CN113336844B - Shark single domain antibody targeting novel coronavirus N protein, and preparation method and application thereof - Google Patents
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Abstract
The invention discloses a shark single domain antibody targeting a novel coronavirus N protein, a preparation method and application thereof, belonging to the field of biotechnology; the amino acid sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 3 or SEQ ID NO. 4; meanwhile, the invention also provides a preparation method of the shark single domain antibody targeting the novel coronavirus N protein, which comprises the steps of separating immune streak zebra shark peripheral blood lymphocytes, extracting total RNA, and performing reverse transcription to obtain cDNA; amplifying stripe bamboo shark vNAR fragments by taking cDNA as a template, and connecting the cDNA with a carrier to construct a phage library; panning positive clones from the phage library that recognize the new coronavirus N protein; constructing an expression vector, and inducing expression of the novel coronavirus N protein single domain antibody to finally obtain the shark single domain antibody targeting the novel coronavirus N protein.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a shark single domain antibody targeting a novel coronavirus N protein, and 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; the first virus found in 1937 has identified a variety of coronaviruses to date, belonging to the genera α, β, γ and δ, of which 7 coronaviruses can infect humans, namely HCoV-229E, HCoV-NL63 of the genera α and novel coronavirus SARS-CoV-2 identified at the end of 2019 by HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, respectively.
SARS-CoV-2 is a respiratory virus carried by droplets, and unlike any of the six previously known coronaviruses capable of infecting humans, the virus involves amino acid changes at least 17 positions on different proteins, which are very easy to pass by humans in closely contacted people, resulting in explosive overall spread, and thus rapid diagnostic assays are needed to control the outbreak and spread of disease.
The SARS-CoV-2 genome consists of approximately 30000 nucleotides and encodes four structural proteins, including S protein (Spike protein), E protein (Envelope protein), M protein (Membrane protein) and N protein (Nucleocapsid protein), which are the most abundant proteins in coronaviruses and are also a highly immunogenic phosphoprotein, expressed in large amounts during viral infection, with nearly 2000N protein monomers present in each viral RNA genome copy. During the period of SARS-CoV in 2003-2004, the presence of N protein was detected in the serum sample of patients at the early stage of viral infection, and thus N protein detection was considered as a reliable basis for detecting SARS-CoV. Research data from the pandemic of SARS-CoV-2 suggests that a large amount of SARS-CoV-2 virus N protein is detected in nasopharyngeal swab samples of new coronavirus infected persons. The novel coronavirus N protein has high immunogenicity, high copy number in the body of an infected person and good self protein stability, and is an important detection target for detecting SARS-CoV-2 virus.
Currently, the detection method of new coronaviruses mainly relies on nucleic acid detection, i.e. the detection of viral genomic RNA present in respiratory tract samples by qRT-PCR (quantitative reverse transcriptase polymerase chain reaction). The nucleic acid detection has the advantages of high sensitivity, large parallel detection quantity and the like, but also faces the challenges of high sample pretreatment difficulty, high cost of reverse transcription reagents, advanced real-time thermal cycler for detection and the like. The detection of the antibody can effectively make up the risk of missed detection of the nucleic acid detection, and plays a role in timely diagnosis, treatment, prevention and control of the novel coronary pneumonia. Specific IgM and IgG are the most commonly used new coronavirus detection antibodies at present, but more false positives are reflected in clinic, which afflicts clinical decisions.
