Method for displaying nano antibody library by using escherichia coli and application thereof

Abstract

The invention discloses a nano antibody expression vector, which is characterized in that pET28a (+) plasmid is used as a vector skeleton, a nano antibody gene is connected to the downstream of a T7 promoter of the vector skeleton, and a maltose protein gene is connected or not connected between the T7 promoter and the nano antibody gene; the vector expressed by using the nano antibody expression vector can have excellent living activity without purification. The invention further provides a method for displaying the nano-antibody library by using escherichia coli based on the nano-expression vector, and compared with the existing method for displaying the nano-antibody library by using bacteriophage, the method for displaying the nano-antibody library has the advantages that the yield is high; the library diversity is higher, the amino acid loss rate is low, the efficiency is high, the cost is low, the period is short, and the specific nano antibody can be obtained without screening by a flow instrument.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for displaying a nanobody library using Escherichia coli and its application. Background Technology

[0002] Influenza A virus can be classified into four types—A, B, C, and D—based on the differences in its matrix protein and nucleoprotein. Among them, influenza A virus (IAV) infection is the most common and most dangerous form of influenza during the flu season. IAV can be further divided into several subtypes based on different combinations of HA and NA proteins. H7N9 is one such subtype, capable of infecting humans, poultry, birds, and other animals.

[0003] Prevention and control strategies against influenza viruses mainly include antiviral vaccines, antiviral drugs, antibody therapy, and novel small-molecule drugs. However, due to the high pathogenicity and rapid mutation characteristics of the H7N9 avian influenza virus, this poses certain challenges to the protective efficacy and development of vaccines. Furthermore, the virus may develop resistance to antiviral drugs, necessitating continuous monitoring of changes in viral resistance during treatment. Therefore, developing highly effective, low-immunogenic, and high-affinity antibodies is of paramount importance.

[0004] Nanobodies (Nb) have attracted widespread attention due to their unique molecular structure and small molecular weight. These antibodies lack the light chain (VL) and heavy chain variable region (VH) of conventional antibodies, containing only one heavy chain variable region (VHH) and two conventional heavy chain constant regions (CH2 and CH3 regions). Their molecular weight is approximately 15 kDa, about one-tenth that of conventional antibodies. Nanobodies exhibit great application potential in multiple fields, including disease diagnosis and treatment, due to their small molecular weight, low cost, ease of modification, high permeability, short half-life, and low immunogenicity. Currently, in the field of tumor therapy, Nb, as a targeting module in drug delivery systems, shows the potential for precise targeting of tumor cells. In cellular and molecular imaging, the small size and high affinity of Nb make it an ideal imaging probe; when combined with radionuclides or fluorescent probes, it optimizes imaging techniques.

[0005] The acquisition and screening of highly specific Nb usually requires the acquisition of nanobody libraries. Currently, phage display of nanobody libraries is commonly used. However, phage display of nanobody libraries suffers from problems such as low display level, high background noise, and artificial loss of diversity in nanobody libraries. Escherichia coli is a Gram-negative bacterium with an inner membrane (IM) and an outer membrane (OM). The presence of the outer membrane of E. coli has always been a major obstacle to the development of surface display for E. coli. Therefore, it is currently impossible to develop an effective E. coli display system targeting Ab / Nb fragments using E. coli.

[0006] Therefore, there is still a need for a highly efficient, low-cost, and short-cycle method for displaying nanobody libraries. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method for displaying nanobody libraries using Escherichia coli and its application.

[0008] The primary objective of this invention is to provide a nanobody expression vector.

[0009] A second objective of this invention is to provide the application of the above-mentioned nanobody expression vector in displaying nanobody libraries.

[0010] A third objective of this invention is to provide a method for displaying a nanobody library using Escherichia coli.

[0011] A fourth objective of this invention is to provide the application of the nanobody library constructed by the above method in screening specific nanobodies.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] A nanobody expression vector uses pET28a(+) plasmid as the vector backbone, with a nanobody gene linked downstream of the T7 promoter of the vector backbone, and a maltose-binding protein gene (MBP) linked or not linked between the T7 promoter and the nanobody gene.

[0014] Preferably, when the nanobody expression vector is linked to a maltose-binding protein gene, the maltose-binding protein gene is linked between the NcoI site and the BamHI site downstream of the T7 promoter, and the nanobody gene is linked between the BamHI site and the XhoI site downstream of the T7 promoter.

[0015] When the nanobody expression vector is not linked to the maltose-binding protein gene, the nanobody gene is linked between the NcoI site and the XhoI site downstream of the T7 promoter.

[0016] Preferably, the nucleotide sequence of the maltose-binding protein gene (MBP) shown is as shown in SEQ ID NO: 1.

[0017] More preferably, the connection is a seamless clone connection.

[0018] Preferably, the nanobody gene is the VHH gene obtained by PCR amplification after immunizing a camel with an antigen.

[0019] More preferably, the camel is an alpaca.

[0020] More preferably, the PCR amplification is nested PCR amplification.

[0021] More preferably, after immunizing camelids at least three times with the antigen, peripheral blood mononuclear cells (PBMCs) of the immunized camelids are isolated, RNA is extracted from the PBMCs and reverse transcribed into cDNA, and the VHH gene is amplified by PCR using the cDNA as a template.

[0022] More preferably, the antigen is the H7N9 avian influenza virus antigen.

[0023] More preferably, the H7N9 avian influenza virus antigen is an H7N9 inactivated vaccine.

[0024] This invention also claims protection for the use of any of the above-described nanobody expression vectors in displaying nanobody libraries.

[0025] This invention also claims protection for a method for displaying a nanobody library using Escherichia coli, comprising the following steps:

[0026] S1. Transform any of the above-described nanobody expression vectors into competent Escherichia coli to obtain a recombinant strain;

[0027] S2. The recombinant strain obtained in step S1 is cultured to OD. 450 =0.6-0.8, using IPTG as an inducer, induced at 16-18℃, 200-220r / min for 12-15h to obtain the induced recombinant strain;

[0028] S3. Perform solid-liquid separation on the recombinant strain obtained after induction in step S2, collect the bacterial cells and resuspend them to obtain a bacterial resuspension, freeze and thaw the bacterial resuspension to obtain a freeze-thawed bacterial resuspension and perform solid-liquid separation, collect the supernatant to obtain the nanobody library.

[0029] Preferably, the competent Escherichia coli in step S1 is BL21 Escherichia coli.

[0030] More preferably, the BL21 Escherichia coli is BL21(DE3) Escherichia coli.

[0031] Preferably, the conversion described in step S1 is a chemical conversion.

[0032] More preferably, the chemical conversion is carried out using CaCl2.

[0033] Preferably, the culture in step S2 is carried out using kanamycin-resistant (Kan) culture. + They were cultured in LB medium.

[0034] Preferably, the final concentration of the inducer in step S2 is 0.45–0.50 mM.

[0035] More preferably, the final concentration of the inducer in step S2 is 0.50 mM.

[0036] Preferably, step S2 involves induction at 18°C ​​and 220 r / min for 15 h.

[0037] Preferably, the solid-liquid separation method in step S3 is centrifugation.

[0038] Preferably, in step S3, resuspension is performed using PBS.

