change makes its calculating with the interactional Kd value of HLA-A2Tax mixture is become difficult.Yet, the interactional transformation period (t of this of calculating _1/2) and the interactional t of wild-type _1/27.2 compare second is 51.6 minutes (seeing Figure 16).

Embodiment 11

The generation of the carrier of the solvable NY-ESO TCR of coding disulfide linkage

The β chain of the solvable A6TCR of preparation comprises the BglII restriction site (AAGCTT) that is suitable for use as connection site among the embodiment 1 in native sequences.

Carry out PCR mutagenesis as detailed below and introduce BamHI restriction site (GGATCC) with 5 ' end of halfcystine codon new in the α of solvable A6TCR chain.The sequence of describing among Fig. 2 a is as the template of this mutagenesis.Used following primer:

|BamHI|

5’-ATATCCAGAACCCgGAtCCTGCCGTGTA-3’(SEQ ID 135)

5’-TACACGGCAGGAaTCcGGGTTCTGGATAT-3’(SEQ ID 136)

100 nanogram plasmids mix with 5 microlitre 10mM dNTP, 25 microlitre 10xPfu damping fluids (Stratagene), 10 Pfu of unit polysaccharases (Stratagene) and use H ₂O is adjusted to 240 microlitres with final volume.Adding primer in this mixture of 48 microlitres, to make its final concentration dilution in 50 microlitre end reaction volumes be 0.2 μ M.After 95 ℃ of initial denaturing steps of 30 seconds, reactant has carried out 15 sex change of taking turns (95 ℃, 30 seconds), annealing (55 ℃, 60 seconds) and has extended (73 ℃, 8 minutes) on Hybaid PCR express PCR instrument.Digested 5 hours for 37 ℃ with 10 DpnI of unit Restriction Enzymes (New England Biolabs) with after product.10 microlitre digestion reaction thing transformed competence colibacillus XL-1-Blue bacterial strains and 37 ℃ growth 18 hours.Picking list bacterium colony and at 5 milliliters of TYP+ penbritins (16 grams per liter Tryptoness, 16 grams per liter yeast extracts, 5 grams per liter NaCl, 2.5 grams per liter K ₂HPO ₄, 100 mg/litre penbritins) in grow overnight.According to manufacturer specification on Qiagen mini-prep post purifying plasmid DNA and the automatization sequence verification by on the sequencing equipment of Oxford University department of biochemistry this sequence.

The cDNA that from the T cell, has separated coding NY-ESO TCR according to known technology.By handle the cDNA that mRNA has generated coding NY-ESO TCR with reversed transcriptive enzyme.

In order to produce the solvable NY-ESO TCR that coding has merged new disulfide linkage, the A6TCR that will contain α chain BamHI and β chain BglII restriction site is as template.Used following primer:

|NdeI|

5’-GGAGATATACATATGCAGGAGGTGACACAG-3’(SEQ ID 137)

5’-TACACGGCAGGATCCGGGTTCTGGATATT-3’(SEQ ID 138)

|BamHI|

|NdeI|

5’-GGAGATATACATATGGGTGTCACTCAGACC-3’(SEQ ID 139)

5’-CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3’(SEQ ID 140)

|BglII|

NY-ESO TCR α and β chain member have been obtained by following PCR clone.Use above-mentioned primer and the template that contains natural NY-ESO TCR chain to carry out the PCR reaction.The PCR product has carried out restrictive diges-tion with corresponding Restriction Enzyme, and is cloned into pGMT7 to obtain expression plasmid.The plasmid insertion sequence confirms by the automatization dna sequencing.Figure 17 a and 17b show the NY-ESO TCR α of sudden change and the dna sequence dna of β chain respectively, and Figure 18 a and 18b show final aminoacid sequence.

Embodiment 12

The dna clone of Construction of Vector for Phage Display and will encode NY-ESO TCR α and β chain advances this phagemid carrier

According to the merging that produces described in the embodiment 11 new halfcystine codon to promote to form the coding solvable NY-ESO TCR α of non-natural interchain disulfide bond and the following phagemid carrier pEX746 that incorporates into of DNA of β chain.

Use following primer respectively the DNA of two the NY-ESO TCR chains of encoding to be carried out PCR to introduce and the compatible cloning site of pEX746 phagemid carrier (DNA that contains coding A6TCR clone 7):

Primer at NY-ESO TCR α chain

TRAV21

GCCG GCCATGGCCAAACAGGAGGTGACGCAGATTCCT(SEQ ID 141)

YOL6

CTTCTTAAAGAATTCTTAATTA ACCTAGGTTATTAGGAACTTTCTGGGCTG

GGGAAG(SEQ ID 142)

Primer at NY-ESO TCR β chain

TRBV6-1/2/3/5/6/7/8/9

TCAC AGCGCGCAGGCTGGTGTCACTCAGACCCCAAA(SEQ ID 143)

RT1

CGAGAGCCCGTAGAACTGGACTTG(SEQ ID 144)

Molecular cloning method in order to carrier construction has been described in J.Sambrook and D.W.Russell " molecular cloning: laboratory manual ".The primer of enumerating in the table 1 is used for the structure of described carrier.An example of PCR program is 94 ℃ of 1 circulations in 2 minutes, 94 ℃ 5 seconds, 53 ℃ 5 seconds and 72 ℃ of 25 circulations in 90 seconds subsequently, then 72 ℃ of 1 circulations in 10 minutes, and 4 ℃ of insulations subsequently.High-fidelity Taq archaeal dna polymerase is available from Roche.

By from pEX746, remove the DNA of coding clone 7 A6 TCR β chains with the digestion of Restriction Enzyme BssHII and BglII.Replace into this phagemid by the PCR DNA of connection subsequently the coding NY-ESO β chain of corresponding digestion.The sequence of clone's product is verified by automatic sequencing.

Similarly, by from pEX746, remove the DNA of coding clone 7A6TCR α chain with the digestion of Restriction Enzyme NcoI and AvrII.Replace phagemid by connecting PCR DNA with the coding NY-ESO α chain of corresponding digestion subsequently into the DNA that comprises coding NY-ESO TCR β chain.The sequence of clone's product is verified by automatic sequencing.Figure 19 a and 19b have described the DNA and the aminoacid sequence of NY-ESO TCR α and β chain respectively in detail and have incorporated correlated series around its of phage (pEX746:NY-ESO) into.Sequence before the NcoI site is identical with pEX746.

Embodiment 13

The expression of fusion rotein in intestinal bacteria of bacterium coat protein and allos dimerization NY-ESO TCR

Use describedly is used to produce the antibody scFv that phage particle shows and is following improved method (Li etc. previous, 2000, Journal of Immunological Methods 236:133-146) prepared and showed the allos dimerization NY-ESO TCR that contains the non-natural interchain disulfide bond.E. coli tg1 cell inoculation to the 10 milliliter 2xTY (containing 100 mcg/ml penbritins and 2% glucose) that will contain pEX746:NY-ESO phagemid (promptly encode solvable NY-ESO TCR α chain and the phagemid method generation as described in example 12 above and the NT-ESO TCR β chain fusion of phage gIII albumen), the overnight incubation (16 hours) of 37 ℃ of vibrations of culture subsequently.50 microlitre overnight culture are inoculated in 10 milliliters of 2xTY (containing 100 mcg/ml penbritins and 2% glucose), and 37 ℃ of vibrations of culture are subsequently hatched until OD (600nm) and are reached 0.8.Adding final concentration in culture is 5 * 10 ⁹The HYPERPHAGE helper phage of pfu/ml.Culture leaves standstill for 37 ℃ and hatched 30 minutes and hatched 30 minutes with the further vibration of 200rpm subsequently.Use 2xTY (containing 100 mcg/ml penbritins and 25 mcg/ml kantlex) that the culture volume of above-mentioned culture is adjusted into 50 milliliters subsequently, 25 ℃ of 250rpm vibrations were subsequently hatched 36 to 48 hours.4 ℃ of 4000rpm of culture centrifugal 30 minutes subsequently.Supernatant liquor filters also 4 ℃ of preservations further to concentrate through 0.45 micron syringe filter.Supernatant liquor concentrates by the PEG precipitation with the PBS of 10% original preservation volume is resuspended subsequently.

Embodiment 14

Contain the detection of the functional allos dimerization NY-ESOT CR of non-natural interchain disulfide bond on the filobactivirus particle

Use the phage E LISA method described in the embodiment 4 that functional (HLA-A2-NY-ESO bonded) the NY-ESO TCR that shows on the phage particle in the concentrated suspension prepared among the embodiment 13 is detected.The NY-ESO TCR that Figure 20 explanation is showed at phage E LISA assay method pnagus medius particle combines with the specificity of HLA-A2-NY-ESO.

Embodiment 15

The structure of the plasmid of expressing in order to the HLA-DRA gene cell

Use polymerase chain reaction (PCR) and be designed to comprise that the synthetic dna primer of BglII restriction site is right, from separating the dna sequence dna of HLA-DRA chain born of the same parents outside part of encoding that increased from healthy human blood's cDNA.

Use 3 ' the terminal DNA that add coding Fos leucine zipper of PCR mutagenesis subsequently at this extension increasing sequence.

Above-mentioned DNA operation and clone are according to Sambrook, J etc. show " molecular cloning-laboratory manual " the 2nd edition, and (Molecular Cloning-A Laboratory Manual.Second Edit ion.Cold Spring HarborLaboratory Press carries out described in USA).

Figure 21 provides the dna sequence dna of the HLA-DR β chain that is suitable for inserting two-cistron expression vector.This figure has pointed out the position of the codon of coding Fos leucine zipper peptide and biotin label.

Amino acid is numbered (Kabat, 1991, " sequence of protein of interest on the immunology " the 5th edition (Sequences of Proteins of Immunological Interest, 5 on the basis that is based upon the mouse sequence ^ThEdition, US Dept of Health﹠amp; Human Services, Public Health Service, NIH, Bethesda, MD 1-1137).

Subsequently this dna sequence dna is inserted baculovirus vector pAcAB3 (seeing the sequence of this carrier shown in Figure 22) with in the Sf9 expressed in insect cells together with the DNA of the corresponding II class HLA β chain of coding.This carrier is used in any II class of expressed in insect cells HLA-peptide complex.

Embodiment 16

Structure in order to the plasmid of HLA-DRB wild-type and mutant gene cell expressing

Use polymerase chain reaction (PCR) and be designed to comprise that the synthetic dna primer of BamHI restriction site is right, from separating the dna sequence dna of HLA-DRA chain born of the same parents outside part of encoding that increased from healthy human blood's cDNA.

Use PCR mutagenesis at 3 ' the terminal DNA that adds coding Jun leucine zipper of this extension increasing sequence and at 5 ' the terminal DNA that adds coding by the Flu HA peptide of HLA-DR1 molecule load of this sequence subsequently.

Above-mentioned DNA operation and clone are according to Sambrook, J etc. show " molecular cloning-laboratory manual " the 2nd edition (Molecular Cloning-A Laboratory Manual.Second Edit ion.Cold Spring HarborLaboratory Press, USA, 1999) carry out described in.

Figure 23 provides the dna sequence dna of the HLA-DR β chain that is suitable for inserting two-cistron expression vector.This figure has pointed out the position of the codon of coding Jun leucine zipper peptide and Flu HA peptide.

Amino acid is numbered (Kabat, 1991, " sequence of protein of interest on the immunology " the 5th edition (Sequences of Proteins of Immunological Interest, 5 on the basis that is based upon the mouse sequence ^ThEdition, US Dept of Health﹠amp; Human Services, Public Health Service, NIH, Bethesda, MD 1-1137).

