CN106905435A - It is a kind of to prepare the protein-bonded method based on albumin A mutant - Google Patents

Abstract

The protein-bonded method based on albumin A mutant is prepared the present invention relates to a kind of.In addition, the present invention provides the nucleic acid libraries and corresponding protein library of the coding heteromultimeric protein A mutant, disclose the method that the albumin A mutant that there is binding ability to predetermined target molecule is screened from the mutant library, the method for separating the nucleotides of encoding said proteins A mutant, the method for identifying and analyzing the albumin A mutant, and the method for producing the albumin A mutant.The method of the present invention has the advantages that the binding proteins specific for producing binding ability higher.

Description

It is a kind of to prepare the protein-bonded method based on albumin A mutant

Technical field

Method the present invention relates to prepare the heteromultimeric protein A mutant with new specific binding capacity.This Invention provides the nucleic acid libraries and corresponding protein library of the coding heteromultimeric protein A mutant, disclose from The method that the albumin A mutant for having binding ability to predetermined target molecule is screened in the mutant library, separates coding described The method of the nucleotides of albumin A mutant, the method for identifying and analyzing the albumin A mutant, and the production albumin A is dashed forward The method of variant.Additionally, the present invention further provides can with high-affinity be specifically bound to it is on predetermined target molecule, with heterologous New conjugated protein based on multimeric protein A.

Background technology

Antibody being capable of specific recognition and all kinds of exogenous materials of combination, such as micromolecular compound, pollen, virus, bacterium Deng haptens and antigen.On two bifurcated tops of antibody molecule Y types, respectively there is a complementary region being made up of six ring type polypeptides Domain (CDR) forms lock shape structure, and the amino acid at these positions is and anti-by hydrogen bond, Van der Waals force, charge effect and hydrophobic effect The epitope of original surface forms non-covalent interaction.In antibody gene, the DNA of coding for antigens binding site can be with random groups Close and be mutated, the amino acid of this hypervariable region can occur the change enriched very much, and each specific change can be assigned anti- The ability that body has and a certain predetermined antigens are combined.

With the outer affine triage techniques of gene recombination technology, protein engineering and recombinant antibodies the library and structure biology Development, increasing antibody three-dimensional crystalline structure parsed, and the molecule mechanism of antibodies bind antigen is best understood from. The limitation of antibody, such as molecular weight are big, complex structure etc., promote researcher to derive from the " land in antibody CDR region domain This concept of domain ", is transplanted to the albumen (such as the heteromultimeric protein A that the present invention is used) of some highly stable non-antibody classes Surface.Some neighbouring amino acid of these protein surfaces are highly mutated, can be artificial mould appropriate calmodulin binding domain CaM, So as to assign the ability that these albumen are combined with all kinds of predetermined target molecules.

WO 95/19374 describes the associated proteins (Affibody) based on a domain monomer of albumin A Produce.Albumin A is derived from a kind of protein on aureus cell surface, by 5 domain groups of very high homology Into its immunoglobulin for combining many mammalian species (including people).Due to albumin A single structure domain molecular weight very Small (about 6kDa), the area of the calmodulin binding domain CaM that its surface can provide is relatively small, limits its binding ability.According to prior art Information, the protein-bonded affinity K produced in WO 95/19374_DScope is generally 10^-6-10^-8M scopes, in most cases exist 10^-7The protein-bonded affinity of the combination EGFR that M, such as primary dcreening operation are obtained in 130nM-185nM, with reference to the combination egg of H-Ras White affinity in 79nM-283nM, with reference to rVIII protein-bonded affinity in 100nM-200nM.In addition, albumin A The calmodulin binding domain CaM in single structure domain forms a flat faying face by two Amino acid profiles of α spirals side arranged side by side, Which also limits the type of its target molecule epitope that can be combined.

The content of the invention

It is an object of the present invention to provide the more different types of targets of identification with binding ability higher, can be universal point The method of the multimeric protein A mutant of sublist position.Further object of the present invention is to provide with novel heteromultimers egg Based on white A, by initial screening, you can generally prepare the protein-bonded method of high-affinity.

More specifically, preparing the heteromultimeric protein A with new specific binding capacity the invention provides a kind of The method of mutant, including following key step：

Storehouse of the monomer by the heteromultimers mutant of the albumin A of modification is provided, the storehouse includes heteromultimers egg White matter, the heteromultimeric-protein includes two or more albumin A monomers linked together in the way of head and the tail are arranged, Monomer described at least two of wherein described heteromultimeric-protein is by being pointed to SEQ ID No：In 19,10,11, 13rd, the replacement of at least five amino acid at 14,17,18,24,25,27,28,32,35 and the modification that carries out, the modification Monomeric protein has at least 80% Amino acid sequence identity with unmodified albumin A；

For the storehouse of the albumin A by modification provides potential target molecule；

The storehouse of the protein of the modification is set to be contacted with the target molecule；

Heteromultimeric protein A mutant is obtained by screening technique.

For the definition of term important in this application

Term " albumin A monomer " refers to a domain protein of albumin A, including with SEQ ID NO:1 is completely the same, Or protein of the Amino acid sequence identity more than 80%.

In this specification, term " heteromultimeric protein " is the albumin A monomer comprising two or more different modifyings Protein.Therefore, " heteromultimers " of the invention have two eggs of at least two different modifyings of independent calmodulin binding domain CaM The fusion protein of white A monomers, preferably heterodimer or heterotrimer.

