LU101600B1 - Single-chain antibody TRSAB1 against TORSV and preparation method thereof - Google Patents
Single-chain antibody TRSAB1 against TORSV and preparation method thereof Download PDFInfo
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- LU101600B1 LU101600B1 LU101600A LU101600A LU101600B1 LU 101600 B1 LU101600 B1 LU 101600B1 LU 101600 A LU101600 A LU 101600A LU 101600 A LU101600 A LU 101600A LU 101600 B1 LU101600 B1 LU 101600B1
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- trsab1
- chain antibody
- torsv
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- 238000002360 preparation method Methods 0.000 title description 5
- 241000723681 Tomato ringspot virus Species 0.000 claims abstract description 41
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
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- 125000000539 amino acid group Chemical group 0.000 claims abstract description 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 36
- 230000014509 gene expression Effects 0.000 claims description 26
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
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- 239000004327 boric acid Substances 0.000 claims description 6
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- 238000000502 dialysis Methods 0.000 claims description 6
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 6
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 6
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 6
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
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- 239000011684 sodium molybdate Substances 0.000 claims description 6
- 235000015393 sodium molybdate Nutrition 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 3
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- 238000004519 manufacturing process Methods 0.000 claims description 2
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 9
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
Abstract
Provided is a single-chain antibody TRsAbl against ToRSV, having an amino acid sequence shown in SEQ ID NO.1, and consisting of a 15aa histidine tag region, a 180aa ToRSV heavy chain variable region, a 15aa linker peptide, and a reverse-linked 117aa ToRSV light chain variable region, connected in sequence, with a length of 327 amino acid residues. The sequences of the 180aa heavy chain variable region and the 117aa light chain variable region are derived from the sequence of a monoclonal antibody produced by the hybridoma cell line CGMCC18321. The yield of TRsAbl, obtained by a whole gene synthesis method, is 200-2000mg/L, with a purity of 82-95%. The single-chain antibody TRsAbl has the characteristics of selectively identifying the peptide QAAQQAGKNPFGRG in the ToRSV coat protein, and has a positive response to natural ToRSV particles. The invention has broad application prospects in the Entry-Exit inspection and quarantine of the ToRSV.
Description
SINGLE-CHAIN ANTIBODY TRSAB1 AGAINST TORSV AND PREPARATION METHOD LU101600
[0001] Technical Field
[0002] The invention relates to the field of biopolymer research, and in particular to . a single-chain antibody against TORSV and a preparation method thereof.
[0003] Description of Related Art
[0004] Tomato Ringspot virus (TORSV) belongs to the Comoviridae family and is a high-risk plant virus. Its genome contains two sense single-stranded RNAs that encode replicase, kinesin (MP), and coat protein (CP), respectively. TORSV is widely distributed in Europe, America and Oceania, and can infect more than 150 crops such as soybean, apple, cherry, tomato and tobacco plants. At present, the methods used for quarantine of ToRSV at home and abroad include electron microscope observation, identification of host responses (biological methods), serological tests and molecular biological methods. ToRSV is a globular virus with a diameter of 28nm, and the virus content in samples is low, which makes it difficult to observe and identify under electron microscopy; vaccination identification hosts require a special isolation greenhouse, which takes a long time; and moreover the masking of symptom occurring in the virus is likely to affect the identification result. Serological methods are a necessary means for rapid detection of plant viruses. At present, virus detection at ports relies on the purchase of imported antiserum, which is expensive. However, there are no reports on use of TORSV serum test kits for imported plant seedlings in China.
