WO2024027083A1 - Procédé d'immuno-pcr quantitative multiple médié par l'affichage de phages et phage recombiné à cet effet - Google Patents

Procédé d'immuno-pcr quantitative multiple médié par l'affichage de phages et phage recombiné à cet effet Download PDF

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WO2024027083A1
WO2024027083A1 PCT/CN2022/141387 CN2022141387W WO2024027083A1 WO 2024027083 A1 WO2024027083 A1 WO 2024027083A1 CN 2022141387 W CN2022141387 W CN 2022141387W WO 2024027083 A1 WO2024027083 A1 WO 2024027083A1
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phage
sequence
recombinant phage
recombinant
rbd
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Chinese (zh)
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王建勋
陈汉祎
李申
王伽利
和似琦
雒晨祎
王栋
钱朝晖
胡克平
胡丹丹
祈方昉
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深圳北京中医药大学研究院
北京中医药大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the invention relates to the field of biological detection, and in particular to a phage display-mediated immune multiplex quantitative PCR method and its recombinant phage.
  • SARS-CoV-2 pandemic Since the emergence of coronavirus disease 2019 (COVID-19) in late 2019, the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has triggered a global public health crisis. Due to its importance in viral tropism and infectivity, the S protein has been the target of most vaccines and antibody drugs. However, as a single-stranded positive-sense RNA virus with a high mutation rate, mutations accumulated during the SARS-CoV-2 pandemic and resulted in variants with higher adaptability and potential to evade immune responses.
  • Luminex can simultaneously analyze antibodies against multiple different SARS-CoV-2 antigens, such as S1, RBD, and nucleocapsid proteins.
  • S1, RBD and nucleocapsid proteins.
  • S1, RBD nucleocapsid proteins
  • BSFA color-based barcode labeling flow cytometry
  • Phage immunoprecipitation sequencing was first reported in 2011, with phage-displayed antigen libraries encoded by synthetic oligonucleotides. Following immunoprecipitation of serum samples, deep DNA sequencing allows the quantification of antibody-dependent enrichment of each peptide. In this way, humoral immunoassays can be used for DNA sequencing, significantly increasing the throughput of the assay. However, due to the limited length of synthetic oligonucleotide libraries, this phage display method can only detect linear epitope-directed antibodies. In 2021, Stoddard et al. captured immunogenic peptides spanning the entire SARS-CoV-2 proteome in a phage-displayed antigen library. S, N, and ORF1ab were identified as highly immunogenic regions through humoral immunoassays in 19 COVID-19 patients. However, this study failed to detect an antibody response targeting the RBD region because the displayed fragment was insufficient to form an effective conformation.
  • the present invention provides a phage display-mediated immune multiplex quantitative PCR method, which is characterized in that the method includes the following steps: Step 1: Coat the capture antibody on the bottom of the well plate; Step 2: Add the blocking solution to the well plate coated with the antibody to block; Step 3: Add the recombinant phage so that the recombinant phage fully combines with the capture antibody; the antigen sequence and the sequence for probe recognition are simultaneously inserted into the recombinant phage genome, The sequence recognized by the probe is used to detect different recombinant phages at the same time, the antigen sequence is used to react with the capture antibody, and the antigen sequence is at least two or more; step 4: between the recombinant phage and the After the capture antibody is fully combined, the combined recombinant phage is lysed, and the eluate is collected as a multiplex quantitative PCR reaction template; and step 5: perform a real-time fluorescence multiplex quantitative
  • the antigen sequence is inserted between the signal peptide and structural region of the pIII protein of the phage.
  • the sequence recognized by the probe is inserted after the structural region of the phage.
  • the SARS-CoV-2 RBD sequence is inserted between the signal peptide and structural region of the pIII protein of the M13KO7 phage, and the SARS-CoV-2 RBD sequence includes wild-type and mutant sequences.
  • step 2 prepare 2% BSA with PBST as the blocking solution.
  • the invention provides a recombinant phage.
  • An antigen sequence and a sequence for probe recognition are simultaneously inserted into the recombinant phage genome.
