CN116063474A - Shark single domain antibody targeting SARS-CoV-2-S1-RBD, application and kit thereof - Google Patents
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Abstract
The invention discloses a shark single domain antibody targeting SARS-CoV-2-S1-RBD, application and a kit thereof. The single shark domain antibody comprises a monovalent single shark domain antibody with a coding nucleotide sequence shown as SEQ ID No.1 and a bivalent single shark domain antibody with a coding nucleotide sequence shown as SEQ ID No.3, or shown as SEQ ID No.4, or shown as SEQ ID No.5, or shown as SEQ ID No. 6. The shark single domain antibody has good specific binding force and strong affinity, can better prevent SARS-CoV-2-S1-RBD from being combined with ACE2, and has wide application prospect in the preparation of novel crown antibody medicines and detection kits.
Description
Technical Field
The invention relates to the field of antibodies, in particular to a shark single domain antibody targeting SARS-CoV-2-S1-RBD, application and a kit thereof.
Background
Covd-19 is transmitted by inhalation or contact with infectious droplets, with latency periods of 2 to 14 days or even longer, common clinical features including fever, coughing, fatigue, myalgia, dyspnea, and the like. Acute respiratory distress syndrome or multiple organ dysfunction, etc., may develop rapidly after infection and may even lead to death. SARS-CoV-2 is an enveloped single-stranded positive-stranded RNA virus belonging to the family Coronaviridae. SARS-CoV-2 genome is about 29.8kb in size and contains 14 ORFs, which encode 27 proteins, including 4 more conserved structural proteins: spike protein S, envelope protein E, membrane glycoprotein M and nucleocapsid protein N, and 8 accessory proteins 3a,3b, p6,7a,7b,8b,9b and orf14, etc.
SARS-CoV-2 entry into the host cell is mediated by the spike glycoprotein S (spike glycoprotein, S protein). The total length of the S protein is about 1273 to 1285 amino acids, comprising two subunits S1 and S2, wherein the S1 subunit at the N-terminus is a receptor binding domain, and the extracellular domain comprises a conserved receptor-binding domain (RBD) that mediates binding of the virus to the host cell surface angiotensin converting enzyme 2 (ACE 2) receptor; the S2-intracellular domain at the C end can be rivet protein, is taken as a membrane fusion subunit and is responsible for fusing a host cell and a viral membrane, so that a viral genome can enter the host cell for replication to cause diseases. The S protein gene of SARS-CoV-2 encodes a longer spike glycoprotein than SARS-CoV, which contains 2 important mutations: first, unlike most beta coronaviruses, the 2 subunits of the SARS-CoV-2S protein have multiple base cleavage sites at their junctions, which allow for increased intercellular fusion while invading the cells. Secondly, 5 of the 6 major amino acids in the RBD region are mutated, which results in higher affinity for RBD binding to ACE 2. ACE2 is a transmembrane glycoprotein, which is mainly distributed on endothelial cells and smooth muscle cells of organ tissues, has high expression in various tissues and organs such as oral mucosa, myocardial cells, lungs, small intestines, kidneys, bile ducts, bladder epithelial cells, esophageal ileum and the like of human beings, and also has high expression in partial immune cells such as macrophages, dendritic cells and monocytes. Thus, SARS-CoV-2, which is a receptor for SARS-CoV-2, is bound to ACE2 and invades the human body, and causes various diseases such as myocardial injury, respiratory system, digestive system, circulatory urinary system and immune system. Therefore, the difference of the S protein and the great specificity of the S protein combined with ACE2 determine the importance of the S protein as a drug target, and the blocking of the S protein combined with the ACE2 can directly block SARS-CoV-2 from entering host cells, thereby preventing a series of diseases caused by virus infection of human bodies.
The shark neoantigen receptor (Immunoglobulin new antigen receptor, igNAR) is a new type of natural antibody that can be widely recognized and bound to antigens. IgNAR is structurally a homodimer consisting of two heavy chains, each chain contains 1 variable domain and 5 constant domains, and compared with classical antibody molecular structures, the variable domain exerting antigen binding lacks CDR2 regions, and the corresponding CDR3 region exerting key recognition binding is longer, so that some hidden epitopes can be better recognized, and the antigen has high binding capacity. Subsequently, the researchers obtained single domain antibodies (sdAbs) containing only the antigen binding domain of the heavy chain variable region from the library of shark neoantigen receptor variable region fragments (VNARs). Because of its unique structure, significant progress has been made in the development of antibody-based drugs designed and engineered based on heavy chain antibody structures. Thus, screening for shark antibodies with good activity is potentially valuable.