[0005] The present invention adopts the strategy of expressing single-chain antibodies in E. coli, and uses the sequences of the heavy chain variable region and the light chain variable region in a monoclonal antibody that specifically identifies the peptide QAAQQAGKNPFGRG in the ToRSV coat protein as a template to prepare a recombinant single-chain antibody. The single-chain antibody of the present invention, named TRsAb1, consists of four peptides, a 15aa histidine tag region (6xHis tag), a 180aa ToRSV heavy 1chain variable region (igH180), a 15aa linker peptide, and a reverse-linked 117aa ToRSV LU101600 light chain variable region (igk117), connected in sequence, with a length of 327 amino acid residues. The sequences of the 180aa heavy chain variable region and the 117aa light chain variable region are derived from the sequence of a monoclonal antibody produced by the hybridoma cell line CGMCC18321 (deposited by: China General Microbiological Culture Collection Center). The single-chain antibody TRsAb1 of the present invention has the characteristics of low preparation cost, simple preparation method, specificity in binding to the ToRSV coat protein QAAQQAGKNPFGRG peptide, and positive reaction to natural ToRSV particles. The invention has broad application prospects in the inspection and quarantine of imported and exported seeds and seedlings.
[0006] An objective of the invention is to provide a single-chain antibody TRsAb1 against ToRSV, which can be used for detection of TORSV. The single-chain antibody, having an amino acid sequence as shown in SEQ ID NO.1, consists of four peptides, a 15aa histidine tag region (6xHis tag), a 180aa ToRSV heavy chain variable region (igH180), a 15aa linker peptide, and a reverse-linked 117aa ToRSV light chain variable region (igK117), connected in sequence, with a length of 327 amino acid residues. The sequences of the 180aa heavy chain variable region and the 117aa light chain variable region are derived from the sequence of a monoclonal antibody produced by the hybridoma cell line CGMCC18321.
[0007] Further, the coding gene sequence of the single-chain antibody TRsAb1 is shown in SEQ ID NO.2. The coding gene sequence of the single-chain antibody TRsAb1 has a length of 981bp, in which the coding gene sequence of the histidine tag region is 45bp in length, the coding gene sequence of the 180aa ToRSV heavy chain variable region is 540bp in length, the coding gene sequence of the linker peptide is 45bp in length, and the coding gene sequence of the 117aa ToRSV light chain variable region is 351bp.
[0008] Further, the upstream of the coding gene sequence of the single-chain 2antibody TRsAb1 further includes expression elements: Lac promoters, Lac operons, and LU101600 ribosome binding sites (RBSs).
[0009] Further, a single-chain antibody TRsAb1 expression vector is PUC57, its insertion site is an EcoRV site, and its expression host strain is an E. coli DH5alpha strain.
[0010] The invention also discloses a method for producing a single-chain antibody TRsAb1, comprising the following steps:
[0011] (1) construction of a single-chain antibody TRsAb1 expression vector: inserting a gene sequence shown in SEQ ID NO. 2 into an EcoRV site on a PUC57 vector to obtain the single-chain antibody TRsAb1 expression vector; | [0012] (2) introducing the single-chain antibody TRsAb1 expression vector into E. coli DH5a to obtain a single-chain antibody TRSAB1 expression strain;
[0013] (3) self-induced expression of the single-chain antibody TRsAb1: inoculating the single-chain antibody TRSAB1 expression strain into a self-inducing expression medium, and fermenting at a temperature of 16-40 °C for 12-24 hours to obtain a product fermentation broth, wherein one liter of the self-induction expression medium comprises 1.0-5.0g of yeast extract, 5.0-10.0g of tryptone, 5.0-15.0g of corn steep powder,
0.1-1.5g of sucrose, 4.0-10.0g of dipotassium phosphate, 3.5-9.0g of potassium dihydrogen phosphate, 2.0-6.0g of diammonium hydrogen phosphate, 0.2-1.6g of magnesium sulfate, 2.0-5.0mg of calcium chloride, 0.5-3.5mg of cobalt chloride,
0.5-1.5mg of copper chloride, 1.2-5.0mg of manganese sulfate, 3.2-8.0mg of sodium molybdate, 0.1-0.5mg of boric acid, 1.0-8.0mg of ferric chloride, 0.5-3.0mg of zinc sulfate,
1.0-10.0g of glycerol, and 20-300mg of isopropylthio-B-D-galactoside (IPTG); and
[0014] (4) separation and purification of the single-chain antibody TRsAb1: centrifuging the fermentation broth obtained in step (3), collecting the bacterial cells, resuspending in a phosphate buffer solution, carrying out ultrasonication, and then recentrifuging, collecting supernatant, loading the supernatant on a nickel ion column, eluting with gradient-concentration imidazole, collecting single-chain antibody TRsAb1 3eluate, and carrying out dialysis and freeze-drying to obtain the single-chain antibody LU101600 TRsAb1.
