CN115058431A - mEOS nano antibody and preparation method and application thereof - Google Patents

mEOS nano antibody and preparation method and application thereof Download PDF

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CN115058431A
CN115058431A CN202210604378.7A CN202210604378A CN115058431A CN 115058431 A CN115058431 A CN 115058431A CN 202210604378 A CN202210604378 A CN 202210604378A CN 115058431 A CN115058431 A CN 115058431A
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meos
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CN115058431B (en
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容益康
吴靖
李凯丽
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University of South China
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention discloses a mEOS nano antibody and a preparation method and application thereof, and relates to the technical field of biological engineering, wherein a mChery or mEOS nano antibody library is constructed to screen out a specific positive monoclonal nano antibody, two mChery nano antibodies capable of being combined with mChery protein are detected, the nucleotide sequences of the two mChery nano antibodies are shown as SEQ ID No.1 and SEQ ID No.3, and seven mEOS nano antibodies capable of being combined with mEOS protein are shown as SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15 and SEQ ID No. 17. Compared with the traditional antibody, the mCherry or mEOS nano antibody constructed by the invention can improve the yield, reduce the cost and obtain better stability.

Description

mEOS nano antibody and preparation method and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to an mEOS nano antibody and a preparation method and application thereof.
Background
The mcherry is a monomeric red fluorescent protein molecule widely used in biotechnology as a tracer, and comprises the labeling of molecules, the positioning of cell components and the like. Because of its color and light stability of monomer molecules, it is more excellent as a protein tag than other fluorescent protein tags in the biomedical research field.
moeos is a light-activated green to red fluorescent protein molecule. mEOS emits intense green fluorescence which irreversibly converts to red under 390 nm UV irradiation. The fluorescent label is commonly used as a protein label in the biomedical research field, is used for multicolor marking of proteins, carries out positioning marking on the proteins and tracks the movement of the proteins in living cells.
The traditional mCherry antibody and the mEOS antibody are obtained by immunizing animals, the cost is high, the yield is low, the stability among batches is poor, and the traditional antibody is a tetramer consisting of two heavy chains and two light chains, so that the acid-base stability is poor and the variability is easy.
The nano antibody is a single-domain antibody which is obtained by cloning a variable region of a heavy chain antibody and only consists of a heavy chain variable region, the heavy chain antibody (HCAb) exists in camel serum such as camel, a natural deleted light chain and a conventional antibody heavy chain constant region CH1 are reported by Hamers et al for the first time in 1993, and the crystal structure of the nano antibody has the diameter of 2.5 nm, the length of 4 nm and very small molecular weight. Compared with the conventional antibody, the nano antibody has a plurality of advantages, and the preparation of the nano antibody can be one of effective means for solving the defects of the conventional antibody.
Disclosure of Invention
The invention aims to provide an mEOS nano antibody so as to improve the yield, reduce the cost and obtain better stability compared with the traditional antibody.
In order to solve the technical problems, the invention adopts the following technical scheme: the mchery or mEOS nano antibody comprises two coding genes, and the nucleotide sequences of the mchery or mEOS nano antibody are respectively shown as a sequence table SEQ ID NO.1 and a sequence table SEQ ID NO. 3; the mEOS nano antibody comprises seven coding genes, and nucleotide sequences of the seven coding genes are respectively shown as a sequence table SEQ ID NO.5, a sequence table SEQ ID NO.7, a sequence table SEQ ID NO.9, a sequence table SEQ ID NO.11, a sequence table SEQ ID NO.13, a sequence table SEQ ID NO.15 and a sequence table SEQ ID NO. 17.
In addition, the invention also provides two types of mcherry nano antibodies, and the amino acid sequences of the two types of mcherry nano antibodies are respectively shown as the sequence tables SEQ ID NO.2 and SEQ ID NO. 4.
And also provides seven mEOS nano antibodies, and the amino acid sequences of the seven mEOS nano antibodies are respectively shown as SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.18 of the sequence table.
Meanwhile, an expression vector of the mcherry nano-antibody is also provided, and comprises two expression vectors of the mcherry nano-antibody, wherein the expression vectors respectively contain coding genes of the two mcherry nano-antibodies; and also provides an expression vector of the mEOS nano antibody, wherein the expression vector comprises expression vectors of seven mEOS nano antibodies, and the expression vectors respectively contain coding genes of the seven mEOS nano antibodies.
Further, the expression vector is an Escherichia coli plasmid expression vector or pADL-10 b.
A biomaterial related to the aforementioned mcherry nanobody or moeos nanobody, which is any one of (a 1) to (a 8):
(a1) nucleic acid molecules encoding the above mcherry nanobodies or moeos nanobodies;
(a2) an expression cassette comprising the nucleic acid molecule of (a 1).
(a3) A recombinant vector comprising the nucleic acid molecule of (a 1).
(a4) A recombinant vector comprising the expression cassette of (a 2).
(a5) A transgenic animal cell line comprising the nucleic acid molecule of (a 1).
(a6) A transgenic animal cell line comprising the expression cassette of (a 2).
(a7) A transgenic animal cell line comprising the recombinant vector of (a 3).
