CN114702575A - Nano antibody for resisting SARS-CoV-2S protein, recombinant nano antibody, recombinant vector, recombinant bacterium and application - Google Patents
Nano antibody for resisting SARS-CoV-2S protein, recombinant nano antibody, recombinant vector, recombinant bacterium and application Download PDFInfo
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- CN114702575A CN114702575A CN202210077156.4A CN202210077156A CN114702575A CN 114702575 A CN114702575 A CN 114702575A CN 202210077156 A CN202210077156 A CN 202210077156A CN 114702575 A CN114702575 A CN 114702575A
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Abstract
The invention provides a nano antibody, a recombinant vector, a recombinant bacterium and application of anti-SARS-CoV-2S protein, relating to the technical field of biological medicine. The nano antibody comprises C4 and C9, and both C4 and C9 are camel sources and can specifically recognize SARS-CoV-2S protein. The invention fuses the C4 and the C9 with human IgG Fc segment respectively to obtain two recombinant nano antibodies of recombinant C4 and recombinant C9, and 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 coronavirus.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a nano antibody, a recombinant vector, a recombinant bacterium and application of anti-SARS-CoV-2S protein.
Background
2019 the novel coronavirus (SARS-CoV-2) is a new strain of coronavirus found in human in 2019. SARS-CoV-2 virus invades a host cell by binding its spike protein, namely S protein, to human angiotensin converting enzyme-2 (ACE 2). After SARS-CoV-2 virus infection, alveolar macrophage cells and epithelial cells release a large amount of proinflammatory cytokines and chemokines; monocytes and neutrophils will be recruited to the site of infection and clear the exudate containing viral particles and infected cells, leading to a runaway inflammatory response. In this process, adaptive immunity is difficult to effectively initiate due to significant lymphocyte reduction and dysfunction. Uncontrolled viral infection can lead to severe macrophage infiltration, further exacerbating lung injury. Meanwhile, the spread SARS-CoV-2 virus can also directly attack other organs, the immune reaction can cause systemic inflammation storm, and simultaneously, the microcirculation disturbance also exists, and the factors act together to finally cause viral sepsis.
The nanobody is the first report in Nature by belgium scientist in 1993 that a naturally light chain-deleted antibody (VHH) exists in the peripheral blood of an alpaca, and is found in 1995 in nurse shark, mackerel and other cartilaginous fish, and is the currently known minimum unit capable of binding a target antigen. VHH has a molecular weight of only 15KD and is therefore also called Nanobody (Nb). The nano antibody has unique advantages of small molecular weight, good solubility, strong stability, high affinity, low immunogenicity, good in vivo tissue permeability, easy to pass through blood vessels or tissues to reach target sites and the like, has a large development space, is widely applied, and can be clinically used for tumor treatment and also used as a diagnostic tool.
Therefore, the search for more effective antibodies for the treatment of SARS-CoV-2 virus infection is particularly urgent.
Disclosure of Invention
In view of the above, the present invention aims to provide a nanobody against SARS-CoV-2S protein, a recombinant nanobody, a recombinant vector, a recombinant bacterium and applications thereof, wherein both the nanobody and the recombinant nanobody are capable of specifically recognizing 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, which comprises C4 and C9, wherein the amino acid sequence of C4 is shown as SEQ ID NO.1, and the amino acid sequence of C9 is shown as SEQ ID NO. 2.
The invention also provides a group of recombinant nano antibodies for resisting SARS-CoV-2S protein, wherein the recombinant nano antibodies comprise any one of the nano antibodies and 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 nano antibody, and the recombinant vector comprises a nucleotide sequence for coding the recombinant nano antibody and a basic vector.
Preferably, the nucleotide sequence for coding the recombinant C4 is shown as SEQ ID NO.8, and the nucleotide sequence for coding the recombinant C9 is shown as SEQ ID NO. 9.
Preferably, the base vector comprises pET-30 a.