[0015] The invention mainly has the following inventive innovations: . [0016] (1) The two main sequences constituting the single-chain antibody of the invention, namely the heavy chain variable region and the light chain variable region of the monoclonal antibody against TORSV, are derived from the monoclonal hybridoma cell line (deposited under the number CGMCC18321) obtained by immunizing mice with a TORSV coat protein QAAQQAGKNPFGRG peptide. The amino acid sequence of the single-chain antibody obtained by the fusion design is as shown in SEQ ID NO. 1, and the single-chain antibody is named TRsAb1. The amino acid sequence of TRsAb1 is submitted to the NCBI database for BLASTp alignment analysis. It shows that there is a region in TRsAb1 that is highly similar to the heavy chain of the mouse antibody. In the case of the highest coverage rate of 53%, the similarity is 89.83%; no highly similar protein with a coverage rate of more than 53% is found. In addition, the heavy chain variable region and the light chain variable region constituting the single-chain antibody are derived from a monoclonal hybridoma cell line (deposited under the number CGMCC18321) obtained by the inventor of the present application through immunizing mice with a ToRSV coat protein QAAQQAGKNPFGRG peptide, and the obtained sequence is not found in any other database, so the single-chain antibody according to the invention has a specific amino acid sequence.
[0017] (2) The gene sequence used to prepare the single-chain antibody TRsAb1 against TORSV is not a natural gene sequence, but a brand-new gene sequence that has been codon-modified and artificially synthesized. Due to a large number of E. coli rare codons in the gene sequence encoding the four peptides of the single-chain antibody TRsAb1, it is not suitable for direct expression in E. coli. Therefore, the invention optimizes the codons of the gene encoding the single-chain antibody TRsAb1, removes all rare codons, and finally obtains a novel artificial single-chain antibody TRsAb1 gene sequence by the whole gene synthesis method (shown in SEQ ID NO. 2). Through BLASTn 4alignment analysis with the NCBI database, no sequence significantly similar to the LU101600 sequence of the single-chain antibody TRsAb1 gene is found.
[0018] (3) A Lac gene expression element is designed at the upstream of the single-chain antibody TRsAb1 gene so that it can be expressed in E. coli DH5a strain under the induction of IPTG. Finally, the fusion protein of the invention is obtained by cell disruption and nickel ion column affinity chromatography. The E. coli here was purchased from Takara Biotech Co., Ltd. (Dalian, China) and deposited under the number GIM1.571; the plasmid PUC57 is a public plasmid, provided by GenScript (Nanjing) Co., Ltd. for free.
[0019] All percentage contents in the invention, unless otherwise specified, refer to mass percentage contents.
[0020] The single-chain antibody against TORSV according to the invention can specifically identify the TORSV coat protein and is used for detection of TORSV.
[0021] Fig. 1 is a schematic structural diagram of a single-chain antibody TRSAB1 protein.
[0022] The following describes the embodiments of the invention in detail. The embodiments are implemented on the premise of the technical solutions of the invention. Detailed implementations and specific operation processes are given. However, the protection scope of the invention is not limited to the following embodiments.