(a8) A transgenic animal cell line comprising the recombinant vector of (a 4).
In addition, a derivative antibody is provided, wherein the derivative antibody is the following A) or B) or C) or D) or E):
A) a single-chain antibody containing the nano-antibody;
B) a fusion antibody comprising A) the single-chain antibody;
C) a fusion antibody comprising the aforementioned nanobody;
D) fab containing the above nanobody;
E) and (3) a complete antibody containing the nano antibody.
The invention also provides a preparation method of the mcherry nano antibody or the mEOS nano antibody, which comprises the following steps: constructing a mcherry nano antibody library or a mEOS nano antibody library, then screening, enriching the antibody library capable of combining with the antigen, then continuously screening a specific positive monoclonal nano antibody from the antibody library capable of combining with the antigen by an ELISA measuring method, and finally detecting the mcherry nano antibody capable of combining with the mcherry protein or the mEOS nano antibody capable of combining with the mEOS protein by Native binding.
Preferably, the mcherry nanobody library or the mEOS nanobody library is obtained from a mherry protein or a mhos protein clone expressed by escherichia coli.
More preferably, when obtaining the mcherry nanobody library or the mEOS nanobody library from the mherry protein or the mEOS protein clone expressed by escherichia coli, the primers for the first round of amplification by PCR are: the upstream primer is as follows: GGTGGTCCTGGCTGC, respectively; the downstream primer is: GGTACGTGCTGTTGAACTGTTCC are provided.
More preferably, the primers obtained by performing the second round of PCR amplification by using the primers of the first round of amplification as templates are:
an upstream primer:
CTCGCGGCCCAGCCGGCCATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGG；
a downstream primer:
GTGTTGGCCTCCCGGGCCGCGTGCGCCTGAGGAGACGGTGACCTGGGTCC
more preferably, a monoclonal antibody is picked from the constructed mcherry nano antibody library or the mEOS nano antibody library, and the VHH sequence insertion rate is detected by PCR, and the obtained primers are:
CACAGGAAACAGCTATGACCATGATTA/GCGTAACGATCTAAAGTTTTGTCG。
more preferably, the screening is performed by phage display technology to enrich the antibody library for binding to the antigen.
More preferably, after screening out specific positive monoclonal nanobodies, PCR amplification is performed, then Hinf1 is used for enzyme digestion, and clones with non-repetitive sequences are picked for subsequent steps of experiments.
In addition, the invention also provides an application of the mCherry nano antibody or the mEOS nano antibody in preparation of a protein tag.
Compared with the traditional mcherry antibody and the traditional moes antibody, the mcherry nano antibody or the mEOS nano antibody provided by the invention has the advantages of smaller molecular weight, stronger penetrating power, higher specificity, stronger affinity, simpler structure, better stability, easiness for recombination and expression, capability of being produced by mass fermentation of bacteria or yeast, high yield and low cost, and obvious advantages in the diagnosis and treatment of diseases.
Drawings
FIG. 1 is a diagram showing the first round of PCR amplification results in the construction of a mcherry nanobody library in the example;
FIG. 2 is a diagram showing the second round of PCR amplification results in the construction of a mcherry nanobody library in the example;
FIG. 3 is a schematic diagram showing the color development of the coated mcherry plate in the case of screening a specific positive monoclonal by ELISA in the examples;
FIG. 4 is a diagram showing the color development of a BSA-coated ELISA plate in the case of screening specific positive monoclonal by ELISA in the examples;
FIG. 5 is a schematic diagram showing the reaction of the expression supernatant of each monoclonal with mcherry protein in the non-denatured protein gel electrophoresis in the examples;
FIG. 6 is a schematic diagram showing the reaction of expression supernatants of D1, E1, F1, and H1 monoclonals with mEOS protein in the non-denatured protein gel electrophoresis in the examples;
FIG. 7 is a diagram showing the reaction of the expression supernatants of the A2, B9, E8, and G2 monoclonals with mEOS protein in the non-denatured protein gel electrophoresis in the examples.
Detailed Description
In order to facilitate understanding of those skilled in the art, the present invention will be further described with reference to the following examples and drawings, which are not intended to limit the present invention. It should be noted that, unless otherwise specified in the examples, it is understood that the steps may be performed according to conventional experimental conditions, or according to the manufacturer's instructions (e.g., experiments performed with reference to the PrimeScript II 1st Strand cDNA Synthesis Kit instructions).
The preparation method comprises the steps of immunizing alpaca with mCherry protein expressed by escherichia coli, constructing an immunized alpaca nano antibody library, screening specific positive monoclonal nano antibodies from the antibody library by utilizing a phage display technology, finally detecting two mCherry nano antibodies capable of being combined with mChery protein, respectively naming D10 and C1, and obtaining nucleotide sequences of the two mChery nano antibodies through sequencing, wherein the nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.3 in a sequence table, and the corresponding amino acid sequences are shown as SEQ ID NO.2 and SEQ ID NO.4 in the sequence table.
