CN116143913A - Shark source single domain antibody combined with SARS-CoV-2RBD, preparation method and application thereof - Google Patents
Shark source single domain antibody combined with SARS-CoV-2RBD, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a shark source single domain antibody combined with SARS-CoV-2RBD, the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 2 or SEQ ID NO. 4; the nucleotide for encoding the shark source single domain antibody is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 3; the invention also provides a preparation method of the shark source single domain antibody combined with SARS-CoV-2RBD, which comprises the steps of sequentially carrying out immunization, constructing phage antibody library, panning the phage antibody library to obtain positive clone, finally amplifying culture, adding inducer to induce expression, and collecting purified antibody protein to obtain the shark source single domain antibody; the shark source single domain antibody prepared by the invention has good application prospect in developing new coronavirus disinfection spray and new coronavirus antigen detection kit.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and in particular relates to a shark source single domain antibody combined with SARS-CoV-2RBD, a preparation method and application thereof.
Background
Coronaviruses belonging to the genus coronaviridae, the family coronaviridae, are a class of single-stranded positive sense RNA viruses. SARS-CoV-2 belongs to the coronavirus. The coronavirus genome encodes, in order, a Spike protein (Spike protein), an Envelope protein (Envelope protein), a Membrane protein (Membrane protein) and a Nucleocapsid protein (nucleoapsid). Among them, spike protein (Spike protein) is the most important surface membrane protein of coronavirus, and contains two subunits S1 and S2. Wherein S1 mainly comprises a receptor binding domain (receptorbinding domain, RBD) responsible for recognizing the receptor of the cell; SARS-CoV-2 enters the cell after binding to angiotensin converting enzyme 2 (ACE 2) on the surface of epithelial cells via its Receptor Binding Domain (RBD) of surface spike protein (spike), and completes invasion.
Shark source single domain antibody vNAR (variable New Antigen Receptor) is derived from a specific immunoglobulin-heavy chain antibody IgNAR (Ig New Antigen Receptor) in vivo and is the smallest antibody fragment capable of binding antigen and has a molecular weight of about 12-18kDa. The shark source single domain antibody can be matched with the traditional monoclonal antibody in affinity and specificity, and has the advantages of high physical and chemical stability, good tissue permeability, strong stress resistance, high stability and easy transformation. Meanwhile, in the aspect of production, the vNAR prokaryotic cells or the yeast expression system can be expressed in a large quantity, so that the production period is short, and the production cost is low. At present, there is no shark source single domain antibody of SARS-CoV-2RBD for detection or prevention of new coronavirus infection.
Disclosure of Invention
In order to solve some of the problems of the prior art or at least to alleviate some of the problems of the prior art, the present invention provides a method for preparing a shark source single domain antibody that binds SARS-CoV-2RBD, which will be used for the detection or prevention of SARS-CoV-2 virus.
The technical scheme of the invention is as follows:
the invention provides a shark source single domain antibody combined with SARS-CoV-2RBD, the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 2 or SEQ ID NO. 4.
In addition, the invention also provides a nucleotide for encoding the single domain antibody, and the nucleotide sequence of the single domain antibody is shown as SEQ ID NO. 1 or SEQ ID NO. 3.
The invention also provides a preparation method of the shark source single domain antibody for combining SARS-CoV-2RBD, which comprises the following steps:
s1, immunization is carried out by taking striped zebra shark as an immune object and adopting SARS-CoV-2RBD recombinant protein as an antigen; after the multiple immunity is completed, extracting total RNA of immune shark peripheral blood lymphocytes, performing reverse transcription to obtain cDNA, and amplifying a vNAR gene fragment by taking the cDNA as a template;
s2, inserting the purified vNAR gene fragment into a phagemid vector pComb3XSS, constructing a recombinant phagemid vector, converting the recombinant phagemid vector into E coli TG-1 cells, and carrying out expansion culture and then carrying out infection by an auxiliary phage VCS-M13 to obtain a phage antibody library;
s3, screening the phage antibody library to obtain positive clones, namely S-RBD-55 and S-RBD-61, and sequencing and analyzing the gene sequences of the positive clones;
s4: amplifying the vNAR gene fragments contained in S-RBD-55 and S-RBD-61, cloning the vNAR gene fragments into an expression vector PET30a, constructing a recombinant expression vector, transforming E coli Shuffle T7 cells by the recombinant expression vector, screening positive strains, amplifying and culturing, adding an inducer to induce expression, and collecting and purifying antibody proteins, namely the single domain antibody of SARS-CoV-2 RBD.
