CN114478761A - Green fluorescent protein shark source nano antibody, preparation method and application thereof - Google Patents
Green fluorescent protein shark source nano antibody, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a green fluorescent protein shark-derived nano antibody, a preparation method and application thereof. The nucleotide sequence of the nano antibody is shown as SEQ ID NO: 1-6, the amino acid sequence is shown as SEQ ID NO: 7-12; the preparation method comprises extracting total RNA of peripheral blood lymphocytes from green fluorescent protein immune striped bamboo shark, and performing reverse transcription to obtain cDNA; amplifying the VNAR fragment, and connecting the VNAR fragment with the phagemid to construct a phage antibody library; and (3) screening a green fluorescent protein specific nano antibody sequence from the library and carrying out recombinant expression to obtain the shark-derived nano antibody targeting the green fluorescent protein. The nano antibody is derived from striped spot bamboo shark, has small molecular weight, strong stability and strong tissue penetration capability, can identify hidden epitope, and can be used in the fields of affinity purification of green fluorescent protein, immunological detection, molecular imaging, immunosensor probes and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a green fluorescent protein antibody and a preparation method thereof, in particular to a green fluorescent protein shark source nano antibody and a preparation method thereof.
Background
Heavy chain-only antibodies (HcAbs) with a naturally occurring light chain deletion were first discovered in camelids in 1993. In 1995, immunoglobulins with similar heavy chain antibody structures, called immunoglobulin neo-antigen receptors (IgNAR), were found in nurse shark (Ginglymostoma cirratum). Similar to the variable regions of traditional immunoglobulin scaffolds, the shark novel antigen receptor variable region (VNAR) was identified as having 4 highly conserved Framework Regions (FRs) and 3 highly variable Complementarity Determining Regions (CDRs), which are referred to as single domain antibodies (sdabs). The large deletion of FR2-CDR2 made VNAR the smallest antibody fragment found to have a binding function, with a molecular weight of about 12 kDa. Single domain antibody proteins are less than 10 nanometers in diameter and are therefore also referred to as nanobodies. Compared with the traditional monoclonal antibody, the nano antibody is easy to produce and modify, the practical applicability of the nano antibody is increased to a certain extent, and the specific advantages of the nano antibody cannot be separated by wide application. VNAR has the characteristics of small molecular weight, strong stability, high affinity and specificity, good solubility, strong tissue penetration capability, capability of identifying hidden epitopes and the like, so that VNAR has the advantages of low production cost, stable quality and the like when being applied. Has potential application prospect in the fields of molecular imaging, disease diagnosis, immunodetection, environmental monitoring and the like.
Green Fluorescent Protein (GFP) was found in Victoria multicell luminescent jellyfish in 1962 by Nomuraea repair et al. GFP is a bioluminescent protein existing in the body of a marine coelenterate, consists of 238 amino acids, and emits green fluorescence under the irradiation of blue light. The green fluorescent protein is basically non-toxic and harmless to biological cells and tissues, and the fluorescent signal of the green fluorescent protein is stable and easy to detect, so that the green fluorescent protein is widely applied to the fields of immunological detection, cell imaging, affinity purification, protein engineering and the like.
The anti-GFP nanobody can be combined with GFP in vivo and in vitro, so that the anti-GFP nanobody can be widely applied to the aspects of subcellular localization, protein activity, protein interaction and the like. The GFP nanobody can be used as a purified affinity ligand for purifying GFP label protein. The high-affinity anti-GFP nano antibody can be coupled with horseradish peroxidase to be used as a detection secondary antibody, and compared with the existing detection secondary antibody in the market, the sensitivity is higher. The nano antibody can also be used as a novel immunosensor probe, and a fluorescence resonance energy transfer nano sensor based on the fluorescence nano antibody is developed by comprehensively applying the immunoassay and the fluorescence resonance energy transfer principle. At present, no shark-derived nano antibody sequence patent for resisting green fluorescent protein exists.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a shark-derived heavy chain antibody variable region sequence (VNAR) capable of binding green fluorescent protein with high affinity, wherein the variable region sequence is also called nano antibody and can be used in the fields of affinity purification of green fluorescent protein, immunological detection, cell imaging, immunosensor probe and the like.