The invention also provides a construction method of the recombinant vector, which comprises the following steps: and inserting the nucleotide sequence for coding the recombinant nano antibody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
The invention also provides a recombinant bacterium for expressing the recombinant nano antibody, and the basic bacterium of the recombinant bacterium comprises escherichia coli.
Preferably, the strain of escherichia coli comprises Arctic Express.
The invention also provides the application of the nano antibody, the recombinant vector or the recombinant bacterium in preparing antiviral drugs or virus infection diagnostic reagents.
Has the advantages that: the invention provides a group of nano antibodies for resisting SARS-CoV-2S protein, the nano antibodies comprise C4 and C9, and C4 and C9 are camel sources and can specifically recognize SARS-CoV-2S protein. The invention fuses the C4 and the C9 with human IgG Fc segment respectively to obtain two recombinant nano antibodies of recombinant C4 and recombinant C9, and 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 anti-virus drugs of new coronavirus.
Drawings
FIG. 1 is a first round DNA electrophoresis diagram of the nanobody, the DNA bands from left to right of the gel hole are: the sixth lane is Marker of 1000bp (the sizes of the bands are 1000bp, 700bp, 500 bp, 400bp, 300 bp, 200 bp and 100bp in sequence), the first lane, the second lane, the third lane and the fifth lane are PCR products, the bands are about 700bp, and the fourth lane is empty;
FIG. 2 is a second round DNA electrophoresis diagram of the nanobody, the DNA bands from left to right of the gel hole are: the first lane is a Marker of 1000bp (the size of the band is the same as 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 of specific single positive clones by phage enzyme-linked immunosorbent assay (phase-ELISA): wherein 1, SARS-CoV-2S protein is coated on an enzyme label plate, 2 is phage supernatant, 3 is a mouse anti-km 13107 antibody, 4 is a goat anti-mouse IgG (AP) antibody, and 5 is TMB color development liquid;
FIG. 4 is a SDS-PAGE electrophoresis staining pattern of a treated sample, an effluent sample and an eluted sample after the recombinant C4 nanobody is broken, wherein the left image is a purified recombinant nanobody; the right picture is an SDS-PAGE electrophoresis staining picture of a treated sample, an effluent sample and an elution sample after the recombinant C9 nano antibody is broken;
FIG. 5 is a western blot of purified recombinant nanobody, wherein the left is recombinant C4 nanobody, and the right is 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 first antibody in the left panel is recombinant C4 nano antibody, and the first antibody in the right panel is recombinant C9 nano antibody.
Detailed Description
The invention provides a group of nano antibodies for resisting SARS-CoV-2S protein, which comprises C4 and C9, wherein the amino acid sequence of C4 is shown as SEQ ID NO.1, and the amino acid sequence of C9 is shown as SEQ ID NO. 2.
The amino acid sequence of the C4 is shown as SEQ ID NO.1, and the C4 comprises 4 framework regions FR (FR1, FR2, FR3 and FR4) and 3 antigenic determinant complementary regions CDR (CDR1, CDR2 and CDR 3). The amino acid sequence of the framework region FR of C4 according to the present 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 sequences are 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 CDR of the epitope complementary region of C4 according to 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 in the present invention is shown in SEQ ID No.2, and preferably includes 4 framework regions FR (FR1, FR2, FR3 and FR4) and 3 epitope-complementary regions CDR (CDR1, CDR2 and CDR 3).
