A method for constructing a phage display peptide library with positive electrostatic amino acid expansion stability

Abstract

The application belongs to the field of biotechnology, and discloses a method for constructing a phage display peptide library with positive electric amino acid amplification stability. It is found that inserting a negative electric amino acid at the N end of a display polypeptide or a polypeptide mutation site can improve the stability of a phage containing a positive electric amino acid in amplification, thereby improving the construction quality of the phage display peptide library and the success rate of panning of a sequence containing a positive electric amino acid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a phage display peptide library with positively charged amino acid amplification stability. Background Technology

[0002] Phage display peptide technology involves inserting a foreign polypeptide gene into the N-terminus of the gene encoding a phage capsid protein (typically the p3 and p8 capsid proteins of filamentous M13 phage) (usually located between the signal peptide and the mature capsid protein domain). Provided the reading frame is correct, the foreign polypeptide fuses with the phage capsid protein and is displayed on the phage surface during progeny phage assembly. By performing several rounds of affinity panning (binding-elution-amplification) on the phage display peptide library using target proteins, polypeptide sequences that specifically recognize the target protein can be obtained. This technology has achieved widespread success in the discovery of functional peptides. However, both the construction of the phage display peptide library and the affinity panning procedure involve phage amplification. The amino acid composition of the displayed polypeptide can affect the amplification efficiency of the corresponding phage, causing unexpected sequence composition changes. This not only reduces the quality of the phage display peptide library construction but also interferes with the affinity enrichment of the target sequence, affecting the success rate of the panning.

[0003] Of the 20 naturally occurring amino acids, positively charged amino acids, especially arginine (R), are crucial for forming non-covalent bonds. R participates in almost all major forms of non-covalent interactions, such as strong electrostatic interactions, dense hydrogen bond networks, and unique π-interactions. Furthermore, R possesses a long, flexible fatty chain, enabling it to adapt to imperfect geometric fits. These characteristics make R, despite its moderate abundance in the proteome, the second most enriched amino acid in hotspot regions of protein-protein interactions. Phage amplification involves a transmembrane transport process mediated by the *E. coli* Sec pathway, used to transport intracellularly produced capsid proteins to the inner membrane or periplasm for progeny phage assembly. Substrate proteins transported via the Sec pathway typically have a cleavable signal peptide at their N-terminus for translocase recognition and initiation of transport. After transport, the signal peptide is cleaved by a signal peptidase, releasing the mature domain. The positive-inside rule of positively charged amino acids (especially R) inhibits the amplification of phages displaying positively charged amino acids in their polypeptides. This results in a low initial frequency of positively charged amino acids in phage display peptide libraries, and also leads to the loss of potential target sequences during amplification. Although a few studies have found that negatively charged amino acids can "neutralize" the inhibition of Sec transport by positively charged amino acids in certain situations, the "neutralization" mechanism of negatively charged amino acids on positively charged amino acids has not yet been elucidated, and there is no research on how to construct phage display peptide libraries with amplification stability of positively charged amino acids. Summary of the Invention

[0004] Based on the above, the present invention aims to provide a method for constructing a phage display peptide library, which can maintain the stability of the positively charged amino acid frequency in the displayed peptides during the amplification steps involved in the construction and affinity panning of the display peptide library, thereby improving the construction quality and panning success rate of the phage display peptide library.

[0005] In one aspect, this application provides a method for constructing a phage display peptide library with positively charged amino acid amplification stability, namely, inserting a negatively charged amino acid at the N-terminus of the displayed peptide or peptide mutation site.

[0006] Furthermore, the insertion site of the negatively charged amino acid can be any position between the signal peptide and the display polypeptide or between the signal peptide and the polypeptide mutation site.

[0007] Furthermore, the insertion of the negatively charged amino acid includes, but is not limited to, inserting a codon encoding the negatively charged amino acid at the N-terminus of a gene displaying a polypeptide gene or a gene with a polypeptide mutation site.

[0008] Furthermore, the insertion type of the negatively charged amino acid includes aspartic acid, glutamic acid, or a mixture of both.

[0009] Furthermore, the number of negatively charged amino acids inserted is one or more.

