A mycobacterium tuberculosis small peptide display library and its construction method and application

Abstract

The application provides a mycobacterium tuberculosis small peptide display library and a construction method and application thereof, and the construction method comprises the following steps: S1, small peptide sequence information is obtained by intercepting according to mycobacterium tuberculosis proteome sequence information; S2, the amino acid sequence of the small peptide sequence information is converted into a codon sequence, sequence optimization is carried out, and after a consensus sequence is added, oligonucleotide sequence information is obtained; S3, an oligonucleotide physical object is synthesized in a large scale and in parallel by using a chemical method; S4, PCR amplification is carried out by taking the oligonucleotide physical object as a template, and after being digested by a restriction endonuclease, an inserted nucleic acid fragment is obtained; and S5, the inserted nucleic acid fragment is used to construct the small peptide display library by using a display system. The mycobacterium tuberculosis small peptide display library constructed by the application provides important tools and resources for systematically understanding mycobacterium tuberculosis immunological epitopes, vaccine development, new drug research and development and the like.

Description

Technical Field

[0001] This invention relates to the field of library construction technology, and more specifically, to a Mycobacterium tuberculosis small peptide display library, its construction method, and its application. Background Technology

[0002] Tuberculosis is caused by Mycobacterium tuberculosis. According to the latest report from the World Health Organization (Global Tuberculosis Report 2024), tuberculosis has once again become the leading cause of death from a single infectious disease. Furthermore, the proportion of drug-resistant Mycobacterium tuberculosis remains at a high level. These factors pose significant challenges to the prevention, diagnosis, and treatment of tuberculosis.

[0003] Systematic and in-depth exploration of the biological characteristics of Mycobacterium tuberculosis, particularly the screening and identification of key epitopes that trigger humoral immunity, could potentially provide clues and support for controlling or even ending tuberculosis. However, current strategies for exploring the patterns of humoral immunity are still limited by factors such as throughput and cost. Several teams have already constructed Mycobacterium tuberculosis proteome microarrays (Proc. Natl. Acad. Sci. USA, 2010, 107: p.14703-14708; Cell Rep, 2014, 9: p.2317-2329; Theranostics, 2017, 7: p.4057-4070), which can provide important tools for basic research on Mycobacterium tuberculosis. While Mycobacterium tuberculosis proteome microarrays are also used to explore its humoral immune patterns, they are difficult to apply to screening and identifying key epitopes that trigger humoral immunity.

[0004] In recent years, with the development of synthetic biology, oligonucleotide synthesis technology has matured, enabling the simultaneous synthesis of 1,000,000 oligonucleotides, each 300 nt in length. Further, combined with display technology, small peptide display libraries have been constructed for the screening and identification of serum biomarkers. Currently, oligonucleotide synthesis technology has been applied to the construction of multiple small peptide display libraries and for the screening of serum biomarkers (Mol Cell Proteomics, 2024, 23:p.100831). In all constructed libraries, the peptides are generally longer than 35 amino acids, limiting the use of these libraries for identifying key epitopes that trigger humoral immunity.

[0005] Therefore, if large-scale parallel oligonucleotide synthesis technology can be used to obtain physical oligonucleotides and construct small peptide display libraries with short lengths (no more than 30 amino acid residues), it will provide key materials for basic research on Mycobacterium tuberculosis, especially for identifying key epitopes that trigger humoral immunity. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies, the present invention aims to provide a Mycobacterium tuberculosis small peptide display library, its construction method, and its applications. By integrating large-scale oligonucleotide synthesis technology and display technology, a small peptide display library targeting Mycobacterium tuberculosis is constructed, providing important tools and resources for basic research related to Mycobacterium tuberculosis, such as systematically understanding the immune epitopes of Mycobacterium tuberculosis, vaccine development, and new drug research.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for constructing a Mycobacterium tuberculosis small peptide display library, comprising the following steps: S1. Based on the Mycobacterium tuberculosis proteome sequence information, small peptide sequence information is obtained by truncation; the length of each small peptide sequence is a predetermined length of a amino acids, where a is selected from any value between 10 and 30. S2. The amino acid sequence of the small peptide sequence information is converted into a codon sequence according to the central dogma, and the sequence is optimized. After adding the common sequence, the oligonucleotide sequence information is obtained. S3. Based on the oligonucleotide sequence information, large-scale parallel synthesis of oligonucleotides was carried out using chemical methods; S4. Using the oligonucleotide sample as a template, PCR amplification was performed, and the inserted nucleic acid fragment was obtained after digestion with restriction endonuclease. S5. Construct a small peptide display library by using a display system to insert the nucleic acid fragments; In step S1, the length of each small peptide sequence is a predetermined length of a amino acids, where a is selected from any value between 10 and 30.