[0039] Preferably, the freeze-thaw process in step S3 is as follows: after placing the bacterial resuspension at -40 to -80°C for 30 to 60 minutes, it is transferred to 28 to 30°C and placed until completely dissolved.

[0040] More preferably, in step S3, there are at least three freeze-thaw cycles.

[0041] More preferably, the bacterial resuspension is placed at -80°C for 30 minutes, and then transferred to 30°C until completely dissolved.

[0042] This invention also claims protection for the use of nanobody libraries constructed by any of the methods described above in screening for specific nanobodies.

[0043] Preferably, the screening of specific nanobodies is based on ELISA.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention provides a nanobody expression vector using the pET28a(+) plasmid as a vector backbone. A nanobody gene is linked downstream of the T7 promoter of the vector backbone, and a maltose protein gene may or may not be linked between the T7 promoter and the nanobody gene. The vector expressed using this nanobody expression vector exhibits excellent biological activity without purification. Furthermore, based on this nanobody expression vector, a method for displaying a nanobody library using *E. coli* is provided. Compared to existing phage-based nanobody library display methods, this method offers higher library diversity, lower amino acid loss rate, higher efficiency, lower cost, shorter cycle time, and the ability to obtain specific nanobodies without flow cytometry screening. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the recombinant vector in Example 1;

[0047] Figure 2 A flowchart illustrating the soluble strategy and its screening process;

[0048] Figure 3 The images show the agarose gel electrophoresis results of the amplification and enzyme digestion products in Example 1; A is the electrophoresis result of the amplification product of pET28a-MBP-SD36; B is the electrophoresis result of the enzyme digestion product of pET28a-SD36; C is the electrophoresis result of the enzyme digestion product of pET22b-pelB-SD36; and D is the electrophoresis result of the enzyme digestion product of pET22b-pelB-MBP-SD36.

[0049] Figure 4 Figure A shows the expression verification results of the supernatant of the lysate of each recombinant strain and the precipitate of each strain in Example 1; Figure B shows the Coomassie Brilliant Blue staining results of SDS-PAGE; Figure B shows the Western Blotting detection results.

[0050] Figure 5 Figure A shows the binding activity verification results of the recombinant vector expressing nanobodies in Example 1; Figure B shows the ELISA detection results; Figure C shows the EC50 results.

[0051] Figure 6Figure A shows the feasibility test results in Example 2; Figure B shows the ELISA results of experimental group 1, positive control group 1, and negative control group 1 when H7N9-AH13-HA was used as the antigen; Figure C shows the EC50 values ​​of experimental group 1, positive control group 1, and negative control group 1 when H7N9-AH13-HA and recombinant live virus H7N9-AH13 were used as antigens; Figure D shows the ELISA results of experimental group 2 and positive control group 2; Figure E shows the ratio of positive serum to negative serum in experimental group 2; Figure F shows the ratio of positive serum to negative serum in positive control group 2.

[0052] Figure 7 This is a graph showing the results of the ELISA detection method in Example 2;

[0053] Figure 8 Figure A shows the results of H7N9 immunization of alpacas and the construction and identification of recombinant strains; Figure B shows the agarose gel electrophoresis results of PBMC cDNA amplification products; Figure C shows the agarose gel electrophoresis results of VHH-CH2 amplification products; Figure D shows the growth of the bacterial culture after dilution following resuscitation; Figure E shows the agarose gel electrophoresis results of 48 colony amplification products.

[0054] Figure 9 This is a diagram showing the results of Example 4, which displays a nanobody library based on E. coli; A represents the OD values ​​of 35 specific antibodies in different strains. 450 Scatter plot; B is a phylogenetic tree analysis of specific antibodies;

[0055] Figure 10 The image shows the agarose gel electrophoresis results of the Q1G9, Q2C4, Q2D10, Q2C6, Q2G2, and Q2H2 amplification products. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0057] The antibody Protein SD36 involved in this embodiment of the invention is: synthesizing the SD36 gene with the nucleotide sequence shown in SEQ ID NO: 3 and inducing protein expression, and collecting the induced expressed protein is the purified antibody protein SD36;

[0058] The human conventional antibody 70C3 is the 70C3 antibody shown in the prior art (DOI: 10.27043 / d.cnki.ggzyc.2023.000735);

[0059] The novel coronavirus specific antibody D1F6 is the D1F6 antibody shown in the prior art (CN117143228A);

[0060] Antibody 8852 is the influenza A virus antibody MEDI8852.

[0061] Example 1: Construction and Validation of a Prokaryotic Expression Vector for H7N9 Influenza Virus Nanoantibody

[0062] I. Experimental Methods

[0063] 1. Construction of recombinant vectors

[0064] The MBP gene, with nucleotide sequences as shown in SEQ ID NO: 1, the pelB gene, with nucleotide sequences as shown in SEQ ID NO: 2, and the SD36 gene, with nucleotide sequences as shown in SEQ ID NO: 3, were constructed. The amino acid sequence of the MBP protein encoded by the MBP gene is shown in SEQ ID NO: 4, the amino acid sequence of the pelB protein encoded by the pelB gene is shown in SEQ ID NO: 5, and the amino acid sequence of the SD36 protein encoded by the SD36 gene is shown in SEQ ID NO: 6.

[0065] Construction of the recombinant vector pET28a-SD36: The pET28a(+) plasmid and the SD36 gene with nucleotide sequences as shown in SEQ ID NO: 3 were double-digested with NcoI restriction endonuclease and XhoI restriction endonuclease, respectively, to obtain the linearized pET28a(+) plasmid and the digested SD36 gene. The two were then ligated with recombinase (Exnase II) to obtain the recombinant vector pET28a-SD36. The SD36 gene was ligated downstream of the T7 promoter of the pET28a(+) plasmid. The C-terminus of the pET28a(+) plasmid carries a 6*His tag.

[0066] Construction of the recombinant vector pET28a-MBP-SD36: The pET28a(+) plasmid and the MBP gene with the nucleotide sequence shown in SEQ ID NO: 1 were double-digested with NcoI restriction endonuclease and BamHI restriction endonuclease, respectively, to obtain the linearized pET28a(+) plasmid and the digested MBP gene. These were then ligated using Exnase II to obtain the pET28a-MBP recombinant vector. The pET28a-MBP recombinant vector and the nucleotide sequence shown in SEQ ID NO: 1 were then... The SD36 gene shown in NO:3 was double-digested with BamHI restriction endonuclease and XhoI restriction endonuclease to obtain the linearized pET28a-MBP recombinant vector and the digested SD36 gene. The two were then ligated using recombinase (Exnase II) to obtain the recombinant vector pET28a-MBP-SD36. The MBP gene and SD36 gene were sequentially ligated downstream of the T7 promoter of the pET28a(+) plasmid. The C-terminus of the pET28a(+) plasmid carries a 6*His tag.