The invention further provides the use of a shark source single domain antibody that binds to SARS-CoV-2RBD in the manufacture of a kit or medicament for preventing, treating and/or diagnosing SARS-CoV-2 infection.
The invention further provides the use of a shark source single domain antibody that binds to SARS-CoV-2RBD in the preparation of a spray for preventing SARS-CoV-2 infection.
The invention further provides a spray for preventing SARS-CoV-2 infection, which comprises the following components in percentage by mass:
further, the antibacterial agent is ethanol or silver ion antibacterial agent; the stabilizer is sodium polyphosphate, sodium percarbonate or sodium sulfite; the dispersing agent is sodium dodecyl benzene sulfonate.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a shark source single domain antibody capable of combining SARS-CoV-2RBD protein, which is identified by ELISA, the prepared 2 single domain antibody has good affinity to SARS-CoV-2RBD protein, and IC thereof 50 1.826+ -0.03 nM and 2.107+ -0.045 nM, respectively;
2. the invention prepares the shark source single domain antibody which can combine SARS-CoV-2RBD protein, and has good stability between normal temperature and 50 ℃ through the experiment identification of storage stability; through round two-color spectrum identification, the half-dissolution temperature is above 75 ℃, so that the single-domain antibody can get rid of cold chain transportation, and the transportation and storage cost is effectively reduced; therefore, the shark source single domain antibody capable of combining SARS-CoV-2RBD protein has better application prospect in developing new coronavirus disinfection spray and new coronavirus antigen detection kit.
Drawings
FIG. 1 shows the result of SDS-PAGE gel of shark source single domain antibodies prepared according to the present invention;
FIG. 2 shows the results of stability test of shark source single domain antibodies prepared according to the present invention stored at 37℃for 14 days;
FIG. 3 shows the results of circular dichroism spectrum detection of shark source single domain antibodies prepared by the present invention;
FIG. 4 shows the result of Western blot detection of specific recognition of SARS-CoV-2RBD by the shark source single domain antibody prepared by the present invention;
FIG. 5 is a schematic diagram showing ELISA for identifying the binding of a shark source single domain antibody prepared by the present invention to SARS-CoV-2RBD protein;
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments and the accompanying drawings, but the present invention is not limited thereto.
The striped bamboo shark (Chiloscyllium plagiosum) in the following examples is a small shark, the adult human body is not more than 1 meter, the experimental operation is convenient, and the striped bamboo shark is the research model most commonly used for developing single domain antibodies at present.
EXAMPLE 1 preparation of Single Domain antibodies that bind SARS-CoV-2RBD
1. Construction of phage antibody library:
1) The specific method for immunization by using adult striped bamboo shark as immunization object and SARS-CoV-2RBD recombinant protein as antigen is as follows: immunization was performed by multipoint subcutaneous injection after emulsifying the antigen with Freund's adjuvant at a dose of 5 nM/kg. 6 times of immunization are carried out, and the immunization interval is 14 days;
2) After 14 days of completion of the 6 th immunization, blood of immunostriped zebra shark was collected, and peripheral blood mononuclear fineness was separated by density gradient centrifugation. Extracting total RNA by adopting a TRIZOL method, and performing reverse transcription to obtain cDNA;
3) The designed specific primer for amplifying the stripe bamboo shark vNAR gene is utilized to amplify vNAR gene fragments by PCR, and the purified vNAR gene fragments are cloned into a phagemid vector pComb3XSS to construct a recombinant phagemid vector, wherein the primer sequences are shown as follows:
an upstream primer: CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT;
a downstream primer: CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG;
4) Transferring the constructed recombinant phagemid vector into TG-1 competent cells by means of electrotransformation to obtainPrimary antibody Gene libraries (library capacity of at least 10) 8 pfu/mL or more);
5) After the primary antibody gene library is subjected to amplification culture, VCS-M13 helper phage is added to construct a phage antibody library;