[0008] As some specific embodiments of the present invention, in step S1, the proteomic sequence information of the Mycobacterium tuberculosis is derived from a public database, or is the protein sequence information obtained by genome sequencing of Mycobacterium tuberculosis isolated and identified in a laboratory. The public database includes, but is not limited to, NCBI, Uniprot, or Mycobrowser databases. In this specific embodiment of the present invention, the Mycobacterium tuberculosis is Mycobacterium tuberculosis strain H37Rv, and its proteomic sequence information is derived from the Mycobrowser database, specifically from tuberculist.epfl.ch.

[0009] In some specific implementations, after obtaining the proteomic sequence information of Mycobacterium tuberculosis, the process also includes removing proteins with recorded errors, including proteins whose protein sequences contain illegal characters. Illegal characters refer to characters other than the abbreviations of the 20 amino acids, such as V and *.

[0010] As some specific embodiments of the present invention, in step S1, the method for obtaining small peptide sequence information based on proteomic sequence information is as follows: The first case: if the length of the protein's amino acid sequence is less than 'a', it will not be considered. The second case: if the length of the amino acid sequence of a protein is equal to 'a', then the amino acid sequence of the protein is a small peptide sequence. The third case: If the length of the protein's amino acid sequence is greater than 'a', then starting from the first amino acid of the protein, extract 'a' amino acids to form the first small peptide; starting from the second small peptide, extract 'a' amino acids starting from the 'b'th amino acid of the previous small peptide, where 'b' can be any value from 2 to 16, and 'b' is less than 'a'; if the length of the last small peptide is less than 'a', then start from the last amino acid of the protein sequence and move forward 'a' amino acids to form the last small peptide of the protein. The duplicate sequences were summarized and removed, and only one sequence of each small peptide sequence was retained.

[0011] In some specific implementations, the short peptide sequence is 30 amino acids long. The strategy for obtaining short peptide sequence information based on proteomic sequence information is as follows: The first case: if the amino acid sequence length of the protein is less than 30 amino acids, it will not be considered. The second case: If the length of the protein's amino acid sequence is equal to 30 amino acids, then the protein's amino acid sequence is a small peptide sequence. The third scenario: If the protein's amino acid sequence is longer than 30 amino acids, then starting from the first amino acid, 30 amino acids are extracted to form the first peptide. Starting with the second peptide, 30 amino acids are extracted from the 16th amino acid of the previous peptide, resulting in a 15-amino acid overlap. If the last peptide is shorter than 30 amino acids, then starting from the last amino acid of the protein sequence, 30 amino acids are moved forward to form the last peptide of the protein. Only one peptide sequence of each type is retained.

[0012] As some specific embodiments of the present invention, step S2 specifically includes the following steps: S21. According to the central dogma, convert each amino acid in the small peptide sequence into its corresponding codon, and arrange the codons in the order from the amino terminus to the carboxyl terminus according to the amino acid sequence of the small peptide. S22. Perform sequence optimization and label the small peptides with corresponding oligonucleotides; the sequence optimization includes: a. optimizing codon preferences to match the expression system or expression host; b. balancing the GC content between 40% and 60%; c. removing restriction endonuclease recognition sites; S23. Add common nucleotide sequence 1 and common nucleotide sequence 2 to the 5' and 3' ends of the corresponding oligonucleotide of the small peptide, respectively, to obtain the oligonucleotide sequence to be synthesized; the common nucleotide sequence 1 is shown as SEQ ID NO.1 (5'-GGGAATTCCGCTGCG-3'), and the common nucleotide sequence 2 is shown as SEQ ID NO.2 (5'- GATTACAAGGATGAC-3').

[0013] As some specific embodiments of the present invention, the expression system or expression host used in the present invention is Escherichia coli. Therefore, in step S22a above, codon preference optimization is performed to match Escherichia coli; the restriction endonuclease recognition sites in step c are the recognition sites of restriction endonucleases EcoR I and Hind III.

