Construction method of flag immunobead display antibody library, flag antibody scFv1 and application thereof

Abstract

The application provides a KLH-Flag immune phage display antibody library construction method, and steps of the method comprise the following steps: (1) immunizing a New Zealand white rabbit with a KLH-Flag protein, extracting total RNA of lymphocytes, and reversely transcribing the total RNA into cDNA; (2) amplifying heavy chain variable region and light chain variable region fragments and splicing into scFv genes by using a PCR technology; (3) constructing a pCANTAB5E-2sfi1-scFv recombinant plasmid, electrically transforming the recombinant plasmid into TG1 competent cells, and constructing a phage single-chain antibody library; (4) specifically enriching the phage single-chain antibody library, and screening positive clones from the phage single-chain antibody library. And monoclonal antibody amino acid sequences finally obtained for application are disclosed. The application screens a rabbit source single-chain antibody with high affinity by using a phage display antibody library technology, the antibody has high affinity to the Flag protein and high specificity, and the antibody can be used for preparing an antibody of Anti-Flag affinity gel and can be used for WB detection of proteins with a Flag label.

Description

Technical Field

[0001] This invention patent relates to a method for constructing a Flag immune phage display antibody library, as well as the screened Flag antibody scFv1 and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] A FLAG-tag is an 8-amino acid residue, N-DYKDDDDK-C (10¹² Da), typically used as a labeling tag. In protein expression and purification, genetic engineering techniques can be used to link the target gene and the FLAG-tag gene sequence, attaching the flag tag to the C-terminus or N-terminus of the target protein. The integrated gene is then transferred into prokaryotic or eukaryotic cells. Subsequent detection and purification primarily rely on the specific binding of the immunodeterminant cluster formed by the FLAG-tag peptide chain to its monoclonal antibody. Detection methods include immunofluorescence and Western blotting.

[0003] Because Flag tags are among the most commonly used and stable tag proteins in the quality control of protein biopharmaceutical production, they are widely used in scientific research and industrial production of protein expression and purification, and have a large market demand. Therefore, it is essential to screen for an antibody with high affinity for Flag tags. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a Flag immune phage display antibody library.

[0005] A method for constructing a Flag-based phage display antibody library, comprising the following steps:

[0006] (1) Immunize rabbits with Flag protein, extract total RNA from lymphocytes, and reverse transcribe it into cDNA;

[0007] (2) The heavy chain variable region and light chain variable region fragments were amplified by PCR technology and spliced ​​into the scFv gene;

[0008] (3) Construct the pCANTAB5E-2SFi1-scFv recombinant plasmid, electroporate it into TG1 competent cells, and construct a phage single-chain antibody library;

[0009] (4) Specific enrichment of phage single-chain antibody library, from which positive clones are screened.

[0010] The primers used in step (2) of the PCR technique are as follows:

[0011]

[0012]

[0013] In step (2), the upstream primer IgG-sfi1-VH-F and the downstream primer IgG-VH-R of the heavy chain VH are cross-paired and mixed for PCR. The upstream primer IgG-VL-F and the downstream primer IgG-sfi1-VL-R of the light chain VL are paired one-to-one and mixed for PCR. The obtained PCR products of light chain VL and heavy chain VH are recovered and mixed in equimolar amounts as templates. The corresponding upstream primer of the heavy chain and the downstream primer of the light chain in the primer table are used for PCR.

[0014] In step (4), the specific enrichment of the phage single-chain antibody library refers to taking the antigen and mixing it with PBS, coating it onto a 96-well plate, using 5% BSA as the blocking solution, adding the phage single-chain antibody library and incubating at room temperature.

[0015] Elution: Add 100 mM HCl, incubate at room temperature for 5 min, then collect by forcefully blowing with a pipette tip. Add 1 M Tris-HCl to the collection tube to neutralize the pH, and then add BSA solution to dilute.

[0016] Elution product amplification: The elution product was added to TG1 bacterial culture and cultured. Helper phages were added and cultured for a longer period. The bacterial culture was centrifuged and the precipitate was resuspended in 2×YT medium and cultured overnight with shaking. The next day, the bacterial culture was centrifuged and the supernatant was transferred to a new centrifuge tube. PEG-NaCl was added, and the mixture was allowed to stand on ice to precipitate. After centrifugation, the supernatant was removed, and the precipitate was resuspended in BSA solution. The precipitate was transferred to another EP tube, centrifuged, and the precipitate was removed. The supernatant was the amplified and enriched product.