Heavy chain antibodies are useful as naturally deleted light chain antibody molecules with greater advantages over conventional IgG antibody molecules in genetic engineering. The single domain antibody (single domain antibody) engineered from heavy chain antibody has the ability to bind antigen in a natural state, and has better affinity retention than the single domain antibody of human or murine origin, and the single domain antibody has various advantages. In recent years, single domain antibodies attract great research enthusiasm, and great progress is made in exploring antigen receptor origins, developing vaccines, therapeutic drugs, diagnostic reagents, biotechnology research tools and the like. The best currently developed in terms of single domain antibodies is camel single domain antibodies, and following camel discovery of single domain antibodies, greenberg et al have successively discovered a naturally deleted light chain, antibody IgNAR (Ig New antigen Receptor) consisting of heavy chain homodimers alone, each chain consisting of 1 variable region (vNAR) and 5 constant regions (cNAR), from the cartilage fish such as nurse shark. The variable region vNAR of IgNAR is recombined and expressed to obtain the antibody molecule fragment with complete function, which is called shark single domain antibody. The Chinese patent with publication number CN106831981A discloses a single-domain antibody protein skeleton and a preparation method thereof, in particular discloses a single-domain antibody derived from striped bamboo shark, the single-domain antibody protein skeleton sequence composition of the single-domain antibody protein skeleton is FR1, CDR1, FR2, CDR3 and FR3 areas, wherein the FR1, FR2 and FR3 areas are fixed amino acid sequences, the CDR1 and CDR3 areas are antibody complementarity determining areas, the amino acid sequences are variable, the CDR1 and CDR3 areas determine the combination with different antigens, and the shark single-domain antibody has an antigen binding area, can realize the combination of antigen and antibody, and is used for diagnosis and treatment; furthermore, WO2010033913A1 discloses an antibody, mimetic and use thereof, in particular shark and camelid heavy chain antibodies and analogues thereof for use in diagnosis, therapy and simultaneous diagnosis and therapy.
Therefore, the detection of the novel coronavirus antigen protein by utilizing the shark single-domain antibody and the preparation of the antibody detection kit are completely feasible and have higher clinical application value.
Disclosure of Invention
In order to solve the problems that the detection of the novel coronavirus N protein with high sensitivity needs to be further improved and the development and application of antibodies in the prior art, the invention creatively provides a shark single domain antibody targeting the novel coronavirus N protein, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
a shark single domain antibody of targeting novel coronavirus N protein has an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2.
A shark single domain antibody of targeting novel coronavirus N protein has a nucleotide sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4.
The preparation method of the shark single domain antibody targeting the novel coronavirus N protein specifically comprises the following steps:
(1) Separating immune streak zebra shark peripheral blood lymphocytes, extracting total RNA, and performing reverse transcription to obtain cDNA;
(2) Amplifying stripe bamboo shark vNAR fragments by taking cDNA as a template, and connecting the cDNA with a carrier to construct a phage library: nest PCR is adopted, and two rounds of PCR amplification are carried out to obtain stripe bamboo shark single domain antibody vNAR genes; enzyme grafting is carried out on the vNAR gene fragment and a vector to construct a phage library;
(3) Panning positive clones from phage library that recognize the new coronavirus N protein: performing amplification culture, enrichment, screening and identification on the phage library, sequencing and analyzing all positive clones with OD450 value greater than 1, and finally obtaining 2 single-domain antibodies, namely an N05 antibody and an N38 antibody of the shark single-domain antibody after eliminating repetition; sequencing to obtain the vNAR gene sequences of the N05 antibody and the N38 antibody;
(4) Constructing an expression vector, and inducing expression of a novel coronavirus N protein single domain antibody: cloning the vNAR gene sequences of the N05 antibody and the N38 antibody onto PET30a to obtain the expression vectors of the N05 antibody and the N38 antibody, converting the expression vectors into E.coli BL21 strain, performing expansion culture, adding IPTG to induce expression, and collecting and purifying recombinant antibody protein by using broken bacteria.
Further, the immune striped bamboo shark in the step (1) is the recombinant new coronavirus N protein subcutaneously injected in the striped bamboo shark, the dosage is 5nM/kg, the interval period is 14 days, and the striped bamboo shark immunized by the recombinant new coronavirus N protein is obtained after completing 6 immunization for 14 days.
Further, in the enzyme grafting process in the step (2), sfi I is used for respectively carrying out enzyme digestion on the stripe bamboo shark vNAR gene obtained by PCR and the pComb3XSS vector, and after enzyme digestion recovery, T4 ligase is connected; the ligation product was transformed into XL1-Blue competent cells, and when OD660 reached 0.6, helper phage VCSM13 was added, and after overnight incubation, the culture supernatant was collected to obtain a primary phage library.
A nucleic acid molecule encoding a shark single domain antibody as described above.
A vector comprising a nucleic acid molecule encoding a shark single domain antibody as described above.
A method for detecting a novel coronavirus N protein for non-diagnostic purposes comprising the steps of:
s1, infecting HEK293T cells by using a novel coronavirus N protein pseudovirus;
s2, extracting total cell proteins and transferring the proteins to a PVDE membrane through electrophoresis and separation;
s3, carrying out Western Blot detection when the affinity purified shark single domain antibody according to claim 1 or 2 is contacted with PVDE membrane.