[0014] As some specific embodiments of the present invention, in step S3, the large-scale parallel synthesis of oligonucleotides is performed using either the TWIST or CustomArray platform. The CustomArray platform is preferred.

[0015] As some specific embodiments of the present invention, in step S4, the PCR is of the type of extension PCR; The required DNA polymerase is a hot-start high-fidelity DNA polymerase, specifically NEB's Q5 hot-start high-fidelity DNA polymerase, which has the characteristics of high fidelity and hot start. The required primers are preferably primer 1 and primer 2. The nucleotide sequence of primer 1 is shown in SEQ ID NO.39, and the nucleotide sequence of primer 2 is shown in SEQ ID NO.40. SEQ ID NO.39: 5'-GTGCTAATGAGGCTCAGCAATGATACGGCGGGAATTCCGCTGCG -3', SEQ ID NO.40: 5'-CAAGCAGAAGAAAGCTTTTACTTACTTATCGTCGTCATCCTTGTAATC -3'.

[0016] As some specific embodiments of the present invention, in step S4, the restriction endonuclease includes restriction endonuclease A and restriction endonuclease B, wherein restriction endonuclease A and restriction endonuclease B are different types of restriction endonucleases, and the sticky ends produced after digestion by the two restriction endonucleases are different.

[0017] Based on the obtained oligonucleotide material, an extension PCR combined with restriction enzyme digestion strategy was used to prepare the insert nucleic acid fragment. Using the oligonucleotide material as a shorter template, a longer PCR product was obtained through primers. The obtained PCR product was then digested with restriction enzymes to obtain the insert nucleic acid fragment.

[0018] As some specific embodiments of the present invention, in step S4, the obtained PCR product includes the following sequence: (1) The nucleotide sequence corresponding to the small peptide sequence; (2) The upstream of the nucleotide sequence corresponding to the small peptide contains the recognition sequence of restriction endonuclease A and protective bases; (3) Downstream of the nucleotide sequence corresponding to the small peptide, the same reading frame contains the nucleotide sequence corresponding to the tag sequence (the tag sequence cannot be too long, within 6-20 amino acids in length, otherwise it cannot be introduced by primers); the tag includes, but is not limited to, any one of FLAG, HA, oligohistidine, and myc; (4) The downstream of the tag sequence contains a stop codon in all three reading frames (introduce a stop codon sequence that exists in all three reading frames, as long as it is a stop codon that the host can recognize. The reason is that in the central dogma, three bases correspond to one amino acid. During the synthesis of oligonucleotides, sometimes a base is mistakenly inserted or missing, which will produce different reading frames. In order to deal with this situation, it is required that there is a stop codon in all reading frames: one for normal termination, one for the insertion of a base, and one for the deletion of a base, so that translation can be terminated in time). (5) Restriction endonuclease B recognition sequence and its protective bases.

[0019] As some specific embodiments of the present invention, in step S4, the PCR product is digested with restriction enzymes, and a protective base sequence of the restriction endonuclease is removed from each end, and the obtained product is an inserted nucleic acid fragment.

[0020] As some specific embodiments of the present invention, in step S5, the display system includes, but is not limited to, a T7 phage display system, a λ phage display system, an Escherichia coli display system, a yeast display system, a cell display system, or a ribosome display system, in order to maintain the correspondence between nucleotides and small peptides.

[0021] Preferably, in step S5, the display system used to construct the small peptide display library is the T7 phage display system. Specifically, a T7 phage packaging kit is used to construct the small peptide library.

[0022] Secondly, the present invention provides a Mycobacterium tuberculosis small peptide display library, which is constructed using any of the above-described construction methods.