[0010] In another aspect, this application provides the use of a negatively charged amino acid at the N-terminus of a positively charged amino acid for neutralizing the inhibitory effect of the positively charged amino acid on the corresponding phage amplification.

[0011] In another aspect, this application provides the use of inserting a negatively charged amino acid at the N-terminus of a display peptide or a peptide mutation site for stabilizing the frequency of positively charged amino acids in the construction of a phage display peptide library.

[0012] In another aspect, this application provides a comparison of the effects of inserting a negatively charged amino acid at the N-terminus of a display peptide or a peptide mutation site, inserting a negatively charged amino acid at the C-terminus, and inserting a similar amino acid at the N-terminus on stabilizing the frequency of positively charged amino acids in a phage-displayed peptide library.

[0013] In another aspect, this application provides the use of a phage display peptide library with positively charged amino acid amplification stability for improving the success rate of panning sequences containing positively charged amino acids.

[0014] The beneficial effects of the technical solution of this invention are as follows:

[0015] (1) A method was proposed to inhibit the amplification of the corresponding bacteriophage by neutralizing the positively charged amino acid with the negatively charged amino acid at the N-terminus of the positively charged amino acid.

[0016] (2) A method was proposed to construct a phage display peptide library with positively charged amino acid amplification stability by inserting negatively charged amino acids at the N-terminus of the display peptide or the peptide mutation site;

[0017] (3) The phage display peptide library construction method provided by the present invention can stabilize the frequency of positively charged amino acids in the phage amplification steps involved in the construction and panning of the display library, thereby improving the construction quality and panning success rate of the phage display peptide library. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 The amino acid frequency determination results in Example 2 show the ratio of amino acid frequencies at mutation sites in the phage display peptide library and DNA synthesis fragments. Solid black circles represent R, solid black squares represent K, and solid black triangles represent two negatively charged amino acids. A represents C. X PWP X R XX C is the display library, B is DC. X PWP X R XX C is the display library, C is for DDC X PWP X R XX C is the display library, and D is C. X PWP X R XX CD display library.

[0020] Figure 2 The amino acid frequency determination results in Example 3 show the ratio of amino acid frequencies at mutation sites in the phage display peptide library and DNA synthesis fragment. Black solid circles represent R, and black solid squares represent K. A represents C. XXXX GSATC Display Library, B for DC XXXX GSATC Display Library, C for NC XXXX GSATC Display Library, D for C XXXX GSATCD display library, E for CGSAT XXXX C is the display library, F is DCGSAT XXXX C is the display library, G is NCGSAT XXXX C is the display library, H is for CGSAT. XXXX CD display library.

[0021] Figure 3 The positive clone identification results of Example 4. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0023] Example 1: Neutralization of the inhibitory effect of negatively charged amino acids on the amplification of bacteriophages displaying positively charged amino acids

[0024] Phage display peptide library construction: Oligonucleotide chains (5'-CAT GCC CGG GTACCT TTC TAT TCT CAC TCT GCT TGT) were constructed using the DNA polymerase Klenow. NNK CCG TGG CCG NNK CGT NNK NNK TGC GGT GGAGGT TCG GCC GAA ACA TG-3') and universal extension primer (5'-CATGCCCGGGTACCTTTCTATTCTC-3') were used to synthesize a product that can translate into Cys Xxx Pro Trp Pro Xxx Arg Xxx Xxx Cys(C X PWP X R XX C, X The DNA double strands (with any amino acid sequence) were digested with Kpn I and Eag I, respectively. The digested DNA double strands and vector (purchased from NEB) were purified separately and then ligated using T4 DNA ligase. The ligation products were purified using a PCR purification kit. 1–2 µg of the purified ligation product was electroporated 20 times into ER2738 highly efficient electroporation competent cells. After each electroporation, 1 mL of SOC medium was added and the cells were incubated at 37 °C and 250 rpm for 30 minutes. All the recovered electroporation products were mixed, and 10 µL was used for positive clone count determination. The remaining electroporation products were transferred to 1 L of ER2738 (OD) medium. 600nmIn LB medium (containing 20 µg / mL tetracycline) with a concentration of approximately 0.01–0.05 μg / mL, cultured at 37°C and 250 rpm for 4.5 hours. Centrifuge the cultured solution at 12000g at 4°C for 20 minutes. Add 1 / 5 volume of 20% PEG8000 / 2.5 M NaCl to the supernatant, mix well, and incubate on ice for at least 2 hours. Centrifuge the supernatant at 12000g at 4°C for 20 minutes and discard the entire supernatant. Resuspend the phage pellet in 5 mL of sterile PBS, filter through a 0.22 µm filter to remove residual E. coli, add an equal volume of glycerol, mix well, and aliquot at -20°C for storage.