[0023] The host bacteria in all the following embodiments were E. coli DH5a, the expression vector framework was a PUC57 plasmid, and the single-chain antibody TRsAb1 expression cassette insertion site was an EcoRV site. E. coli DH5a, purchased from Takara Biotech Co., Ltd. (Dalian, China), having a classification name; Escherichia coli DH5a, Latin scientific name: Escherichia coli DH5a, deposited by Guangdong Culture Collection Center under the accession number of GIM1.571. The plasmid PUC57, 2710bp,
derived from E. coli, was provided by GenScript (Nanjing) Co., Ltd. for free. LU101600
[0024] In the following embodiments, a more representative embodiment is taken as an Embodiment for description, but the protection scope of the invention is not limited to the following embodiments.
[0025] Embodiment 1
[0026] Step 1: a biotech company was entrusted to perform a whole gene synthesis of the single-chain antibody TRSAB1 expression cassette sequence (SEQ ID NO. 2), and the sequence was then inserted into the EcoRV site on PUC57 by restriction enzyme ligation method, and then introduced in an E. coli DH5a strain using a chemical method or electric shock method, and carbenicillin-resistant medium scanning and DNA sequencing verification were carried out to obtain a single-chain antibody TRsAb1 expression strain.
[0027] Step 2: The single-chain antibody TRsAb1 expression strain was cloned and inoculated into 200mL of self-induction medium, cultured at 16 °C for 12 hours at a shaking speed of 220rpm, and then centrifuged at 6000xg for 5 minutes at room temperature, and bacterial cells were collected. One liter of the self-inducing medium here comprised: 1.0g of yeast extract, 5.0g of tryptone, 5.0g of corn steep powder, 0.1g of sucrose, 4.0g of dipotassium phosphate, 3.5g of potassium dihydrogen phosphate, 2.0g of diammonium hydrogen phosphate, 0.2g of magnesium sulfate, 2.0mg of calcium chloride,
0.5mg of cobalt chloride, 0.5mg of copper chloride, 1.2mg of manganese sulfate, 3.2mg of sodium molybdate, 0.1mg of boric acid, 1.0mg of ferric chloride, 0.5mg of zinc sulfate,
1.0g of glycerol, and 20mg of isopropylthio-B-D-galactoside (IPTG).
[0028] Step 3: The bacterial cells collected by centrifugation in step 2 were suspended in 20mL of a cell disruption buffer solution; after being fully vortex mixed and resuspended, the cells were sonicated for 30 minutes at 130 W, 5 seconds each time with a pause of 5 seconds. The cells were then centrifuged at 12000xg for 30 minutes at room temperature; the supernatant was transferred to a new centrifuge tube, filtered with a
0.45um hydrophilic filter membrane, and then loaded to a nickel ion column (taking 6
GenScript High Affinity Ni-Charged Resin as an Embodiment); the nickel ion column was LU101600 then eluted with a gradient imidazole solution, and the target eluate was collected.
[0029] Step 4: The collected eluate was dialyzed three times in ultrapure water in a dialysis bag with an exclusion molecular weight of 14kDa, and then freeze-dried to obtain the target single-chain antibody TRsAb1.
[0030] Based on Embodiment 1, a single-chain antibody TRsAb1 was obtained , with a yield of 200 mg/L fermentation broth and a purity of 82%. The single-chain antibody TRsAb1 was tested positive by indirect ELISA for the synthetic peptide QAAQQAGKNPFGRG and tomato samples carrying TORSV.
[0031] Embodiment 2
[0032] Step 1: The method of step 1 in Embodiment 1 was used to obtain a single-chain antibody TRsAb1 expression strain.
[0033] Step 2: The single-chain antibody TRsAb1 expression strain was cloned and inoculated into 200mL of self-induction medium, cultured at 40 °C for 24 hours at a shaking speed of 220rpm, and then centrifuged at 6000xg for 5 minutes at room temperature, and bacterial cells were collected. One liter of the self-inducing medium here comprised: 5.0g of yeast extract, 10.0g of tryptone, 15.0g of corn steep powder, 1.5g of sucrose, 10.0g of dipotassium phosphate, 9.0g of potassium dihydrogen phosphate, 6.0g of diammonium hydrogen phosphate, 1.69 of magnesium sulfate, 5.0mg of calcium chloride, 3.5mg of cobalt chloride, 1.5mg of copper chloride, 5.0mg of manganese sulfate,
8.0mg of sodium molybdate, 0.5mg of boric acid, 8.0mg of ferric chloride, 3.0mg of zinc sulfate, 10.0g of glycerol, and 300mg of isopropylthio-B-D-galactoside (IPTG).
