Anti-dextran single-chain antibody and application

Abstract

The invention discloses an anti-dextran single-chain antibody and an application thereof. The amino acid sequence of the anti-dextran single-chain antibody is as shown in SEQ ID NO.6, further, a segment of COMP sequence is added to the C end of the single-chain antibody to form a derivative variant A48-COMP, and the amino acid sequence of the derivative variant of the single-chain antibody is as shown in SEQ ID NO.8. The derivative variant A48-COMP can generate turbidity with dextran, and can be directly applied to determination of dextran by immunoturbidimetry. Compared with the common immunoturbidimetry, the method uses the genetically engineered antibody, is easy to prepare and low in detection cost, and has the advantages of simplicity, convenience and rapidness.

Description

Technical Field

[0001] The invention relates to the field of sugar production, and in particular to the preparation and application of variable region sequences of anti-dextran antibodies and recombinant single-chain antibodies thereof. Background Art

[0002] Dextran is a poly-D-glucose, whose glucose residues are connected to the main chain by α-1,6-glycosidic bonds, and the side chains are connected to the main chain by glucose residues or glucose chains by α-1,2-, α-1,3- or α-1,4-glycosidic bonds. Dextran is an impurity commonly found in the sugar production process, which has a variety of adverse effects on the sugar production process. The detection of dextran is of great significance for evaluating the quality and application range of sugar, the severity of dextran problems in sugar production, and the effectiveness of prevention and removal measures.

[0003] Previously, the methods for detecting dextran included the alcohol Haze method, GPC method, Robert-Copper method, immunoturbidimetry, etc. The alcohol Haze method and GPC method are non-specific and easily affected by common substances in the sugar-making process such as starch and pectin; the Robert-Copper method is complicated and time-consuming, and there is interference from other colors during colorimetry.

[0004] The immunoturbidimetric method detects antigen-antibody complexes through light scattering, and has the advantages of high specificity, simple operation and rapid detection. However, due to the high cost of preparing monoclonal antibodies based on hybridoma technology, this detection method is costly and has not been widely used in the sugar industry. The use of exogenous protein expression systems to prepare recombinant antibodies can reduce production costs. However, recombinant single-chain antibodies often have the characteristics of low affinity and small molecules. Although they can bind to antigens, they cannot produce turbidity of sufficient intensity, so they cannot be used in immunoturbidimetric methods. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an anti-dextran single-chain antibody and its derivative variants. The present invention is the first to use an immunoturbidimetric method based on the derivative variants of the anti-dextran single-chain antibody to determine dextran. Compared with the general immunoturbidimetric method, it uses genetically engineered antibodies, has a small amount of antibodies, and has a low detection cost.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] The present invention first obtains anti-dextran monoclonal antibodies through hybridoma technology. The antibody gene is cloned and the gene sequence is obtained. The corresponding single-chain antibody is obtained by exogenous expression through genetic engineering. Although the single-chain antibody has a high titer and can specifically bind to dextran, the single-chain antibody cannot react with dextran to produce turbidity and cannot be directly used in the immunoturbidimetric method to determine dextran.

[0008] A derivative variant A48-COMP is formed by adding a COMP sequence to the C-terminus of the single-chain antibody. The COMP sequence is used to form a polymer, thereby bringing together the single-chain antibodies that originally existed separately. The derivative variant A48-COMP can produce turbidity with dextran and can be directly used in the immunoturbidimetric method to determine dextran.

[0009] In one aspect, the present invention provides an anti-dextran single-chain antibody comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0010] Accordingly, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.3; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.1.

[0011] The light chain variable region and the heavy chain variable region of the single-chain antibody of the present invention are connected via a connecting polypeptide, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.6.

[0012] Correspondingly, the nucleotide sequence of the single-chain antibody is shown in SEQ ID NO.5.

[0013] On the other hand, the present invention also provides a derivative variant of an anti-dextran single-chain antibody, which comprises the above-mentioned anti-dextran single-chain antibody and an amino acid sequence COMP, whose amino acid sequence is shown in SEQ ID NO.8.