The amino acid sequence of the framework region FR of C9 according to the present 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 CDR of the epitope complementary region of C9 according to 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 invention is not limited to the method for obtaining C4 and C9, and in the embodiment, the natural camel source nano antibody phage display library is preferably constructed by using the phage surface display technology, and then screening is carried out on the basis of the biotinized SARS-CoV-2S protein, so as to obtain the specific nano antibody gene sequence of the SARS-CoV-2S protein. In the present invention, the method for constructing the natural camel-derived nanobody phage display library preferably comprises the following steps: 1) extracting total RNA of camel spleen tissues and reversely transcribing the total RNA to obtain cDNA; 2) performing nested PCR amplification by using the cDNA as a template to obtain a variable region fragment of the heavy chain antibody; 3) respectively carrying out enzyme digestion on the variable region fragment of the heavy chain antibody and a pcantab5e phage vector, and then connecting to obtain a connection product; 4) and transferring the ligation product into a competent cell to obtain a natural camel source nano antibody phage display library. The method for extracting total RNA and the method for reverse transcription in step 1) are not particularly limited in the present invention, and the extraction and reverse transcription may be performed by a conventional method in the art. After the cDNA is obtained, the cDNA is taken as a template to carry out nested PCR amplification to obtain a variable region fragment of a heavy chain antibody, and the nested PCR preferably comprises two rounds of PCR; the first round of PCR is used for amplifying a fragment between the heavy chain antibody leader peptide and the antibody CH2, and the primer sequence of the first round of PCR is preferably shown as SEQ ID NO.38 and SEQ ID NO. 39; a second round of PCR, the primer sequences of which are preferably shown in SEQ ID NO.40 and SEQ ID NO.41, was used to amplify the fragment between the FR1 region and the long and short hinge regions of the heavy chain antibody. After obtaining the variable region fragment of the heavy chain antibody, the invention respectively enzyme-cleaves the variable region fragment of the heavy chain antibody and a pcantab5e phage vector, and then connects to obtain a ligation product. After the ligation product is obtained, the ligation product is transferred into competent cells to obtain a natural camel source nano antibody phage display library. The competent cell of the invention is preferably Escherichia coli competent cell TG 1; the method of transformation is preferably electrotransformation. The present invention preferably further comprises helper phage rescue after the transformation, and the specific methods of the electric transformation and helper phage rescue are not particularly limited by the present invention, and can be performed by a conventional method in the art.
The invention also provides a gene sequence 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 shown in SEQ ID NO. 4.
The invention also provides a group of recombinant nano antibodies for resisting SARS-CoV-2S protein, wherein the recombinant nano antibodies comprise any one of the nano antibodies and human IgG Fc segment.
The invention connects C4 with human IgG Fc segment to obtain recombinant C4 nano antibody (recombinant C4 for short); c9 is connected with a human IgG Fc segment to obtain a recombinant C9 nano antibody (recombinant C9 for short), the connection is preferably carried out by utilizing a connecting fragment, and the nucleotide sequence of the coding gene of the connecting fragment is preferably shown as SEQ ID NO. 7: GGTGGTGGTGGTAGT are provided. The nucleotide sequence of the encoding gene of the human IgG Fc segment 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 for coding the recombinant C4 is preferably shown as 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 for coding the recombinant C9 is preferably shown as SEQ ID NO. 9.
The invention also provides a recombinant vector for expressing the recombinant nano antibody, and the recombinant vector comprises a nucleotide sequence for coding the recombinant nano antibody and a basic vector.
The basic vector of the present invention preferably includes pET-30a, and the gene represented by SEQ ID NO.8 or the gene represented by SEQ ID NO.9 described above are inserted between the NdeI and XhoI sites of pET-30 a.
The invention also provides a construction method of the recombinant vector, which comprises the following steps: and inserting the nucleotide sequence for coding the recombinant nano antibody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
The method of insertion is not particularly limited in the present invention, and a double-enzyme digestion method is preferably used.
The invention also provides a recombinant bacterium for expressing the recombinant nano antibody, and the basic bacterium of the recombinant bacterium comprises escherichia coli.
The strain of Escherichia 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 strain. The transfer method is not particularly limited, and the conventional transfer method in the field can be adopted.
The invention also provides the application of the nano antibody, the recombinant vector or the recombinant bacterium in the preparation of antiviral drugs or virus infection diagnostic reagents.
The recombinant bacterium can be used for preparing the recombinant nano antibody, and the nano antibody and the recombinant nano antibody can specifically recognize SARS-CoV-2S protein and can be used for preparing antiviral drugs or virus infection diagnostic reagents.