Disclosure of Invention
The invention aims to provide a shark single domain antibody targeting SARS-CoV-2-S1-RBD, and application and a kit thereof. The shark single domain antibodies include one monovalent shark single domain antibody and four bivalent shark single domain antibodies.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a monovalent shark single domain antibody targeting SARS-CoV-2-S1-RBD, the coding nucleotide sequence of which is shown as SEQ ID No. 1.
Further, the amino acid sequence of the monovalent shark single domain antibody is shown as SEQ ID No. 2.
Further, the monovalent shark single domain antibody comprises an epitope complementarity region CDR and a framework region FR.
Further, the epitope complementarity region CDRs comprise DR1 having amino acid sequence DSSCALDSC and CDR3 having amino acid sequence RAYAGMDCRWDG.
Further, the framework region FR comprises FR1 having the amino acid sequence VEQTPTTTTKEAGESLTINCVLR; the amino acid sequence is: FR2 of SAWYFTKKGATKKE; FR3 with amino acid sequence SLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHC.
Further, the preparation steps of the shark single domain antibody are as follows:
(1) Immunization of the zebra sharks with recombinant SARS-CoV-2-S1-RBD antigen protein;
(2) Collecting immunized shark peripheral blood, and separating lymphocytes;
(3) Extracting total RNA of shark peripheral blood, and carrying out reverse transcription to obtain cDNA; amplifying by using primers BamVF1 and BamVR1 to obtain a shark single domain antibody library fragment;
(4) Cutting and connecting the shark single-domain antibody library fragments by enzyme, inserting the fragments into pHEN-2 phagemid vector, and converting the fragments into escherichia coli TG1 to construct phage display antibody immune library; randomly picking 10 clones from the library, and carrying out sequence determination and analysis to ensure that more than 99% of the clones in the library contain target insertion sequences;
(5) Screening the antibody immune library for shark single domain antibodies by a 3-round panning method;
(6) The polyclonal phage ELISA method verifies that the screened shark single domain antibody is combined with SARS-CoV-2-S1-RBD antigen;
(7) Monoclonal phage ELISA method to verify the binding of the screened shark single domain antibody with SARS-CoV-2-S1-RBD antigen;
(8) Sequencing the monoclonal containing the phage expressing the shark single domain antibody to obtain the amino acid sequence of the shark single domain antibody.
The invention also provides a bivalent shark single domain antibody targeting SARS-CoV-2-S1-RBD, which is formed by connecting the monovalent shark single domain antibody with a linker; the coding nucleotide sequence is shown as SEQ ID No.3, or as SEQ ID No.4, or as SEQ ID No.5, or as SEQ ID No. 6.
Further, the amino acid sequence of the bivalent shark single domain antibody is shown as SEQ ID No.7, or as SEQ ID No.8, or as SEQ ID No.9, or as SEQ ID No. 10.
The invention also provides application of the monovalent shark single domain antibody or the bivalent shark single domain antibody in preparing medicines for treating new coronavirus infection.
The invention also provides a kit for detecting or diagnosing the novel coronavirus, which comprises the monovalent shark single domain antibody or the bivalent shark single domain antibody.
Furthermore, the kit also comprises a detection antibody, an enzyme-labeled antibody, a standard substance, a coating buffer solution, a sealing solution, a diluent, a washing solution, a color development solution and a termination solution.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The monovalent shark single domain antibody R1C2 for resisting SARS-CoV-2-S1-RBD has the characteristics of small molecular weight, single structure, strong specificity, high affinity and the like. The shark antibody is not provided with a complete antibody structure, lacks an Fc end and a Y-type structure of an IgG antibody, so that the shark antibody is not easy to recognize, can easily escape from the capture of an immune system, has lower immunogenicity, and can enable long-term injection of antibody proteins. In addition, the binding site of the shark single domain antibody R1C2 and the antigen is also different from other antibodies, so that the shark single domain antibody R1C2 can be more tightly bound to the antigen and can be bound to places where the traditional antigen cannot be bound, thereby enhancing the affinity and the specificity of the shark single domain antibody.