[0067] Construction of the recombinant vector pET22b-pelB-SD36:

[0068] Using the recombinant vector pET28a-MBP-SD36 as a template, amplification was performed using pET22b-pelB-SD36-F (nucleotide sequence as shown in SEQ ID NO: 7) and pET22b-pelB-SD36-R (nucleotide sequence as shown in SEQ ID NO: 8) according to the pET22b-pelB-SD36 amplification system shown in Table 1. The amplification products were obtained. The amplification products and pET22b plasmid were digested with NcoI restriction endonucleases and XhoI restriction endonucleases to obtain linearized pET22b plasmid and digested amplification products. The plasmids were then ligated using recombinase (Exnase II) to obtain the recombinant vector pET22b-pelB-SD36.

[0069] Table 1. pET22b-pelB-SD36 amplification system

[0070] Components Added amount / μL Primer STAR max premix (2×) 25 pET28a-MBP-SD36 (template), 10 ng / μL 1.0 pET22b-pelB-SD36-F (SEQ ID NO: 7), 10 μM 1.0 pET22b-pelB-SD36-R (SEQ ID NO: 8), 10 μM 1.0 <![CDATA[ddH2O]]> Increase to 50 Total 50

[0071] PCR amplification program: 98℃, 3 min; 98℃, 10 s, 55℃, 15 s, 72℃, 5 s / kb, 35 cycles; 72℃, 3 min.

[0072] Construction of the recombinant vector pET22b-pelB-MBP-SD36:

[0073] Using the recombinant vector pET28a-MBP-SD36 as a template, pET22b-pelB-SD36-F and pET22b-pelB-SD36-R in the amplification system shown in Table 1 were replaced with pET22b-pelB-MBP-SD36-F and pET22b-pelB-MBP-SD36-R, respectively, with nucleotide sequences as shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Amplification was then performed to obtain amplification product 1. Amplification product 1 and pET22b plasmid were digested with NcoI restriction endonuclease and XhoI restriction endonuclease to obtain linearized pET22b plasmid and digested amplification product 1. These were then ligated using recombinase (Exnase II) to obtain the recombinant vector pET22b-pelB-MBP-SD36.

[0074] The structural diagrams of the recombinant vectors pET28a-SD36, pET28a-MBP-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 are shown below. Figure 1 As shown.

[0075] 2. Identification and expression verification of recombinant vectors

[0076] (1) Identification of recombinant vectors

[0077] Using the pET28a-MBP-SD36 constructed in step 1 as a template, the PCR amplification was performed according to the PCR amplification system shown in Table 2 using the T7 primer with the nucleotide sequence shown in SEQ ID NO: 11 and the T7-TER primer with the nucleotide sequence shown in SEQ ID NO: 12. The amplification products of pET28a-MBP-SD36 were collected and detected by agarose gel electrophoresis.

[0078] Table 2 PCR amplification system

[0079]

[0080]

[0081] PCR amplification program: 98℃, 3 min; 98℃, 10 s, 55℃, 15 s, 72℃, 5 s / kb, 35 cycles; 72℃, 3 min.

[0082] Using the recombinant vectors pET28a-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 obtained in step 1 as templates, each template was double-digested with NcoI and XhoI restriction endonucleases to obtain the digested products of pET28a-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36. These products were then subjected to agarose gel electrophoresis, with undigested recombinant vectors pET28a-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 serving as controls. The electrophoresis results were recorded.

[0083] (2) Expression verification of recombinant vector

[0084] The pET28a-SD36 strain obtained in step 1 was chemically transformed (CaCl2) into BL21(DE3) Escherichia coli (competent Escherichia coli) to obtain the pET28a-SD36 recombinant strain;

[0085] The pET28a-SD36 recombinant strain was inoculated into 48-well plates at a rate of 1 strain per well. Then, 2 mL of kanamycin-resistant (Kan) antibiotic was added to each well of the 48-well plate inoculated with the pET28a-SD36 recombinant strain. + LB medium was incubated at 37°C and 220 rpm until the OD of a 48-well plate was reached. 450 =0.6~0.8; Using IPTG (isopropyl-β-D-thiogalactoside) as an inducer, IPTG was added to each well of a 48-well plate until the final IPTG concentration was 0.5mM. The plate was induced and cultured at 18℃ and 220r / min for 15h to obtain the induced pET28a-SD36 recombinant strain.

[0086] The induced pET28a-SD36 recombinant strain was centrifuged at 3750 rpm for 30 min, and the bacterial cells (precipitate) were collected by solid-liquid separation. The precipitate was diluted with 500 μL of PBS to obtain a bacterial resuspension. The bacterial resuspension was subjected to three freeze-thaw cycles (the bacterial resuspension was frozen at -80℃ for 30 min, and then completely thawed at 30℃ to obtain a freeze-thaw bacterial resuspension). Solid-liquid separation was performed to obtain the pET28a-SD36 recombinant strain lysis supernatant and pET28a-SD36 recombinant strain precipitate. The lysis supernatant contained nanobodies expressed by the pET28a-SD36 recombinant strain.

[0087] Replace the pET28a-SD36 obtained in step 1 with pET28a-MBP-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 obtained in step 1, respectively, and process them according to the method shown above to obtain the lysate supernatant of pET28a-MBP-SD36 recombinant strain, the precipitate of pET28a-MBP-SD36 recombinant strain, the lysate supernatant of pET22b-pelB-SD36 recombinant strain, the precipitate of pET22b-pelB-SD36 recombinant strain, the lysate supernatant of pET22b-pelB-SD36 recombinant strain, and the precipitate of pET22b-pelB-MBP-SD36 recombinant strain, respectively.

[0088] Using the lysate supernatant of the pET28a-SD36 recombinant strain as an example, SDS-PAGE gel electrophoresis was performed for detection:

[0089] 50 μL of the lysate supernatant of the pET28a-SD36 recombinant strain was mixed with 10 μL of 6*Loading Buffer and boiled at 100℃ for 10 min to obtain the loading solution. Then, the loading solution was subjected to 10% (w / w) SDS-PAGE electrophoresis at a loading volume of 12 μL / well. The electrophoresis parameters were set to 80V. Electrophoresis was stopped when the bromophenol blue in the loading solution (containing in 6*Loading Buffer) reached the bottom of the gel, and the post-electrophoretic gel was obtained. The post-electrophoretic gel was placed in Coomassie Brilliant Blue staining solution for 1 h. After staining, it was boiled in boiling water until decolorized, and the Coomassie Brilliant Blue staining results were observed.

[0090] The supernatant of pET28a-SD36 recombinant strain lysis was replaced with the supernatant of pET28a-SD36 recombinant strain lysis, the supernatant of pET28a-MBP-SD36 recombinant strain lysis, the supernatant of pET28a-MBP-SD36 recombinant strain lysis, the supernatant of pET22b-pelB-SD36 recombinant strain lysis, the supernatant of pET22b-pelB-SD36 recombinant strain lysis, the supernatant of pET22b-pelB-MBP-SD36 recombinant strain lysis, and the supernatant of pET22b-pelB-MBP-SD36 recombinant strain lysis, and the supernatant of pET22b-pelB-MBP-SD36 recombinant strain lysis, and the supernatant of pET22b-pelB-MBP-SD36 recombinant strain lysis, respectively, and the Coomassie brilliant blue staining results were observed after the same treatment.