Use of a shark single domain antibody targeting a novel coronavirus N protein in the preparation of a novel coronavirus detection product or pharmaceutical composition.
Compared with the prior art, the invention has the beneficial effects that:
1. the novel coronavirus N protein shark single domain antibody provided by the invention can specifically recognize endogenous novel coronavirus N protein, can be used as a quality control antibody of a novel coronavirus detection kit, and fills the blank that no shark single domain antibody exists in an ELISA detection kit in a novel coronavirus serological detection market; the shark single domain antibody provided by the invention has higher binding affinity of N05 antibody, N38 antibody and novel coronavirus N protein, and IC thereof, as measured by ELISA method 50 1.415nM and 1.749nM, respectively.
2. Compared with the traditional monoclonal antibody and camel single domain antibody, the shark single domain antibody provided by the invention has the unique advantages in detection of the N protein of the novel coronavirus, firstly, the CDR3 (complementary-determining Region 3) structural domain of the shark single domain antibody is in a convex ring shape, and can recognize the hidden epitope of the N protein of the novel coronavirus, so that the binding with antigen is superior to that of the traditional monoclonal antibody; in the aspect of production, the traditional antibody plays a role in recognition and combination, glycosylation modification is usually needed, expression is needed through mammalian cells, and the cost is high.
3. The N05 antibody and the N38 antibody of the shark single-domain antibody provided by the invention can be further modified to provide the affinity of the antibody, and can be directly used for developing a high-sensitivity novel crown antigen detection kit and also can be used for debugging the novel crown antibody detection kit.
Drawings
FIG. 1 shows phage display screening results according to example 1 of the present invention; wherein, FIG. 1A shows the change in phage enrichment after three rounds of panning; FIG. 1B shows Phage-ELISA detection of Phage polyclonal supernatants;
FIG. 2 is a schematic diagram showing the result of SDS-PAGE gel electrophoresis of shark single domain antibodies according to example 2 of the present invention;
FIG. 3 is a schematic diagram showing the binding of shark single domain antibodies to novel coronavirus N protein prepared by ELISA assay according to example 3 of the present invention;
FIG. 4 is a schematic representation of the recognition of endogenous novel coronavirus N proteins by shark single domain antibodies prepared using Western Blot detection according to example 3 of the present invention.
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.
Example 1
A shark single domain antibody of targeting novel coronavirus N protein is named as N05 antibody, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of which is shown as SEQ ID NO. 3.
A shark single domain antibody targeting the N protein of a novel coronavirus is named as an N38 antibody, the amino acid sequence of which is SEQ ID NO. 2, and the nucleotide sequence of which is SEQ ID NO. 4.
Example 2
The preparation method of the shark single domain antibody targeting the novel coronavirus N protein specifically comprises the following steps:
(1) Separating immune streak zebra shark peripheral blood lymphocytes, extracting total RNA, and performing reverse transcription to obtain cDNA;
immunization of the recombinant novel coronavirus N protein against striped bamboo shark: immunization of striped bamboo shark with purified prokaryotic expressed recombinant new coronavirus N protein is carried out by the following specific method: according to the dose of 5nM/kg, emulsifying the antigen with Freund's adjuvant, performing multipoint subcutaneous injection on the striped bamboo shark for immunization, wherein the immunization interval is 14 days, and after the 14 th immunization is completed, 5mL of the striped bamboo shark tail vein is taken into a heparin sodium anticoagulation tube to complete immunization of the striped bamboo shark;
obtaining peripheral blood lymphocytes of striped bamboo sharks: equally split charging 5mL of blood obtained through venous blood sampling into 5 15mL centrifuge tubes, respectively adding 10 times of physiological saline for diluting the blood sample, and slowly and uniformly mixing; sucking the diluted blood sample on the liquid surface of the separating liquid, centrifuging for 30min at 300 g/min; after centrifugation, the lymphocytes are in an off-white band shape and are positioned between a light yellow plasma layer and a transparent separation liquid layer, the lymphocytes are carefully sucked into another 15mL centrifuge tube by a suction tube, 10mL PBS buffer solution is added for resuspension and washing, 350g/min is carried out, centrifugation is carried out for 10min, 3 times are repeated, and the peripheral blood lymphocytes of the immune streak zebra shark are obtained by collection;