[0017] Repeat the above steps for four rounds of screening, increasing the rigor of each round to enrich phages with affinity.

[0018] This invention also discloses a Flag monoclonal antibody scFv, whose light chain variable region sequence is as follows:

[0019] DMTQTPSSKSVPVGDTVTINCQSSQSVYLNNDLAWFQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCVGYKSSSIDGVAFGGGTELEIL(SEQ IDNo.1),

[0020] The amino acid sequence of the light chain variable region CDR1 is: QSSQSVYLNNDLA.

[0021] The amino acid sequence of the light chain variable region CDR2 is: YASTLAS.

[0022] The amino acid sequence of the CDR3 variable region of the light chain is: VGYKSSSIDGVA;

[0023] The heavy chain variable region sequence is as follows:

[0024] QQQLVESGGRLVTPGTPLTLTCTASGFSLHSYAMSWVRQAPGKGLEWIGTIEFNDNTY

[0025] YASWPKGRFTISKTSTAVDLKMTSLTAADTGTYFCVRSSNGWSQTIWGPGTLVTVSS

[0026] (SEQ ID No. 2),

[0027] The amino acid sequence of the heavy chain variable region CDR1 is: SYAMS.

[0028] The amino acid sequence of the heavy chain variable region CDR2 is: TIEFNDNTYYASWPKG.

[0029] The amino acid sequence of the heavy chain variable region CDR3 is: SSNGWSQTI.

[0030] Name it scFv1.

[0031] The method for constructing a Flag-based phage display antibody library of the present invention can screen for rabbit-derived single-chain antibodies with high affinity, and can construct a library with a capacity of 2.4 × 10⁻⁶. 9 The recombinant phage titer was 1.0 × 10⁻⁶. 13 A phage single-chain antibody library with pfu / ml and 100% recombination rate. The single-chain antibodies of this invention can be obtained by expression in Escherichia coli BL21. Attached Figure Description

[0032] Figure 1 This is an electrophoresis diagram of the extracted total RNA.

[0033] Figure 2 This is a gel electrophoresis image of the PCR products of the light chain VL and heavy chain VH genes.

[0034] Figure 3 This is a gel electrophoresis image of the PCR product of the scFv gene.

[0035] Figure 4 These are electrophoresis images of the pCANTAB5E-2sfi1 plasmid before and after double enzyme digestion.

[0036] Figure 5 It is a flat plate diagram of the electroconversion of the connected products.

[0037] Figure 6 This is a colony PCR identification image.

[0038] Figure 7 This is the affinity test result for the scFv antibody (IgM-Flag-middle).

[0039] Figure 8 This is the affinity test result (IgM-Flag-C-terminus) of the scFv antibody.

[0040] Figure 9 This is a graph showing the results of Western blot (WB) testing using the final selected antibodies. Detailed Implementation

[0041] 1. Biomaterials

[0042] New Zealand White Rabbit, Brilliant Farm

[0043] 2. Experimental reagents and consumables

[0044] Table 1 Experimental Reagents and Consumables

[0045]

[0046]

[0047] Example 1: Construction of a phage display antibody library

[0048] 1.1 Extraction of total RNA from animal immune cells and spleen cells

[0049] New Zealand white rabbits weighing approximately 2 kg were immunized subcutaneously every two weeks with Flag-KLH. For the initial immunization, Flag-KLH was emulsified with Freund's complete adjuvant; for booster immunizations, it was emulsified with incomplete adjuvant. The initial immunization dose was 0.5 mg of protein, followed by 0.25 mg for each subsequent immunization. Two months after immunization, three days before spleen harvesting, a 0.25 mg protein was administered subcutaneously as a pulse immunization. Three days after the pulse immunization, a 30-50 mg spleen tissue block was harvested. Total RNA was extracted using a DNase I ultrapure RNA extraction kit. The sample was thoroughly homogenized in liquid nitrogen with 1 ml of TRIzon Reagent added, or 1 ml of TRIzon Reagent was added to the spleen tissue before homogenization. After adding TRIzon Reagent, the sample was repeatedly pipetted several times to ensure complete lysis. The sample was incubated at room temperature for 5 minutes to allow complete separation of the protein and nucleic acid complexes. Add chloroform at a ratio of 200 μl of chloroform per 1 ml of TRIzon Reagent, cap the tube, shake vigorously for 15 seconds, and incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm (~13,400 × g) at 4℃ for 10 minutes. At this point, the sample will separate into three layers: a red organic phase, a middle layer, and a colorless aqueous phase on top. RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube (self-provided).

