LU101793B1 - Single-chain antibody TRsAb2 against ToRSV and preparation method thereof - Google Patents
Single-chain antibody TRsAb2 against ToRSV and preparation method thereof Download PDFInfo
- Publication number
- LU101793B1 LU101793B1 LU101793A LU101793A LU101793B1 LU 101793 B1 LU101793 B1 LU 101793B1 LU 101793 A LU101793 A LU 101793A LU 101793 A LU101793 A LU 101793A LU 101793 B1 LU101793 B1 LU 101793B1
- Authority
- LU
- Luxembourg
- Prior art keywords
- trsab2
- chain antibody
- torsv
- sequence
- variable region
- Prior art date
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- 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
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- 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)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Peptides Or Proteins (AREA)
Abstract
Provided is a single-chain antibody TRsAb2 against ToRSV, having an amino acid sequence shown in SEQ ID NO.l, and consisting of a 15 aa histidine tag region, a 184 aa ToRSV heavy chain variable region, a 15 aa linker peptide, and a reverse-linked 102 aa ToRSV light chain variable region, connected in sequence, with a length of 316 aa residues. The sequences of the 184 aa heavy chain variable region and the 102 aa light chain variable region are derived from the sequence of a monoclonal antibody produced by the hybridoma cell line CGMCCl 8320. The TRsAb2 gene is obtained by a whole gene synthesis method, and the codon composition is optimized. The single-chain antibody TRsAb2 of the invention has the characteristics of selectively identifying the peptide AFGKGVEEIEQTST in the ToRSV coat protein, and has a positive response to natural ToRSV particles.
Description
SINGLE-CHAIN ANTIBODY TRSAB2 AGAINST TORSV AND
[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
| - 2 AFGKGVEEIEQTST (SEQ-ID NO. 3) in the ToRSV coat protein as a template to prepare LU101793 a recombinant single-chain antibody. The single-chain antibody of the present invention, named TRsAb2, consists of four peptides, a 15aa histidine tag region (6xHis tag), a 184aa ToRSV heavy chain variable region (igH184), a 15aa linker peptide, and a reverse-linked 102aa ToRSV light chain variable region (igK 102), connected in sequence, with a length of 316 amino acid residues. The sequences of the 184aa heavy chain variable region and the 102aa light chain variable region are derived from the sequence of a monoclonal antibody produced by the hybridoma cell line CGMCC18320 (deposited by: China General Microbiological Culture Collection Center). The single-chain antibody TRsAb2 of the present invention has the characteristics of low preparation cost, simple preparation method, specificity in binding to the ToRSV coat protein AFGKGVEEIEQTST 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.
BRIEF SUMMARY OF THE INVENTION |
[0006] An objective of the invention is to provide a single-chain antibody TRsAb2 | 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 184aa ToRSV heavy chain variable region / (igH184), a 15aa linker peptide, and a reverse-linked 102aa ToRSV light chain variable [ region (igK 102), connected in sequence, with a length of 316 amino acid residues. The | sequences of the 184aa heavy chain variable region and the 102aa light chain variable | region are derived from the sequence of a monoclonal antibody produced by the / hybridoma cell line CGMCC18320. /
[0007] Further, the coding gene sequence of the single-chain antibody TRsAb2 is |shown in SEQ ID NO.2. The coding gene sequence of the single-chain antibody TRsAb2 LU101793 has a length of 948bp, in which the coding gene sequence of the histidine tag region is 45bp in length, the coding gene sequence of the 184aa ToRSV heavy chain variable region is 552bp in length, the coding gene sequence of the linker peptide is 45bp in length, and the coding gene sequence of the 102aa ToRSV light chain variable region is 306bp.
[0008] Further, the upstream of the coding gene sequence of the single-chain antibody TRsAb2 further includes expression elements: Lac promoters, Lac operons, and ribosome binding sites (RBSs).
