CN110055594A - A kind of phasmid display systems building polypeptide libraries method - Google Patents
A kind of phasmid display systems building polypeptide libraries method Download PDFInfo
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- CN110055594A CN110055594A CN201910388317.XA CN201910388317A CN110055594A CN 110055594 A CN110055594 A CN 110055594A CN 201910388317 A CN201910388317 A CN 201910388317A CN 110055594 A CN110055594 A CN 110055594A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
A kind of phasmid display systems building polypeptide libraries method, it is after connecting polypeptide with fixed sequence program, at both ends, insertion restriction enzyme site is connect with carrier, it greatly improves the segment rate of recovery and shows efficiency, one section of flexibility linker is added between polypeptide and fixed sequence program simultaneously, so that displayed polypeptides is maintained better flexibility, reduces steric hindrance, do not influence it and function.The present invention constructs polypeptide libraries method and is not limited by polypeptide size, after increasing by one section of fixed sequence program, it is equal to the length that indirect increases aim sequence, simultaneously because the effect of flexible connection linker, so that the random peptide shown is not influenced by fixed sequence program.Several to tens polypeptide libraries buildings can be carried out, can be applied to drug target screening, epitope analysis etc..
Description
Technical field
The present invention relates to biotechnologys, field of biological pharmacy, are related to one kind by phage display system and prepare polypeptide text
The method in library.
Background technique
Rondom polypeptide library has in research protein ligands/acceptor interaction, the analysis of zymolyte, searching
The screening etc. of the simulating peptide and novel drugs of the protein of biological function plays increasing effect.There are two main classes
The method for constructing polypeptide libraries.First kind method is iii vitro chemical synthesis, and the second class method is the oligomerization core random using synthesis
Thuja acid is expressed as soluble fusion protein in vivo, but above-mentioned two classes method has its limitation.It is external artificial synthesized more
Although peptide design is got up relatively simple, to use expensive instrument and complicated method, work time-consuming and laborious, and cost compared with
The library of height, synthesis cannot recycle.Though preferably being solved with the method that the oligonucleotide of synthesis expresses polypeptide in vivo
The above problem, but it is there are also intrinsic disadvantage needs to consider letter if oligonucleotide sequences design is got up more complex
And codon, terminator codon and palindrome is avoided the formation of as far as possible etc.；It also anneals during testing and carrying out, filling-in end
End such as connects at the processes with digestion, these can all cause very big influence to the success of experiment.In addition, constructed by above method
The length of random peptide library is substantially confined in 6-8 amino acid, and a kind of method can only construct a kind of library (horse of length
Element ginseng etc. constructs nearly rondom polypeptide library, bioengineering journal, Vol.21No.2March2005 using tobacco gene group DNA).
And display technique of bacteriophage has been widely used to each research neck of biological industry since the Smith invention in 1985
Domain, including epitope analysis, drug screening etc., the drawbacks of particularly for overcoming traditional technology in the building of polypeptide libraries, more
It is simple and cheap.Market has had commercialized phagedisplay peptide library product to sell at present, will be more but be all based on
Bacteriophage full price of the peptide library gene integration into phage genome is shown, builds library and screening operation process is relative complex, no
Conducive to the affinity screening and sequence analysis of polypeptide, while being difficult visual supervisory control operating process.Phasmid display systems pass through by
Target gene is fused to after phage coat protein gene through helper phage coinfection Escherichia coli, and it is purposeful to show band
The bacteriophage of gene protein sequence is simpler in operation, cheap.But since phasmid display systems are needed target gene Cook
It is grand on phagemid vector, to the restricted requirement of target fragment size, if segment is too small to will lead to fragment purification and connection effect
Rate is low, the storage capacity diversity not reached requirement, and limits the micromolecule polypeptide (such as several to more than ten polypeptide) of arbitrary size
Library construction.