(2) Compared with a monovalent single-domain antibody, the bivalent shark single-domain antibody has higher affinity and stronger neutralization capacity.
(3) The invention adopts phage display technology, inserts the sequence with specific gene into phagemid vector, adds auxiliary phage, makes the expression product of exogenous gene fusion present on the protein P III on phage surface, forms phage display library; three to four rounds of panning to obtain a monoclonal antibody containing phage expressing the single domain antibody, and sequencing to obtain the single domain antibody; the screening method has simple operation and good repeatability, can screen in a short time to obtain effective results, has high screening efficiency, ensures that the obtained shark single domain antibody can be efficiently expressed, can better prevent SARS-CoV-2-S1-RBD from being combined with ACE2, and has wide application prospect in the preparation of new crown antibody medicines and detection kits.
Drawings
FIG. 1 is a flow chart of a method for preparing monovalent and bivalent shark single domain antibodies against SARS-CoV-2-S1-RBD;
FIG. 2 is a graph showing ELISA titer detection results of antibodies specific for SARS-CoV-2-S1-RBD antigen protein in shark serum;
FIG. 3 is a diagram of agarose gel electrophoresis of shark single domain antibodies;
FIG. 4 is a library capacity assay of shark single domain antibodies;
FIG. 5 is a diagram of agarose gel electrophoresis of a PCR analysis of library identification bacterial liquid;
FIG. 6 is a graph of helper phage titer determinations;
FIG. 7 is a library capacity determination of shark single domain antibodies after helper phage rescue;
FIG. 8 is a polyclonal ELISA identification of specific phages;
FIG. 9 is a diagram showing the detection result of purified SDS-PAGE of shark single domain antibody R1C 2;
FIG. 10 is a graph showing the results of the R1C2 SPR affinity assay for shark single domain antibodies;
FIG. 11 is a graph showing the results of indirect non-competitive ELISA detection of shark single domain antibody R1C 2;
FIG. 12 is a graph showing the results of shark single domain antibody R1C2 HTRF (SARS-CoV-2-S1-RBD & ACE-2);
FIG. 13 is a diagram showing the detection result of purified SDS-PAGE of shark single domain antibody R1C 2-A;
FIG. 14 is a diagram showing the detection result of purified SDS-PAGE of shark single domain antibody R1C 2-G;
FIG. 15 is a diagram showing the detection result of purified SDS-PAGE of shark single domain antibody R1C 2-2G;
FIG. 16 is a diagram showing the detection result of SDS-PAGE for purification of shark single domain antibody R1C2-3G;
FIG. 17 is a graph showing the results of the affinity detection of the shark single domain antibody R1C2-ASPR provided by the present invention;
FIG. 18 is a graph showing the results of the detection of R1C2-G SPR affinity of the shark single domain antibody provided by the present invention;
FIG. 19 is a graph showing the results of the detection of R1C2-2G SPR affinity of the shark single domain antibody provided by the present invention;
FIG. 20 is a graph showing the results of the detection of R1C2-3G SPR affinity of the shark single domain antibody provided by the present invention;
FIG. 21 is a graph showing the results of indirect non-competitive ELISA detection of shark single domain antibody R1C2-A provided by the invention;
FIG. 22 is a graph showing the results of indirect non-competitive ELISA detection of shark single domain antibodies R1C2-G provided by the present invention;
FIG. 23 is a graph showing the results of indirect non-competitive ELISA detection of shark single domain antibody R1C2-2G provided by the present invention;
FIG. 24 is a graph showing the results of indirect non-competitive ELISA detection of shark single domain antibodies R1C2-3G provided by the present invention;
FIG. 25 is a preliminary detection result of a double-sandwich ELISA kit for detecting human SARS-CoV-2-S1 based on monoclonal antibodies.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The invention selects the striped zebra shark (Chiloscyllium plagiosum) as an antibody preparation material for preparing the shark single domain antibody, which does not belong to endangered shark species, has small size, is easy to artificially breed, and is suitable for antibody development. The flow chart of the method for preparing the monovalent and bivalent shark single domain antibody for resisting SARS-CoV-2-S1-RBD is shown in figure 1, and is specifically as follows:
example 1 immunization and immune response of shark
1. Striped bamboo shark immunity
1) Primary immunization, namely mixing immune striped bamboo shark with 240 mug SARS-CoV-2-S1-RBD recombinant protein and Freund' S complete adjuvant according to the ratio of 1:1 for the first time;
2) After three weeks, 5 mug SARS-CoV-2-S1-RBD recombinant protein is taken for direct intravenous injection of striped bamboo shark for the second immunization;
3) After three weeks, 4 mug SARS-CoV-2-S1-RBD recombinant protein is taken for direct intravenous injection of striped bamboo shark, and the third immunization is carried out;
4) After three weeks, 4 mug SARS-CoV-2-S1-RBD recombinant protein is taken for direct intravenous injection into striped bamboo shark for the fourth immunization;
5) After three weeks, 4 mug SARS-CoV-2-S1-RBD recombinant protein is taken for direct intravenous injection to striped bamboo shark for the fifth immunization;
2. detection of antibody titers in serum
The laboratory-made rabbit polyclonal anti-shark antibody is used for detecting the antibody titer in the post-5-immune shark serum by an indirect ELISA method. As shown in FIG. 2, the immunization effect was enhanced with the increase of the number of immunization, and the peripheral blood lymphocytes obtained by the fifth separation were selected for the construction of the shark single-domain antibody phage library.
