WO2024027083A1

WO2024027083A1 - Phage display-mediated immune multiple quantitative pcr method and recombinant phage therefor

Info

Publication number: WO2024027083A1
Application number: PCT/CN2022/141387
Authority: WO
Inventors: 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
Original assignee: 深圳北京中医药大学研究院; 北京中医药大学
Priority date: 2022-08-01
Filing date: 2022-12-23
Publication date: 2024-02-08
Also published as: CN115537456A

Abstract

A phage display-mediated immune multiple quantitative PCR method. The method comprises the following steps: coating the bottom of wells of a well plate with a capture antibody; adding a blocking solution to the well plate coated with the antibody for blocking; adding a recombinant phage, so that the recombinant phage fully binds to the capture antibody; after the recombinant phage fully binds to the capture antibody, lysing the bound recombinant phage; collecting an eluate to serve as a multiple quantitative PCR template; and performing a real-time fluorescent multiple quantitative PCR reaction.

Description

Phage display-mediated immune multiplex quantitative PCR method and its recombinant phage

Technical field

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.

Background technique

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.

In the context of increasing vaccination rates and the emergence of variants, assessing the humoral immunity status of the population against different variants and adjusting countermeasures will play an important role in combating the spread of the virus. Since the beginning of the COVID-19 pandemic, a number of serological tests based on the spike, nucleocapsid, and other proteins of SARS-CoV-2 have been developed. These assays use different techniques such as ELISA, lateral flow immunoassay (LFIA), and chemiluminescent enzyme immunoassay (CLIA). However, most of these techniques only allow singleplex detection of antibody levels targeting a certain protein. Based on fluorescence immunoassays, Luminex can simultaneously analyze antibodies against multiple different SARS-CoV-2 antigens, such as S1, RBD, and nucleocapsid proteins. In 2021, Niklas et al. Wild-type (WT) or mutant S protein was transfected into the Ramos human B lymphoma cell line, and a color-based barcode labeling flow cytometry (BSFA) was constructed, which can compare the expression of wild-type SARS-S protein and Antibody levels to its variants. However, multiplexed beads and stably transfected cell lines for fluorescent immunoassays require stringent production and storage conditions, while flow cytometry-based assays will still be throughput limited.

Phage immunoprecipitation sequencing (PhIP-Seq) 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.

Contents of the invention

In order to solve the above problems, 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 PCR reaction.

In one embodiment, the antigen sequence is inserted between the signal peptide and structural region of the pIII protein of the phage.

In one embodiment, the sequence recognized by the probe is inserted after the structural region of the phage.

In one embodiment, 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.

In one embodiment, in 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.

In the present invention, 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. By using phage display-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.

Description of the drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the embodiments recorded in the present application. , for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 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.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with examples. Obviously, the described embodiments are only some of the embodiments of this application, not all of them. . Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of this application.

Example 1 Construction of M13KO7-SARS-CoV-2 RBD-Probe-1 recombinant phage

The most potent neutralizing antibodies against SARS-CoV-2—both those used clinically and those dominant in polyclonal sera—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. In order to simultaneously detect different To enrich the level of recombinant phage, a sequence Probe1 that can be recognized by the probe is inserted into the phage genome, see Figure 1. Based on this structure, phage display-mediated immune multiplex quantitative PCR (Pi-mqPCR) 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. In high-throughput assays, phages with specific barcodes can be used for immunoprecipitation with human sera before and after vaccination. Following this, 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 μM working solution. The sequences of each primer are shown in Table 1 below.

Table 1 Primer sequence list

1. Construction of recombinant phage genome

1. Amplification of vector fragments containing probe sequences

1.1. 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.

1.2. Use M13KO7-1-F and M13KO7-Probe-1-R pairing, M13KO7-Probe-1-F and M13KO7-2-R pairing respectively to amplify Probe1-containing vector fragments M13KO7-P1-1 and M13KO7 in two sections. -P1-2.

The amplification system is shown in Table 2, and the amplification conditions are shown in Table 3.

