CN114702575B - Nanometer antibody, recombinant vector, recombinant bacterium for resisting SARS-CoV-2S protein and application thereof - Google Patents
Nanometer antibody, recombinant vector, recombinant bacterium for resisting SARS-CoV-2S protein and application thereof Download PDFInfo
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- CN114702575B CN114702575B CN202210077156.4A CN202210077156A CN114702575B CN 114702575 B CN114702575 B CN 114702575B CN 202210077156 A CN202210077156 A CN 202210077156A CN 114702575 B CN114702575 B CN 114702575B
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Abstract
The invention provides a nano antibody for resisting SARS-CoV-2S protein, a recombinant nano antibody, a recombinant vector, recombinant bacteria and application thereof, and relates to the technical field of biological medicine. The nano antibody provided by the invention comprises C4 and C9, wherein both C4 and C9 are camel sources, and SARS-CoV-2S protein can be specifically identified. The invention fuses the C4 and C9 with human IgG Fc segment respectively, can obtain two recombinant nano antibodies of recombinant C4 and recombinant C9, and through verification, the recombinant C4 and the recombinant C9 can specifically recognize SARS-CoV-2S protein, and can be applied to the preparation of diagnostic reagents or antiviral drugs of new coronaviruses.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a nano antibody for resisting SARS-CoV-2S protein, a recombinant nano antibody, a recombinant vector, recombinant bacteria and application thereof.
Background
2019 novel coronavirus (SARS-CoV-2) is a novel strain of coronavirus found in humans in 2019. The SARS-CoV-2 virus invades host cells by binding of its spike protein, the S protein, to human angiotensin converting enzyme-2 (ACE 2). After infection with SARS-CoV-2 virus, alveolar macrophagocytes and epithelial cells release a large number of pro-inflammatory cytokines and chemokines; monocytes and neutrophils can be recruited to the site of infection and clear exudates containing viral particles and infected cells, leading to a deregulated inflammatory response. In this process, adaptive immunity is difficult to effectively initiate due to significant lymphocyte count reduction and dysfunction. Uncontrolled viral infection can lead to severe macrophage infiltration, further exacerbating lung injury. Meanwhile, the scattered SARS-CoV-2 virus can directly attack other organs, immune response can cause systemic inflammatory storm and microcirculation disturbance, and the factors act together to finally cause viral sepsis.
Nanobodies were first reported by belgium scientists in 1993 in Nature, and were a naturally deleted light chain antibody (VHH) present in alpaca peripheral blood, which was found in cartilage fish such as nurse shark, mackerel shark, scottish mackerel, and the like in 1995, and were the smallest unit known to bind to the antigen of interest. VHH has a molecular weight of only 15KD and is therefore also called Nanobody (Nb). The nanometer antibody has unique advantages, including small molecular weight, good solubility, strong stability, high affinity, low immunogenicity, good in vivo tissue permeability, easy passage through blood vessels or tissues to target sites, etc., has large development space, and has wide application, and can be clinically used for tumor treatment and also used as a diagnostic tool.
Thus, it is particularly urgent to find more effective antibodies for use in the treatment of SARS-CoV-2 virus infection.
Disclosure of Invention
In view of the above, the present invention aims to provide a nanobody, a recombinant vector, a recombinant bacterium and an application of the anti-SARS-CoV-2S protein, wherein both the nanobody and the recombinant nanobody can specifically recognize SARS-CoV-2S protein.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a group of nano antibodies for resisting SARS-CoV-2S protein, wherein the nano antibodies comprise C4 and C9, the amino acid sequence of the C4 is shown as SEQ ID NO.1, and the amino acid sequence of the C9 is shown as SEQ ID NO. 2.
The invention also provides a group of recombinant nanobodies against SARS-CoV-2S protein, comprising any of the above nanobodies and a human IgG Fc segment.
Preferably, the recombinant nanobody comprises recombinant C4 and recombinant C9, the amino acid sequence of the recombinant C4 is shown as SEQ ID NO.5, and the amino acid sequence of the recombinant C9 is shown as SEQ ID NO. 6.
The invention also provides a recombinant vector for expressing the recombinant nanobody, wherein the recombinant vector comprises a nucleotide sequence for encoding the recombinant nanobody and a basic vector.
Preferably, the nucleotide sequence for encoding recombinant C4 is shown as SEQ ID NO.8, and the nucleotide sequence for encoding recombinant C9 is shown as SEQ ID NO. 9.
Preferably, the base vector comprises pET-30a.