[0014] The present invention also provides a gene encoding a derivative variant of the above-mentioned anti-dextran single-chain antibody, and the nucleotide sequence of the gene is shown in SEQ ID NO.7.

[0015] The present invention also provides a vector, which contains the coding gene of the derivative variant.

[0016] The present invention also provides a recombinant cell, which contains the above-mentioned vector.

[0017] The present invention also provides the use of the derivative variant of the anti-dextran single-chain antibody in the preparation of a dextran detection kit.

[0018] Preferably, the kit is an immunoturbidimetry kit.

[0019] The present invention also provides a kit, which comprises the derivative variant of the above-mentioned anti-dextran single-chain antibody.

[0020] The present invention has the following beneficial effects: the present invention discloses for the first time a derivative variant of an anti-dextran single-chain antibody, which can be applied to immunoturbidimetric method for detecting dextran. Compared with the general immunoturbidimetric method, it uses genetically engineered antibodies, is easy to prepare, has low detection cost, and has the advantages of being simple and rapid. DETAILED DESCRIPTION

[0021] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0022] Example 1 Generation of anti-dextran monoclonal antibodies

[0023] 1. Immunization of Mice

[0024] Dextran T-40 and Dextran T-40-BSA crosslinked products were mixed with an equal amount of complete Freund's adjuvant and fully emulsified, and injected intradermally into mice at a dose of 50 μg / mouse each time. Two weeks later, the second immunization was performed with an equal amount of incomplete Freund's adjuvant, and then booster immunization was performed every two weeks. After three immunizations, blood was collected to determine the titer of the antibody in the serum.

[0025] 2. Serum antibody testing

[0026] Indirect ELISA was used. Dextran T-40-OVA crosslink was diluted with carbonate buffer and coated on a 96-well plate at 100 μL / well at 4°C overnight. The plate was blocked with 1% gelatin for 2 hours, washed 3 times with PBST, and 100 μL of serially diluted serum was added to each well. Positive and negative controls were set up at the same time. The plate was placed at 37°C for 1 hour, washed 3 times with PBST washing solution, and then sheep anti-mouse enzyme-labeled secondary antibody was added. The plate was placed at 37°C for 1 hour, washed 3 times with PBST, and OPD substrate solution was added. The plate was placed at 37°C in the dark for 15 minutes, and the reaction was terminated by adding stop solution. The results were read at 490 nm by an enzyme reader.

[0027] 3. Cell fusion, screening and cloning

[0028] 3 days after shock immunization, mouse spleen cell suspension was prepared and SP2 / 0 cells in the logarithmic growth phase were collected. Splenocytes and SP2 / 0 cells were mixed at a ratio of 5:1, fused by 50% PEG, and screened and cultured with HAT medium. Hybridoma clones were formed 1 week later, and hybridoma supernatants were screened by indirect ELISA. Positive cells were subcloned three times in a row by limiting dilution. After indirect ELISA screening and cloning, hybridoma cell lines that stably secreted antibodies were obtained.

[0029] 4. Antibody Preparation

[0030] Liquid paraffin was inoculated intraperitoneally, 0.5 mL per mouse. Diluted hybridoma cells were inoculated intraperitoneally 7 days later. After an interval of 5 days, the ascites of the mice was observed every day. If the abdomen was obviously swollen, the ascites was collected with a needle. The ascites was centrifuged at 3000 rpm for 5 minutes, the supernatant was collected, the titer was determined, and the ascites was frozen at -20℃.

[0031] 5. Antibody Purification

[0032] Affinity chromatography:

[0033] (1) Equilibrate the column with Buffer A (0.05 mol / L boric acid, 4.0 mol / L NaCl, pH 9.0, solvent: water).

[0034] (2) Add a small amount of ascites and rinse the column with Buffer A.

[0035] (3) Elute with Buffer B (0.05 mol / L sodium phosphate, 0.05 mol / L sodium citrate, 0.3 mol / L NaCl, pH 3.0, solvent: water).

