CN113336844A - Shark single-domain antibody targeting new coronavirus N protein, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a shark single domain antibody of a targeted new coronavirus N protein, a preparation method and application thereof, belonging to the technical field of biology; 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; the invention also provides a preparation method of the shark single domain antibody of the targeted new coronavirus N protein, which comprises the steps of separating immune striped bamboo shark peripheral blood lymphocytes, extracting total RNA (ribonucleic acid), and performing reverse transcription to obtain cDNA (complementary deoxyribonucleic acid); amplifying a striped bamboo shark vNAR fragment by taking cDNA as a template, and connecting the striped bamboo shark vNAR fragment with a carrier to construct a phage library; panning and identifying positive clones of the N protein of the new coronavirus from a phage library; constructing an expression vector, and inducing and expressing the new coronavirus N protein single domain antibody to finally obtain the shark single domain antibody targeting the new 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
The coronavirus belongs to the family of coronavirus and the genus coronavirus, and is a single-stranded positive-sense RNA virus; since the first virus was discovered in 1937, several coronaviruses were identified, belonging to the genus α, β, γ and δ, among which 7 coronaviruses were able to infect humans, HCoV-229E, HCoV-NL63 of the genus α and HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV and the novel coronaviruses SARS-CoV-2 identified in 2019.
SARS-CoV-2 is respiratory virus carried by droplet, different from any one of the six kinds of coronavirus capable of infecting human being known in the past, the virus has at least 17 sites related to amino acid change on different protein, and is easy to be propagated by people in close contact people, thus causing full spread of outbreak, and needing rapid diagnosis and detection to control outbreak and spread of disease.
The SARS-CoV-2 genome is composed of about 30000 nucleotides, and encodes four structural proteins, including S protein (Spike protein, spinous process protein), E protein (Envelope protein), M protein (Membrane protein) and N protein (Nucleocapsid protein), N protein, which is the most abundant protein in coronavirus, and is also a highly immunogenic phosphoprotein, and is expressed in large quantities during virus infection, and nearly 2000N protein monomers exist in each viral RNA genome copy. During SARS-CoV in 2003-2004, the presence of N protein was detectable in serum samples from patients at the early stage of viral infection, and N protein detection was considered as a reliable basis for the detection of SARS-CoV. Data from studies since the SARS-CoV-2 pandemic indicate that large amounts of SARS-CoV-2 virus N protein are detected in nasopharyngeal swab samples from newly coronavirused individuals. The new coronavirus N protein has high immunogenicity, high copy number in infected body and high protein stability, and is important target for detecting SARS-CoV-2 virus.
At present, the detection method of the new coronavirus mainly relies on nucleic acid detection, namely qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) to detect the virus genome RNA in respiratory tract samples. Nucleic acid detection has the advantages of high sensitivity, large parallel detection amount and the like, but also faces the challenges of large sample pretreatment difficulty, high reverse transcription reagent cost, 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 nucleic acid detection, and plays a role in the timely diagnosis, treatment, prevention and control of the novel coronary pneumonia. Specific IgM and IgG are the most common new coronavirus detection antibodies at present, but clinical application also reflects that more false positives appear, which troubles clinical decision.
Heavy chain antibodies, as antibody molecules that naturally lack a light chain, have greater advantages over traditional IgG antibody molecules in genetic engineering. The single domain antibody (single domain antibody) modified from the heavy chain antibody has the capability of combining with antigen in a natural state, has better affinity retention compared with the single domain antibody of human or mouse source, and has various advantages. In recent years, single domain antibodies have attracted tremendous research enthusiasm, and have made great progress in exploring the origin of antigen receptors, developing vaccines, therapeutic drugs, diagnostic reagents, biotechnological research tools, and the like. The best research and development in terms of single domain antibodies is at present the camel single domain antibody, and following the discovery of the single domain antibody by the camel, Greenberg et al have successively discovered from the body of cartilaginous fish such as nurse shark, an 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). The variable region vNAR of the IgNAR is recombined and expressed to obtain the antibody molecule fragment with complete function, which is called shark single-domain antibody. Chinese patent publication No. CN106831981A discloses a single domain antibody protein scaffold and a preparation method thereof, and specifically discloses a single domain antibody derived from striped bamboo shark, the single domain antibody protein scaffold sequence composition of which is FR1, CDR1, FR2, CDR3 and FR3 regions in sequence, wherein FR1, FR2 and FR3 regions are fixed amino acid sequences, CDR1 and CDR3 regions are antibody complementarity determining regions, the amino acid sequences thereof are variable, and the CDR1 and CDR3 regions determine to be combined with different antigens, proving that the shark single domain antibody has an antigen binding region, can realize antigen-antibody combination, and is used for diagnosis and treatment; in addition, WO2010033913A1 discloses antibodies, mimetics and uses thereof, and specifically discloses shark and camelid heavy chain antibodies and analogs thereof that are useful for diagnosis, therapy and both.