Meanwhile, the method also uses the mEOS protein expressed by escherichia coli to immunize alpaca, constructs an immunized alpaca nano antibody library, screens specific positive monoclonal nano antibodies from the antibody library by using a phage display technology, finally detects seven mEOS nano antibodies capable of being combined with the mEOS protein and respectively names A2, B9, D1, E1, E8, G2 and H1, obtains the nucleotide sequences of the seven mEOS nano antibodies by sequencing, wherein the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.17 in a sequence table, and the corresponding amino acid sequences are shown as SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.18 in the sequence table.
Example 1
The preparation method of the mcherry nano antibody comprises the following steps:
(1) constructing a mcherry nano antibody library:
(1.1) the protein used for immunization is mCherry protein expressed by escherichia coli, 1mg of antigen protein (0.25 mg of mCherry protein among the proteins) is taken to be vibrated, uniformly mixed and emulsified with equal volume of Freund complete adjuvant, the mixture is injected into the neck of an alpaca in a portionwise manner, the immunization is performed for four times in total, the immunization is performed once every two weeks, the dosage of each immunization protein is 0.5mg (0.125 mg of mCherry protein among the proteins), and the adjuvants used for enhancing the immunization are Freund incomplete adjuvants. At least 20mL of alpaca whole blood was collected about four days after the four-immunization, and PBMCs were separated using lymphocyte separation medium.
(1.2) PBMC total RNA was extracted (using trizol method).
(1.3) reverse transcription of RNA into cDNA, the procedure was performed according to the PrimeScript ™ II 1st Strand cDNA Synthesis Kit (available from takara) instructions.
(1.4) performing nested PCR amplification on VHH fragments by using cDNA as a template, wherein primers of the first round of PCR are as follows:
the upstream primer is as follows: GGTGGTCCTGGCTGC, respectively;
the downstream primer is: GGTACGTGCTGTTGAACTGTTCC are provided.
The annealing temperature is 56 ℃, the cycle number is 23, two fragments, namely a VH fragment with the size of about 1000bp and a VHH fragment with the size of 700bp can be amplified, the amplification result is shown in figure 1, and the VHH gene fragment with the size of 700bp can be recovered by cutting gel.
And (3) carrying out second round PCR by taking the first round PCR product as a template to obtain second round PCR primers:
an upstream primer:
CTCGCGGCCCAGCCGGCCATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGG；
a downstream primer:
GTGTTGGCCTCCCGGGCCGCGTGCGCCTGAGGAGACGGTGACCTGGGTCC。
the annealing temperature was 58 ℃ and the number of cycles was 12, and the size of the target fragment was about 450bp, and as a result, the fragment was purified by using a PCR product purification kit, as shown in FIG. 2.
(1.5) digesting and recovering a target gene fragment and a pADL-10b vector by using Bgl1 endonuclease, connecting the vector and the VHH fragment by using T4 ligase, transforming an escherichia coli SS320 competent cell into a connection product, namely a constructed mcherry nano antibody bacterial library, and calculating to obtain the library capacity of 1 multiplied by 10 8 Selecting 24 monoclonals, and detecting the VHH sequence insertion rate by using PCR (polymerase chain reaction) with the primers as follows:
CACAGGAAACAGCTATGACCATGATTA/GCGTAACGATCTAAAGTTTTGTCG。
(2) screening of mcherry nanobody libraries:
(2.1) to a 5mL immune tube, 200. mu.g of mcherry protein dissolved in PBS was added and coated overnight at 4 ℃ while a BSA-coated negative control was set.
(2.2) Add 2 mL of 3% BSA for 2h at room temperature blocking, and add 500. mu.L phage solution (phage display library titer 1.48X 10) 13 cfu), at room temperature for 1 h.
(2.3) washing the immune tube 10 times by using PBST (tween concentration is 0.05 percent) to remove the non-combined phage, adding 0.25mg/mL trypsin to elute the phage specifically combined with the protein for 30min, infecting the phage with Escherichia coli SS320 in logarithmic growth phase, and preparing the phage required by the next round of screening and detecting the screening effect.
(2.4) the screening process needs to be repeated for 3 rounds, the amount of the coated protein is properly reduced along with the increase of the number of screening rounds, and the cleaning conditions are also set to be more severe.
(2.5) the screening results are shown in Table 1, after two rounds of screening, the antibody library capable of binding the antigen is enriched, and subsequent experiments can be carried out to find monoclonal antibody binding the antigen.
input titer mCherry potency BSA control titer
First round of screening 1.6×10 12 1.08×10 7 1×10 6
Second round of screening 1.84×10 10 2.4×10 5 8×10 3
Third round of screening 1.16×10 11 1.64×10 9 4×10 3
TABLE 1
(3) ELISA screening of specific Positive monoclonals
(3.1) after 3 rounds of screening, 96 SS320 monoclonals infected with phage were picked and inoculated into 200. mu.L of 2 XYT medium (Amp, Tet resistance), after logarithmic growth phase was reached, helper phage M13k07, kana and IPTG were added, overnight culture was carried out at 30 ℃ and centrifuged to remove the bacterial pellet and the phage supernatant was used.