Subsequently this dna sequence dna is inserted baculovirus vector pAcAB3 (seeing the sequence of this carrier shown in Figure 22) with in the Sf9 expressed in insect cells together with the DNA of the corresponding II class HLA β chain of coding.This carrier is used in any II class of expressed in insect cells HLA-peptide complex.

Embodiment 17

The expression and the renaturation of II class HLA-DR1-Flu HA mixture

Use as the two-cistron expression vector that contains II class HLA-DR1 α and β chain and Flu HA peptide of embodiment 15 and 16 described methods generations and realized that II class MHC expresses.Described in the expression of using and purification process such as Gauthier (1998) PNAS USA 95 11828-11833.In brief, by being used in rhabdovirus system, having expressed soluble HLA-DR1 from the leucine zipper dimerization structural domain replacement DR α that transcribes factor Fos and Jun and the hydrophobicity membrane spaning domain and the kytoplasm fragment of β chain.In expressing member, covalently bound and CR α chain comprises the biotinylation label utilizes the double function ligand of vitamin H/streptavidin multimerization method with promotion formation to the Flu HA peptide sequence of the II class MHC load that needs with the N-terminal of ripe DR β chain.With the Sf9 emiocytosis recombinant protein of recombinate shape virus infection, and recombinant protein is carried out purifying by affinity chromatography.This albumen further carries out purifying by anionresin HPLC.

The structure of the solvable peptide of embodiment 18-I class-HLA molecule

In order further to study high-affinity A6TCR clone 134 specificity, generated following solvable I class peptide-HLA molecule:

HLA-A2-peptide (LLGRNSFEV) (SEQ ID 23)

HLA-A2-peptide (KLVALGINAV) (SEQ ID 24)

HLA-A2-peptide (LLGDLFGV) (SEQ ID 25)

HLA-B8-peptide (FLRGRAYGL) (SEQ ID 26)

HLA-B27-peptide (HRCQAIRKK) (SEQ ID 27)

HLA-Cw6-peptide (YRSGIIAVV) (SEQ ID 28)

HLA-A24-peptide (VYGFVRACL) (SEQ ID 29)

HLA-A2-peptide (ILAKFLHWL) (SEQ ID 30)

HLA-A2-peptide (LTLGEFLKL) (SEQ ID 31)

HLA-A2-peptide (GILGFVFTL) (SEQ ID 33)

HLA-A2-peptide (SLYNTVATL) (SEQ ID 34)

These solvable peptides-HLA uses the method generation of implementing described in 10.

Embodiment 19-clones the BIAcore surface plasma resonance of 134 high-affinity A6 TCR and peptide-HLA binding specificity and measures

(BIAcore 3000 to have used the surface plasma resonance biological inductor ^TM) analyze the binding specificity of high-affinity clone 134A6TCR (seeing the global DNA and the aminoacid sequence of the mutation T CR β chain that Figure 15 a and 15b represent respectively).This is achieved by the I class peptide-HLA mixture implemented in 18 that is set forth in that uses as the II class HLA-DR1-peptide of generation as described in the embodiment 15-17 and use the method that describes in detail among the embodiment 10 to produce.Following table has been enumerated the peptide-HLA mixture that adopts:

1.HLA-A2-peptide (LLGRNSFEV) (SEQ ID 23)

2.HLA-A2-peptide (KLVALGINAV) (SEQ ID 24)

3.HLA-A2-peptide (LLGDLFGV) (SEQ ID 25)

4.HLA-B8-peptide (FLRGRAYGL) (SEQ ID 26)

5.HLA-B27-peptide (HRCQAIRKK) (SEQ ID 27)

6.HLA-Cw6-peptide (YRSGIIAVV) (SEQ ID 28)

7.HLA-A24-peptide (VYGFVRACL) (SEQ ID 29)

8.HLA-A2-peptide (ILAKFLHWL) (SEQ ID 30)

9.HLA-A2-peptide (LTLGEFLKL) (SEQ ID 31)

10.HLA-DR1-peptide (PKYVKQNTLKLA) (SEQID 32)

11.HLA-A2-peptide (GILGFVFTL) (SEQ ID 33)

12.HLA-A2-peptide (SLYNTVATL) (SEQ ID 34)

Above peptide-HLA is fixed on BIAcore 3000 in the semi-directional mode ^TMOn the mating surface of the streptavidin bag quilt in the flow cell.

BIAcore 3000 ^TMAllow simultaneously solvable TXi Baoshouti is detected with 4 kinds of combinations with interior different pMHC (it is interior to be fixed on different flow cells).In flow cell 2-4, fix 3 kinds of different HLA-peptides and flow cell 1 in the experiment and given over to blank.The manual HLA-of injection peptide complex allows the accurate level of fixed member is handled.

After the binding ability of preceding 3 kinds of HLA-peptide complex in high-affinity A6TCR clone 134 and the above tabulation was assessed, ensuing 3 kinds of mixtures directly were fixed in these flow cells on the last reply compound.Continuing this process assesses with combining of all 12 kinds of HLA-peptide complex until high-affinity A6TCR clone 134.

The 5 microlitre high-affinity A6TCR clone 134 who injects with between 4.1 nanograms/milliliter to the flow velocity of the concentration of 2.1 mg/ml and 5 mul/min by each flow cell (seeing Figure 24-28).

As last contrast, high-affinity A6TCR clone 134 is by containing the flow cell of fixed HLA-A2Tax (LLFGYPVYV) (SEQ ID 21) (the similar part of this TCR).

Only found the specificity combination of high affinity A6TCR clone's 134 similar parts (HLA-A2Tax (LLFGYPVYV) (SEQ ID21)) with it.These data further specify high-affinity A6TCR clone 134 specificity (seeing Figure 24-28).

The mutagenesis in embodiment 20-NY-ESO TCR CDR3 district

The CDR3 district of NY-ESO TCR is as introducing sudden change produces high-affinity mutant possibility with research target.This by be used in combination NY-ESO TCR specific PCR primer with embodiment 7 in the fully identical method of the method that describes in detail be achieved.

The separation of embodiment 21-high-affinity A6TCR mutant

First method in use embodiment 8 described two kinds of methods has realized the separation of high-affinity NY-ESO TCR mutant.

Differentiated single high-affinity NY-ESO TCR.

Embodiment 22

Generation with solvable high-affinity allos dimerization NY-ESO TCR that contains the variable domains sudden change of non-natural disulfide linkage between the constant domain

(Quiagen UK) has separated the phage DNA of the high-affinity NY-ESO TCR mutant of being differentiated among the coding embodiment 20 from corresponding Bacillus coli cells to use the Mini-Prep test kit.

Use with phagemid dna as the pcr amplification of template and following primer be used to the to increase solvable NY-ESOTCR β chain variable domains dna sequence dna of sudden change

NY-ESO β chain forward primer

Tctctc attaatgaatgctggtgtcactcagacccc(SEQ ID 145)

(the AseI restriction site is represented with underscore)

General β chain reverse primer

Tagaa accggtggccaggcacaccagtgtgg(SEQ ID 146)

(the AgeI restriction site is represented with underscore)

The PCR product advances pEX821 with the NdeI/AgeI cutting (as generation as described in the embodiment 11) with AgeI/AseI digestion and clone subsequently.

Kuo Zeng sudden change NY-ESO TCR β chain DNA sequence and be used for subsequently producing as mentioned above as WO 03/020763 described solvable high-affinity NY-ESO TCR as the NY-ESOTCR α chain of generation as described in the embodiment 11.In brief, these two chains are expressed in different culture of Escherichia coli with the form of inclusion body.Subsequently inclusion body is carried out renaturation outside purifying, sex change and the community.

The BIAcore surface plasma resonance feature description of embodiment 23-and HLA-A2NY-ESO bonded high-affinity NY-ESO TCR

(Biacore 3000 to have used the surface plasma resonance biological inductor ^TM) analyze combining of high-affinity NY-ESOTCR and HLA-A2NY-ESO part.Thereby this is allowed effectively to detect solvable TCR simultaneously on mating surface of streptavidin bag quilt and is promoted with interior different pMHC (being fixed in the different flow cells) bonded pMHC mixtures (as described in embodiment 10) with 4 kinds by being fixed in the semi-directional mode of generating.The manual HLA of injection mixture makes the accurate level of fixed I quasi-molecule is carried out easy manipulation.

Also as described in example 10 above, at Biacore 3000 ^TMAnalyzed the interaction between the high-affinity NY-ESO TCR that contains new interchain key and HLA-A2NY-ESO mixture or the irrelevant HLA-A2Tax composition (having described it among the embodiment 10 produces) on surface plasma resonance (SPR) biological inductor.

The interactional Kd value of solvable high-affinity NY-ESO and HLA-A2NY-ESO is calculated as 4.1 μ M (seeing Figure 29 a and 29b), and this and wild-type interaction Kd value are that 15.7 μ M (seeing Figure 30 a and 30b) form contrast.

Generation and the detection of the more high-affinity A6TCR of embodiment 24-

Use the method that is described in detail among the embodiment 9, produced and contain following solvable TCR corresponding to the sudden change of being differentiated in clone's 89,1,111 and 71 (seeing embodiment 8).Using the Biacore assay method that describes in detail among the embodiment 10 that these solvable TCR are combined with HLA-A2Tax subsequently assesses.

A6TCR β chain mutant

The clone	The CDR3 sequence
		Wild-type	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGAGCAGTAG (SEQ ID 83) A S R P G L A G G R P E Q Y (SEQ ID 84)
89	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCAGAGGATTAG (SEQ ID 91) A S R P G L A G G R P E D X (SEQ ID 92)
		1	GCCTCGAGGCCGGGGCTGGTTCCGGGGCGACCAGAGCAGTAG (SEQ ID 97) A S R P G L V P G R P E Q X (SEQ ID 98)
111	GCCTCGAGGCCGGGACTAGCGGGAGGGCGACCACATCCGTAG (SEQ ID 101) A S R P G L A G G R P H P X (SEQ ID 102)

A6TCR α chain mutant

The clone	CDR3
		Wild-type	GCCGTTACAACTGACAGCTGGGGGAAGCTTCAG (SEQ ID 107) A V T T D S W G K L Q (SEQ ID 108)
71	GCCGTTACAACTGACAGCTGGGGGGAGCTTCAT (SEQ ID 117) A V T T D S W G E L H (SEQ ID 118)

The sudden change of combination is as producing the solvable A6TCR that suddenlys change:

Clone 89 sudden change+clones 134 sudden changes

Clone 71 sudden change+clones 134 sudden changes

Clone 71 sudden change+clones 89 sudden changes

Clone 1 sudden change+β G102 → A sudden change

The result:

Following form has compared above soluble sudden change A6TCR and has contained the avidity that not mutated variable domains obtains with use.Note, with interactional half life (T _1/2) represent the avidity of high-affinity mutant.As interact by it lower Kd value or longer T _1/2Proved that these solvable sudden change A6TCR have the higher avidity than not mutated solvable A6TCR to HLA-A2Tax.

Figure 31-37 has shown and has been used to calculate the Biacore record of these solvable mutation T CR to HLA-A2Tax avidity.Figure 38 a-e has shown the aminoacid sequence of sudden change A6TCR chain.