RNA extraction: adding 1mL Tripure Reagent into a centrifuge tube filled with lymphocyte sample, performing ultrasonic disruption for 30s, adding 1/5 volume of chloroform, standing at room temperature for 5min,12000rpm, and centrifuging at 42 ℃ for 15min; carefully transferring the supernatant into another centrifuge tube, adding equal volume of isopropanol, mixing, standing at room temperature for 20min, centrifuging at 12000rpm and 42 ℃ for 5min; slowly sucking the supernatant, adding 75% ethanol for washing, centrifuging at 42 ℃ for 5min at 12000 rpm; carefully pouring out the supernatant, precipitating air-dried RNA, adding DEPC water for dissolution, measuring the concentration of RNA by a nucleic acid measuring instrument, and simultaneously taking a proper amount of RNA for 1% agarose gel electrophoresis to identify the quality of the RNA;
reverse transcription to obtain cDNA:
first, the following mixture was added to a 0.2mL PCR tube on ice:
| Oligoc(dT)(10μM) | 1μL |
| total RNA | 5μg |
Secondly, heating the mixture at 65 ℃ for 10min, and then immediately placing the mixture on ice;
then, under ice bath conditions, the following reagents were added to the PCR tube:
| 5×M-Mμlv Reaction Buffer | 8μL |
| dNTP Mixture(10mM) | 5μL |
| M-Mμlv Reverse Transcriptase | 1μL |
| DEPC water | 20μL |
| Total | 40μL |
finally, after slightly centrifuging the mixture, immediately incubating for 90min at 37 ℃ to synthesize first strand cDNA, heating for 5min at 65 ℃, and terminating the reaction;
(2) Amplifying stripe bamboo shark vNAR fragments by taking cDNA as a template, and connecting the cDNA with a carrier to construct a phage library;
amplification of stripe bamboo shark single domain antibody vNAR gene: nest PCR is adopted, and two rounds of PCR amplification are carried out to obtain stripe bamboo shark single domain antibody vNAR genes; wherein, the primer used in the first round of PCR is the upstream primer (F1): ATGAATATTTTCTTGTTTTCGGGCC the number of the individual pieces of the plastic,
downstream primer (R1): ATAGTATCCGCTAATTAGACAAA;
the first round PCR amplification system is as follows:
| 5×Premix Taq | 25μL |
| F1(10mM) | 0.5μL |
| R1(10mM) | 0.5μL |
| cDNA | 2μL |
| ddH 2 O | to50μL |
the first round PCR amplification procedure was:
a second round of nested PCR was performed using the first round of PCR products, with the primers used for the second round of PCR being the upstream primer (F1'): CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT; downstream primer (R1'):
CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG；
the second round PCR amplification system is as follows:
| 5×Premix Taq | 25μL |
| F1’(10mM) | 0.5μL |
| R1’(10mM) | 0.5μL |
| first round PCR amplification products | 2μL |
| ddH 2 O | to50μL |
The second round of PCR amplification procedure was:
after the second round of PCR products were electrophoresed using a 2% agarose gel, the vNAR fragment (about 500 bp) was purified by cutting gel and purified using gel-extraction purification kit; detecting by agarose gel electrophoresis after purification, wherein the band is unique (about 500 bp) and clear, thus obtaining the vNAR gene fragment;
the vNAR gene is linked to a vector: the vNAR gene fragment and the vector pComb3xss are respectively subjected to double enzyme digestion by using endonuclease Sfi I, and the enzyme digestion reaction system is as follows:
| Sfi I | 120U |
| CutSmart | 5μL |
| DNA | 5μg |
| ddH 2 O | to 50μL |
the enzyme digestion reaction conditions are as follows: reacting for 6 hours at 55 ℃; after the enzyme digestion reaction is finished, a DNA purification recovery kit is used for recovering the vNAR gene fragment and the pComb3xss vector, and after recovery, the ligation is carried out by using T4 ligase, wherein the ligation system is as follows:
| pComb3xss vector | 1μg |
| vNAR fragment | 300μg |
| 10X T4 buffer | 4μL |
| T4 Ligase | 2μL |
| ddH 2 O | to 50μL |
After 3h of connection at 16 ℃, purifying and recycling the connection product, and dissolving ddH 2O;
construction of phage library: pre-cooling the electric shock cup on ice, taking XL1-Blue electric conversion competence to be dissolved on the ice, adding 5 mu L of the connecting product after the electric shock cup is dissolved, lightly mixing the connecting product, and placing the mixture on the ice for 5min; transfer to pre-chilled electrocuvette, electrometer program: 1800kV,5ms, electric shock conversion; immediately after electrotransformation, 1mL of SOCG culture medium is added, and the culture is carried out for 60min at 37 ℃ and 180 rpm; taking 100uL of cultured bacterial liquid, coating an ampicillin-resistant SOC plate after gradient dilution according to the proportion of 1:10, 1:100 and 1:1000, culturing overnight at 37 ℃, calculating the colony count on the plate, and calculating the reservoir capacity (the reservoir capacity should at least reach 10 7 pfu/ml); the remaining bacteria liquid after transformation was spread on ampicillin-resistant SOC plates, incubated overnight at 37℃and then incubated at-70℃with 15% final glycerol.