2. affinity screening of phage antibodies against SARS-CoV-2RBD
Three rounds of affinity screening and enrichment are carried out on the phage primary antibody library according to the modes of combination, washing, elution and amplification, and the first round of affinity screening and enrichment method comprises the following steps:
1) Dissolving 10 mug SARS-CoV-2RBD recombinant protein in PBS buffer solution, coating immune tube, and coating at 4deg.C overnight;
2) The next day, the coating solution is poured out, washed 3 times by PBS, added with 2% BSA solution, and blocked by shaking at room temperature for 2 hours;
3) Discarding the blocking solution, washing with PBS, adding the phage antibody obtained in the step 1, and slowly shaking at room temperature for incubation for 2 hours;
4) Discarding antibody liquid, washing by PBST, adding Glycine-HCl Glycine hydrochloride (pH=2.0) into an immune test tube for eluting, slowly shaking at room temperature for 15 minutes, adding 0.2mL Tris-HCl (pH=9.0) for neutralization, adjusting the pH to about 7.5, then adding TG-1 bacterial liquid (OD 600-0.5), uniformly mixing, transferring into a 50mL centrifuge tube, and shaking and culturing at 37 ℃ and 150rpm for 1h;
5) Centrifuging at 4000rpm for 5min, and coating SOC plates containing ampicillin resistance and tetracycline after re-suspending and precipitating; the next day the cells on the plates were scraped and glycerol was saved for the next round of screening. The affinity screening was carried out for 3 rounds, the procedure was the same as the first round, and the antigen coating amount was decreased round by round, with 5. Mu.g and 2.5. Mu.g in sequence. After three rounds of elutriation, the enrichment and recovery rate of phage antibodies of the specific targeting SARS-CoV-2RBD are gradually improved;
3. positive recombinant antibody screening
Screening antigen positive recombinant antibodies by using Phage ELISA, the specific method is as follows:
1) Taking 96-well culture plate, adding 2XYTG into each well amp+tet+kana A culture medium;
2) Randomly picking single antibody colonies on SOCamp+tet plates coated with a three-stage library (namely SARS-CoV-2RBD phage antibody library obtained by three rounds of screening), inoculating the single antibody colonies into the culture Plate, and performing shaking culture at 250rpm and 37 ℃ for overnight;
3) Another 96-well plate was used, and 0.4mL of 2XYTG containing 1x 1010pfu VCSM13 helper phage was used amp +tet+kana Culture medium to each well; adding 50 mu L of culture solution from each well of the Master Plate in the step 2) into the corresponding well, marking as P1 Plate, and carrying out shaking culture at 150rpm and 37 ℃ for 2 hours; centrifuging at 4000rpm for 20min, discarding supernatant, and adding 0.4mL 2XYT per well amp+tet+kana Shaking culture medium at 37 deg.C and 250rpm for overnight; the next day, centrifuging at 4000rpm for 20min, taking supernatant, and preserving at 4 ℃ for standby, thus completing phage recombinant antibody preparation;
4) Diluting SARS-CoV-2RBD recombinant protein to 1 mug/mL with PBS, coating 100 mug/hole on 96-hole ELISA plate, pouring coating liquid, and coating at 4deg.C overnight; the next day, the coating solution is poured out, after PBS washing, 2% BSA solution is added, and the room temperature is closed for 2 hours; discarding the blocking solution, correspondingly adding the phage recombinant antibody prepared in the step 3), and slowly shaking and incubating for 2 hours at room temperature; discarding the recombinant antibody liquid, washing by PBST, adding enzyme-labeled secondary antibody-M13-HRP (1:5000 ratio dilution), and slowly shaking at room temperature for incubation for 1 hour; after the enzyme-labeled secondary antibody liquid is abandoned and PBST is washed, 100 mu L of TBM chromogenic liquid is added into each hole, and after the solution is placed for 10min in a dark place, 100 mu L of 2M H is added into each hole 2 SO 4 Terminating the color development, using an enzyme-labeled instrument at OD 450 Reading the value; in experimental group OD 450 Screening out corresponding positive clones with the value which is more than 2 times higher than that of a negative control group as a judgment standard, and carrying out sequencing analysis on the positive clones, and finally obtaining positive clones named S-RBD-55 and S-RBD-61 after repeated elimination;