[0050] Add an equal volume of 70% ethanol (prepared with RNase-free water), approximately 600-700 μl, to the obtained aqueous solution and mix thoroughly by inverting. Add the entire solution obtained in the previous step to the adsorption column (Spin Columns RM) already loaded into the collection tube. If the solution cannot be added all at once, it can be added in multiple batches. Centrifuge at 12,000 rpm for 20 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Prepare the DNase I mixture: Take 52 μl of RNase-Free Water, add 8 μl of 10×Reaction Buffer and 20 μl of DNase I (1 U / μl), mix well, and prepare a reaction solution with a final volume of 80 μl. Add 80 μl of DNase I mixture directly to the adsorption column and incubate at 20-30℃ for 15 min. Add 500 μl of Buffer RW2 (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 min, and discard the waste liquid in the collection tube. Let the adsorption column air dry completely at room temperature for 3 min. Place the adsorption column in a new RNase-free centrifuge tube, add 30-50 μl of RNase-Free Water to the middle of the adsorption column, incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and run 1 μl of the sample for electrophoresis to detect the extraction effect. The results are as follows. Figure 1 The remaining RNA samples were stored at -80°C for later use.

[0051] 1.2 Cloning and splicing of the scFv gene

[0052] This invention uses a reverse transcription kit from Yisheng Biotechnology Co., Ltd., with RNA extracted from New Zealand white rabbit spleen cells as a template. The reverse transcription reaction system is shown in Table 2-1, and RNA template, RNase-free water, and... III. SuperMix was used for incubation at 25℃ for 5 min, followed by reverse transcription at 55℃ for 15 min, and then heating at 85℃ for 5 min to obtain cDNA. Primers in Table 2-2 were synthesized by Shanghai Sangon Biotech. Both the light chain variable region gene and the heavy chain variable region gene used rabbit-derived cDNA as templates. PCR was performed using a mixture of upstream and downstream primers for the light chain and a mixture of upstream and downstream primers for the heavy chain. The PCR reaction components are shown in Table 2-3, and the reaction conditions are as shown in Table 2-4. The obtained PCR products of the light chain VL and heavy chain VH were loaded onto a 1.2% agarose gel, and the amplification results were observed by electrophoresis. The results are shown below. Figure 2The product was recovered using the OMEGA gel recovery kit following the kit's experimental procedures, and stored at -20°C.

[0053] The recovered heavy and light chain genes from the previous step were mixed in equimolar amounts and used as a template. PCR was performed using the corresponding upstream primer for the heavy chain VH gene and the downstream primer for the light chain, and the resulting fragments were spliced ​​into scFv. The PCR reaction system is shown in Table 2-5, and the reaction conditions were the same as those for VH gene amplification in Table 2-4. After the reaction, the samples were loaded and detected by agarose gel electrophoresis. The results are shown below. Figure 3 The product was recovered using the OMEGA gel recovery kit following the kit's experimental procedures, and stored at -20°C.

[0054] Table 2-1 Reverse Transcription Reaction System

[0055]

[0056] Table 2-2 Primers used

[0057]

[0058]

[0059] Table 2-3 PCR Reaction System

[0060] Component Amount used Prime STAR GXL Premix (2x) 25 μl Primer F (10 μM) 1 μl Primer R (10 μM) 1 μl Template (cDNA) 1 μl Sterile water 22 μl Total 50 μl

[0061] Table 2-4 PCR Amplification Conditions

[0062] Fragment of interest Denaturation Annealing Extension End of reaction Number of cycles VL 98℃10s 55℃15s 68℃42s 4℃ 30 VH 98℃10s 60℃15s 68℃42s 4℃ 30

[0063] Table 2-5 PCR Reaction System

[0064]

[0065]

[0066] 1.3 Construction of pCANTAB5E-2sfi1-scFv recombinant plasmid

[0067] pCANTAB5E-2sfi1 was transformed into DH5α for amplification, and plasmids were prepared in large batches. Following the restriction enzyme digestion system of Takara Pharmaceuticals (Japan), the pCANTAB5E-2sfi1 plasmid and scFv fragment were digested with Sfi1, as shown in Table 2-6 below. The mixture was thoroughly mixed and placed in a 50℃ water bath for 2 hours. After digestion, the pCANTAB5E-2sfi1 plasmid was observed by 1.2% agarose gel electrophoresis. The results are shown below. Figure 4The product was recovered using an OMEGA gel extraction kit. The enzyme-digested scFv gene fragment and pCANTAB5E-2sfi1 plasmid were ligated using YEASEN T4 ligase according to the reaction system in Table 2-7, and ligated at 20°C for 2 hours.

