CN115537456A - Phage Display-Mediated Immune Multiplex Quantitative PCR Method and Its Recombinant Phage - Google Patents

Abstract

The invention provides a phage display-mediated immune multiplex quantitative PCR method, the method comprising the following steps: coating the capture antibody on the well bottom of a well plate, adding a blocking solution to the well plate coated with the antibody to seal, adding recombinant phage, so that The recombinant phage is fully combined with the capture antibody, after the recombinant phage is fully combined with the capture antibody, the combined recombinant phage is lysed, the eluate is collected, and used as a multiplex quantitative PCR reaction template, and real-time fluorescence multiplex Quantitative PCR reaction. The method of the invention converts the antigen-antibody reaction into DNA detection, and significantly improves the throughput of detection.

Description

Phage Display-Mediated Immune Multiplex Quantitative PCR Method and Its Recombinant Phage

technical field

The invention relates to the field of biological detection, in particular to an immune multiple quantitative PCR method mediated by phage display and its recombinant phage.

Background technique

Since the emergence of coronavirus disease 2019 (COVID-19), 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 variants with higher fitness and possible immune response evasion were generated.

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 start of the COVID-19 pandemic, a number of serological assays based on the spike, nucleocapsid, and other proteins of SARS-CoV-2 have been developed. These assays employ 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 fluorescent 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. The 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 wild-type SARS-S protein and the Antibody levels of its variants. However, multiplexed beads and stably transfected cell lines for fluorescent immunoassays require stringent manufacturing and storage conditions, while flow cytometry-based assays will still be throughput-limited.

Phage immunoprecipitation sequencing (PhIP-Seq) was first reported in 2011, in which phage-displayed antigen libraries are encoded by synthetic oligonucleotides. Deep DNA sequencing allowed quantification of antibody-dependent enrichment of each peptide following immunoprecipitation from serum samples. In this way, humoral immunoassays can be used for DNA sequencing, significantly increasing assay throughput. 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 proteome of SARS-CoV-2 in a phage-displayed antigen library. S, N, and ORF1ab were identified as highly immunogenic regions by humoral immunoassays in 19 COVID-19 patients. However, this study failed to detect an antibody response targeting the RBD region because the displayed fragments were insufficient to form a valid 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, characterized in that the method comprises the following steps: Step 1: coating the capture antibody on the bottom of the hole of the well plate; Step 2: Adding blocking solution to the antibody-coated well plate; step 3: adding recombinant phage to fully combine the recombinant phage with the capture antibody; inserting the antigen sequence and the sequence for probe recognition into the recombinant phage genome 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 to react with the capture antibody, and the antigen sequence is at least two or more; Step 4: After the recombinant phage and the After the capture antibody is fully bound, the combined recombinant phage is lysed, and the eluate is collected as a multiplex quantitative PCR reaction template; and step 5: performing real-time fluorescence multiplex quantitative PCR reaction.

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

In one embodiment, in step 2, 2% BSA is prepared in PBST as a blocking solution.

The present invention provides a recombinant phage, wherein an antigen sequence and a sequence for probe recognition are simultaneously inserted into the genome of the recombinant phage, and the sequence recognized by the probe is used for simultaneous detection of different recombinant phages, and the antigen sequence is used for Reactive with the capture antibody, the antigen sequence is 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 in this study showed that, in addition to detecting antibodies targeting linear epitopes, recombinant antigens displayed on the phage surface displayed and spatial conformation, 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 between antibodies and different SARS-CoV-2 variants can be compared in the same amplification reaction. In this way, the antigen-antibody response can be converted to DNA detection, and the throughput of detection can be significantly improved.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the accompanying drawings that need to be used in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments recorded in the present application , those skilled in the art can also obtain other drawings based on these drawings without creative work.