[0036] (4) Collect the protein and add 5% volume of 1.0 mol / L Tris-HCL, pH 8.0 to adjust the pH value. The purified antibody was analyzed by SDS-PAGE, and the molecular weight of its heavy chain was about 50 kDa, and the molecular weight of its light chain was about 23 kDa.

[0037] 6. Antibody titer determination

[0038] Indirect ELISA method to determine antibody titer

[0039] Coating: Dextran T-40-OVA cross-linker was dissolved in buffer (0.05 M carbonate buffer, pH 9.6) to a final concentration of 5 μg / mL and incubated in a 96-well plate at 4°C overnight.

[0040] Blocking: After washing three times with 0.01 M PBST (containing 0.05% Tween-20, pH 7.4), the wells were blocked with 2% BSA (200 μL per well) at 37° C. for 2 hours and washed again with PBST.

[0041] Primary antibody incubation: Monoclonal antibody (MAb) diluted in PBS was added to each well (100 μL per well), incubated at 37°C for 1 hour, and then washed.

[0042] Secondary antibody incubation: Add goat anti-mouse IgG-HRP secondary antibody diluted to 1:10,000, incubate at 37℃ for 1 hour, and then wash.

[0043] Color development: Add OPD substrate solution (50 μL per well) and incubate at 37° C. for 10 minutes. Terminate the reaction by adding 2 M sulfuric acid solution (50 μL per well), and then measure the OD value at 492 nm on a microplate reader (Model 680, Bio-Rad).

[0044] The antibody titer was determined by indirect ELISA, and the highest titer of the antibody A48 was 5.12×10 6 .

[0045] Example 2 Cloning and determination of anti-dextran monoclonal antibody gene

[0046] 1. Use RNA extraction kit (RNAiso Plus, Takara) to extract and purify total RNA from hybridoma cells. According to the instructions of the kit (PrimeScript 1st Strand cDNA Synthesis Kit, Takara), use Oligo dT Primer to reverse transcribe RNA to synthesize the first strand cDNA chain.

[0047] The cDNA synthesis reaction solution was directly used as a template for PCR reaction, and PCR amplification was performed using high-fidelity polymerase TransGen HiFi (Beijing Quanshijin Biotechnology Co., Ltd.).

[0048] in,

[0049] Antibody heavy chain PCR primers:

[0050] Upstream primer: 5′-GCCGCCACAGTTCCTGAAGACACTG-3′;

[0051] Downstream primer: 5′-CATGGTCCCAGGCATTGCTGGGTGCT-3′;

[0052] PCR conditions were as follows: denaturation at 95°C for 5 min; denaturation at 94°C for 1 min; annealing at 50°C for 1 min; extension at 72°C for 2 min, 31 cycles; and total extension at 72°C for 10 min.

[0053] Antibody light chain PCR primers:

[0054] Upstream primer: 5′-GGCACCATGGAGWCACAKWCTCAGGTCTTTRTA-3′;

[0055] Downstream primer: 5′-TCAAGTGCAAAGACTCACTTTATTGAA-3′;

[0056] PCR conditions were as follows: denaturation at 95°C for 5 min; denaturation at 94°C for 1 min; annealing at 50°C for 1 min; extension at 72°C for 1 min, 31 cycles; and total extension at 72°C for 10 min.

[0057] After PCR is completed, Taq polymerase is added and incubated at 72°C for 10 minutes. The reaction products are subjected to agarose gel electrophoresis, and the target fragments are cut and recovered, and the PCR products are purified. The PCR products are connected to T vectors (TA cloning kit, full-form gold) respectively, and positive clones are selected for sequence determination and analysis.

[0058] 2. Use the IMGT website (https: / / www.imgt.org / ) to analyze the sequenced antibody nucleic acid sequence. Obtain the sequence information of the antibody heavy chain and light chain variable regions:

[0059] The heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.1:

[0060] CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGG

[0061] TGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGA

[0062] GGCCTGGACAGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTGAC

[0063] TACAATGAGAGGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTG

[0064] CCTACATGCACCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG

[0065] GGGTAGCTTACTATGGTACACCTCTCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0066] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2:

[0067] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0068] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT VSS.