According to the present invention, the heteromultimers associated proteins energy being made up of at least two different albumin A modified proteins With reference to predetermined antigens.Albumin A modified protein therein can be combined in various ways together, form melting for " headtotail " Hop protein, such as Gene Fusion etc..This fusion can enable the calmodulin binding domain CaM group on multiple different albumin A modified proteins Altogether, big, globality a calmodulin binding domain CaM is formed.This fusion, similar to multiple CDR region domains group of antibody variable region Altogether, together form paratope.Corresponding, the calmodulin binding domain CaM on multiple different albumin A modified proteins can The different parts of same specific antigen are respectively incorporated to, these part combinations get up, and together form epitope.

Term " connected head-to-tail fusion " describes multiple different albumin A modified proteins to couple together to form fusion egg White form.Multiple different albumin A modified proteins according to (aminoterminal-c-terminus)-(aminoterminal-c-terminus) n connection Direction, couples together to form heteromultimeric-fusion albumen, and n depends on the albumin A modified protein that needs are coupled together here Quantity.In this connected head-to-tail type of attachment, two adjacent albumin A modified proteins can be directly linked together, Can be by flexible polypeptide linker, such as, amino acid sequence is the connecting peptides of SGGGG, or other connecting peptides, such as KPEVIDASELTPAVT, GGGGS etc..Other connecting peptides for being generally used for Gene Fusion can also be used herein.In a word, Heteromultimers can be by two or more different albumin A modified proteins, one being connected to by end to end mode Rise, form one by two and the fusion protein that constitutes of more than two albumin A modified proteins.By flexible polypeptide by two or Calmodulin binding domain CaM on multiple albumin A modification monomers is coupled together, and described two or more calmodulin binding domain CaMs are provided with more structures As, there is provided the possibility of the further types of target molecule epitope of identification.

Term " group " or " storehouse ", " library " refer in particular to be mixed by the heteromultimers mutant of the albumin A by different modifying Gather obtained from conjunction.The mixing of the DNA of the heteromultimers mutant of the coding albumin A by modification is also known as core Thuja acid group.Term " group " or " storehouse ", " library " are all synonymous and can used interchangeablies.

" specific bond area " of the present invention represents：Formed polymer each albumin A monomer between, mutational site due to The reasons such as the hydrophobicity/hydrophily of electric charge, space structure and side chain, the amino acid of mutation and the environmental interaction of surrounding, shape Into a continuous surface-exposed region, the continuum can combine with pre-determined target molecular specificity, and the environment can be molten Agent, usually water, or other molecules.

In the present invention, " amino acid modified " refers to amino acid substitution, insertion, missing or chemical modification；It is preferred that amino Acid is replaced.In a particular embodiment, it is amino acid modified by albumin A monomer the 9th, 10,11,13,14,17,18, 24th, the replacement of at least five amino acid at 25,27,28,32,35 is realized.Term " modification " or " mutation " refer to using 20 kinds Other any amino acid of amino acid are replaced to the amino acid of institute's select location.Both of which is synonymous and can exchange and make With.

The heteromultimeric protein library that the albumin A modified protein produced above by mutation is constituted, can be by biting Phage display, ribosomal display, mRNA displayings or cell surface display, yeast display or bacterium surface displaying method, Jin Ertong The mode of in-vitro directed screening is crossed, is screened for any predetermined target molecule, enable the heterologous of specific binding target molecule Polyprotein A mutant is enriched with, so as to obtain such associated proteins.According to the present invention it is possible to pass through one or more of side Method, it is determined that whether described albumin A mutant has the binding ability that can be quantified, such as Enzyme-linked Immunosorbent Assay with certain target molecules Experiment (ELISA), surface plasmon resonance (SPR), immunofluorescence spectroscopy (IF), flow cytometry (FACS), isothermal titration amount Hot method (ITC) and analysis centrifugal etc..

In phage display method of the present invention, recombinant protein A mutant is illustrated in M13 filobactiviruses On, while the coding DNA of the mutant is packaged in bacteriophage coating with single stranded form.Therefore, in affine screening, Ke Yicong The mutant with some characteristics is selected in library, its hereditary information can respectively by bacterial infection and affine in next round Expanded in screening.Mutant protein by being fused to phage capsid protein, preferably piii protein, such that it is able to Phage expression is shown.Additionally, the fusion protein of coding can also contain other functions element, such as it is easy to by parent With chromatography detection or the affinity tag for purifying etc..

As the described protein-bonded target molecule according to produced by the present invention, it is possible to use all biology and medical science are lived Property related molecule, including but not limited to antigen and haptens.Antigen refer to can by immune system be identified as foreign substance (it is non-from Body) any material.In most of immune response situations, immune response can be caused.Haptens is typically simple, low point The compound of son amount, it can not cause immune response, but after being bonded on carrier protein, can become complete antigen. Preferably, target molecule can be biological acceptor, preferably g protein coupled receptor (GPCR, such as Human epidermal growth factor receptor, HER2, HER3, VEGF/ R1-4, EpCAM) or its part or its domain, tumor necrosis factor α (TNF-α), tumor necrosis factor β (TNF-β), Bai Jie Plain (such as IL-2, IL-6, IL-11, IL-12), growth factor (such as nerve growth factor) and its precursor (ProNGF), BMP, EGF, kinases, integrin (such as glycoprotein receptor IIb/IIIa), human serum albumin HSA, T and B cell antigen, preferably CD4, CD11, CD14, CD16, CD20, CD22, CD25, CD34, CD47, CD56, CD137, CD154, CTLA-4 etc..