Therefore, the shark single-domain antibody is used for detecting the new coronavirus antigen protein and preparing the antibody detection kit, so that the method is completely feasible and has high clinical application value.
Disclosure of Invention
In order to solve the problems that high-sensitivity detection of a new coronavirus N protein needs to be further improved and antibodies need to be developed and used in the prior art, the invention creatively provides a shark single-domain antibody targeting the new coronavirus N protein, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
a shark single-domain antibody targeting new coronavirus N protein has an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2.
A shark single-domain antibody targeting a new coronavirus N protein has a nucleotide sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4.
A preparation method of a shark single-domain antibody targeting a novel coronavirus N protein comprises the following steps:
(1) separating peripheral blood lymphocytes of the immune striped bamboo shark, extracting total RNA, and performing reverse transcription to obtain cDNA;
(2) amplifying a striped bamboo shark vNAR fragment by using cDNA as a template, and connecting the amplified striped bamboo shark vNAR fragment with a carrier to construct a phage library: performing two rounds of PCR amplification by adopting nested PCR to obtain a striped bamboo shark single-domain antibody vNAR gene; carrying out enzyme grafting on the vNAR gene fragment and a vector to construct a phage library;
(3) panning from the phage library identified positive clones of the new coronavirus N protein: performing amplification culture, enrichment, panning and identification on a phage library, performing sequencing analysis on all positive clones with OD450 values greater than 1, and eliminating repetition to finally obtain 2 strains of single-domain antibodies, namely an N05 antibody and an N38 antibody of a shark single-domain antibody; obtaining vNAR gene sequences of the N05 antibody and the N38 antibody through sequencing;
(4) constructing an expression vector, and inducing and expressing the new coronavirus N protein single domain antibody: cloning vNAR gene sequences of the N05 antibody and the N38 antibody to PET30a to obtain expression vectors of the N05 antibody and the N38 antibody, transforming the expression vectors to an E.coli BL21 strain, performing amplification culture, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, breaking bacteria, collecting and purifying recombinant antibody protein.
Further, the immune striped bamboo shark in the step (1) is obtained by injecting recombinant new coronavirus N protein subcutaneously into the striped bamboo shark, the dose is 5nM/kg, the interval period is 14 days, and 14 days after 6 times of immunization are finished.
Further, in the enzyme grafting process in the step (2), firstly, carrying out enzyme digestion on the striped mottled bamboo shark vNAR gene obtained by PCR and the pComb3XSS vector by using Sfi I, and connecting T4 ligase after enzyme digestion recovery; the ligation product was transformed into XL1-Blue competent cells, cultured until OD660 reached 0.6, and helper phage VCSM13 was added, and after overnight culture, the culture medium 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 the N protein of a novel coronavirus, for non-diagnostic purposes, comprising the steps of:
s1, infecting HEK293T cells by the new coronavirus N protein pseudovirus;
s2, extracting total cell protein, and carrying out electrophoresis, separation and transfer on the protein to a PVDE membrane;
s3, Western Blot detection by contacting the affinity-purified shark single domain antibody of claim 1 or 2 with PVDE membrane.
An application of shark single domain antibody of targeting new coronavirus N protein in preparing new coronavirus antibody detection product or medicinal composition is provided.
Compared with the prior art, the invention has the beneficial effects that:
1. the shark single-domain antibody of the new coronavirus N protein provided by the invention can specifically recognize the endogenously generated new coronavirus N protein, can be used as a quality control antibody of a new coronavirus antibody detection kit, and fills the blank that no shark single-domain antibody exists in an ELISA detection kit in the serological detection market of the new coronavirus; the shark single-domain antibody provided by the invention has higher binding affinity of the N05 antibody and the N38 antibody with the N protein of the novel coronavirus measured by an ELISA method, and the IC of the antibodies501.415nM and 1.749nM, respectively.