The present invention occurs in construction and screening polypeptide libraries for phage display system and phasmid display systems
These difficult points and problem, a kind of method for having developed phasmid displayed polypeptides library not limited by small fragment, can show one
To the phage-displayed library of multiple polypeptides, can be applied in practical screening case.
Summary of the invention
The problems such as present invention is small for segment in current phage-displayed polypeptides technology, not easy to operate develops one kind not
By the polypeptide library constructing method for showing that clip size is limited.The phasmid displayed polypeptides library constructing method are as follows:
Objective gene sequence uses restriction enzyme site-polypeptide gene sequence-fixation gene order-restriction enzyme site sequence in the gene
Mode, carries out the target gene fragment synthesis in phasmid displayed polypeptides library, polypeptide gene sequence therein using (NNB) n,
(NNM) it is mixed after the synthesis of the coding modes such as n, (NNV) n (n can be arbitrary number)；Fixed gene order uses linker-
The mode of sumo label protein is synthesized, and (linker-sumo refers to that one section of flexible amino acid and sumo dissolution label protein connect
The fixation gene order connect)；SfiI restriction enzyme site is participated in polypeptide gene Sequences upstream, polypeptide gene sequence downstream participates in
Linker base fragment is realized and overlapping (overlaping) of fixed gene order；Fixed gene order downstream participates in and upstream
The different sfiI restriction enzyme site of cohesive end sequence.
Target gene is connect with pComb3x expression vector, and target gene fragment and pcomb3x carrier are used sfiI enzyme respectively
After cutting digestion, successfully target gene fragment is connected with carrier through T4 ligase, and convert XL1-blue competence bacteria.
Bacterium after the conversion coated plate after 100 times of dilutions obtains bacterium colony and grows number, at the same random 8 bacterium colonies of picking into
Row sequence verification (as shown in SEQIDNO:4-SEQIDNO:11).Storage capacity effect is calculated by clump count and sequencing result accuracy
Valence.
In some embodiments, the link position of polypeptide gene sequence and fixed gene order linker-sumo be with
Machine, i.e., polypeptide gene sequence can be in the N-terminal of fixed gene orresponding amino acid, can be in the C of fixed gene orresponding amino acid
End, can also be between the N-terminal and C-terminal of fixed gene orresponding amino acid.Thus, it is possible to make the polypeptide libraries potency of building more
It is high.
In some embodiments, the fixed gene order of linker-sumo is as shown in SEQIDNO:3, this sequence is in polypeptide
After designing one section of flexibility linker (as shown in seqid no:1) between sumo (as shown in SEQIDNO:2), polypeptide is not by space
Steric hindrance limitation, while sumo label is dissolution type prokaryotic expression label, increases and shows efficiency and stability, height may be implemented
Effect easily constructs polypeptide libraries.
In some embodiments, the fixed gene order of linker-sumo can be the fixation gene of other sequences substitution
Sequence, and then this polypeptide libraries has more fully use scope.
In some embodiments, the vector plasmid connecting with target gene is pComb3x carrier.Thus, it is possible to make to connect
It connects that efficiency is more efficient, and then the building of polypeptide libraries can be made more efficient.
In some embodiments, polypeptide-linker-sumo target gene fragment and the connection type of carrier can be enzyme
Connection perhaps homologous recombination connection or other modes connection are cut, it is possible thereby to improve being applicable in for polypeptide libraries building system
Property.
In some embodiments, the polypeptide libraries constructed by this method, polypeptide sequence can be it is random, should be with
The polypeptide sequence gene coding mode of machine can be the degenerate codes such as NNB, NNM, NNV mode or combination instructs whole amino
Acid synthesis.
In some embodiments, the polypeptide libraries constructed by this method, polypeptide sequence can be fixed form
Amino acid sequence.