The technical scheme adopted by the invention is as follows:
a green fluorescent protein shark-derived nano antibody:
the nucleotide sequence of the shark source nano antibody is shown as SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO. 6; the amino acid sequence of the shark source nano antibody is shown as SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 or SEQ ID NO.10 or SEQ ID NO.11 or SEQ ID NO. 12.
The shark-derived nano antibody is applied to the fields of affinity purification of green fluorescent protein, immunological detection, cell imaging and the like.
Secondly, a preparation method of a green fluorescent protein shark-derived nano antibody, wherein the preparation method of the shark-derived nano antibody comprises the following steps:
1) preparing green fluorescent protein;
2) immune striped bamboo shark;
3) constructing a phage antibody library;
4) screening specific nano antibodies;
5) and (4) recombinant expression of the antibody.
The green fluorescent protein is prepared by the recombinant expression and purification of the gene of the green fluorescent protein by a prokaryotic or eukaryotic system.
The immune striped bamboo shark is specifically as follows:
s1, mixing the green fluorescent protein solution with Freund' S complete adjuvant at a volume ratio of 1:1, and after complete emulsification, injecting the mixture into the striped bamboo shark lateral fin, wherein the first immunization is carried out;
s2, boosting every 7-28 days, wherein each boosting is to mix a green fluorescent protein solution and Freund' S incomplete adjuvant in a volume ratio of 1:1, and after complete emulsification, injecting the mixture into the striped bamboo shark lateral fin subcutaneously;
s3, repeating the immunization times for 4-6 times, wherein the single immunization dose is 25-50 mu g of each striped bamboo shark.
The construction of the phage antibody library specifically comprises the following steps:
3.1) spraying 0.1% anesthetic MS-222 to the oral nose and gills of the striped bamboo shark 3-14 days after the end of the last immunization to anaesthetize the striped bamboo shark, collecting peripheral blood of the striped bamboo shark through the tail vein, centrifuging the peripheral blood to collect lymphocyte solution, extracting total RNA from the lymphocyte solution and performing reverse transcription to obtain cDNA;
3.2) obtaining VNAR fragments by using high-fidelity enzyme amplification by taking the cDNA as a template;
3.3) using pR2 phagemid as a template, amplifying pR2 phagemid by using high-fidelity enzyme, and utilizing endonuclease to cut pR2 phagemid;
and 3.4) connecting the VNAR fragment with the digested pR2 phagemid through seamless cloning, electrically transforming competent bacteria, diluting and coating the bacteria, and freezing and storing a bacterial scraper to obtain the phage antibody library.
In one embodiment, the library size of the phage antibody library is counted and if the library size is less than a predetermined threshold, reprocessing preparations are discarded.
The specific screening of the specific nano antibody is as follows:
4.1) activating a bacterial strain of a phage antibody library, adding auxiliary phage, collecting thalli through static culture and centrifugation, and performing shake culture on the thalli overnight;
4.2) enriching the phage by using a PEG precipitation method to obtain the enriched phage, and calculating the titer of the enriched phage;
4.3) coating 0.1mg/mL of green fluorescent protein in one hole of a 96-hole immune plate to be used as a green fluorescent protein group, setting one hole without coating protein as a negative control hole, adding enriched phage into the two holes according to the titer of the phage to combine the phage with the protein on the immune plate, washing for multiple times, and eluting by pancreatin to obtain specifically combined phage;
in particular, green fluorescent protein is added into the wells of the immunoplate, and then the enriched phage is added into the wells of the immunoplate with green fluorescent protein.
4.4) infecting TG1 bacteria with specifically bound phage, diluting and coating the plate, culturing to obtain a single colony, and counting the colonies of the green fluorescent protein group and the negative control group to make the colonies of the green fluorescent protein group larger than the colonies of the negative control group;
the negative colonies refer to the colonies in the wells of the plate without the green fluorescent protein treatment, and the positive colonies refer to the colonies in the wells of the plate with the green fluorescent protein treatment.