wherein, the amino acid sequence of the S-RBD-55 is shown as SEQ ID NO. 2: MNIFLLSGLLAWLPNVFSQRIEQTPTTTTKEAGESLTINCVLKGSSCAVSSTYRYFTKKGATKKARLSAGGRYWDTKNATSKSFSLRISDLRVEDSGTYHCEGYTGTAGCVLFWKMAIEGGGTILTVKPGK;
the nucleotide sequence encoding the positive clone designated S-RBD-55 is shown in SEQ ID NO: 1: ATGAATATTTTCTTGCTTTCGGGCCTGTTAGCCTGGTTACCAAATGTCTTTAGTCAACGGATTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCAGTGAGTAGCACGTACAGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGATTATCAGCTGGCGGAAGATATTGGGACACAAAGAATGCGACATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGGGTATACTGGTACAGCTGGATGTGTGCTGTTCTGGAAAATGGCCATTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;
wherein the amino acid sequence of the S-RBD-61 is shown as SEQ ID NO. 4
MNIFLLSGLLAWLPNVFSQRIEQTPTTTTKEAGESLTINCVLKGSSCAVSSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYTRTAGCVLFSKLAIEGGGTILTVKPGK；
The nucleotide sequence encoding the positive clone designated S-RBD-61 is shown in SEQ ID NO: 3: ATGAATATTTTCTTGCTTTCGGGCCTTTTAGCCTGGTTACCAAATGTCTTTAGTCAACGGATTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCAGTGAGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATACTCGTACAGCTGGATGTGTACTGTTCTCCAAATTAGCCATTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;
4. expression and purification of single domain antibodies
Amplifying the vNAR gene fragments of positive clones S-RBD-55 and S-RBD-61 according to the sequencing result, and cloning into a PET-30a expression vector; transforming E.coli Shuffle T7 competent cells with recombinant expression vector plasmid, determining positive strain, inoculating to LB culture medium containing kanamycin resistance, shake culturing at 200rpm to OD 600 Adding inducer IPTG solution with final concentration of 0.5mmol/L between 0.6 and 1.0, and oscillating at 200rpm and 18 ℃ for inducing expression for 18h; after the induction expression is finished, centrifugally collecting thalli, crushing thalli by ultrasonic waves, centrifugally collecting supernatant, purifying antibody proteins by adopting a conventional His-Tag affinity chromatography method to obtain the protein purity of S-RBD-55 and S-RBD-61 single domain antibodies>The result of SDS-PAGE electrophoresis is shown in FIG. 1.
Example 2 storage stability test
The shark source single domain antibodies S-RBD-55 and S-RBD-61 prepared according to example 1 were placed in an incubator, stored at 37℃and 50℃respectively, and their degradation was detected at 3,6,9, 12, 15 days of placement, respectively, and as a result, as shown in FIG. 2, after 15 days of placement at 37℃the residual amounts of the shark source single domain antibodies S-RBD-55 and S-RBD-61 were both kept at 100%; after being placed at 50 ℃ for 15 days, the rest of the shark source single domain antibodies S-RBD-55 and S-RBD-61 still maintain above 90%, which shows that the shark source single domain antibodies prepared by the invention have good storage stability under the condition of 50 ℃.
Example 3 round two chromatography detection of thermal stability of Single-Domain antibodies
The concentration of the shark source single domain antibodies S-RBD-55 and S-RBD-61 prepared according to example 1 was diluted to 1mg/mL, and the temperature was varied in the range of 20-85℃at a temperature variation rate of 2.5 ℃/min and the detection wavelength was 205nm, as shown in FIG. 3, the dissolution temperatures (Tm values) of the single domain antibodies S-RBD-55 and S-RBD-61 were 72℃and 77.5℃respectively, indicating that the single domain antibodies S-RBD-55 and S-RBD-61 had good thermal stability.
EXAMPLE 4Western Blot detection of specific recognition of the SARS-CoV-2RBD protein by Single-domain antibodies
Carrying out SDS-PAGE electrophoresis on SARS-CoV-2RBD recombinant protein, and transferring the protein to a PVDF membrane by a semi-dry transfer method; sealing with 5% skimmed milk powder, and incubating at room temperature for 1h; after TBST washing, purified S-RBD-55 and S-RBD-61 single domain antibodies (the concentration of the antibodies is 1 mu g/mL) are respectively added, and the mixture is incubated for 1h at 37 ℃; after TBST washing, adding HRP-coupled rabbit anti-striped bamboo shark IgNAR secondary antibody, and incubating for 45min at room temperature by shaking; after TBST washing, developing by using a chemiluminescent gel imager; the experimental results are shown in fig. 4: a specific band at about 35kDa indicates that both the single domain antibodies S-RBD-55 and S-RBD-61 specifically recognize the SARS-CoV-2RBD protein.