In conclusion the present invention constructs bacteriophage in such a way that random/immobilized polypeptide is connect with fixed amino acid sequence
Displayed polypeptides library is not limited by polypeptide size, and (sequence is such as using the form of linker-sumo for fixed gene order
Shown in SEQIDNO:3), and one section of flexibility linker is designed between polypeptide and sumo (gene order is as shown in SEQIDNO:2)
After (gene order is as shown in seqid no:1), polypeptide is not limited by steric hindrance, while sumo label is dissolution type prokaryotic expression
Label increases and shows efficiency and stability, may be implemented it is efficient, easily construct polypeptide libraries, be very suitable to targeting peptides, control
The screenings such as the property treated pharmaceutical polypeptide.
Detailed description of the invention
Fig. 1 is the gene chemical synthesis PCR electrophoretogram of fixed amino acid sequence linker-sumo；
Fig. 2 is dodecapeptide-linker-sumo genetic fragment PCR electrophoretogram；
Fig. 3 is that dodecapeptide-linker-sumo genetic fragment connects PCR electrophoresis and restriction enzyme digestion and electrophoresis result with pComB3X carrier
Figure.
Specific embodiment
The present invention describes a kind of phasmid display systems building polypeptide libraries method, which can be for 1 extremely
Multiple amino acid compositions.
One, case study on implementation embodiment 1: is configured to the construction method combination specific steps and data with dodecapeptide library
It is explained name, phasmid shows the construction method in random dodecapeptides library:
1), the gene chemical synthesis of random dodecapeptides:
12 random peptides using the coding modes such as (NNB) 12, (NNM) 12, (NNV) 12 (N indicates tetra- kinds of bases of A, T, G, C,
B indicates tri- kinds of bases of G, T, C, and M indicates two kinds of bases of A, C, and V indicates tri- kinds of bases of G, A, C), in 12 random peptide gene sequences
Upstream be added sfiI restriction enzyme site number of base as synthesize target gene fragment upstream primer connector (adaptor),
12 random peptide gene sequences downstream be added connector (linker) upstream portion base sequence as with linker-sumo
The adaptor (base pair complementarity sequence) of connection, ultimate sequence form be 5 '-CCAAGCGGCC- (NNB) 12 (NNM) 12
(NNV)12-GGCGGCGGTAGCG。
2), the gene chemical synthesis of the fixed gene order of linker-sumo:
The fixed gene order of linker-sumo by directly synthesize linker primer and sumo gene order (SEQIDNO:
2) linker-sumo spliced after overlapped (overlaping) PCR reaction, while being mixed in the downstream sumo design primer
SfiI restriction enzyme site.Linker primer sequence is 5 '-GGCGGCGGTAGCGGTGGTGGTAGTGGTGGTGGCAGCatgtcgga
Ctcag-3 ', downstream sfiI restriction enzyme site primer are 5 '-GAGGAGGAGGGCCGACGGGGCCACCAATCTGTTCTC-3 ',
Sumo gene (sequence SEQIDNO:2) template comes from PET28a-sumo carrier, linker gene order such as SEQIDNO:1 institute
Show.PCR program is 98 DEG C of denaturation 10s, and 55 DEG C of annealing 10s, 68 DEG C of extension 1min, 30 recycle.PCR product electrophoresis result is shown in figure
1。
3), dodecapeptide-linker-sumo target gene fragment synthesizes:
By the random dodecapeptides genetic fragment of synthesis, linker-sumo fixed genetic fragment, upstream primer 5 '-
CTGCTGCTGGGCCCAAGCGGCC-3 ', downstream primer 5 '-GAGGAGGAGGGCCGACGGGGCCACCAATCTGTTCTC-3 '
Mixing obtains overall length target gene fragment after 30 PCR cycles react.PCR reaction condition are as follows: 94 DEG C of 2min, 98 DEG C of 10S,
55 DEG C of 10S, 68 DEG C of 1min, 68 DEG C of 5min, 10 circulations.All primers are closed by Suzhou Jin Weizhi Biotechnology Co., Ltd
At.Dodecapeptide-linker-sumo genetic fragment PCR electrophoresis product is shown in Fig. 2.