[0091] Western Blot analysis:

[0092] The gel obtained during SDS-PAGE gel electrophoresis was immersed in the electrotransfer solution; the polyvinylidene fluoride (PVDF) membrane was immersed in formaldehyde for 5 min to obtain the activated PVDF membrane. The membrane was then transferred at 200 mA for 90 min according to the structure of sponge pad-filter paper-gel after electrophoresis-activated PVDF membrane-filter paper-sponge pad.

[0093] After the transfer, the PVDF membrane was obtained. The membrane was washed three times with 1×PBST for 5 min each time. Then, the washed PVDF membrane was immersed in 5% (w / w) skim milk powder and blocked on a shaker for 1 h. After blocking, the membrane was washed three times with 1×PBST for 5 min each time. Then, it was immersed in primary antibody (0.01% HRP, v / v) and incubated at 4℃ for 15 h. After incubation, the membrane was washed three times with 1×PBST for 5 min each time. Then, ECL colorimetric solution was added to immerse the PVDF membrane, and it was developed and photographed in a fluorescence spectrometer to observe the results.

[0094] (3) Verification of the binding activity of nanobodies expressed on recombinant vectors

[0095] The flowchart for verifying the binding activity of nanobodies expressed on recombinant vectors is as follows: Figure 2 As shown, the details are as follows:

[0096] As shown in step (2), replace the pET28a-SD36 constructed in step 1 with pET22b-pelB-SD36 and pET22b-pelB-MBP-SD36 constructed in step 1, respectively, and perform induction culture. Centrifuge the induced pET22b-pelB-SD36 recombinant strain at 3750 rpm for 30 min and collect the supernatant to obtain the pET22b-pelB-SD36 bacterial cell LB medium. Centrifuge the induced pET22b-pelB-MBP-SD36 recombinant strain at 3750 rpm for 30 min and collect the supernatant to obtain the pET22b-pelB-MBP-SD36 bacterial cell LB medium.

[0097] The pET28a-SD36 recombinant strain lysate supernatant, pET28a-MBP-SD36 recombinant strain lysate supernatant, pET22b-pelB-SD36 recombinant strain lysate supernatant, pET22b-pelB-SD36 recombinant strain lysate supernatant, pET22b-pelB-SD36 bacterial cell LB medium and pET22b-pelB-MBP-SD36 bacterial cell LB medium obtained in step (2) were used as experimental groups. The purified antibody Protein SD36 was used as a positive control group, and the blank strain BL21(DE3) was used as a negative control group.

[0098] Using the H7N9 / AH13 strain (A / Anhui / 1 / 2013(H7N9)) as the antigen, the hemagglutination titer of the antigen was adjusted to 2. 3 The antigen was coated onto the ELISA plate at a concentration of 100 μL / well and incubated at 4°C for 16 h. The plate was then discarded, washed once with PBST (phosphate-buffered saline) at a concentration of 300 μL / well, and blotted dry. Blocking buffer (DPBS containing 5% skim milk powder) was added to the ELISA plate at a concentration of 200 μL / well, and the plate was blocked for 2 h. The blocking buffer was then discarded, and the plate was washed three times with PBST at a concentration of 300 μL / well, and blotted dry. Antibody was added to the ELISA plate at a concentration of 100 μL / well, and the plate was incubated at 37°C for 2 hours. After 1 hour, wash the plate three times with PBST at 300 μL / well and blot dry. Add secondary antibody (0.01% HRP, v / v) to the plate at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate six times with PBST at 300 μL / well and blot dry. Add TMB chromogenic solution at 50 μL / well and incubate in the dark for 15 minutes. Then, stop the reaction by adding TMB stop solution at 50 μL / well. Measure the OD of each well in the plate using a microplate reader. 450 value;

[0099] The antibodies added to the ELISA plates were substances from each experimental group, positive control group, and negative control group, with a concentration of 10 μg / mL.

[0100] II. Experimental Results

[0101] 1. Identification results of the recombinant vector:

[0102] The agarose gel electrophoresis results of the amplification products and enzyme digestion products are shown in the figure below. Figure 3 As shown, Figure 3 Figure A shows the electrophoresis results of the amplification products of pET28a-MBP-SD36. Figure 3 B shows the electrophoresis results of the pET28a-SD36 enzyme digestion products. Figure 3 Image C shows the electrophoresis results of the pET22b-pelB-SD36 enzyme digestion products. Figure 3 D is the electrophoresis result of the pET22b-pelB-MBP-SD36 enzyme digestion product.

[0103] The results show: Figure 3 In result A, a protein band appeared at 1766bp, which is consistent with the theoretical value of the specific primers (i.e., the band size of the theoretical amplification products of the T7 primer and the T7-TER primer); Figure 3 B~ Figure 3 The sizes of the electrophoretic bands in the electrophoresis results shown in D are consistent with the theoretical values ​​of the enzyme digestion products (i.e., the digestion products of NcoI restriction endonuclease and XhoI restriction endonuclease); among them Figure 3 B~ Figure 3 Lane 1 in D shows the electrophoresis results of the enzyme digestion products, and lane 2 shows the electrophoresis results of the undigested recombinant vector.

[0104] This indicates that the recombinant vectors pET28a-SD36, pET28a-MBP-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 were successfully constructed.

[0105] 2. Results of expression verification of recombinant vectors

[0106] The expression verification results of the lysate supernatant and precipitate of each recombinant strain are shown in the figure below. Figure 4 As shown, Figure 4 Image A shows the results of Coomassie Brilliant Blue staining using SDS-PAGE. Figure 3 B represents the results of the Western Blot analysis. Figure 4 A and Figure 4 Lanes 1-8 in B represent the results of the lysate supernatant of the pET28a-SD36 recombinant strain, the lysate supernatant of the pET28a-MBP-SD36 recombinant strain, the lysate supernatant of the pET22b-pelB-SD36 recombinant strain, the pellet of the pET22b-pelB-MBP-SD36 recombinant strain, the pellet of the pET28a-SD36 recombinant strain, the pellet of the pET28a-MBP-SD36 recombinant strain, the pellet of the pET22b-pelB-SD36 recombinant strain, and the pellet of the pET22b-pelB-MBP-SD36 recombinant strain, respectively. M represents the result of the marker.

[0107] The results showed that the recombinant strains constructed using the recombinant vectors pET28a-SD36, pET28a-MBP-SD36, pET22b-pelB-SD36, and pET22b-pelB-MBP-SD36 could all effectively express SD36 antibodies and could be recognized by His-tagged antibodies (HRPs).

[0108] 3. Validation results of the binding activity of nanobodies expressed on recombinant vectors

[0109] The binding activity verification results of nanobodies expressed on recombinant vectors are shown in the figure below. Figure 5 As shown, Figure 5 Figure A shows the ELISA test results for each experimental group, positive control group, and negative control group. Figure 5 B shows the EC50 results for each experimental group, positive control group, and negative control group.

[0110] The results showed that the lysate supernatant (containing crude antibody) of the recombinant strains constructed using recombinant vectors pET28a-SD36 and pET28a-MBP-SD36 exhibited strong binding activity with the H7N9 strain. Furthermore, the binding levels of the lysate supernatant (containing crude antibody) of the recombinant strains constructed using pET28a-MBP-SD36 were closer to those of the purified antibody Protein SD36. Additionally, the binding titer (EC50 value) of the lysate supernatant (containing crude antibody) of the recombinant strains constructed using pET28a-SD36 and pET28a-MBP-SD36 were closer to those of the purified antibody Protein SD36, with the lysate supernatant of the recombinant strains constructed using pET28a-MBP-SD36 showing a higher binding titer and better binding effect.