[0069] The light chain variable region nucleic acid sequence is shown in SEQ ID NO.3:

[0070] AACATTCTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACC

[0071] TTGAGCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTATCCTGGTTTCAACAGAAACC

[0072] AGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCG

[0073] ATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGGTGCAG

[0074] GCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACAGCTATCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0075] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4:

[0076] NILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQSPKLLIYGASNRYTGVPDR FTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEIK.

[0077] Example 3 Expression and purification of single-chain antibodies

[0078] 1. Construction of single-chain antibody A48HL

[0079] Based on the sequence information of the antibody heavy chain and light chain obtained above, a single-chain antibody was designed, and the overall structure was: heavy chain variable region-linker-light chain variable region.

[0080] The nucleic acid sequence of the synthetic single-chain antibody A48HL is shown in SEQ ID NO.5: CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTGACTACAATGAGAGGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCACCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAGGGGTAGCTTACTATGGTACACCTCTCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGTGGTTCTGGAGGTGGTGGATCTGGTGGAGGTGGATCTAACATTCTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTATCCTGGTTTCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACAGCTATCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACATCATCATCATCATCATTGA。

[0081] The amino acid sequence of the single-chain antibody A48HL is shown in SEQ ID NO.6:

[0082] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0083] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT

[0084] VSSGGGGSGGGGSGGGGSNILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQ

[0085] SPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEIKHHHHHH.

[0086] The synthesized sequence was cloned into the vector pUC57.

[0087] Using the synthetic sequence as a template, PCR amplification was performed.

[0088] Upstream primer: 5′-TCTCTCGAGAAAAGACAGGTCCAGCTGCAGCAGTCT-3′;

[0089] Downstream primer: 5′-TGCTCTAGATCAATGATGATGATGATGATGTTTTAT-3′;

[0090] The conditions were: denaturation at 95°C for 5 min; denaturation at 94°C for 1 min; annealing at 53°C for 1 min; extension at 72°C for 2 min, 30 cycles; total extension at 72°C for 10 min. The PCR product was recovered by gel chromatography to obtain the gene fragment.

[0091] The obtained gene fragment and plasmid pGAPZαA (purchased from Invitrogen) were double-digested with XhoI and XbaI, respectively. The digested products were ligated with T4 DNA ligase and transformed into competent Escherichia coli JM109 (Beijing Quanshijin Co., Ltd.), and positive clones were screened on LB resistance plates (containing 25 μg / mL bleomycin). Several clones were selected for colony PCR identification and sequencing identification to obtain the recombinant expression vector.

[0092] The recombinant expression vector was digested with AvrII endonuclease at 37°C overnight, and the digestion product was purified using a DNA fragment purification kit (purchased from Shanghai Bioengineering Co., Ltd.) to obtain a linearized vector. Competent Pichia pastoris KM71H was prepared according to the Pichia pastoris expression manual of Invitrogen. The obtained linearized vector was mixed with 80 μL competent Pichia pastoris KM71H, placed in a 1mm electroporation transformation cup and electroporated for 5 minutes after ice bath. The electroporation parameters were: 1600V, 25μF, 200Ω. After yeast cell transformation, it was spread on a YPD resistance plate containing 100μg / mL bleomycin (containing 100μg / mL bleomycin, 1% yeast extract, 2% peptone, 2% agar, and the solvent was water), and cultured at 30°C for 3-4 days. Positive clones were selected, and the engineered bacteria were obtained by colony PCR and sequencing verification.

[0093] Shake flask culture of engineered bacteria: The activated strain in step (1) is transferred into a liquid culture medium (culture medium ingredients: yeast powder 10 g / L, peptone 20 g / L, 5% (w / v) glycerol, solvent is water) for culture. The culture conditions are: 40 mL of liquid in a 250 mL Erlenmeyer flask, temperature 30°C, rotation speed 250 r / min, and culture for 120 hours.