2. Compared with the traditional monoclonal antibody and the camel single domain antibody, the shark single domain antibody provided by the invention has unique advantages in detecting the N protein of the new coronavirus, firstly, the CDR3 (complementary-determining Region 3) structural domain of the shark single domain antibody is in a convex ring shape and can identify the hidden epitope of the N protein of the new coronavirus, so that the shark single domain antibody is superior to the traditional monoclonal antibody in the aspect of combining with the antigen; secondly, in the production aspect, the traditional antibody plays a recognition and binding function, usually needs glycosylation modification, needs to be expressed by mammalian cells, and is expensive in manufacturing cost, while the shark single domain antibody provided by the invention can play a role without glycosylation modification, can be expressed in a large amount by a prokaryotic cell or yeast expression system, and greatly reduces the production cost.
3. The N05 antibody and the N38 antibody of the shark single-domain antibody provided by the invention can be continuously reformed, the affinity of the antibody is provided, and the antibody can be directly used for developing a high-sensitivity new crown antigen detection kit and can also be used for debugging the new crown antibody detection kit.
Drawings
FIG. 1 shows the results of phage display screening according to example 1 of the present invention; wherein, FIG. 1A shows the enrichment change of phage after three rounds of panning; FIG. 1B shows the Phage polyclonal supernatant detected by Phage-ELISA;
FIG. 2 is a diagram showing the result of SDS-PAGE gel electrophoresis of a shark prepared single domain antibody according to example 2 of the present invention;
FIG. 3 is a schematic diagram showing the binding of shark single domain antibody prepared by ELISA detection to the novel coronavirus N protein according to example 3 of the present invention;
FIG. 4 is a schematic diagram showing the recognition of the endogenous novel coronavirus N protein by the shark single domain antibody prepared by Western Blot detection according to example 3 of the present invention.
Detailed Description
In order to facilitate understanding of the present invention, the technical solutions of the present invention will be further described with reference to the following detailed description and the accompanying drawings, but the present invention is not limited thereto.
Example 1
A shark single-domain antibody targeting a new coronavirus N protein is named as an N05 antibody, the amino acid sequence of the shark single-domain antibody is shown as SEQ ID NO. 1, and the nucleotide sequence of the shark single-domain antibody is shown as SEQ ID NO. 3.
A shark single-domain antibody targeting a new coronavirus N protein is named as an N38 antibody, the amino acid sequence of the shark single-domain antibody is SEQ ID NO. 2, and the nucleotide sequence of the shark single-domain antibody is SEQ ID NO. 4.
Example 2
A preparation method of a shark single-domain antibody targeting a novel coronavirus N protein comprises the following steps:
(1) separating peripheral blood lymphocytes of the immune striped bamboo shark, extracting total RNA, and performing reverse transcription to obtain cDNA;
immunizing the striped bamboo shark with the recombinant new coronavirus N protein: the purified prokaryotic expression recombinant new coronavirus N protein is used for immunizing striped bamboo shark, and the specific method comprises the following steps: emulsifying the antigen with Freund's adjuvant according to a dose of 5nM/kg, and performing multi-point subcutaneous injection on the striped spot bamboo shark, wherein the immunization interval is 14 days each time, and after 14 days of completing the 6 th immunization, collecting blood from the tail vein of the striped spot bamboo shark to 5mL in a heparin sodium anticoagulation tube to complete the immunization on the striped spot bamboo shark;
obtaining of striped bamboo shark peripheral blood lymphocytes: 5mL of blood obtained by venous blood collection is equally divided into 5 centrifuge tubes with 15mL, physiological saline with the volume 10 times that of the blood sample is respectively added for dilution, and the mixture is slowly and uniformly mixed; sucking the diluted blood sample on the liquid surface of the separation liquid, centrifuging for 30min at a speed of 300 g/min; after centrifugation, the lymphocytes are in a grey-white belt 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 pipette, 10mL PBS buffer solution is added for resuspension and washing, the solution is 350g/min and centrifuged for 10min, the operation is repeated for 3 times, and the peripheral blood lymphocytes of the immune striped bamboo shark are collected;