[0025] Amino acid frequency analysis: Using double-stranded DNA synthesized by DNA polymerase Klenow as a template, and F1 (5'-TTTGGAGCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTGGTACCTTTCTATTCTCACTCTGCT-3') and R1 (5'-TCGTCTTTCCAGACGTTAGTAAATGAATTTTCTGTATGGGATTTTGCTAAACAACTTTCACATGTTTCGGCCGAACCTCCACC-3') as primers, a ~200bp DNA fragment containing the polypeptide gene was amplified using Q5 high-fidelity DNA polymerase (purchased from NEB). After the target fragment was purified by agarose gel, PCR-free library construction and PE150 sequencing (commissioned by Novogene) were performed for amino acid frequency analysis of mutation sites before library construction and amplification. Using amplified phages as templates, and F2 (5'-TTTGGAGCCTTTTTTTTGGAG-3') and R2 (5'-tcgtctttccagacgttagta-3') primers, DNA fragments containing the polypeptide gene were amplified. After fragment purification, next-generation sequencing (NGS) was performed for amino acid frequency analysis of mutation sites after library construction and amplification. DNA sequences with corresponding sequence characteristics were extracted from the NGS data, translated into amino acid sequences, and identical amino acid sequences were merged and counted. The frequencies of 21 amino acids at each mutation site were calculated (including the stop codon corresponding to TAG, which is translated as Gln in ER2738).

[0026] The results showed that, compared with the synthesized DNA fragment before library construction and amplification, the ratio of the actual frequency (P(AB)) to the theoretical frequency (P(A) × P(B)) of the combination with the negatively charged amino acid at the beginning of the phage fragment after library construction and amplification was significantly higher than that of the combination with the negatively charged amino acid at the end (Table 1). This indicates that when a negatively charged amino acid is present at the N-terminus of a positively charged amino acid in a sequence, the sequence is more likely to be retained during amplification. To eliminate the interference of negatively charged amino acids at the remaining two mutation sites, sequences containing only one negatively charged amino acid were extracted. Table 2 lists the ratio of the abundance of the corresponding sequence in the phage display peptide library to its abundance in the synthesized DNA fragment when the negatively charged amino acid is located at different sites. Obviously, only when the negatively charged amino acid is located at the N-terminus of at least one positively charged amino acid can the sequence be effectively retained during amplification. When the negatively charged amino acid is located at the C-terminus of a positively charged amino acid, the abundance of the corresponding sequence in the phage display peptide library is reduced by more than 90%. This indicates that only negatively charged amino acids located at the N-terminus can neutralize the amplification inhibition of positively charged amino acids on phages. Table 3 lists the neutralization effects of the two negatively charged amino acids. The results show that both Asp (D) and Glu (E) can effectively maintain the amplification stability of positively charged amino acids, but D, which has a lower pI value, is slightly better than E.

[0027] Table 1. Ratio of actual to theoretical frequencies of charged amino acid combinations before and after amplification.

[0028]

[0029] a : (R7) indicates the R contained in the fixed sequence of the polypeptide.

[0030] Table 2. Abundance ratio of sequences in phage display peptide libraries to DNA synthesis fragments.

[0031] Site X2=D / E X6=D / E X8=D / E X9=D / E R2(R7) --- 0.606 0.117 0.078 R6(R7) 0.773 --- 0.035 0.029 (R7)R8 0.759 1.004 --- 0.044 (R7)R9 1.214 1.472 1.097 ---

[0032] Table 3. Abundance ratio of two negatively charged amino acid sequences in the phage display peptide library to the DNA synthesis fragment.