[0034] Step 3: The bacterial cells collected by centrifugation in step 2 were suspended in 20mL of a cell disruption buffer solution; after being fully vortex mixed and resuspended, the cells were sonicated for 30 minutes at 130 W, 5 seconds each time with a pause of 5 seconds. The cells were then centrifuged at 12000xg for 30 minutes at room temperature; the supernatant was transferred to a new centrifuge tube, filtered with a 0.45 yum hydrophilic filter membrane, and then loaded to a nickel ion column (taking GenScript 7
High Affinity Ni-Charged Resin as an Embodiment); the nickel ion column was then eluted LU101600 with a gradient imidazole solution, and the target eluate was collected.
[0035] The cell disruption buffer solution comprised 50mM of sodium dihydrogen phosphate and 300mM of sodium chloride and had a pH of 8.0; the nickel ion column affinity chromatography pre-elution buffer solution comprised 10mM of imidazole, 50mM of sodium dihydrogen phosphate, and 300mM of chloride Sodium and had a pH of 8.0; the elution buffer solution comprised 100 mM of imidazole, 50 mM of sodium dihydrogen phosphate, and 300 mM of sodium chloride and had a pH of 8.0.
[0036] Step 4: The collected eluate was dialyzed three times in ultrapure water in a dialysis bag with an exclusion molecular weight of 14kDa, and then freeze-dried to obtain the target single-chain antibody TRsAb1.
[0037] Based on Embodiment 2, a single-chain antibody TRsAb1 was obtained with a yield of 1100mg/L fermentation broth and a purity of 87%. The single-chain antibody TRsAb1 was tested positive by indirect ELISA for the synthetic peptide QAAQQAGKNPFGRG and tomato samples carrying TORSV.
[0038] Embodiment 3
[0039] Step 1: The method of step 1 in Embodiment 1 was used to obtain a single-chain antibody TRsAb1 expression strain.
[0040] Step 2: The single-chain antibody TRsAb1 expression strain was cloned and inoculated into 200mL of self-induction medium, cultured at 28 °C for 18 hours at a shaking speed of 220rpm, and then centrifuged at 6000xg for 5 minutes at room temperature, and bacterial cells were collected. One liter of the self-inducing medium here comprised: 3.0g of yeast extract, 7.5g of tryptone, 10.0g of corn steep powder, 0.75g of sucrose, 7.0g of dipotassium phosphate, 6.0g of potassium dihydrogen phosphate, 4.0g of diammonium hydrogen phosphate, 1.0g of magnesium sulfate, 3.5mg of calcium chloride, | 2.0 mg of cobalt chloride, 1.0 mg of copper chloride, 3.1mg of manganese sulfate, 5.6mg of sodium molybdate, 0.3 mg of boric acid, 4.5mg of ferric chloride, 1.7mg of zinc sulfate,
5.0 g of glycerol, and 160mg of isopropylthio-B-D-galactoside (IPTG). 8
[0041] Step 3: The bacterial cells collected by centrifugation in step 2 were LU101600 suspended in 20mL of a cell disruption buffer solution; after being fully vortexed and resuspended, the cells were sonicated for 30 minutes at 130 W, 5 seconds each time with | a pause of 5 seconds. The cells were then centrifuged at 12000xg for 30 minutes at room temperature; the supernatant was transferred to a new centrifuge tube, filtered with a 0.45 um hydrophilic filter membrane, and then loaded to a nickel ion column (taking GenScript High Affinity Ni-Charged Resin as an Embodiment); the nickel ion column was then eluted with a gradient imidazole solution, and the target eluate was collected.