RNA extraction: adding 1mL of Tripure Reagent into a centrifugal tube filled with a lymphocyte sample, carrying out ultrasonic disruption for 30s, adding 1/5 volumes of chloroform, standing at room temperature for 5min, and centrifuging at 12000rpm and 42 ℃ for 15 min; carefully transferring the supernatant into another centrifuge tube, adding isopropanol with the same volume, mixing uniformly, standing at room temperature for 20min, centrifuging at 12000rpm and 42 ℃ for 5 min; slowly sucking supernatant, adding 75% ethanol, washing, centrifuging at 12000rpm and 42 deg.C for 5 min; carefully pouring out the supernatant, drying the RNA precipitate in the air, adding DEPC water for dissolving, determining the RNA concentration by a nucleic acid determination quantitative instrument, and simultaneously taking a proper amount of RNA for 1% agarose gel electrophoresis to identify the quality of the RNA;
obtaining cDNA through reverse transcription:
first, add the following mixture to a 0.2mL PCR tube on ice:
| Oligoc(dT)(10μM) | 1μL |
| total RNA | 5μg |
Secondly, the mixture is heated at 65 ℃ for 10min and then immediately placed on ice;
then, under ice-bath conditions, the following reactions were further 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 the mixture is slightly centrifuged, the mixture is immediately placed at 37 ℃ for incubation for 90min to synthesize first strand cDNA, and the first strand cDNA is heated at 65 ℃ for 5min to terminate the reaction;
(2) amplifying a striped bamboo shark vNAR fragment by taking cDNA as a template, and connecting the striped bamboo shark vNAR fragment with a carrier to construct a phage library;
amplification of the zona striata shark single-domain antibody vNAR gene: performing two rounds of PCR amplification by adopting nested PCR to obtain a striped bamboo shark single-domain antibody vNAR gene; the primers used in the first round of PCR are an upstream primer (F1): ATGAATATTTTCTTGTTTTCGGGCC, downstream primer (R1): ATAGTATCCGCTAATTAGACAAA, respectively;
the first round of PCR amplification system is:
| 5×Premix Taq | 25μL |
| F1(10mM) | 0.5μL |
| R1(10mM) | 0.5μL |
| cDNA | 2μL |
| ddH2O | to50μL |
the first round of PCR amplification procedure was:
second round of nested PCR was performed using the first round of PCR products, and the primers used in the second round of PCR were, the upstream primer (F1'): CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT, respectively; downstream primer (R1'): CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG, respectively;
the second round of PCR amplification system is:
| 5×Premix Taq | 25μL |
| F1’(10mM) | 0.5μL |
| R1’(10mM) | 0.5μL |
| first round PCR amplification product | 2μL |
| ddH2O | to50μL |
The second round of PCR amplification procedure was:
after the second round PCR products were electrophoresed using 2% agarose gel, the vNAR fragment (about 500bp) was gel-purified using gel-extraction purification kit; detecting the purified agarose gel electrophoresis, wherein the band is unique (about 500bp) and clear, and obtaining the vNAR gene fragment;
the vNAR gene is linked to a vector: carrying out double enzyme digestion on the vNAR gene fragment and the vector pComb3xss by using an endonuclease Sfi I respectively, wherein the enzyme digestion reaction system is as follows:
| Sfi I | 120U |
| CutSmart | 5μL |
| DNA | 5μg |
| ddH2O | to 50μL |
the enzyme digestion reaction conditions are as follows: reacting for 6 hours at 55 ℃; after the enzyme digestion reaction is finished, recovering the vNAR gene fragment and the pComb3xss vector by using a DNA purification recovery kit, and connecting the recovered vNAR gene fragment and the pComb3xss vector by using T4 ligase, wherein the connecting system is as follows:
| pComb3xss vector | 1μg |
| vNAR fragment | 300μg |
| 10X T4 buffer | 4μL |
| T4 Ligase | 2μL |
| ddH2O | to 50μL |
After connecting for 3h at 16 ℃, purifying and recovering a connecting product, and dissolving ddH 2O;
construction of phage library: placing the electric shock cup on ice for precooling, taking XL1-Blue electric conversion competence to melt on ice, adding 5 mu L of the ligation product after the mixture is dissolved, gently mixing the mixture evenly, and placing the mixture on ice for 5 min; transferring into a pre-cooled electrotransfer cup, and carrying out electrotransfer instrument program: 1800kV, 5ms, and electric shock conversion; immediately adding 1mL of SOCG culture medium after electrotransformation, culturing at 37 ℃ and 180rpm for 60 min; diluting the cultured bacterial liquid 100uL in a gradient manner according to the proportion of 1:10, 1:100 and 1:1000, coating the diluted bacterial liquid on an SOC plate containing ampicillin resistance, culturing at 37 ℃ overnight, calculating the colony number on the plate, and calculating the library capacity (the library capacity at least needs to reach 10)7pfu/ml); the remaining post-transformation cultured bacterial solution was spread on SOC plates containing ampicillin resistance and cultured overnight at 37 ℃ and glycerol was added to a final concentration of 15% after scraping and the bacterial solution was stored at-70 ℃.