The present invention will be described in detail with reference to examples, but it should not be construed as limiting the scope of the present invention.
Example 1
Construction of natural camel source nano antibody gene bank
(1) Total RNA of camel spleen tissue is extracted by a Trizol method, purified by an RNA purification kit provided by TIANGEN company, and reverse transcribed according to a Thermo Scientific reverse aid First Strand cDNA Synthesis kit to obtain cDNA.
(2) Using cDNA as template, using nested PCR amplification to obtain variable region fragment of 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 μ L reaction: cDNA2 mu L, Mix 10 mu L, forward primer 1 mu L, reverse primer 1 mu L and the balance ddH2O；
PCR amplification reaction: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃, 45s at 72 ℃ and 32 cycles; 10min at 72 ℃.
The amplification result is shown in FIG. 1, and the gene electrophoresis band of the nano antibody is about 700 bp.
And (3) performing second round PCR by taking the first round PCR product as a template:
an upstream primer: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGT CTGGGG-3' (SEQ ID NO. 40);
a downstream primer: 5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCT GGGTCCCC-3' (SEQ ID NO. 41);
50 μ L reaction: 700bp product 2. mu. L, Mix 25. mu.L, upstream primer 1. mu.L, downstream primer 1. mu.L and the balance ddH2O；
PCR amplification reaction: 5min at 94 ℃; 94 ℃ for 40s, 55 ℃ for 40s, 72 ℃ for 40s, 25 cycles; 10min at 72 ℃.
The amplification result is shown in FIG. 2, and the gene electrophoresis band of the nano antibody is about 400 bp.
(3) The pcantab5e phage vector and the VHH fragment (VHH fragment SEQ ID NO.1 for C4; VHH fragment SEQ ID NO.2 for C9) were cut with restriction enzymes (purchased from Takara Inc.) SifI and Not I, and the two fragments were ligated with T4 DNA ligase (purchased from Takara Inc.).
The ligation product is electrically transformed into an electrotransformation competent cell TG1 to construct a natural camel source nano antibody phage display library, and the library capacity reaches 9.0 multiplied by 10 after the phage display library is rescued by auxiliary phage13。
The helper phage rescue steps are as follows:
firstly, 100 mu L of library is inoculated into 50mL of 2 XYT/Amp/Glu culture medium, the temperature is 37 ℃, the rpm is 200, and the shake culture is carried out until the logarithmic phase OD is reached600About 0.4 to about 0.5.
② adding the helper phage M13KO7 with the multiplicity of infection of 20:1 into the culture solution, mixing uniformly and standing for 30min at 37 ℃.
③ the culture solution is centrifuged at 9000rpm for 10min at room temperature, the supernatant is discarded to precipitate the thalli, 200mL of 2 XYT/Amp/Kana culture solution is used for resuspension, and the culture is carried out at 200rpm at 37 ℃ overnight.
Fourthly, the culture solution is centrifuged for 10min at 9000rpm at 4 ℃, the supernatant is taken, and 1/5 volume of PEG/NaCl is added, and the mixture is kept stand for 6h at 4 ℃.
Fifthly, centrifuging at 9000rpm for 20min, discarding the supernatant, resuspending the precipitate with PBS (1mL) to obtain a recombinant phage antibody library, subpackaging in 1.5mL Ep tubes, and storing at 4 ℃.
At the same time, the library was tested for insertion by colony PCR using primers from the second round of PCR primers at 55 ℃ and showed an insertion rate of 95% or more (target fragment insertion rate ═ number of colonies containing the target fragment/number of all colonies).