(3.2) two enzyme-labeled plates are taken to coat mCherry protein and BSA protein respectively, and after 3% BSA is blocked, 50 mu L of phage supernatant is added to act for 1h at room temperature.
(3.3) washing with TBST, adding Anti-fd bacteriophagae antibody (from sigma), and reacting at room temperature for 1 h.
(3.4) after TBST washing, Goat anti-rabbitIgG HRP conjugated was added and the reaction was carried out at room temperature for 1 hour.
And (3.5) washing with TBST, adding a TMB color development liquid for color development, and adding a stop solution to stop the reaction.
(3.6) the color development of the coated mCherry enzyme label plate is shown in FIG. 3, the color development of the coated BSA enzyme label plate is shown in FIG. 4, the color development values of all clones are higher, 48 single clones are selected, enzyme digestion is carried out by Hinf1 after PCR amplification, and clones with non-repetitive sequences are selected for subsequent experiments, wherein the clones are as follows: a1, D10, C1, D1, B3 and E3.
(4) Native binding detection of Nanobody binding to antigen protein
(4.1) A1, D10, C1, D1, B3 and E3 were picked up and cultured in 2 XYT medium (Amp, Tet-resistant) at 37 ℃.
(4.2) when OD600 reached 0.6, IPTG was added for induction for 6 h.
(4.3) centrifugation was performed to collect the pellet, and CelLytic ™ B lysate (available from sigma) was added thereto, and the lysate was lysed at room temperature for 15min, followed by centrifugation to collect the supernatant.
And (4.4) sucking 18 mu L of supernatant, adding purified mcherry protein, uniformly mixing, standing at room temperature for reaction for 20min, and then carrying out non-denaturing protein gel electrophoresis.
(4.5) the results are shown in FIG. 5, and the expressed supernatants D10, C1, B3 and E3 reacted with mcherry protein, so that the mixture of the expressed supernatant and the mcherry protein has a great difference in electrophoresis speed with the mcherry protein when subjected to non-denaturing protein gel electrophoresis, and displacement is generated, which indicates that these nanobodies can be combined with the mcherry.
Finally, the sequences of D10 and C1 are determined by sequencing, and two strains of mCherry nanobodies are obtained.
This example provides 2 anti-mcherry nanobodies.
Example 2
The preparation method of the mEOS nano antibody comprises the following steps:
(1) constructing an mEOS nano antibody library:
(1.1) the protein used for immunization is mEOS protein expressed by escherichia coli, 0.5mg of mEOS protein is taken to vibrate, uniformly mix and emulsify with equivalent Freund's complete adjuvant during first immunization, the mixture is injected into the neck of the alpaca in points, immunization is performed for four times in total, immunization is performed once every two weeks, the dosage of each immune protein is 0.5mg, and adjuvants used for enhancing the immunization are Freund's incomplete adjuvants. At least 20mL of alpaca whole blood was collected four days or so after the four-immunization, and PBMCs were separated using a lymphocyte separation medium.
(1.2) PBMC total RNA was extracted (using trizol method).
(1.3) reverse transcription of RNA into cDNA, the procedure was performed with reference to the PrimeScript ™ II 1st Strand cDNA Synthesis Kit (available from takara) instructions.
(1.4) performing nested PCR amplification on VHH fragments by using cDNA as a template, wherein primers of the first round of PCR are as follows:
the upstream primer is as follows: GGTGGTCCTGGCTGC, respectively;
the downstream primer is: GGTACGTGCTGTTGAACTGTTCC are provided.
The annealing temperature is 56 ℃, the cycle number is 23, two fragments can be amplified, the sizes of the two fragments are respectively about 1000bp VH fragment and 700bp VHH fragment, and the VHH gene fragment with the size of 700bp can be recovered by cutting gel.
And (3) carrying out second round PCR by taking the first round PCR product as a template, wherein the primers are as follows:
an upstream primer:
CTCGCGGCCCAGCCGGCCATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGG；
a downstream primer:
GTGTTGGCCTCCCGGGCCGCGTGCGCCTGAGGAGACGGTGACCTGGGTCC。
the annealing temperature was 58 ℃ and the number of cycles was 12, the size of this fragment of interest was about 500bp, and this fragment was purified using a PCR product purification kit.
(1.5) digesting and recovering a target gene fragment and a pADL-10b vector by using Bgl1 endonuclease, connecting the vector and the VHH fragment by using T4 ligase, transforming Escherichia coli SS320 competent cells into a connection product, namely a constructed mEOS nano antibody bacterial library, and calculating to obtain a library capacity of 1 multiplied by 10 8 Selecting 24 monoclonals, and detecting the VHH sequence insertion rate by using PCR (polymerase chain reaction) with the primers as follows:
CACAGGAAACAGCTATGACCATGATTA/GCGTAACGATCTAAAGTTTTGTCG, the results show that the insertion rate is: 79 percent.
(2) Screening of the moes nanobody library:
(2.1) to a 5mL immune tube, 200. mu.g mEOS protein dissolved in PBS was added and coated overnight at 4 ℃ while a BSA coated negative control was set.
(2.2) Add 2 mL of 3% BSA for 2h at room temperature blocking, and add 500. mu.L phage solution (phage display library titer 1.48X 10) 13 cfu), at room temperature for 1 h.