[0023] Thirdly, this invention provides an application of the Mycobacterium tuberculosis small peptide display library described above in screening serum biomarkers, identifying drug targets, identifying protein interactions, and identifying small peptides or vaccine development. It can provide resources and tools for basic research on Mycobacterium tuberculosis, including but not limited to screening serum biomarkers, identifying drug targets, identifying protein interactions, and identifying small peptides or vaccine development.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1) The short length of small peptide sequences results in lower costs for synthesizing the corresponding oligonucleotide sequences, thereby reducing the production cost of small peptide display libraries; 2) The correspondence between DNA and small peptides and proteins can be used to identify small peptides and proteins through sequencing in subsequent applications, which can significantly improve the throughput of sample analysis; 3) Bacteriophages, as a small peptide library display system, have the ability to proliferate, which can continuously provide small peptide materials for their applications and reduce the cost of using small peptide display libraries. 4) The Mycobacterium tuberculosis small peptide display library constructed in this invention can be used to analyze antigenic epitopes that trigger humoral immunity at the proteomic level, thereby enabling its application in the diagnosis and vaccine development of Mycobacterium tuberculosis. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The figures below show the construction and quality control results of the phage-displayed small peptide library in Example 1; where a is a schematic diagram of the amplification products; b is a copy of the dot blot quality control results of the small peptide library; c is a statistical graph of each small peptide and its frequency of occurrence in the NGS quality control; d is a graph showing the quality results of the phage-displayed small peptide library at the small peptide abundance level in the NGS quality control; and e and f are graphs showing the quality results of the phage-displayed small peptide library at the protein level in the NGS quality control. Figure 2 The figures below show the experimental results of applying the phage display peptide library in Example 2. Figure a is a flowchart of the experimental plan for immunizing, challenging, and collecting blood from mice in Example 2; Figure b shows the response of mouse humoral immunity to Mycobacterium tuberculosis H37Rv. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0027] Example 1: Construction of a Mycobacterium tuberculosis small peptide display library 1. Obtain the proteomic sequence information of Mycobacterium tuberculosis. (1) Download all protein sequences of Mycobacterium tuberculosis strain H37Rv from the website https: / / mycobrowser.epfl.ch / , totaling 4,031 sequences; (2) Remove protein sequences with errors, i.e. sequences containing illegal characters (characters other than the 20 amino acid abbreviations, such as V and *), leaving 4,027 sequences.

[0028] 2. Obtain small peptide sequence information based on the Mycobacterium tuberculosis proteome sequence information. The small peptide sequence was obtained using the following procedure: Case 1: After removing proteins with an amino acid sequence length of less than 30 amino acids, 4,025 sequences remain. The second case: a protein with an amino acid length of 30 amino acids has a small peptide sequence that is a protein sequence. The third scenario: If the protein's amino acid sequence is longer than 30 amino acids, then starting from the first amino acid, 30 amino acids are extracted to form the first peptide. Starting from the second peptide, 30 amino acids are extracted from the 16th amino acid of the previous peptide, resulting in a 15-amino acid overlap with the previous peptide. If the last peptide is shorter than 30 amino acids, then starting from the last amino acid of the protein sequence, 30 amino acids are moved forward to form the last peptide of the protein. All obtained small peptide sequences were summarized, duplicate sequences were removed, and only one of each small peptide sequence was retained, resulting in a total of 86,287 different small peptide sequences.

[0029] 3. Obtain the oligonucleotide sequence information to be synthesized based on the small peptide sequence information. (1) According to the central dogma, convert small peptide sequences into nucleotide sequences; (2) Optimize the nucleotide sequence, specifically including: Codon optimization based on E. coli codon usage preferences; The GC content should be adjusted to be between 40% and 60%. Remove the recognition sites of restriction endonucleases EcoR I and Hind III; (3) Add common nucleotide sequence 1 to the 5' end of each of the above nucleotide sequences and common nucleotide sequence 2 to the 3' end, which are the oligonucleotide sequences to be synthesized, and their lengths are all 120 nt. The common nucleotide sequence 1 is 5'-GGGAATTCCGCTGCG-3' (SEQ ID NO.1); The common nucleotide sequence 2 is 5'-GATTACAAGGATGAC-3' (SEQ ID NO.2).

[0030] 4. Synthesize the oligonucleotide physical product based on the sequence information of the oligonucleotide to be synthesized. The above-mentioned oligonucleotide sequence information was submitted to Peking University, and large-scale parallel synthesis was carried out using chemical methods on the CustomArray platform to obtain the oligonucleotide physical product. It was dissolved in 200 μL of ultrapure water and labeled as nucleic acid sample A, which is the oligonucleotide physical product.

[0031] 5. Based on the oligonucleotide sample, prepare the inserted nucleic acid fragment: (1) PCR amplification was performed using oligonucleotide samples as templates. The PCR reaction system is shown in Table 1 below: Table 1. PCR reaction system for amplifying oligonucleotides

[0032] Among them, Q5 reaction buffer and Q5 pol are derived from NEB's Q5® ultra-fidelity DNA polymerase product, catalog number M0493L.