(4) The screening process aiming at the anti-SARS-CoV-2S protein nano antibody comprises the following steps:
phage library (1X 10)12Phage) and 50 mu L streptavidin magnetic beads are incubated for 1h at room temperature on a rotating platform, and then phage antibodies are collected; in 2 has 2% PBSM blocked 1ml centrifugal tube, respectively adding 500 u L pre-cut phage antibody, then to one centrifugal tube adding 500 u L PBS diluted 5 u g biotinylated SARS-CoV-2S protein, another centrifugal tube adding 500 u L PBS buffer as negative control, in the rotating table at room temperature incubation for 1h, then adding pre-closed 50 u L streptavidin magnetic beads, in the rotating table at room temperature incubation for 30min, collectingAnd collecting the magnetic beads. 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 used for neutralization. The above neutralized solution was added to 5mL of TG1 (OD) in the logarithmic growth phase6000.5), the generated and purified phage is used for the next round of screening, and the positive clones are continuously enriched after 4 rounds of screening, thereby achieving the purpose of screening the specific antibody of SARS-CoV-2S protein in the antibody library by using the phage display technology.
Screening of specific single positive clones by enzyme-linked immunosorbent assay (phase-ELISA) of the phages:
the screening principle mode diagram is shown in fig. 3, and the specific method is as follows:
first, a monoclonal supernatant of VHH phage was prepared: from the 3-4 screening solid plate, randomly picking 90 single colony and inoculating in 100 u g/mL ampicillin and 2% glucose 2 XYT medium, culturing at 220rpm and 37 ℃ overnight, taking 50 mu L of bacterial liquid the next day to a new 96-deep-well plate, each well was added 800. mu.L of 2 XYT medium containing 100. mu.g/mL ampicillin and 2% glucose, grown to a logarithmic phase, adding helper phage M13K07 with the multiplicity of infection of 20:1, infecting for 30min at 37 ℃, centrifuging for 5min at 10000rpm, discarding the supernatant, resuspending the thallus with 800 μ L of fresh 2 XYT culture medium containing 100 μ g/mL ampicillin and 50 μ g/mL kanamycin, culturing for 12h at 37 ℃, 220rpm, centrifuging for 5min the next day at 10000rpm of bacterial liquid, and obtaining the supernatant which is the monoclonal supernatant of the VHH phage.
SARS-CoV-2S protein was diluted to 10. mu.g/mL with coating solution, 100. mu.L was added to each well, coated overnight at 4 ℃, and negative and positive controls were set up. The next day, three washes with PBST, blocking with 2% PBSM at 37 ℃ for 2h, three washes with PBST, addition of 200. mu.L of pretreated VHH phage monoclonality supernatant, and incubation at 37 ℃ for 1 h. Add 1:5000 secondary antibody of murine anti-M13 KO7/HRP diluted with 0.1% PBST, incubate 1h at 37 deg.C, wash away unbound antibody, add TMB developing solution, read absorbance values at 450nm wavelength on a microplate reader. And when the OD value of the sample hole is more than twice of the OD value of the control hole, determining the sample hole as a positive control hole, and taking the positive bacterial liquid to perform gene sequencing.
Using DNAMAN software for sequence analysis and blast alignment, strains with identical CDR1, CDR2, and CDR3 sequences were considered to be identical clones. Finally, the nano antibody sequences shown by the amino acid sequences SEQ ID NO.1 and SEQ ID NO.2 are adopted to carry out subsequent experiments.
Example 2
Expression and purification of recombinant nano antibody in host bacterium escherichia coli
(1) Connecting the nano antibody sequence obtained by sequencing analysis in example 1 with a human IgG Fc segment and subcloning into a pET-30a plasmid vector, converting into Escherichia coli Arctic Express, selecting a single clone on a conversion plate, inoculating into a test tube containing 3mL LB culture solution of 50 ug/mLKan, and shaking at 37 ℃ and 220rpm overnight; (2) the next day, the cells were inoculated into 30mL LB medium at a ratio of 1:100 and 50. mu.g/mLKan, and shaken at 37 ℃ and 220rpm until the OD of the cells6000.6-0.8, adding IPTG to a final concentration of 0.5mM, shaking at 20 ℃ and 220rpm overnight, and inducing expression of the fusion protein; (3) collecting thalli, carrying out ultrasonic crushing to obtain an inclusion body protein crude body fluid, and then carrying out Ni column affinity purification to obtain the fusion protein.