Example 5 binding affinity assay of Single Domain antibodies to SARS-CoV-2RBD protein
Diluting SARS-CoV-2RBD recombinant protein to 4 mug/mL with PBS according to optimal antigen coating concentration obtained by orthogonal experiment, adding 100 mug coated ELISA plate in each hole, sealing, diluting single domain antibody S-RBD-55 and S-RBD-61 in 1:2 ratio gradient, sequentially adding into corresponding holes, incubating at 37deg.C for 1.5hThe method comprises the steps of carrying out a first treatment on the surface of the After washing, adding an HRP-coupled rabbit anti-striped bamboo shark vNAR antibody, and incubating for 1h at 37 ℃; after washing, 100. Mu.L of TBM chromogenic substrate was added to each well, and after 10min of exposure to light, 100. Mu.L of 2M H was added to each well 2 SO 4 Terminating the color development, using an enzyme-labeled instrument at OD 450 Reading at nm, and detecting the combination condition; as shown in FIG. 5, the single domain antibodies S-RBD-55 and S-RBD-61 bind with good affinity to SARS-CoV-2RBD protein, and have good EC 50 1.256 nM.+ -. 0.03nM and 0.832.+ -. 0.045nM, respectively.
EXAMPLE 6 use of shark source single domain antibodies
Optionally, the single domain antibody S-RBD-55 or S-RBD-61 prepared according to the method of example 1, is used to prepare a spray for preventing SARS-CoV-2 infection, said spray comprising the following components in mass percent:
the preparation method of the spray comprises the following steps: adding triethanolamine, sodium dodecyl benzene sulfonate and sodium polyphosphate in the above formula into ethanol, heating to 55 ℃, continuously and rapidly stirring, homogenizing and uniformly mixing, then adding a shark source single domain antibody S-RBD-55 or S-RBD-61 and floral essential oil, inactivating viruses by a Pasteur disinfection method, and filtering and sterilizing by a film of 0.22 mu m to obtain the spray for preventing SARS-CoV-2 infection.
Example 7
A spray for preventing SARS-CoV-2 infection, the spray comprising the following components in mass percent:
example 8
A spray for preventing SARS-CoV-2 infection, the spray comprising the following components in mass percent:
the foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (7)
1. A shark source single domain antibody combined with SARS-CoV-2RBD is characterized in that the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 2 or SEQ ID NO. 4.
2. A nucleotide sequence encoding a single domain antibody of shark origin as claimed in claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 1 or SEQ ID No. 3.
3. A method for preparing a shark source single domain antibody binding to SARS-CoV-2RBD as claimed in claim 1, comprising the steps of:
s1, immunization is carried out by taking striped zebra shark as an immune object and adopting SARS-CoV-2RBD recombinant protein as an antigen; after the multiple immunity is completed, extracting total RNA of immune shark peripheral blood lymphocytes, performing reverse transcription to obtain cDNA, and amplifying a vNAR gene fragment by taking the cDNA as a template;
s2, inserting the purified vNAR gene fragment into a phagemid vector pComb3XSS, constructing a recombinant phagemid vector, converting the recombinant phagemid vector into E coli TG-1 cells, and carrying out expansion culture and then carrying out infection by an auxiliary phage VCS-M13 to obtain a phage antibody library;
s3, screening the phage antibody library to obtain positive clones, namely S-RBD-55 and S-RBD-61, and sequencing and analyzing the gene sequences of the positive clones;
s4: amplifying the vNAR gene fragments contained in S-RBD-55 and S-RBD-61, cloning the vNAR gene fragments into an expression vector PET30a, constructing a recombinant expression vector, transforming E coli Shuffle T7 cells by the recombinant expression vector, screening positive strains, amplifying and culturing, adding an inducer to induce expression, and collecting and purifying antibody proteins, namely the single domain antibody of SARS-CoV-2 RBD.
4. Use of a shark source single domain antibody binding to SARS-CoV-2RBD as claimed in claim 1 in the manufacture of a kit or medicament for the prevention, treatment and/or diagnosis of SARS-CoV-2 infection.
5. Use of a shark source single domain antibody binding to SARS-CoV-2RBD as claimed in claim 1 in the preparation of a spray for preventing SARS-CoV-2 infection.
6. A spray for preventing SARS-CoV-2 infection, comprising the following components in percentage by mass:
0.5 to 1 weight percent of shark source single domain antibody S-RBD-55 or S-RBD-61;
8-10wt% of antibacterial agent;
0.5 to 1 weight percent of flower essential oil;
0.5 to 0.8 weight percent of triethanolamine;
3-5 wt% of stabilizer;
3-5 wt% of dispersing agent;
sterile water was made up to 100wt%.
7. The spray for preventing SARS-CoV-2 infection as claimed in claim 6, wherein said antibacterial agent is ethanol or silver ion antibacterial agent; the stabilizer is sodium polyphosphate, sodium percarbonate or sodium sulfite; the dispersing agent is sodium dodecyl benzene sulfonate.
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