EXAMPLE 2 construction of anti-SARS-CoV-2-S1-RBD recombinant protein shark phage antibody library
1. cDNA acquisition
1) Blood is collected from the tail veins of the five-free shark, and the supernatant and the milky lymphocytes in the middle layer are collected by gradient centrifugation; extracting total RNA by TRIZOL method, and reverse transcribing to obtain cDNA.
2. Amplification of shark single domain antibody genes
1) The single domain antibody gene was amplified using primers Bam VF1 and Bam VR1 using the synthesized cDNA as a template, wherein the sequences of the primers Bam VF1 and Bam VR1 were as follows:
Bam VF1：CGCGGCCCAGCCGGCCATGGCCGCCSMACGGSTTGAACAAACACC(SEQ ID NO.11)；
Bam VR1：GAACCGCCTCCACCAGCGGCCGCCACAGTCASARKGGTSCC，(SEQ ID NO.12)；
2) The PCR amplification reaction system is as follows: bacterial liquid 0.5 mu L, upstream primer 0.2 mu L, downstream primer 0.2 mu L,2x HIieff Robust PCR Master Mix 5 mu L, ddH 2 O replenishmentTo 10 μl.
3) The amplification conditions for PCR were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 10s, annealing at 55℃for 20s, extension at 72℃for 10s, and repeating 30 cycles; final extension at 72℃for 5min; preserving at 4 ℃.
The stripe size of the stripe bamboo shark VNAR region coding sequence obtained by PCR amplification is shown in figure 3.
3. Library construction
The coding sequence of the VNAR region of the PCR product is connected into pHEN2 phagemid vector, and the connection product is electrically transformed into escherichia coli TG1 competent to form an original phage library. The bacterial liquids are respectively diluted 10 2 、10 3 、10 4 、10 5 100. Mu.L of each of the gradient-diluted bacterial solutions was applied to a freshly prepared 2 XYT/A100 solid medium (16 g of tryptone, 10g of yeast extract, 5g of NaCl, respectively, dissolved in 900mL of double distilled water, adjusted to pH 7.0 with 1M NaOH solution, and then subjected to constant volume to 1L with double distilled water, sterilized, and stored at room temperature) and incubated overnight at 37 ℃. Statistical dilution according to colony growth 10 5 The number of colonies on the plate was doubled and the library capacity of the resulting library of shark single domain antibodies was calculated.
The calculation of the reservoir capacity is performed according to the following formula:
shark single domain antibody phage library titer = colony count x 10 x dilution gradient x library volume.
By calculation, as shown in FIG. 4, a storage capacity of 2X 10 was finally obtained 8 Phage library of CFU (color-forming units). 10 single clones were randomly picked and PCR identified, and the results are shown in FIG. 5, wherein 9 bands were about 400bp in size, so that the phage library fragment insertion rate was over 95%.