(3) Panning and identifying positive clones of the N protein of the new coronavirus from a phage library;
amplification of phage library: 1ml of the above-mentioned cultured bacterial suspension (containing about 10 parts of the bacterial suspension)8Individual transformed cells) were added to 100mL of 2XYT medium, cultured at 37 ℃ and 250rpm until OD600 reached 0.6; adding helper phage VCSM13 at MOI of 1:20, culturing at 37 deg.C and 220rpm for 1h, adding ampicillin with final concentration of 100 μ g/ml and 0.1mM IPTG, culturing overnight at 37 deg.C, centrifuging overnight cultured bacterial liquid at 4 deg.C and 10000rpm for 15min, and collecting supernatant; adding 1/4 volume of PEG/NaCl into the supernatant, settling for 30 minutes on ice at 4 ℃, 10000rpm, centrifuging for 10min, discarding the supernatant, adding 2mL of PBS for heavy suspension precipitation, and storing at 4 ℃;
affinity screening of single domain antibodies against the new coronavirus N protein: diluting the new coronavirus N protein to 25 mu L/mL, coating an immune tube (2 mL/tube, 10 mu L in total), and incubating overnight at 4 ℃; the next day, PBS wash 3 times, blocking the immune tube with blocking solution (2% skim milk PBS, MPBS), incubating for 2h at 37 ℃; after 3 washes with PBS, 1 × 10 was added per well11phages above pfu (dissolved in 200. mu.L MPBS) were incubated for 2h at 37 ℃; PBST (PBS + 0.5% Tween-20)) After washing 3 times with PBS each, 2ml of Glycine-HCl (pH 2.5) elution buffer was added, and after gentle running elution for 10min, the pH was neutralized to 7.0 by adding an equal volume of Tris-HCl (pH 7.4); the phage obtained by elution is treated according to the method 10-1To 10-8Performing gradient dilution, adding fresh XL1-blue bacterial liquid, and incubating for 30min with gentle shaking at 37 ℃; coating 100uL of each gradient infected bacterial liquid on an SOC (system on chip) plate containing benzyl amine resistance, and calculating the output titer of the phage; adding an equal volume of XL1-blue (OD600 is 0.5) bacterial liquid into the rest phage eluate, culturing at 37 ℃ and 150rpm for 1h, centrifuging at 4000rpm for 5min, resuspending the precipitate, and coating an SOC plate containing aminobenzyl resistance; scraping thallus on the plate the next day, and storing glycerol for the next round of screening; affinity screening is carried out for 3 rounds, the coating amount of the antigen is reduced from round to round, the coating amount of the second round is 25 mu g, and the coating amount of the third round is 12.5 mu g; referring to fig. 1, in the three rounds of affinity panning, the enrichment amount and recovery rate of the phage specific to the new coronavirus N protein are increased round by round;
phage Elisa screens positive monoclonal antibodies against the N protein single domain of the new coronavirus: taking a 72-hole culture plate, adding 400 mu L of 2 XYT-Gamp+kan+Selecting monoclonal antibody on the output Plate after affinity screening, inoculating the monoclonal antibody into a hole, marking as Master Plate, culturing at 37 ℃ and 200rpm overnight; taking another 72-hole culture plate, and taking 400 μ L of culture plate containing 1 × 10102XYT of pfu VCSM13 helper phage-Gamp+kan+To each well, labeled P1 Plate; adding 40ul of culture solution from each Master Plate to the corresponding hole of the P1 Plate, culturing at 37 deg.C and 150rpm for 2h with shaking; centrifuging at 4000rpm for 20min, discarding the supernatant, and adding 400 μ L of 2XYT to each wellamp+kan+The culture solution is subjected to shaking culture at 37 ℃ and 250rpm overnight; centrifuging at 4000rpm for 20min, mixing 320 μ L of supernatant with 80 μ L of MPBS, and storing at 4 deg.C for use, to complete preparation of phage recombinant antibody;