[0009] Further, a single-chain antibody TRsAb2 expression vector is PUC57, its insertion site is an EcoRV site, and its expression host strain is an Æ. coli DH5a strain. |
[0010] | The invention also discloses a method for producing a single-chain | antibody TRsAb2, comprising the following steps: |
[0011] (1) construction of an single-chain antibody TRsAb2 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 TRsAb2 expression vector; |
[0012] (2) introducing the single-chain antibody TRsAb2 expression vector into E. | coli DH5a to obtain a single-chain antibody TRSAB2 expression strain; |
[0013] (3) self-induced expression of the single-chain antibody TRsAb2: | inoculating the single-chain antibody TRSAB2 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 1.0-5.0 g of yeast extract, 5.0-10.0 g of tryptone, 5.0-15.0 g of corn steep powder, 0.1-1.5 g of sucrose, 4.0-10.0 g of dipotassium phosphate, 3.5-9.0 g of potassium | dihydrogen phosphate, 2.0-6.0 g of diammonium hydrogen phosphate, 0.2-1.6g of : magnesium sulfate, 2.0-5.0 mg of calcium chloride, 0.5-3.5 mg of cobalt chloride, 0.5-1.5 | mg of copper chloride, 1.2-5.0mg of manganese sulfate, 3.2-8.0 mg of sodium molybdate, |
| 4
0.1-0.5mg of boric acid, 1.0-8.0 mg of ferric chloride, 0.5-3.0 mg of zinc sulfate, 1.0-10.0 LU101793 g of glycerol, and 20-300 mg of isopropylthio-B-D-galactoside (IPTG);
[0014] (4) separation and purification of the single-chain antibody TRsAb2: 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 TRsAb2 eluate, and carrying out dialysis and freeze-drying to obtain the single-chain antibody TRsAb2.
[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 ToRSYV, are derived from the monoclonal hybridoma cell line (deposited under the number CGMCC18320) obtained by immunizing mice with a ToRSV coat protein AFGKGVEEIEQTST 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 TRsAb2. The amino acid sequence of TRsAb2 is 8 submitted to the NCBI database for BLASTp alignment analysis. It shows that there is a | region in TRsAb2 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 | CGMCC18320) 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. LU101793
[0017] (2) The gene sequence used to prepare the single-chain antibody TRsAb2 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 TRsAb2, it is not suitable for direct expression in E. coli. Therefore, the invention optimizes the codons of the gene encoding the single-chain antibody TRsAb2, removes all rare codons, and finally obtains a novel artificial single-chain antibody TRsAb2 gene sequence by the whole gene synthesis method (shown in SEQ ID NO. 2). Through BLASTn alignment analysis with the NCBI database, no sequence significantly similar to the sequence of the single-chain antibody TRsAb2 gene is found.
[0018] (3) A Lac gene expression element is designed at the upstream of the single-chain antibody TRsAb2 gene so that it can be expressed in E. coli DH50, 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 Æ. coli here was purchased from Takara Biotech Co., Ltd. (Dalian, China) and deposited under the number GIM1.571; the plasmid PUCS57 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. | BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS |
[0021] Fig. 1 is a schematic structural diagram of a single-chain antibody TRSAB2 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 TRsAb2 expression cassette insertion site was an EcoRV site. E. coli DH5a, purchased from Takara Biotech Co., Ltd. (Dalian, China), having a classification name: E. 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.