EXAMPLE 3 panning of anti-recombinant SARS-CoV-2-S1-RBD antigen protein specific phage
1. Amplification and rescue of helper phage
Helper phage M13K07 was infected with E.coli TG1 in log phase and plaques were counted to determine helper phage titer. The results are shown in FIG. 6, and the helper phage titer was measured to be 7X 10 12 pfu/mL. The obtained shark single domain antibodyThe library bacteria were mixed with M13K07 helper phage and the titers of the shark single domain antibody library phage after helper phage rescue were calculated. The results are shown in FIG. 7, and the phage titer of the shark single domain antibody library is determined to be 7.4X10 12 pfu/mL。
2. Specific phage panning
(1) The recombinant SARS-CoV-2-S1-RBD antigen protein was diluted to 100. Mu.g/mL with PBS, the immune tube was coated, PBS was the antigen-free control, coated overnight at 4deg.C, then washed 3 times with PBS, 5% nonfat milk powder, and blocked overnight at 4deg.C. PBST (0.1% Tween 20) was washed 3 times, the library phage prepared above was taken and added to the coated immune tube, incubated for 2h at room temperature, phage samples were discarded, and PBST was washed 10 times. 1mL of freshly prepared 0.1M triethylamine solution was added to each well, and the mixture was allowed to stand at room temperature for 10min, and the eluate was rapidly neutralized with an equal volume of 1M Tris-HCl (pH=7.4).
(2) First round panning: 1mL of the eluted phage solution was added to 10mL of TG1 E.coli in the logarithmic growth phase (OD 600 = 0.5) for infection. And (5) taking part of infected bacterial liquid to perform 10-time gradient multiple ratio dilution, and calculating phage titer. After centrifugation, the remaining infected bacterial liquid was resuspended in 2 XYT liquid medium and spread on 2 XYT/A100/G2% plates and incubated overnight at 30 ℃. The following day colonies on the plates were scraped with a spreading bar, a suitable amount of bacterial liquid was added to 2 XYT/A100/G2% liquid medium, shaken to logarithmic phase (OD 600 = 0.5), helper phage M13K07 was added at a multiplicity of infection of 20:1, centrifuged, the supernatant was discarded, 2 XYT/A100/K50/G0.1% medium was resuspended, and cultured overnight at 30℃at 200 rpm. Centrifuging at 4 ℃, collecting the supernatant, adding 1/4 volume of precooled PEG/NaCl solution, and ice-bathing for 1h to precipitate phage; and (3) after centrifugation, the phage precipitate is resuspended by PBS, and the phage single-domain antibody library of the first round is obtained.
(3) Second round panning: the immune tube is coated with SARS-CoV-2-S1-RBD antigen protein of 10 mug/mL, the phage library amplified after the first round of screening is added after the sealing treatment at 4 ℃ overnight, the second round of screening is carried out according to the step of the first round of screening, and similarly, the titer of the phage library after the second round of screening is measured and stored.
(4) Third round panning: coating immune tube with 1 mug/mL SARS-CoV-2-S1-RBD antigen protein, closing, adding phage library amplified after second round of screening, screening according to the second round of screening, measuring titer of phage library after third round of screening, keeping size, screening to obtain phage capable of combining with SARS-CoV-2-S1-RBD antigen protein.
After three rounds of panning, phage titers were obtained as shown in table 1 below:
TABLE 1 phage titers from three rounds of panning
3. Identification of positive clones by phage ELISA
(1) Polyclonal ELISA identification of phages
The absorbance values were measured by indirect ELISA to determine the binding capacity of phage to antigen from three rounds of panning and to determine the enrichment effect of specific phage panning.
As a result, as shown in FIG. 8, the absorbance value was gradually increased as the panning times were increased, compared to the original phage single domain antibody library, indicating that the amount of antigen-binding specific phage was gradually increased as panning proceeded, and a significant increase in the third round, indicating that phage against SARS-CoV-2-S1-RBD was effectively enriched.
(2) Identification of phage monoclonal ELISA
To obtain high affinity single phages we picked 200 single colonies from the plates obtained from the third round of panning for identification. And (3) selecting 23 positive monoclonal antibodies from the 200 selected monoclonal antibodies, and selecting the 23 selected monoclonal antibodies for secondary identification, so that the 17 selected monoclonal antibodies are finally determined to meet the requirements. Sequencing the bacterial liquid of 17 single colonies after verification, selecting sequences consistent with the expected size for sequence comparison, and finally obtaining 6 sequences, wherein the amino acid sequences are respectively shown as SEQ ID No.2 and SEQ ID No. 13-17.