Figure 1 is used to express the schematic diagram of the phage vector of multivalent display phage;

Figure 2 is the electrophoresis diagram of the vector and the insert;

Fig. 3 is colony PCR electrophoresis figure;

Figure 4 is a schematic diagram of the enrichment results of recombinant phage in antibodies against SARS-CoV-2 and myc tags;

Figure 5 is a schematic diagram showing the results of enrichment of RBD phage and wild-type M13KO7 by ACE2;

Figure 6 is a schematic diagram of the binding activity results between recombinant phages with different mutations in RBD construction and two commercially available anti-RBD antibodies, wherein 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 phage 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 Pi-mqPCR detection of a polyclonal antibody (A) and three monoclonal antibodies (B-D) to the recombinant phage display of wild-type SARS-CoV-2 and different variants of the RBD construction results, wherein 9A is polyclonal antibody, 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 Pi-mqPCR detection of the RBD construction of 6 anti-SARS-CoV-2 RBD antibodies to wild-type SARS-CoV-2 and recombinant phages of different variants.

detailed description

In order to enable those skilled in the art to better understand the technical solutions in the application, the present invention will be further described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all embodiments . Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall 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—including those used clinically and those that predominate in polyclonal sera—are antibodies against the receptor-binding domain (RBD) of the spike protein.

The phage vector sequence and 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 the structural region of the M13KO7 phage by means of homologous recombination. For the enrichment level of recombinant phage, the 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 activity of recombinant phage displaying different antigens in the same amplification reaction. Short DNA sequences, such as probes used in Pi-mqPCR or synthetic barcodes, were inserted into the genome of M13 phage and used to measure phage numbers. In high-throughput assays, phage with specific barcodes can be used for immunoprecipitation with human sera before and after inoculation. After this, high-throughput DNA sequencing can analyze the enrichment of phage DNA to measure the humoral immune response of individual samples. Therefore, combined with high-throughput DNA sequencing technology, the 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, PEG-NaCl precipitation is used to obtain recombinant phages that display the corresponding antigen fragments in a multivalent manner.

The primer sequences were all synthesized as dry powder by Anshengda Company. After centrifugation at 4000r/min for 1min, a certain amount of enzyme-free water was added to prepare a 10μM working solution. The primer sequences are shown in Table 1 below.

Table 1 Primer sequence list

1. Construction of recombinant phage genome

1. Amplification of the vector fragment containing the probe sequence

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 to amplify Probe1-containing vector fragments M13KO7-P1-1 and M13KO7 in two segments - 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 Fragment

Table 3 Amplification conditions of phage vector fragments

2. Amplification of Antigen Sequences

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

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

CCGAACATCACCAACCTGTGCCCGTTTGGCGAGGTTTTCAACGCGACCCG TTTCGCGAGCGTGTACGCGTGGAACCGTAAACGTATCAGCAACTGCGTTG CGGACTATAGCGTGCTGTACAACAGCGCGAGCTTCAGCACCTTTAAGTGC TATGGTGTGAGCCCGACCAAACTGAACGATCTGTGCTTTACCAACGTTTAC GCGGATAGCTTCGTGATTCGTGGCGACGAGGTTCGTCAGATCGCGCCGGG TCAAACCGGCAAGATTGCGGACTACAACTATAAACTGCCGGACGATTTCA CCGGTTGCGTTATCGCGTGGAACAGCAACAACCTGGATAGCAAAGTGGGT GGCAACTACAACTATCTGTACCGTCTGTTTCGTAAGAGCAACCTGAAACC GTTCGAGCGTGACATTAGCACCGAAATCTACCAGGCGGGTAGCACCCCGT GCAACGGTGTTGAGGGCTTTAACTGCTATTTCCCGCTGCAGAGCTACGGCT TCCAACCGACCAACGGTGTGGGCTATCAACCGTACCGTGTGGTTGTGCTG AGCTTTGAACTGCTGCATGCGCCG

The sequence of the RBD coding gene of SARS-COV2 with N501Y, E484K mutations:

CCGAACATCACCAACCTGTGCCCGTTTGGCGAGGTTTTCAACGCGACCCG TTTCGCGAGCGTGTACGCGTGGAACCGTAAACGTATCAGCAACTGCGTTG CGGACTATAGCGTGCTGTACAACAGCGCGAGCTTCAGCACCTTTAAGTGC TATGGTGTGAGCCCGACCAAACTGAACGATCTGTGCTTTACCAACGTTTAC GCGGATAGCTTCGTGATTCGTGGCGACGAGGTTCGTCAGATCGCGCCGGG TCAAACCGGCAAGATTGCGGACTACAACTATAAACTGCCGGACGATTTCA CCGGTTGCGTTATCGCGTGGAACAGCAACAACCTGGATAGCAAAGTGGGT GGCAACTACAACTATCTGTACCGTCTGTTTCGTAAGAGCAACCTGAAACC GTTCGAGCGTGACATTAGCACCGAAATCTACCAGGCGGGTAGCACCCCGT GCAACGGTGTTAAGGGCTTTAACTGCTATTTCCCGCTGCAGAGCTACGGCT TCCAACCGACCTACGGTGTGGGCTATCAACCGTACCGTGTGGTTGTGCTGA GCTTTGAACTGCTGCATGCGCCG

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

CCGAACATCACCAACCTGTGCCCGTTTGGCGAGGTTTTCAACGCGACCCG TTTCGCGAGCGTGTACGCGTGGAACCGTAAACGTATCAGCAACTGCGTTG CGGACTATAGCGTGCTGTACAACAGCGCGAGCTTCAGCACCTTTAAGTGC TATGGTGTGAGCCCGACCAAACTGAACGATCTGTGCTTTACCAACGTTTAC GCGGATAGCTTCGTGATTCGTGGCGACGAGGTTCGTCAGATCGCGCCGGG TCAAACCGGCAAGATTGCGGACTACAACTATAAACTGCCGGACGATTTCA CCGGTTGCGTTATCGCGTGGAACAGCAACAACCTGGATAGCAAAGTGGGT GGCAACTACAACTATCGGTACCGTCTGTTTCGTAAGAGCAACCTGAAACC GTTCGAGCGTGACATTAGCACCGAAATCTACCAGGCGGGTAGCAAGCCGT GCAACGGTGTTGAGGGCTTTAACTGCTATTTCCCGCTGCAGAGCTACGGCT TCCAACCGACCAACGGTGTGGGCTATCAACCGTACCGTGTGGTTGTGCTG AGCTTTGAACTGCTGCATGCGCCG

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

CCGAACATCACCAACCTGTGCCCGTTTGGCGAGGTTTTCAACGCGACCCG TTTCGCGAGCGTGTACGCGTGGAACCGTAAACGTATCAGCAACTGCGTTG CGGACTATAGCGTGCTGTACAACAGCGCGAGCTTCAGCACCTTTAAGTGC TATGGTGTGAGCCCGACCAAACTGAACGATCTGTGCTTTACCAACGTTTAC GCGGATAGCTTCGTGATTCGTGGCGACGAGGTTCGTCAGATCGCGCCGGG TCAAACCGGCAAGATTGCGGACTACAACTATAAACTGCCGGACGATTTCA CCGGTTGCGTTATCGCGTGGAACAGCAACAACCTGGATAGCAAAGTGGGT GGCAACTACAACTATCAGTACCGTCTGTTTCGTAAGAGCAACCTGAAACC GTTCGAGCGTGACATTAGCACCGAAATCTACCAGGCGGGTAGCACCCCGT GCAACGGTGTTGAGGGCTTTAACTGCTATTCCCCGCTGCAGAGCTACGGCT TCCAACCGACCAACGGTGTGGGCTATCAACCGTACCGTGTGGTTGTGCTG AGCTTTGAACTGCTGCATGCGCCG

具有L452R、E484Q突变的SARS-COV2的RBD编码基因的序列(B.1.617.1,卡帕变体)：CCGAACATCACCAACCTGTGCCCGTTTGGCGAGGTTTTCAACGCGACCCG TTTCGCGAGCGTGTACGCGTGGAACCGTAAACGTATCAGCAACTGCGTTG CGGACTATAGCGTGCTGTACAACAGCGCGAGCTTCAGCACCTTTAAGTGC TATGGTGTGAGCCCGACCAAACTGAACGATCTGTGCTTTACCAACGTTTAC GCGGATAGCTTCGTGATTCGTGGCGACGAGGTTCGTCAGATCGCGCCGGG TCAAACCGGCAAGATTGCGGACTACAACTATAAACTGCCGGACGATTTCA CCGGTTGCGTTATCGCGTGGAACAGCAACAACCTGGATAGCAAAGTGGGT GGCAACTACAACTATCGGTACCGTCTGTTTCGTAAGAGCAACCTGAAACC GTTCGAGCGTGACATTAGCACCGAAATCTACCAGGCGGGTAGCACCCCGT GCAACGGTGTTcAGGGCTTTAACTGCTATTTCCCGCTGCAGAGCTACGGCT TCCAACCGACCAACGGTGTGGGCTATCAACCGTACCGTGTGGTTGTGCTG AGCTTTGAACTGCTGCATGCGCCG