In the present invention, based on heterodimer albumin A, its mutant modified in specific site is obtained, These mutant obtain its original without target molecule binding capacity.In the present invention, the target as heterodimer albumin A point The example of son is Procalcitonin (PCT), human serum albumins (HSA), Calcium ionorphore 16 (CDH16).By initial screening institute The protein-bonded affinity of albumin A heterodimer of acquisition is distributed in 3.6nM-57.6nM, hence it is evident that provided higher than prior art Protein-bonded affinity in material according to prepared by WO 95/19374, there is nearly 10 times or so of raising.

The mutant protein of the heteromultimeric protein A that the present invention is obtained in the cell, all have to combine in external environment and live Property, have a wide range of applications field.Protein of the invention can be used to detecting, quantitative determination, separate and extract corresponding target point Son, and it is related to disease that corresponding target molecule is participated in etc. for diagnosing and treating.For example, can by ELISA, Direct detection target molecule is tested in the bioanalysis such as western blot, be can be used for treatment tumour or communicable disease, be can be used to eat Product and nutraceutical industry, nutritious supplementary pharmaceutical, cosmetics, medical science and medical diagnostic and analysis field.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.

The beneficial effects of the invention are as follows：Compared with prior art, the method have the advantages that:

1. the associated proteins based on albumin A heterodimer or polymer can provide greater area of calmodulin binding domain CaM, and then Binding ability higher can be provided；

2. two or more albumin A monomers are connected by flexible connecting peptides, make to be distributed on two albumin A monomers Calmodulin binding domain CaM is flexible and changeable on space conformation, so that there is provided the ability for recognizing more target molecule epitopes.

Brief description of the drawings

Fig. 1 is newborn binding site region after wild type albumin A monomer surface is modified；

Substituted by amino acid randomization on surface and modified, so as to obtain the wild-type protein A of new binding site The crystal structure schematic diagram of monomer.The three-dimensional that Secondary structural elements are carried out with PyMOL softwares (DeLano Scientific) shows Show.The site 9,10,11,13,14,17,18,24,25,27,28,32,35 of the generation random replacement in library is prepared as example Represented with side chain.

Fig. 2 is the structural representation of heterodimer albumin A；

Two albumin A monomers have by the end to end connection of flexible polypeptide chain as being formed in the present invention The basis of the heterodimer albumin A of new binding activity.Oval coil represents the target molecule calmodulin binding domain CaM of protein surface, Formed by the modification mode being replaced to specific amino acids.

Fig. 3 is the phasmid matter for screening the modified protein based on heteromultimeric protein A with new binding characteristic Grain pCan-dPA；

PCan-dPA is used to prepare phage library, under the transcriptional control of Lac operators, coding g3p protein signal peptides, The sequence of albumin A dimer, E-tag labels and phage capsid protein III is expressed as fusion protein, and then makes albumin A two Aggressiveness is illustrated in phage surface.

Fig. 4 represents the modified protein PCT-P1G07 and its for detecting the heterodimer albumin A screened through phage display The ELISA experimental results of target molecule PCT binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Fig. 5 represents the modified protein PCT-P2A08 and its for detecting the heterodimer albumin A screened through phage display The ELISA experimental results of target molecule PCT binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Fig. 6 represents the modified protein PCT-P2A12 and its for detecting the heterodimer albumin A screened through phage display The ELISA experimental results of target molecule PCT binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Fig. 7 represents the modified protein HSA-P1A02 and its for detecting the heterodimer albumin A screened through phage display The ELISA experimental results of target molecule HSA binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Fig. 8 represents the modified protein HSA-P1F11 and its for detecting the heterodimer albumin A screened through phage display The ELISA experimental results of target molecule HSA binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Fig. 9 represents the modified protein CDH16-2H03-2 of the heterodimer albumin A that detection is screened through phage display With the ELISA experimental results of its target molecule CDH16 binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Figure 10 represents the modified protein CDH16-2H07-1 of the heterodimer albumin A that detection is screened through phage display With the ELISA experimental results of its target molecule CDH16 binding affinities.The K under equilibrium condition is determined by nonlinear regression_DValue.

Figure 11 represents unmodified dPA, dPA mutant library and each modified heterodimer albumin A combination egg White sequence information and comparison.Gray background shows the amino acid sequence of unmutated conservative region, and white background shows The selected amino acid sites modified.X is represented carries out randomization replacement to the amino acid of position, to prepare modification text Storehouse.

Specific embodiment

Embodiment 1：Unmodified albumin A dimer (dPA-wt) gene of synthesis

Present embodiments provide and unmodified albumin A dimer nucleotides (DNA) is obtained by way of gene chemical synthesis The mode of fragment.

Following genetic engineering operation is carried out with standard method well known by persons skilled in the art.To prepare coding dPA-wt eggs The DNA sequence dna (Seq ID No.3) of (Seq ID No.2), as the starting point and template that prepare dPA mutant libraries, uses in vain Following operating procedure：It is synthetic gene fragment, performing PCR reaction is entered in 50 μ l volumes, is made using primer (0.1 μM of every kind of concentration) For template carries out bridging PCR reactions, every kind of 1 μ l.Primer length is generally 30-59 base, the primer 3 ' being connected before and after each pair 15-20 base is about overlapped with 5 ' ends.PCR primer is identified by agarose gel electrophoresis, is used in combinationSV Gel and PCR Clean-Up kits (Promega) reclaim target stripe.Above PCR is reacted according to following program, 94 DEG C Predegeneration 5 minutes, subsequently into 28 circulations.94 DEG C of each circulation is reacted 30 seconds, and 55 DEG C are reacted 30 seconds, and 68 DEG C are reacted 1 minute. Last 68 DEG C are incubated 10 minutes.