[0094] The fermentation product was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and purified using a histidine tag protein purification kit HisTrap FFCrude (GE Healthcare) to obtain the single-chain antibody A48HL.

[0095] Example 4 Expression and purification of single-chain antibody variants

[0096] Construction of single-chain antibody A48HL-COMP:

[0097] Based on the aforementioned single-chain antibody A48HL, the helical domain of human cartilage oligomeric matrix protein (COMP48, Asp29-Gln76) is connected to the C-terminus of the single-chain antibody, and its amino acid sequence is (DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGMQQ). The overall structure of A48HL-COMP is: heavy chain variable region-linker-light chain variable region-linker-COMP.

[0098] Artificially synthesized sequence (SEQ ID NO.7):

[0099] TCTCTCGAGAAAAGACAAGTCCAGCTTCAGCAATCCGGTGCCGAATTGGTTAGACCTGG

[0100] TACATCAGTCAAAGTTTCCTGCAAAGCAAGTGGTTACGCATTTACCAACTACTTGATTGA

[0101] ATGGGTTAAGCAAAGACCTGGTCAGGGACTTGAGTGGATTGGTGTCATCAACCCTGGAT

[0102] CTGGTGGAACAGATTACAACGAAAGATTCAAGGGTAAAGCTACCTTGACTGCCGACAAA

[0103] TCTTCCTCAACCGCTTACATGCATTTGAGTTCTCTTACTTCTGATGACTCCGCCGTTTATT

[0104] TTTGTGCAAGAGGTGTCGCTTACTATGGAACTCCATTGTGGTACTTCGATGTTTGGGGTG

[0105] CCGGAACTACAGTTACAGTCTCCTCAGGTGGAGGTGGATCTGGTGGAGGTGGTTCCGGT

[0106] GGAGGTGGATCAAATATCTTGATGACTCAATCTCCTAAGTCCATGTCAATGAGTGTTGGT

[0107] GAAAGAGTCACACTTTCATGCAAAGCTAGTGAGAACGTTGGTACCTATGTCAGTTGGTT

[0108] CCAACAGAAGCCAGAACAGTCTCCTAAATTGCTTATCTACGGTGCATCCAATAGATATAC

[0109] TGGAGTTCCAGATAGATTCACTGGTTCTGGTTCCGCTACAGACTTCACATTGACCATCAG

[0110] TTCTGTTCAAGCCGAGGATCTTGCAGACTACCACTGTGGACAGTCATATAGTTACCCTAC

[0111] CTTCGGATCAGGAACAAAGTTGGAAATCAAGGGTGGTGGTGGTTCAGGTGGTGGTGGT

[0112] TCAGGTGGTGGTGGTTCCGATTTGGGTCCTCAGATGCTTAGAGAGCTTCAAGAAACAAA

[0113] CGCTGCCTTGCAAGATGTTAGAGAATTGCTTAGACAACAGGTCAGAGAGATTACTTTTCT

[0114] TAAGAACACAGTTATGGAATGCGACGCCTGCGGAATGCAACAAGGTAGTCATCATCATCACCACCACTAATCTAGAGCA.

[0115] The synthesized sequence was cloned into the vector pUC57.

[0116] The amino acid sequence of the single-chain antibody A48HL-COMP is shown in SEQ ID NO.8:

[0117] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0118] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT

[0119] VSSGGGGSGGGGSGGGGSNILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQ

[0120] SPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEI

[0121] KGGGGSGGGGSGGGGSDLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGMQQGSHHHHHH.

[0122] The synthesized gene fragment and plasmid pGAPZαA (Invitrogen) were double-digested with XhoI and XbaI, respectively. The digested products were ligated with T4 DNA ligase and transformed into competent Escherichia coli JM109 (purchased from Beijing Quanshijin Co., Ltd.), and positive clones were screened on LB resistance plates (containing 25 μg / mL bleomycin). Several clones were selected for colony PCR identification and sequencing identification to obtain the recombinant expression vector.