Table 2 Amplification system containing Probe vector fragments

Table 3 Phage vector fragment amplification conditions

2. Amplification of antigen sequences

2.1 Check the RBD antigen sequence from the literature as shown below (5’-3’) and entrust Anhui General Biotechnology Company to synthesize the puc57 plasmid corresponding to the antigen sequence.

The sequence of the RBD encoding gene of SARS-COV2 wild type:

The sequence of the RBD encoding gene of SARS-COV2 with N501Y and E484K mutations:

Sequence of the RBD encoding gene of SARS-COV2 with L452R and T478K mutations (B.1.617.2, delta variant):

Sequence of the RBD encoding gene of SARS-COV2 with L452Q and F490S mutations (C37, lambda variant):

Sequence of the RBD encoding gene of SARS-COV2 with L452R and E484Q mutations (B.1.617.1, kappa variant):

Sequence of the RBD encoding gene of the SARS-COV2 Omicron variant (B.1.1.529):

2.2. Take the wild type as an example. Using the plasmid as a template, design the corresponding primers M13KO7-RBD-F and M13KO7-RBD-R containing homology arms, and PCR amplify the antigen sequence M13KO7-RBD; the amplification system is shown in Table 4, and the PCR amplification reaction conditions are shown in Table 5. The PCR product obtained from the reaction was stored at 4°C until subsequent agarose gel electrophoresis.

Table 4 M13KO7-RBD fragment amplification system

Table 5 M13KO7-RBD fragment amplification conditions

3. Construction of recombinant phage genome containing probe sequence

Add the PCR products of M13KO7-P1-1, M13KO7-P1-2, and antigen fragment M13KO7-RBD to 10 μL of 6× Loading Buffer to prepare a 1% agarose gel (containing 0.6 μL of ethidium bromide/10mL), and proceed The conditions were 150V voltage, 30 minutes of agarose gel electrophoresis, and then a gel imaging system was used to confirm the electrophoresis agarose gel electrophoresis. The results are shown in Figure 2.

As shown in Figure 2, by designing primers, 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.

Use a clean surgical blade to cut the gel of the desired fragment, use a gel recovery kit to recover the DNA fragments in the gel, and use an ultramicro spectrometer to measure the concentration of the recovered product.

Use 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.

Table 6 M13KO7-RBD-Probe-1 recombinant phage genome homologous recombination system

4. Preparation and purification of recombinant phage

4.1. Preparation of bacterial strains infected with recombinant phage

(1) Take out the DH5α competent cells from the -80°C refrigerator and place them in an ice bath. Add 5 μL of the recombinant product to 50 μL of the newly melted DH5α competent cells. Use a pipette to gently mix evenly and let stand in the ice bath for 30 minutes. Move the centrifuge tube to a 42°C water bath and place it for 75 seconds, and then keep it in ice bath for 3 minutes again. Add 700 μL of antibiotic-free SOB culture medium to each centrifuge tube, mix well, and incubate for 1 hour on a shaker at 37°C and 200 r/min. Then, spread the competent cells onto a solid LB medium plate containing 50 μg/mL KANA. , incubate overnight at 37°C for 12 hours.

(2) The next day, pick a single clone colony of appropriate size and add it to 800 μL of 2×YT medium containing 50 μg/mL KANA. Place the centrifuge tube containing the bacterial solution with a sealing film and place it in a 37°C constant-temperature shaker. Cultivate at 200 r/min for 10 hours. Use the cultured bacterial liquid as a template and design primers M13KO7-seq-F and M13KO7-seq-R (see Table 1) for colony PCR. The system is shown in Table 7 and the PCR conditions are shown in Table 8. The colony PCR products were subjected to agarose gel electrophoresis as described above, and imaged using a gel imaging system to determine the bacterial solution corresponding to the PCR product with the correct size of the target fragment.

As shown in Figure 3, 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.

Expand and culture the bacterial liquid verified by colony PCR overnight. Use the plasmid mini-prep mid-volume kit to extract the recombinant phage genome from the expanded cultured bacterial liquid. Use an ultra-micro spectrophotometer to measure the concentration and A260/A280 of the extracted product to confirm Extraction effect.