Expected PCR primer separates target stripe by agarose gel electrophoresis, is used in combinationSV Gel and PCR Clean-Up kits (Promega) reclaim target stripe.DNA fragmentation after recovery uses sfiI/NotI (New respectively England Biolabs) double digestion, purify the DNA fragmentation after reclaiming and be inserted into the same phasmid through sfiI/NotI double digestions Carrier pCANTAB, obtains plasmid pCan-dPA, structure as shown in figure 3, as the template for building dPA mutant libraries.

Embodiment 2：Prepare dPA modification mutant libraries

Method set up by this area and known is modified, and can obtain dPA modification mutant libraries.In this hair In bright, " modification mutant library " refers to that the nucleotides or amino acid sequence of selecting site are arbitrarily modified, this to repair Decorations cause the replacement completely random of nucleotides or amino acid, and its final expression product is the average mark with different aminoacids The sequence of the completely random matched somebody with somebody.In the present invention, mutant library (Seq ID No.4) is modified front and rear two as the dPA for building 11 decorating sites are contained on individual albumin A monomer respectively, respectively positioned at monomer the 9th, 10,13,14,17,18,24,27,28, 32nd, 35 amino acid sites.

According to the present invention, preferably modified by gene level, randomization is carried out preferably through to amino acid Replace, it is amino acid modified so as to obtain so as to cause to undergo mutation.Preferably, it is right to be realized by the technological means of genetic engineering The modification of albumin A.Preferably, in order to the sequence of randomization is rightly introduced into dPA genetic fragments, can be by synthesizing and making With the primer being mutated with randomization, mutant library is produced by many wheel bridging PCR.For example can be desired restricted interior First group of forward and reverse primer is built around enzyme cutting recognition site, and in second group of the upstream and downstream structure of codon to be mutated Primer comprising randomization mutation, i.e. forward and reverse mutant primer.Appropriate use these primers, can build in the middle of first Fragment and the second intermediate segment.Two PCR reactions generate linear intermediate product fragment.Each fragment comprises at least one The consensus sequence that selected codon mutation is merged with upstream and downstream intermediate segment.The most upstream of the complete fragment after fusion and most Downstream further comprises restriction enzyme site and protection base, by forming cohesive end after digestion, to be connected by DNA Connect enzyme to be connected with carrier segments, produce the oligonucleotide product of ring-type, such as cyclic plasmid.This area can using other method and The use other method that can replace.

Specifically, after obtaining the intermediate segment comprising mutational site, after whole intermediate segment equimolars are mixed, add The primer pair (Seq ID No.5 and Seq ID No.6) of most upstream and most downstream comprising restriction enzyme site, carries out last PCR and expands Increase reaction, then useAfter SV Gel and PCR Clean-Up kits reclaim the DNA fragmentation, SfiI is used Double digestion is carried out to fragment with NotI, and the DNA fragmentation of about 400bp is separated and recovered with agarose gel electrophoresis.

To prepare phagemid vector, plasmid pCANTAB is carried out according to manufacturer specification using SfiI and NotI respectively Digestion, and separate larger carrier segments with agarose gel electrophoresis.WithSV Gel and PCR Clean-Up are tried Agent box cmy vector DNA, it is finally soluble in water with 50fmol/ul.To be attached reaction, 100 10 × T4DNA of μ l connections Described in enzyme buffer liquid, 3pmol through the dPA library DNA fragments of SfiI and NotI double digestions, 9pmol pCANTAB carrier segments and 8000U T4DNA ligases (New England Biolabs) are incubated 24 hours for 16 DEG C in 1ml reaction volumes.65 DEG C of heating Inactivate 10 minutes T4DNA ligases, Ran HouyongThe connection of SV Gel and PCR Clean-Up kits is produced Thing, finally preserves DNA with the aseptic water elutions of 50ul, for electric transformation experiment.

In order to connection product is transformed into Escherichia coli XL1Blue (Stratagene), Gene Pulser II are used (Bio-rad) electricity conversion cup (Bio-rad) of electric conversion instrument and 2mm spacing.Above-mentioned connection product resulting solution and electricity conversion sense After by state Escherichia coli XL1Blue mixing, transformation experiment is carried out according to manufacturer specification.Gained cell mixture is coated onto 10 pieces On plate containing 2 × YT/ ampicillin solid mediums, through 37 DEG C of incubated overnight plates, and to through the life after gradient dilution Bacterium colony long is counted, it is found that constructed library includes 3.1 × 10¹⁰Individual independent cloning.Scraped from plate with 2 × YT culture mediums Lower bacterium colony, adds glycerine to make final concentration of 20% (v/v), is divided into every pipe 1ml, is stored in -80 DEG C.50 Dan Ke of random picking It is grand, through sequencing analysis, it is found that 26 clones have functional target DNA sequence, there is property completely not in predetermined mutational site Same amino acid substitution, and DNA sequence dna is different, it was demonstrated that the accuracy in the library is about 52%.

