Benzonase immune phage display antibody library construction method, monoclonal antibodies, encoding genes and kits

Abstract

The present invention provides a method for constructing a Benzonase immune phage display antibody library, the steps of which include: (1) immunizing rabbits with Benzonase protein, extracting total RNA from lymphocytes, and reverse transcribing into cDNA; (2) amplifying heavy chain variable region and light chain variable region fragments using PCR technology and splicing them into scFv genes; (3) constructing a pCANTAB5E-scFv recombinant plasmid, electrotransforming it 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 therefrom. The screened monoclonal antibodies and their encoding genes are disclosed. The present invention obtains high-affinity rabbit-derived single-chain antibodies by screening through phage display antibody library technology. The single-chain antibodies of the present invention can be expressed by Escherichia coli BL21, providing optional raw materials for the development of immune detection kits.

Description

Technical Field

[0001] The patent of this invention relates to a method for constructing a Benzonase immune phage display antibody library, a Benzonase monoclonal antibody scFv and its encoding gene. Background Art

[0002] Benzonase is a genetically engineered endonuclease derived from the bacterium Serratia marcescens. Benzonase can degrade double-stranded, single-stranded, linear, and circular DNA and RNA, completely degrading nucleic acids into 3-5 base-long 5'-monophosphate oligonucleotides. Because it can efficiently degrade all forms of DNA and RNA, it is also known as the "universal nuclease." Whether in laboratory research or industrial-scale production, Benzonase is an effective enzyme for removing all forms of DNA and RNA from biological products. Its efficient nucleic acid removal significantly improves the effectiveness and yield of subsequent experiments and production, outperforming other nucleic acid removal methods.

[0003] Benzonase is a recombinant protein expression product and cannot be left in biological products. After using Benzonase to remove nucleic acids during the production process of biological products, Benzonase must be removed and residual detection must be performed to ensure that the product meets production standards. Previous studies have shown that Benzonase has poor immunogenicity in mice immunized with mice, making it difficult to obtain high-affinity mouse monoclonal antibodies. The purpose of the present invention is to obtain high-affinity rabbit-derived single-chain antibodies through phage display antibody library technology. The single-chain antibody of the present invention can be expressed in Escherichia coli BL21, providing an optional raw material for the development of immunoassay kits. Summary of the Invention

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

[0005] A method for constructing a Benzonase immune phage display antibody library, characterized in that the method comprises the following steps:

[0006] (1) Rabbits were immunized with Benzonase protein, total RNA of lymphocytes was extracted, and reverse transcribed into cDNA;

[0007] (2) Using PCR technology to amplify the heavy chain variable region and light chain variable region fragments and splice them into scFv genes;

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

[0009] (4) Specifically enrich the phage single-chain antibody library and screen out positive clones.

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

[0011]

[0012]

[0013] Preferably, in step (2), the heavy chain VH upstream primers (VH-F1, VH-F2, VH-F3, VH-F4) and downstream primers (VH-R) are mixed in pairs for PCR, the light chain VLK upstream primers (VLK-F1, VLK-F2) and downstream primers (VLK-R1, VLK-R2, VLK-R3) are mixed in pairs for PCR, and the light chain VLlamda-F and VLlamade-R primers are mixed for PCR. The obtained PCR products of the light chain VL and heavy chain VH are recovered and mixed in equimolar amounts as templates, and PCR is performed using the corresponding heavy chain upstream primers and light chain downstream primers in the primer table.

[0014] Preferably, the specific enrichment of the phage single-chain antibody library in step (4) specifically refers to taking the antigen with PBS, mixing it and coating it in an immunotube, using blocking solution, adding the phage single-chain antibody library, and incubating it with BSA solution;

[0015] Elution: Add pH 2.2 Gly-HCl, let it stand, then collect the sample by vigorous pipetting with a pipette tip. Add pH 8.5 Tris-HCl to the collection tube to neutralize the pH, and then add BSA solution to dilute.

[0016] Amplification of eluted product: The eluted product was added to TG1 bacterial solution and cultured, and M13K07 helper phage was added to continue the culture. The bacterial solution was centrifuged, and the precipitate was resuspended in 2×YT medium and cultured overnight with shaking. On the next day, the bacterial solution was centrifuged, and the supernatant was transferred to a new centrifuge tube. PEG-NaCl was added, and the tube was allowed to stand on ice for precipitation and then centrifuged. The supernatant was removed, and the precipitate was resuspended in BSA solution, transferred to another EP tube, centrifuged, and the precipitate was removed. The supernatant was the amplified enriched product.

[0017] Repeat the above steps and perform multiple pannings to increase the severity of each round of panning and enrich for phages with affinity.

[0018] The present invention also discloses a Benzonase monoclonal antibody scFv, the sequence of which is any one of SEQ ID No. 1 to 6, and is named scFv1, scFv2, scFv3, scFv4, scFv5, and scFv6 in sequence.

[0019] The present invention also discloses a Benzonase monoclonal antibody scFv encoding gene, the sequence of which is any one of SEQ ID No. 7 to 12. The DNA sequence shown in SEQ ID No. 7 encodes scFv1, and so on.

[0020] The invention also discloses a Benzonase detection kit containing the Benzonase monoclonal antibody scFv.