[0068] Table 2-6 Enzyme digestion reaction system

[0069] Reaction components Volume 10 x M buffer 16 μl Sfi 1 enzyme 8 μl pCANTAB5E-2 sfi 1 / scFv 8 μg Sterile water Make up to 160 μl

[0070] Table 2-7 Enzyme ligation system

[0071] Reaction components Volume 10 x T4 DNA Ligase buffer 80 μl T4 DNA Ligase 80 μl pCANTAB5E-2 sfi 1 (S) 4 μg ScFv (S) 2 μg Sterile water Make up to 800 μl

[0072] 1.4 Determination of TG1, library capacity, and recombination rate of the ligation product conversion competent cells

[0073] After thawing TG1 competent cells on ice, 1 μl of pCANTAB5E-2sfi1-scFv ligation product was added and gently mixed. The mixture was then poured along the tube wall into an ice-cold electroporation cuvette. After incubation for 5-10 minutes, the cuvette was placed in an electroporator. The voltage was set to 2.5 kV, and the electroporation time was approximately 5.0 ms. Immediately after electroporation, 1 ml of preheated SOC liquid culture medium was added, and the cuvette was incubated at 37°C with shaking for 1 hour. This experiment involved 20 parallel transformations, with 1 μl of cells per transformation, for a total of 20 transformations, yielding 20 ml of transformed competent cells.

[0074] After transformation and resuscitation, 100 μl of the bacterial culture was mixed and diluted 10-fold serially. Another 100 μl of each diluted culture was then plated onto a 2×YT-C plate (containing 50 mg / ml Carb). The plate was incubated overnight at 37°C with the plate inverted. The library volume was calculated, and the transformation results of the ligation product were as follows: Figure 5 The following day, 20 single colonies from well-dispersed dilution plates were picked and incubated overnight at 37°C with shaking at 200 rpm in 2×YT-C (containing 50 mg / ml Carb) liquid medium with dilution. 1 μl of bacterial culture from each of the 20 expanded single colonies was used as a template for PCR according to the scFv gene splicing PCR system. The PCR products were analyzed by electrophoresis and gel imaging, and the recombination rate of the antibody library was calculated. The PCR identification results are as follows: Figure 6 .

[0075] Calculation formula: Storage capacity = Number of single colonies × Dilution factor × Total volume (Formula 1-1)

[0076] Recombination rate (%) = positive clones / number of single clones picked (Formula 1-2).

[0077] 1.5 Construction of phage scFv antibody library

[0078] 1.5.1 Assisting in the preparation of bacteriophage strains

[0079] Inoculate the overnight cultured TG1 bacterial suspension into 5 ml of SOB medium at a ratio of 1:100. Incubate at 37°C with shaking at 200 rpm until the OD600 reaches 0.4-0.6. Then add 5 μl of M13KO7 helper phage and incubate at 37°C with shaking at 200 rpm for 1 h. Next, add 5 μl of kanamycin (50 mg / ml) and incubate at 37°C with shaking at 200 rpm for 1 h. Transfer the cultured suspension to 100 ml of 2×YT liquid medium, add kanamycin (50 mg / ml) to a final concentration of 50 μg / ml, and incubate overnight at 37°C with shaking at 200 rpm. The following day, the overnight culture was aliquoted into 50ml centrifuge tubes and centrifuged at 12000rpm / min for 20min at 4℃. The supernatant was transferred to a sterile 500ml Erlenmeyer flask, and PEG / NaCl (1 / 5 volume of the initial culture, 20ml in this operation) was added. After mixing, the mixture was aliquoted into new 50ml centrifuge tubes and centrifuged on ice for 2 hours. The mixture was then centrifuged at 12000rpm / min for 10min at 4℃. The supernatant was discarded, and the precipitate was resuspended in 2ml (1 / 50 volume) of 1×PBS. The precipitate was transferred to sterile centrifuge tubes and centrifuged again at 12000rpm / min at 4℃ for 5min. The supernatant was carefully collected, filtered through a 0.22μM filter for sterilization, and 50% glycerol was added to the final concentration. The mixture was then aliquoted into sterile centrifuge tubes and stored at -20℃.