Embodiment 3：Prepare dPA modified protein phage display libraries

To have produced in surface display the bacteriophage of different dPA mutant, in 400ml 2 × YT/ ampicillin cultures Base inoculation makes cell density reach OD by the glycerol stock obtained in embodiment 2₆₀₀=0.05, shaken under the conditions of 37 DEG C and 200rmp Culture is swung until cell density reaches OD₆₀₀=0.6.With 10¹²The helper phage M13KO7 infection of cfu, at 30 DEG C and 50rpm Under the conditions of cultivate 30 minutes.Add after 50mg/l kanamycins that shaken cultivation after 30 minutes, is led under the conditions of 30 DEG C and the 200rmp Centrifugation (15 minutes, 1,600 × g, 4 DEG C) precipitation and separation is crossed, 2 × YT/ of 400ml ampicillins/kanamycins culture is resuspended in Base, the shaken cultivation 16 hours under the conditions of 30 DEG C and 200rmp.Last cell is by being centrifuged (20 minutes, 8,000 × g, 4 DEG C) point From precipitating and abandoning, supernatant adds 1/4 volume 20% (w/v) PEG8000,2.5M NaCl with after 0.45 μm of specification membrane filtration Solution and 1 hour precipitating phage of ice bath.Precipitation (20 minutes, 12,000 × g, 4 DEG C) then is collected by centrifugation, bacteriophage is resuspended In 25ml precoolings PBS (137mM NaCl, 2.7mM KCl, 8mM Na₂HPO₄, 2mM KH₂PO₄) in, kept for 30 minutes on ice (5 minutes, 20,000 × g, 4 DEG C) are centrifuged afterwards.Add 1/4 volume 20% (w/v) PEG8000,2.5M NaCl molten to supernatant Liquid, and the precipitating phage again of ice bath 30 minutes.Centrifugation (30 minutes, 20,000 × g, 4 DEG C), again by phages It is resuspended in 4ml precoolings PBS, is kept for 30 minutes on ice and be centrifuged (30 minutes, 17,000 × g, 4 DEG C).Supernatant and closing Liquid is with 1:1 mixing, is placed on impeller, is incubated 10 minutes at room temperature, is then directly used for screening.

Embodiment 4：The phasmid of energy specific bond target molecule is enriched with by in-vitro screening

It is that from the phage display library of various dPA mutant is contained, filtering out can specifically bind pre-determined target point The dPA mutant of son, using the phage display library prepared in above-described embodiment 3, respectively for predetermined different target molecules (antigen or haptens), implements screening and enrichment experiment.

After target molecule is diluted with PBS, 4 DEG C are coated with overnight in immune pipe.After second day abandons supernatant, 5ml PBS-B are added (B represents confining liquid) and it is incubated at room temperature 90 minutes, so as to close the combination not occupied by antigen on immune pipe internal surface Site.Then the phage solution prepared by 4ml is drawn, is added in immune pipe, and be incubated at room temperature 2 hours.Use respectively PBST (T represents 0.1%Tween-20) and PBS is washed five times, and removes uncombined bacteriophage.Finally use 2ml glycine-salt Acid is incubated at room temperature 5 minutes, and the bacteriophage elution with the antigen binding being fixed on immune pipe is got off, and adds 300 μ l Concentration is the Tris solution of 1M.The phage solution that each case is eluted and 10ml cell densities OD₆₀₀=0.6 culture In thing, with ehec infection XL1blue, and cultivated 30 minutes in 37 DEG C of 220rpm.These cell suspensions are taped against containing 2 × On the plate of YT/ ampicillin solid mediums, through 37 DEG C of incubated overnight plates, and the growth bacterium after gradient dilution is counted Fall.Bacterium colony is scraped from plate with 2 × YT culture mediums, adds glycerine to make final concentration of 20% (v/v), be divided into every pipe 1ml, preserved In -20 DEG C.

It is the protein-bonded enrichment degrees of dPA in increase screening system, the directed screening for target molecule carries out 3-5 wheels altogether Circulation, usually 4 wheel circulation.Since the second wheel screening, culture is carried out in 100ml culture mediums every time and bacteriophage is rescued, And use harsher wash conditions, i.e., washed with PBST and PBS 15 times and 5 times respectively in the second wheel screening, third round difference Washing 25 times and 5 times, fourth round is washed 35 times and 5 times respectively.

Embodiment 5：It is identified and isolated from the phasmid specifically bound from different target molecules

After 3-5 wheels are directed to the external affine screening of different predetermined target molecules, the random picking from the clone for obtaining 96 clones, use the identification of single phage ELISA methods and the combination situation of corresponding target molecule.Concrete operation step is such as Under：Each single bacterium colony is inoculated into 600 μ l 2 × YT/ ampicillin mediums, is cultivated 16 hours in 37 DEG C of 220rpm.Next day 30 μ l cultures are respectively taken, 800 fresh μ l 2 × YT/ ampicillin mediums are inoculated into, it is small in 37 DEG C of 220rpm cultures 2 When, 10 are then added per hole⁹Cfu M13KO7 helper phages.After 37 DEG C of 200rpm are cultivated 30 minutes, 50ml/l cards are added That mycin, then proceedes to be cultivated 16 hours in 37 DEG C of 220rpm.30 minutes (3,000 × g, 4 DEG C) precipitums are finally centrifuged, take Supernatant is transferred to new 96 well culture plates for being previously added isometric 20% (w/v) PEG8000,2.5M NaCl solution, in ice Phage particle in 1 hour precipitation supernatant of upper insulation, centrifugation (5,000 × g, 4 DEG C, 30 minutes), heavy by precipitation again 200 μ l PBS solutions are suspended from, are tested for follow-up ELISA.