phage Elisa detection: diluting the new coronavirus N protein to 0.5 mu g/mL, coating a 96-well enzyme label plate with 200 mu L/well, and incubating for 1h at 37 ℃; washing with PBS for 3 times, adding 1% BSA, and blocking at 37 deg.C for 1.5 h; correspondingly adding the prepared phage recombinant antibody, and incubating for 2h at 37 ℃; PBST and PBS were washed 3 times, and enzyme-labeled Anti-M13-HRP (diluted with MPBS at a ratio of 1: 4000) was added) Incubating at 37 deg.C for 1 hr, washing PBST and PBS for 3 times, adding 150 μ L TBM chromogenic substrate to each well, standing in dark for 10min, and adding 150 μ L2M H to each well2SO4Terminating color development, reading at OD450nm by using an enzyme-labeling instrument, and analyzing sequencing of all positive clones with OD450 values greater than 1 as shown in figure 1B, and removing repetition to finally obtain 2 positive clones which are respectively named as an N05 antibody and an N38 antibody;
the amino acid sequence of the N05 antibody is:
MNIFLFSFLLAWLPNVFTQWVEQTPTTTTKEAGESLTINCVLKGSSYGLCNTN WYFTKKSVTKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKPSM GWDETGYCLGLGEGGGTILTVKPGK(SEQ ID NO:1)；
the nucleotide sequence of the N05 antibody is:
AATATTTTCTTGTTTTCGGTCCTTTTAGCCTGGTTACCAAATGTCTTTACTCA ATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCA CTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTATGGATTGTGTAACACG AACTGGTATTTCACAAAAAAGAGCGTTACAAAGAAGGAGAGCTTATCAAA TGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTT TGCGAATTAGCGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAC CGTCTATGGGCTGGGATGAGACCGGTTACTGTCTGGGATTGGGGGAAGGA GGCGGCACCATTCTGACTGTGAAACCTGGCAAA(SEQ ID NO:3)
the amino acid sequence of the N38 antibody is:
MNIFLFSFLLAWLPNVFTQWVEQTPRTTTKEAGESLTINCVLKGSSYVLCNTY WYFTKKGATKKETLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYS RYSWDGCSVILLATGSDYYEGGGTILTVKPGK(SEQ ID NO:2)；
the nucleotide sequence of the N38 antibody is:
AATATTTTCTTGTTTTCGTTCCTTTTAGCCTGGTTACCAAATGTCTTTACTCA ATGGGTTGAACAAACACCGAGAACGACAACAAAGGAGGCAGGCGAATCA CTGACCATCAATTGCGTCCTAAAAGGTTCCAGTTATGTATTGTGTAATACGT ACTGGTATTTCACAAAAAAGGGCGCTACAAAGAAGGAGACCTTATCAAAT GGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTT GCGAATCAGTGACCTGCGAGTTGAAGACAGTGGTACATATTACTGTAAAGC GTATAGTCGGTACAGCTGGGATGGGTGTAGTGTTATACTGTTAGCGACAGG TTCCGACTATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAA A(SEQ ID NO:4)。
(4) constructing an expression vector, and inducing and expressing the 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 the primer F: CCATGGTCCAATGGGTTGAACAAACACCGA, primer R: CTCGAGTTTGCCAGGTTTCACAGTCAG, respectively; purifying the PCR product by using a purification kit; after purification, agarose gel electrophoresis detection shows that the band is unique (about 400bp) and clear, NotI and xHoI are used for double enzyme digestion of the vNAR fragment and the expression vector Pet30a, and the enzyme digestion reaction system is as follows:
| DNA | 5μL |
| 10x H Buffer | 5μL |
| Nde I | 2μL |
| xHoI | 2μL |
| ddH2O | to 50μL |
carrying out enzyme digestion reaction at 37 ℃ for 1h, carrying out 2% agarose gel electrophoresis, cutting out gel respectively, and recovering corresponding vNAR genes and vectors; after the recovered vNAR gene and the vector were ligated using T4 ligase, BL21(DE3) competent cells were transformed, plated with LB plates containing kanamycin resistance, and cultured overnight at 37 ℃; the next day, after the positive clones are preliminarily screened out by PCR, sequencing analysis is carried out to determine positive strains;
prokaryotic expression and purification of the anti-new coronavirus N protein single domain antibody: selecting the obtained positive strain, inoculating the positive strain into LB liquid culture medium containing kanamycin, culturing overnight at 37 ℃ with a shaker at 200rpm, and extracting plasmids; transforming the obtained recombinant plasmid into Escherichia coli BL21, culturing at 37 deg.C under shaking at 200rpm until OD600 is 0.6-1.0, adding IPTG solution with final concentration of 0.5mM, and performing induced expression at 25 deg.C under shaking at 200rpm for 12-18 h; after induction expression is finished, centrifugally collecting thalli, crushing the thalli by ultrasonic waves, centrifugally collecting supernate, purifying the antibody protein by adopting a conventional His-tag affinity chromatography method, wherein the purity of the obtained purified recombinant antibody protein is more than 95%, and the SDS-PAGE electrophoresis detection result is shown in figure 2.