[0123] The recombinant expression vector was digested with AvrII endonuclease at 37°C overnight, and the digestion product was purified using a DNA fragment purification kit (purchased from Shanghai Bioengineering Co., Ltd.) to obtain a linearized vector. Competent Pichia pastoris KM71H was prepared according to the Pichia pastoris expression manual of Invitrogen. The obtained linearized vector was mixed with 80 μL competent Pichia pastoris KM71H, placed in a 1mm electroporation transformation cup and electroporated for 5 minutes after ice bath. The electroporation parameters were: 1600V, 25μF, 200Ω. After yeast cell transformation, it was spread on a YPD resistance plate containing 100μg / mL bleomycin (containing 100μg / mL bleomycin, 1% yeast extract, 2% peptone, 2% agar, and the solvent was water), and cultured at 30°C for 3-4 days. Positive clones were selected, and the engineered bacteria were obtained by colony PCR and sequencing verification.

[0124] Shake flask culture of engineered bacteria: The activated strain in step (1) is transferred into a liquid culture medium (culture medium ingredients: yeast powder 10 g / L, peptone 20 g / L, 5% (w / v) glycerol, solvent is water) for culture. The culture conditions are: 40 mL of liquid in a 250 mL Erlenmeyer flask, temperature 30°C, rotation speed 250 r / min, and culture for 120 hours.

[0125] The fermentation product was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and purified using a histidine tag protein purification kit HisTrap FFCrude (GE Healthcare) to obtain the single-chain antibody A48HL-COMP.

[0126] Example 5: Single-chain antibody titer verification

[0127] Dextran T-40-OVA crosslinker was diluted with carbonate buffer and coated on a 96-well plate at 100 μL / well at 4°C overnight. The plate was blocked with 1% gelatin for 2 hours, washed 3 times with PBST, and 100 μL of the antibody to be tested was added to each well in a doubling dilution. At the same time, positive and negative controls were set up, placed at 37°C for 1 hour, washed 5 times with PBST washing solution, added with mouse anti-6×His antibody, placed at 37°C for 1 hour, washed 3 times with PBST, added with goat anti-mouse enzyme-labeled secondary antibody, placed at 37°C for 45 minutes, washed 5 times with PBST, added with OPD substrate solution, placed at 37°C in the dark for 15 minutes, added with stop solution to terminate the reaction, and read the results with an enzyme reader at 490nm.

[0128] The titers of single-chain antibody A48HL and single-chain antibody A48HL-COMP are shown in Table 1:

[0129] Table 1. Validation of the potency of single-chain antibodies

[0130] Antibody concentration (μg / mL) A48HL(OD490) A48HL-COMP(OD490) 100 2.275 2.718 50 2.024 2.200 25 1.755 2.026 12.5 1.242 1.780 6.25 0.805 1.353 3.125 0.677 1.027 1.56 0.434 0.716 0.781 0.209 0.404 0.390 0.122 0.231 0 0.098 0.110

[0131] Example 6 Turbidimetric reaction of single-chain antibody

[0132] Dextran standard stock solution (1 mg / mL): weigh dextran standard ([C6H 10 O5]n, CAS number: 9004-54-0, purity> 99%, molecular weight of 1500000~2800000) about 2g (weighed to 0.1mg) in a weighing bottle with constant weight, put in a drying oven at 105℃ for 3h, take out, put in a desiccator, cool to room temperature, weigh (accurate to 0.1mg), and calculate the water content in the dextran standard. According to the water content of the dextran standard, quickly weigh the undried dextran to make it contain about 0.1000g of water-free dextran, add about 2mL of water to dissolve into a paste, leave for about 10min, stir from time to time to make the particles evenly hydrated, when there is gel, add a small amount of water to about 25mL several times to dissolve the gel, transfer to a 100mL volumetric flask, add water to the scale, and mix well. This solution is prepared and used immediately.

[0133] Take the dextran standard stock solution and prepare dextran solutions with concentrations of 100, 200, 400, 600, 800, and 1000 ppm.