  • the sequence for probe recognition is used to simultaneously detect different recombinant phages.
  • the antigen sequence is used for To react with the capture antibody, the antigen sequence must be at least two or more.
  • a multivalent phage display system based on M13 phage is disclosed.
  • the RBD region of SARS-CoV-2 was fused to protein III of the M13 phage and displayed on the phage surface.
  • the data from this study show that in addition to detecting antibodies targeting linear epitopes, recombinant antigens displayed on the phage surface display the correct folding based on and spatial conformational functions, including reducing the binding efficiency of existing antibodies and binding to the receptor ACE2 through mutations.
  • phage display-mediated immune multiplex quantitative PCR Pieris-mediated immune multiplex quantitative PCR (Pi-mqPCR)
  • the binding efficiency of antibodies to different SARS-CoV-2 variants can be compared in the same amplification reaction. In this way, antigen-antibody reactions can be converted into DNA detection and significantly increase the throughput of detection.
  • FIG. 1 Schematic diagram of a phage vector used to express multivalent display phage
  • Figure 2 is an electrophoresis diagram of the vector and insert
  • Figure 3 is a colony PCR electrophoresis diagram
  • Figure 4 is a schematic diagram of the enrichment results of recombinant phage in antibodies against SARS-CoV-2 and myc tag;
  • Figure 5 is a schematic diagram showing the results of RBD phage and wild-type M13KO7 being enriched by ACE2;
  • Figure 6 is a schematic diagram of the binding activity results between recombinant phages with different mutations in the RBD construction and two commercially available anti-RBD antibodies, where 6A is the R007 antibody and 6B is the R118 antibody;
  • Figure 7 is a schematic diagram showing the binding specificity results of recombinant phages from different viruses after immunoprecipitation with corresponding labeled antibodies;
  • Figure 8 is a schematic diagram showing the binding specificity results of recombinant phages after immunoprecipitation of antigens from different regions of SARS-CoV-2 with corresponding labeled antibodies;
  • Figure 9 is a schematic diagram of the enrichment results of RBD construction displayed by Pi-mqPCR detection of one polyclonal antibody (A) and three monoclonal antibodies (B-D) against wild-type SARS-CoV-2 and recombinant phages of different variants, in which 9A They are polyclonal antibodies, 9B is R007 monoclonal antibody, 9C is R118 monoclonal antibody, and 9D is MM48 monoclonal antibody.
  • Figure 10 is a schematic diagram of the enrichment results of RBD construction displayed by Pi-mqPCR detection of 6 anti-SARS-CoV-2 RBD antibodies against wild-type SARS-CoV-2 and recombinant phages of different variants.
  • the most potent neutralizing antibodies against SARS-CoV-2 are those directed against the receptor-binding domain (RBD) of the spike protein.
  • the phage vector sequence and the SARS-CoV-2 RBD (hereinafter referred to as RBD) sequence were amplified by PCR, and the RBD sequence was inserted between the pIII protein signal peptide and structural region of the M13KO7 phage by homologous recombination.
  • RBD SARS-CoV-2 RBD
  • a sequence Probe1 that can be recognized by the probe is inserted into the phage genome, see Figure 1.
  • phage display-mediated immune multiplex quantitative PCR can compare the binding activities of recombinant phages displaying different antigens in the same amplification reaction.
  • Short DNA sequences such as the probes used in Pi-mqPCR or synthetic barcodes, are inserted into the genome of the M13 phage and used to measure phage numbers.
  • phages with specific barcodes can be used for immunoprecipitation with human sera before and after vaccination.
  • high-throughput DNA sequencing can analyze the enrichment of phage DNA to measure the humoral immune response in individual samples. Therefore, combined with high-throughput DNA sequencing technology, this phage display system can be further applied to monitor the humoral immune response of a large number of people before and after vaccination. After transformation, sequencing verification, and expanded culture, the recombinant phage multivalently displaying the corresponding antigen fragment was obtained using the PEG-NaCl precipitation method.
  • the primer sequences were all synthesized as dry powder by Ascenda Company. After centrifugation at 4000 r/min for 1 min, a certain amount of enzyme-free water was added to prepare a 10 ⁇ M working solution.