[0080] 1.5.2 Assisted phage titration

[0081] The prepared phage solution was serially diluted 10-fold using 1% BSA (in 1×PBS) as the diluent, and the diluted product was taken as the final product. -4 10 -6 10 -8 10 -10 10 -12 10 μl of bacterial suspension was added to TG1 with an OD600 of 0.4-0.6. The diluted solution and TG1 served as negative controls. The mixture was incubated at 37°C for 30 min, followed by shaking for 30 min. After incubation, the bacterial suspensions of the five dilution gradients and the two negative controls were plated onto a plate containing kanamycin (50 mg / ml) and incubated at 37°C. The following day, the number of clones on the plate was calculated, and the phage titer (pfu / ml) was calculated using the following formula. Generally, a titer of 10 pfu / ml is required. 12 Only then can subsequent experiments be conducted. Calculation formula: Phage titer = Number of single clones × Dilution factor × 10 (Formula 1-3).

[0082] 1.5.3 Construction of the primary phage antibody library

[0083] Take 20 ml of the remaining culture from the transformation and preservation and add it to 380 ml of 2×YT-C (25 μg / ml Carb) medium. Incubate at 37°C and 200 rpm for 1 h using a shaker. Add Carb to a final concentration of 50 μg / ml and continue incubating for another h. Add 10 μl of helper phage M13KO7 to the above culture at a multiplicity of infection (MOI) of 20:1. Gently shake at 37°C and 60 rpm for 30 min to facilitate phage infection, then incubate on a shaker for 30 min. Transfer the culture to a sterile centrifuge tube in a sterile laminar flow hood and centrifuge at 5000 rpm for 15 min. Carefully discard the supernatant. Resuspend the bacterial pellet in 400 ml of 2×YT-CK (50 mg / ml Carb and 50 mg / ml Kan) medium under aseptic conditions and incubate overnight with shaking. The next day, transfer all the overnight bacterial culture to a sterile 50 ml centrifuge tube and centrifuge at 12000 rpm at 4°C for 20 min.

[0084] Transfer the supernatant to a new sterile Erlenmeyer flask, add PEG-NaCl (80 ml) equal to 1 / 5 volume of the initial bacterial culture, and incubate on ice for 1 hour to precipitate. Centrifuge at 12000 rpm and 4°C for 20 min, then remove the supernatant. Invert the centrifuge tube for 2-5 min to remove as much excess liquid as possible. Resuspend the precipitate in 1% BSA (in 1×PBS) equal to 1 / 50 volume of the initial bacterial culture, transfer to a 1.5 ml sterile tube, centrifuge at 12000 rpm for 5 min, remove the precipitate, and transfer the supernatant to a new 1.5 ml sterile tube. The supernatant is the primary antibody library; add glycerol to 50% and store at -20°C. Determine the titer of the recombinant phage using the same method as in 1.5.2.