[0033] R2_(D / E)6 R6_(D / E)2 R8_(D / E)2 R8_(D / E)6 R9_(D / E)2 R9_(D / E)6 R9_(D / E)8 D 0.637 0.886 0.893 1.057 1.228 1.473 1.403 E 0.574 0.665 0.630 0.950 1.200 1.471 0.781

[0034] Example 2: Construction of a phage display peptide library with positively charged amino acid amplification stability 1

[0035] Phage display peptide library construction: Synthesis that can be translated into Asp Cys Xxx Pro Trp Pro Xxx Arg Xxx Xxx Cys (DC) X PWP X R XX C, X(any amino acid), Asp, Asp, Cys Xxx Pro Trp Pro Xxx Arg Xxx Xxx Cys (DDC) X PWP X R XX C) and Cys Xxx Pro Trp Pro Xxx Arg Xxx Xxx Cys Asp (C X PWP X R XX The DNA double strands with CD sequence characteristics are obtained, and the other steps are the same as in Example 1.

[0036] Amino acid frequency analysis: Same as in Example 1.

[0037] The results show that: Figure 1 As shown, compared to C X PWP X R XX In a C-terminal display peptide library, inserting one D terminus at the N-terminus significantly improved the amplification stability of positively charged amino acids R and Lys(K). When two D terms were inserted at the N-terminus, the frequencies of R and K were almost identical to those in the DNA synthesis fragment. This indicates that to completely neutralize the amplification inhibition of positively charged amino acids, the negatively charged amino acid inserted at the N-terminus needs to make the net charge of the peptide chain neutral. However, inserting a D terminus at the C-terminus had no effect on improving the amplification stability of R and K. Furthermore, the D terminus did not cause significant changes in the frequencies of other amino acids; instead, it further eliminated the amplification advantage of negatively charged amino acids within the displayed peptide.

[0038] Example 3: Construction of a phage display peptide library with positively charged amino acid amplification stability 2

[0039] Phage display peptide library construction: synthesis of peptides that can be translated into Cys Xxx Xxx Xxx Xxx Gly Ser Ala ThrCys (C XXXX GSATC, X (any amino acid), Cys Xxx Xxx Xxx Xxx Gly Ser Ala Thr Cys Asp (C XXXX GSATCD), Asp Cys Xxx Xxx Xxx XxxGly Ser Ala Thr Cys (DC) XXXX GSATC), AsnCys Xxx Xxx Xxx Xxx Gly Ser Ala Thr Cys (NC) XXXX GSATC), Cys Gly Ser Ala Thr Xxx Xxx Xxx Xxx Cys (CGSAT) XXXX C), Cys Gly Ser Ala Thr Xxx Xxx Xxx Xxx Cys Asp (CGSAT) XXXX CD), Asp Cys Gly Ser Ala Thr Xxx Xxx Xxx Xxx Cys (DCGSAT) XXXX C) and AsnCys Gly Ser Ala Thr Xxx Xxx Xxx Xxx Cys (NCGSAT) XXXX C) DNA double strands with sequence characteristics; other steps are the same as in Example 1. To ensure NGS can fully cover all sequence types in the display library and obtain more accurate results, a segmented phage display peptide library is constructed here, and the fixed amino acids in this example are replaced with those corresponding to C. X PWP X R XX C contains amino acids of completely different properties.

[0040] Amino acid frequency analysis: Same as in Example 1.

[0041] The results show that: Figure 2 As shown, among the four treatments—unmodified display peptide (conventional construction method), insertion of D at the C-terminus, insertion of D at the N-terminus, and insertion of Asn at the N-terminus—only the insertion of D at the N-terminus maintained the stability of positively charged amino acids during amplification. Furthermore, when the display peptide did not contain additional positively charged amino acids, although the frequency of positively charged amino acids at the mutation site also showed a significant decrease, the degree of decrease was far less than that of the C-terminus. X PWP X R XXThe C-display library indicates that for sequences containing two consecutive positively charged amino acids, if there is no negatively charged amino acid at the N-terminus, the sequence is likely to be completely lost during amplification.

[0042] Example 4: Affinity panning of phage display peptide libraries with positively charged amino acid amplification stability

[0043] Phage display peptide library construction: Synthesizing peptides that can be translated into Cys Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx Cys(CX8C, X (any amino acid) and Asp Cys Xxx Xxx Xxx Xxx Xxx Xxx Xxx Xxx The DNA double strand is characterized by the Cys (DCX8C) sequence, and the other steps are the same as in Example 1.