(3) Panning positive clones from the phage library that recognize the new coronavirus N protein;
amplification of phage library: 1ml of the cultured bacterial liquid (containing about 10 8 The transformed cells) were added to 100mL of 2XYT medium, and cultured at 37℃and 250rpm until the OD600 reached 0.6; adding helper phage VCSM13 at MOI=1:20, culturing at 37deg.C and 220rpm for 1 hr, adding ampicillin with final concentration of 100 μg/ml and 0.1mM IPTG, culturing overnight at 37deg.C, centrifuging overnight culture broth at 4deg.C and 10000rpm for 15min, and collecting supernatant; adding 1/4 volume of PEG/NaCl into the supernatant, settling on ice for 30min, centrifuging at 4deg.C and 10000rpm for 10min, discarding the supernatant, adding 2mL of PBS, suspending the precipitate, and preserving at 4deg.C;
affinity screening of anti-novel coronavirus N protein single domain antibodies: the new coronavirus N protein was diluted to 25. Mu.L/mL, the immune tube (2 mL/tube, 10. Mu.L total) was coated and incubated overnight at 4 ℃; the next day, PBS was washed 3 times, the immune tubes were blocked with blocking solution (2% skim milk PBS, MPBS) and incubated for 2h at 37 ℃; after 3 washes with PBS, 1X10 wells were added 11 phage above pfu (dissolved in 200. Mu.L MPBS), incubated for 2h at 37 ℃; after 3 washes of PBST (PBS+0.5% Tween-20) and PBS, 2ml of Glycine-HCl (pH=2.5) elution buffer was added, and after 10min of gentle spin elution, an equal volume of Tris-HCl (pH=7.4) was added to neutralize the pH to 7.0; phage obtained by elution was eluted according to 10 -1 To 10 -8 Gradient dilution, adding fresh XL1-blue bacterial liquid, and light shaking and incubation at 37 ℃ for 30min; coating 100uL bacterial liquid after each gradient infection on an ampicillin resistance SOC plate, and calculating phage output titer; adding an equal volume XL1-blue (OD 600 = 0.5) bacterial liquid into the residual phage eluent, culturing for 1h at 37 ℃ and 150rpm, centrifuging at 4000rpm for 5min, and coating an ampicillin-resistant SOC plate after re-suspending and precipitating; the next day the thalli on the plate are scraped, and the glycerol is saved for the next round of screening; the affinity screening is carried out for 3 rounds, the coating amount of the antigen is gradually decreased round by round, the coating amount of the second round is 25 mug, and the third round is 12.5 mug; referring to fig. 1, the enrichment and recovery of phage specific for the new coronavirus N protein was increased round by round in three rounds of affinity panning;
phage Elisa screening positive monoclonal against New coronavirus N protein single domain antibody: taking 72-well culture plate, adding 400 μL of 2 XYT-G amp+kan+ Culture medium, the monoclonal on the output Plate after affinity screening is inoculated into a hole, marked as Master Plate, and cultured overnight at 37 ℃ and 200 rpm; another 72 well plate was taken and 400. Mu.L of the plate containing 1X10 10 2XYT of pfu VCSM13 helper phage - G amp+kan+ To each well, labeled P1 Plate; 40ul of culture solution is taken from each hole of the Master Plate and added into the corresponding hole of the P1 Plate, and the culture solution is cultured for 2 hours at 37 ℃ and 150rpm in a shaking way; centrifuging at 4000rpm for 20min, discarding supernatant, adding 400 μl of 2XYT per well amp+kan+ Culture medium, 37 ℃,250rpm, shaking culture overnight; centrifuging at 4000rpm for 20min, mixing 320 μl supernatant with 80 μl MPBS, and preserving at 4deg.C to obtain phage recombinant antibody;