[0085] Example 2: Screening of phage antibody library

[0086] 2.1 Screening of Specific Antibodies Using ELISA Plate Screening Method

[0087] Add 10 μg / ml of scFv-Flag fusion protein to each well of an ELISA plate, one protein per well per round, 100 μL per well, and incubate overnight at 4°C. Remove the coating buffer from the 96-well plate, add 200 μL of 5% BSA to each well, and set up a blank control by adding 200 μL of 5% BSA directly to the blank well as the blocking buffer. Incubate on a shaker at room temperature for 2 h. Remove the BSA blocking buffer by inverting the plate, wash 10 times with 200 μL of PBST, and add 100 μL of phage antibody library to both the antigen well and the BSA control well. Incubate on a shaker at room temperature for 2 h. Discard the antibody library liquid, add 200 μL of PBST to each well, wash 10 times, and blot dry. Add 100 μL of 100 mM HCl to each well, incubate at room temperature for 5 min, elute the product, and neutralize with 40 μL of 1M Tris-HCl. Mix well. Add the obtained elution products to each well containing 1 mL of HCl. In TG1 (OD600 = 0.4-0.6), after incubation at 37℃ and 220 rpm for 1 h, 20 μL of each solution was diluted appropriately and titrated on LB agar plates containing Carb resistance. The plates were then incubated overnight at 37℃. Finally, 1 μL of helper phage was added to each protein tube (ensuring a final concentration of 10⁻⁶). 10 Incubate with helper phage (pfu / ml), at 37°C, 220 rpm for 1 hour; transfer approximately 1 ml of the culture medium from the above EP tube into 50 ml of 2×YT-CK (50 mg / ml Carb and 50 mg / ml Kan) medium, incubate overnight at 37°C, 220 rpm. The next day, collect phages according to the library construction method. Repeat the above operation four times. The first round uses the original antibody library solution; the antibody libraries added in the second and third rounds are collected from the overnight culture from the previous day. Obtain the results of the titer experiment in each round and compare whether there is enrichment in each round of screening; verify the success of screening based on the titer results, and then select single clones for PCR colony identification.

[0088] Table 3-1 4-round screening criteria

[0089] First round of screening Second round of screening Third round of screening Fourth round of screening Antigen coating concentration (μg / ml) 10 10 10 10 Number of washes 8 8 8 8

[0090] Table 3-2 Results of 4 Rounds of Screening

[0091]

[0092] 2.2 Gator Prime detection of antibodies expressed in bacterial lysates

[0093] Based on the PCR colony identification of the selected and enriched single clones, 96 single clones were obtained. Plasmids from these 96 single clones were extracted and transformed into HB2151 competent cells for induction. After expression, the expression of antibodies in the bacterial lysate was detected using Gator Prime. The specific procedures were as follows: Single clones were picked and cultured overnight, with 3 negative controls containing empty vectors. The overnight culture was diluted 1:100 with 2YT-C (Carb 50mg / ml) to OD600 = 0.1, and cultured at 37℃ with shaking at 220 rpm until OD600 = 0.6-0.8. 0.2M IPTG (final concentration 0.1mM) was added, and the culture was incubated overnight at 16℃ with shaking at 220 rpm. The next day, the induced bacterial culture was collected at 5000 rpm for 5 min. The collected bacterial cells were added to lysis buffer (PBS + lysozyme), and then sonicated for 15 minutes. After centrifugation at 12000 rpm for 20 min, the supernatant was collected for later use. The Flag antigen was labeled with biotin, dialyzed overnight, and its concentration was measured. The biotin-Flag was first immobilized using an SA probe, and then bound to the prepared lysis buffer. The experimental results for the six positive clones after subtracting the response values ​​of three negative controls are shown in Table 3-3.

[0094] Table 3-3 Antibody expression levels in lysis buffer

[0095] Sample Name Response (nm) scFv-1# 1.35 scFv-2# 2.18 scFv-3# 0.44 scFv-4# 0.15 scFv-5# 0.73 scFv-6# 1.3

[0096] Example 3: Expression, purification, ELISA identification, and affinity detection of scFv Escherichia coli

[0097] The above six positive phage clones were sent for sequencing. The gene sequences and corresponding amino acid sequences of scFv1-6 were obtained. After obtaining the scFv gene sequence, the CDR1, CDR2, and CDR3 regions were analyzed using Immunoglobulin BLAST and Nucleotide.nucleotide BLAST software in NCBI BLAST (Numbering Scheme: Kabat). Based on the scFv sequencing results, the pET-Duet1-scFv expression vector was constructed. The primer design sequences are shown in Table 3-4 below.

[0098] Table 3-4 Primers for constructing the scFv expression vector

[0099]

[0100] Using plasmids extracted from positive clones as templates, PCR amplification yielded the scFv fragment, and PCR amplification yielded the pET-Duet1 fragment. PET-Duet1-scFv was constructed using a one-step cloning method. After positive colony PCR identification, plasmids were extracted and sequenced to verify correct construction.