4.5) selecting a single colony of the green fluorescent proteome in a 96-well plate, adding an auxiliary phage, and amplifying to obtain a monoclonal phage;
4.6) coating the green fluorescent protein with low concentration of 1 mu g/mL in the immune plate again, setting the hole without coating the protein as a negative control hole, adding monoclonal phage into the two groups, and combining the phage with the protein on the immune plate; adding a phage specific antibody marked by horseradish peroxidase, developing by using a substrate, measuring the absorbance of a solution after the development, and screening out a positive colony to obtain an antibody sequence with green fluorescent protein specificity. Positive here means OD450Greater than 1.0.
The specific implementation also utilizes a sequencing primer to perform sequencing analysis on the positive bacterial colony, performs multi-sequence comparison and eliminates repeated sequences, and determines the specific antibody sequence of the green fluorescent protein, which is shown as SEQ ID NO. 1-SEQ ID NO. 6.
The sequencing primer specifically comprises: 5'-CCCTCATAGTTAGCGTAACGA-3', as shown in SEQ ID NO. 13.
The endonuclease is specifically Nco I and Not I.
The seamless clonal connection specifically comprises the following steps: a total of 100. mu.L of ligation system comprising 50. mu.L of seamless clonal ligase, 0.5pmol of pR2 phagemid, 2pmol of VNAR fragment, wherein the volume of pR2 phagemid and VNAR fragment is 50. mu.L; the ligation reaction was carried out in a water bath at 50 ℃ for 1 h.
The helper phage is specifically KM13, M13K07, VCSM13, R408 and the like.
The antibody recombinant expression is specifically as follows: constructing the antibody sequence into a prokaryotic or eukaryotic expression vector, inducing and expressing recombinant protein, and purifying the antibody by using an affinity chromatography column.
In the specific implementation, the interaction relation between the green fluorescent protein and the antibody is further researched, and the affinity of the antibody is measured.
The nucleotide sequence of the nano antibody is shown as SEQ ID NO: 1-6, the amino acid sequence is shown as SEQ ID NO: 7-12; the preparation method comprises extracting total RNA of peripheral blood lymphocyte from green fluorescent protein immune striped bamboo shark, and reverse transcribing into cDNA; amplifying the VNAR fragment, and connecting the VNAR fragment with the phagemid to construct a phage antibody library; and (3) screening a green fluorescent protein specific nano antibody sequence from the library and carrying out recombinant expression to obtain the shark-derived nano antibody targeting the green fluorescent protein.
Compared with the prior art, the invention has the following advantages:
the nano antibody is derived from striped spot bamboo shark, has small molecular weight, strong stability and strong tissue penetration capability, can identify hidden epitope, and can be used in the fields of affinity purification of green fluorescent protein, immunological detection, molecular imaging, immunosensor probes and the like.
The shark source nano antibody screening only needs 3-5 sharks, the nano antibody protein can be obtained in a large amount through a recombinant expression system, the antibody expression amount is high, the preparation cost is low, and the stability is strong. Therefore, the invention overcomes the defects of large molecular weight, difficult preparation, complex operation and the like of the traditional humanized monoclonal antibody.
Drawings
FIG. 1 is a schematic diagram of green fluorescent protein-immunized shark format.
FIG. 2 is a graph showing the results of the monoclonal phage ELISA.
FIG. 3 is a diagram showing the alignment of the amino acid sequences of the nanobodies.
FIG. 4 is a diagram of the result of SDS-PAGE gel electrophoresis of the Nanobody Fc fusion protein. Lane M is the standard protein.
Fig. 5 is a schematic diagram of the affinity between the nanobody and green fluorescent protein characterized by BLI. Where the solid line is the real-time monitored kinetic curve and the dashed line is the software fitted curve. The kinetic curves of the different green fluorescent protein concentration gradients correspond in sequence from top to bottom to the top to bottom concentrations indicated on the right.