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

CCCAATATCACAAACCTGTGCCCTTTTGACGAGGTGTTCAACGCAACCAG GTTCGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCCAACTGCGTGG CCGACTATTCTGTGCTGTACAACCTGGCCCCCTTCTTCACCTTTAAGTGCT ATGGCGTGAGCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTGTAC GCCGATTCCTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCAGG ACAGACAGGCAACATCGCAGACTACAATTATAAGCTGCCTGACGATTTCA CCGGCTGCGTGATCGCCTGGAACTCTAACAAGCTGGATAGCAAAGTGAGC GGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGTCTAATCTGAAGCC ATTCGAGAGGGACATCTCCACAGAGATCTACCAGGCCGGCAACAAGCCCT GCAATGGCGTGGCCGGCTTTAACTGTTATTTCCCTCTGCGGAGCTACAGCT TCCGGCCAACCTACGGCGTGGGCCACCAGCCCTACCGCGTGGTGGTGCTG TCTTTTGAGCTGCTGCACGCACCT

2.2. Take the wild type as an example. Using the plasmid as a template, design the corresponding primers M13KO7-RBD-F, 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 for subsequent agarose gel electrophoresis.

Table 4 M13KO7-RBD fragment amplification system

Table 5 M13KO7-RBD fragment amplification conditions

3. Construction of recombinant phage genomes containing probe sequences

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

As shown in Figure 2, the Probe sequence was successfully amplified in the vector fragment by designing primers. The sizes of M13KO7-P1-1, M13KO7-P1-2, and M13KO7-RBD were 4393bp, 4363bp, and 614bp, respectively, which were consistent with the expected size.

Use a clean scalpel blade to cut the target fragment gel, use a gel recovery kit to recover the DNA fragments in the gel, and measure the concentration of the recovered product with an ultramicro spectrophotometer.

Use a homologous recombination kit to perform homologous recombination between the phage vector and the corresponding antigen, and obtain the genome of the recombinant phage containing different probe sequences. The homologous recombination system is shown in Table 6, and the conditions are 50°C, 30min.

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

4. Preparation and purification of recombinant phage

4.1. Preparation of strains infected with recombinant phage

(1) Take out the DH5α competent cells from the -80°C refrigerator and place them in an ice bath, absorb 5 μL of the recombinant product and add them to 50 μL of freshly melted DH5α competent cells, use a pipette to mix gently, and put them in the ice bath for 30 minutes. The centrifuge tube was moved to a 42°C water bath for 75 seconds, and then ice-bathed again for 3 minutes. Add 700 μL of antibiotic-free SOB medium to each centrifuge tube, mix well, place in a shaker at 37°C and 200 r/min for 1 hour, and then spread the competent cells on a solid LB medium plate containing 50 μg/mL KANA , Cultivate overnight at 37°C for 12h.

(2) The next day, pick a moderate-sized monoclonal colony and add it to 800 μL of 2×YT medium containing 50 μg/mL KANA, seal the centrifuge tube containing the bacterial solution, and place it in a constant temperature shaker at 37°C. Cultivate at 200r/min for 10 hours, take the cultured bacterial solution as a template, design primers M13KO7-seq-F, M13KO7-seq-R (see Table 1) for colony PCR, see Table 7 for the system, and see Table 8 for the PCR conditions. Perform agarose gel electrophoresis on the PCR products of the colony using the above method, and use a gel imaging system for imaging to determine the bacterial solution corresponding to the PCR product with the correct size of the target fragment.