  • the sequences of each primer are shown in Table 1 below.
  • M13KO7 helper phage genome containing c-myc tag Take the commercially purchased M13KO7 helper phage genome containing c-myc tag as a template, and design vector universal primers M13KO7-1-F, M13KO7-2-R and primers containing Probe sequences: M13KO7-Probe-1-F, M13KO7 -Probe-1-R.
  • the amplification system is shown in Table 2, and the amplification conditions are shown in Table 3.
  • the Probe sequence was successfully amplified in the vector fragment.
  • the sizes of M13KO7-P1-1, M13KO7-P1-2, and M13KO7-RBD were 4393bp, 4363bp, and 614bp respectively, consistent with the expected sizes.
  • the homologous recombination kit uses a homologous recombination kit to perform homologous recombination between the phage vector and the corresponding antigen to obtain the genome of the recombinant phage containing different probe sequences.
  • the homologous recombination system is shown in Table 6. The conditions are 50°C, 30 minutes.
  • the size of the colony PCR products of clones 1, 2, 4, 6, 7, 8, and 9 is 836 bp, which is consistent with the expected size and can be used for subsequent sequencing experiments.
  • c-myc antibody as the primary antibody (1:1000 dilution) into the incubation box, place it on a rocker shaker for 5 minutes to mix the antibody evenly, and move it to a 4°C refrigerator for overnight incubation.
  • the next day after washing the membrane three times with TBST solution, use goat anti-mouse polyclonal antibody as the secondary antibody (diluted 1:5000) and incubate it at 37°C for 1 hour. Wash the membrane again, mix the chromogenic solution in equal proportions and immediately develop the NC membrane. , and imaged using an imaging system.
  • Coating of protein Take the 10 ⁇ ELISA coating solution stored in the refrigerator at 4°C, return it to normal temperature and dilute it with enzyme-free water to the working concentration. Take another ACE2 protein stored at -80°C. After thawing, use a pipette to blow evenly. Dilute with 1 ⁇ coating solution and mix by inverting or vortexing. Take an appropriate amount of the detachable high-adsorption ELISA 96-well plate, add 50 ⁇ L of diluted coating protein to each well, ensure that the capture antibody sinks to the bottom of the well, attach a sealing film, and coat the well plate in a 4°C refrigerator overnight.
  • Blocking of the well plate The next day, prepare 2% (2g/100mL) BSA with PBST as the blocking solution. Take the coated well plate, pour out the capture antibody that is not coated on the well plate, add 300 ⁇ L of PBST buffer to each well, wash the plate 5 times, and pat dry each time to avoid cross-wells. After washing the plate, add 300 ⁇ L of blocking solution to each well, attach the sealing film, and place it in a 37°C constant-temperature incubator for blocking for 2 hours.
  • qPCR sample preparation After the binding is completed, pour out the unbound recombinant phage in the well plate, wash the plate 10 times with PBST to reduce non-specific binding as much as possible, add 100 ⁇ L of enzyme-free water to each well, and heat at 95°C for 15 minutes. The phage is fully lysed, and the eluate is collected as a qPCR template.
  • the recombinant phage can bind to human ACE2 protein in a concentration-dependent manner, see Figure 5.
  • the binding activity between ACE2 and phages displaying the SARS-CoV-2 RBD region suggests that the phage-displayed antigens have similar properties to proteins found on the viral membrane. Based on this result, phage-displayed antigens can be used not only to detect linear antibodies but also for some studies that require the correct structure.
  • Coating of protein Take the 10 ⁇ ELISA coating solution stored in the refrigerator at 4°C, return it to normal temperature and dilute it with enzyme-free water to the working concentration. Take another anti-RBD antibody (R007 and R118) stored at -80°C. After thawing, use a pipette to blow evenly. Dilute with 1 ⁇ coating solution and mix by inverting or vortexing. Take an appropriate amount of the removable high-adsorption ELISA 96-well plate, add 50 ⁇ L of diluted coating antibody to each well, ensure that the capture antibody sinks to the bottom of the well, attach a sealing film, and coat the well plate in a 4°C refrigerator overnight.