[0111] In summary, the recombinant strains constructed using the recombinant vectors pET28a-SD36 or pET28a-MBP-SD36 can effectively express nanobodies, and the expressed antibodies exhibit excellent binding activity with the virus.

[0112] Example 2: Establishment of an ELISA method based on the recombinant vector pET28a-MBP-SD36

[0113] I. Experimental Methods

[0114] 1. Feasibility test

[0115] ELISA tests were performed on experimental group 1, positive control group 1, and negative control group 1 using the pure H7N9 viral structural protein HA (hemagglutinin) H7N9-AH13-HA and the recombinant live virus H7N9 / AH13 (A / Anhui / 1 / 2013(H7N9)) as antigens, respectively.

[0116] Experimental Group 1: Using Protein SD36, a specific antibody against the H7N9 virus, as the antibody, an enzyme-linked immunosorbent assay (ELISA) was performed: the hemagglutination titer of the antigen was adjusted to 2. 3The antigen was coated onto the microplate at 100 μL / well, and after incubation at 4°C for 16 h, the antigen was discarded and the plate was washed once with PBST (phosphate buffer) at 300 μL / well, then patted dry. Blocking buffer (DPBS containing 5% skim milk powder) was added to the microplate at 200 μL / well, and after blocking for 2 h, the blocking buffer was discarded. The plate was washed three times with PBST at 300 μL / well, then patted dry. Different concentrations of Protein SD36 (initial concentration 1) were added to different wells of the microplate at 100 μL / well. 0 μg / mL (4-fold serial dilution), incubated at 37℃ for 2 h, then washed 3 times with PBST at 300 μL / well and blotted dry; added secondary antibody (0.01% HRP, v / v) at 100 μL / well to the plate, incubated at 37℃ for 1 h, then washed 6 times with PBST at 300 μL / well and blotted dry; added TMB chromogenic solution at 50 μL / well, incubated in the dark for 15 min, then stopped the reaction by adding TMB stop solution at 50 μL / well, and the OD of each well in the plate was measured using a microplate reader. 450 The EC50 value was calculated using GraphPadPrism 9.0 software.

[0117] Positive control group 1: The difference between positive control group 1 and experimental group 1 is that the antibody was replaced with human conventional antibody 70C3, while the other treatments remained the same.

[0118] Negative control group 1: The difference between negative control group 1 and experimental group 1 is that the antibody was replaced with the novel coronavirus specific antibody D1F6, while the other treatments remained the same.

[0119] 2. ELISA detection of nanobodies expressed by recombinant vectors

[0120] Using recombinant live virus H7N9 / AH13 as the antigen, ELISA was performed on experimental group 2 and positive control group 2, respectively.

[0121] Experimental Group 2: Using the lysate supernatant of the pET28a-MBP-SD36 recombinant strain obtained in step 2 of Example 1 (containing the nanobody expressed by the pET28a-MBP-SD36 recombinant strain) as the antibody, an enzyme-linked immunosorbent assay (ELISA) was performed. The ELISA detection process was the same as that shown in Experimental Group 1. The OD values ​​of each well in the ELISA plate after processing the lysate supernatant of the pET28a-MBP-SD36 recombinant strain were obtained. 450 The values ​​were calculated and the average value was used as the positive value (P0.05) of the lysate supernatant of the pET28a-MBP-SD36 recombinant strain. MBP ).

[0122] Negative Control Group 2: The difference between Negative Control Group 2 and Experimental Group 2 is that the antibody was replaced with an equal amount of PBS. The OD values ​​of each well in the ELISA plate of Negative Control Group 2 were then obtained. 450 The values ​​are calculated, and the average value is taken as the negative value (N).

[0123] Positive control group 2: The difference between positive control group 2 and experimental group 2 is that the antibody was replaced with Protein SD36, a specific antibody against the H7N9 virus, while other treatments remained unchanged. The positive value of Protein SD36 (P) was obtained. SD36 ).

[0124] Calculate the P / N values ​​for experimental group 1 and positive control group 2, respectively, where N is the negative value of negative control group 2.

[0125] 3. Investigation of ELISA detection method for nanobodies expressed by recombinant vectors

[0126] Using the lysate supernatant of the pET28a-MBP-SD36 recombinant strain obtained in step 2 of Example 1 (containing nanobodies expressed by the pET28a-MBP-SD36 recombinant strain) as an antibody, the effect of secondary antibody (HRP) dilution on ELISA results during ELISA detection was investigated:

[0127] Following the enzyme-linked immunosorbent assay (ELISA) method shown in Experimental Group 1 in Step 1, replace Protein SD36 (initial concentration of 10 μg / mL, 4-fold serial dilution) with the lysate supernatant of the pET28a-MBP-SD36 recombinant strain obtained in Step 2 of Example 1 (initial concentration of 10 μg / mL, 4-fold serial dilution, i.e., when the dilution is 1:1, the concentration is 10 μg / mL), perform the ELISA reaction, and calculate the P / N value;

[0128] In the ELISA reaction, the dilutions of the secondary antibody added to the enzyme-labeled plate were 1:5000 (0.02%, v / v), 1:10000 (0.01%, v / v), 1:20000 (0.005%, v / v), 1:40000 (0.0025%, v / v), and 1:80000 (0.00125%, v / v), respectively, to obtain the P / N value of the ELISA reaction after adding secondary antibody at different dilutions.

[0129] II. Experimental Results

[0130] Feasibility test results diagram as shown Figure 6 As shown, Figure 6 A~ Figure 6 C represents the feasibility test result shown in step 1. Figure 6 D~ Figure 6 F represents the feasibility test result shown in step 2; Figure 6Figure A shows the ELISA results of experimental group 1, positive control group 1, and negative control group 1 when H7N9-AH13-HA was used as the antigen. Figure 6 B shows the ELISA results of experimental group 1, positive control group 1, and negative control group 1 when recombinant live virus H7N9-AH13 was used as the antigen. Figure 6 C represents the EC50 values ​​of experimental group 1, positive control group 1, and negative control group 1 when H7N9-AH13-HA and recombinant live virus H7N9-AH13 were used as antigens. Figure 6 D shows the ELISA test results of experimental group 2 and positive control group 2; Figure 6 E represents the ratio of positive serum to negative serum in experimental group 2; Figure 6 F represents the ratio of positive serum to negative serum in the positive control group 2.

[0131] The results showed that Protein SD36 antibody exhibited binding activity against both H7N9-AH13-HA and recombinant live virus H7N9 / AH13, and there was no significant difference in the binding activity of Protein SD36 antibody against the two antigens. Furthermore, the EC50 values ​​of Protein SD36 antibody against H7N9-AH13-HA and recombinant live virus H7N9 / AH13 were similar, indicating that Protein SD36 has excellent binding activity against both H7N9-AH13-HA and recombinant live virus H7N9 / AH13.