The invention also provides a construction method of the recombinant vector, which comprises the following steps: inserting a nucleotide sequence encoding the recombinant nanobody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
The invention also provides recombinant bacteria for expressing the recombinant nano antibody, and the basic bacteria of the recombinant bacteria comprise escherichia coli.
Preferably, the strain of E.coli includes Arctic Express.
The invention also provides application of the nano-antibody, the recombinant vector or the recombinant bacterium in preparation of antiviral drugs or virus infection diagnostic reagents.
The beneficial effects are that: the invention provides a group of nano antibodies for resisting SARS-CoV-2S protein, wherein the nano antibodies comprise C4 and C9, both C4 and C9 are camel sources, and SARS-CoV-2S protein can be specifically identified. The invention fuses the C4 and C9 with human IgG Fc segment respectively, can obtain two recombinant nano antibodies of recombinant C4 and recombinant C9, and through verification, both the recombinant C4 and the recombinant C9 can specifically recognize SARS-CoV-2S protein, and can be applied to the preparation of diagnostic reagents or antiviral drugs of new coronaviruses.
Drawings
FIG. 1 is a first round of DNA electrophoresis of nanobodies, DNA bands of gel wells from left to right are respectively: the sixth lane is a Marker of 1000bp (the sizes of the bands are 1000, 700, 500, 400, 300, 200 and 100bp in sequence), the first, second, third and fifth lanes are PCR products, the band is about 700bp, and the fourth lane is empty;
FIG. 2 is a second round of DNA electrophoresis of nanobodies, DNA bands of gel wells from left to right are respectively: the first lane is a Marker of 1000bp (the band size is the same as the above), the second, fourth and sixth lanes are PCR products, the band is about 400bp, and the third and fifth lanes are empty;
FIG. 3 is a schematic diagram of screening specific single positive clones by phage enzyme-linked immunosorbent assay (phage-ELISA): wherein 1 is SARS-CoV-2S protein coated on an ELISA plate, 2 is phage supernatant, 3 is mouse anti-km 13107 antibody, 4 is goat anti-mouse IgG (AP) antibody, 5 is TMB chromogenic solution;
FIG. 4 is a diagram showing SDS-PAGE electrophoresis staining of a treated sample, an effluent sample and an eluted sample after disruption of the recombinant C4 nanobody; the right figure is SDS-PAGE electrophoresis staining diagram of the treated sample, the effluent sample and the eluted sample after disruption of the recombinant C9 nanobody;
FIG. 5 is a western blot diagram of purified recombinant nanobodies, wherein the left panel is a recombinant C4 nanobody and the right panel is a recombinant C9 nanobody (M: protein molecular mass standard 1: purified anti-SARS-CoV-2S protein nanobody);
FIG. 6 is a western blot of SARS-CoV-2S protein, wherein the left panel is a recombinant C4 nanobody and the right panel is a recombinant C9 nanobody.
Detailed Description
The invention provides a group of nano antibodies for resisting SARS-CoV-2S protein, wherein the nano antibodies comprise C4 and C9, the amino acid sequence of the C4 is shown as SEQ ID NO.1, and the amino acid sequence of the C9 is shown as SEQ ID NO. 2.
The amino acid sequence of the C4 is shown in SEQ ID NO.1, and the C4 comprises 4 framework regions FR (FR 1, FR2, FR3 and FR 4) and 3 epitope complementary regions CDR (CDR 1, CDR2 and CDR 3). The amino acid sequence of the framework region FR of C4 according to the invention is preferably as follows:
FR1:QVQLQESGGGSVQAGGSIVLSCAAF(SEQ ID NO.10)；
FR2:MAWWRQGSDKVREQVAN(SEQ ID NO.11)；
FR3:SYSRSVEGRFTISRDNAKNTLTLQMNELKPEDTDMYYC(SEQ ID NO.12)；
FR4:WGPGTQVTVSS(SEQ ID NO.13)；
the corresponding nucleotide sequence is preferably as follows:
FR1:CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGC TGGAGGGTCTATAGTACTCTCCTGCGCGGCCTTT(SEQ ID NO.14)；
FR2:ATGGCCTGGTGGCGCCAGGGTTCAGACAAGGTGCGCGAACA GGTCGCAAAT(SEQ ID NO.15)；
FR3:AGCTACTCACGCTCCGTAGAGGGCCGATTCACCATCTCCCGA GACAACGCCAAGAACACTCTGACTCTCCAAATGAACGAATTGAAACCT GAGGACACTGACATGTACTACTGT(SEQ ID NO.16)；
FR4:TGGGGCCCGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.17)。
the amino acid sequence of the CDRs of the epitope complementary region of C4 of the present invention is preferably as follows:
CDR1:GYSGTPLC(SEQ ID NO.18)；
CDR2:IDSDGTT(SEQ ID NO.19)；
CDR3:AAVEGSAGEIYCSGGY(SEQ ID NO.20)；
the corresponding nucleotide sequence is preferably as follows:
CDR1:GGATACAGCGGCACGCCCTTGTGC(SEQ ID NO.21)；
CDR2:ATTGATAGTGATGGGACGACA(SEQ ID NO.22)；
CDR3:GCAGCTGTGGAGGGCTCCGCCGGCGAAATTTATTGCAGTGG TGGTTAC(SEQ ID NO.23)。
the amino acid sequence of C9 of the present invention is shown in SEQ ID No.2, which preferably includes 4 framework regions FR (FR 1, FR2, FR3 and FR 4) and 3 epitope-complementary regions CDR (CDR 1, CDR2 and CDR 3).