  • Blocking of the well plate The next day, prepare 2% (2g/100mL) BSA with PBST as the blocking solution. Take the coated well plate, pour out the capture antibody that is not coated on the well plate, add 300 ⁇ L of PBST buffer to each well, wash the plate 5 times, and pat dry each time to avoid cross-wells. After washing the plate, add 300 ⁇ L of blocking solution to each well, attach the sealing film, and place it in a 37°C constant-temperature incubator for blocking for 2 hours.
  • recombinant phage According to the calculated phage titer, recombinant phage displaying four different RBD variants were diluted with PBS to the same titer. Remove the well plate after incubation, pour out the unbound protein solution in the well plate, wash the plate five times with PBST, add 50 ⁇ L of diluted recombinant phage to the well plate, seal the plate with a membrane, and place it in a 37°C constant temperature incubator. 1h to allow the recombinant phage to fully bind to the protein to be tested.
  • qPCR sample preparation After the binding is completed, pour out the unbound recombinant phage in the well plate, wash the plate 10 times with PBST to reduce non-specific binding as much as possible, add 100 ⁇ L of enzyme-free water to each well, and heat at 95°C for 15 minutes. The phage is fully lysed, and the eluate is collected as a qPCR template.
  • NTD N-terminal domain
  • N protein the C-terminal truncated version of the nucleocapsid protein
  • H3N2 the hemagglutinin HA1 subunit of influenza viruses A/Perth/16/2009
  • H1N1 the hemagglutinin HA1 subunit of influenza viruses A/Perth/16/2009
  • H1N1 the hemagglutinin HA1 subunit of influenza viruses A/Perth/16/2009
  • H1N1N1 the hemagglutinin HA1 subunit of influenza viruses A/Perth/16/2009
  • H1N1 A/WSN/1933
  • Blocking of the well plate The next day, prepare 2% (2g/100mL) BSA with PBST as the blocking solution. Take the coated well plate, pour out the capture antibody that is not coated on the well plate, add 300 ⁇ L of PBST buffer to each well, wash the plate 5 times, and pat dry each time to avoid cross-wells. After washing the plate, add 300 ⁇ L of blocking solution to each well, attach the sealing film, and place it in a 37°C constant-temperature incubator for blocking for 2 hours.
  • qPCR sample preparation After the binding is completed, pour out the unbound recombinant phage in the well plate, wash the plate 10 times with PBST to reduce non-specific binding as much as possible, add 100 ⁇ L of enzyme-free water to each well, and heat at 95°C for 15 minutes. The phage is fully lysed, and the eluate is collected as a qPCR template.
  • Coating of protein Take the 10 ⁇ ELISA coating solution stored in the refrigerator at 4°C, return it to normal temperature and dilute it with enzyme-free water to the working concentration. In addition, take four commercial new coronavirus antibodies and six nanobodies stored at -80°C. After thawing, use a pipette to blow evenly. Use 1 ⁇ coating solution to dilute to the corresponding concentration, mix by inverting or vortexing. Take an appropriate amount of the detachable high-adsorption ELISA 96-well plate, add 50 ⁇ L of diluted coating protein to each well, ensure that the capture antibody sinks to the bottom of the well, attach a sealing film, and coat the well plate in a 4°C refrigerator overnight.
  • Blocking of the well plate The next day, prepare 2% (2g/100mL) BSA with PBST as the blocking solution. Take the coated well plate, pour out the capture antibody that is not coated on the well plate, add 300 ⁇ L of PBST buffer to each well, wash the plate 5 times, and pat dry each time to avoid cross-wells. After washing the plate, add 300 ⁇ L of blocking solution to each well, attach the sealing film, and place it in a 37°C constant-temperature incubator for blocking for 2 hours.
  • qPCR sample preparation After the binding is completed, pour out the unbound recombinant phage in the well plate, wash the plate 10 times with PBST to reduce non-specific binding as much as possible, add 100 ⁇ L of enzyme-free water to each well, and heat at 95°C for 15 minutes. The phage is fully lysed, and the eluate is collected as a qPCR template.