[0033] Primer 1: (SEQ ID NO.39) 5'-GTGCTAATGAGGCTCAGCAATGATACGGCGGGAATTCCGCTGCG -3'; Primer 2: (SEQ ID NO.40) 5'-CAAGCAGAAGAAAGCTTTTACTTACTTATCGTCGTCATCCTTGTAATC -3'.

[0034] The PCR amplification reaction procedure is as follows: .

[0035] The obtained amplification products are as follows Figure 1As shown in Figure a, the template includes an oligonucleotide (120 bp in length). After PCR amplification to introduce the elements (upstream restriction endonuclease EcoRI, downstream FLAG tag, and restriction endonuclease HindIII), the theoretical length is 182 bp.

[0036] The size and purity of the amplified products were identified by 2.5% agarose gel electrophoresis. The bands of about 200 bp in size were recovered by gel cutting using the QIAquick Gel Extraction Kit (QIAGEN, Germany, catalog number 28706). The DNA fragments obtained were labeled as nucleic acid sample B and used for enzyme digestion.

[0037] (2) Digestion to obtain inserted nucleic acid fragments: ①According to the protocol provided by NEB, the restriction endonucleases EcoR I and Hind III were used simultaneously for overnight digestion at 37°C; ② The digestion products were subjected to 2.5% agarose gel electrophoresis to identify their size and purity. The 150 bp bands were extracted using the QIAquick Gel Extraction Kit (QIAGEN, Germany, catalog number 28706). The resulting DNA fragment was labeled as nucleic acid sample C, which is the obtained inserted nucleic acid fragment. This fragment was used to insert into the phage genome and construct a small peptide display library.

[0038] 6. Constructing a small peptide display library (1) Quality control of nucleic acid sample C: A portion of nucleic acid sample C was aspirated and inserted into the T vector strictly according to the procedure in the T vector kit (Takara Bio Engineering (Dalian) Co., Ltd., catalog number 6011, product name pMD™ 18-T Vector Cloning Kit). The ligated T vector was transformed into E. coli DH5α competent cells strictly according to the transformation procedure and plated on LB solid medium containing 100 μg / mL ampicillin. The medium was incubated overnight at 37°C with the cells inverted. Twenty single clones were randomly selected for Sanger sequencing. In clones containing the inserted fragment, there should be no undigested EcoRI or HindIII sequences, which means that nucleic acid sample C is qualified.

[0039] (2) Construction of phage display peptide libraries was performed using the T7 phage packaging kit (Merck, catalog number 70550-M, product name T7Select® 10-3 Cloning Kit): ① Strictly follow the procedures in the phage packaging kit to construct the phage display library. The ratio of nucleic acid sample C to vector is 3:1 for phage packaging; ② Test the titer of the bacteriophage. Based on the titer of the bacteriophage, use double-layer plates to amplify the packaged bacteriophage using the host E. coli strain BLT5403; ③ Following the procedure in the T7 phage packaging kit, collect and concentrate the phage display peptide library to complete the construction of the phage display peptide library.

[0040] 7. Perform titer testing and quality control on the constructed phage display peptide library. Quality control was performed using dot blot and NGS methods.

[0041] (1) Dot blot quality control: Dot blot experiments were performed strictly following the procedures in the phage packaging kit, using FLAG antibodies to determine whether the small peptide was displayed on the phage surface: the presence of a FLAG signal indicated that the phage had successfully displayed the small peptide; otherwise, the phage was empty or displayed an incorrect small peptide sequence.

[0042] The quality of the phage display library was initially and rapidly assessed using dot blot. Figure 1 Figure b shows the photocopy results of the small peptide library dot blot, where black spots represent phages with FLAG tags on their surface, and gray spots represent phages without FLAG tags on their surface; the results show that about 50% of the phages showed FLAG tag-related signals, indicating that about 50% of the phages successfully displayed the target small peptide.

[0043] (2) NGS quality inspection NGS quality control was performed based on the NGS sequencing library construction and labeling strategy described in patent ZL201710943109.2. The specific steps included are as follows: ① First round of PCR: using the constructed Mycobacterium tuberculosis small peptide display library as a template The upstream and downstream primers for the first round of PCR are shown in Tables 2 and 3. Select one primer from each of Tables 2 and 3 as the upstream and downstream primers. Table 2. Upstream primer sequences used in the first round of PCR amplification.