phage Elisa assay: diluting the novel coronavirus N protein to 0.5 mug/mL, coating a 96-well ELISA plate with 200 mug/well, and incubating for 1h at 37 ℃; washing 3 times with PBS, adding 1% BSA, and blocking at 37 ℃ for 1.5h; correspondingly adding the prepared phage recombinant antibody, and incubating for 2 hours at 37 ℃; PBST and PBS were washed 3 times, and enzyme-labeled secondary antibody-M13-HRP (diluted 1:4000 with MPBS) was added, incubated at 37℃for 1 hour, and after PBST and PBS were washed 3 times, 150. Mu.L of TBM chromogenic substrate was added to each well, and after 10min of exposure to light, 150. Mu.L of 2M H was added to each well 2 SO 4 Stopping color development, reading the value at the OD450nm by using an enzyme-labeled instrument, sequencing and analyzing all positive clones with the OD450 value more than 1 as shown in the result of FIG. 1B, and finally obtaining 2 positive clones which are respectively named as an N05 antibody and an N38 antibody after repeated amplification is eliminated;
the amino acid sequence of the N05 antibody is:
MNIFLFSFLLAWLPNVFTQWVEQTPTTTTKEAGESLTINCVLKGSSYGLCNTNWYFTKKSVTKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKPSMGWDETGYCLGLGEGGGTILTVKPGK(SEQ ID NO:1)；
the nucleotide sequence of the N05 antibody is as follows:
AATATTTTCTTGTTTTCGGTCCTTTTAGCCTGGTTACCAAATGTCTTTACTCAATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGGATTGTGTAACACGAACTGGTATTTCACAAAAAAGAGCGTTACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGCGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAACCGTCTATGGGCTGGGATGAGACCGGTTACTGTCTGGGATTGGGGGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA(SEQ ID NO:3)
the amino acid sequence of the N38 antibody is:
MNIFLFSFLLAWLPNVFTQWVEQTPRTTTKEAGESLTINCVLKGSSYVLCNTYWYFTKKGATKKETLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYSRYSWDGCSVILLATGSDYYEGGGTILTVKPGK(SEQ ID NO:2)；
the nucleotide sequence of the N38 antibody is:
AATATTTTCTTGTTTTCGTTCCTTTTAGCCTGGTTACCAAATGTCTTTACTCAATGGGTTGAACAAACACCGAGAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGTTATGTATTGTGTAATACGTACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGACCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATCAGTGACCTGCGAGTTGAAGACAGTGGTACATATTACTGTAAAGCGTATAGTCGGTACAGCTGGGATGGGTGTAGTGTTATACTGTTAGCGACAGGTTCCGACTATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA(SEQ ID NO:4)。
(4) Constructing an expression vector, and inducing expression of a new coronavirus N protein single domain antibody;
construction of single domain antibody prokaryotic expression strains: designing a primer to amplify the vNAR gene according to the sequencing result; wherein primer F: CCATGGTCCAATGGGTTGAACAAACACCGA, primer R: CTCGAGTTTGCCAGGTTTCACAGTCAG; purifying the PCR product by using a purification kit; after purification, agarose gel electrophoresis detection shows that the band is unique (about 400 bp) and clear, and the NotI and xHoI are used for carrying out double digestion on the vNAR fragment and the expression vector Pet30a, and the digestion reaction system is as follows:
| DNA | 5μL |
| 10x H Buffer | 5μL |
| Nde I | 2μL |
| xHoI | 2μL |
| ddH 2 O | to 50μL |
enzyme digestion reaction is carried out at 37 ℃ for 1h, 2% agarose gel electrophoresis is used, gel is respectively cut, and the corresponding vNAR gene and vector are recovered; after ligating the recovered vNAR gene and vector using T4 ligase, BL21 (DE 3) competent cells were transformed, plated on LB plates containing kanamycin resistance, and incubated overnight at 37 ℃; the next day, after the positive clones are primarily screened out by PCR, sequencing analysis is carried out to determine positive strains;
prokaryotic expression and purification of anti-novel coronavirus N protein single domain antibody: selecting the obtained positive strain, inoculating the positive strain into LB liquid medium containing the kanamycin, culturing overnight at 37 ℃ with a shaking table of 200rpm, and extracting plasmids; transforming the obtained recombinant plasmid into escherichia coli BL21, and when the recombinant plasmid is subjected to shaking culture at the temperature of 37 ℃ and at the speed of 200rpm until the OD600 = 0.6-1.0, adding an IPTG solution with the final concentration of 0.5mM, and performing shaking induction expression at the temperature of 25 ℃ and at the speed of 200rpm for 12-18 hours; after the induction expression is finished, the thalli are collected by centrifugation, the thalli are crushed by ultrasonic waves, the supernatant is collected by centrifugation, the antibody protein is purified by adopting a conventional His-tag affinity chromatography method, the purity of the obtained purified recombinant antibody protein is more than 95%, and the detection result of SDS-PAGE electrophoresis is shown in figure 2.