[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 I
[0026] Step 1: a biotech company was entrusted to perform a whole gene synthesis of the single-chain antibody TRSAB2 expression cassette sequence (SEQ ID NO. 2), and the sequence was then inserted into the EcoRV site on PUCS7 by restriction enzyme ligation method, and then introduced in an Æ. coli DHS 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 TRsAb2 expression strain. |
[0027] Step 2: The single-chain antibody TRsAb2 expression strain was cloned | and inoculated into 200 mL of self-induction medium, cultured at 16 °C for 12 hours at a |
| shaking speed of 220 rpm, and then centrifuged at 6000xg for 5 minutes at room LU101793 temperature, and bacterial cells were collected. One liter of the self-inducing medium here | comprised: 1.0 g of yeast extract, 5.0 g of tryptone, 5.0 g of corn steep powder, 0.1 g of | sucrose, 4.0 g of dipotassium phosphate, 3.5 g of potassium dihydrogen phosphate, 2.0 g | of diammonium hydrogen phosphate, 0.2 g of magnesium sulfate, 2.0 mg of calcium | chloride, 0.5 mg of cobalt chloride, 0.5 mg of copper chloride, 1.2 mg of manganese sulfate, 3.2 mg of sodium molybdate, 0.1 mg of boric acid, 1.0 mg of ferric chloride, 0.5 mg of zinc sulfate, 1.0 g of glycerol, and 20 mg of isopropylthio-B-D-galactoside (IPTG). | [0028] Step 3: The bacterial cells collected by centrifugation in step 2 were | suspended in 20 mL 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 | 0.45pm 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.
| [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 TRsAb2.
| [0030] Based on Embodiment 1, a single-chain antibody TRsAb2 was obtained | with a yield of 200 mg/L fermentation broth and a purity of 82%. The single-chain | antibody TRsAb2 was tested positive by indirect ELISA for the synthetic peptide | AFGKGVEEIEQTST 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 TRsAb 2 expression strain.
| 8
[0033] Step 2: The single-chain antibody TRsAb2 expression strain was cloned LU101793 and inoculated into 200 mL of self-induction medium, cultured at 40 °C for 24 hours at a shaking speed of 220 rpm, 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: 5.0 g of yeast extract, 10.0 g of tryptone, 15.0 g of corn steep powder, 1.5 g of sucrose, 10.0 g of dipotassium phosphate, 9.0 g of potassium dihydrogen phosphate, 6.0 g of diammonium hydrogen phosphate, 1.6 g of magnesium sulfate, 5.0 mg of calcium chloride, 3.5 mg of cobalt chloride, 1.5 mg of copper chloride, 5.0 mg of manganese sulfate, 8.0 mg of sodium molybdate, 0.5 mg of boric acid, 8.0 mg of ferric chloride, 3.0 mg of zinc sulfate, 10.0 g 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 20 mL 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 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.
[0035] The cell disruption buffer solution comprised 50 mM of sodium dihydrogen phosphate and 300 mM 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 LU101793 the target single-chain antibody TRsAb2.
| [0037] Based on Embodiment 2, a single-chain antibody TRsAb2 was obtained | with a yield of 1100mg/L fermentation broth and a purity of 87%. The single-chain | antibody TRsAb2 was tested positive by indirect ELISA for the synthetic peptide | AFGKGVEEIEQTST 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 TRsAb2 expression strain.
| [0040] Step 2: The single-chain antibody TRsAb2 expression strain was cloned | and inoculated into 200 mL of self-induction medium, cultured at 28 °C for 18 hours at a | shaking speed of 220 rpm, 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: 3.0 g of yeast extract, 7.5 g of tryptone, 10.0 g of corn steep powder, 0.75 g of { sucrose, 7.0 g of dipotassium phosphate, 6.0 g of potassium dihydrogen phosphate, 4.0 g | of diammonium hydrogen phosphate, 1.0 g of magnesium sulfate, 3.5 mg of calcium | chloride, 2.0 mg of cobalt chloride, 1.0 mg of copper chloride, 3.1 mg of manganese sulfate, 5.6 mg of sodium molybdate, 0.3 mg of boric acid, 4,5 mg of ferric chloride, 1.7 | mg of zinc sulfate, 5.0 g of glycerol, and 160 mg of isopropylthio-B-D-galactoside (IPTG).