A6TCR	Kd(μM)	T 1/2(second)
			Wild-type	1.9	7
Clone 1		810
			Clone 89	0.41
Clone 111	1.18
			Clone 71	1.37
Clone 89+ clone 134 sudden changes		114 (time mutually 1) 4500 (time mutually 2)
			Clone 71+ clone 134 sudden changes		882
Clone 71+ clone 89 sudden changes	0.35
			Clone 1+ β G102 → A sudden change		738

Embodiment 25-uses high-affinity A6TCR tetramer and monomeric cell dyeing

T2 antigen presenting cell and β 2m (3 mcg/ml) were hatched 90 minutes for 37 ℃, simultaneously with the pulse mode transportation concentration between 10 ^-5M-10 ^-9The Tax peptide of M.For the contrast of also using the T2 cell of hatching, carry 10 with pulse mode with β 2m (3 mcg/ml) ^-5The Flu peptide of M or do not hatch (no pulse) jointly with peptide.After pulse was carried, cell was used serum-free RPMI cleaning and 2 * 10 ⁵Cell and usefulness phycoerythrin (PE) (Molecular probes, TheNetherlands) the continuous high-affinity of the streptavidin of (10 mcg/ml) mark is cloned the 134A6TCR tetramer or was cloned 134A6TCR monomer incubated at room 10 minutes with the high-affinity of Alexa 488 (Molecular probes, The Netherlands) mark.After cell cleans, use FACSVantage SE (Becton Dickinson) to check the TCR tetramer and the monomeric combination of mark by flow cytometer.

The result

Shown in Figure 39 a, when the Tax peptide concentration is lower than 10 ^-9During M, can observe the T2 cell by the tetrameric specific stain of high-affinity A6TCR.

Shown in Figure 39 b, when the Tax peptide concentration is lower than 10 ^-8During M, can observe the T2 cell by the monomeric specific stain of high-affinity A6TCR.

The applicant of company

----------------------

Street: Milton park 57C

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: martin.green@avidex.com

＜110〉Business Name: Avidex Co., Ltd

Individual application people

----------------------

Street: Milton park 57C, Avidex Co., Ltd

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: bent.jakobsen@avidex.com

＜110〉surname: Jacobson

＜110〉name: Ben Te

＜110〉middle name: K

＜110〉note: doctor

Individual application people

----------------------

Street: Milton park 57C, Avidex Co., Ltd

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: torben.andersen@avidex.com

＜110〉surname: Anderson

＜110〉name: Tuo Ben

＜110〉middle name: B

＜110〉note: doctor

Individual application people

---------------------

Street: Milton park 57C, Avidex Co., Ltd

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: peter.molloy@avidex.com

＜110〉surname: Mo Luoyi

＜110〉name: Bi Te

＜110〉middle name: E

＜110〉note: doctor

Individual application people

----------------------

Street: Milton park 57C, Avidex Co., Ltd

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: jonathan.boulter@avidex.com

＜110〉surname: Bai Erte

＜110〉name: Jonathan

＜110〉middle name: M

＜110〉note: doctor

Individual application people

----------------------

Street: Milton park 57C, Avidex Co., Ltd

City: A Bindun

State: Oxfordshire

Country: Britain

Postcode: OX14 4RX

Telephone number :+44 1,235 438600

Fax number :+44 1,235 438601

E-mail address: yi.li@avidex.com

＜110〉surname: Lee

＜110〉name: virtuous

＜110〉middle name:

＜110〉note: doctor

The application scheme

----------------------

＜120〉denomination of invention: material

＜130〉application documents numbering: Case No 19

＜140〉current application number: GB 0226227.7

＜141〉the current applying date: 2002-11-09

In first to file

----------------------

＜150〉application number: GB 0226227.7 formerly

＜151〉applying date: 2002-11-09 formerly

In first to file

＜150〉application number: GB 0301814.0 formerly

＜151〉applying date: 2003-01-25 formerly

In first to file

----------------------

＜150〉application number: GB 0304067.2 formerly

＜151〉applying date: 2003-02-22 formerly

In first to file

---------------------

＜150〉application number: GB 0311397.4 formerly

＜151〉applying date: 2003-05-16 formerly

In first to file

----------------------

＜150〉application number: GB 0316356.5 formerly

＜151〉applying date: 2003-07-11 formerly

In first to file

----------------------

＜150〉application number: us 60/463,323 formerly

＜151〉applying date: 2003-04-16 formerly

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

DSDVYITDKT VLDMRSMDFK 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 1

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

QSKDSDVYIT DKTVLDMRSM 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 2

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

DI QNPDPAVY QLRDSKSSDK 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 3

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

DPAVYQLRDS KSSDKSVCLF 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 4

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

NGKEVHSGVS TDPQPLKEQP 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 5

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

NGKEVHSGVSTDPQPLKEQP 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 5

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ALNDSRYALSSRLRVSATFW 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 6

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

PPEVAVFEPSEAEISHTQKA 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 7

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

KEVHSGVSTDPQPLKEQPAL 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 8

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

VFPPEVAVFPSEAEISHTQ 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 9

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKS 60

NGAIAWSNQTSFTCQDIFKETNATYPSSDVP 91

＜212〉type: PRT

＜211〉length: 91

Sequence title: SEQ ID 10

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGREVHSGVSTDP 60

QAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAE 120

AWGRAD 126

＜212〉type: PRT

＜211〉length: 126

Sequence title: SEQ ID 11

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ESGTFITDKTVLDMKAMDSK 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 12

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

KTMESGTFITDKTVLDMKAM 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 13

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

YIQNPEPAVYQLKDPRSQDS 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 14

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVYQLKDPRSQDSTLCLFTD 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 15

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

NGREVHSGVSTDPQAYKESN 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 16

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

KESNYSYCLSSRLRVSATFW 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 17

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

PPKVSLFPSKAEIANKQKA 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 18

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

REVHSGVSTDPQAYKESNYS 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 19

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

VTPPKVSLFEPSKAEIANKQ 20

＜212〉type: PRT

＜211〉length: 20

Sequence title: SEQ ID 20

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

LLFGYPVYV 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 21

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

SLLMITQC 8

＜212〉type: PRT

＜211〉length: 8

Sequence title: SEQ ID 22

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

LLGRNSFEV 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 23

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

KLVALGINAV 10

＜212〉type: PRT

＜211〉length: 10

Sequence title: SEQ ID 24

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

LLGDLFGV 8

＜212〉type: PRT

＜211〉length: 8

Sequence title: SEQ ID 25

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

FLRGRAYGL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 26

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

HRCQAIRKK 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 27

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

YRSGIIAVV 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 28

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

VYGFVRACL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 29

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ILAKFLHWL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 30

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

LTLGEFLKL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 31

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

PKYVKQNTLKLA 12

＜212〉type: PRT

＜211〉length: 12

Sequence title: SEQ ID 32

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

GILGFVFTL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 33

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

SLYNTVATL 9

＜212〉type: PRT

＜211〉length: 9

Sequence title: SEQ ID 34

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cacagacaaa tgtgtgctag acat 24

＜212〉type: DNA

＜211〉length: 24

Sequence title: SEQ ID 35

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 35

Sequence

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgtctagca cacatttgtc tgtg 24

＜212〉type: DNA

＜211〉length: 24

Sequence title: SEQ ID 36

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 36

Sequence

＜213〉organism title: artificial sequence

＜400〉presequence string:

cagtggggtc tgcacagacc c 21

＜212〉type: DNA

＜211〉length: 21

Sequence title: SEQ ID 37

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 37

Sequence

＜213〉organism title: artificial sequence

＜400〉presequence string:

gggtctgtgc agaccccact g 21

＜212〉type: DNA

＜211〉length: 21

Sequence title: SEQ ID 38

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 38

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

taataatacg tataataata ttctatttca aggagacagt c 41

＜212〉type: DNA

＜211〉length: 41

Sequence title: SEQ ID 39

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 39

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

caatccagcg gctgccgtag gcaataggta tttcattatg actgtctcct tgaaatag 58

＜212〉type: DNA

＜211〉length: 58

Sequence title: SEQ ID 40

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 40

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctacggcagc cgctggattg ttattactcg cggcccagcc ggccatggcc cag 53

＜212〉type: DNA

＜211〉length: 53

Sequence title: SEQ ID 41

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 41

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gttctgctcc acttccttct gggccatggc cggctgggcc g 41

＜212〉type: DNA

＜211〉length: 41

Sequence title: SEQ ID 42

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 42

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cagaaggaag tggagcagaa c 21

＜212〉type: DNA

＜211〉length: 21

Sequence title: SEQ ID 43

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 43

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cttcttaaag aattcttaat taacctaggt tattaggaac tttctgggct ggggaag 57

＜212〉type: DNA

＜211〉length: 57

Sequence title: SEQ ID 44

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 44

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gttaattaag aattctttaa gaaggagata tacatatgaa aaaattatta ttcgcaattC 60

＜212〉type: DNA

＜211〉length: 60

Sequence title: SEQ ID 45

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 45

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cgcgctgtga gaatagaaag gaacaactaa aggaattgcg aataataatt ttttcatatG 60

＜212〉type: DNA

＜211〉length: 60

Sequence title: SEQ ID 46

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 46

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctttctattc tcacagcgcg caggctggtg tcactcagac 40

＜212〉type: DNA

＜211〉length: 40

Sequence title: SEQ ID 47

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 47

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgatgtcta gatgcggccg cgtctgctct accccaggcc tc 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 48

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 48

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcatctagac atcatcacca tcatcactag actgttgaaa gttgtttagc aaaac 55

＜212〉type: DNA

＜211〉length: 55

Sequence title: SEQ ID 49

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 49

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctagagggta ccttattaag actccttatt acgcagtatg 40

＜212〉type: DNA

＜211〉length: 40

Sequence title: SEQ ID 50

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 50

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

agctgcagct aatacgactc actataggaa caggccacca tggcgtcgat tatgctgagt 60

gatatccttg tccggtggta ccctag 86

＜212〉type: DNA

＜211〉length: 86

Sequence title: SEQ ID 51

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 51

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

agctgcagct aatacgactc actataggaa caggccacca tgg 43

＜212〉type: DNA

＜211〉length: 43

Sequence title: SEQ ID 52

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 52

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gatcccatgg tggcctgttc ctatagtgag tcgtattagc tgc 43

＜212〉type: DNA

＜211〉length: 43

Sequence title: SEQ ID 53

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 53

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ccaccatggg ccagaaggaa gtggagcaga actc 34

＜212〉type: DNA

＜211〉length: 34

Sequence title: SEQ ID 54

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 54

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cgagagcccg tagaactgga cttg 24

＜212〉type: DNA

＜211〉length: 24

Sequence title: SEQ ID 55

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 55

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gtggatccgg cggtggcggg tcgaacgctg gtgtcactca gacccc 46

＜212〉type: DNA

＜211〉length: 46

Sequence title: SEQ ID 56

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 56

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ccggatccac ctccgcctga accgcctcca ccggtgacca caacctgggt ccctg 55

＜212〉type: DNA

＜211〉length: 55

Sequence title: SEQ ID 57

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 57

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctgagaattc ttatgactct ccgcggttga agctc 35

＜212〉type: DNA

＜211〉length: 35

Sequence title: SEQ ID 58

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 58

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgacgaattc tgactctccg cggttgaagc tc 32

＜212〉type: DNA

＜211〉length: 32

Sequence title: SEQ ID 59

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 59

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

agctgcagct aatacgactc actatagg 28

＜212〉type: DNA

＜211〉length: 28

Sequence title: SEQ ID 60

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 60

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggccaccatg ggcaacgctg gtgtcactca gacccc 36

＜212〉type: DNA

＜211〉length: 36

Sequence title: SEQ ID 61

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 61

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgaaccgcct ccaccgtctg ctctacccca ggcctcggcg 40