FIG. 6 is a graph of the results of characterization of the nanobody binding to green fluorescent protein using pull-down experiments.
FIG. 7 is a graph of the results of characterizing the epitope competition relationship of the nanobody using BLI.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific examples, which are illustrative of the present invention and are not to be construed as limiting the present invention.
The examples of the invention are as follows:
example 1 Green fluorescent protein immune shark
The green fluorescent protein which is expressed by recombination is used for immunizing sharks, and the immunization scheme is shown in figure 1. The invention carries out four times of immunization, and all the immunization is subcutaneous injection. The time interval between immunizations was 10 days, and 5 days after the fourth immunization, shark peripheral blood was collected via tail vein. The invention uses 3 striped bamboo sharks, and the dosage of each shark per immune green fluorescent protein is 25 mug.
Example 2 Green fluorescent protein Nanobody screening
1) Slowly adding the extracted shark peripheral blood to the upper layer of an equal volume of 30% (m/v) sucrose solution, horizontally centrifuging for 30min at 350g, taking the intermediate lymphocyte layer, washing twice by PBS, and centrifuging to collect cell precipitates. Total RNA is extracted by an RNA extraction kit and is reversely transcribed into cDNA.
2) Taking cDNA as a template, amplifying a VNAR sequence by using a specific primer, wherein DNA Polymerase used for amplification is high fidelity enzyme PrimeSTAR Max DNA Polymerase, and the amplification program is as follows: cycling at 98 deg.C, 10s, 57 deg.C, 15s, 72 deg.C, 25s for 30 times. The amplified VNAR fragments were recovered using a kit. Using pR2 phagemid as a template, amplifying pR2 phagemid by using specific primers, wherein the DNA Polymerase used for amplification is high fidelity enzyme PrimeSTARGXL DNA Polymerase, and the amplification program is as follows: cycling at 98 deg.C, 10s, 53 deg.C, 15s, 68 deg.C, 4min, 15s, 30 times. The amplified pR2 phagemid was digested with Nco I and Not I as a template, and then the amplified pR2 phagemid was recovered with a kit. The pR2 phagemid and VNAR fragments were ligated using seamless cloning at a molar ratio of 1: 4. The ligation product was recovered using a kit and the solution used for elution was sterile water. The ligation products were electrically transformed into TG1 competent cells, cultured at 37 ℃ and 200rpm for 12 hours, 0.2. mu.L and 0.02. mu.L (dilution method) of the bacterial solution were applied to a 10cm solid plate, and the number of colonies was counted after 12 hours of culture, and the size of the constructed antibody library was calculated. And (3) centrifuging the residual bacterial liquid, coating the bacterial liquid on a 5-piece 150mm solid plate, culturing at 37 ℃ for 12h, scraping lawn, quickly freezing by liquid nitrogen, and storing at-80 ℃ to obtain the phage antibody library.
3) The cryopreserved antibody library bacteria were activated and KM13 helper phage was added. The bacterial culture supernatant was taken and the phage titer was measured, which was the amplified phage. Coating green fluorescent protein to immune plate at 0.1mg/mL concentration, adding 1X 1011The phage amplified above pfu were incubated for 1h at room temperature. The phages specifically bound to the green fluorescent protein were eluted with pancreatin and infected into TG1 bacteria. 50 mul and 5 mul of infected bacterial liquid were taken respectively to coat a solid plate and the total number of colonies was recorded.
4) From the above plates, 95 single colonies were randomly picked and activated overnight. KM13 helper phage was added and the lysed supernatant was collected by centrifugation, which was a monoclonal phage. Green fluorescent protein was coated at 1. mu.g/mL concentration to a 96-well immunoplate, and the monoclonal phage solution prepared above was added to each well and incubated at room temperature for 1 h. Phage bound to green fluorescent protein were captured using HRP-anti M13 antibody and developed with TMB substrate for 5min at 1M H2SO4The solution stops the reaction. Recording OD by enzyme-linked immunosorbent assay450Numerical values and collated as shown in FIG. 2A histogram.