The amino acid sequence of the framework region FR of C9 according to the invention is preferably as follows:
FR1:QVQLQESGGGSVGLGGSMRLTCTIS(SEQ ID NO.24)；
FR2:MGWYRQAPGKWREGVAD(SEQ ID NO.25)；
FR3:GYIDSVRGRFIISRDNDKNILYLQMNSLKPEDTAMYYC(SEQ ID NO.26)；
FR4:WGQGTQVTVSS(SEQ ID NO.27)；
the corresponding nucleotide sequence is preferably as follows:
FR1:CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGGGGCT TGGAGGGTCTATGAGACTCACCTGCACAATCTCC(SEQ ID NO.28)；
FR2:ATGGGTTGGTACCGCCAGGCTCCAGGGAAATGGCGCGAGGG GGTCGCAGAC(SEQ ID NO.29)；
FR3:GGCTACATAGACTCCGTGAGGGGCCGATTCATCATCTCCCGGG ACAACGACAAGAACATTCTGTATCTCCAAATGAACAGCCTAAAACCTG AGGACACCGCCATGTACTACTGT(SEQ ID NO.30)；
FR4:TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.31)。
the amino acid sequence of the CDRs of the epitope-complementary region of C9 of the present invention is preferably as follows:
CDR1:AVIDDYC(SEQ ID NO.32)；
CDR2:IAESGTP(SEQ ID NO.33)；
CDR3:AARLRGSCSTDPHNFGY(SEQ ID NO.34)；
the corresponding nucleotide sequence is preferably as follows:
CDR1:GCCGTAATCGACGACTACTGT(SEQ ID NO.35)；
CDR2:ATTGCCGAATCTGGCACCCCA(SEQ ID NO.36)；
CDR3:GCGGCTAGATTACGTGGCTCGTGTAGCACGGACCCCCACAA CTTTGGTTAC(SEQ ID NO.37)。
the method for obtaining the C4 and the C9 is not particularly limited, and in the embodiment, a natural camel nano-antibody phage display library is preferably constructed by using a phage surface display technology, and then the nano-antibody gene sequence specific to SARS-CoV-2S protein is obtained by screening based on biotinylated SARS-CoV-2S protein. In the invention, the construction method of the natural camelid nanobody phage display library preferably comprises the following steps: 1) Extracting camel spleen tissue total RNA, and carrying out reverse transcription on the total RNA to obtain cDNA; 2) Performing nested PCR amplification by taking the cDNA as a template to obtain a variable region fragment of the heavy chain antibody; 3) Respectively enzyme-cutting the variable region fragment of the heavy chain antibody and the pcantab5e phage vector, and then connecting to obtain a connection product; 4) Transferring the connection product into competent cells to obtain the natural camel nano-antibody phage display library. The method for extracting the total RNA and the method for reverse transcription in the step 1) are not particularly limited, and the extraction and reverse transcription can be carried out by using a conventional method in the art. After the cDNA is obtained, performing nested PCR amplification by taking the cDNA as a template to obtain a variable region fragment of a heavy chain antibody, wherein the nested PCR preferably comprises two rounds of PCR; the first round of PCR is used for amplifying fragments between the heavy chain antibody guide peptide and the antibody CH2, and the primer sequences of the first round of PCR are preferably shown as SEQ ID NO.38 and SEQ ID NO. 39; the second round of PCR is used to amplify fragments between the FR1 region and the long and short hinge regions of the heavy chain antibody, and the primer sequences of the second round of PCR are preferably shown in SEQ ID NO.40 and SEQ ID NO. 41. After the variable region fragment of the heavy chain antibody is obtained, the variable region fragment of the heavy chain antibody and the pcantab5e phage vector are respectively digested, and then are connected to obtain a connection product. After the connection product is obtained, the connection product is transferred into competent cells to obtain a natural camel-source nanobody phage display library. The competent cells of the invention are preferably E.coli competent cells TG1; the method of transfer is preferably electrotransformation. The present invention preferably further includes helper phage rescue after the transformation, and the specific method of the present invention for the electric transformation and helper phage rescue is not particularly limited, and conventional methods in the art may be adopted.