(2.3) washing the immune tube 10 times by using PBST (tween concentration is 0.05 percent) to remove the non-combined phage, adding 0.25mg/mL trypsin to elute the phage specifically combined with the protein for 30min, infecting the phage with Escherichia coli SS320 in logarithmic growth phase, and preparing the phage required by the next round of screening and detecting the screening effect.
(2.4) the screening process needs to be repeated for 2 rounds, the amount of the coated protein is properly reduced along with the increase of the number of screening rounds, and the cleaning conditions are also set to be more severe.
(2.5) the screening results are shown in Table 2, after two rounds of screening, the antibody library capable of binding the antigen is enriched, and subsequent experiments can be carried out to find monoclonal antibodies binding the antigen.
input titer mEOS potency BSA control titer
First round of screening 1.48×10 13 1.36×10 6 1.84×10 4
Second round of screening 1×10 13 4.5×10 8 1.36×10 5
TABLE 2
(3) ELISA screening of specific Positive monoclonals
(3.1) after 2 rounds of selection, 94 phage-infected SS320 monoclonals were picked, and two uninfected phage SS320 were picked as controls. Inoculating to 200 μ L2 XYT medium (Amp, Tet resistance), adding helper phage M13k07, kana and IPTG after logarithmic growth phase, culturing overnight at 30 deg.C, centrifuging to remove thallus precipitate, and collecting phage supernatant.
(3.2) taking an enzyme label plate to coat mEOS protein, blocking by 3% BSA, adding 50 mu L of phage supernatant, and acting for 1h at room temperature.
(3.3) washing with TBST, adding Anti-fd bacteriophagae antibody (from sigma), and reacting at room temperature for 1 h.
(3.4) after TBST washing, Goat anti-rabbitIgG HRP conjugated was added and the reaction was carried out at room temperature for 1 hour.
And (3.5) washing with TBST, adding TMB color development liquid for color development, and reading at 450nm by using a microplate reader after termination.
(3.6) results Table 3 shows that the italic values indicate that the color rendering values are high, the binding ability with antigen is strong, single clones with high color rendering values are selected, enzyme digestion is carried out by Hinf1 after PCR amplification, and clones with non-repetitive sequences are selected for subsequent experiments, wherein the clones are as follows: a2, B9, D1, E1, E8, F1, G2 and H1.
<> 1 2 3 4 5 6 7 8 9 10 11 12
A 2.461 3.093 0.116 0.151 0.169 2.497 2.812 0.135 0.431 2.812 2.761 0.106
B 0.329 3.198 3.198 3.004 0.184 2.365 0.148 0.142 3.336 0.298 0.142 0.148
C 2.862 0.489 0.139 0.515 3.914 0.307 0.543 0.263 3.388 0.323 3.914 3.25
D 3.234 0.186 3.234 3.898 3.898 1.974 3.234 3.898 3.898 3.566 0.143 0.923
E 3.249 3.581 0.53 3.912 0.358 3.249 3.581 3.249 2.917 3.142 0.103 3.249
F 3.049 3.575 0.188 0.262 3.907 0.291 3.575 3.136 0.268 3.136 3.907 0.126
G 3.161 3.161 0.177 0.198 3.931 3.931 3.405 3.931 3.931 3.931 3.6 0
H 3.08 0.215 2.277 2.919 3.187 3.85 2.992 0.282 2.448 1.347 2.748 0
TABLE 3
(4) Native binding detection of Nanobody binding to antigen protein
(4.1) A2, B9, D1, E1, E8, F1, G2 and H1 were picked up and cultured in 2 XYT medium (Amp, Tet-resistant) at 37 ℃.
(4.2) when OD600 reached 0.6, IPTG was added for induction for 6 h.
(4.3) centrifugation was carried out to collect the pellet of the cells, CelLytic ™ B lysate (from sigma) was added thereto, lysis was carried out at room temperature for 15min, and centrifugation was carried out to collect the supernatant.
And (4.4) sucking 18 mu L of supernatant, adding 16 mu g of purified mEOS protein, uniformly mixing, standing at room temperature for reaction for 20min, and then carrying out non-denatured protein gel electrophoresis.
(4.5) As shown in FIGS. 6 and 7, the expression supernatants of the individual clones reacted with mEOS protein, so that the mixture of the expression supernatants and the mEOS protein was subjected to non-denaturing protein gel electrophoresis at a rate that was significantly different from the rate of mEOS electrophoresis, resulting in a shift, indicating that these nanobodies were both bound to mEOS.
Finally, the sequencing results showed that the amino acid sequences of E1 and F1 were identical, so there were a total of seven different nanobody sequences, respectively: a2, B9, D1, E1, E8, G2 and H1. This example provides seven anti-mEOS nanobodies.
It will be appreciated by those skilled in the art that the encoding gene (DNA molecule) of the present invention may also be present in the form of an "expression cassette" or "recombinant vector". An "expression cassette" refers to a nucleic acid molecule, linear or circular, encompassing DNA and RNA sequences capable of directing the expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter is included that is operably linked to a nucleotide of interest, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest in the sense or antisense orientation, e.g., an antisense RNA or an untranslated RNA. An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component. The expression cassette may also be naturally occurring but obtained with efficient recombinant formation for heterologous expression.