[0044] Table 3. Downstream primer sequences used in the first round of PCR amplification.

[0045] The upstream primer sequences in Table 2 contain a common nucleotide sequence 3: 5'-ATGCTCGGGGATCCGAATTC-3' (SEQ ID NO. 35); The downstream primer sequences in Table 3 contain a common nucleotide sequence 4: 5'-GTTACTCGAGTGCGGCCGCAAGC-3' (SEQ ID NO. 36).

[0046] ② Second round of PCR: After the first round of PCR amplification, the gel was cut and recovered, serving as a template for the second round of PCR. The second round of PCR used a pair of upstream and downstream primers, S502 and N701, from the NexteraXT Index Kit v2.

[0047] ③ After the second round of PCR amplification, the size and purity of the amplification products were identified by 2.5% agarose gel electrophoresis. The 300 bp band was extracted by cutting the gel using the QIAquick Gel Extraction Kit (QIAGEN, Germany, catalog number 28706). The obtained DNA sample is the constructed NGS sequencing library 1. ④ Quality control of NGS sequencing library 1: NGS sequencing library 1 was extracted and inserted into the T vector strictly following the procedure in the T vector kit (Takara Bio Engineering (Dalian) Co., Ltd., catalog number: 6011). Then, following the transformation procedure of E. coli strain DH5α competent cells (Beijing TransGen Biotech Co., Ltd., catalog number CD201-01), the ligated T vector was transformed into E. coli DH5α, plated on LB agar containing 100 μg / mL ampicillin, and incubated overnight at 37°C inverted position. Twenty single clones were randomly selected for Sanger sequencing to analyze the structure and composition of the NGS sequencing library.

[0048] ⑤ NGS sequencing and sequencing result analysis: NGS sequencing library 1 was delivered to Beijing Novogene Technology Co., Ltd., and NGS sequencing was performed using 2 × 250 bp paired-end sequencing mode.

[0049] The quality of the phage-displayed peptide library was assessed by statistically analyzing the frequency of each detectable small peptide. The results showed a total of 38,112,072 reads, of which 54.20% were completely correct, 35.56% contained deletion mutations, 8.03% contained substitution mutations, and 2.21% contained insertion mutations (e.g., ...). Figure 1 (As shown in c); at the small peptide level: of the 86,287 designed small peptides, 82,529 could be detected by NGS (e.g., ... Figure 1As shown in c), accounting for 95.64%. In assessing the quality of the phage-displaying small peptide library at the level of small peptide abundance, 81.08% of the small peptides had an abundance of 5–500 (e.g., ...). Figure 1 (As shown in d). The quality of the phage-displayed small peptide library was assessed at the protein level; 62% of the proteins, including all involved small peptides, were detectable (e.g., ...). Figure 1 As shown in Figure e), 85% of the protein can be completely covered (e.g. Figure 1 (As shown in f). There were 3,758 small peptides that were not detected by NGS, distributed across 1,513 protein sequences, of which 586 proteins contained regions not covered by the detected small peptides.

[0050] The quality of the constructed library was comprehensively evaluated through NGS quality control, and the results showed that the Mycobacterium tuberculosis small peptide display library was successfully constructed.

[0051] Example 2: Application of phage-displayed small peptide libraries 1. Immunization blood collection from mice Four female mice of strain C57BL6, aged 6-8 weeks, each weighing approximately 20 g, were used. In a P2+ laboratory, each mouse was individually... Figure 2 The procedure shown in Figure a involved immunization, viral challenge, and blood collection, resulting in a total of 24 serum samples. The specific procedure is as follows: (1) Blood was collected through the orbital vein and marked as week 0. Immunization was performed using normal saline. (2) Blood was drawn from the orbital vein before immunization with saline at weeks 3 and 6; (3) Blood was drawn via the orbital vein before the administration of Mycobacterium tuberculosis H37Rv challenge in week 10; (4) Finally, blood was collected via the orbital vein at weeks 14 and 24 respectively; Blood was collected via the orbital vein using Microvette® 100 / 200 micro-volume blood collection tubes. After each blood collection was allowed to stand at room temperature for 1 hour, it was centrifuged at 1800 rpm to obtain serum, which was then frozen and stored at -80°C for later use.