The invention also provides gene sequences for coding the C4 and the C9, wherein the nucleotide sequence of the gene for coding the C4 is preferably shown as SEQ ID NO. 3; the nucleotide sequence of the gene encoding C9 is preferably as shown in SEQ ID NO. 4.
The invention also provides a group of recombinant nanobodies against SARS-CoV-2S protein, comprising any of the above nanobodies and a human IgG Fc segment.
The invention connects C4 with human IgG Fc segment to obtain recombinant C4 nanometer antibody (recombinant C4 for short); c9 is connected with a human IgG Fc segment to obtain a recombinant C9 nano antibody (short for recombinant C9), and the connection is preferably carried out by using a connection fragment, and the nucleotide sequence of a coding gene of the connection fragment is preferably shown as SEQ ID NO. 7: GGTGGTGGTGGTAGT. The nucleotide sequence of the coding gene of the Fc segment of the human IgG is preferably shown as SEQ ID NO. 42.
The amino acid sequence of the recombinant C4 is preferably shown as SEQ ID NO. 5; the nucleotide sequence of the gene encoding the recombinant C4 is preferably as shown in SEQ ID NO. 8.
The amino acid sequence of the recombinant C9 is preferably shown as SEQ ID NO. 6; the nucleotide sequence of the gene encoding the recombinant C9 is preferably as shown in SEQ ID NO. 9.
The invention also provides a recombinant vector for expressing the recombinant nanobody, wherein the recombinant vector comprises a nucleotide sequence for encoding the recombinant nanobody and a basic vector.
The base vector of the present invention preferably comprises pET-30a, and either the gene described in SEQ ID NO.8 or the gene described in SEQ ID NO.9 is inserted between the NdeI and XhoI sites of pET-30a.
The invention also provides a construction method of the recombinant vector, which comprises the following steps: inserting a nucleotide sequence encoding the recombinant nanobody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
The method of the insertion is not particularly limited, and a method using double cleavage is preferable.
The invention also provides recombinant bacteria for expressing the recombinant nano antibody, and the basic bacteria of the recombinant bacteria comprise escherichia coli.
The strain of E.coli of the present invention preferably includes Arctic Express. The recombinant vector is preferably transferred into the escherichia coli Arctic Express to obtain the recombinant bacterium. The method of transferring is not particularly limited, and a transfer method conventional in the art may be used.
The invention also provides application of the nano-antibody, the recombinant vector or the recombinant bacterium in preparation of antiviral drugs or virus infection diagnostic reagents.
The recombinant nanometer antibody can be prepared by utilizing the recombinant bacteria, and both the nanometer antibody and the recombinant nanometer antibody can specifically identify SARS-CoV-2S protein, thus being applicable to preparing antiviral drugs or virus infection diagnostic reagents.
The nanobody, recombinant vector, recombinant bacterium and application of the anti-SARS-CoV-2S protein provided by the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of natural camel-derived nanobody gene libraries
(1) Trizol method is used for extracting camel spleen tissue total RNA, RNA purification kit provided by TIANGEN company is used for purification, and cDNA is obtained through reverse transcription according to Thermo Scientific ReverAid First Strand cDNA Synthesis Kits kit.
(2) Using cDNA as a template, and amplifying by nested PCR to obtain a variable region fragment of the heavy chain antibody;
first round PCR:
an upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID No. 38)
A downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID No. 39)
20 mu L reactionThe system comprises: cDNA 2. Mu. L, mix 10. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L and the balance ddH 2 O；
PCR amplification reaction: 95 ℃ for 5min;95 ℃ 30s,55 ℃ 30s,72 ℃ 45s,32 cycles; and at 72℃for 10min.
The amplification result is shown in FIG. 1, and the nano antibody gene electrophoresis band is about 700bp.