(3) Entrust Suzhou Anshengda Company to perform Sanger sequencing on the extracted plasmid using primers M13KO7-seq-F and M13KO7-seq-R. Add the correctly sequenced bacterial solution to 200 μL/mL glycerol and place it at -80°C. save.

Table 7 Colony PCR system

Table 8 Colony PCR reaction conditions

4.2. Preparation and purification of recombinant phage

Add 10 μL of each correctly sequenced strain to 10 mL 2×YT culture medium containing 50 μg/mL KANA, place it in a 37°C constant-temperature shaker, and culture at 250 r/min overnight. Centrifuge the bacterial solution that was cultured overnight until it becomes turbid at 8000r/min and 4°C for 15min. Carefully aspirate the supernatant and discard the sediment. Place the supernatant into a new 15mL centrifuge tube and centrifuge again at 8000r/min and 4°C for 15min. Make sure that there is no obvious bacterial sedimentation during the second centrifugation. Transfer the supernatant after centrifugation twice to a new 15 mL centrifuge tube, add 2 mL of PEG-NaCl solution, mix thoroughly by inverting, and keep on ice for 1 hour. Centrifuge the supernatant after ice bathing at 10000 r/min and 4°C for 30 min. Carefully discard the supernatant to avoid losing the phage. Use 1 mL of PBS buffer to resuspend the white precipitate attached to the tube wall that is easily soluble in the buffer. Take a 0.22 μm filter membrane, use a 2 mL sterile syringe to filter the phage dissolved in PBS buffer, and collect the filtrate in a 5 mL centrifuge tube. The filtrate is the recombinant phage containing the antigen fragment. Store the filtered recombinant phage at 4°C. .

Example 2 Western-blot verification of fusion protein display effect

Use PBS to dilute the recombinant phage and wild-type M13KO7 helper phage (without c-myc tag) to 5×10 ⁸ copies/μL, add protein loading buffer, and heat in a boiling 100°C water bath for 15 minutes to prepare a sample. Take the electrophoresis buffer instant particles to prepare the electrophoresis buffer and pour it into the electrophoresis tank. Then add 30 μL of each sample into the polyacrylamide electrophoresis prefabricated gel hole, add 10 μL protein marker to the gel hole next to the sample, and perform SDS-PAGE under the conditions of 160V voltage for 20 minutes. Take out the gel, cut the NC membrane of appropriate size, use instant particles to prepare transfer buffer, and perform wet transfer in the transfer tank at 100V voltage for 2 hours. Place the wet-transferred NC membrane in an incubation box, and wash the membrane three times with TBST solution, 5 minutes each time. Add 2g of skimmed milk powder to 40mL of TBST to prepare a blocking solution. Add 10 mL of blocking solution to the cleaned NC membrane, place it on a rocker shaker and block at room temperature for 1 hour. After blocking, add 10 μL of 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.

Western blot results show that the molecular weight of the fused recombinant protein III is approximately 65kDa, consistent with the expected size, while protein III of wild-type M13KO7 cannot bind to the anti-myc-tag antibody.

Implementation of functional verification of triple recombinant RBD

(A) To test the function of recombinant RBD, we coated human ACE2 protein, anti-RBD antibody and anti-myc-tag antibody in microplates and determined the enrichment of recombinant phage and wild-type M13KO7 phage after immunoprecipitation.

1. 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 μL 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.

2. 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 μL 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 μL 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.

3. Addition of recombinant phage: According to the calculated phage titer, dilute the recombinant phage and wild-type M13KO7 phage with PBS to the appropriate 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 μL 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.

4. 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 μL 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.

5. Real-time fluorescence quantitative PCR: Design the primers QPCR-SYBR-F and QPCR-SYBR-R (see Table 1), and perform a qPCR reaction using SYBR as the dye. The system is shown in Table 9. The reaction conditions are shown in Table 10. Record after the reaction is completed. CT value, copy number was calculated based on the standard curve.