It can be seen from Figure 3 that the colony PCR products of clones 1, 2, 4, 6, 7, 8, and 9 have a size of 836 bp, which is consistent with the expected size and can be used for subsequent sequencing experiments.

The colony PCR-verified bacterial solution was expanded and cultivated overnight, and the expanded bacterial solution was extracted using a plasmid mini-extraction kit to extract the recombinant phage genome, and the concentration and A260/A280 of the extracted product were measured with an ultra-micro spectrophotometer to confirm Extraction effect.

(3) Entrust Suzhou Anshengda Company to use primers M13KO7-seq-F and M13KO7-seq-R to perform Sanger sequencing on the extracted plasmids, add 200 μL/mL glycerol to the sequenced bacteria liquid, 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

Take 10 μL of the strains with correct sequencing and add them to 10 mL of 2×YT medium containing 50 μg/mL KANA, put them in a constant temperature shaker at 37°C, and culture at 250 r/min overnight. Centrifuge the bacterial solution cultivated overnight to turbidity at 8000r/min, 4°C for 15min, carefully absorb the supernatant, and discard the precipitate. Put the supernatant into a new 15mL centrifuge tube, centrifuge again at 8000r/min, 4°C for 15min, and make sure that there is no obvious bacterial precipitation in the second centrifugation. Transfer the supernatant after twice centrifugation to a new 15mL centrifuge tube, add 2mL of PEG-NaCl solution, mix well by inverting, and ice-bath for 1h. Centrifuge the supernatant after ice bathing at 10,000 r/min, 4°C for 30 min, discard the supernatant carefully to avoid loss of phage, and use 1 mL of LPBS buffer to resuspend the white precipitate that is easily soluble in buffer attached to the tube wall. Take a 0.22μm filter membrane, filter the phage dissolved in PBS buffer with a 2mL sterile syringe, collect the filtrate with a 5mL centrifuge tube, the filtrate is the recombinant phage containing antigen fragments, and store the filtered recombinant phage at 4°C .

Example 2 Western-blot verification of fusion protein display effect

Dilute recombinant phage and wild-type M13KO7 helper phage (without c-myc tag) to 5×10 ⁸ copies/μL with PBS, add protein loading buffer, place in a boiling 100°C water bath and heat for 15 minutes to prepare samples. Take the electrophoresis buffer instant particles to prepare the electrophoresis buffer and pour it into the electrophoresis tank. Then take 30 μL of each sample and load it into the polyacrylamide electrophoresis precast gel well, add 10 μL protein marker to the well next to the sample, and perform SDS-PAGE under the condition of 160V voltage for 20min. Take out the gel, cut the appropriate size of the NC membrane and use the instant granules to prepare the transfer buffer, and perform wet transfer in the transfer tank under the condition of 100V voltage for 2h. Place the wet-transferred NC membrane in an incubation box, and wash the membrane 3 times with TBST solution, 5 min each time. Add 2 g of skim milk powder to 40 mL of TBST to prepare blocking solution. Add 10 mL of blocking solution to the washed NC membrane and place it on a rocker shaker at room temperature for blocking for 1 h. After blocking, 10 μL of c-myc antibody was added to the incubation box as the primary antibody (diluted 1:1000), placed on a rocker shaker for 5 minutes to mix the antibody evenly, and then moved to a refrigerator at 4°C for overnight incubation. The next day, after washing the membrane with TBST solution for 3 times, incubate at 37°C for 1 hour with goat anti-mouse polyclonal antibody (1:5000 dilution) as the secondary antibody, wash the membrane again, mix the chromogenic solution in equal proportions, and immediately develop the NC membrane color , and imaged using an imaging system.

The results of Western blot showed that the molecular weight of the fused recombinant protein III was about 65kDa, which was consistent with the expected size, while the protein III of wild-type M13KO7 could not bind to the anti-myc-tag antibody.

Implementing functional verification of the triplex RBD

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

1. Protein coating: Take the 10×ELISA coating solution stored in the refrigerator at 4°C, return to room temperature and dilute to the working concentration with enzyme-free water. Take another ACE2 protein stored at -80°C, and blow it evenly with a pipette after it is thawed. Dilute with 1× coating solution, mix by inversion or vortex. Take another appropriate amount of detachable high-adsorption ELISA 96-well plate, add 50 μL of diluted coating protein to each well to ensure that the capture antibody sinks to the bottom of the well, paste the sealing film, and put the well plate in a 4°C refrigerator to coat overnight.