The bacteriophage of each clone to be analyzed, need to simultaneously for its corresponding screening target molecule and negative control protein (usually bovine serum albumin(BSA) or lysozyme) carries out ELISA detections.Correspondence antigen and lysozyme are coated on 96 orifice plates, And closed using confining liquid, make the remaining binding site saturation of frosting.After washing three times with PBST, above-mentioned preparation is taken Good Phage samples are added to each hole of elisa plate.After being incubated 2 hours at room temperature, washed with PBST three times.In order to detect combination Bacteriophage, per hole add 100 μ l with 1:Antibody-peroxidase conjugate (the GE of the anti-M13 bacteriophages of 5000 dilutions Healthcare), after being incubated 1 hour at room temperature, washed respectively three times with PBST and PBS.50 μ l tmb substrates are finally drawn to add Enter in hole, and develop the color 10 minutes at room temperature, the 2M H of 50 μ l are then added per hole₂SO₄Color development stopping is reacted.With Microplate reader (Bio-Rad) measure absorption value in 450nm.In ELISA experiments, relatively strong knot is shown to target molecule Signal is closed, with bacteriophage of the reference protein without binding signal, its corresponding monoclonal is sequenced with primer Seq ID No.7. Be derived from and further analyze dPA modified proteins amino acid sequence, it is exemplary in table 1 to list the ammonia wherein replaced There is the amino acid for replacing modification in having marked out first albumin A monomer of dPA albumen in base acid position and sequence, wherein 9-35 Position；9 ' -35 ' amino acid position modified is replaced in generation in being labelled with second albumin A monomer.

In the embodiment with PCT as target molecule, resulting albumin A heterodimeric binding protein precursor PCT-P1G07 The amino acid substitution of (Seq ID No.8), PCT-P2A08 (Seq ID No.9) and PCT-P2A12 (Seq ID No.10) is in table It is exemplary in 1 to list.In the embodiment with HSA as target molecule, resulting albumin A heterodimeric binding protein precursor HSA- The amino acid substitution of P1A02 (Seq ID No.11) and HSA-P1F11 (Seq ID No.12) is exemplary in table 1 to be listed. In embodiment with CDH16 as target molecule, resulting albumin A heterodimeric binding protein precursor CDH16-2H03-2 (Seq ID No.13) exemplary in table 1 listed with the amino acid substitution of CDH16-2H07-1 (Seq ID No.14).

Embodiment 6：Prepare and purify the modified protein based on dPA

The dPA modified proteins gene with target molecule specific binding activity for obtaining will be screened and pass through primer pair (Seq ID No.15 and Seq ID No.16) enter performing PCR amplification after, be cloned into respectively in expression vector pET22b (+), and by plasmid turn Enter to e. coli bl21 (DE3).Shaking flask and conventional isopropyl-beta D-thio galactopyranoside are used in laboratory level (IPTG) induced expression is carried out, can great expression dPA modified proteins.It is available after Escherichia coli after induced expression are through cracking Six histidine polypeptides of protein carboxyl terminal fusion, the albumen is purified by immobilized metal affinity chromatography method (IMAC).

It is dPA modified protein of the production with new binding activity, picking single bacterium colony is inoculated into 5ml 2 × YT/ ammonia benzyl moulds Plain culture medium, in 37 DEG C, 220rpm concussion and cultivates 16 hours.Culture is with 1:100 are transferred to 200ml 2 × YT/ ammonia benzyl moulds Plain culture medium, and in 37 DEG C of 220rpm concussion and cultivates, until cell density reaches about OD₆₀₀=0.6.Addition 1mM isopropyls-β- D- Thiogalactopyranosides (IPTG) induction exogenous gene is expressed, in 30 DEG C, 220rpm concussion and cultivates 6 hours.Centrifugation (4, 000 × g, 4 DEG C, 15 minutes) cell precipitation is collected, and it is resuspended in 15ml NPI-20 solution (50mM MaH₂PO₄,150mM NaCl, 20mM imidazoles, pH 8.0).200 μ g/ml lysozymes and 80U Benzonase is added to mix at room temperature 30 minutes, with Sonicated cells are used afterwards, is often worked in ice-water bath 15 seconds and is spaced 30 seconds, be repeated 5 times altogether.Centrifugation (15,000 × g, 4 DEG C, 30 minutes) pellet cell debris are removed, Soluble target albumen is present in supernatant, can be directly used for ensuing immobilization Metal affinity chromatography.

Using 5 times of NPI-20 buffer solutions of column volume, then pre-balance 5ml HisTrap HP purification columns make State cell cracking supernatant and flow through purification column.With 8 times of NPI-50 buffer solutions (50mM MaH of column volume₂PO₄,500mM NaCl, 50mM imidazoles, pH 8.0) uncombined foreign protein is washed away, then with NPI-250 buffer solutions (50mM MaH₂PO₄,150mM NaCl, 250mM imidazoles, pH 8.0) elution of bound destination protein.Protein sample is carried out dialysis into PBS, it is poly- by SDS The component and purity of acrylamide gel electrophoresis analysis albumen after purification, and determine protein concentration with BCA methods.In Shaking culture Under the conditions of, the yield of dPA modified proteins is generally 10-150mg/l cell cultures.