Example 3
Nucleic acids, vectors, compositions or complexes
The present invention relates to nucleic acid molecules encoding shark single domain antibodies of the invention, which may be RNA, DNA or cDNA.
The nucleic acid 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 providing for the 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, and the like). 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 antibody produced according to the invention to New coronavirus N protein
Diluting the recombinant new coronavirus N protein to 4 mu g/mL by PBS according to the optimal antigen coating concentration obtained by orthogonal experiment, adding 100 mu L of coated enzyme label plate in each hole, and conventionally sealing by 1% BSA; diluting the purified recombinant antibody according to the proportion of 1:2 in a gradient manner, sequentially adding the diluted recombinant antibody into corresponding holes, and incubating for 1.5h at 37 ℃; after washing, HRP-coupled rat anti-striped mottled bamboo is addedIncubating shark vNAR antibody at 37 deg.C for 1h, washing, adding 150 μ L TBM chromogenic substrate per well, standing in dark for 10min, and adding 150 μ L2M H per well2SO4The color development is stopped, and the binding condition is detected by reading the value at OD450nm by using an enzyme-labeling instrument; referring to FIG. 3, the results of the experiment showed that the N05 antibody and the N38 antibody bound to the N protein of the novel coronavirus with high affinity, and their IC50 was 1.826. + -. 0.03nM and 2.107. + -. 0.045nM, respectively.
2. Detection of endogenous novel coronavirus N protein by shark single-domain antibody prepared according to invention
2.1, infection of HEK293T cells by the new coronavirus N protein pseudovirus: HEK293T cells were plated in 6-well plates in DMEM + 10% FBS at 37 ℃ and 5% CO2Culturing for 48 hours; new coronavirus N gene pseudovirus (Lenti-EF1 alpha-SARS-COV-2-Nucleocapsid-Flag/CMV-Puro) was diluted to 10 with DMEM + 1% FBS5pfu/mL, adding 500uL into 6-well plate, setting control group without infecting pseudovirus, shaking gently, mixing, and reacting at 37 deg.C with 5% CO2Incubating for 6 h; the incubation solution was aspirated, washed 3 times with PBS, and 1mL DMEM (10% FBS + 0.5% methylcellulose) was added to each well at 37 ℃ with 5% CO2Culturing for 48 h;
2.2, extraction of total cell protein: sucking out the culture solution, washing with PBS for 2 times, adding 0.05% pancreatin digestive juice, treating for 10min, sucking out HEK293T cells of the infected group and the control group, transferring to a 1.5ml centrifuge tube, centrifuging at 4000rpm for 5min, collecting the cells, washing with PBS for 3 times, and removing the residual pancreatin; adding 200uL of cell lysate into a centrifugal tube, carrying out ultrasonic disruption for 30s to obtain a crude extract of total cell protein, and measuring the protein concentration by using a BCA method;
2.3, Western Blot detection: diluting protein samples of infected group and control group cells to 100 mu g/mL by using PBS, wherein the loading amount of the protein is 1 mu g; SDS-PAGE electrophoresis: separating gel at 80V for 20 min; concentrating the gel at 120V for 80 min; transferring the membrane by a semi-dry transfer method: transferring the protein to a PVDF membrane, soaking the PVDF membrane in methanol for more than 15S before use, and then soaking in a transfer buffer for 15 min; film transferring conditions: 23V, 30 min; sealing with 5% skimmed milk powder, and incubating at 37 deg.C for 2 hr; adding affinity purified N05 and N38 antibodies (the antibodies are diluted to 0.5 mu g/mL by TBST), and incubating for 1h at 37 ℃; washing with TBST for 5 times, adding HRP-coupled antibody of mouse anti-striped bamboo shark vNAR, and incubating at 37 deg.C for 1 h; washing with TBST for 5 times, mixing ECL chemiluminescence solution A and solution B at equal volume, spraying onto membrane, and exposing with chemiluminescence gel imager (BIORAD ChemiDoc XRS); the results of the experiment are shown in FIG. 4: the prepared single domain antibodies N05 and N38 for resisting the new coronavirus N protein can specifically recognize endogenous new coronavirus N protein generated in cells infected by pseudoviruses.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
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 shark single domain antibody targeting a novel coronavirus N protein, characterized in that: the nucleotide sequence of the shark single domain antibody is shown as SEQ ID NO. 3 or SEQ ID NO. 4.