5) Picking OD450And (5) carrying out sequencing analysis on the holes with the value larger than 1, wherein the sequencing primers are as follows: 5'-CCCTCATAGTTAGCGTAACGA-3' are provided.
6) The measured antibody sequences were analyzed by alignment, and after eliminating duplicate clones, a total of six different nanobody sequences were obtained (fig. 3). SEQ ID NO.1-6 show the nucleotide sequence of the nano antibody, so that the amino acid sequence of the nano antibody shown in SEQ ID NO.7-12 can be obtained.
Example 3 expression and purification of the resulting Green fluorescent protein Nanobody Fc fusion protein
The sequence of the nano antibody is constructed into a mammalian expression vector pTT5 with signal peptide, so that the sequence is fused with human IgG1 Fc for expression, the Fc fragment is expressed at the C terminal, and the nano antibody can be obtained by TEV enzyme digestion. And transfecting the recombinant plasmid into HEK 293 cells, collecting culture supernatant, and purifying the nano antibody Fc fusion protein by using an rProtein A affinity chromatography column. As shown in FIG. 4, we obtained a high-purity green fluorescent protein nanobody Fc fusion protein.
Example 4 characterization of the Green fluorescent protein Nanobodies
1) And (3) adopting BLI to characterize the affinity of the nano antibody and green fluorescent protein. In order to characterize the affinity of the nano antibody and the green fluorescent Protein, firstly, the nano antibody Fc fusion Protein is solidified on a Protein A biosensor, different green fluorescent Protein concentration gradients are set, and the affinity of the green fluorescent Protein and the nano antibody is detected. The results are shown in fig. 5, and the binding force of the six nanobodies and the green fluorescent protein is as follows: g10 > G12 > G6 > G4 > G2 > G29, and the affinity constant K thereofDValues were 5.88, 8.52, 11.1, 19.3, 19.9, 21.8nM, respectively.
2) And (3) representing the combination condition of the nano antibody and the green fluorescent protein by adopting a pull-down experiment. And (3) incubating the Ni-coupled magnetic beads with the recombinant expressed green fluorescent protein with the His tag for 1h, washing the unbound protein, incubating with the G10 antibody, the G12 antibody, the control nano antibody or the Fc fragment for 1h, washing for multiple times, eluting the bound protein on the magnetic beads, and carrying out SDS-PAGE analysis. As can be seen from fig. 6, the G10 antibody and the G12 antibody specifically bind to green fluorescent protein, while neither the control nanobody nor the Fc fragment binds to green fluorescent protein.
3) And characterizing the epitope competition relationship of the nano antibody by adopting BLI. Firstly, the biotinylated green fluorescent protein is solidified on the SA biosensor, and then the solidified green fluorescent protein reacts with the G10 antibody and the G12 antibody in sequence to detect the change condition of the combined signal. As can be seen from fig. 7, after the G10 antibody binds to green fluorescent protein, the G12 antibody can further bind to it, indicating that the G10 antibody and the G12 antibody bind to different epitopes of green fluorescent protein, i.e., there is no epitope competition relationship between the G10 antibody and the G12 antibody.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The sequence related by the invention is as follows:
SEQ ID NO.1：
name: nucleotide sequence of green fluorescent protein shark source nano antibody G2
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaaaaggttccagctatgcattgtgtgacacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgggagctgggtactgcaatagctgggataggggtgactactattatgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.2：
Name: nucleotide sequence of green fluorescent protein shark source nano antibody G4
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgtatccacagctgggatgccctactgttcgagctggaattgctggctatattgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.3：
Name: nucleotide sequence of green fluorescent protein shark source nano antibody G6
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctgagagttgaagacagtggtacatatcactgtaaagcgtatttcagctgggatgactgttcccatctggaattgcggtatagctatattgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.4：