The first round PCR product is used as a template, and the second round PCR is carried out:
an upstream primer: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGT CTGGGG-3' (SEQ ID NO. 40);
a downstream primer: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCT GGGTCCCC-3' (SEQ ID NO. 41);
50. Mu.L of reaction system: 700bp product 2. Mu. L, mix 25. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L and the balance ddH 2 O；
PCR amplification reaction: 94 ℃ for 5min;94 ℃ for 40s,55 ℃ for 40s,72 ℃ for 40s,25 cycles; and at 72℃for 10min.
The amplification result is shown in FIG. 2, and the nano antibody gene electrophoresis band is about 400bp.
(3) The pcantab5e phage vector and the VHH fragment (VHH fragment of C4 SEQ ID NO.1; VHH fragment of C9 SEQ ID NO. 2) were digested with SifI and Not I using restriction enzymes (available from Takara) and the two fragments were ligated using T4 DNA ligase (available from Takara).
Electrotransformation of the connected product into electrotransformation competent cell TG1 to constitute natural camel source nanometer antibody phage display library with library capacity up to 9.0×10 after supplementary phage rescue 13 。
Wherein the steps of the auxiliary phage rescue are as follows:
(1) 100. Mu.L of library was inoculated into 50mL of 2 XYT/Amp/Glu medium, shake-cultured at 37℃and 200rpm to logarithmic phase OD 600 About 0.4 to about 0.5.
(2) Adding helper phage M13KO7 with a complex infection number of 20:1 into the culture solution, mixing, and standing at 37deg.C for 30min.
(3) The culture was centrifuged at 9000rpm for 10min at room temperature, the supernatant was discarded to precipitate cells, and 200mL of the 2 XYT/Amp/Kana culture broth was used for resuspension, and the culture was carried out at 37℃at 200rpm overnight.
(4) The culture broth was centrifuged at 9000rpm at 4℃for 10min to obtain a supernatant, and 1/5 volume of PEG/NaCl was added thereto, followed by standing at 4℃for 6h.
(5) The supernatant was discarded after centrifugation at 9000rpm for 20min, and the pellet was resuspended in PBS (1 mL) to give a recombinant phage antibody library, which was dispensed into 1.5mL Ep tubes and stored at 4 ℃.
At the same time, the insertion rate of the library was measured by colony PCR, and the primer was used for the second round of PCR at an annealing temperature of 55 ℃ and showed that the insertion rate reached 95% or more (target fragment insertion rate=colony number containing target fragment/all colony numbers).
(4) Screening process of anti-SARS-CoV-2S protein nanobody:
phage library (1X 10) 12 Phage) were incubated with 50 μl of streptavidin beads on a rotating table for 1h at room temperature, phage antibodies were collected; 500. Mu.L of the pre-cut phage antibody was added to each of 21 ml centrifuge tubes that had been blocked with 2% PBSM, 500. Mu.L of 5. Mu.g biotinylated SARS-CoV-2S protein diluted with PBS was added to one centrifuge tube, 500. Mu.L of PBS buffer was added to the other centrifuge tube as a negative control, incubated for 1h at room temperature on a rotating table, 50. Mu.L of pre-blocked streptavidin magnetic beads were added, and incubated for 30min at room temperature on the rotating table, and the magnetic beads were collected. The beads were washed 7 times with PBST, 2 times with PBSM and 1 time with PBS. Glycine at pH2.7 was added for elution and 1mol/L Tris-HCl at pH9.1 was neutralized. The above-mentioned neutralization solution was added to 5mL of TG1 (OD) 600 0.5), phage are generated and purified for the next round of screening, and positive clones are continuously enriched after 4 rounds of screening, so that the aim of screening SARS-CoV-2S protein specific antibodies in an antibody library by using phage display technology is fulfilled.
ELISA method of phages (phage-ELISA) screening specific single positive clones:
the screening principle pattern is shown in fig. 3, and the specific method is as follows:
VHH phage monoclonal supernatants were first prepared: from 3-4 rounds of screening solid plate, randomly picking 90 single colonies and inoculating in 100 mug/mL ampicillin and 2% glucose 2 XYT medium, 220rpm 37 ℃ culture overnight, the next day 50 mug bacterial liquid to a new 96 deep well plate, each hole adding 800 mug 2 XYT medium containing 100 mug/mL ampicillin and 2% glucose, after growing to logarithmic phase, adding 20:1 complex infection of helper phage M13K07, 37 ℃ infection 30min,10000rpm centrifugation 5min, discarding supernatant, 800 mug fresh 2 XYT medium containing 100 mug/mL ampicillin and 50 mug/mL kanapigenin resuspension thalli, 37 ℃,220rpm culture 12h, next day 10000rpm bacterial liquid, centrifugation 5min, the supernatant is VHH phage monoclonal supernatant.