3. A method for preparing a shark single domain antibody targeting the N protein of the novel coronavirus according to claim 1 or 2, comprising the following steps:
(1) separating peripheral blood lymphocytes of the immune striped bamboo shark, extracting total RNA, and performing reverse transcription to obtain cDNA;
(2) amplifying a striped bamboo shark vNAR fragment by using cDNA as a template, and connecting the amplified striped bamboo shark vNAR fragment with a carrier to construct a phage library: performing two rounds of PCR amplification by adopting nested PCR to obtain a striped bamboo shark single-domain antibody vNAR gene; carrying out enzyme grafting on the vNAR gene fragment and a vector to construct a phage library;
(3) panning from the phage library identified positive clones of the new coronavirus N protein: performing amplification culture, enrichment, panning and identification on a phage library, performing sequencing analysis on all positive clones with OD450 values greater than 1, and eliminating repetition to finally obtain 2 strains of single-domain antibodies, namely an N05 antibody and an N38 antibody of a shark single-domain antibody; obtaining vNAR gene sequences of the N05 antibody and the N38 antibody through sequencing;
(4) constructing an expression vector, and inducing and expressing the new coronavirus N protein single domain antibody: cloning vNAR gene sequences of the N05 antibody and the N38 antibody to PET30a to obtain expression vectors of the N05 antibody and the N38 antibody, transforming the expression vectors to an E.coliBL21 strain, performing amplification culture, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, breaking bacteria, collecting and purifying recombinant antibody protein.
4. The method for preparing a shark single domain antibody targeting the N protein of a novel coronavirus according to claim 3, wherein the shark single domain antibody comprises: in the step (1), the immune striped bamboo shark is a striped bamboo shark immunized by the recombinant new coronavirus N protein, which is obtained after 14 days of 6 times of immunization, wherein the dose of the immune striped bamboo shark is 5nM/kg, the interval period is 14 days, and the recombinant new coronavirus N protein is injected subcutaneously into the striped bamboo shark.
5. The method for preparing a shark single domain antibody targeting the N protein of a novel coronavirus according to claim 3, wherein the shark single domain antibody comprises: in the enzyme grafting process in the step (2), firstly, carrying out enzyme digestion on the striped mottled bamboo shark vNAR gene obtained by PCR and the pComb3XSS vector respectively by using Sfi I, and carrying out T4 ligase after enzyme digestion recovery; the ligation product was transformed into XL1-Blue competent cells, cultured until OD660 reached 0.6, and helper phage VCSM13 was added, and after overnight culture, the culture medium supernatant was collected to obtain a primary phage library.
6. A nucleic acid molecule encoding a shark single domain antibody as claimed in any of claims 1 or 2.
7. A vector comprising a nucleic acid molecule encoding a shark single domain antibody according to any of claims 1 or 2.
8. A method for the detection of the N protein of a novel coronavirus, for non-diagnostic purposes, characterized in that: the method comprises the following steps:
s1, infecting HEK293T cells by the new coronavirus N protein pseudovirus;
s2, extracting total cell protein, and carrying out electrophoresis, separation and transfer on the protein to a PVDE membrane;
s3, Western Blot detection by contacting the affinity-purified shark single domain antibody of claim 1 or 2 with PVDE membrane.
9. Use of a shark single domain antibody targeting the N protein of neocoronavirus according to any of claims 1 or 2 for the preparation of a detection product or a pharmaceutical composition for neocoronavirus.
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