EXAMPLE 4 cloning expression and purification of SARS-CoV-2-S1-RBD shark single domain antibody
(1) The 6 shark single domain antibody gene VNAR fragments of example 3 were ligated to pET28a vector, respectively, and the ligation reaction system is shown in table 2 below:
TABLE 2 connection reaction System
(2) Preparing the reaction system, gently mixing, rapidly centrifuging, and connecting at 16 ℃ overnight; the ligation product was used directly for the next conversion.
(3) Directly converting the connection product into E.coli BL21 (DE 3) competent cells, and then carrying out bacterial liquid PCR to verify whether the recombinant expression vector is constructed successfully.
(4) Purification by using a His tag and a nickel ion chelating filler revealed that only shark single domain antibody R1C2 could obtain purified antibody protein in the supernatant, the purified protein was shown in FIG. 9, and stored at-80 ℃. The nucleotide sequence of the shark single domain antibody R1C2 is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.
Example 5 affinity assay of shark Single Domain antibody with SARS-CoV-2-S1-RBD
The affinity of the specific shark single domain antibody R1C2 purified in SPR detection example 4 for SARS-CoV-2-S1-RBD was determined, and the measured affinity and kinetic parameters are shown in FIG. 10 and Table 3, respectively, with a dissociation constant KD for binding of the single domain antibody R1C2 to SARS-CoV-2-S1-RBD of 9.64X10 -9 mol/L, it shows that the shark single domain antibody R1C2 has strong affinity with SARS-CoV-2-S1-RBD protein.
TABLE 3 kinetic parameters of shark single domain antibodies R1C2
Example 6 indirect ELISA detection of shark Single-Domain antibodies
The recombinant SARS-CoV-2-S1-RBD protein is diluted to 5 mug/mL by PBS, 100 mug/hole of ELISA plate is added for coating, and the temperature is 4 ℃ overnight; discarding the supernatant coating liquid, and washing by PBST; blocking with PBST containing 5% skimmed milk at 37deg.C for 2 hr; discarding the sealing liquid in the hole, and washing by PBST; different concentrations of shark single domain antibody R1C2 were added and incubated for 1h at 37 ℃. Discarding the liquid, and washing with PBST; diluted anti-his primary antibody was added to each well and incubated at 37℃for 1h. Discarding the liquid, and washing with PBST; adding diluted HRP secondary antibody into each hole, and incubating for 1h at 37 ℃; discarding the liquid, and washing with PBST; TMB color development liquid is added into each hole, reaction is carried out for 10-15min at room temperature, stop solution is added to stop the reaction, and OD value at 450nm is read.
As a result, the EC50 of R1C2 and the antigen SARS-CoV-2-Spike-RBD protein was 15.86nM, as shown in FIG. 11.
Example 7 shark Single Domain antibody influences the interaction between SARS-CoV-2-S1 and ACE2
Using HTRF technology, based on Time Resolved Fluorescence (TRF) and Fluorescence Resonance Energy Transfer (FRET) principles, it was tested whether the shark single domain antibody R1C2 could affect the interaction between SARS-CoV-2-S1 and ACE 2.
As shown in FIG. 12, R1C2 can affect the binding of the antigen SARS-CoV-2-Spike protein to ACE2 with an EC50 of 14.51nM.
As described above, the shark single domain antibody R1C2 has good specific binding force with the novel coronavirus SARS-CoV-2spike protein S1-RBD, high affinity, and can prevent the SARS-CoV-2-S1-RBD from binding with ACE 2.
Example 8 design and recombinant expression and purification of bivalent shark single domain antibodies
(1) By adding different linker in 4 (as shown in table 4) between two identical R1C2 antibody sequences, 4 diabody sequences were designed;
TABLE 4 linker sequence
(2) The bivalent shark single domain antibody gene obtained by the design is fully synthesized, the nucleotide sequences of R1C2-A, R1C2-G, R C2-2G, R1C2-3G are respectively shown as SEQ ID No.3-6, and the amino acid sequences are shown as SEQ ID No. 7-10.
(3) The designed four shark single domain antibody genes VNAR fragments R1C2-A, R1C2-G, R1C2-2G, R1C2-3G are respectively connected with pET28a vector.
(4) The reaction system as in example 4 was prepared, gently mixed, rapidly centrifuged and connected at 16℃overnight. The ligation product was used directly for the next conversion.