[0044] Phage display peptide library selection: Monoclonal antibodies recognizing methyl parathion were coated onto ELISA plates. After blocking the ELISA plates with 5% skim milk powder, phage display peptide libraries with and without positively charged amino acid amplification stability were added, respectively. After incubation at room temperature with shaking for 1 h, unbound phages were washed away. A standard solution of methyl parathion was added, and incubation at room temperature with shaking for 1 h was performed to competitively elute bound phages. The eluted phages were then added to 20 mL of ER2738 (OD2738) solution. 600nm The phages were cultured in LB medium (containing 20 µg / mL tetracycline) at 37°C and 250 rpm for 4.5 hours (approximately 0.01–0.05 μg / mL). The amplified phages were precipitated from the culture medium using PEG / NaCl, resuspended in sterile PBS, and filtered through a 0.22 µm filter to remove residual E. coli. An equal volume of glycerol was added, and the mixture was stored at -20°C. The above "adsorption-washing-elution-amplification" process was performed three times. From the elution products of the third round, 23 single clones were randomly selected for amplification, and positive clones were verified using phage enzyme-linked immunosorbent assay (ELISA).

[0045] Amino acid frequency analysis: NGS analysis was performed on the CX8C and DCX8C phage display libraries, as well as the third-round eluted phages from the two phage display libraries, following the same procedure as in Example 1.

[0046] The results showed that although the amount of NGS data could not completely cover the number of sequence types in the display library, it also demonstrated that the frequency of positively charged amino acids in the DCX8C display library was higher than that in the CX8C display library (Table 4). The CX8C display library had a 96% positive rate for random clones, containing 6 different positive sequences. The DCX8C display library had a 100% positive rate for random clones, containing 13 different positive sequences. Figure 3 (and Table 5). This firstly shows that the N-terminal insertion of D does not affect the affinity enrichment process. Since the phage display peptide in this example has a theoretical sequence class of 20... 8 =2.56 × 10 10 The number of sequences identified in the DCX8C library is 200 times the actual number of sequences in the display library, so it is normal that there is no overlap between the positive sequences identified in the two libraries. However, it is clear that the sequence richness identified in the DCX8C library is higher, and there is a positive sequence containing two consecutive Rs. According to the NGS data of the third round of elution products of the two libraries, among the top 30 most abundant sequences (Table 6), 5 sequences in DCX8C contain two Rs, while 0 sequences in CX8C contain two Rs. This indicates that it is possible to obtain multi-positively charged amino acid sequences from phage display peptide libraries with positively charged amino acid amplification stability, which cannot be obtained from conventional phage display peptide libraries.

[0047] Table 4. Amino acid frequencies in phage-displayed CX8C and DCX8C peptide libraries

[0048]

[0049] Table 5. Positive sequences identified from the phage-displayed CX8C and DCX8C peptide libraries.

[0050]

[0051] Table 6. Top 30 most abundant sequences in the final panning products of the phage display CX8C and DCX8C peptide libraries