[0132] The binding activity of the pET28a-MBP-SD36 recombinant strain lysate supernatant obtained in step 2 of Example 1 with the recombinant live virus H7N9 / AH13 reached over 1 at OD450nm without dilution. This indicates that the pET28a-MBP-SD36 recombinant strain lysate supernatant (nanobody expressed by pET28a-MBP-SD36 recombinant strain) has excellent binding activity for the recombinant live virus. Furthermore, the large P / N value (ratio of positive serum to negative serum) indicates that the nanobody expressed by pET28a-MBP-SD36 recombinant strain has high sensitivity.

[0133] The results of the evaluation of the ELISA detection method are shown in the figure below. Figure 7 As shown, the results indicate that the P / N values ​​of the nanobodies expressed by the pET28a-MBP-SD36 recombinant strain were relatively high at all dilutions (1:1 on the x-axis, 10 μg / mL). As the dilution of the nanobodies expressed by the pET28a-MBP-SD36 recombinant strain gradually increased to 256-fold, the HRP secondary antibody showed the highest P / N values ​​at dilutions of 1:5000 and 1:10000, and was also more stable than other dilutions.

[0134] Example 3: A method for constructing an E. coli nanobody display library expressing anti-H7N9 virus nanobodies.

[0135] I. Experimental Methods

[0136] 1. H7N9 immunization of alpacas and construction and identification of recombinant strains

[0137] Alpacas were immunized with an H7N9 inactivated vaccine, once every 28 days for a total of 3 immunizations. Seven days after the third immunization, anticoagulated blood was collected from the jugular vein of the alpacas for PBMC (peripheral blood mononuclear cells) isolation. RNA was extracted from the isolated PBMCs using the TRIzoI extraction method, and the extracted RNA was then processed using a reverse transcription kit. TM The RTreagent Kit (Perfect Real Time) was used to reverse transcribe cDNA according to the instructions to obtain the cDNA of PBMCs.

[0138] Using PBMC cDNA as a template, amplification was performed using the H1R primer with nucleotide sequence as shown in SEQ ID NO: 13 and the CALL002 primer with nucleotide sequence as shown in SEQ ID NO: 14, according to the cDNA amplification system shown in Table 3. The cDNA amplification products of PBMC were collected and detected by agarose gel electrophoresis.

[0139] Table 3 cDNA amplification system

[0140]

[0141]

[0142] PCR amplification program: 98℃, 5 min; 98℃, 10 s, 62℃, 30 s, 68℃, 30 s, 35 cycles; 68℃, 7 min.

[0143] The 600bp VHH-CH2 fragment was isolated from the cDNA amplification product of PBMC. Using the VHH-CH2 fragment as a template, amplification was performed using primers VHH-For2 (nucleotide sequence shown in SEQ ID NO: 15), VHH-For3 (nucleotide sequence shown in SEQ ID NO: 16), VHH-For4 (nucleotide sequence shown in SEQ ID NO: 17), and VHH-Rev (nucleotide sequence shown in SEQ ID NO: 18) according to the VHH-CH2 amplification system shown in Table 4. The VHH-CH2 amplification products were collected and detected by agarose gel electrophoresis.

[0144] Table 4 VHH-CH2 amplification system

[0145] Components Added amount KOD One™ PCR Master Mix 12.5 VHH-CH2 fragment (template), 50 ng / μL 1.0 VHH-For2 primers (SEQ ID NO: 15), 10 μM 0.25 VHH-For3 primers (SEQ ID NO: 16), 10 μM 0.25 VHH-For4 primers (SEQ ID NO: 17), 10 μM 0.25 VHH-Rev primers (SEQ ID NO: 18), 10 μM 0.75 <![CDATA[ddH2O]]> Increase to 25 Total 25

[0146] PCR amplification program: 98℃, 5 min; 98℃, 10 s, 62℃, 30 s, 68℃, 30 s, 35 cycles; 68℃, 7 min.

[0147] A 500bp VHH fragment was collected from the VHH-CH2 amplification product. Using the VHH fragment as a template, amplification was performed according to the VHH amplification system shown in Table 5 using the following sequences: pET28a-MBP-VHH-For-1 (SEQ ID NO: 19), pET28a-MBP-VHH-For-2 (SEQ ID NO: 20), pET28a-MBP-VHH-For-3 (SEQ ID NO: 21), and pET28a-MBP-VHH-Rev (SEQ ID NO: 22). The VHH amplification products were collected and detected by agarose gel electrophoresis. The VHH amplification product is the VHH fragment (approximately 500bp) containing homologous arms.

[0148] Table 5 VHH Amplification System

[0149] Components Added amount KOD One™ PCR Master Mix 12.5 VHH fragment (template), 50 ng / μL 1.0 pET28a-MBP-VHH-For1 primers (SEQ ID NO: 19), 10 μM 0.25 pET28a-MBP-VHH-For2 primers (SEQ ID NO: 20), 10 μM 0.25 pET28a-MBP-VHH-For3 primers (SEQ ID NO: 21), 10 μM 0.25 pET28a-MBP-VHH-Rev primers (SEQ ID NO: 22), 10 μM 0.75 <![CDATA[ddH2O]]> Increase to 25 Total 25

[0150] PCR amplification program: 98℃, 5 min; 98℃, 10 s, 62℃, 30 s, 68℃, 30 s, 35 cycles; 68℃, 7 min.

[0151] As shown in step one of Example 1, the pET28a-MBP recombinant vector was constructed. The pET28a-MBP recombinant vector was digested with NcoI restriction endonuclease and XhoI restriction endonuclease to obtain a linearized pET28a-MBP recombinant vector. The linearized pET28a-MBP recombinant vector and the VHH fragment containing homologous arms were recombinantly ligated with recombinase (Exnase II) to obtain the pET28a-MBP-VHH recombinant vector. 100 ng of the pET28a-MBP-VHH recombinant vector was chemically transformed (CaCl2) into 50 μL of BL21(DE3) Escherichia coli (competent Escherichia coli) to obtain the pET28a-MBP-VHH recombinant strain.

[0152] The pET28a-MBP-VHH recombinant strain was revived in LB medium at 37°C and 220 rpm for 1 h to obtain the revived bacterial solution. The revived bacterial solution was diluted 10 times, 100 times and 1000 times, respectively. The diluted revived bacterial solutions were plated and incubated at 37°C and 220 rpm for 1 day. The growth of the revived bacterial solutions at different dilutions was observed and recorded.

[0153] The pET28a-MBP-VHH recombinant strain was coated onto a substrate containing kanamycin-resistant (Kan) bacteria. + The culture was carried out on agar plates at 37°C for 14 hours. Forty-eight single-clone colonies that grew on the agar plates were selected. The 48 single-clone colonies were identified by PCR using primers with nucleotide sequences as shown in SEQ ID NO: 23 (VHH-F) and nucleotide sequences as shown in SEQ ID NO: 12 (T7-TER). The amplification products were then detected by agarose gel electrophoresis.

[0154] Colonies that showed a positive result in agarose gel electrophoresis (i.e., a protein band appeared at 581 bp) were selected for sequencing, and the sequencing results were recorded.