Name: nucleotide sequence of green fluorescent protein shark source nano antibody G10
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttccttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgtattacagctgggatgccaactgttggaacctacgatatagctatattgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.5：
Name: nucleotide sequence of green fluorescent protein shark source nano antibody G12
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacagcgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgtatcgacagctgggatgctcggactggtcaatccatagctatattgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.6：
Name: nucleotide sequence of green fluorescent protein shark source nano antibody G29
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
actcaacgggttgaacaaacaccgacaacgacaacaaaggaggcaggcgaatcactgaccatcaattgcgtcctaagagattccagctgtgcattggatagcacgtactggtatttcacaaaaaagggcgcaacaaagaaggagagcttatcaaatggcggacgatacgcggaaacagtgaacaaggcatcaaagtccttttctttgcgaattagtgacctaagagttgaagacagtggtacatatcactgtaaagcgtatcccagctgggactgtgggaagtccatatatagctatattgaaggaggcggcaccattctgactgtaaaacca
SEQ ID NO.7：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G2
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAGAGYCNSWDRGDYYYEGGGTILTVKP
SEQ ID NO.8：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G4
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPQLGCPTVRAGIAGYIEGGGTILTVKP
SEQ ID NO.9：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G6
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYFSWDDCSHLELRYSYIEGGGTILTVKP
SEQ ID NO.10：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G10
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYYSWDANCWNLRYSYIEGGGTILTVKP
SEQ ID NO11：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G12
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTATTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYRQLGCSDWSIHSYIEGGGTILTVKP
SEQ ID NO.12：
Name: amino acid sequence of green fluorescent protein shark source nano antibody G29
The source is as follows: striped bamboo shark (Cihiloscyllium platiasum)
TQRVEQTPTTTTKEAGESLTINCVLRDSSCALDSTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPSWDCGKSIYSYIEGGGTILTVKP
SEQ ID NO.13：
Name: sequencing primer
The source is as follows: artificial Sequence (Artificial Sequence)
ccctcatagttagcgtaacga。
Sequence listing
<110> college university
<120> green fluorescent protein shark-derived nano antibody, preparation method and application thereof
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 336
<212> DNA
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 1
actcaacggg ttgaacaaac accgacaacg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaaaagg ttccagctat gcattgtgtg acacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttctttgcga attagtgacc taagagttga agacagtggt 240
acatatcact gtaaagcggg agctgggtac tgcaatagct gggatagggg tgactactat 300
tatgaaggag gcggcaccat tctgactgta aaacca 336
<210> 2
<211> 342
<212> DNA
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 2
actcaacggg ttgaacaaac accgacaacg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaagaga ttccagctgt gcattggata gcacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttctttgcga attagtgacc taagagttga agacagtggt 240
acatatcact gtaaagcgta tccacagctg ggatgcccta ctgttcgagc tggaattgct 300
ggctatattg aaggaggcgg caccattctg actgtaaaac ca 342
<210> 3
<211> 342
<212> DNA
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 3
actcaacggg ttgaacaaac accgacaacg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaagaga ttccagctgt gcattggata gcacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttctttgcga attagtgacc tgagagttga agacagtggt 240
acatatcact gtaaagcgta tttcagctgg gatgactgtt cccatctgga attgcggtat 300
agctatattg aaggaggcgg caccattctg actgtaaaac ca 342
<210> 4
<211> 339
<212> DNA
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 4
actcaacggg ttgaacaaac accgacaacg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaagaga ttccagctgt gcattggata gcacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttccttgcga attagtgacc taagagttga agacagtggt 240
acatatcact gtaaagcgta ttacagctgg gatgccaact gttggaacct acgatatagc 300
tatattgaag gaggcggcac cattctgact gtaaaacca 339
<210> 5
<211> 336
<212> DNA