2. Blocking of the well plate: the next day, 2% (2 g/100 mL) BSA was prepared in PBST as a 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 stringing wells. After washing the plate, add 300 μL of blocking solution to each well, stick the plate sealing film, and place in a constant temperature incubator at 37°C for 2 hours to seal.

3. Addition of recombinant phage: According to the calculated phage titer, the recombinant phage and wild-type M13KO7 phage were diluted to an appropriate titer with PBS. Take out 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 the diluted recombinant phage to the well plate, seal the plate film, and put it in a 37°C constant temperature incubator 1h, fully combine the recombinant phage with 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 enzyme-free water to each well, and heat at 95°C for 15 minutes The phage was fully lysed, and the eluate was collected as a template for qPCR.

5. Real-time fluorescence quantitative PCR: design primers QPCR-SYBR-F, QPCR-SYBR-R (see Table 1), carry out qPCR reaction with SYBR as dye, system is shown in Table 9, reaction conditions are shown in Table 10, record after the reaction CT value, copy number was calculated according to the standard curve.

Table 9 SYBR qPCR system

Table 10 SYBR qPCR reaction conditions

The results of real-time fluorescent quantitative PCR showed that the two microwell plates 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 phage and can be recognized by antibodies.

Compared with M13KO7, the recombinant phage can bind to human ACE2 protein in a concentration-dependent manner, see Figure 5. The binding activity of ACE2 to phages displaying the SARS-CoV-2 RBD region indicated that the phage-displayed antigens had similar properties to proteins found on the viral membrane. Based on this result, phage-displayed antigens can be used not only for detection of linear antibodies, but also for some studies that require the correct structure.

(ii) We further investigated whether mutations in the recombinant RBD would affect the recognition of anti-RBD antibodies. We introduced mutants with L452R, T478K mutation (B.1.617.2, delta variant), L452Q, F490S mutation (C37, lambda variant), L452R, E484Q mutation (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-immunoprecipitate with four recombinant phages separately, and the enrichment degree of corresponding phages was detected by quantitative PCR.

1. Protein coating: Take the 10×ELISA coating solution stored in the refrigerator at 4°C, return to room temperature and dilute to the working concentration with enzyme-free water. Take another anti-RBD antibody (R007 and R118) stored at -80°C, and after it is thawed, blow it evenly with a pipette gun. Dilute with 1× coating solution, mix by inversion or vortex. Take another appropriate amount of detachable high-adsorption ELISA 96-well plate, add 50 μL of diluted coating antibody to each well to ensure that the capture antibody sinks to the bottom of the well, paste the sealing film, and put the well plate in a 4°C refrigerator to coat overnight.

2. Blocking of the well plate: the next day, 2% (2 g/100 mL) BSA was prepared in PBST as a 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 stringing wells. After washing the plate, add 300 μL of blocking solution to each well, stick the plate sealing film, and place in a constant temperature incubator at 37°C for 2 hours to seal.

3. Addition of recombinant phages: According to the calculated phage titers, recombinant phages displaying four different RBD variants were diluted with PBS to the same titer. Take out 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 the diluted recombinant phage to the well plate, seal the plate film, and put it in a 37°C constant temperature incubator 1h, fully combine the recombinant phage with 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 enzyme-free water to each well, and heat at 95°C for 15 minutes The phage was fully lysed, and the eluate was collected as a template for qPCR.

5. Real-time fluorescent quantitative PCR: design primers QPCR-SYBR-F and QPCR-SYBR-R, and carry out qPCR reaction with SYBR as dye. The system is shown in Table 9, and the reaction conditions are shown in Table 10. After the reaction, record the CT value. According to the standard The curve calculates the 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 reduced the binding efficiency between recombinant phage and anti-RBD antibodies, especially L452R, T478K and N501Y , E484K mutation, see Figure 6. These data suggest that the recombinant RBD displayed on the surface of phage has more functions similar to its original structure than the linear peptide.