Example 3
Nucleic acids, vectors, compositions or complexes
The present invention relates to nucleic acid molecules encoding the shark single domain antibodies of the present invention, where the nucleic acids of the present invention may be RNA, DNA or cDNA.
The nucleic acids of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid, or YAC. The vector may in particular be an expression vector, i.e. a vector that provides for expression of the shark single domain antibody in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art.
Example 4
1. Detection of binding of shark single domain antibodies prepared according to the present invention to novel coronavirus N protein
Diluting recombinant novel coronavirus N protein to 4 mug/mL by PBS according to the optimal antigen coating concentration obtained by orthogonal experiments, adding 100 mug of coated ELISA plate into each hole, and conventionally blocking by 1% BSA; the purified recombinant antibody is diluted in a gradient way according to the proportion of 1:2, and is sequentially added into corresponding holes for incubation for 1.5h at 37 ℃; after washing, HRP-conjugated antibody against striped bamboo shark vNAR was added, incubated at 37℃for 1h, after washing, 150. Mu.L of TBM chromogenic substrate was added to each well, and after 10min of exposure to light, 150. Mu.L of 2M H was added to each well 2 SO 4 Terminating the color development, reading a value at an OD450nm by using an enzyme-labeled instrument, and detecting the combination condition; referring to FIG. 3, the experimental results show that the N05 antibody and the N38 antibody have higher affinity for binding to the N protein of the novel coronavirus, and the IC50 of the N05 antibody and the N38 antibody are respectively 1.826+ -0.03 nM and 2.107+ -0.045 nM.
2. Detection of endogenous New coronavirus N protein by shark Single-domain antibodies prepared according to the present invention
2.1 infection of HEK293T cells with novel coronavirus N protein pseudovirus: HEK293T cells were seeded in 6-well plates in DMEM+10% FBS at 37deg.C in 5% CO 2 Culturing for 48 hours; new coronavirus N-gene pseudovirus (Lenti-EF 1. Alpha. -SARS-COV-2-nucleocasid-Flag/CMV-Puro) was diluted to 10 with DMEM+1% FBS 5 pfu/mL, 500uL was added to 6-well plate, control group without pseudovirus infection was set at the same time, and after mixing by shaking gently, 37℃and 5% CO 2 Incubating for 6 hours; the incubation was aspirated, washed 3 times with PBS, 1mL of DMEM (10% FBS+0.5% methylcellulose) was added to each well, 37℃and 5% CO 2 Culturing for 48 hours;
2.2, extraction of total cell proteins: sucking out the culture solution, washing for 2 times by using PBS, adding 0.05% pancreatin digestive juice for treatment for 10min, sucking out HEK293T cells in the infected group and the control group, transferring the HEK293T cells to a 1.5ml centrifuge tube, centrifuging at 4000rpm for 5min, collecting cells, washing for 3 times by using PBS, and removing residual pancreatin; adding 200uL of cell lysate into a centrifuge tube, performing ultrasonic disruption for 30s to obtain a crude cell total protein extract, and determining the protein concentration by a BCA method;