[0042] The cell disruption buffer solution comprised 50mM of sodium dihydrogen phosphate and 300mM of sodium chloride and had a pH of 8.0; the nickel ion column affinity chromatography pre-elution buffer solution comprised 10mM of imidazole, 50mM of sodium dihydrogen phosphate, and 300mM of chloride Sodium and had a pH of 8.0; the elution buffer solution comprised 76mM of imidazole, 50mM of sodium dihydrogen phosphate, and 300mM of sodium chloride and had a pH of 8.0.
[0043] Step 4: The collected eluate was dialyzed three times in ultrapure water in a dialysis bag with an exclusion molecular weight of 14 kDa, and then freeze-dried to obtain the target single-chain antibody TRsAb1.
[0044] Based on Embodiment 3, a single-chain antibody TRsAb1 was obtained with a yield of 1500mg/L fermentation broth and a purity of 93%. The single-chain antibody TRsAb1 was tested positive by indirect ELISA for the synthetic peptide QAAQQAGKNPFGRG and tomato samples carrying TORSV.
[0045] Embodiment 4
[0046] Step 1: The method of step 1 in Embodiment 1 was used to obtain a single-chain antibody TRsAb1 expression strain.
[0047] Step 2: The single-chain antibody TRsAb1 expression strain was cloned and inoculated into 200mL of self-induction medium, cultured at 34 °C for 16 hours at a shaking speed of 220rpm, and then centrifuged at 6000xg for 5 minutes at room temperature, and bacterial cells were collected. One liter of the self-inducing medium here 9comprised: 4.0g of yeast extract, 6.5g of tryptone, 8.0g of corn steep powder, 0.5g of LU101600 sucrose, 8.0g of dipotassium phosphate, 7.0g of potassium dihydrogen phosphate, 3.0g of diammonium hydrogen phosphate, 1.0g of magnesium sulfate, 4.2mg of calcium chloride,
2.4mg of cobalt chloride, 0.8 mg of copper chloride, 2.8mg of manganese sulfate, 4.5mg of sodium molybdate, 0.3mg of boric acid, 5.4mg of ferric chloride, 1.2mg of zinc sulfate,
5.0g of glycerol, and 120mg of isopropylthio-B-D-galactoside (IPTG).
[0048] Step 3: The bacterial cells collected by centrifugation in step 2 were suspended in 20mL of a cell disruption buffer solution; after being fully vortex mixed and resuspended, the cells were sonicated for 30 minutes at 130 W, 5 seconds each time with a pause of 5 seconds. The cells were then centrifuged at 12000xg for 30 minutes at room temperature; the supernatant was transferred to a new centrifuge tube, filtered with a 0.45 pm hydrophilic filter membrane, and then loaded to a nickel ion column (taking GenScript High Affinity Ni-Charged Resin as an Embodiment); the nickel ion column was then eluted with a gradient imidazole solution, and the target eluate was collected.
[0049] The cell disruption buffer solution comprised 50mM of sodium dihydrogen phosphate and 300mM of sodium chloride and had a pH of 8.0; the nickel ion column affinity chromatography pre-elution buffer solution comprised 10mM of imidazole, 50mM of sodium dihydrogen phosphate, and 300mM of sodium chloride and had a pH of 8.0; the elution buffer solution comprised 80mM of imidazole, 50 mM of sodium dihydrogen phosphate, and 300mM of sodium chloride and had a pH of 8.0.
[0050] Step 4: The collected eluate was dialyzed three times in ultrapure water in a dialysis bag with an exclusion molecular weight of 14kDa, and then freeze-dried to obtain the target single-chain antibody TRsAb1.
[0051] Based on Embodiment 2, a single-chain antibody TRsAb1 was obtained with a yield of 2000mg/L fermentation broth and a purity of 95%. The single-chain antibody TRsAb1 was tested positive by indirect ELISA to the synthetic peptide QAAQQAGKNPFGRG and tomato samples carrying ToRSV.