(3) Directly converting the connection product into a shuffle-T7-B and a shuffle-T7-K12 competent cell, and determining whether the connection product is successfully converted by using bacterial liquid sequencing.
(4) And (3) expressing and purifying the 4 bivalent shark single domain antibodies by using His labels and nickel ion chelating fillers, wherein the purified proteins are respectively shown in figures 13-16, and are stored at-80 ℃ after subpackaging to obtain the bivalent shark single domain antibodies R1C2-A, R C1C 2-G, R C2-G, R1C2-3G.
Example 9 affinity assay of bivalent shark single domain antibodies to SARS-CoV-2-S1-RBD
The affinity of specific shark single domain antibody R1C2-A, R1C2-G, R C2-2G, R1C2-3G purified in example 8 for SARS-CoV-2-S1-RBD was detected using SPR, and the affinity assay results and kinetic parameters of the detection are shown in FIGS. 17-20 and Table 5, respectively, and the dissociation constant KD values for binding of bivalent shark single domain antibody R1C2-A, R1C2-G, R1C2-2G, R C2-3G to SARS-CoV-2-S1-RBD are 1.72X10, respectively -8 mol/L，1.03×10 -8 mol/L，6.9×10 -9 mol/L，5.6×10 -9 mol/L, it shows that the shark single domain antibodies R1C2-A, R1C2-G, R1C2-2G, R1C2-3G have stronger affinity with SARS-CoV-2-S1-RBD protein.
TABLE 5 kinetic parameters of bivalent shark single domain antibodies
Example 10 indirect ELISA detection of bivalent shark single domain antibodies
SARS-CoV-2-S1-RBD recombinant protein is diluted to 5 mug/mL by PBS, 100 mug/hole of ELISA plate is added for coating, and the temperature is 4 ℃ overnight; discarding the supernatant coating liquid, and washing by PBST; sealing with PBST containing 5% skimmed milk at 37deg.C for 2 hr; discarding the sealing liquid in the hole, and washing by PBST; different concentrations of bivalent shark single domain antibodies R1C2-A, R1C2-G, R1C2-2G, R1C2-3G were added and incubated for 1h at 37 ℃. Discarding the liquid, and washing with PBST; diluted anti-his primary antibody was added to each well and incubated at 37℃for 1h. Discarding the liquid, and washing with PBST; adding diluted HRP secondary antibody into each hole, and incubating for 1h at 37 ℃; discarding the liquid, and washing with PBST; TMB color development liquid is added into each hole, reaction is carried out for 10-15min at room temperature, stop solution is added to stop the reaction, and OD value at 450nm is read.
The results are shown in FIGS. 21-24, with EC50 s of 12.03nM,9.04nM,4.92nM,6.34nM for R1C2-A, R1C2-G, R1C2-2G, R1C2-3G and the antigen SARS-CoV-2-Spike-RBD protein, respectively.
As described above, the bivalent shark single domain antibody R1C2-A, R1C2-G, R C2-2G, R1C2-3G has better specific binding force with the novel coronavirus SARS-CoV-2spike protein S1-RBD, higher affinity and better resistance to the binding of SARS-CoV-2-S1-RBD with ACE 2.
Example 11 application of double-sandwich ELISA detection kit
The embodiment provides a double-sandwich ELISA kit for detecting novel coronavirus SARS-CoV-2-S1 protein based on bivalent shark single-domain antibody.
1. The kit comprises the following components: the kit comprises a capture antibody, a detection antibody, an enzyme-labeled antibody, a standard substance, a coating buffer solution, a blocking solution, a sample and detection antibody diluent, an ELISA (enzyme-linked immunosorbent assay) plate, a washing solution, a color development solution and a termination solution;
wherein: the capture antibody is a bivalent shark single domain antibody R1C2-3G;
the detection antibody is a mouse anti-human SARS-CoV-2Spike RBD monoclonal antibody;
the enzyme-labeled antibody is a horseradish peroxide-labeled goat anti-mouse IgG antibody;
the standard is recombinant SARS-CoV-2-S1-RBD protein;
the coating buffer was a 0.05M carbonate buffer, pH 9.6;
the sealing liquid is phosphate buffer solution containing 5% of nonfat milk powder by volume fraction, and the pH value is 7.4;
the sample and detection antibody diluent is phosphate buffer solution containing calf serum with the volume fraction of 1%, and the pH value is 7.4;
the ELISA plate washing liquid is phosphate buffer solution containing 0.10% Tween-20 by volume fraction, and the pH value is 7.4;
the color development liquid is TMB;
the stop solution is a concentrated sulfuric acid solution with the molar concentration of 2M.