2.3, western Blot detection: protein samples of cells of the infected group and the control group are diluted to 100 mug/mL by PBS, and the protein loading amount is 1 mug; SDS-PAGE electrophoresis: separating gel 80V for 20min; concentrating the gel 120V for 80min; transferring film by semi-dry transfer method: transferring the protein to a PVDF membrane, soaking the PVDF membrane in methanol for more than 15S before using, and soaking the PVDF membrane in a transfer buffer for 15min; transfer conditions: 23V,30min; sealing with 5% skimmed milk powder, and incubating at 37deg.C for 2 hr; adding the N05 and N38 antibodies after affinity purification (the antibodies are diluted to 0.5 mug/mL by TBST), and incubating at 37 ℃ for 1h; after 5 times of TBST washing, adding an HRP-coupled mouse anti-striped bamboo shark vNAR antibody, and incubating for 1h at 37 ℃; TBST is washed for 5 times, ECL chemiluminescent liquid A and ECL chemiluminescent liquid B are mixed in equal volume and then sprayed on a film, and a chemiluminescent gel imager (BIORAD ChemiDoc XRS) is used for exposure and color development; the experimental results are shown in fig. 4: the prepared anti-novel coronavirus N protein single-domain antibodies N05 and N38 can specifically recognize endogenous novel coronavirus N proteins generated in pseudovirus infected cells.
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 (5)
1. A shark single domain antibody targeting a novel coronavirus N protein, characterized in that: the amino acid sequence of the shark single domain antibody is shown as SEQ ID NO. 1 or SEQ ID NO. 2.
2. A nucleic acid molecule encoding the shark single domain antibody of claim 1.
3. A vector comprising a nucleic acid molecule encoding the shark single domain antibody of claim 2.
4. A method for detecting a novel coronavirus N protein for non-diagnostic purposes, characterized by: the method comprises the following steps:
s1, infecting HEK293T cells by using a novel coronavirus N protein pseudovirus;
s2, extracting total cell proteins and transferring the proteins to a PVDE membrane through electrophoresis and separation;
s3, carrying out Western Blot detection by contacting the affinity purified shark single domain antibody according to claim 1 with PVDE membrane.
5. Use of a shark single domain antibody targeting a novel coronavirus N protein as claimed in claim 1 in the manufacture of a novel coronaantibody detection product or a pharmaceutical composition.
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| WO2023077287A1 (en) * | 2021-11-03 | 2023-05-11 | City university of hong kong shenzhen research institute | High-affinity anti-egfp and anti-sars-cov-2 vnar single domain antibodies and use thereof |
| WO2023108666A1 (en) * | 2021-12-19 | 2023-06-22 | 中国人民解放军总医院 | Ultra-high affinity small protein targeting s protein of covid-19 virus and use |
| CN114106187B (en) * | 2022-01-05 | 2023-01-24 | 中国海洋大学 | Specific shark single-domain antibody targeting OGT (one glass solution) and preparation method and application thereof |
| CN117088973A (en) * | 2022-01-28 | 2023-11-21 | 集美大学 | Shark-derived nanobody targeting SARS-CoV-2RBD protein and application |
| CN115716867B (en) * | 2022-11-24 | 2023-08-08 | 扬州大学 | V-type secretion system MisL exhibiting expression novel coronavirus receptor binding domain B cell epitope antigen and application |
| CN115724963B (en) * | 2022-12-07 | 2024-04-30 | 复旦大学 | Nanometer single domain antibody targeting novel coronavirus S-NTD protein and application thereof |
| CN116063474A (en) * | 2023-02-09 | 2023-05-05 | 中国海洋大学 | Shark single domain antibody targeting SARS-CoV-2-S1-RBD, application and kit thereof |
| CN117129596B (en) * | 2023-08-29 | 2024-04-23 | 首都医科大学附属北京儿童医院 | Establishment of quantitative detection method of STING agonist cyclic dinucleotide cGAMP based on high performance liquid technology |
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| CN112062839A (en) * | 2020-09-22 | 2020-12-11 | 石河子大学 | Nano antibody based on novel coronavirus S protein S1 subunit and application thereof |
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