SARS-CoV-2S protein was diluted to 10. Mu.g/mL with coating solution, 100. Mu.L per well was added, coated overnight at 4℃and negative and positive controls were established. The following day, three washes with PBST, 2% PBSM blocked at 37℃for 2h, three washes with PBST, 200. Mu.L of pre-treated VHH phage single-clone supernatant was added and incubated for 1h at 37 ℃. The secondary murine anti-M13 KO7/HRP antibody diluted with 0.1% PBST was added at 1:5000, incubated at 37℃for 1h, unbound antibody was washed away, TMB chromogenic solution was added and absorbance was read on a microplate reader at a wavelength of 450 nm. And when the OD value of the sample hole is more than twice that of the control hole, judging the sample hole as a positive control hole, and taking positive bacterial liquid for gene sequencing.
Sequence analysis and blast alignment were performed using DNAMAN software, and strains with identical CDR1, CDR2 and CDR3 sequences were considered as the same clone. Finally, the nanometer antibody sequences shown in the amino acid sequences SEQ ID NO.1 and SEQ ID NO.2 are adopted for subsequent experiments.
Example 2
Expression and purification of recombinant nanobody in host bacterium escherichia coli
(1) The sequence of the nanobody obtained by sequencing analysis in example 1 was ligated to a humanized IgG Fc segment and subcloned into a pET-30a plasmid vector, and transformed into E.coli Arctic Express, and the monoclonal on the transformed plate was picked up and inoculated into a test tube containing 50. Mu.g/mL Kan of 3mL LB culture solution, and shaken at 37℃for 220rpm overnight; (2) The next day was inoculated at 1:100 into 30mL LB medium of 50. Mu.g/mLKan, and shaken at 37℃and 220rpmShaking to thallus OD 600 Adding IPTG to a final concentration of 0.5mM at 0.6-0.8, shaking at 20 ℃ and 220rpm overnight, and inducing the expression of the fusion protein; (3) And collecting thalli, performing ultrasonic crushing to obtain crude body fluid containing the body protein, and performing Ni column affinity purification to obtain fusion protein.
FIG. 4 is a purified recombinant nanobody, wherein the left panel shows SDS-PAGE electrophoresis staining during purification of recombinant C4: wherein lane M is a protein molecular standard, lanes 1-2 are a crushed post-treatment sample and an effluent sample, and lanes 3-4 are elution samples, respectively; the right panel shows SDS-PAGE electrophoresis staining during recombinant C9 purification: wherein lane M is a protein molecular standard, lanes 1-2 are the crushed post-treatment sample and the effluent sample, respectively, and lanes 3-4 are the elution samples. FIG. 5 is a western blot plot of purified recombinant nanobodies, wherein the left plot is recombinant C4 and the right plot is recombinant C9.
Example 3
Specificity verification of recombinant nanobody:
SARS-CoV-2S protein is used as a western blot (wherein the primary antibody is a purified recombinant nanobody and the secondary antibody is an anti-human IgG Fc/HRP).
The western blot results are shown in fig. 6, the left primary antibody is recombinant C4, and the right primary antibody is recombinant C9): wherein lane M is a protein molecular standard, and lanes 1 and 2 are both SARS-CoV-2S protein. Therefore, the recombinant nano antibody provided by the invention can specifically recognize SARS-CoV-2S protein.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the present invention, and such modifications and variations should also be considered as being within the scope of the present invention.