[0101] The constructed PET-Duet1-scFv plasmid was transformed into BL21(DE3). Single clones were selected and amplified to 1L. When the OD600 was 0.6-0.8, IPTG was added to a final concentration of 0.1mM and the mixture was incubated overnight at 16℃ for expression induction. After induction, the bacterial culture was centrifuged, the supernatant was discarded, and the bacterial cells obtained from each 1L of bacterial culture were resuspended in 50ml of PBS. The cells were homogenized in an ice bath for 5min, then transferred to centrifuge tubes and centrifuged at 12000rpm / min at 4℃ for 30min. The supernatant was transferred to a new centrifuge tube. The supernatant of the bacterial lysate was purified by nickel affinity chromatography. After incubating the supernatant with 1ml of nickel affinity medium at 4℃ for 1 hour, the supernatant was loaded into a gravity chromatography column. After flow-through (FT), the cells were washed with 20ml of PBS containing 30mM imidazole, and eluted with PBS containing 10mM, 30mM, 50mM, 100mM, and 250mM imidazole. The elution products were concentrated using a 10kD concentration tube. Finally, the protein concentration of the elution product at 250 mM was determined using a Nanodrop OD280. The amounts of scFv1, scFv2, scFv3, scFv4, scFv5, and scFv6 were 0.72 mg, 0.061 mg, 0.13 mg, 0.92 mg, 0.47 mg, and 0.055 mg, respectively.

[0102] scFv samples were diluted to 1 μg / ml and then serially diluted twofold. Flag-tag samples were coated onto plates, blocked with BSA, and the diluted scFv samples were then tested. HRP-labeled anti-flag-tag antibody was used as the secondary antibody. BSA coating and blocking served as controls. The ELISA results are shown in the table below. All six scFv samples bound to the Flag-tag antibody but not to the BSA control, with scFv1 exhibiting the strongest affinity.

[0103] Table 1-10 ELISA Results

[0104]

[0105]

[0106] The purified scFv-1 antibody was used, and its affinity was detected using Gator Prime. The specific procedure was as follows: scFv-1 was labeled with biotin and dialyzed overnight, and its concentration was measured. The biotin-Flag fusion protein (IgM antibodies with Flag tags at the middle and C-terminus positions) was first immobilized using an SA probe, and then bound to the labeled scFv-1. The affinity was then measured, and the antibody affinity results are as follows. Figure 7 , Figure 8 As shown.

[0107] Example 4: The Flag antibody scFv1, which was finally screened out, was used for Western blotting to detect the fusion protein A with the Flag tag at different positions.

[0108] 4.1 Sample preparation

[0109] 4.1.1 Take 100 ng of each of the three fusion proteins A with Flag tags at different locations (the Flag tags are located at the N-terminus, C-terminus, and middle part of protein A, respectively) and add 5×SDS loading buffer to a final concentration of 1×SDS Buffer. For reduction electrophoresis, add DTT to the loading buffer to a final concentration of 10 mM and mix well.

[0110] 4.1.2 Place the sample in a metal bath and heat at 100°C for 5 minutes to denature the protein before loading.

[0111] 4.2 Electrophoresis:

[0112] 4.2.1 Pipette 20 μL of sample into the sample well.

[0113] 4.2.2 After sample loading, close the cap, connect the electrophoresis apparatus, and turn it on. Before the sample enters the gel, maintain the voltage at 100–200V for approximately 15–20 minutes. Once the bromophenol blue indicator in the sample reaches the separating gel, increase the voltage to 200V and maintain a stable voltage throughout the electrophoresis process. Stop electrophoresis when the bromophenol blue indicator migrates to a point 1–2 cm from the leading edge, approximately 0.5–1 hour. 4.3 Staining and destaining of Cochlear stain:

[0114] 4.3.1 After electrophoresis, turn off the power, remove the glass plates, and gently pry them apart with a knife in the gap between the lower corners of the two glass plates of different lengths. Remove the gel and soak it in water.

[0115] 4.3.2 Turn on the power of the transfer apparatus, immerse one piece of absorbent paper in the transfer solution, and immerse another piece of absorbent paper in the dyeing solution for later use;

[0116] 4.3.3 Take out the transfer box, place absorbent paper soaked in transfer solution, electrophoresis gel, and absorbent paper soaked in staining solution on the negative electrode side in sequence. Carefully use a small porcelain trowel to repeatedly press out the air bubbles between the gel and the absorbent paper. Cover the positive electrode side, tighten it, and insert the transfer box into the transfer apparatus.

[0117] 4.3.4 Adjust the voltage to 30V, set the time to 5 minutes, and click the staining button to start staining;

[0118] 4.3.5 After staining, remove the gel, immerse it in water, take a picture, and analyze it;

[0119] 4.4WB transfer:

[0120] 4.4.1 After electrophoresis, turn off the power, remove the glass plates, and gently pry them apart with a knife in the gap between the lower corners of the two glass plates of different lengths. Remove the gel and soak it in water.