FIG. 4 is a purified recombinant nanobody, wherein the left image is SDS-PAGE electrophoresis staining pattern during the purification of recombinant C4: wherein lane M is a protein molecule standard, lanes 1-2 are a treated sample and an effluent sample after disruption, respectively, and lanes 3-4 are elution samples; the right panel is the SDS-PAGE staining pattern during purification of recombinant C9: wherein lane M is a protein molecule standard, lanes 1-2 are a treated sample after disruption and an effluent sample, respectively, and lanes 3-4 are elution samples. FIG. 5 is a western blot of purified recombinant nanobodies, in which the left panel is recombinant C4 and the right panel is recombinant C9.
Example 3
Specificity verification of the recombinant nano antibody:
the SARS-CoV-2S protein is used as western blot (the primary antibody is purified recombinant nano antibody, and the secondary antibody is anti-human IgG Fc/HRP).
The results of western blot are shown in fig. 6, with the left antibody being recombinant C4 and the right antibody being recombinant C9): wherein lane M is a protein molecule 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 only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Guangdong university of medical science
<120> nano antibody for resisting SARS-CoV-2S protein, recombinant nano antibody, recombinant vector, recombinant bacterium and application
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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
115 120
<210> 2
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
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 nano antibodies for resisting SARS-CoV-2S protein, the nano antibodies include C4 and C9, the amino acid sequence of C4 is shown as SEQ ID NO.1, the amino acid sequence of 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 of claim 2, which comprises recombinant C4 and recombinant C9, wherein the amino acid sequence of the recombinant C4 is shown in SEQ ID No.5, and the amino acid sequence of the recombinant C9 is shown in SEQ ID No. 6.
4. The recombinant vector for 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-30 a.
7. The method for constructing the recombinant vector according to any one of claims 4 to 6, comprising the steps of: and inserting the nucleotide sequence for coding the recombinant nano antibody between NdeI and XhoI sites of the basic vector to obtain the recombinant vector.
8. The recombinant strain for expressing the recombinant nano-antibody of claim 2 or 3, wherein the basic strain of the recombinant strain comprises Escherichia coli.
9. The recombinant bacterium of claim 8, wherein the strain of E.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 for the preparation of an antiviral drug or a diagnostic agent for viral infection.
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| CN111153997A (en) * | 2020-01-16 | 2020-05-15 | 佛山汉腾生物科技有限公司 | anti-CTLA-4 nano antibody and application thereof in tumor treatment |
| CN111574629A (en) * | 2020-06-01 | 2020-08-25 | 宁夏医科大学 | CD 20-resistant nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant strain and application |
| CN111825762A (en) * | 2020-06-17 | 2020-10-27 | 武汉华美生物工程有限公司 | Nano antibody of S protein RBD structure domain of anti SARS-COV-2 virus and its use |
| 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 |
| CN112940111A (en) * | 2020-09-22 | 2021-06-11 | 石河子大学 | Nano antibody based on novel coronavirus S protein and application thereof |
| CN112961238A (en) * | 2020-09-22 | 2021-06-15 | 石河子大学 | Nano antibody based on novel coronavirus S protein S1 subunit and application thereof |
| CN112724209A (en) * | 2021-01-18 | 2021-04-30 | 广东华南疫苗股份有限公司 | Coronavirus recombinant protein capable of forming nano-particles and carrier and application thereof |
| CN112394180A (en) * | 2021-01-19 | 2021-02-23 | 南京立顶医疗科技有限公司 | Detection method and detection kit for SARS-CoV-2 neutralizing antibody |
| CN113121680A (en) * | 2021-04-12 | 2021-07-16 | 华南农业大学 | H5 subtype avian influenza resisting nano antibody protein and encoding gene and application thereof |
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