[0052] 2. Take 1 μL of each serum sample and add it to each well of the PCR plate. Each well contains 180 μL of TBST-I solution (containing 0.5% Tween 20 TBS solution) as a control. One well does not contain serum.

[0053] 3. Add 10 μL of the phage display peptide library constructed in Example 1 to each well, which is about 1000 times more diverse than the library, and incubate overnight at 4°C; add 10 μL of G protein magnetic beads (Thermo Fisher Scientific Inc., catalog number 10004D) and immunoprecipitate at 4°C for 4 h.

[0054] 4. Use a 96-well magnetic rack to separate the magnetic beads; wash three times with 200 μL of TBST-I solution, then wash once with 200 μL of ultrapure water to remove as much residual ultrapure water as possible, and then suspend the magnetic beads in 15 μL of ultrapure water; seal and incubate at 95℃ for 10 min, which is the template used for the first round of PCR to construct the NGS sequencing library 2.

[0055] 5. Constructing NGS sequencing library 2: Based on the NGS sequencing library construction and labeling strategy in patent ZL201710943109.2, NGS library construction was performed. The specific steps are as follows: ① First round of PCR: The upstream and downstream primers for the first round of PCR are shown in Tables 2 and 3 above. Different combinations of upstream and downstream primers should be used for different samples.

[0056] ② Second round of PCR: After the first round of PCR amplification, all PCR products were mixed, excised from the gel, and used as a template for the second round of PCR. The second round of PCR used a pair of upstream and downstream primers, S502 and N701, from the Nextera XT Index Kit v2. After PCR amplification, 2.5% agarose gel electrophoresis was used to identify the size and purity of the amplified products. Bands of 300 bp were excised from the gel and recovered. This constitutes NGS sequencing library 2.

[0057] ③ Quality control of NGS sequencing library 2: The NGS sequencing library was extracted and inserted into the T-vector strictly following the procedure outlined in the T-vector kit (Takara Bio Engineering (Dalian) Co., Ltd., catalog number: 6011). The ligated T-vector was transformed into E. coli strain DH5α competent cells (Beijing TransGen Biotech Co., Ltd., catalog number CD201-01) strictly following the transformation procedure, and the cells were plated on LB agar containing 100 μg / mL ampicillin. The culture was incubated overnight at 37°C with the plates inverted. Twenty single clones were randomly selected for Sanger sequencing to analyze the structure and composition of the NGS sequencing library.

[0058] ④ NGS sequencing and sequencing result analysis: NGS sequencing library 2 was delivered to Beijing Novogene Technology Co., Ltd., and NGS sequencing was performed using 2 × 250 bp paired-end sequencing mode.

[0059] For each serum sample, the frequency of each small peptide was counted, and samples with low sequencing reads were removed. Additionally, for the same mouse, at least one sample before and after challenge had a higher sequencing read count. The remaining 8 samples met the requirements, and the sequencing read count of each sample was proportionally normalized to 10,000 reads.

[0060] The results are as follows Figure 2 As shown in Figure b, small peptides that enhance humoral immunity were screened and obtained, including peptide Rv2931-19 derived from protein Rv2931, with the amino acid sequence TACSSSLVAIHLACQSLRTQDCHLAIAAGV (SEQ ID NO.37); and peptide Rv2787-17 derived from protein Rv2787, with the amino acid sequence DAPRTNTTAEQAGSPQPKFRHPYDDGRDLL (SEQ ID NO.38). After challenge with Mycobacterium tuberculosis H37Rv, the reaction intensity increased in all three mice, identifying key antigenic epitopes that induce humoral immunity. Therefore, the Mycobacterium tuberculosis small peptide display library of this invention can be directly used to identify specific small peptides with key epitopes, i.e., key regions of proteins that can induce humoral immunity. Subsequently, based on this, the corresponding small peptides can be obtained in large quantities through chemical synthesis to further verify their value in tuberculosis diagnosis, vaccine development, and other fields.