| [0041] Step 3: The bacterial cells collected by centrifugation in step 2 were suspended in 20 mL 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 theneluted with a gradient imidazole solution, and the target eluate was collected. LU101793 | [0042] The cell disruption buffer solution comprised 50 mM of sodium dihydrogen phosphate and 300 mM of sodium chloride and had a pH of 8.0; the nickel ion column | affinity chromatography pre-elution buffer solution comprised 10 mM of imidazole, 50 | mM of sodium dihydrogen phosphate, and 300 mM of chloride Sodium and had a pH of | 8.0; the elution buffer solution comprised 75 mM of imidazole, 50 mM of sodium | dihydrogen phosphate, and 300 mM 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 TRsAb2.
| [0044] Based on Embodiment 3, a single-chain antibody TRsAb2 was obtained | with a yield of 1500 mg/L fermentation broth and a purity of 93%. The single-chain | antibody TRsAb2 was tested positive by indirect ELISA for the synthetic peptide | AFGKGVEEIEQTST 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 TRsAb2 expression strain.
[0047] Step 2: The single-chain antibody TRsAb2 expression strain was cloned | and inoculated into 200 mL of self-induction medium, cultured at 34 °C for 16 hours at a [ shaking speed of 220 rpm, 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: 4.0 g of yeast extract, 6.5 g of tryptone, 8.0 g of corn steep powder, 0.5 g of | sucrose, 8.0 g of dipotassium phosphate, 7.0 g of potassium dihydrogen phosphate, 3.0 g | of diammonium hydrogen phosphate, 1.0 g of magnesium sulfate, 4.2 mg of calcium | chloride, 2.4 mg of cobalt chloride, 0.8 mg of copper chloride, 2.8 mg of manganese | sulfate, 4.5 mg of sodium molybdate, 0.3 mg of boric acid, 5.4 mg of ferric chloride, 1.2
| mg of zinc sulfate, 5.0 g of glycerol, and 120 mg of isopropylthio-B-D-galactoside (IPTG). | LU101793 | [0048] Step 3: The bacterial cells collected by centrifugation in step 2 were | suspended in 20 mL 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 [ 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.
| [0049] The cell disruption buffer solution comprised 50 mM of sodium dihydrogen | phosphate and 300 mM of sodium chloride and had a pH of 8.0; the nickel ion column | affinity chromatography pre-elution buffer solution comprised 10 mM of imidazole, | 50mM of sodium dihydrogen phosphate, and 300 mM of sodium chloride and had a pH of | 8.0; the elution buffer solution comprised 80 mM of imidazole, 50 mM of sodium | dihydrogen phosphate, and 300 mM 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 i dialysis bag with an exclusion molecular weight of 14kDa, and then freeze-dried to obtain ' the target single-chain antibody TRsAb2.
[0051] Based on Embodiment 2, a single-chain antibody TRsAb2 was obtained with a yield of 2000 mg/L fermentation broth and a purity of 95%. The single-chain antibody TRsAb2 was tested positive by indirect ELISA to the synthetic peptide AFGKGVEEIEQTST and tomato samples carrying ToRSV.