Example Four 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 recognition ability of the system to different antibodies, we constructed the N-terminal domain (NTD) displaying SARS-CoV-2, S protein ), the C-terminal truncated version of nucleocapsid protein (N protein), and the hemagglutinin HA1 subunit of influenza virus A/Perth/16/2009 (H3N2) and A/WSN/1933 (H1N1) to construct recombinant phage. After coating the microplates with the corresponding antibodies, equal amounts of different recombinant phages were mixed for immunoprecipitation.

1. Protein coating: Take the 10×ELISA coating solution stored in the refrigerator at 4°C, return to room temperature and dilute to the working concentration with enzyme-free water. Another target, the N-terminal domain (NTD) of the S protein, the C-terminal truncated version of the nucleocapsid protein (N protein), and the influenza virus A/Perth/16/2009 (H3N2) and A /WSN/1933 (H1N1) commercial antibody against the hemagglutinin HA1 subunit, after thawing, use a pipette to mix well. Dilute with 1× coating solution, mix by inversion or vortex. Take another appropriate amount of detachable high-adsorption ELISA 96-well plate, add 50 μL of diluted coating protein to each well to ensure that the capture antibody sinks to the bottom of the well, paste the sealing film, and put the well plate in a 4°C refrigerator to coat overnight.

2. Blocking of the well plate: the next day, 2% (2 g/100 mL) BSA was prepared in PBST as a 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 stringing wells. After washing the plate, add 300 μL of blocking solution to each well, stick the plate sealing film, and place in a constant temperature incubator at 37°C for 2 hours to seal.

3. Addition of recombinant phages: According to the calculated phage titers, equal amounts of three different recombinant phages were mixed and diluted with PBS to an appropriate titer. Take out 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 the diluted recombinant phage to the well plate, seal the plate film, and put it in a 37°C constant temperature incubator 1h, fully combine the recombinant phage with 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 was fully lysed, and the eluate was collected as a template for qPCR.

5. Multiplex real-time fluorescent quantitative PCR: design primers and probe sequences in Table 11, perform qPCR reaction with SYBR as dye, see Table 12 for the system, and see Table 13 for the reaction conditions, record the CT value after the reaction, and calculate the copy according to the standard curve number.

Table 11 Multiplex PCR primers 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 were only enriched in microwell plates coated with corresponding labeled antibodies, see Figure 7 and Figure 8. This result suggests that the system can be used to identify 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 system has been used for phage display selection; compared with this, multivalent display system can reduce non-specific binding, which is more advantageous in diagnostic analysis. In this study, our system could 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, which is more suitable for clinical serological diagnosis.

(2) Next, we verified whether the Pi-mqPCR system can identify the immune escape responses produced by different mutant strains of the new coronavirus. Using the same primers M13KO7-RBD-F and M13KO7-RBD-R, we constructed RBD regions displaying delta variant (B.1.617.2), omicron variant (B.1.1.529) and mutations of N501Y, E484K 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. Protein coating: Take the 10×ELISA coating solution stored in the refrigerator at 4°C, return to room temperature and dilute to the working concentration with enzyme-free water. Take another four kinds of commercial COVID-19 antibodies and six kinds of nanobodies stored at -80°C, and blow them evenly with a pipette after they are thawed. Dilute to the corresponding concentration with 1× coating solution, mix by inversion or vortex. Take another appropriate amount of detachable high-adsorption ELISA 96-well plate, add 50 μL of diluted coating protein to each well to ensure that the capture antibody sinks to the bottom of the well, paste the plate sealing film, and place the well plate in a 4°C refrigerator to coat overnight.

2. Blocking of the well plate: the next day, 2% (2 g/100 mL) BSA was prepared in PBST as a 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 stringing wells. After washing the plate, add 300 μL of blocking solution to each well, stick the plate sealing film, and place in a constant temperature incubator at 37°C for 2 hours to seal.

3. Addition of recombinant phages: According to the calculated phage titers, the four recombinant phages were mixed in equal amounts and diluted to an appropriate titer. Take out 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 the diluted recombinant phage to the well plate, seal the plate film, and put it in a 37°C constant temperature incubator 1h, fully combine the recombinant phage with 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 enzyme-free water to each well, and heat at 95°C for 15 minutes The phage was fully lysed, and the eluate was collected as a template for qPCR.