Embodiment 7:The binding activity of dPA modified protein of the identification with new binding characteristic

Tested by concentration gradient ELISA, determine binding activity of the dPA modified proteins to different target molecules.For this purpose, Use 0.1M NaHCO₃(pH 9.6) coating buffer dilutes target molecule, and 200ng is coated with per hole, and 50 μ l/ holes, 4 DEG C of coatings overnight, are used in combination PBST containing confining liquid is closed 2 hours at room temperature.Then rinse flat board three times with PBST and remove clean.Then, to each Orifice plate adds a series of measure of the PBST solution of each dPA modified proteins of the 100 μ l containing concentration, each sample to use parallel three hole Analysis.After 37 DEG C are incubated 2 hours, rinsed three times with PBST, be subsequently added 1:The HRP mark anti-E-tag antibody of mouse of 5000 dilutions (being purchased from Jin Sirui) 100 μ l/ holes, 37 DEG C are reacted 1 hour.In order to detect, Kong Sanci is rinsed with PBST, then rinse three with PBS It is secondary, it is eventually adding TMB and shows 15 minutes, with the 2M H of the μ l of every hole 50₂SO₄Color development stopping is reacted, and uses enzyme-linked immunosorbent assay instrument (Bio-Rad) absorbance value is measured in 450nm.With the photon absorbing intensity value obtained by Sigma Plot software evaluations, calculating antibody Bond strength.For this purpose, the extinction value measured under each case is mapped to corresponding associated proteins concentration, and with following Nonlinear regression is fitted to curve obtained.

Wherein identifying the combination/dissociation equilibrium between fixed target molecule and dPA modified proteins is：

The concentration of x=dPA modified proteins

The concentration (being measured indirectly by light absorption value after chromogenic reaction) of y=target molecules/dPA modified protein complexs

The total concentration of a=immobilization target molecules

B=dissociation constants (K_DValue)

SEQUENCE LISTING

<110>Wuhan sea sand hundred obtains Bioisystech Co., Ltd

<120>It is a kind of to prepare the protein-bonded method based on albumin A mutant

<130> 2017-03-10

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 58

<212> PRT

<213>Artificial sequence

<400> 1

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 2

<211> 126

<212> PRT

<213>Artificial sequence

<400> 2

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala

65 70 75 80

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn

85 90 95

Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu

100 105 110

Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 3

<211> 378

<212> DNA

<213>Artificial sequence

<400> 3

gtagacaaca aattcaacaa agaacaacaa aacgcgttct atgagatctt acatttacct 60

aacttaaacg aagaacaacg aaacgccttc atccaaagtt taaaagatga cccaagccaa 120

agcgctaacc ttttagcaga agctaaaaag ctaaatgatg ctcaggcgcc gaaatctggt 180

ggtggcggta gtggaggtgg tggagtggac aataaattta acaaggagca gcagaacgct 240

ttctacgaaa tcctgcacct gccgaacctg aacgaagaac agcgtaacgc gttcattcag 300

tctctgaagg acgacccgtc gcagtctgcc aacctgctgg ctgaagccaa gaaactgaac 360

gatgcgcagg ccccgaaa 378

<210> 4

<211> 128

<212> PRT

<213>Artificial sequence

<220>

<221> misc_feature

<222> (9)..(10)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (13)..(14)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (17)..(18)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (27)..(28)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (79)..(80)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (83)..(84)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (87)..(88)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (94)..(94)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (97)..(98)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (102)..(102)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (105)..(105)

<223> Xaa can be any naturally occurring amino acid

<400> 4

Val Asp Asn Lys Phe Asn Lys Glu Xaa Xaa Asn Ala Xaa Xaa Glu Ile

1 5 10 15

Xaa Xaa Leu Pro Asn Leu Asn Xaa Glu Gln Xaa Xaa Ala Phe Ile Xaa

20 25 30

Ser Leu Xaa Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Xaa Xaa

65 70 75 80

Asn Ala Xaa Xaa Glu Ile Xaa Xaa Leu Pro Asn Leu Asn Xaa Glu Gln

85 90 95

Xaa Xaa Ala Phe Ile Xaa Ser Leu Xaa Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 5

<211> 35

<212> DNA

<213>Artificial sequence

<400> 5

tcatggccca gccggccatg gtagacaaca aattc 35

<210> 6

<211> 26

<212> DNA

<213>Artificial sequence

<400> 6

agatgcggcc gctttcgggg cctgcg 26

<210> 7

<211> 24

<212> DNA

<213>Artificial sequence

<400> 7

caacgtgaaa aaattattat tcgc 24

<210> 8

<211> 128

<212> PRT

<213>Artificial sequence

<400> 8

Val Asp Asn Lys Phe Asn Lys Glu Tyr Leu Asn Ala Phe Phe Glu Ile

1 5 10 15

Ser Ile Leu Pro Asn Leu Asn Arg Glu Gln Lys His Ala Phe Ile Arg

20 25 30

Ser Leu Gly Asp Asp Leu Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Thr Gly

65 70 75 80

Asn Ala Leu Thr Glu Ile Val Leu Leu Pro Asn Leu Asn Ile Glu Gln

85 90 95

Ile Leu Ala Phe Ile Val Ser Leu Gly Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 9

<211> 128

<212> PRT

<213>Artificial sequence

<400> 9

Val Asp Asn Lys Phe Asn Lys Glu Leu Tyr Asn Ala Gly Leu Glu Ile

1 5 10 15

Asn Arg Leu Pro Asn Leu Asn Arg Glu Gln Trp Val Ala Phe Ile Val

20 25 30

Ser Leu Leu Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Arg Asp

65 70 75 80

Asn Ala Leu Val Glu Ile Thr Gln Leu Pro Asn Leu Asn Ile Glu Gln

85 90 95

Arg Lys Ala Phe Ile Arg Ser Leu Ile Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 10