[0052] SEQID NO.1:
[0053] MGSSHHHHHHGGGGSACRPVQLQESGTVLARPGASVKMSCKASG
YPLAPGCGDTTGSSVTLGCLVRGYFPESVTVTWNSGSLSSSVHTFPDSCSCTG |
| GGGSGGGGSGGGGSRTARVLKTPDGAQEWCRVHAHRGELQKNGAQDWCR = LU101793 | VHASCYIRSLLLLSGSGWGGSQQDQTHFRDRIWQWQVQRPGWFIPQFSQLDP | [0055] ATGGGTTCTAGCCATCATCATCACCATCATGGTGGCGGCGGTTCTG | CATGCCGTCCTGTACAGCTGCAGGAAAGCGGTACTGTTCTGGCACGTCCG | GGCGCTAGCGTGAAAATGTCTTGCAAAGCTTCCGGCTATTCTTTCACTTCTT | ACTGGATGCACTGGGTCAAACAGCGCCCAGGCCAAGGTCTGG
| AATGGATCGGTGCTATTTATCCGGGTAACTCTGATACCAACTACAACCAAA | AATTCAAAGGCAAAGCAAAACTGACTGCAGTTACGTCCGCATCCACTGCA | TACATGGAACTGTCCTCTCTGACCAATGAAGACAGCGCGGTTTATTATTGT | ACCCGCCTGTCTATGCTGGGTCGCTCCTACTATTTCGATTACTGGGGCCAGG | GTACCACCCTGACTGTCTCCTCTGCAAAAACTACTCCACCGTCTGTCTACC | CACTGGCACCTGGTTGTGGCGATACCACTGGTTCTTCCGTTACTCTGGGTT | GCCTGGTGCGCGGTTACTTTCCGGAGTCCGTTACCGTTACCTGGAACTCCG | GTTCCCTGTCCTCTTCCGTTCACACCTTTCCTGACTCTTGTTCTTGTACCGG | TGGTGGTGGCTCTGGTGGTGGTGGTTCTGGTGGTGGCGGTTCTCGTACTGC | ACGTGTGCTGAAAACCCCTGACGGCGCACAGGAATGGTGCCGTGTACATG | CGCATCGTGGCGAACTGCAGAAAAATGGCGCCCAGGACTGGTGCCGTGTT | CATGCATCTTGCTATATCCGCTCCCTGCTGCTGCTGTCTGGCTCTGGTTGGG | GTGGCAGCCAACAGGACCAGACCCACTTCCGTGACCGCATCTGGCAGTGG | CAAGTTCAACGCCCGGGCTGGTTCATTCCGCAGTTCTCTCAACTGGATCCG | GCAAAAAGCGTACCTCACGCGCGTTTTCGTTGGCCTCGTTGCGCA
| SEQUENCE LISTING LU101793 | <118> CHINA JILIANG UNIVERSITY | ZHEJIANG ACADEMY OF SCIENCE & TECHNOLOGY FOR INSPECTION & | QUARANTINE | <120> SINGLE-CHAIN ANTIBODY TRSAB2 AGAINST TORSV AND PREPARATION METHOD THEREOF | <130> PTO730LU | <160> 3 | <170> BiSSAP 1.3.6 | <210> 1 | <211> 316 | <212> PRT | <213> Artificial Sequence | <220> | <223> single-chain antibody TRsAb2 against ToRSV | <400> 1 | Met Gly Ser Ser His His His His His His Gly Gly Gly Gly Ser Ala | 1 5 10 15 | Cys Arg Pro Val Gln Leu Gln Glu Ser Gly Thr Val Leu Ala Arg Pro | - 20 25 30 | Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr | 35 40 45 Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 50 55 60 Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asn Tyr Asn Gln 65 70 75 80 Lys Phe Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr 85 90 95 Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr : 100 105 118 Tyr Cys Thr Arg Leu Ser Met Leu Gly Arg Ser Tyr Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro 138 135 148 Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser 145 150 155 169 Ser Val Thr Leu Gly Cys Leu Val Arg Gly Tyr Phe Pro Glu Ser Val N 165 a 178 175 Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe 180 185 190 Pro Asp Ser Cys Ser Cys Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly 195 : 208 - 205 Ser Gly Gly Gly Gly Ser Arg Thr Ala Arg Val Leu Lys Thr Pro Asp 218 “215 220 Gly Ala Gln Glu Trp Cys Arg Val His Ala His Arg Gly Glu Leu Gln 225 : 230 235 240
| Lys Asn Gly Ala Gln Asp Trp Cys Arg Val His Ala Ser Cys Tyr Ile LU101793
| 245 250 255
| Arg Ser Leu Leu Leu Leu Ser Gly Ser Gly Trp Gly Gly Ser Gln Gln
| 260 265 270
Asp Gln Thr His Phe Arg Asp Arg Ile Trp Gln Trp Gln Val Gln Arg