＜212〉type: DNA

＜211〉length: 40

Sequence title: SEQ ID 62

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 62

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgactctccg cggttgaagc tc 22

＜212〉type: DNA

＜211〉length: 22

Sequence title: SEQ ID 63

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 63

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cagctggggg aagcttcagt ttggagcag 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 64

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 64

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctgctccaaa ctgaagcttc ccccagctg 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 65

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 65

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gtacttctgt gcctcgaggc cgggactag 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 66

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 66

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctagtcccgg cctcgaggca cagaagtac 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 67

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 67

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cattttcagg gatagcaagc 20

＜212〉type: DNA

＜211〉length: 20

Sequence title: SEQ ID 68

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 68

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tcacacagga aacagctatg tcacacagga aacagctatg 40

＜212〉type: DNA

＜211〉length: 40

Sequence title: SEQ ID 69

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 69

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggnnknnknn knnknnknnk cgaccagagc agtacttcg 49

＜212〉type: DNA

＜211〉length: 49

Sequence title: SEQ ID 70

Sequence description:

Feature

--------

Sequence: SEQ ID 70

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 49

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 70

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggccgnnknn knnknnknnk nnkccagagc agtacttcgg gc 52

＜212〉type: DNA

＜211〉length: 52

Sequence title: SEQ ID 71

Sequence description:

Feature

--------

Sequence: SEQ ID 71

＜221〉characteristic key words: misc_feature

＜222〉zero position:

＜222〉final position:

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 71

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 52

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 71

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggccgnnknn knnknnknnk nnkcgaccag agcagtactt cg 52

＜212〉type: DNA

＜211〉length: 52

Sequence title: SEQ ID 72

Sequence description:

Feature

--------

Sequence: SEQ ID 72

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 52

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 72

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggccgnnknn knnknnknnk nnkggagggc gaccagagca g 51

＜212〉type: DNA

＜211〉length: 51

Sequence title: SEQ ID 73

Sequence description:

Feature

--------

Sequence: SEQ ID 73

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 51

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 73

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggccgggann knnknnknnk nnknnkgggc gaccagagca gtac 54

＜212〉type: DNA

＜211〉length: 54

Sequence title: SEQ ID 74

Sequence description:

Feature

--------

Sequence: SEQ ID 74

＜221〉characteristic key words: misc_feature

＜222〉zero position:

＜222〉final position:

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 74

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 54

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 74

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggnnknnknn knnknnknnk ccagagcagt acttcgggc 49

＜212〉type: DNA

＜211〉length: 49

Sequence title: SEQ ID 75

Sequence description:

Feature

--------

Sequence: SEQ ID 75

＜221〉characteristic key words: misc_feature

＜222〉zero position:

＜222〉final position:

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 75

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 49

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 75

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggnnknnknn knnknnknnk gagcagtact tcgggccg 48

＜212〉type: DNA

＜211〉length: 48

Sequence title: SEQ ID 76

Sequence description:

Feature

--------

Sequence: SEQ ID 76

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 48

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 76

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggnnknnknn knnknnknnk cagtacttcg ggccgggc 48

＜212〉type: DNA

＜211〉length: 48

Sequence title: SEQ ID 76

Sequence description:

Feature

--------

Sequence: SEQ ID 77

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 48

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 77

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tgtgcctcga ggccgnnknn knnknnkggg cgaccagagc agtacttcg 49

＜212〉type: DNA

＜211〉length: 49

Sequence title: SEQ ID 78

Sequence description:

Feature

--------

Sequence: SEQ ID 78

＜221〉characteristic key words: misc_feature

＜222〉zero position:

＜222〉final position:

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 78

＜221〉characteristic key words: miscfeature

＜222〉zero position: 1

＜222〉final position: 49

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 78

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

aaactgaagc ttmnnmnnmn nmnnmnntgt aacggcacag aggtag 46

＜212〉type: DNA

＜211〉length: 46

Sequence title: SEQ ID 79

Sequence description:

Feature

--------

Sequence: SEQ ID 79

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 46

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 79

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

aaactgaagc ttmnnmnngc tgtcmnntgt aacggcacag aggtag 46

＜212〉type: DNA

＜211〉length: 46

Sequence title: SEQ ID 80

Sequence description:

Feature

--------

Sequence: SEQ ID 80

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 46

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 80

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

aaactgaagc ttmnnmnnmn ngctgtcmnn tgtaacggca cagaggtag 49

＜212〉type: DNA

＜211〉length: 49

Sequence title: SEQ ID 81

Sequence description:

Feature

--------

Sequence: SEQ ID 81

＜221〉characteristic key words: misc_feature

＜222〉zero position:

＜222〉final position:

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 81

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 49

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 81

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

aaactgaagc ttmnnmnngc tgtcmnnaac ggcacagagg tag 43

＜212〉type: DNA

＜211〉length: 43

Sequence title: SEQ ID 82

Sequence description:

Feature

--------

Sequence: SEQ ID 82

＜221〉characteristic key words: misc_feature

＜222〉zero position: 1

＜222〉final position: 43

Out of Memory: m is a or c; N is a or t or g or c; K is g or t

CDS connects: do not have

Habitual codon

--------

Sequence title: SEQ ID 82

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccagagcagt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 83

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 83

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 84

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cggggctgat gagtgcgtag ccagagcagt ac 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 85

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 85

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLMSAX PEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 86

Sequence description:

Feature

--------

Sequence: SEQ ID 86

＜221〉characteristic key words: MISC FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cggggctgag gtcggcgtag ccagagcagt ac 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 87

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 87

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLRSAX PEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 88

Sequence description:

Feature

--------

Sequence: SEQ ID 88

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccagaggcgt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 89

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 89

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPEAX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 90

Sequence description:

Feature

--------

Sequence: SEQ ID 90

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 90

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccagaggatt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 91

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 91

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPEDX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 92

Sequence description:

Feature

--------

Sequence: SEQ ID 92

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccagatcagt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 93

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 93

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPDQX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 94

Sequence description:

Feature

--------

Sequence: SEQ ID 94

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 94

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggtctgta ggctgggcga ccagagcagt ac 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 95

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 95

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLX AGRPEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 96

Sequence description:

Feature

--------

Sequence: SEQ ID 96

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 96

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cggggctggt tccggggcga ccagagcagt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 97

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 97

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLVPGRPEQX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 98

Sequence description:

Feature

--------

Sequence: SEQ ID 98

＜221〉characteristic key words: MISC FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 98

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cggggcttgt gtctgcttag ccagagcagt ac 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 99

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 99

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLVSAX PEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 100

Sequence description:

Feature

--------

Sequence: SEQ ID 100

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccacatccgt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 101

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 101

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPHPX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 101

Sequence description:

Feature

--------

Sequence: SEQ ID 102

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 102

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggactagc gggagggcga ccagatgcgt ag 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 103

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 103

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLAGGRPDAX 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 104

Sequence description:

Feature

--------

Sequence: SEQID 104

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gcctcgaggc cgggtctgat tagtgcttag ccagagcagt ac 42

＜212〉type: DNA

＜211〉length: 42

Sequence title: SEQ ID 105

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 105

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ASRPGLISAX PEQY 14

＜212〉type: PRT

＜211〉length: 14

Sequence title: SEQ ID 106

Sequence description:

Feature

--------

Sequence: SEQ ID 106

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 14

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaagctt cag 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 107

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 107

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKLQ 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 108

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggccgctt cag 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 109

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 109

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGPLQ 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 110

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaagatg cag 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 111

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 111

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKMQ 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 112

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaagttg cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 113

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 113

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKLH 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 114

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggtagctt cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 115

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 115

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGXL H 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 116

Sequence description:

Feature

--------

Sequence: SEQ ID 116

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position:

＜222〉final position:

Out of Memory: out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Feature

--------

Sequence: SEQ ID 116

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 11

＜223〉out of Memory: x represents amber codon amino acids coding residue

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg gggggagctt cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 117

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 117

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGELH 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 118

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaggctg cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 119

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 119

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGRLH 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 120

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggcagctt cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 121

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 121

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGQLH 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 122

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaaggtt cat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 123

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 123

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKVH 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 124

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaaggtg aat 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 125

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 125

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKVN 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 126

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccgttacaa ctgacagctg ggggaagctt ctg 33

＜212〉type: DNA

＜211〉length: 33

Sequence title: SEQ ID 127

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 127

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AVTTDSWGKLL 11

＜212〉type: PRT

＜211〉length: 11

Sequence title: SEQ ID 128

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggaattcatc gatgcagaag gaagtggagc ag 32

＜212〉type: DNA

＜211〉length: 32

Sequence title: SEQ ID 129

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 129

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gtacacggcc gggtcagggt tctggatata c 31

＜212〉type: DNA

＜211〉length: 31

Sequence title: SEQ ID 130

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 130

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tctctcatta atgaatgctg gtgtcactca gacccc 36

＜212〉type: DNA

＜211〉length: 36

Sequence title: SEQ ID 131

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 131

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tagaaaccgg tggccaggca caccagtgtg gc 32

＜212〉type: DNA

＜211〉length: 32

Sequence title: SEQ ID 132

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 132

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctgctctggt tccgcactc 19

＜212〉type: DNA

＜211〉length: 19

Sequence title: SEQ ID 133

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 133

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gagtgcggaa ccagagcag 19

＜212〉type: DNA

＜211〉length: 19

Sequence title: SEQ ID 134

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 134

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atatccagaa cccggatcct gccgtgta 28

＜212〉type: DNA

＜211〉length: 28

Sequence title: SEQ ID 135

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 135

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tacacggcag gaatccgggt tctggatat 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 136

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 136

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggagatatac atatgcagga ggtgacacag 30

＜212〉type: DNA

＜211〉length: 30

Sequence title: SEQ ID 137

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 137

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tacacggcag gatccgggtt ctggatatt 29

＜212〉type: DNA

＜211〉length: 29

Sequence title: SEQ ID 138

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 138

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggagatatac atatgggtgt cactcagacc 30

＜212〉type: DNA

＜211〉length: 30

Sequence title: SEQ ID 139

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 139

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cccaagctta gtctgctcta ccccaggcct cggc 34

＜212〉type: DNA

＜211〉length: 34

Sequence title: SEQ ID 140

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 140

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gccggccatg gccaaacagg aggtgacgca gattcct 37

＜212〉type: DNA

＜211〉length: 37

Sequence title: SEQ ID 141

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 141

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cttcttaaag aattcttaat taacctaggt tattaggaac tttctgggct ggggaag 57

＜212〉type: DNA

＜211〉length: 57

Sequence title: SEQ ID 142

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 142

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tcacagcgcg caggctggtg tcactcagac cccaaa 36

＜212〉type: DNA

＜211〉length: 36

Sequence title: SEQ ID 143

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 143

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

cgagagcccg tagaactgga cttg 24

＜212〉type: DNA

＜211〉length: 24

Sequence title: SEQ ID 144

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 144

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tctctcatta atgaatgctg gtgtcactca gacccc 36

＜212〉type: DNA

＜211〉length: 36

Sequence title: SEQ ID 145

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 145

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

tagaaaccgg tggccaggca caccagtgtg g 31

＜212〉type: DNA

＜211〉length: 31

Sequence title: SEQ ID 146

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 146

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgcagaagg aagtggagca gaactctgga cccctcagtg ttccagaggg agccattgcc 60

tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 120

tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180

aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240

cagcccagtg attcagccac ctacctctgt gccgttacaa ctgacagctg ggggaaattg 300

cagtttggag cagggaccca ggttgtggtc accccagata tccagaaccc tgaccctgcc 360

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaatgt 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttccta a 621