[0155] 2. E. coli-based display of nanobody libraries

[0156] Select 200 pET28a-MBP-VHH recombinant strains obtained in step 1 and inoculate them evenly into 48-well plates (containing 2 mL / well of Kans). + LB medium was used to incubate the culture at 37°C and 220 rpm until OD500. 450 =0.6~0.8, add IPTG (isopropyl-β-D-thiogalactoside) to each well to a final concentration of 0.5mM, and incubate at 18℃ for 15h. After incubation, centrifuge at 3750rpm for 30min to collect pET28a-MBP-VHH cells and resuspend in PBS to obtain pET28a-MBP-VHH bacterial resuspension. Repeat the freeze-thaw cycle of pET28a-MBP-VHH bacterial resuspension 3 times (freeze the bacterial resuspension at -80℃ for 30min, then thaw it completely at 30℃ to obtain the freeze-thawed pET28a-MBP-VHH bacterial resuspension). Separate the solid and liquid and collect the supernatant to obtain the nanobody library.

[0157] II. Experimental Results

[0158] 1. Figures showing the results of H7N9 immunization of alpacas and the construction and identification of recombinant strains. Figure 8 As shown.

[0159] Figure 8 A shows the agarose gel electrophoresis results of the cDNA amplification product of PBMC. The results show that the cDNA amplification product of PBMC contains two fragments of different sizes: the VH-CH1-CH2 fragment (approximately 900bp, conventional antibody) and the VHH-CH2 fragment (approximately 600bp, heavy chain antibody).

[0160] Figure 8B is the agarose gel electrophoresis result of the VHH-CH2 amplification product. The result shows that there is a 500bp fragment in the VHH-CH2 amplification product, which is consistent with the size of the target fragment VHH.

[0161] Figure 8 C shows the agarose gel electrophoresis results of the VHH amplification product. The results show a clear protein band at 500bp, which is similar in size to the VHH fragment containing homologous arms.

[0162] Figure 8 Figure D shows the growth of the bacterial culture after dilution following resuscitation. The results show that colonies grew in the undiluted, 10-fold, and 100-fold diluted bacterial cultures after one day of incubation. The 100-fold diluted culture produced 13 colonies after one day of incubation, while no colonies were observed in the 1000-fold diluted culture after one day of incubation. This indicates that the constructed recombinant strain can still grow after a 100-fold dilution, suggesting that its library capacity is 1.0 × 10⁻⁶. 3 .

[0163] Figure 8 E represents the agarose gel electrophoresis results of 48 colony amplification products. The results show that only 4 of the 48 colony amplification products did not show a band at 581 bp, and the library insertion rate reached 91.7%. Furthermore, the sequencing results of the monoclonal colonies that showed positive results in the agarose gel electrophoresis showed that the VHH gene could be successfully detected in each monoclonal colony, and there was no sequence duplication between different positive monoclonal colonies, indicating excellent monoclonal colony diversity.

[0164] Example 4: Method and Validation for Screening Specific Nanobodies Based on E. coli Displayed Nanobody Library

[0165] I. A method for screening specific nanobodies based on E. coli-displayed nanobody libraries

[0166] 1. Experimental Methods

[0167] The pET28a-MBP-VHH recombinant strain constructed in step 1 of Example 3 was used with Kan + The cultures were cultured in LB medium, and 200 single colonies were collected from each culture. Each single colony was then inoculated into different 48-well plates (containing 2 mL / well of Kansas). + LB medium was used to incubate the culture at 37°C and 220 rpm until OD500. 450=0.6~0.8, add IPTG (isopropyl-β-D-thiogalactoside) to each well of each ELISA plate to a final concentration of 0.5mM, incubate at 18℃ for 15h, centrifuge at 3750rpm for 30min after incubation, collect the bacterial cells and resuspend them in PBS, repeat the freeze-thaw cycle 3 times (freeze at -80℃ for 30min, then thaw completely at 30℃ to complete the freeze-thaw cycle), separate the solid and liquid and collect the supernatant to obtain the lysate supernatant of 200 single colonies (200 nanobodies).

[0168] Using H7N9 / AH13 (A / Anhui / 1 / 2013(H7N9)) as the strain, the strain was diluted with PBS to a hemagglutination titer of 2. 3 The diluted viral strain was used as antigen and coated onto the ELISA plate at 100 μL / well. The plate was incubated at 4°C for 10 h. The antigen was then discarded, and the plate was washed once with PBST (phosphate-buffered saline) at 300 μL / well, and the plate was blotted dry. Blocking buffer (DPBS containing 5% skim milk powder) was added to the ELISA plate at 200 μL / well, and the plate was blocked for 2 h. The blocking buffer was then discarded, and the plate was washed once with PBST at 300 μL / well, and the plate was blotted dry. Next, the supernatant from the lysis of 200 single-clone colonies was used as antibody, and antibody (10 μg / mL) was added to the ELISA plate at 100 μL / well, and the plate was incubated at 37°C for 2 h. The liquid in the ELISA plate was then discarded, and the plate was washed three times with PBST at 300 μL / well, and the plate was blotted dry. The Anti-His tag was then applied. The HRP antibody was diluted 1:10000 (v / v) with PBS to obtain a fluorescent secondary antibody. 100 μL / well of the fluorescent secondary antibody was added to the microplate and incubated at 37°C for 1 h. After incubation, the secondary antibody was discarded, and the plate was washed 6 times with 300 μL / well of PBST and patted dry. Then, 50 μL / well of TMB chromogenic solution was added to the microplate, and the plate was incubated at 30°C in the dark for 15 min. Finally, 50 μL / well of TMB stop solution was added to the microplate, and the OD values ​​of each well were measured using a microplate reader. 450nm value.

[0169] An antibody-free ELISA plate was used as a negative sample, and the OD value of each well in the negative sample-treated ELISA plate was detected using an ELISA reader. 450nm The average value was used to calculate the OD value of each well in the ELISA plate treated with negative samples. 450nm The average value (X) 平均 ) and standard deviation (SD), and determine OD according to Formula I. 450nm The critical value; OD in the ELISA plate 450nm Value greater than OD 450nm When the threshold value is reached, it is judged as positive and the positive enzyme-labeled plate is recorded.

[0170] Formula I: OD 450nm The critical value = X 平均 +3×SD.

[0171] Replace H7N9 / AH13(A / Anhui / 1 / 2013(H7N9)) with H7N9 / SF003(A / Guangdong / 17SF003 / 2016), perform the same treatment, and record the number of positive ELISA plates when H7N9 / SF003(A / Guangdong / 17SF003 / 2016) is used as the strain.

[0172] Single colonies from the positive ELISA plates were selected and sequenced. Duplicate single colonies from the positive ELISA plates were removed and MEGA phylogenetic tree analysis was performed.

[0173] 2. Experimental Results

[0174] Based on the E. coli display nanobody library, when using H7N9 / AH13 (A / Anhui / 1 / 2013(H7N9)) and H7N9 / SF003 (A / Guangdong / 17SF003 / 2016) as strains to screen for specific antibodies, 35 specific nanobodies were screened in both cases. The OD values ​​of these 35 specific antibodies in different strains were... 450 Scatter plot as follows Figure 9 As shown in Figure A, after removing duplicates from the 35 specific nanobodies, a total of 27 specific nanobodies were obtained; the phylogenetic tree analysis diagram of the 27 specific nanobodies is shown below. Figure 9 As shown in B.