Table 9 SYBR qPCR system

Table 10 SYBR qPCR reaction conditions

Real-time fluorescence quantitative PCR results showed that both microplates of anti-RBD antibody and anti-myc-tag antibody showed significant enrichment of phage DNA, see Figure 4. This result shows that the recombinant protein has been displayed on the surface of the phage and can be recognized by the antibody.

Compared with M13KO7, 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.

(2) We further studied whether mutations in recombinant RBD would affect the recognition of anti-RBD antibodies. We introduced genes harboring the L452R, T478K mutations (B.1.617.2, delta variant), L452Q, F490S mutations (C37, lambda variant), L452R, E484Q mutations (B.1.617.1, kappa variant), and N501Y , E484K mutation, which is characteristic of alpha and beta variants. Two commercially available anti-RBD antibodies (R007 and R118) were used to coat microwell plates and co-immunoprecipitated with four recombinant phages separately, and the enrichment degree of the corresponding phages was detected by quantitative PCR.

1. 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 μL 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.

3. Addition of 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 μL 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.

5. Real-time fluorescence quantitative PCR: Design primers QPCR-SYBR-F and QPCR-SYBR-R, and perform qPCR reaction using SYBR as dye. The system is shown in Table 9. The reaction conditions are shown in Table 10. After the reaction, the CT value is recorded. According to the standard Curves to calculate copy number.

After immunoprecipitation with two commercially available anti-RBD antibodies (R007 and R118), quantitative PCR showed that point mutations in the RBD region significantly reduce the binding efficiency between recombinant phage and anti-RBD antibodies, especially L452R, T478K, and N501Y , E484K mutation, see Figure 6. These data indicate that the recombinant RBD displayed on the phage surface has more functions similar to its original structure than the linear peptide fragment.

Example 4 Phage display-mediated immune multiplex quantitative PCR (Pi-mqPCR) detection

(1) In order to determine the binding specificity between the recombinant phage and different antibodies, we first verified the system's ability to recognize different antibodies. We constructed a N-terminal domain (NTD) displaying SARS-CoV-2, S protein. ), the C-terminal truncated version of the nucleocapsid protein (N protein) and the hemagglutinin HA1 subunit of influenza viruses A/Perth/16/2009 (H3N2) and A/WSN/1933 (H1N1) to construct recombinant phages. After coating the microplate with the corresponding antibodies, equal amounts of different recombinant phages were mixed for immunoprecipitation.

1. 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, the targets stored at -80°C include the N-terminal domain (NTD) of the S protein, the C-terminal truncated version of the nucleocapsid protein (N protein), and influenza viruses A/Perth/16/2009 (H3N2) and A /WSN/1933 (H1N1) commercial antibody of hemagglutinin HA1 subunit, 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 μL 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.

3. Addition of recombinant phage: According to the calculated phage titer, mix three different recombinant phages in equal amounts and dilute them with PBS to the appropriate 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 μL 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.

5. Multiplex real-time fluorescence quantitative PCR: Design the primer and probe sequences as shown in Table 11. Perform a qPCR reaction using SYBR as the dye. The system is shown in Table 12 and the reaction conditions are shown in Table 13. After the reaction, the CT value is recorded and the copies are calculated based on the standard curve. number.

Table 11 Multiplex PCR primer and probe sequences

Table 12 Probe method multiplex qPCR system

Table 13 Probe method multiplex qPCR amplification conditions

The results of Pi-mqPCR showed that all types of recombinant phages showed enrichment only in microwell plates coated with corresponding labeled antibodies, see Figures 7 and 8. This result shows that this system can be used to identify different antibodies targeting different viruses or even different domains of the same virus. In order to isolate biomolecules with high affinity, dual plasmid-assisted phage display systems have been used for phage display selection; in contrast, multivalent display systems can reduce non-specific binding and are more advantageous in diagnostic analysis. In this study, our system can identify antibodies against more than three antigens in the same multiplex real-time PCR. This method can significantly improve the specificity of detection and provide a more comprehensive picture of the humoral immune response. Compared with flow cytometry-based methods, Pi-mqPCR-based detection can be combined with nucleic acid amplification detection and is more suitable for clinical serology diagnosis.