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 5
actcaacggg ttgaacaaac accgacagcg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaagaga ttccagctgt gcattggata gcacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttctttgcga attagtgacc taagagttga agacagtggt 240
acatatcact gtaaagcgta tcgacagctg ggatgctcgg actggtcaat ccatagctat 300
attgaaggag gcggcaccat tctgactgta aaacca 336
<210> 6
<211> 333
<212> DNA
<213> striped spotted bamboo shark (Cihiloscyllium platiasum)
<400> 6
actcaacggg ttgaacaaac accgacaacg acaacaaagg aggcaggcga atcactgacc 60
atcaattgcg tcctaagaga ttccagctgt gcattggata gcacgtactg gtatttcaca 120
aaaaagggcg caacaaagaa ggagagctta tcaaatggcg gacgatacgc ggaaacagtg 180
aacaaggcat caaagtcctt ttctttgcga attagtgacc taagagttga agacagtggt 240
acatatcact gtaaagcgta tcccagctgg gactgtggga agtccatata tagctatatt 300
gaaggaggcg gcaccattct gactgtaaaa cca 333
<210> 7
<211> 112
<212> PRT
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 7
Thr Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Tyr Ala Leu
20 25 30
Cys Asp Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Gly Ala Gly Tyr Cys Asn Ser Trp Asp Arg
85 90 95
Gly Asp Tyr Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys Pro
100 105 110
<210> 8
<211> 114
<212> PRT
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 8
Thr Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Arg Asp Ser Ser Cys Ala Leu
20 25 30
Asp Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Tyr Pro Gln Leu Gly Cys Pro Thr Val Arg
85 90 95
Ala Gly Ile Ala Gly Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val
100 105 110
Lys Pro
<210> 9
<211> 114
<212> PRT
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 9
Thr Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Arg Asp Ser Ser Cys Ala Leu
20 25 30
Asp Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Tyr Phe Ser Trp Asp Asp Cys Ser His Leu
85 90 95
Glu Leu Arg Tyr Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val
100 105 110
Lys Pro
<210> 10
<211> 113
<212> PRT
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 10
Thr Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Arg Asp Ser Ser Cys Ala Leu
20 25 30
Asp Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Tyr Tyr Ser Trp Asp Ala Asn Cys Trp Asn
85 90 95
Leu Arg Tyr Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys
100 105 110
Pro
<210> 11
<211> 112
<212> PRT
<213> striped bamboo shark (Cihiloscyllium platiasum)
<400> 11
Thr Gln Arg Val Glu Gln Thr Pro Thr Ala Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Arg Asp Ser Ser Cys Ala Leu
20 25 30
Asp Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Tyr Arg Gln Leu Gly Cys Ser Asp Trp Ser
85 90 95
Ile His Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys Pro
100 105 110
<210> 12
<211> 111
<212> PRT
<213> striped spotted bamboo shark (Cihiloscyllium platiasum)
<400> 12
Thr Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly
1 5 10 15
Glu Ser Leu Thr Ile Asn Cys Val Leu Arg Asp Ser Ser Cys Ala Leu
20 25 30
Asp Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu
35 40 45
Ser Leu Ser Asn Gly Gly Arg Tyr Ala Glu Thr Val Asn Lys Ala Ser
50 55 60
Lys Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly
65 70 75 80
Thr Tyr His Cys Lys Ala Tyr Pro Ser Trp Asp Cys Gly Lys Ser Ile
85 90 95
Tyr Ser Tyr Ile Glu Gly Gly Gly Thr Ile Leu Thr Val Lys Pro
100 105 110
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ccctcatagt tagcgtaacg a 21
Claims (9)
1. A green fluorescent protein shark-derived nano antibody is characterized in that: the nucleotide sequence of the shark source nano antibody is shown as SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO. 6.
2. The use of the green fluorescent protein shark-derived nanobody of claim 1, characterized in that: the shark-derived nano antibody is applied to the fields of affinity purification of green fluorescent protein, immunological detection, cell imaging and the like.
3. A method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 1 or 2, which comprises: the preparation method of the shark-derived nano antibody comprises the following steps:
1) preparing green fluorescent protein;
2) immune striped bamboo shark;
3) constructing a phage antibody library;
4) screening specific nano antibodies;
5) and (4) recombinant expression of the antibody.