The above embodiments are preferred implementations of the present invention, and the present invention can be implemented in other ways without departing from the spirit of the present invention.
Some of the drawings and descriptions of the present invention have been simplified to facilitate the understanding of the improvements over the prior art by those skilled in the art, and some other elements have been omitted from this document for the sake of clarity, and it should be appreciated by those skilled in the art that such omitted elements may also constitute the subject matter of the present invention.
Sequence listing
<110> university of southern China
<120> mEOS nano antibody and preparation method and application thereof
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 384
<212> DNA
<213> Lama pacos
<400> 1
gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggagtctct gagactctcc 60
tgtgatgcct cagaattccg gttcaattat tatgccatag gttggttccg ccaggcccca 120
ggaaaggggc gtgagggggt ctcatgtatt tctatgaatg acggtagtac aaagtatgga 180
gactccgtga aggaccgatt caccatctcc aaagacaaca ccaagaacac agtgtatctg 240
caaatgaaca gcccgaagcc tgaggacacg gccgtttatt cctgtgcagc aaaacgaggc 300
ccgatttgta ccttcgtaga atcggcctat gactcctggg gccaggggac ccaggtcacc 360
gtctcctcag gcgcacgcgg cccg 384
<210> 2
<211> 128
<212> PRT
<213> Lama pacos
<400> 2
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu Ser
1 5 10 15
Leu Arg Leu Ser Cys Asp Ala Ser Glu Phe Arg Phe Asn Tyr Tyr Ala
20 25 30
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ser
35 40 45
Cys Ile Ser Met Asn Asp Gly Ser Thr Lys Tyr Gly Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ser Lys Asp Asn Thr Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Pro Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Ala
85 90 95
Ala Lys Arg Gly Pro Ile Cys Thr Phe Val Glu Ser Ala Tyr Asp Ser
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Ala Arg Gly Pro
115 120 125
<210> 3
<211> 378
<212> DNA
<213> Lama pacos
<400> 3
gtgcagctgg tggagtctgg gggaggattg gtgcaggctg ggggctctct gagactctcc 60
tgtacagcct ctggaggcac cttcagtacc tatgccatgg gctggttccg ccaggctcca 120
gggaaggagc gtgagtttgt agcagctatt acctggagtg gtggtgacac atactatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
caaatggaca gcctgaaacc tgaggacacg gccgtttatt actgtgcagc agacccgtac 300
ggtagtagct ggcacccatt atgggcccga actcagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 4
<211> 126
<212> PRT
<213> Lama pacos
<400> 4
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Ala Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Thr Ala Ser Gly Gly Thr Pro Ser Thr Thr Ala
20 25 30
Met Gly Thr Pro Ala Gly Ala Pro Gly Leu Gly Ala Gly Pro Val Ala
35 40 45
Ala Ile Thr Thr Ser Gly Gly Ala Thr Thr Thr Ala Ala Ser Val Leu
50 55 60
Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Thr Val Thr Leu
65 70 75 80
Gly Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Ala
85 90 95
Ala Ala Pro Thr Gly Ser Ser Thr His Pro Leu Thr Ala Ala Thr Gly
100 105 110
Thr Ala Thr Thr Gly Gly Gly Thr Gly Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 366
<212> DNA
<213> Lama pacos
<400> 5
gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggggtctct gagactctcc 60
tgtgcagcct ctggattcac tttggattat tatgccatag gctggttccg ccaggcccca 120
gggaaggagc gtgagggggt ctcatgtatt agtcgtagcg atattaccac gcactatgca 180
gactccgtga aaggccgatt caccatctcc agagacaacg ccaagaacac agtgtatctg 240
caaatgaaca gcctgaaacc tgaggacaca gccgtttatt actgtgcagc acgtatgaat 300
tacttctgta caatctctat gtcctatgac tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 6
<211> 122
<212> PRT
<213> Lama pacos
<400> 6
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Ala Ser Gly Pro Thr Leu Ala Thr Thr Ala
20 25 30
Ile Gly Thr Pro Ala Gly Ala Pro Gly Leu Gly Ala Gly Gly Val Ser
35 40 45
Cys Ile Ser Ala Ser Ala Ile Thr Thr His Thr Ala Ala Ser Val Leu
50 55 60
Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Thr Val Thr Leu
65 70 75 80
Gly Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Ala
85 90 95
Ala Ala Met Ala Thr Pro Cys Thr Ile Ser Met Ser Thr Ala Thr Thr
100 105 110
Gly Gly Gly Thr Gly Val Thr Val Ser Ser
115 120
<210> 7
<211> 366
<212> DNA
<213> Lama pacos