| 275 280 285
| Pro Gly Trp Phe Ile Pro Gln Phe Ser Gln Leu Asp Pro Ala Lys Ser
| 290 295 300
| Val Pro His Ala Arg Phe Arg Trp Pro Arg Cys Ala
| 305 310 315
| <210> 2
| <211> 948
| <212> DNA
| <213> Artificial Sequence
| <220>
| <223> coding gene sequence of the single-chain antibody TRsAb2
| <400> 2
| atgggttcta gccatcatca tcaccatcat ggtggeggeg gttctgecatg ccgtectgta 60
| cagctgcagg aaagcggtac tgttctggea cgtecgggeg ctagegtgaa aatgtettge 126
| aaagcttecg getattettt cacttettac tggatgcact gggtcaaaca gcgcccaggc 188
| caaggtctgg aatggatcgg tectatttat cecgggtaact ctgataccaa ctacaaccaa 240
| aaattcaaag gcaaagcaaa actgactgca gttacgtccg catccactge atacatggaa 300
| ctgtectetec tgaccaatga agacagegeg gtttattatt gtacecgect gtectatgetg 360
| ggtcgeteect actatttcga ttactggggc cagggtacca ccctgactgt ctectctgca 420
| aaaactactc caccgtctgt ctacccactg gecacctggtt gtggcgatac cactggttct 480 tccgttactc tgggttgeet ggtgegeggt tactttecgg agtecgttac cgttacctgg 540 aactccggtt ccectgteete ttccgttcac acctttectg actettgtte ttgtaccggt 698 getgetgect ctggtggtgg tggttctggt ggtggcggtt ctcgtactge acgtgtgetg 660 aaaacccctg acggcgcaca ggaatggtgc cgtgtacatg cgcatcgtgg cgaactgcag 728 aaaaatggcg cccaggactg gtgeegtgtt catgcatett gctatatccg ctecctgctge 780 ctgetgtetg getetggttg ggetggcagc caacaggacc agacccactt ccgtgaccge 840 atctggcagt ggcaagttca acgcceggge tggttcattc cgcagttetc tcaactggat 9080 Cccggcaaaaa gcgtacctca cgegegtttt cgttggecte gttgegea 948
| 211s 1 LU101793 | <211> 14
| <212> PRT
| <213> Artificial Sequence
| <220>
| <223> ToRSV coat protein
| <400> 3
| Ala Phe Gly Lys Gly Val Glu Glu Ile Glu Gln Thr Ser Thr
| 1 5 10
Claims (2)
1.0-5.0 g of yeast extract, 5.0-10.0 g of tryptone, 5.0-15.0 g of corn steep powder, 0.1-1.5 g of sucrose, 4.0-10.0 g of dipotassium phosphate, 3.5-9.0 g of potassium dihydrogen | phosphate, 2.0-6.0 g of diammonium hydrogen phosphate, 0.2-1.6 g of magnesium sulfate,
2.0-5.0 mg of calcium chloride, 0.5-3.5 mg of cobalt chloride, 0.5-1.5 mg of copper chloride, 1.2-5.0 mg of manganese sulfate, 3.2-8.0 mg of sodium molybdate, 0.1-0.5 mg of boric acid, 1.0-8.0 mg of ferric chloride, 0.5-3.0 mg of zinc sulfate, 1.0-10.0 g of | glycerol, and 20-300mg of isopropylthio-B-D-galactoside (IPTG); (4) separation and purification of the single-chain antibody TRsAb2: 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 TRsAb2 eluate, and carrying out dialysis and freeze-drying to obtain the single-chain antibody TRsAb2. |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101793A LU101793B1 (en) | 2020-05-15 | 2020-05-15 | Single-chain antibody TRsAb2 against ToRSV and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101793A LU101793B1 (en) | 2020-05-15 | 2020-05-15 | Single-chain antibody TRsAb2 against ToRSV and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| LU101793B1 true LU101793B1 (en) | 2020-09-21 |
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Effective date: 20200921 |