＜212〉type: DNA

＜211〉length: 621

Sequence title: SEQ ID 147

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 147

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgaacgctg gtgtcactca gaccccaaaa ttccaggtcc tgaagacagg acagagcatg 60

acactgcagt gtgcccagga tatgaaccat gaatacatgt cctggtatcg acaagaccca 120

ggcatggggc tgaggctgat tcattactca gttggtgctg gtatcactga ccaaggagaa 180

gtccccaatg gctacaatgt ctccagatca accacagagg atttcccgct caggctgctg 240

tcggctgctc cctcccagac atctgtgtac ttctgtgcca gcaggccggg actagcggga 300

gggcgaccag agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360

aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420

caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480

tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540

gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600

accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660

gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720

gcctggggta gagcagacta a 741

＜212〉type: DNA

＜211〉length: 741

Sequence title: SEQ ID 148

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 148

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDG 60

RFTAQLNKASQYVSLLIRDSQPSDSATYLCAVTTDSWGKLQFGAGTQVVVTPDIQNPDPA 120

VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNK 180

SDFACANAFNNSIIPEDTFFPSPESS 206

＜212〉type: PRT

＜211〉length: 206

Sequence title: SEQ ID 149

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGRPEQYFGPGTRLTVTEDLK 120

NVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLK 180

EQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE 240

AWGRAD 246

＜212〉type: PRT

＜211〉length: 246

Sequence title: SEQ ID 150

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60

tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120

ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180

ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240

tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300

gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360

gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420

acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480

tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540

caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600

gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660

cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720

tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780

agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840

tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900

ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960

acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020

ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080

aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140

aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200

aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260

ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320

ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380

aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440

gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500

cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800

ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280

ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340

accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400

acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta taataatatt 2460

ctatttcaag gagacagtca taatgaaata cctattgcct acggcagccg ctggattgtt 2520

attactcgcg gcccagccgg ccatggccca gaaggaagtg gagcagaact ctggacccct 2580

cagtgttcca gagggagcca ttgcctctct caactgcact tacagtgacc gaggttccca 2640

gtccttcttc tggtacagac aatattctgg gaaaagccct gagttgataa tgtccatata 2700

ctccaatggt gacaaagaag atggaaggtt tacagcacag ctcaataaag ccagccagta 2760

tgtttctctg ctcatcagag actcccagcc cagtgattca gccacctacc tctgtgccgt 2820

tacaactgac agctggggga aattgcagtt tggagcaggg acccaggttg tggtcacccc 2880

agatatccag aaccctgacc ctgccgtgta ccagctgaga gactctaaat ccagtgacaa 2940

gtctgtctgc ctattcaccg attttgattc tcaaacaaat gtgtcacaaa gtaaggattc 3000

tgatgtgtat atcacagaca aatgtgtgct agacatgagg tctatggact tcaagagcaa 3060

cagtgctgtg gcctggagca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag 3120

cattattcca gaagacacct tcttccccag cccagaaagt tcctaataac ctaggttaat 3180

taagaattct ttaagaagga gatatacata tgaaaaaatt attattcgca attcctttag 3240

ttgttccttt ctattctcac agcgcgcagg ctggtgtcac tcagacccca aaattccagg 3300

tcctgaagac aggacagagc atgacactgc agtgtgccca ggatatgaac catgaataca 3360

tgtcctggta tcgacaagac ccaggcatgg ggctgaggct gattcattac tcagttggtg 3420

ctggtatcac tgaccaagga gaagtcccca atggctacaa tgtctccaga tcaaccacag 3480

aggatttccc gctcaggctg ctgtcggctg ctccctccca gacatctgtg tacttctgtg 3540

ccagcaggcc gggactagcg ggagggcgac cagagcagta cttcgggccg ggcaccaggc 3600

tcacggtcac agaggacctg aaaaacgtgt tcccacccga ggtcgctgtg tttgagccat 3660

cagaagcaga gatctcccac acccaaaagg ccacactggt gtgcctggcc acaggcttct 3720

accccgacca cgtggagctg agctggtggg tgaatgggaa ggaggtgcac agtggggtct 3780

gcacagaccc gcagcccctc aaggagcagc ccgccctcaa tgactccaga tacgctctga 3840

gcagccgcct gagggtctcg gccaccttct ggcaggaccc ccgcaaccac ttccgctgtc 3900

aagtccagtt ctacgggctc tcggagaatg acgagtggac ccaggatagg gccaaacccg 3960

tcacccagat cgtcagcgcc gaggcctggg gtagagcaga cgcggccgca tctagacatc 4020

atcaccatca tcactagact gttgaaagtt gtttagcaaa accccataca gaaaattcat 4080

ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggttgtc 4140

tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 4200

gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 4260

ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 4320

ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 4380

accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 4440

agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 4500

aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 4560

atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgagg 4620

atccattcgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 4680

ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 4740

gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 4800

attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 4860

aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 4920

ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 4980

gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 5040

taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 5100

ttgtctttag cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 5160

tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 5220

ttgctaacat actgcgtaat aaggagtctt aataaggtac cctctagtca aggcctatag 5280

tgagtcgtat tacggactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 5340

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 5400

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcttcgc 5460

ttggtaataa agcccgcttc ggcgggcttt ttttt 5495

＜212〉type: DNA

＜211〉length: 5495

Sequence title: SEQ ID 151

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 151

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

agctgcagct aatacgactc actataggaa caggccacca tgggccagaa ggaagtggag 60

cagaactctg gacccctcag tgttccagag ggagccattg cctctctcaa ctgcacttac 120

agtgaccgag gttcccagtc cttcttctgg tacagacaat attctgggaa aagccctgag 180

ttgataatgt ccatatactc caatggtgac aaagaagatg gaaggtttac agcacagctc 240

aataaagcca gccagtatgt ttctctgctc atcagagact cccagcccag tgattcagcc 300

acctacctct gtgccgttac aactgacagc tgggggaaat tgcagtttgg agcagggacc 360

caggttgtgg tcaccggtgg aggcggttca ggcggaggtg gatccggcgg tggcgggtcg 420

aacgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 480

ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct ggtatcgaca agacccaggc 540

atggggctga ggctgattca ttactcagtt ggtgctggta tcactgacca aggagaagtc 600

cccaatggct acaatgtctc cagatcaacc acagaggatt tcccgctcag gctgctgtcg 660

gctgctccct cccagacatc tgtgtacttc tgtgccagca ggccgggact agcgggaggg 720

cgaccagagc agtacttcgg gccgggcacc aggctcacgg tcacagagga cctgaaaaac 780

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 840

aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 900

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 960

cagcccgccc tcaatgactc cagatacgct ctgagcagcc gcctgagggt ctcggccacc 1020

ttctggcagg acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 1080

aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 1140

tggggtagag cagacggtgg aggcggttca ctcagcagca ccctgacgct gagcaaagca 1200

gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagttcgccc 1260

gtcacaaaga gcttcaaccg cggagagtca taagaattct cag 1303

＜212〉type: DNA

＜211〉length: 1303

Sequence title: SEQ ID 152

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 152

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MGQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKED 60

GRFTAQLNKASQYVSLLIRDSPSDSATYLCAVTTDSWGKLQFGAGTQVVVTGGGGSGGGG 120

SGGGGSNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGI 180

TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGRPEQYFGPGTRLTV 240

TEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTD 300

PQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 360

IVSAEAWGRADGGGGSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES 416

＜212〉type: PRT

＜211〉length: 416

Sequence title: SEQ ID 153

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ctgctttccc ggagcactat gcggataaaa atatccaatt acagtactat tattaccaaa 60

gaatctgcag tccaccgtga aaagcccctt tacacgcgcc ttggggataa acaaataaaa 120

agatttatgt aagtttatac ataggcgagt actctgttat tgggactatt tacgaagtta 180

ttataacttt ttccttctca tactcataag ttgtaaaggc acagcgggaa taagggaaaa 240

aacgccgtaa aacggaagga caaaaacgag tgggtctttg cgaccacttt cattttctac 300

gacttctagt caacccacgt gctcacccaa tgtagcttga cctagagttg tcgccattct 360

aggaactctc aaaagcgggg cttcttgcaa aaggttacta ctcgtgaaaa tttcaagacg 420

atacaccgcg ccataatagg gcataactgc ggcccgttct cgttgagcca gcggcgtatg 480

tgataagagt cttactgaac caactcatga gtggtcagtg tcttttcgta gaatgcctac 540

cgtactgtca ttctcttaat acgtcacgac ggtattggta ctcactattg tgacgccggt 600

tgaatgaaga ctgttgctag cctcctggct tcctcgattg gcgaaaaaac gtgttgtacc 660

ccctagtaca ttgagcggaa ctagcaaccc ttggcctcga cttacttcgg tatggtttgc 720

tgctcgcact gtggtgctac ggacatcgtt accgttgttg caacgcgttt gataattgac 780

cgcttgatga atgagatcga agggccgttg ttaattatct gacctacctc cgcctatttc 840

aacgtcctgg tgaagacgcg agccgggaag gccgaccgac caaataacga ctatttagac 900

ctcggccact cgcacccaga gcgccatagt aacgtcgtga ccccggtcta ccattcggga 960

gggcatagca tcaatagatg tgctgcccct cagtccgttg atacctactt gctttatctg1020

tctagcgact ctatccacgg agtgactaat tcgtaaccat tgacagtctg gttcaaatga1080

gtatatatga aatctaacta aattttgaag taaaaattaa attttcctag atccacttct1140

aggaaaaact attagagtac tggttttagg gaattgcact caaaagcaag gtgactcgca1200

gtctggggca tcttttctag tttcctagaa gaactctagg aaaaaaagac gcgcattaga1260

cgacgaacgt ttgttttttt ggtggcgatg gtcgccacca aacaaacggc ctagttctcg1320

atggttgaga aaaaggcttc cattgaccga agtcgtctcg cgtctatggt ttatgacagg1380

aagatcacat cggcatcaat ccggtggtga agttcttgag acatcgtggc ggatgtatgg1440

agcgagacga ttaggacaat ggtcaccgac gacggtcacc gctattcagc acagaatggc1500

ccaacctgag ttctgctatc aatggcctat tccgcgtcgc cagcccgact tgccccccaa1560

gcacgtgtgt cgggtcgaac ctcgcttgct ggatgtggct tgactctatg gatgtcgcac1620

tcgatactct ttcgcggtgc gaagggcttc cctctttccg cctgtccata ggccattcgc1680

cgtcccagcc ttgtcctctc gcgtgctccc tcgaaggtcc ccctttgcgg accatagaaa1740

tatcaggaca gcccaaagcg gtggagactg aactcgcagc taaaaacact acgagcagtc1800

cccccgcctc ggataccttt ttgcggtcgt tgcgccggaa aaatgccaag gaccggaaaa1860

cgaccggaaa acgagtgtac aagaaaggac gcaatagggg actaagacac ctattggcat1920

aatggcggaa actcactcga ctatggcgag cggcgtcggc ttgctggctc gcgtcgctca1980

gtcactcgct ccttcgcctt ctcgcgggtt atgcgtttgg cggagagggg cgcgcaaccg2040

gctaagtaat tacgtcgacc gtgctgtcca aagggctgac ctttcgcccg tcactcgcgt2100

tgcgttaatt acactcaatc gagtgagtaa tccgtggggt ccgaaatgtg aaatacgaag2160

gccgagcata caacacacct taacactcgc ctattgttaa agtgtgtcct ttgtcgatac2220

tggtactaat gcggttcgac gtcgattatg ctgagtgata tccttgtccg gtggtaccct2280

aggggcccat ggctcgagct taagtgaccg gcagcaaaat gttgcagcac tgaccctttt2340

gggaccgcaa tgggttgaat tagcggaacg tcgtgtaggg ggaaagcggt cgaccgcatt2400

atcgcttctc cgggcgtggc tagcgggaag ggttgtcaac gcgtcggact taccgcttac2460

cgcggactac gccataaaag aggaatgcgt agacacgcca taaagtgtgg cgtataccac2520

gtgagagtca tgttagacga gactacggcg tatcaattcg gtcggggctg tgggcggttg2580

tgggcgactg cgcgggactg cccgaacaga cgagggccgt aggcgaatgt ctgttcgaca2640

ctggcagagg ccctcgacgt acacagtctc caaaagtggc agtagtggct ttgcgcgct 2699

＜212〉type: DNA

＜211〉length: 2699

Sequence title: SEQ ID 154

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 154

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ccatgggcca gaaggaagtg gagcagaact ctggacccct cagtgttcca gagggagcca 60