[0134] Pipette 1.5mL of single-chain antibody solution (0.2mg / mL) into the test dish, put it into the turbidity meter, cover it, and accurately read the turbidity value (N) at 1 minute. 样0); Take out the test dish, add 15μL of the sample solution to the antibody solution with a pipette, and start the stopwatch. Cover the test dish with a lid, turn it upside down 6 times to mix it evenly, and avoid generating bubbles during the mixing process. Put the test dish into the turbidity meter, cover it with the lid, and accurately read the turbidity value (N) at 1 minute. 样 ), calculate the turbidity increment of the reaction system (△NTU 样 =N 样 -N 样0 ).

[0135] Table 2. Turbidimetric response of single-chain antibody

[0136] Dextran concentration (ppm) A48HL(△NTU) A48HL-COMP(△NTU) 0 0.2 0.3 100 0.2 1.5 200 0.3 5.4 400 0.4 12.4 600 0.3 39.7 800 0.5 46.3 1000 0.9 50.2

[0137] Determination of Dextran Content in Example 7 Samples

[0138] Dextran standard series solution: Pipette 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL of dextran standard stock solution (1mg / mL) into a 10mL volumetric flask and dilute to volume. The mass concentrations of this dextran standard series solution are 0mg / L, 20mg / L, 40mg / L, 60mg / L, 80mg / L and 100mg / L respectively. Dextran standard series solutions of corresponding mass concentrations can be prepared according to the actual content of dextran in the sample, and the mass concentration shall not exceed 600mg / L.

[0139] Pipette 1.5 mL of single-chain antibody (A48HL-COMP) solution (0.2 mg / mL) into the test dish, put it into the turbidity meter, cover it, and accurately read the turbidity value (N) at 1 min. 标0 ); Take out the test dish, add 15 μL of the standard series of dextran solution to the antibody solution with a pipette, and start the stopwatch. Cover the test dish with a lid and mix well. Avoid bubbles during the mixing process. Put the test dish into the turbidity meter, cover it with the lid, and accurately read the turbidity value (N) at 1 minute. 标 ), calculate the turbidity increment of the reaction solution system (△NTU 标 ). The standard curve was obtained with the concentration of dextran as the horizontal axis and the turbidity increment as the vertical axis.

[0140] Pipette 1.5mL of single-chain antibody solution (0.2mg / mL) into the test dish, put it into the turbidity meter, cover it, and accurately read the turbidity value (N) at 1 minute. 样0 ); Take out the test dish, add 15μL of the sample solution to the antibody solution with a pipette, and start the stopwatch. Cover the test dish with a lid, turn it upside down 6 times to mix it evenly, and avoid generating bubbles during the mixing process. Put the test dish into the turbidity meter, cover it with the lid, and accurately read the turbidity value (N) at 1 minute. 样 ), calculate the turbidity increment of the reaction system (△NTU样 =N 样 -N 样0 ).

[0141] Table 3. Determination of dextran content in samples

[0142] Test samples Dextran content (ppm) maltose Not detected Isomaltose Not detected fructose Not detected glucose Not detected BSA Not detected OVA Not detected white sugar 182 brown sugar 335 Sugar making juice 90

[0143] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An anti-dextran single-chain antibody, characterized in that: Include: A light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.4; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

2. The anti-dextran single-chain antibody according to claim 1, characterized in that: The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

6.

3. A derivative variant of an anti-dextran single-chain antibody, characterized in that: The invention comprises the anti-dextran single-chain antibody according to claim 1 or 2 and an amino acid sequence COMP, wherein the amino acid sequence is shown in SEQ ID NO.

8.

4. A gene encoding a derivative variant of the anti-dextran single-chain antibody according to claim 3.

5. The gene according to claim 4, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

7.

6. A carrier, characterized in that Containing the gene according to claim 4.

7. A recombinant cell, characterized in that Containing the vector according to claim 6.

8. Use of the derivative variant of the anti-dextran single-chain antibody according to claim 3 in the preparation of a dextran detection kit.

9. The use according to claim 8, characterized in that: The kit is an immunoturbidimetry kit.

10. A kit, characterized in that: A derivative variant comprising the anti-dextran single-chain antibody according to claim 3.