[0121] 4.4.2 Turn on the power of the transfer apparatus, soak two absorbent papers in the transfer buffer, and soak a PVDF membrane the size of a gel in the membrane buffer for later use; keep the transfer buffer on ice for half an hour before transfer.

[0122] 4.4.3 Take out the transfer box, and place absorbent paper soaked in transfer solution, PVDF membrane, electrophoresis gel, and another absorbent paper soaked in transfer solution on the negative electrode side in sequence. After each layer, carefully press out the air bubbles repeatedly with a small porcelain trowel. Cover the positive electrode side, tighten it, insert the transfer box into the transfer apparatus, and tighten the hose connected to the corresponding pump.

[0123] 4.4.4 Click "Rinse" to remove air bubbles from the pipes, then adjust the voltage to 30V and set the time to 15 minutes. Click the "Transfer" button to start the transfer process.

[0124] 4.4.5 After the transfer is complete, remove the PVDF membrane, immerse it in clean water to cool it down, and rinse it.

[0125] 4.5WB hybridization and color development:

[0126] 4.5.1 Prepare 50 mL of 5% skim milk powder using PBST buffer for later use;

[0127] 4.5.2 Immerse the PVDF membrane in 15 mL of 5% skim milk, place it in a petri dish, and seal it on a horizontal decolorizing shaker at room temperature for 1 hour;

[0128] 4.5.3 Add scFv1 (with His tag) as the primary antibody to the sealed skim milk at a ratio of 1:5000, and hybridize at room temperature for 1 hour;

[0129] 4.5.4 Discard the hybridization solution, soak and shake with PBST, and wash the PVDF membrane 5 times, 5 minutes each time;

[0130] 4.5.5 Soak the cleaned PVDF membrane in 15 mL of 5% skim milk, add anti-His tag antibody as secondary antibody at a ratio of 1:5000 to the skim milk, and hybridize at room temperature for 1 hour;

[0131] 4.5.6 Discard the hybridization solution, soak and shake with PBST, and wash the PVDF membrane 5 times, 5 minutes each time;

[0132] 4.5.7 Take 200 μL of each of the A and B solutions of the colorimetric reagent, mix them in equal proportions, and set aside for later use;

[0133] 4.5.8 Take out the cleaned PVDF membrane, and in a dark room, evenly drop the mixed color developing solution onto the membrane, and let it stand in the dark for 5 minutes;

[0134] 4.5.9 Remove the developed PVDF membrane, expose it using a gel imaging system, take a photograph, and analyze the results. Figure 9 As shown.

[0135] In summary, this Flag antibody exhibits high affinity and specificity for Flag tags and can be used for Western blotting to detect Flag-tagged proteins.

Claims

1. A Flag antibody scFv-1, characterized in that: The light chain variable region sequence is as follows: DMTQTPSSKSVPVGDTVTINCQSSQSVYLNNDLAWFQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCVGYKSSSIDGVAFGGGTELEIL, The amino acid sequence of the light chain variable region CDR1 is: QSSQSVYLNNDLA. The amino acid sequence of the light chain variable region CDR2 is: YASTLAS. The amino acid sequence of the CDR3 variable region of the light chain is: VGYKSSSIDGVA; The heavy chain variable region sequence is as follows: QQQLVESGGRLVTPGTPLTLTCTASGFSLHSYAMSWVRQAPGKGLEWIGTIEFNDNTYYASWPKGRFTISKTSTAVDLKMTSLTAADTGTYFCVRSSNGWSQTIWGPGTLVTVSS, The amino acid sequence of the heavy chain variable region CDR1 is: SYAMS. The amino acid sequence of the heavy chain variable region CDR2 is: TIEFNDNTYYASWPKG. The amino acid sequence of the heavy chain variable region CDR3 is: SSNGWSQTI.

2. The expression plasmid of the Flag antibody scFv-1 according to claim 1, wherein the plasmid vector is pET-Duet1.

3. The prokaryotic expression vector of the Flag antibody scFv-1 according to claim 1, wherein the vector is Escherichia coli.

4. The use of the Flag antibody scFv-1 according to claim 1 in the purification or precipitation of Flag-tagged fusion proteins or protein complexes.

Images