Sequence listing
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
1 5 10 15
Ser Met Arg Leu Thr Cys Thr Ile Ser Ala Val Ile Asp Asp Tyr Cys
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
35 40 45
Asp Ile Ala Glu Ser Gly Thr Pro Gly Tyr Ile Asp Ser Val Arg Gly
50 55 60
Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Asn Ile Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
85 90 95
Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 3
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg cctttggata cagcggcacg cccttgtgca tggcctggtg gcgccagggt 120
tcagacaagg tgcgcgaaca ggtcgcaaat attgatagtg atgggacgac aagctactca 180
cgctccgtag agggccgatt caccatctcc cgagacaacg ccaagaacac tctgactctc 240
caaatgaacg aattgaaacc tgaggacact gacatgtact actgtgcagc tgtggagggc 300
tccgccggcg aaatttattg cagtggtggt tactggggcc cggggaccca ggtcaccgtc 360
tcctca 366
<210> 4
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctccgccgt aatcgacgac tactgtatgg gttggtaccg ccaggctcca 120
gggaaatggc gcgagggggt cgcagacatt gccgaatctg gcaccccagg ctacatagac 180
tccgtgaggg gccgattcat catctcccgg gacaacgaca agaacattct gtatctccaa 240
atgaacagcc taaaacctga ggacaccgcc atgtactact gtgcggctag attacgtggc 300
tcgtgtagca cggaccccca caactttggt tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 5
<211> 359
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Ile Val Leu Ser Cys Ala Ala Phe Gly Tyr Ser Gly Thr Pro Leu
20 25 30
Cys Met Ala Trp Trp Arg Gln Gly Ser Asp Lys Val Arg Glu Gln Val
35 40 45
Ala Asn Ile Asp Ser Asp Gly Thr Thr Ser Tyr Ser Arg Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Thr Leu
65 70 75 80
Gln Met Asn Glu Leu Lys Pro Glu Asp Thr Asp Met Tyr Tyr Cys Ala
85 90 95
Ala Val Glu Gly Ser Ala Gly Glu Ile Tyr Cys Ser Gly Gly Tyr Trp
100 105 110
Gly Pro Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu
115 120 125
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 6
<211> 359
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
1 5 10 15
Ser Met Arg Leu Thr Cys Thr Ile Ser Ala Val Ile Asp Asp Tyr Cys
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
35 40 45
Asp Ile Ala Glu Ser Gly Thr Pro Gly Tyr Ile Asp Ser Val Arg Gly
50 55 60
Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Asn Ile Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
85 90 95
Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu
115 120 125
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 7
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ggtggtggtg gtagt 15
<210> 8
<211> 1077
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg cctttggata cagcggcacg cccttgtgca tggcctggtg gcgccagggt 120
tcagacaagg tgcgcgaaca ggtcgcaaat attgatagtg atgggacgac aagctactca 180
cgctccgtag agggccgatt caccatctcc cgagacaacg ccaagaacac tctgactctc 240
caaatgaacg aattgaaacc tgaggacact gacatgtact actgtgcagc tgtggagggc 300
tccgccggcg aaatttattg cagtggtggt tactggggcc cggggaccca ggtcaccgtc 360
tcctcaggtg gtggtggtag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 420
tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 480
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 540
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 600
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 720
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 780
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 840
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 900
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 960
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1020
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaa 1077
<210> 9
<211> 1077
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctccgccgt aatcgacgac tactgtatgg gttggtaccg ccaggctcca 120
gggaaatggc gcgagggggt cgcagacatt gccgaatctg gcaccccagg ctacatagac 180
tccgtgaggg gccgattcat catctcccgg gacaacgaca agaacattct gtatctccaa 240
atgaacagcc taaaacctga ggacaccgcc atgtactact gtgcggctag attacgtggc 300
tcgtgtagca cggaccccca caactttggt tactggggcc aggggaccca ggtcaccgtc 360
tcctcaggtg gtggtggtag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 420
tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 480
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 540
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 600
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 720
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 780
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 840
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 900
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 960
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1020
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaa 1077
<210> 10
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Ile Val Leu Ser Cys Ala Ala Phe
20 25
<210> 11
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Met Ala Trp Trp Arg Gln Gly Ser Asp Lys Val Arg Glu Gln Val Ala
1 5 10 15
Asn
<210> 12
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Ser Tyr Ser Arg Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Thr Leu Gln Met Asn Glu Leu Lys Pro Glu Asp
20 25 30
Thr Asp Met Tyr Tyr Cys
35
<210> 13
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 14
<211> 75
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tatagtactc 60
tcctgcgcgg ccttt 75
<210> 15
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
atggcctggt ggcgccaggg ttcagacaag gtgcgcgaac aggtcgcaaa t 51
<210> 16
<211> 114
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