[0061] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a Mycobacterium tuberculosis small peptide display library, characterized in that, The steps include the following: S1. Based on the Mycobacterium tuberculosis proteome sequence information, small peptide sequence information is obtained by truncation; the length of each small peptide sequence is a amino acid of a predetermined length, where a is selected from any value between 10 and 30. S2. The amino acid sequence of the small peptide sequence information is converted into a codon sequence according to the central dogma, and the sequence is optimized. After adding the common sequence, the oligonucleotide sequence information is obtained. S3. Based on the oligonucleotide sequence information, large-scale parallel synthesis of oligonucleotides was carried out using chemical methods; S4. Using the oligonucleotide sample as a template, PCR amplification was performed, and the inserted nucleic acid fragment was obtained after digestion with restriction endonuclease. S5. Construct a small peptide display library by inserting nucleic acid fragments using a display system.

2. The construction method according to claim 1, characterized in that, In step S1, the method for obtaining small peptide sequence information based on proteomic sequence information is as follows: The first case: if the length of the protein's amino acid sequence is less than 'a', it will not be considered. The second case: if the length of the amino acid sequence of a protein is equal to 'a', then the amino acid sequence of the protein is a small peptide sequence. The third case: If the length of the protein's amino acid sequence is greater than 'a', then starting from the first amino acid of the protein, extract 'a' amino acids to form the first small peptide; starting from the second small peptide, extract 'a' amino acids starting from the 'b'th amino acid of the previous small peptide, where 'b' can be any value from 2 to 16, and 'b' is less than 'a'; if the length of the last small peptide is less than 'a', then start from the last amino acid of the protein sequence and move forward 'a' amino acids to form the last small peptide of this protein. The duplicate sequences were summarized and removed, and only one sequence of each small peptide sequence was retained.

3. The construction method according to claim 1, characterized in that, In step S1, the Mycobacterium tuberculosis proteome sequence information is obtained from a public database or through sequencing. The public database is selected from any one of the NCBI, Uniprot, or Mycobrowser databases.

4. The construction method according to claim 1, characterized in that, Step S2 specifically includes the following steps: S21. According to the central dogma, convert each amino acid in the small peptide sequence into its corresponding codon, and arrange the codons in the order from the amino terminus to the carboxyl terminus according to the amino acid sequence of the small peptide. S22. Perform sequence optimization and label the small peptides with corresponding oligonucleotides; the sequence optimization includes: a. optimizing codon preferences to match the expression system or expression host; b. balancing the GC content between 40% and 60%; c. removing restriction endonuclease recognition sites; S23. Add common nucleotide sequence 1 and common nucleotide sequence 2 to the 5' end and 3' end of the oligonucleotide corresponding to the small peptide, respectively, to obtain the oligonucleotide sequence to be synthesized; the common nucleotide sequence 1 is shown in SEQ ID NO.1, and the common nucleotide sequence 2 is shown in SEQ ID NO.

2.

5. The construction method according to claim 1, characterized in that, In step S3, the large-scale parallel synthesis of oligonucleotides is performed using the TWIST or CustomArray platform.

6. The construction method according to claim 1, characterized in that, In step S4, the PCR is an extension PCR; the required DNA polymerase is a hot-start high-fidelity DNA polymerase. And / or, in step S4, the restriction endonuclease includes restriction endonuclease A and restriction endonuclease B, wherein restriction endonuclease A and restriction endonuclease B are different types of restriction endonucleases and can produce different sticky ends after digestion.

7. The construction method according to claim 6, characterized in that, In step S4, the obtained PCR product includes the following sequence: (1) The nucleotide sequence corresponding to the small peptide sequence; (2) The upstream of the nucleotide sequence corresponding to the small peptide contains the recognition sequence of restriction endonuclease A and protective bases; (3) Downstream of the nucleotide sequence corresponding to the small peptide, the same reading frame contains the nucleotide sequence corresponding to the tag sequence, wherein the tag includes any one of FLAG, HA, oligohistidine or myc; (4) Downstream of the tag sequence is a sequence containing a stop codon in all three reading frames; (5) Recognition sequence of restriction endonuclease B and its protective bases.

8. The construction method according to claim 1, characterized in that, In step S5, the display system includes a T7 phage display system, a λ phage display system, an Escherichia coli display system, a yeast display system, a cell display system, or a ribosome display system.

9. A small peptide display library of Mycobacterium tuberculosis, characterized in that, It is constructed using the construction method described in any one of claims 1-8.

10. The application of a Mycobacterium tuberculosis small peptide display library as described in claim 9 in screening serum biomarkers, identifying drug targets, identifying protein interactions, identifying small peptides, or in vaccine development.

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