5. Multiplex real-time fluorescent quantitative PCR: design primers and probe sequences in Table 1, perform qPCR reaction using SYBR as dye, see Table 14 for the system, and see Table 13 for reaction conditions. After the reaction, record the CT value and calculate the copy according to the standard curve number.

Table 14 Probe method multiplex qPCR system

We observed that all four types of recombinant phage could bind polyclonal antibody T62 in a concentration-dependent manner. However, recombinant phage displaying RBD mutants showed a greater decrease in enrichment compared to wild type, see Figure 9A). Interestingly, co-immunoprecipitation with three monoclonal antibodies targeting the RBD region revealed significant differences in the enrichment of recombinant phages displaying the RBD of wild-type and different SARS-CoV-2 variants. Recombinant phage displaying the RBD of the delta variant were still recognized by antibodies R007 and R118 in a concentration-dependent manner. However, recombinant phage displaying RBD with N501Y and E484K point mutations could only bind R007 and MM48 at high concentrations, while recombinant phage displaying omicron's RBD region showed little enrichment for all three mAbs ( Figure 9B-D).

Similar to the monoclonal antibodies, the six anti-SARS-CoV-2 RBD Nanobodies showed a greater decline in RBD binding activity from different variants, especially the omicron variant, see Figure 10. These results are consistent with previous studies that mutations in the RBD region reduce the binding efficiency of existing antibodies by altering the spatial structure of the RBD [26-30], whereas omicron variants exhibit neutralizing effects on monoclonal and convalescent plasma antibodies out of the maximum resistance [31]. Based on these data, the phage display antigen system can be used to assess 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. Such equivalents are also covered by the appended claims.

Claims (9)

1. A phage display-mediated multiplex quantitative PCR method for immunization, characterized in that said method comprises the following steps:

step 1: coating the capture antibody on the bottom of the hole plate;

step 2: adding the hole plate coated with the antibody into a sealing liquid for sealing;

and 3, step 3: adding recombinant phage such that the recombinant phage binds well to the capture antibody; the recombinant phage genome is inserted with antigen sequences and sequences for probe recognition, the sequences for probe recognition are used for detecting different recombinant phage simultaneously, the antigen sequences are used for reacting with the capture antibody, and the antigen sequences are at least two or more;

and 4, step 4: after the recombinant phage is fully combined with the capture antibody, the combined recombinant phage is cracked, and eluent is collected to be used as a multiple quantitative PCR reaction template;

and 5: and carrying out real-time fluorescent multiplex quantitative PCR reaction.

2. The method of claim 1, wherein the antigenic sequence is inserted between a pIII protein signal peptide and a structural region of the bacteriophage.

3. The method according to claim 1, wherein the sequence recognized by the probe is inserted after the structural region of the bacteriophage.

4. The method of any one of claims 1 to 3, wherein a SARS-CoV-2RBD sequence is inserted between the pIII protein signal peptide and the structural region of M13KO7 phage, said SARS-CoV-2RBD sequence comprising wild type and mutant sequences.

5. The method according to any one of claims 1 to 3, wherein in step 2, 2% BSA is prepared in PBST as a blocking solution.

6. A recombinant bacteriophage characterized in that an antigen sequence and a sequence for probe recognition are inserted into the genome of the recombinant bacteriophage simultaneously, the sequence for probe recognition is used for detecting different recombinant bacteriophages simultaneously, the antigen sequence is used for reacting with the capture antibody, and the antigen sequence is at least two or more.

7. The recombinant bacteriophage of claim 6, wherein said antigen sequence is inserted between a pIII protein signal peptide and a structural region of said bacteriophage.

8. The recombinant bacteriophage of claim 6, wherein said probe-recognized sequence is inserted after a structural region of said bacteriophage.

9. The recombinant bacteriophage of claim 6, wherein a SARS-CoV-2RBD sequence is inserted between the piii protein signal peptide and the structural region of the M13KO7 bacteriophage, said SARS-CoV-2RBD sequence comprising wild type and mutant sequences.

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