  • the six anti-SARS-CoV-2 RBD Nanobodies had a greater decrease in binding activity to RBD from different variants, especially the omicron variant, see Figure 10. These results are consistent with previous studies showing that mutations in the RBD region reduce the binding efficiency of existing antibodies by changing the spatial structure of the RBD [26-30], and that omicron variants exhibit neutralizing effects on monoclonal and convalescent plasma antibodies. produce the greatest resistance [31]. Based on these data, the phage display antigen system can be used to evaluate the binding ability of antibodies to different SARS-CoV-2 variants.

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Abstract

Procédé d'immuno-PCR quantitative multiple médié par l'affichage de phages. Le procédé comprend les étapes suivantes : revêtement du fond des puits d'une plaque à puits avec un anticorps de capture ; ajout d'une solution de blocage à la plaque à puits revêtue de l'anticorps pour le blocage ; ajout d'un phage recombiné, de sorte que le phage recombiné se lie entièrement à l'anticorps de capture ; après que le phage recombiné se soit entièrement lié à l'anticorps de capture, lyse du phage recombiné lié ; collecte d'un éluat pour servir de matrice de PCR quantitative multiple ; et réalisation d'une réaction PCR quantitative multiple fluorescente en temps réel.
PCT/CN2022/141387 2022-08-01 2022-12-23 Procédé d'immuno-pcr quantitative multiple médié par l'affichage de phages et phage recombiné à cet effet WO2024027083A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1485432A (zh) * 2003-07-30 2004-03-31 中国药科大学 利用噬菌体展示技术展示抗原制备抗体的方法
WO2006060969A1 (fr) * 2004-12-06 2006-06-15 Instituto Finlay. Centro De Investigación -Producción De Vacunas Y Sueros Peptides imitant les regions antigeniques du virus de l'hepatite a
CN1834655A (zh) * 2006-04-21 2006-09-20 中国科学院武汉病毒研究所 噬菌体免疫pcr检测病原的方法
CN101126175A (zh) * 2006-08-14 2008-02-20 长沙三创生物技术有限公司 一种应用噬菌体展示文库筛选鉴定单克隆抗体的方法
CN113563483A (zh) * 2021-08-09 2021-10-29 广州明药科技有限公司 噬菌体展示新冠病毒衣壳蛋白及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1485432A (zh) * 2003-07-30 2004-03-31 中国药科大学 利用噬菌体展示技术展示抗原制备抗体的方法
WO2006060969A1 (fr) * 2004-12-06 2006-06-15 Instituto Finlay. Centro De Investigación -Producción De Vacunas Y Sueros Peptides imitant les regions antigeniques du virus de l'hepatite a
CN1834655A (zh) * 2006-04-21 2006-09-20 中国科学院武汉病毒研究所 噬菌体免疫pcr检测病原的方法
CN101126175A (zh) * 2006-08-14 2008-02-20 长沙三创生物技术有限公司 一种应用噬菌体展示文库筛选鉴定单克隆抗体的方法
CN113563483A (zh) * 2021-08-09 2021-10-29 广州明药科技有限公司 噬菌体展示新冠病毒衣壳蛋白及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN HANYI, LI SHEN, WANG JIALI, HE SIQI, WANG DONG, QIAN ZHAOHUI, HU DANDAN, QI FANGFANG, HU KEPING, LUO CHENYI, WANG JIANXUN: "Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay", FRONTIERS IN MICROBIOLOGY, vol. 13, August 2022 (2022-08-01), XP093048793, DOI: 10.3389/fmicb.2022.968036 *
LI SHEN; CHEN HANYI; YU ZHUOYING; HU KEPING ; WANG JIANXUN: "Construction of an Antibody Detection Method of SARS-CoV-2 based on Phage Display Technology and Observation of its Effectiveness", SHANDONG YIYAO - SHANDONG MEDICAL JOURNAL, SHANDONG SHENG WEISHENGTING,, CN, vol. 62, no. 5, 8 April 2022 (2022-04-08), CN , pages 1 - 5, XP009552468, ISSN: 1002-266X *

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