2. The specific steps of the kit for detecting the novel coronavirus SARS-CoV-2-S1 protein comprise:
(1) Coating ELISA plates: diluting the capture antibody, namely bivalent shark single domain antibody R1C2-3G, to 20 mug/mL, 100 mug/well with coating buffer solution in proportion, and overnight at 4 ℃;
(2) Closing: removing liquid in the hole, adding 200 mu L/hole ELISA plate washing liquid, washing for 3-5 times, adding 200 mu L/hole sealing liquid, washing for 3-5 times at 37 ℃ for 2 hours, and spin-drying;
(3) Adding a sample to be detected: adding a sample to be detected into the ELISA plate, washing for 3-5 times at 37 ℃ for 1h at 100 mu L/hole, and spin-drying;
(4) Adding a detection antibody: adding a detection antibody, namely a mouse anti-human SARS-CoV-2Spike RBD monoclonal antibody, into the ELISA plate, wherein the dilution ratio of the detection antibody to the mouse anti-human SARS-CoV-2Spike RBD monoclonal antibody is 1:5000, 100 mu L/hole, washing for 3-5 times after 1h at 37 ℃, and spin-drying;
(5) Diluting enzyme-labeled antibody, namely horseradish peroxide-labeled goat anti-mouse IgG, according to a volume ratio of 1:2000 to 100 mu L/hole, washing for 5-6 times at 37 ℃ after 1h, and spin-drying;
(6) Color development: adding a color development liquid into the ELISA plate, and developing color for 30 minutes at 37 ℃ in a dark place with 100 mu L/hole;
(7) And (3) detection: after 100. Mu.L/well of the stop solution was added, the mixture was placed in an ELISA reader to detect the absorbance at 450 nm.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the essence of the corresponding technical solutions.
Claims (10)
1. A monovalent shark single domain antibody targeting SARS-CoV-2-S1-RBD, characterized by: the coding nucleotide sequence of the monovalent shark single domain antibody is shown as SEQ ID No. 1.
2. The monovalent shark single domain antibody of claim 1, wherein: the amino acid sequence of the monovalent shark single domain antibody is shown as SEQ ID No. 2.
3. The monovalent shark single domain antibody of claim 2, wherein: the monovalent shark single domain antibody comprises an epitope complementary region CDR and a framework region FR.
4. A monovalent shark single domain antibody according to claim 3, wherein: the epitope complementarity region CDRs comprise CDR1 of amino acid sequence DSSCALDSC and CDR3 of amino acid sequence RAYAGMDCRWDG.
5. A monovalent shark single domain antibody according to claim 3, wherein: the framework region FR comprises FR1 having the amino acid sequence VEQTPTTTTKEAGESLTINCVLR; the amino acid sequence is: FR2 of SAWYFTKKGATKKE; FR3 with amino acid sequence SLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHC.
6. A bivalent shark single domain antibody targeting SARS-CoV-2-S1-RBD, characterized in that: the bivalent shark single domain antibody is formed by connecting the monovalent shark single domain antibody according to claim 1 with a linker; the coding nucleotide sequence is shown as SEQ ID No.3, or as SEQ ID No.4, or as SEQ ID No.5, or as SEQ ID No. 6.
7. The bivalent shark single domain antibody according to claim 6, wherein: the amino acid sequence of the bivalent shark single domain antibody is shown as SEQ ID No.7, or as SEQ ID No.8, or as SEQ ID No.9, or as SEQ ID No. 10.
8. Use of a monovalent shark single domain antibody according to claim 1 or a bivalent shark single domain antibody according to claim 6 in the manufacture of a medicament for the treatment of a novel coronavirus infection.
9. A kit for detecting or diagnosing a novel coronavirus, characterized in that: the kit comprises the monovalent shark single domain antibody of claim 1 or the bivalent shark single domain antibody of claim 6.
10. The kit of claim 9, wherein: the kit also comprises a detection antibody, an enzyme-labeled antibody, a standard substance, a coating buffer solution, a sealing solution, a diluent, a washing solution, a color development solution and a termination solution.
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