4. The method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 3, wherein: the immune striped bamboo shark is specifically as follows:
s1, mixing the green fluorescent protein solution with Freund' S complete adjuvant at a volume ratio of 1:1, and after complete emulsification, injecting the mixture into the striped bamboo shark lateral fin, wherein the first immunization is carried out;
s2, boosting every 7-28 days, wherein each boosting is to mix a green fluorescent protein solution and Freund' S incomplete adjuvant in a volume ratio of 1:1, and after complete emulsification, injecting the mixture into the striped bamboo shark lateral fin subcutaneously;
s3, repeating the immunization times for 4-6 times, wherein the single immunization dose is 25-50 mu g of each striped bamboo shark.
5. The method for preparing a green fluorescent protein shark-derived nanobody according to claim 3, wherein the method comprises the following steps: the construction of the phage antibody library specifically comprises the following steps:
3.1) spraying an anesthetic MS-222 to the oral nose and gill of the striped bamboo shark 3-14 days after the end of the last immunization to anaesthetize the striped bamboo shark, collecting peripheral blood of the striped bamboo shark through the tail vein, centrifuging the peripheral blood to collect lymphocyte solution, extracting total RNA from the lymphocyte solution and carrying out reverse transcription to obtain cDNA;
3.2) obtaining VNAR fragments by using high-fidelity enzyme amplification by taking the cDNA as a template;
3.3) using pR2 phagemid as a template, amplifying pR2 phagemid by using high-fidelity enzyme, and utilizing endonuclease to cut pR2 phagemid;
and 3.4) connecting the VNAR fragment with the digested pR2 phagemid through seamless cloning, electrically transforming competent bacteria, diluting and coating the bacteria, and freezing and storing a bacterial scraper to obtain the phage antibody library.
6. The method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 3, wherein: the specific screening of the specific nano antibody is as follows:
4.1) activating a bacterial strain of a phage antibody library, adding auxiliary phage, collecting thalli through static culture and centrifugation, and performing shake culture on the thalli overnight;
4.2) enriching the phage by using a PEG precipitation method to obtain the enriched phage, and calculating the titer of the enriched phage;
4.3) coating 0.1mg/mL of green fluorescent protein in one hole of a 96-hole immune plate to be used as a green fluorescent protein group, setting one hole without coating protein as a negative control hole, adding enriched phage into the two holes according to the titer of the phage to combine the phage with the protein on the immune plate, washing for multiple times, and eluting by pancreatin to obtain specifically combined phage;
4.4) infecting TG1 bacteria with specifically bound phage, diluting and coating the plate, culturing to obtain a single colony, and counting the colonies of the green fluorescent protein group and the negative control group to make the colonies of the green fluorescent protein group larger than the colonies of the negative control group;
4.5) selecting a single colony of the green fluorescent proteome in a 96-well plate, adding an auxiliary phage, and amplifying to obtain a monoclonal phage;
4.6) coating 1 microgram/mL green fluorescent protein in the immune plate again, setting the hole without coating protein as a negative control hole, adding monoclonal phage into both groups, and combining the phage and the protein on the immune plate; adding a phage specific antibody marked by horseradish peroxidase, developing by using a substrate, measuring the absorbance of a solution after the development, and screening out a positive colony to obtain an antibody sequence with green fluorescent protein specificity.
7. The method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 6, wherein: the endonuclease is specifically NcoI and NotI.
8. The method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 6, wherein: the seamless clonal connection specifically comprises the following steps: a total of 100. mu.L of ligation system comprising 50. mu.L of seamless clonal ligase, 0.5pmol of pR2 phagemid, 2pmol of VNAR fragment, wherein the volume of pR2 phagemid and VNAR fragment is 50. mu.L; the ligation reaction was carried out in a water bath at 50 ℃ for 1 h.
9. The method for preparing a green fluorescent protein shark-derived nanobody as claimed in claim 3, wherein: the antibody recombinant expression is specifically as follows: constructing the antibody sequence into a prokaryotic or eukaryotic expression vector, inducing and expressing recombinant protein, and purifying the antibody by using an affinity chromatography column.
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