(2) Next, we verified whether the Pi-mqPCR system could identify the immune escape responses produced by different new coronavirus mutant strains. Using the same primers M13KO7-RBD-F and M13KO7-RBD-R, we constructed an RBD region showing delta variant (B.1.617.2), omicron variant (B.1.1.529), and N501Y, E484K mutations recombinant phage and observed its binding activity with four commercially available anti-RBD antibodies (SinoBiological), including one polyclonal antibody (T62) and three monoclonal antibodies (R007, R118, and MM48). In addition, six other commercially available SARS-CoV-2 RBD antibodies (N1-N6) were also used for immunoprecipitation with recombinant phage.

1. 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 μL 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.

3. Addition of recombinant phage: According to the calculated phage titer, mix the four recombinant phages in equal amounts and dilute to the appropriate 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 μL 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.

5. Multiplex real-time fluorescence quantitative PCR: Design the primer and probe sequences as shown in Table 1. Perform a qPCR reaction using SYBR as the dye. The system is shown in Table 14 and the reaction conditions are shown in Table 13. After the reaction, the CT value is recorded and the copies are calculated based on the standard curve. number.

Table 14 Probe method multiplex qPCR system

We observed that all four types of recombinant phage could bind to polyclonal antibody T62 in a concentration-dependent manner. However, recombinant phage showing RBD mutants showed more reduced enrichment compared to wild type, see Figure 9A). Interestingly, co-immunoprecipitation results with three monoclonal antibodies targeting the RBD region showed significant differences in the enrichment of recombinant phages displaying RBDs with wild-type and different SARS-CoV-2 variants. Recombinant phage displaying delta variant RBDs were still recognized by antibodies R007 and R118 in a concentration-dependent manner. However, recombinant phage displaying the RBD with N501Y and E484K point mutations could only bind to R007 and MM48 at high concentrations, whereas recombinant phage displaying the RBD region with omicron showed almost no enrichment of all three mAbs ( Figure 9B-D).

Similar to monoclonal antibodies, 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.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also included in the appended claims.

Claims

Phage display-mediated immune multiplex quantitative PCR method, 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 blocking solution to the antibody-coated well plate 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 genome of the recombinant phage, and the sequence recognized by the probe is used to simultaneously detect different The recombinant phage, the antigen sequence is used to react with the capture antibody, and the antigen sequence is at least two or more;

Step 4: After the recombinant phage is fully combined with the capture antibody, the combined recombinant phage is lysed, and the eluate is collected as a multiplex quantitative PCR reaction template;

Step 5: Perform real-time fluorescence multiplex quantitative PCR reaction.
The method according to claim 1, characterized in that the antigen sequence is inserted between the signal peptide and the structural region of the pIII protein of the phage.
The method according to claim 1, characterized in that the sequence recognized by the probe is inserted after the structural region of the phage.
The method according to any one of claims 1-3, characterized in that the SARS-CoV-2 RBD sequence is inserted between the pIII protein signal peptide and the structural region of the M13KO7 phage, and the SARS-CoV-2 RBD sequence includes Wild-type and mutant sequences.
The method according to any one of claims 1 to 3, characterized in that, in step 2, PBST is used to prepare 2% BSA as the blocking solution.
A recombinant phage, characterized in that an antigen sequence and a sequence for probe recognition are inserted into the genome of the recombinant phage at the same time. The sequence recognized by the probe is used to detect different recombinant phages at the same time. The antigen sequence is used For reacting with the capture antibody, the antigen sequence is at least two or more.
The recombinant phage according to claim 6, characterized in that the antigen sequence is inserted between the signal peptide and structural region of the pIII protein of the phage.
The recombinant phage according to claim 6, characterized in that the sequence recognized by the probe is inserted after the structural region of the phage.
The recombinant phage according to claim 6, characterized in that the SARS-CoV-2 RBD sequence is inserted between the pIII protein signal peptide and the structural region of the M13KO7 phage, and the SARS-CoV-2 RBD sequence includes wild type and Mutant sequence.