<211> 128

<212> PRT

<213>Artificial sequence

<400> 10

Val Asp Asn Lys Phe Asn Lys Glu Val Trp Asn Ala Tyr Lys Glu Ile

1 5 10 15

Ser Val Leu Pro Asn Leu Asn Met Glu Gln Thr Ile Ala Phe Ile Val

20 25 30

Ser Leu Val Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Val Leu

65 70 75 80

Asn Ala Leu Leu Glu Ile Thr Arg Leu Pro Asn Leu Asn Ser Glu Gln

85 90 95

Ile Lys Ala Phe Ile Leu Ser Leu Ile Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 11

<211> 128

<212> PRT

<213>Artificial sequence

<400> 11

Val Asp Asn Lys Phe Asn Lys Glu Pro Trp Asn Ala Trp His Glu Ile

1 5 10 15

Ser Lys Leu Pro Asn Leu Asn Leu Glu Gln Glu Phe Ala Phe Ile Phe

20 25 30

Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Phe Ala

65 70 75 80

Asn Ala Val Ser Glu Ile Lys Gln Leu Pro Asn Leu Asn Gly Glu Gln

85 90 95

Arg Val Ala Phe Ile Val Ser Leu His Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 12

<211> 128

<212> PRT

<213>Artificial sequence

<400> 12

Val Asp Asn Lys Phe Asn Lys Glu Phe Thr Asn Ala Thr Tyr Glu Ile

1 5 10 15

Gln Asp Leu Pro Asn Leu Asn Met Glu Gln Glu Asp Ala Phe Ile Trp

20 25 30

Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Met Glu

65 70 75 80

Asn Ala Val Tyr Glu Ile Gly Val Leu Pro Asn Leu Asn Val Glu Gln

85 90 95

His Met Ala Phe Ile Thr Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 13

<211> 128

<212> PRT

<213>Artificial sequence

<400> 13

Val Asp Asn Lys Phe Asn Lys Glu Tyr Gln Asn Ala Asn Ile Glu Ile

1 5 10 15

Ile Ala Leu Pro Asn Leu Asn Phe Glu Gln Ala Gly Ala Phe Ile Tyr

20 25 30

Ser Leu Thr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Ala His

65 70 75 80

Asn Ala Tyr Ser Glu Ile Ile Ile Leu Pro Asn Leu Asn His Glu Gln

85 90 95

Leu Ile Ala Phe Ile Thr Ser Leu Asn Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 14

<211> 128

<212> PRT

<213>Artificial sequence

<400> 14

Val Asp Asn Lys Phe Asn Lys Glu Gly Trp Asn Ala Phe Ile Glu Ile

1 5 10 15

Lys Ile Leu Pro Asn Leu Asn Gln Glu Gln Pro Leu Ala Phe Ile Met

20 25 30

Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Gln Tyr

65 70 75 80

Asn Ala Phe Met Glu Ile Val Thr Leu Pro Asn Leu Asn Tyr Glu Gln

85 90 95

Asn Val Ala Phe Ile Arg Ser Leu Met Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210> 15

<211> 43

<212> DNA

<213>Artificial sequence

<400> 15

taagaaggag atatacatat ggtagacaac aaattcaaca aag 43

<210> 16

<211> 31

<212> DNA

<213>Artificial sequence

<400> 16

tggtggtggt gctcgagttt cggggcctgc g 31

Claims (8)

1. it is a kind of to prepare the protein-bonded method based on albumin A mutant, comprise the following steps：

A) storehouse of the monomer by the heteromultimers mutant of the albumin A of modification is provided, the storehouse includes heteromultimeric protein Matter, the heteromultimeric-protein includes two or more albumin A monomers linked together in the way of head and the tail are arranged, its Described in heteromultimeric-protein at least two described in monomer be by being pointed to SEQ ID No：In 19,10,11, 13rd, the replacement of at least five amino acid at 14,17,18,24,25,27,28,32,35 and the modification that carries out, the modification Monomeric protein has at least 80% Amino acid sequence identity with unmodified albumin A；

B) for the storehouse of the albumin A by modification provides potential target molecule；

C) storehouse of the protein of the modification is made to be contacted with the target molecule；

D) heteromultimeric protein A mutant is obtained by screening technique, the mutant is with K_DScope is 10^-7-10^-12The parent of M The target molecule is incorporated into power.

2. the method described in claim 1 step, wherein the replacement of at least five amino acid is preferably 8-11 amino acid Replace.

3. method according to claim 1, further includes the nucleotides of described heteromultimeric protein A mutant libraries Storehouse.

4. the combination in the nucleotides storehouse of the protein mutant storehouse of claim 1 and claim 3, the protein mutant storehouse Each member with encode the member nucleotides combined for physically by the means being coupled for genotype-Phenotype.

5. the combination of claim 4, wherein the means for genotype-Phenotype coupling include phage display system, ferment Female bacterium displaying, bacteria display, cell surface display or ribosome display system.

6. any one of claim 1-5 described method, wherein the target molecule is antigen or haptens.

7. the protein library that a kind of expression in nucleotides storehouse by described in claim 3 is obtained.

8. the cell or phage library of a kind of protokaryon comprising nucleotides storehouse according to claim 3 or eucaryon.