Claims (7)
1. A single-chain antibody TRsAb1 against ToRSV, having an amino acid sequence as shown in SEQ ID NO.1.
2. The single-chain antibody TRsAb1 according to Claim 1, consisting of a 15aa histidine tag region, a 180aa ToRSV heavy chain variable region, a 15aa linker peptide, and a reverse-linked 117aa ToRSV light chain variable region, connected in sequence, with a length of 327 amino acid residues, wherein the sequences of the 180aa heavy chain variable region and the 117aa light chain variable region are derived from the sequence of a monoclonal antibody produced by thehybridoma cell strain CGMCC18321.
3. The single-chain antibody TRsAb1 according to Claim 1, wherein the coding gene sequence of the single-chain antibody TRsAb1 is as shown in SEQ ID NO.2.
4. The single-chain antibody TRsAb1 according to Claim 3, wherein the coding gene sequence of the single-chain antibody TRsAb1 has a length of 981bp, in which the coding gene sequence of the histidine tag region is 45bp in length, the coding gene sequence of the 180aa ToRSV heavy chain variable region is 540bp in length, the coding gene sequence of the linker peptide is 45bp in length, and the coding gene sequence of the 117aa ToRSV light chain variable region is 351bp in length.
5. The single-chain antibody TRsAb1 according to Claim 3, wherein the upstream of the coding gene sequence of the single-chain antibody TRsAb1 further comprises expression elements: Lac promoters, Lac operons, and ribosome binding sites (RBSs).
6. The single-chain antibody TRsAb1 according to Claim 4, wherein a single-chain antibody TRsAb1 expression vector is PUC57, its insertion site is an EcoR V restriction enzyme cutting site, and its expression host strain is an E. coli DH5alpha strain.
7. The single-chain antibody TRsAb1 according to Claim 1, wherein a method for producing the single-chain antibody TRsAb1 comprises the following steps: (1) construction of a single-chain antibody TRsAb1 expression vector: inserting a gene sequence shown in SEQ ID NO. 2 into an EcoRV site on a PUC57 vector to obtain a single-chain antibody TRsAb1 expression vector, 11
(2) introducing the single-chain antibody TRsAb1 expression vector into E. coli DH5a LU101600 to obtain a single-chain antibody TRSAB1 expression strain; (3) self-induced expression of the single-chain antibody TRsAb1: inoculating the single-chain antibody TRSAB1 expression strain into a self-inducing expression medium, and fermenting at a temperature of 16-40 °C for 12-24 hours to obtain a product fermentation broth, wherein one liter of the self-induction expression medium comprises
1.0-5.0g of yeast extract, 5.0-10.0g of tryptone, 5.0-15.0g of corn steep powder, 0.1-1.5g of sucrose, 4.0-10.0g of dipotassium phosphate, 3.5-9.0g of potassium dihydrogen phosphate, 2.0-6.0g of diammonium hydrogen phosphate, 0.2-1.6g of magnesium sulfate,
2.0-5.0mg of calcium chloride, 0.5-3.5mg of cobalt chloride, 0.5-1.5mg of copper chloride,
1.2-5.0mg of manganese sulfate, 3.2-8.0mg of sodium molybdate, 0.1-0.5mg of boric acid,
1.0-8.0mg of ferric chloride, 0.5-3.0mg of zinc sulfate, 1.0-10.0g of glycerol, and 20-300mg of isopropylthio-B-D-galactoside (IPTG); and (4) separation and purification of the single-chain antibody TRsAb1: centrifuging the fermentation broth obtained in step (3), collecting the bacterial cells, resuspending in a phosphate buffer solution, carrying out ultrasonication, and then re-centrifuging, collecting supernatant, loading the supernatant on a nickel ion column, eluting with gradient-concentration imidazole, collecting single-chain antibody TRsAb1 eluate, and carrying out dialysis and freeze-drying to obtain the single-chain antibody TRsAb1.
12
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