ttgcctctct caactgcact tacagtgacc gaggttccca gtccttcttc tggtacagac 120

aatattctgg gaaaagccct gagttgataa tgtccatata ctccaatggt gacaaagaag 180

atggaaggtt tacagcacag ctcaataaag ccagccagta tgtttctctg ctcatcagag 240

actcccagcc cagtgattca gccacctacc tctgtgccgt tacaactgac agctggggga 300

aattgcagtt tggagcaggg acccaggttg tggtcaccgg tggaggcggt tcaggcggag 360

gtggatccgg cggtggcggg tcgaacgctg gtgtcactca gaccccaaaa ttccaggtcc 420

tgaagacagg acagagcatg acactgcagt gtgcccagga tatgaaccat gaatacatgt 480

cctggtatcg acaagaccca ggcatggggc tgaggctgat tcattactca gttggtgctg 540

gtatcactga ccaaggagaa gtccccaatg gctacaatgt ctccagatca accacagagg 600

atttcccgct caggctgctg tcggctgctc cctcccagac atctgtgtac ttctgtgcca 660

gcaggccggg actagcggga gggcgaccag agcagtactt cgggccgggc accaggctca 720

cggtcacaga ggacctgaaa aacgtgttcc cacccgaggt cgctgtgttt gagccatcag 780

aagcagagat ctcccacacc caaaaggcca cactggtgtg cctggccaca ggcttctacc 840

ccgaccacgt ggagctgagc tggtgggtga atgggaagga ggtgcacagt ggggtcagca 900

cagacccgca gcccctcaag gagcagcccg ccctcaatga ctccagatac gctctgagca 960

gccgcctgag ggtctcggcc accttctggc aggacccccg caaccacttc cgctgtcaag1020

tccagttcta cgggctctcg gagaatgacg agtggaccca ggatagggcc aaacccgtca1080

cccagatcgt cagcgccgag gcctggggta gagcagacgg tggaggcggt tcactcagca1140

gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc tgcgaagtca1200

cccatcaggg cctgagttcg cccgtcacaa agagcttcaa ccgcggagag tcataagaat1260

tc 1262

＜212〉type: DNA

＜211〉length: 1262

Sequence title: SEQ ID 155

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 155

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240

gctccctccc agacatctgt gtacttctgt gccagcaggc cgggactagc gggagggtga 300

ccagagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600

tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag ac 732

＜212〉type: DNA

＜211〉length: 732

Sequence title: SEQ ID 156

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 156

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP 60

NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGXPEQYFGPGTRLTVTEDLKNV 120

FPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQ 180

PALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAW 240

GRAD 244

＜212〉type: PRT

＜211〉length: 244

Sequence title: SEQ ID 157

Sequence description:

Feature

--------

Sequence: SEQ ID 157

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 244

Out of Memory: X represents opal codon amino acids coding residue, and this is translated into tryptophane usually

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60

tagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240

gctccctccc agacatctgt gtacttctgt gccagcaggc cgggactagc gggagggcga 300

ccagagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600

tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag ac 732

＜212〉type: DNA

＜211〉length: 732

Sequence title: SEQ ID 158

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 158

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240

gctccctccc agacatctgt gtacttctgt gcctcgaggc cggggctgat gagtgcggaa 300

ccagagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc caccggtttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600

tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag actaagcttg aattc 745

＜212〉type: DNA

＜211〉length: 745

Sequence title: SEQ ID 159

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 159

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLMSAEPEQYFGPGTRLTVTEDLK 120

NVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLK 180

EQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE 240

AWGRAD 246

＜212〉type: PRT

＜211〉length: 246

Sequence title: SEQ ID 160

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

AGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP 60

NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLMSAQPEQYFGPGTRLTVTEDLKNV 120

FPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQ 180

PALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAW 240

GRAD 244

＜212〉type: PRT

＜211〉length: 244

Sequence title: SEQ ID 161

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgcaggagg tgacacagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60

ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120

gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180

agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240

cagcctggtg actcagccac ctacctctgt gctgtgaggc ccacatcagg aggaagctac 300

atacctacat ttggaagagg aaccagcctt attgttcatc cgtatatcca gaaccctgac 360

cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 420

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480

aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600

ttcttcccca gcccagaaag ttcctaa 627

＜212〉type: DNA

＜211〉length: 627

Sequence title: SEQ ID 162

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 162

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

atgggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 60

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 120

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 180

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 240

gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 300

ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480

gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540

ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600

gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720

gcagactaa 729

＜212〉type: DNA

＜211〉length: 729

Sequence title: SEQ ID 163

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 163

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSG 60

RLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPD 120

PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS 180

NKSDFACANAFNNSIIPEDTFFPSPESS 208

＜212〉type: PRT

＜211〉length: 208

Sequence title: SEQ ID 164

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP 60

NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFP 120

PEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPa 180

LNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR 240

AD 242

＜212〉type: PRT

＜211〉length: 242

Sequence title: SEQID 165

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca aaggaccgga 60

aaacgaccgg aaaacgagtg tacattacac tcaatcgagt ctcattaggc accccaggct 120

ttacacttta tgcttccggc tcgtatgttg gagtaatccg tggggtccga aatgtgaaat 180

acgaaggccg agcatacaac tgtggaattg tgagcggata acaatttcac acaggaaaca 240

gctatgacca acaccttaac actcgcctat tgttaaagtg tgtcctttgt cgatactggt 300

tgattacgcc aagctacgta cttaagtatt ctatttcaag gagacagtca actaatgcgg 360

ttcgatgcat gaattcataa gataaagttc ctctgtcagt taatgaaata cctattgcct 420

acggcagccg ctggattgtt attactcgcg attactttat ggataacgga tgccgtcggc 480

gacctaacaa taatgagcgc gcccagccgg ccatggccaa acaggaggtg acgcagattc 540

ctgcagctct cgggtcggcc ggtaccggtt tgtcctccac tgcgtctaag gacgtcgaga 600

gagtgtccca gaaggagaaa acttggttct caactgcagt ttcactgata ctcacagggt 660

cttcctcttt tgaaccaaga gttgacgtca aagtgactat gcgctattta caacctccag 720

tggtttaggc aggaccctgg gaaaggtctc cgcgataaat gttggaggtc accaaatccg 780

tcctgggacc ctttccagag acatctctgt tgcttattca gtcaagtcag agagagcaaa 840

caagtggaag tgtagagaca acgaataagt cagttcagtc tctctcgttt gttcaccttc 900

acttaatgcc tcgctggata aatcatcagg acgtagtact ttatacattg tgaattacgg 960

agcgacctat ttagtagtcc tgcatcatga aatatgtaac cagcttctca gcctggtgac1020

tcagccacct acctctgtgc tgtgaggccc gtcgaagagt cggaccactg agtcggtgga1080

tggagacacg acactccggg acatcaggag gaagctacat acctacattt ggaagaggaa1140

ccagccttat tgtagtcctc cttcgatgta tggatgtaaa ccttctcctt ggtcggaata1200

tgttcatccg tatatccaga acccggatcc tgccgtgtac cagctgagag acaagtaggc1260

atataggtct tgggcctagg acggcacatg gtcgactctc actctaaatc cagtgacaag1320

tctgtctgcc tattcaccga ttttgattct tgagatttag gtcactgttc agacagacgg1380

ataagtggct aaaactaaga caaacaaatg tgtcacaaag taaggattct gatgtgtata1440

tcacagacaa gtttgtttac acagtgtttc attcctaaga ctacacatat agtgtctgtt1500

atgtgtgcta gacatgaggt ctatggactt caagagcaac agtgctgtgg tacacacgat1560

ctgtactcca gatacctgaa gttctcgttg tcacgacacc cctggagcaa caaatctgac1620

tttgcatgtg caaacgcctt caacaacagc ggacctcgtt gtttagactg aaacgtacac1680

gtttgcggaa gttgttgtcg attattccag aagacacctt cttccccagc ccagaaagtt1740

cctaataacc taataaggtc ttctgtggaa gaaggggtcg ggtctttcaa ggattattgg1800

taggttaatt aagaattctt taagaagggg atatacatat gaaaaaatta atccaattaa1860

ttcttaagaa attcttcccc tatatgtata cttttttaat ttattcgcaa ttcctttagt1920

tgttcctttc tattctcaca gcgcgcaggc aataagcgtt aaggaaatca acaaggaaag1980

ataagagtgt cgcgcgtccg tggtgtcact cagaccccaa aattccaggt cctgaagaca2040

ggacagagca accacagtga gtctggggtt ttaaggtcca ggacttctgt cctgtctcgt2100

tgacactgca gtgtgcccag gatatgaacc atgaatacat gtcctggtat actgtgacgt2160

cacacgggtc ctatacttgg tacttatgta caggaccata cgacaagacc caggcatggg2220

gctgaggctg attcattact cagttggtgc gctgttctgg gtccgtaccc cgactccgac2280

taagtaatga gtcaaccacg tggtatcact gaccaaggag aagtccccaa tggctacaat2340

gtctccagat accatagtga ctggttcctc ttcaggggtt accgatgtta cagaggtcta2400

caaccacaga ggatttcccg ctcaggctgc tgtcggctgc tccctcccag gttggtgtct2460

cctaaagggc gagtccgacg acagccgacg agggagggtc acatctgtgt acttctgtgc2520

cagcagttac gtcgggaaca ccggggagct tgtagacaca tgaagacacg gtcgtcaatg2580

cagcccttgt ggcccctcga gttttttgga gaaggctcta ggctgaccgt actggaggac2640

ctgaaaaacg caaaaaacct cttccgagat ccgactggca tgacctcctg gactttttgc2700

tgttcccacc cgaggtcgct gtgtttgagc catcagaagc agagatctcc acaagggtgg2760

gctccagcga cacaaactcg gtagtcttcg tctctagagg cacacccaaa aggccacact2820

ggtgtgcctg gccacaggct tctaccccga gtgtgggttt tccggtgtga ccacacggac2880

cggtgtccga agatggggct ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg2940

cacagtgggg ggtgcacctc gactcgacca cccacttacc cttcctccac gtgtcacccc3000

tctgcacaga cccgcagccc ctcaaggagc agcccgccct caatgactcc agacgtgtct3060

gggcgtcggg gagttcctcg tcgggcggga gttactgagg agatacgctc tgagcagccg3120

cctgagggtc tcggccacct tctggcagga tctatgcgag actcgtcggc ggactcccag3180

agccggtgga agaccgtcct cccccgcaac cacttccgct gtcaagtcca gttctacggg3240

ctctcggaga gggggcgttg gtgaaggcga cagttcaggt caagatgccc gagagcctct3300

atgacgagtg gacccaggat agggccaaac ccgtcaccca gatcgtcagc tactgctcac3360

ctgggtccta tcccggtttg ggcagtgggt ctagcagtcg gccgaggcct ggggtagagc3420

agacgcggcc gcacggctcc ggaccccatc tcgtctgcgc cggcgt 3466

＜212〉type: DNA

＜211〉length: 3466

Sequence title: SEQ ID 166

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 166

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

KYLLPTAAAGLLLLAAQP AMAKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQD 60

PGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGS 120

YIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT 180

DKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSMKKLLFAIPLV 240

VPFYSHSAQAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGa 300

GITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTV 260

LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTD 420

PQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ 480

IVSAEAWGRADAAa 494

＜212〉type: PRT

＜211〉length: 494

Sequence title: SEQ ID 167

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggatccatgg ccataagtgg agtccctgtg ctaggatttt tcatcatagc tgtgctgatg 60

agcgctcagg aatcatgggc tatcaaagaa gaacatgtga tcatccaggc cgagttctat 120

ctgaatcctg accaatcagg cgagtttatg tttgactttg atggtgatga gattttccat 180

gtggatatgg caaagaagga gacggtctgg cggcttgaag aatttggacg atttgccagc 240

tttgaggctc aaggtgcatt ggccaacata gctgtggaca aagccaacct ggaaatcatg 300

acaaagcgct ccaactatac tccgatcacc aatgtacctc cagaggtaac tgtgctcacg 360

aacagccctg tggaactgag agagcccaac gtcctcatct gtttcatcga caagttcacc 420

ccaccagtgg tcaatgtcac gtggcttcga aatggaaaac ctgtcaccac aggagtgtca 480

gagacagtct tcctgcccag ggaagaccac cttttccgca agttccacta tctccccttc 540

ctgccctcaa ctgaggacgt ttacgactgc agggtggagc actggggctt ggatgagcct 600

cttctcaagc actgggagtt tgatgctcca agccctctcc cagagactac agagaacgtg 660

gatgggggtc tgactgatac actccaagcg gagacagatc aacttgaaga caagaagtct 720

gcgttgcaga ccgagattgc caatctactg aaagagaagg aaaaactaga gttcatcctg 780

gcagcttacg gatctggtgg tggtctgaac gatatttttg aagctcagaa aatcgaatgg 840

catgagtagg atcc 854

＜212〉type: DNA

＜211〉length: 854

Sequence title: SEQ ID 168

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 168

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

aattctactc gtaaagcgag ttgaaggatc atatttagtt gcgtttatga gataagattg 60

aaagcacgtg taaaatgttt cccgcgcgtt ggcacaacta tttacaatgc ggccaagtta 120

taaaagattc taatctgata tgttttaaaa cacctttgcg gcccgagttg tttgcgtacg 180

tgactagcga agaagatgtg tggaccgcag aacagatagt aaaacaaaac cctagtattg 240

gagcaataat cgatttaacc aacacgtcta aatattatga tggtgtgcat tttttgcggg 300

cgggcctgtt atacaaaaaa attcaagtac ctggccagac tttgccgcct gaaagcatag 360

ttcaagaatt tattgacacg gtaaaagaat ttacagaaaa gtgtcccggc atgttggtgg 420

gcgtgcactg cacacacggt attaatcgca ccggttacat ggtgtgcaga tatttaatgc 480

acaccctggg tattgcgccg caggaagcca tagatagatt cgaaaaagcc agaggtcaca 540

aaattgaaag acaaaattac gttcaagatt tattaattta attaatatta tttgcattct 600

ttaacaaata ctttatccta ttttcaaatt gttgcgcttc ttccagcgaa ccaaaactat 660

gcttcgcttg ctccgtttag cttgtagccg atcagtggcg ttgttccaat cgacggtagg 720

attaggccgg atattctcca ccacaatgtt ggcaacgttg atgttacgtt tatgcttttg 780

gttttccacg tacgtctttt ggccggtaat agccgtaaac gtagtgccgt cgcgcgtcac 840

gcacaacacc ggatgtttgc gcttgtccgc ggggtattga accgcgcgat ccgacaaatc 900

caccactttg gcaactaaat cggtgacctg cgcgtctttt ttctgcatta tttcgtcttt 960

cttttgcatg gtttcctgga agccggtgta catgcggttt agatcagtca tgacgcgcgt1020

gacctgcaaa tctttggcct cgatctgctt gtccttgatg gcaacgatgc gttcaataaa1080

ctcttgtttt ttaacaagtt cctcggtttt ttgcgccacc accgcttgca gcgcgtttgt1140

gtgctcggtg aatgtcgcaa tcagcttagt caccaactgt ttgctctcct cctcccgttg1200

tttgatcgcg ggatcgtact tgccggtgca gagcacttga ggaattactt cttctaaaag1260

ccattcttgt aattctatgg cgtaaggcaa tttggacttc ataatcagct gaatcacgcc1320

ggatttagta atgagcactg tatgcggctg caaatacagc gggtcgcccc ttttcacgac1380

gctgttagag gtagggcccc cattttggat ggtctgctca aataacgatt tgtatttatt1440

gtctacatga acacgtatag ctttatcaca aactgtatat tttaaactgt tagcgacgtc1500

cttggccacg aaccggacct gttggtcgcg ctctagcacg taccgcaggt tgaacgtatc1560

ttctccaaat ttaaattctc caattttaac gcgagccatt ttgatacacg tgtgtcgatt1620

ttgcaacaac tattgttttt taacgcaaac taaacttatt gtggtaagca ataattaaat1680

atgggggaac atgcgccgct acaacactcg tcgttatgaa cgcagacggc gccggtctcg1740

gcgcaagcgg ctaaaacgtg ttgcgcgttc aacgcggcaa acatcgcaaa agccaatagt1800

acagttttga tttgcatatt aacggcgatt ttttaaatta tcttatttaa taaatagtta1860

tgacgcctac aactccccgc ccgcgttgac tcgctgcacc tcgagcagtt cgttgacgcc1920

ttcctccgtg tggccgaaca cgtcgagcgg gtggtcgatg accagcggcg tgccgcacgc1980

gacgcacaag tatctgtaca ccgaatgatc gtcgggcgaa ggcacgtcgg cctccaagtg2040

gcaatattgg caaattcgaa aatatataca gttgggttgt ttgcgcatat ctatcgtggc2100

gttgggcatg tacgtccgaa cgttgatttg catgcaagcc gaaattaaat cattgcgatt2160

agtgcgatta aaacgttgta catcctcgct tttaatcatg ccgtcgatta aatcgcgcaa2220

tcgagtcaag tgatcaaagt gtggaataat gttttctttg tattcccgag tcaagcgcag2280

cgcgtatttt aacaaactag ccatcttgta agttagtttc atttaatgca actttatcca2340

ataatatatt atgtatcgca cgtcaagaat taacaatgcg cccgttgtcg catctcaaca2400

cgactatgat agagatcaaa taaagcgcga attaaatagc ttgcgacgca acgtgcacga2460

tctgtgcacg cgttccggca cgagctttga ttgtaataag tttttacgaa gcgatgacat2520

gacccccgta gtgacaacga tcacgcccaa aagaactgcc gactacaaaa ttaccgagta2580

tgtcggtgac gttaaaacta ttaagccatc caatcgaccg ttagtcgaat caggaccgct2640

ggtgcgagaa gccgcgaagt atggcgaatg catcgtataa cgtgtggagt ccgctcatta2700

gagcgtcatg tttagacaag aaagctacat atttaattga tcccgatgat tttattgata2760

aattgaccct aactccatac acggtattct acaatggcgg ggttttggtc aaaatttccg2820

gactgcgatt gtacatgctg ttaacggctc cgcccactat taatgaaatt aaaaattcca2880

attttaaaaa acgcagcaag agaaacattt gtatgaaaga atgcgtagaa ggaaagaaaa2940

atgtcgtcga catgctgaac aacaagatta atatgcctcc gtgtataaaa aaaatattga3000

acgatttgaa agaaaacaat gtaccgcgcg gcggtatgta caggaagagg tttatactaa3060

actgttacat tgcaaacgtg gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg3120