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gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggggtctct gagactctcc 60
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gggaaggagc gtgagggggt cttatgtatt aatggtagag gtagtagcgc gatctatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaatac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacaca gccgtttatt actgtgcatt ccggtacgtc 300
ggctgttcag gctttcaagg ctcctatcag tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 8
<211> 122
<212> PRT
<213> Lama pacos
<400> 8
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Thr Ser Gly Pro Thr Ser Ala Ser Thr Val
20 25 30
Ile Ala Thr Pro Ala Gly Ala Pro Gly Leu Gly Ala Gly Gly Val Leu
35 40 45
Cys Ile Ala Gly Ala Gly Ser Ser Ala Ile Thr Ala Ala Ser Val Leu
50 55 60
Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Thr Val Thr Leu
65 70 75 80
Gly Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Ala
85 90 95
Pro Ala Thr Val Gly Cys Ser Gly Pro Gly Gly Ser Thr Gly Thr Thr
100 105 110
Gly Gly Gly Thr Gly Val Thr Val Ser Ser
115 120
<210> 9
<211> 369
<212> DNA
<213> Lama pacos
<400> 9
gtgcagctgg tggagtctgg gggaggattg gtgcaggctg gggactctct gagactctcc 60
tgtgcagcct ctggaagcac ctttagttgg tatgccatgg gctggttccg ccaggctcca 120
ggaaaggagc gtgagtttgt agcagctacg agccggagta gccctaccac atactatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtttctg 240
caaatgacta gcctgaaacc tgaggacacg gccgtttatt tctgtgcggg gcgtgtagga 300
cctggggtgc cacgtattcc ccaatcatat agctactggg gccaggggac ccaggtcacc 360
gtctcctca 369
<210> 10
<211> 123
<212> PRT
<213> Lama pacos
<400> 10
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Ala Gly Ala Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Ala Ser Gly Ser Thr Pro Ser Thr Thr Ala
20 25 30
Met Gly Thr Pro Ala Gly Ala Pro Gly Leu Gly Ala Gly Pro Val Ala
35 40 45
Ala Thr Ser Ala Ser Ser Pro Thr Thr Thr Thr Ala Ala Ser Val Leu
50 55 60
Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Thr Val Pro Leu
65 70 75 80
Gly Met Thr Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Pro Cys Ala
85 90 95
Gly Ala Val Gly Pro Gly Val Pro Ala Ile Pro Gly Ser Thr Ser Thr
100 105 110
Thr Gly Gly Gly Thr Gly Val Thr Val Ser Ser
115 120
<210> 11
<211> 342
<212> DNA
<213> Lama pacos
<400> 11
gtgcagctgg tggagtctgg gggaggcttg gtgcaggctg ggggatctct aacactctcc 60
tgtgcagcct ctggaagctt cttcagtatc aatggcatcg gctggtaccg ccaggctcca 120
gggaagcagc gcgagttggt cgcaagtatt agtagtgatg gtagcataga ctatacagac 180
tccgtgaagg gccgattcac catctcagga gacaacgcca agcacacggt gtatctgcaa 240
atgaacagcc tgaaacctga ggatacggcc gtctattact gtggtgcacg taggcgttgg 300
gttaattact ggggccaggg gacccaggtc accgtctcct ca 342
<210> 12
<211> 114
<212> PRT
<213> Lama pacos
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Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Ala Gly Gly Ser
1 5 10 15
Leu Thr Leu Ser Cys Ala Ala Ser Gly Ser Pro Pro Ser Ile Ala Gly
20 25 30
Ile Gly Thr Thr Ala Gly Ala Pro Gly Leu Gly Ala Gly Leu Val Ala
35 40 45
Ser Ile Ser Ser Ala Gly Ser Ile Ala Thr Thr Ala Ser Val Leu Gly
50 55 60
Ala Pro Thr Ile Ser Gly Ala Ala Ala Leu His Thr Val Thr Leu Gly
65 70 75 80
Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Gly Ala
85 90 95
Ala Ala Ala Thr Val Ala Thr Thr Gly Gly Gly Thr Gly Val Thr Val
100 105 110
Ser Ser
<210> 13
<211> 342
<212> DNA
<213> Lama pacos
<400> 13
gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggggtctct gagactctcc 60
tgtgcagcct ctagaagcat cgccagtttc tatgccgcgg gctggtaccg ccaggctcca 120
gggaagcagc gcgagttggt cgcaaaaatt actagtggta gtaacccata ctatgcagac 180
tccgtgaagg gccgattcac catctccaca gacagcgcca agaacacggt cgatctgcaa 240
atgaacagcc tgaaacctga ggacacggcc gtctattact gtaatatagc gagtgtgttt 300
cgtgactact ggggccaggg gacccaggtc accgtctcct ca 342
<210> 14
<211> 114
<212> PRT
<213> Lama pacos
<400> 14
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Ala Ser Ala Ser Ile Ala Ser Pro Thr Ala
20 25 30
Ala Gly Thr Thr Ala Gly Ala Pro Gly Leu Gly Ala Gly Leu Val Ala
35 40 45
Leu Ile Thr Ser Gly Ser Ala Pro Thr Thr Ala Ala Ser Val Leu Gly
50 55 60
Ala Pro Thr Ile Ser Thr Ala Ser Ala Leu Ala Thr Val Ala Leu Gly
65 70 75 80
Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Ala Ile
85 90 95