[0175] II. Verification of the binding activity of specific nanobodies

[0176] 1. Experimental Methods

[0177] Six specific nanobodies (Q1G9, Q2C4, Q2D10, Q2C6, Q2G2 and Q2H2) were randomly selected from the 27 specific antibodies obtained in step one to verify their nanobody binding activity.

[0178] Using six specific nanobodies as templates, amplification was performed according to the specific nanobodies amplification system shown in Table 6 using the following nucleotide sequences: pCMV3-CHH-For2 (SEQ ID NO: 24), pCMV3-VHH-For3 (SEQ ID NO: 25), pCMV3-VHH-For4 (SEQ ID NO: 26), and pCMV3-VHH-Rev (SEQ ID NO: 27). The amplification products were Q1G9, Q2C4, Q2D10, Q2C6, Q2G2, and Q2H2. Agarose gel electrophoresis was then performed to detect each amplification product.

[0179] Table 6. Specific Nanobody Amplification System

[0180] Components Added amount KOD One™ PCR Master Mix 12.5 VHH fragment (template), 50 ng / μL 1.0 pCMV3-VHH-For2 primers (SEQ ID NO: 24), 10 μM 0.25 pCMV3-VHH-For3 primers (SEQ ID NO: 25), 10 μM 0.25 pCMV3-VHH-For4 primers (SEQ ID NO: 26), 10 μM 0.25 pCMV3-VHH-Rev primers (SEQ ID NO: 27), 10 μM 0.75 <![CDATA[ddH2O]]> Increase to 25 Total 25

[0181] PCR amplification program: 98℃, 5 min; 98℃, 10 s, 62℃, 30 s, 68℃, 30 s, 35 cycles; 68℃, 7 min.

[0182] The pCMV3 expression vector was digested with AfeI and NheI restriction endonucleases to obtain linearized pCMV3 expression vectors. The linearized pCMV3 expression vectors were then ligated with the amplification products of Q1G9, Q2C4, Q2D10, Q2C6, Q2G2, and Q2H2 using recombinase II to obtain recombinant plasmids pCMV3-Q1G9, pCMV3-Q2C4, pCMV3-Q2D10, pCMV3-Q2C6, pCMV3-Q2G2, and pCMV3-Q2H2.

[0183] Recombinant plasmid pCMV3-Q1G9 was transfected into 293T cells and incubated at 37°C for 72 h. Cell supernatant was collected using a pipette. Using H7N9-AH13-HA, H7N9-SF003-HA, H1N1-CA04 (ACP41105.1), H3N2-HK14 (Hemagglutinin / HAA / HongKong / 4801 / 2014), H5N6 / GZ14, and H10N3 (A / Jiangsu / 428 / 2021(H10N3)(JS428)) as antigens, the cell supernatant and antigens were subjected to an enzyme-linked immunosorbent assay (ELISA) as shown in Experimental Group 1 of Example 2 to measure OD. 450 value.

[0184] The recombinant plasmid pCMV3-Q1G9 was replaced with pCMV3-Q2C4, pCMV3-Q2D10, pCMV3-Q2C6, pCMV3-Q2G2, and pCMV3-Q2H2, respectively, and the OD values ​​of different recombinant plasmids binding to different antigens were tested. 450 Value; and using antibodies Protein SD36, 70C3, and 8852 as positive control groups, the OD value of the antibodies in the positive control groups when binding to the antigen was tested. 450 value.

[0185] 2. Experimental Results

[0186] The agarose gel electrophoresis results of the Q1G9, Q2C4, Q2D10, Q2C6, Q2G2, and Q2H2 amplification products are shown in the figure below. Figure 10 As shown, the results indicate that a 430bp protein band appeared in the electrophoresis results of the Q1G9, Q2C4, Q2D10, Q2C6, Q2G2, and Q2H2 amplification products, which is consistent with the expected theoretical value.

[0187] The binding activity (OD) of recombinant plasmids pCMV3-Q1G9, pCMV3-Q2C4, pCMV3-Q2D10, pCMV3-Q2C6, pCMV3-Q2G2, and pCMV3-Q2H2 for H7N9-AH13-HA, H7N9-SF003-HA, H1N1-CA04, H3N2-HK14, H5N6-GZ14, and H10N3 was measured. 450 The results are shown in Table 7.

[0188] Table 7 shows the combined activity results.

[0189]

[0190] The results showed that the Q2C6 specific antibody exhibited excellent binding ability with a variety of different antigens, making it a broad-spectrum binding antibody. The results also demonstrated that the method of displaying nanobody libraries using E. coli as shown in Example 3 can display a rich diversity of nanobody libraries, which can be used to screen for specific antibodies.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nanobody expression vector, characterized in that, Using pET28a(+) plasmid as the vector backbone, a nanobody gene is linked downstream of the T7 promoter of the vector backbone, and a maltose-binding protein gene is linked or not linked between the T7 promoter and the nanobody gene.

2. The nanobody expression vector according to claim 1, characterized in that, When the nanobody expression vector is linked to a maltose-binding protein gene, the maltose-binding protein gene is linked between the NcoI site and the BamHI site downstream of the T7 promoter, and the nanobody gene is linked between the BamHI site and the XhoI site downstream of the T7 promoter. When the nanobody expression vector is not linked to the maltose-binding protein gene, the nanobody gene is linked between the NcoI site and the XhoI site downstream of the T7 promoter.

3. The nanobody expression vector according to claim 1, characterized in that, The nanobody gene is the VHH gene obtained by PCR amplification after immunizing camel animals with antigens.

4. The use of the nanobody expression vector according to any one of claims 1 to 3 in displaying nanobody libraries.

5. A method for displaying a nanobody library using *E. coli*, characterized in that, Includes the following steps: S1. Transform the nanobody expression vector according to any one of claims 1 to 3 into competent Escherichia coli to obtain a recombinant strain; S2. The recombinant strain obtained in step S1 is cultured to OD. 450 =0.6-0.8, using IPTG as an inducer, at 16-18℃, 200-220 r / min for 12-15 h, to obtain the induced recombinant strain; S3. Perform solid-liquid separation on the recombinant strain obtained after induction in step S2, collect the bacterial cells and resuspend them to obtain a bacterial resuspension, freeze and thaw the bacterial resuspension to obtain a freeze-thawed bacterial resuspension and perform solid-liquid separation, collect the supernatant to obtain the nanobody library.

6. The method according to claim 5, characterized in that, The Escherichia coli mentioned in step S1 is BL21 Escherichia coli.

7. The method according to claim 5, characterized in that, The culture described in step S2 is carried out using LB medium containing kanamycin resistance.

8. The method according to claim 5, characterized in that, The final concentration of the inducer in step S2 is 0.45–0.50 mM.

9. The method according to claim 5, characterized in that, Step S2 involves induction at 18℃ and 220r / min for 15h.

10. The use of the nanobody library constructed by any one of claims 6 to 9 in screening specific nanobodies.

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