ctctgacgca tttctacaac cacgactcca agtgtgtggg tgaagtcatg catcttttaa3180

tcaaatccca agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtcgacaagc3240

tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa3300

aacaattata aatgctaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac3360

atttgtagta ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa3420

atcattttca aatgattcac agttaatttg cgacaatata attttatttt cacataaact3480

agacgccttg tcgtcttctt cttcgtattc cttctctttt tcatttttct cctcataaaa3540

attaacatag ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt3600

gtcataaata tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct3660

gtaatttaca acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa3720

atttatataa tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta3780

cgcagcttct tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc3840

caaaatgttg tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc3900

tgcgagcagt tgtttgttgt taaaaataac agccattgta atgagacgca caaactaata3960

tcacaaactg gaaatgtcta tcaatatata gttgctgatg atccagcatg ataagataca4020

ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa4080

tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttccgagtt4140

tgtcagaaag cagaccaaac agcggttgga ataatagcga gaacagagaa atagcggcaa4200

aaataatacc cgtatcactt ttgctgatat ggttgatgtc atgtagccaa atcgggaaaa4260

acgggaagta ggctcccatg ataaaaaagt aaaagaaaaa gaataaaccg aacatccaaa4320

agtttgtgtt ttttaaatag tacataatgg atttccttac gcgaaatacg ggcagacatg4380

gcctgcccgg ttattattat ttttgacacc agaccaactg gtaatggtag cgaccggcgc4440

tcagctggaa ttcagatctg tgattgtaaa taaaatgtaa tttacagtat agtattttaa4500

ttaatataca aatgatttga taataattct tatttaacta taatatattg tgttgggttg4560

aattaaaggt cccggcatcc tcaaatgcat aatttcatag tcccccttgt tgtaagtgat4620

gcgtatttct gaatctttgt aaaatagcac acaagactcc aacgcgtttg gcgttttatt4680

ttcttgctcg actctagttt attaggcctc tagagatccg tatttatagg tttttttatt4740

acaaaactgt tacgaaaaca gtaaaatact tatttatttg cgagatggtt atcattttaa4800

ttatctccat gatccaataa acctagaata aagggcccga cctttaattc aacccaacac4860

aatatattat agttaaataa gaattattat caaatcattt gtatattaat taaaatacta4920

tactgtaaat tacattttat ttacaatcac agatcccggg gatccggtta ttagtacatt4980

tattaagcgc tagattctgt gcgttgttga tttacagaca attgttgtac gtattttaat5040

aattcattaa atttataatc tttagggtgg tatgttagag cgaaaatcaa atgattttca5100

gcgtctttat atctgaattt aaatattaaa tcctcaatag atttgtaaaa taggtttcga5160

ttagtttcaa acaagggttg tttttccgaa ccgatggctg gactatctaa tggattttcg5220

ctcaacgcca caaaacttgc caaatcttgt agcagcaatc tagctttgtc gatattcgtt5280

tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc ttttgtattt5340

ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa taaagcttgg5400

acatatttaa catcgggcgt gttagcttta ttaggccgat tatcgtcgtc gtcccaaccc5460

tcgtcgttag aagttgcttc cgaagacgat tttgccatag ccacacgacg cctattaatt5520

gtgtcggcta acacgtccgc gatcaaattt gtagttgagc tttttggaat tatttctgat5580

tgcgggcgtt tttgggcggg tttcaatcta actgtgcccg attttaattc agacaacacg5640

ttagaaagcg atggtgcagg cggtggtaac atttcagacg gcaaatctac taatggcggc5700

ggtggtggag ctgatgataa atctaccatc ggtggaggcg caggcggggc tggcggcgga5760

ggcggaggcg gaggtggtgg cggtgatgca gacggcggtt taggctcaaa tgtctcttta5820

ggcaacacag tcggcacctc aactattgta ctggtttcgg gcgccgtttt tggtttgacc5880

ggtctgagac gagtgcgatt tttttcgttt ctaatagctt ccaacaattg ttgtctgtcg5940

tctaaaggtg cagcgggttg aggttccgtc ggcattggtg gagcgggcgg caattcagac6000

atcgatggtg gtggtggtgg tggaggcgct ggaatgttag gcacgggaga aggtggtggc6060

ggcggtgccg ccggtataat ttgttctggt ttagtttgtt cgcgcacgat tgtgggcacc6120

ggcgcaggcg ccgctggctg cacaacggaa ggtcgtctgc ttcgaggcag cgcttggggt6180

ggtggcaatt caatattata attggaatac aaatcgtaaa aatctgctat aagcattgta6240

atttcgctat cgtttaccgt gccgatattt aacaaccgct caatgtaagc aattgtattg6300

taaagagatt gtctcaagct cggatcgatc ccgcacgccg ataacaagcc ttttcatttt6360

tactacagca ttgtagtggc gagacacttc gctgtcgtcg acgtacatgt atgctttgtt6420

gtcaaaaacg tcgttggcaa gctttaaaat atttaaaaga acatctctgt tcagcaccac6480

tgtgttgtcg taaatgttgt ttttgataat ttgcgcttcc gcagtatcga cacgttcaaa6540

aaattgatgc gcatcaattt tgttgttcct attattgaat aaataagatt gtacagattc6600

atatctacga ttcgtcatgg ccaccacaaa tgctacgctg caaacgctgg tacaatttta6660

cgaaaactgc aaaaacgtca aaactcggta taaaataatc aacgggcgct ttggcaaaat6720

atctatttta tcgcacaagc ccactagcaa attgtatttg cagaaaacaa tttcggcgca6780

caattttaac gctgacgaaa taaaagttca ccagttaatg agcgaccacc caaattttat6840

aaaaatctat tttaatcacg gttccatcaa caaccaagtg atcgtgatgg actacattga6900

ctgtcccgat ttatttgaaa cactacaaat taaaggcgag ctttcgtacc aacttgttag6960

caatattatt agacagctgt gtgaagcgct caacgatttg cacaagcaca atttcataca7020

caacgacata aaactcgaaa atgtcttata tttcgaagca cttgatcgcg tgtatgtttg7080

cgattacgga ttgtgcaaac acgaaaactc acttagcgtg cacgacggca cgttggagta7140

ttttagtccg gaaaaaattc gacacacaac tatgcacgtt tcgtttgact ggtacgcggc7200

gtgttaacat acaagttgct aaccggcggc cgacacccat ttgaaaaaag cgaagacgaa7260

atgttggact tgaatagcat gaagcgtcgt cagcaataca atgacattgg cgttttaaaa7320

cacgttcgta acgttaacgc tcgtgacttt gtgtactgcc taacaagata caacatagat7380

tgtagactca caaattacaa acaaattata aaacatgagt ttttgtcgta aaaatgccac7440

ttgttttacg agtagaattc ccagcttggc actggccgtc gttttacaac gtcgtgactg7500

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg7560

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg7620

cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat7680

atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc7740

gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca7800

agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg7860

cgcggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg7920

gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta7980

tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt8040

caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc8100

ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa8160

gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt8220

aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt8280

ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc8340

atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg8400

gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg8460

gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac8520

atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca8580

aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 8640

actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 8700

aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 8760

tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 8820

ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 8880

agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 8940

tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 9000

aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 9060

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 9120

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 9180

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 9240

gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 9300

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 9360

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 9420

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 9480

cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 9540

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 9600

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 9660

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 9720

ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 9780

cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 9840

gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 9900

tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9960

cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 10020

cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 10080

tatgaccatg attacg 10096

＜212〉type: DNA

＜211〉length: 10096

Sequence title: SEQ ID 169

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 169

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

ggtaccggat ccagcatggt gtgtctgaag ctccctggag gctcctgcat gacagcgctg 60

acagtgacac tgatggtgct gagctcccca ctggctttgt ccggagacac cggacctaag 120

tacgtcaagc agaacacact gaaactggct tccggtggcg gatctctagt tccacgcggt 180

agtggaggcg gtggttccgg agacacgcgt ccacgtttct tgtggcagct taagtttgaa 240

tgtcatttct tcaatgggac ggagcgggtg cggttgctgg aaagatgcat ctataaccaa 300

gaggagtccg tgcgcttcga cagcgacgtg ggggagtacc gggcggtgac ggagctgggg 360

cggcctgatg ccgagtactg gaacagccag aaggacctcc tggagcagag gcgggccgcg 420

gtggacacct actgcagaca caactacggg gttggtgaga gcttcacagt gcagcggcga 480

gttgagccta aggtgactgt gtatccttca aagacccagc ccctgcagca ccacaacctc 540

ctggtctgct ctgtgagtgg tttctatcca ggcagcattg aagtcaggtg gttccggaac 600

ggccaggaag agaaggctgg ggtggtgtcc acaggcctga tccagaatgg agattggacc 660

ttccagaccc tggtgatgct ggaaacagtt cctcggagtg gagaggttta cacctgccaa 720

gtggagcacc caagtgtgac gagccctctc acagtggaat ggagagcacg gtctgaatct 780

gcacagagca aggtcgacgg aggcggtggg ggtagaatcg cccggctgga ggaaaaagtg 840

aaaaccttga aagctcagaa ctcggagctg gcgtccacgg ccaacatgct cagggaacag 900

gtggcacagc ttaaacagaa agtcatgaac tactaggatc c 941

＜212〉type: DNA

＜211〉length: 941

Sequence title: SEQ ID 170

Sequence description:

Habitual codon

--------

Sequence title: SEQ ID 170

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGRPEQYFGPGTRLTVT 116

＜212〉type: PRT

＜211〉length: 116

Sequence title: SEQ ID 171

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLMSAXPEQYFGPGTRLTVT 116

＜212〉type: PRT

＜211〉length: 116

Sequence title: SEQ ID 172

Sequence description:

Feature

--------

Sequence: SEQ ID 172

＜221〉characteristic key words: MISC_FEATURE

＜222〉zero position: 1

＜222〉final position: 116

Out of Memory: wherein x is e, q or r

CDS connects: do not have

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGRPEDQYFGPGTRLTVT 117

＜212〉type: PRT

＜211〉length: 117

Sequence title: SEQ ID 173

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEY MSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLVPGRPEQQFGPGTRLTVT 116

＜212〉type: PRT

＜211〉length: 116

Sequence title: SEQ ID 174

Sequence description:

Sequence

--------

＜213〉organism title: artificial sequence

＜400〉presequence string:

MNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGE 60

VPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPGLAGGRPHPQFGPGTRLTVT 116

＜212〉type: PRT

＜211〉length: 116

Sequence title: SEQ ID 175

Sequence description:

Claims (18)

1. the phage particle at a kind of TXi Baoshouti of its surface display (TCR) is characterized in that,

Described TCR is that a kind of people dTCR polypeptide is right, its feature also is, the dTCR polypeptide of this displaying is to comprising the outer constant region Ig structural domain sequence of natural TCR born of the same parents, and the disulfide linkage that connects the amino-acid residue of described constant chain Ig structural domain sequence, wherein said disulfide linkage is between the cysteine residues that replaces following residue:

The Ser57 of the Thr48 of TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1, or

The Ser77 of the Thr45 of TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1, or

The Ser17 of the Tyr10 of TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1, or

The Asp59 of the Thr45 of TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1, or

The Glu15 of the Ser15 of TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1.

2. phage particle as claimed in claim 1 is characterized in that,

(a) member's of described dTCR polypeptide centering C-terminal links to each other with the residue that described phage particle surface exposes by peptide bond; Or

(b) member's of described dTCR polypeptide centering C-terminal links to each other with the cysteine residues that described phage particle surface exposes by disulfide linkage.

3. phage particle as claimed in claim 2 is characterized in that, the dTCR polypeptide of displaying is to being made of following component

Terminal first polypeptide that merges of the outer sequence of N of TCR α chain variable domains sequence and TCR α chain constant domain born of the same parents wherein, and

Terminal second polypeptide that merges of the outer sequence of N of TCR β chain variable domains sequence and TCR β chain constant domain born of the same parents wherein.

4. phage particle as claimed in claim 1 is characterized in that, with respect to described native sequences, the outer constant chain Ig structural domain sequence of described natural TCR born of the same parents is the C-terminal brachymemma, has got rid of the cysteine residues that forms natural interchain disulfide bond like this.

5. phage particle as claimed in claim 3 is characterized in that, with respect to described native sequences, the outer constant chain Ig structural domain sequence of described natural TCR born of the same parents is the C-terminal brachymemma, has got rid of the cysteine residues that forms natural interchain disulfide bond like this.

6. phage particle as claimed in claim 1 is characterized in that, the cysteine residues of the natural interchain disulfide bond of formation outside natural TCR born of the same parents in the constant chain Ig structural domain sequence is replaced by non-cysteine residues.

7. phage particle as claimed in claim 3 is characterized in that, the cysteine residues of the natural interchain disulfide bond of formation outside natural TCR born of the same parents in the constant chain Ig structural domain sequence is replaced by non-cysteine residues.

8. phage particle as claimed in claim 1 is characterized in that, described phage particle is that described polypeptide is to being made of following component at the right filobactivirus particle of its surface display dimer TXi Baoshouti (dTCR) polypeptide

Terminal first polypeptide that merges of the outer sequence of N of TCR α chain variable domains sequence and TCR α chain constant domain born of the same parents wherein, and

Terminal second polypeptide that merges of the outer sequence of N of TCR β chain variable domains sequence and TCR β chain constant domain born of the same parents wherein,

First and second polypeptide are connected by the disulfide linkage between the cysteine residues of the Ser 57 of the Thr 48 that replaces the TRAC*01 exons 1 and TRBC1*01 or TRBC2*01 exons 1,

The member's that the dTCR polypeptide is right C-terminal links to each other with the coat protein of phage by peptide bond.

9. right diverse libraries of dTCR polypeptide that is illustrated on the described phage particle of claim 1.

10. diverse libraries as claimed in claim 9 is characterized in that described diversity is present in the variable domains of dTCR polypeptide.

11. right diverse libraries of dTCR polypeptide that is illustrated on the described phage particle of claim 8.

12. diverse libraries as claimed in claim 11 is characterized in that, described diversity is present in the variable domains of dTCR polypeptide.

13. the nucleic acid of the described phage particle of coding claim 1.

14. the nucleic acid of the described phage particle of coding claim 8.

15. a discriminating has the method for the TCR of particular feature, it is characterized in that, described method comprises stands the described TCR diverse libraries that is illustrated on the phage particle of claim 11

The chosen process that described feature is selected, and separate and show the phage particle with described feature TCR,

And/or

Measure the screening process of described feature, differentiate those displayings have required feature TCR phage particle and with its separation.

16. method as claimed in claim 15 is characterized in that, described particular feature is to TCR part enhanced avidity.

17. the method shown in claim 15 is characterized in that, shown in method carry out amplification procedure to increase isolating particle after also being included in described chosen process.

18. the method shown in claim 15 is characterized in that, shown in method carry out amplification procedure to increase isolating particle after also being included in described screening process.

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