agctactcac gctccgtaga gggccgattc accatctccc gagacaacgc caagaacact 60
ctgactctcc aaatgaacga attgaaacct gaggacactg acatgtacta ctgt 114
<210> 17
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tggggcccgg ggacccaggt caccgtctcc tca 33
<210> 18
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Gly Tyr Ser Gly Thr Pro Leu Cys
1 5
<210> 19
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Ile Asp Ser Asp Gly Thr Thr
1 5
<210> 20
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Ala Ala Val Glu Gly Ser Ala Gly Glu Ile Tyr Cys Ser Gly Gly Tyr
1 5 10 15
<210> 21
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ggatacagcg gcacgccctt gtgc 24
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
attgatagtg atgggacgac a 21
<210> 23
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gcagctgtgg agggctccgc cggcgaaatt tattgcagtg gtggttac 48
<210> 24
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gly Leu Gly Gly
1 5 10 15
Ser Met Arg Leu Thr Cys Thr Ile Ser
20 25
<210> 25
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Trp Arg Glu Gly Val Ala
1 5 10 15
Asp
<210> 26
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Gly Tyr Ile Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn
1 5 10 15
Asp Lys Asn Ile Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 27
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 28
<211> 75
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
caggtccaac tgcaggagtc tgggggaggc tcggtggggc ttggagggtc tatgagactc 60
acctgcacaa tctcc 75
<210> 29
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
atgggttggt accgccaggc tccagggaaa tggcgcgagg gggtcgcaga c 51
<210> 30
<211> 114
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ggctacatag actccgtgag gggccgattc atcatctccc gggacaacga caagaacatt 60
ctgtatctcc aaatgaacag cctaaaacct gaggacaccg ccatgtacta ctgt 114
<210> 31
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tggggccagg ggacccaggt caccgtctcc tca 33
<210> 32
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Ala Val Ile Asp Asp Tyr Cys
1 5
<210> 33
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Ile Ala Glu Ser Gly Thr Pro
1 5
<210> 34
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Ala Ala Arg Leu Arg Gly Ser Cys Ser Thr Asp Pro His Asn Phe Gly
1 5 10 15
Tyr
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gccgtaatcg acgactactg t 21
<210> 36
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
attgccgaat ctggcacccc a 21
<210> 37
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gcggctagat tacgtggctc gtgtagcacg gacccccaca actttggtta c 51
<210> 38
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gtcctggctg ctcttctaca aag 23
<210> 39
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ggtacgtgct gttgaactgt tcc 23
<210> 40
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
tcgcggccca gccggcccag gtccaactgc aggagtctgg gg 42
<210> 41
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ataagaatgc ggccgctgag gagacggtga cctgggtccc c 41
<210> 42
<211> 696
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgggg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaaa 696
Claims (10)
1. A group of nanometer antibodies for resisting SARS-CoV-2S protein, the nanometer antibodies comprise C4 and C9, the amino acid sequence of the C4 is shown as SEQ ID NO.1, and the amino acid sequence of the C9 is shown as SEQ ID NO. 2.
2. A set of recombinant nanobodies against SARS-CoV-2S protein, comprising any one of the nanobodies of claim 1 and a human IgG Fc fragment.
3. The recombinant nanobody according to claim 2, wherein the recombinant nanobody comprises recombinant C4 and recombinant C9, the amino acid sequence of the recombinant C4 is shown as SEQ ID No.5, and the amino acid sequence of the recombinant C9 is shown as SEQ ID No. 6.
4. A recombinant vector expressing the recombinant nanobody of claim 2 or 3, wherein the recombinant vector comprises a nucleotide sequence encoding the recombinant nanobody and a base vector.
5. The recombinant vector according to claim 4, wherein the nucleotide sequence encoding recombinant C4 is shown in SEQ ID NO.8 and the nucleotide sequence encoding recombinant C9 is shown in SEQ ID NO. 9.
6. The recombinant vector according to claim 4, wherein the base vector comprises pET-30a.
7. The method for constructing a recombinant vector according to any one of claims 4 to 6, comprising the steps of: inserting a nucleotide sequence encoding the recombinant nanobody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
8. A recombinant bacterium expressing the recombinant nanobody of claim 2 or 3, the base bacterium of which comprises escherichia coli.
9. The recombinant bacterium according to claim 8, wherein the strain of escherichia coli comprises Arctic Express.
10. Use of the nanobody of claim 1, the recombinant nanobody of claim 2 or 3, the recombinant vector of any one of claims 4 to 6 or the recombinant bacterium of claim 8 or 9 in the preparation of an antiviral drug or a diagnostic reagent for viral infection.
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|---|---|---|---|
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| CN202210077156.4A CN114702575B (en) | 2022-01-24 | 2022-01-24 | Nanometer antibody, recombinant vector, recombinant bacterium for resisting SARS-CoV-2S protein and application thereof |
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| CN114702575B true CN114702575B (en) | 2023-05-23 |
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