[0052] <![CDATA[Sequence (CX8C)]]> Abundance (%) <![CDATA[Sequence (CX8C)]]> Abundance (%) <![CDATA[Sequence (CX8C)]]> Abundance (%) <![CDATA[CAPWPL R PGC]]> 37.53 <![CDATA[CSPWPP R PGC]]> 1.80 <![CDATA[CNPPWPS R AM]]> 0.60 CNPPWPMSWC 7.34 <![CDATA[CAPWPM R AQC]]> 1.57 <![CDATA[CPPWPP R AAC]]> 0.54 CAPWAAPPEC 5.72 <![CDATA[CMPPWPP R HC]]> 1.40 <![CDATA[C R PPWPQNWC]]> 0.53 CAPWGYPGTC 4.38 <![CDATA[CNPPWPM R HC]]> 1.33 <![CDATA[CPPWPP R LSC]]> 0.47 CLSPWPMWAC 3.60 CLPPWPLGWC 1.25 <![CDATA[CSPWPL R PAC]]> 0.45 <![CDATA[CLGPWP R WDC]]> 3.21 <![CDATA[C K SMWPNWNC]]> 1.23 <![CDATA[CPPWPA R TGC]]> 0.42 CSPPWPLSRC 2.37 CPAPWPMNWC 1.21 CPPWAPGPSC 0.41 CAPWAPSPSC 2.27 <![CDATA[CPPWPA R PLC]]> 0.71 CWPYMWASPC 0.40 <![CDATA[CPPWPP R ASC]]> 1.92 <![CDATA[CAPWAPPP R C]]> 0.69 <![CDATA[CPPWPA R SGC]]> 0.35 <![CDATA[CTPPWPP R PC]]> 1.83 CTAPWPMWTC 0.62 <![CDATA[CPPWPF R THING]]> 0.35 <![CDATA[Sequence (DCX8C)]]> Abundance (%) <![CDATA[Sequence (DCX8C)]]> Abundance (%) <![CDATA[Sequence (DCX8C)]]> Abundance (%) CHLWAWPTSC 18.42 CPMWVGTYPC 1.87 CVMWAWPENC 0.99 CLLWAWPVAC 9.46 <![CDATA[CPPWPP R QAC]]> 1.81 <![CDATA[CAPWPM RR EC]]> 0.86 <![CDATA[CPPWPL R PTC]]> 6.62 <![CDATA[CPPWPWP R LC]]> 1.53 CLLWAWPSPC 0.82 <![CDATA[CPPWP RR PSC]]> 5.83 CPPWAPPPEC 1.52 <![CDATA[CSPPWPS R AM]]> 0.76 CFGPWPWWTC 5.78 <![CDATA[CDPPWPP R AM]]> 1.45 CTGPWPMHVC 0.76 <![CDATA[CPPWP RR PAC]]> 4.85 <![CDATA[CPPWPP RR GC]]> 1.39 <![CDATA[CPPWPT R PMC]]> 0.70 <![CDATA[CPPWPA R PTC]]> 3.68 <![CDATA[CPPWPP R MSC]]> 1.29 <![CDATA[CPPWPP R I DO]]> 0.66 CPPWAPGPSC 2.91 CPLWAPPPSC 1.16 <![CDATA[CE R WAFPHHC]]> 0.63 <![CDATA[CV R WAWPPTC]]> 2.84 CPAWAPPPWC 1.05 <![CDATA[CPPWPP R ATC]]> 0.57 CIWAMWPAQC 2.68 <![CDATA[CPPWPL R ESC]]> 1.02 <![CDATA[CSPPWP RR SC]]> 0.55

[0053] The above embodiments are some embodiments of the present invention, provided merely for a better understanding of the embodiments of the present invention, and are not all embodiments of the present invention. In practical applications, by adjusting the length, structure, and sequence characteristics of the polypeptide displayed in the present invention, the specific position, number, and type of negatively charged amino acids inserted, and different phage display systems, numerous different embodiments can be obtained, all of which are within the scope of the present invention.

Claims

1. A method for constructing a phage display peptide library with positively charged amino acid amplification stability, characterized in that, A negatively charged amino acid is inserted at the N-terminus of the peptide or the peptide mutation site.

2. The construction method according to claim 1, characterized in that, The insertion site of the negatively charged amino acid can be any position between the signal peptide and the display polypeptide or between the signal peptide and the polypeptide mutation site.

3. The construction method according to claim 1, characterized in that, The insertion of the negatively charged amino acid includes, but is not limited to, inserting a codon encoding the negatively charged amino acid at the N-terminus of a gene displaying a polypeptide or a polypeptide mutation site.

4. The construction method according to claim 1, characterized in that, The insertion type of the negatively charged amino acid includes aspartic acid, glutamic acid, or a mixture of both.

5. The construction method according to claim 1, characterized in that, The number of negatively charged amino acids inserted is one or more.

6. A reagent kit, characterized in that, Includes a phage display peptide library constructed according to the method of claim 1.

7. A reagent kit, characterized in that, This includes plasmids or gene fragments prepared according to the method of claim 1 for constructing a phage display peptide library.

8. The application of the phage display peptide library of claim 1, or the kit of claim 6 or 7, in peptide screening.

Images