4), dodecapeptide-linker-sumo target gene fragment is connect with carrier:
Dodecapeptide-linker-sumo target gene fragment and pComB3X carrier are digested through sfiI endonuclease digestion respectively
Afterwards, it is connected overnight through T4 ligase, it is final to obtain containing the phagemid vector for showing segment.Connection method: pComb3x plasmid
500ng, full length fragment 105ng, 10*T4buffer5uL, T4 ligase 2.5uL, H2O polishing, 50ul system, connection 15,4
DEG C connection overnight.Dodecapeptide-linker-sumo target gene fragment connects PCR electrophoresis and restriction enzyme digestion and electrophoresis knot with pComB3X carrier
Fruit sees Fig. 3.
5), the competence preparation of XL1-Blue bacterium:
The previous day arrives in 2YT culture dish (containing tetracycline 10ug/mL) scribing line XL1-Blue bacterium, next day picking monoclonal
3mL2YT culture medium, 37 DEG C, 250rpm is incubated overnight.In the 2YT culture medium for the 2YT culture solution injection 1L that 3mL is incubated overnight,
250rpm is cultivated to OD about 0.8, and culture solution is dispensed into the centrifugal barrel of 500mL, and 4 DEG C, 4000rpm is centrifuged 10min.By precipitating weight
It is suspended in the ice-cold sterile water of 100mL, 4 DEG C, 4000rpm is centrifuged 10min, and repetition is washed primary again with sterile water.Precipitating is resuspended
In 10% 100mL ice-cold glycerol, 4 DEG C, 4000rpm is centrifuged 10min.It is finally resuspended in 2mL15% glycerol, liquid nitrogen speed
Freeze.
6), electrotransformation:
In Electrocompetent cells 90ul be added 100ng connection product, with electroporation (Bio-Rad,
GenePluserIIsystem electrotransformation, voltage 1.8kv) are carried out.37 DEG C of recovery 1h of bacterium solution after electricity turns, take 100ul bacterium solution to dilute
Afterwards in 2YT plate (ampicillin and tetracycline) coated plate, storage capacity is calculated.
7), the amplification in library:
2YT (containing ampicillin, tetracycline and 2% glucose) is added in bacterium solution remaining after electrotransformation and continues culture 1
After hour, after addition M13K07 helper phage is incubated for altogether, 37 DEG C, 250rpm is cultivated 1.5 hours, and supernatant is removed in centrifugation, and 2YT is added
Culture medium (contains ampicillin, tetracycline and kanamycins), and 30 DEG C, 250rpm is incubated overnight.Supernatant is through 4% overnight
After PEG8000 and 3%Nacl concentration, 7%DMSO is added, freezes in -80 DEG C.
8), library is identified:
Plate monoclonal is chosen into 500uL2YT culture medium, 37 DEG C are shaken 4h, and 1uL is taken to verify for bacterium solution PCR.Bacterium solution is each
1uL, 10*easyTaqbuffer1uL, dNTP0.8ul, easyTaq enzyme 0.15uL, Ab-F0.06uL, Ab-R0.06uL,
H2O6.93uL, 10uL system, PCR are identified.The correct bacterium solution of PCR identification send sequencing, and (8 bacterium colonies of random picking are surveyed
Sequence verifying, sequencing result are shown in SEQIDNO:4-SEQIDNO:11).
9), dodecapeptide random peptide library is evaluated:
Sequencing result shows that constructed random dodecapeptides library sequence distribution is random, and diversity is high.The coated plate meter after electricity turns
It calculates potency and reaches 1.05E+9, can be applied to phage library screening.