Ala Ser Val Pro Ala Ala Thr Thr Gly Gly Gly Thr Gly Val Thr Val
100 105 110
Ser Ser
<210> 15
<211> 342
<212> DNA
<213> Lama pacos
<400> 15
gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggggtctct gagactctcc 60
tgtgcagcct ctggaagcat ctcgcgtatc gcacccatgg gctggtaccg ccaggctcca 120
gggaagcagc gcgagttggt cgcagctatt tcaagtggtg gtaacacaaa ctatgcagac 180
tccgtgaagg gccgattcac catctccaga gacaacgcca agatcacggt atatctgcaa 240
atgaacagcc tggagtctga ggacacggcc gtgtattact gtaaagcagt gttcttgggc 300
cgatcttact ggggccaggg gacccaggtc accgtctcct ca 342
<210> 16
<211> 114
<212> PRT
<213> Lama pacos
<400> 16
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ala Ile Ala Pro
20 25 30
Met Gly Thr Thr Ala Gly Ala Pro Gly Leu Gly Ala Gly Leu Val Ala
35 40 45
Ala Ile Ser Ser Gly Gly Ala Thr Ala Thr Ala Ala Ser Val Leu Gly
50 55 60
Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ile Thr Val Thr Leu Gly
65 70 75 80
Met Ala Ser Leu Gly Ser Gly Ala Thr Ala Val Thr Thr Cys Leu Ala
85 90 95
Val Pro Leu Gly Ala Ser Thr Thr Gly Gly Gly Thr Gly Val Thr Val
100 105 110
Ser Ser
<210> 17
<211> 351
<212> DNA
<213> Lama pacos
<400> 17
gtgcagctgg tggagtctgg gggaggcttg gtgcagcctg gggggtctct gagactctcc 60
tgtgcagcct ctggattctt cttcagtagc tatcacatga gctggtaccg ccaggctcca 120
gggctggagc gcgagttggt cgcagccatt attggtggtg gtggtggcac aaactatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtatatt actgtaacgc ccgccggtac 300
gcgccaacga gagactactg gggccagggg acccaggtca ccgtctcctc a 351
<210> 18
<211> 117
<212> PRT
<213> Lama pacos
<400> 18
Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser
1 5 10 15
Leu Ala Leu Ser Cys Ala Ala Ser Gly Pro Pro Pro Ser Ser Thr His
20 25 30
Met Ser Thr Thr Ala Gly Ala Pro Gly Leu Gly Ala Gly Leu Val Ala
35 40 45
Ala Ile Ile Gly Gly Gly Gly Gly Thr Ala Thr Ala Ala Ser Val Leu
50 55 60
Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Thr Val Thr Leu
65 70 75 80
Gly Met Ala Ser Leu Leu Pro Gly Ala Thr Ala Val Thr Thr Cys Ala
85 90 95
Ala Ala Ala Thr Ala Pro Thr Ala Ala Thr Thr Gly Gly Gly Thr Gly
100 105 110
Val Thr Val Ser Ser
115

Claims (6)

  1. A coding gene of a mEOS nanobody, characterized in that: the mEOS nano antibody comprises seven coding genes, and nucleotide sequences of the seven coding genes are respectively shown as a sequence table SEQ ID NO.5, a sequence table SEQ ID NO.7, a sequence table SEQ ID NO.9, a sequence table SEQ ID NO.11, a sequence table SEQ ID NO.13, a sequence table SEQ ID NO.15 and a sequence table SEQ ID NO. 17.
  2. An mEOS nanobody characterized by: comprises seven mEOS nano antibodies, and the amino acid sequences of the seven mEOS nano antibodies are respectively shown as a sequence table SEQ ID NO.6, a sequence table SEQ ID NO.8, a sequence table SEQ ID NO.10, a sequence table SEQ ID NO.12, a sequence table SEQ ID NO.14, a sequence table SEQ ID NO.16 and a sequence table SEQ ID NO. 18.
  3. An expression vector of a mEOS nanobody, characterized in that: the expression vector comprises expression vectors of seven mEOS nano antibodies, and the expression vectors respectively contain coding genes of the seven mEOS nano antibodies in claim 1.
  4. 4. The mEOS nanobody expression vector of claim 3, wherein: the expression vector is an escherichia coli plasmid expression vector.
  5. 5. The muos nanobody-related biomaterial of claim 2, characterized in that: the biomaterial is any one of (a 1) to (a 8):
    (a1) a nucleic acid molecule encoding the moeos nanobody of claim 2;
    (a2) an expression cassette comprising the nucleic acid molecule of (a 1);
    (a3) a recombinant vector comprising the nucleic acid molecule of (a 1);
    (a4) a recombinant vector comprising the expression cassette of (a 2);
    (a5) a transgenic animal cell line comprising the nucleic acid molecule of (a 1);
    (a6) a transgenic animal cell line comprising the expression cassette of (a 2);
    (a7) a transgenic animal cell line comprising the recombinant vector of (a 3);
    (a8) a transgenic animal cell line comprising the recombinant vector of (a 4).
  6. 6. Use of the mEOS nanobody of claim 2 for the preparation of a protein tag.
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