Two, case study on implementation embodiment 2: is configured to the construction method combination specific steps sum number with random heptapeptide library
According to name is explained, phasmid shows the construction method in random heptapeptide library:
1), the gene chemical synthesis of random heptapeptide:
The random peptide of heptapeptide uses the coding modes such as (NNB) 7, (NNM) 7, (NNV) 7, is added in heptapeptide gene sequence upstream
Adaptor of the sfiI restriction enzyme site number of base as synthesis target gene fragment upstream primer, heptapeptide gene order downstream adds
Enter the adaptor that linker upstream portion base sequence is connect as downstream with linker-sumo, ultimate sequence form is 5 '-
CCAAGCGGCC-(NNB)7\(NNM)7\(NNV)7-GGCGGCGGTAGCG。
Step 2)-step 9) is referring to step 2)-step 9) in embodiment 1.
Three, case study on implementation embodiment 3: is configured to the construction method combination specific steps sum number with random nonapeptide library
According to being explained, phasmid shows the construction method in random nonapeptide library:
1), the gene chemical synthesis of random nonapeptide:
The random peptide of nonapeptide uses the coding modes such as (NNB) 9, (NNM) 9, (NNV) 9, is added in nonapeptide gene sequence upstream
Adaptor of the sfiI restriction enzyme site number of base as synthesis target gene fragment upstream primer, nonapeptide gene order downstream adds
Enter the adaptor that linker upstream portion base sequence is connect as downstream with linker-sumo, ultimate sequence form is 5 '-
CCAAGCGGCC-(NNB)9\(NNM)9\(NNV)9-GGCGGCGGTAGCG。
Step 2)-step 9) is referring to step 2)-step 9) in embodiment 1.
Four, embodiment 4: case study on implementation is configured to the construction method combination specific steps sum number to fix dodecapeptide
According to being explained, phage display fixes the construction method in dodecapeptide library:
1), the gene chemical synthesis of fixed dodecapeptide:
Fixed dodecapeptide gene order be ATGACGCATGATCCGGTGATTTCTCTTCCTACTACT (such as SEQIDNO:
12) sfiI restriction enzyme site number of base, is added as synthesis target gene fragment upstream in fixed dodecapeptide gene sequence upstream
The adaptor of primer, fixed dodecapeptide gene order downstream be added linker upstream portion base sequence as downstream and
The adaptor of linker-sumo connection, ultimate sequence form are 5 '-CCAAGCGGCC-ATGACGCATGATCCGGTGATTTC
TCTTCCTACTACT-GGCGGCGGTAGCG。
Step 2)-step 9) is referring to step 2)-step 9) in embodiment 1.
Five, embodiment 5: case study on implementation is configured to the construction method combination specific steps and data to fix nonapeptide
It is explained, the construction method in the fixed nonapeptide library of phage display:
1), the gene chemical synthesis of fixed nonapeptide:
Fixed nonapeptide amino acid sequence is CSNRDARRC, and translating into gene order is
SfiI digestion is added in fixed nonapeptide gene sequence upstream in TGCAGCAATCGCGATGCCCGCCGTTGC (such as SEQIDNO:13)
Adaptor of the Post section base as synthesis target gene fragment upstream primer, fixed nonapeptide gene order downstream are added
The adaptor that linker upstream portion base sequence is connect as downstream with linker-sumo, ultimate sequence form are 5 '-
CCAAGCGGCC-TGCAGCAATCGCGATGCCCGCCGTTGC-GGCGGCGGTAGCG。
Step 2)-step 9) is referring to step 2)-step 9) in embodiment 1.
Six, embodiment 6: case study on implementation is configured to the construction method combination specific steps and data to fix heptapeptide
It is explained, the construction method in the fixed nonapeptide library of phage display:
1), the gene chemical synthesis of fixed heptapeptide:
Fixed heptapeptide amino acid sequence is YQFVWHP, translate into gene order be TACCAGTTCGTTTGGCATCCG (such as
SEQIDNO:14), sfiI restriction enzyme site number of base is added as synthesis target gene piece in fixed heptapeptide gene sequence upstream
The adaptor of section upstream primer, fixed heptapeptide gene order downstream be added linker upstream portion base sequence as downstream and
The adaptor of linker-sumo connection, ultimate sequence form are 5 '-CCAAGCGGCC-TACCAGTTCGTTTGGCATCCG-
GGCGGCGGTAGCG。
Step 2)-step 9) is referring to step 2)-step 9) in embodiment 1.
Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not
Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to the protection scope of invention.
Sequence table
<110>Ai Baisen (Jiangsu) Biotechnology Co., Ltd
<120>a kind of phasmid display systems construct polypeptide libraries method
<130> 2019
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<170> SIPOSequenceListing 1.0
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<213>artificial sequence ()
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ggcggcggta gcggtggtgg tagtggtggt ggcagc 36
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<211> 291
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<213>artificial sequence ()
<400> 2
atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60
gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120
aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180
gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240
ttggacatgg aggataacga tattattgag gctcacagag aacagattgg t 291
<210> 3
<211> 327
<212> DNA
<213>artificial sequence ()
<400> 3
ggcggcggta gcggtggtgg tagtggtggt ggcagcatgt cggactcaga agtcaatcaa 60
gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg 120
ccgatggatc ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctgt 180
ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgt 240
attagaattc aagctgatca gacccctgaa gatttggaca tggaggataa cgatattttg 300
aggctcacag agaacagatt ggt 323
<210> 4
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 4
ggtccggagt aggtgcctcc ggatcagaat ctgctg 36
<210> 5
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 5
attcctctgc ctagtacggt taatcgtgcg attggt 36
<210> 6
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 6
ccgttgaatg cgtctgagaa gcagattcct actatt 36
<210> 7
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 7
ctgtatctta ttccgttgca gctttctggt taggtt 36
<210> 8
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 8
tcgtatgagg tttgtctggc gggtgcgtat tcgcct 36
<210> 9
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 9
ttgtatcgtg atcagaggtc gaataatgat ctgttg 36
<210> 10
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 10
actgtggtgg ataggattgt tgggttgacg actatg 36
<210> 11
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 11
ttttggtcgt cggggcttgt ggagccgcgt gatgat 36
<210> 12
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 12
atgacgcatg atccggtgat ttctcttcct actact 36
<210> 13
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 13
tgcagcaatc gcgatgcccg ccgttgc 27
<210> 14
<211> 36
<212> DNA
<213>artificial sequence ()
<400> 14
taccagttcg tttggcatcc g 21
Claims (10)
1. a kind of phasmid display systems construct polypeptide libraries method, which is characterized in that described method includes following steps:
1) synthesis of polypeptide gene sequence；
2) synthesis of the fixed gene order of linker-sumo；
3) polypeptide gene connect synthesis polypeptide-linker-sumo target gene fragment with fixed gene order；
4) polypeptide-linker-sumo target gene fragment connect and converts with carrier；
5) amplification of polypeptide libraries；
6) identification of polypeptide libraries.
2. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
3) link position of polypeptide gene sequence and the fixed gene order of linker-sumo is random in.
3. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
3) the fixed gene order of linker-sumo is as shown in SEQ ID NO:3 in.
4. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
3) the fixed gene order of linker-sumo can be the fixation gene order of other sequences substitution in.
5. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
4) vector plasmid is pComb3x carrier in.
6. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
4) polypeptide-linker-sumo target gene fragment and the connection type of carrier are digestion connection in.
7. phasmid display systems according to claim 1 construct polypeptide libraries method, which is characterized in that the step
4) polypeptide-linker-sumo target gene fragment and the connection type of carrier are homologous recombination connection in.
8. a kind of polypeptide libraries of phasmid display systems building, which is characterized in that described in any item by claim 1-7
Method is prepared.
9. the polypeptide libraries of phasmid display systems building according to claim 8, which is characterized in that the polypeptide sequence
Column are random.
10. the polypeptide libraries of phasmid display systems building according to claim 8, which is characterized in that the polypeptide
Sequence is fixed.
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