[0021] Preferably, the Benzonase detection kit contains the antibody scFv2 having a sequence as shown in SEQ ID No. 2 as a coating antibody, and also contains the antibody scFv1 having a sequence as shown in SEQ ID No. 1, and the scFv1 is biotin-labeled.

[0022] The method for constructing the Benzonase immune phage display antibody library of the present invention can screen and obtain rabbit-derived single-chain antibodies with high affinity, and can construct a library with a capacity of 5.23×10 7 The recombinant phage titer was 2.86×10 14 The single-chain antibody of the present invention can be expressed in Escherichia coli BL21, and the developed immunoassay kit has high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the electrophoresis diagram of the extracted total RNA.

[0024] Figure 2 This is the gel electrophoresis diagram of the PCR products of the light chain VL and heavy chain VH genes.

[0025] Figure 3 This is the gel electrophoresis diagram of the scFv gene PCR product.

[0026] Figure 4 This is the electrophoresis diagram of pCANTAB5E plasmid before and after double enzyme digestion.

[0027] Figure 5 It is a plan view of the electroporation of the connection products.

[0028] Figure 6 This is a colony PCR identification diagram.

[0029] Figure 7 This is a diagram of the purification of recombinant protein scFv.

[0030] Figure 8 is the fitted standard curve. DETAILED DESCRIPTION

[0031] The main materials of the embodiment of the present invention are as follows:

[0032] Freund's complete / incomplete adjuvant was purchased from Sigma, Germany.

[0033] New Zealand white rabbits (2-2.5 kg) were purchased from Shanghai Jiagan Biological Co., Ltd.

[0034] Ultrapure RNA extraction kit (DNase I) was purchased from Kangwei Century Biotechnology Co., Ltd.

[0035] Anhydrous ethanol, chloroform, and glycerol were purchased from Sinopharm Group.

[0036] PCR amplification primers were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0037] Kanamycin and ampicillin were purchased from Solebao Biotechnology Co., Ltd.

[0038] PEG8000 was purchased from Sigma.

[0039] Sfi1, Not1 restriction endonuclease, and T4 DNA ligase were purchased from NEB, UK.

[0040] SanPrep column-based DNA gel recovery / plasmid minipreparation kit / PCR product purification kit, 20× PBS, Tween 20, were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0041] Escherichia coli (E. coli) TG1 Electro competent cells and E. coli DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0042] The pCANTAB5E phagemid vector was purchased from Shanghai Core Biotechnology Co., Ltd.

[0043] M13K07 helper phage was purchased from NEB, UK.

[0044] GXL DNA Polymerase was purchased from Takara, Japan.

[0045] Benzonase, 50x TAE buffer, DEPC-treated Water, agarose, 5000bp DNA marker, YeaRed Nucleic Acid Gel Stain, Ⅲ1st Strand cDNA Synthesis SuperMix Component, bovine serum albumin, sodium chloride, TMB colorimetric solution, and skim milk powder were obtained from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0046] The main culture medium and buffer solution formula of the present invention are:

[0047] 2×YT liquid medium: peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, pH 7.2-7.4, dilute to 1 L, and sterilize at 121°C for 20 min.

[0048] 2×YT-A liquid medium: 2×YT medium containing 50 mg / L ampicillin (AMP).

[0049] SOB liquid medium: peptone 20 g / L, yeast extract 5 g / L, sodium chloride 0.5 g / L, potassium chloride 0.185 g / L, magnesium chloride 0.95 g / L, pH 7.2-7.4, dilute to 1 L with ultrapure water, and sterilize at 121°C and autoclave for 20 min.

[0050] 2×YT-AK liquid medium: 2×YT medium containing 50 mg / L ampicillin (AMP) and 50 mg / L kanamycin (Kan).

[0051] 2×YT solid medium: Add 1.5 g agar powder to every 100 ml of liquid 2×YT medium and sterilize at 121°C for 20 min.

[0052] 2×YT-A solid medium: 2×YT medium containing 100 mg / ml ampicillin (AMP).

[0053] 2×YT-K solid medium: 2×YT medium containing 50 mg / ml kanamycin (Kan).

[0054] 50% glycerol seed preservation solution: Accurately measure 25 ml of glycerol, dilute to 25 ml with ultrapure water, mix well and sterilize at 121℃ and high pressure for 20 min.

[0055] Prepare 50mg / ml ampicillin / kanamycin stock solution. Accurately weigh 0.5g of AMP / Kan solid powder and dissolve it in 10ml of ultrapure water. After fully dissolving, filter sterilize using a 0.22μM filter membrane. Aliquot into 1.5ml sterilized EP tubes and store at -20℃ until used.

[0056] PEG / NaCl: Weigh 146.1 g / L NaCl and 200 g / L PEG8000 using an electronic balance. Add 800 ml of ultrapure water to 1 L. Sterilize at 121°C and autoclave for 20 min.

[0057] PBST buffer: Dilute 20× PBS to 1×, add 1 ml Tween-20 to 1 L of 1× PBS solution, mix well, and sterilize at 121°C and autoclave for 20 min.

[0058] Agarose gel: For 1.2% gel, weigh 0.36 g agarose into 30 ml 1×TAE nucleic acid electrophoresis buffer and boil until completely dissolved. After cooling to about 50°C, add 3 μl nucleic acid staining aqueous solution and mix thoroughly. Pour the mixture into a gel mold and allow to cool to room temperature until solidified. Place the gel in an electrophoresis tank for electrophoresis.

[0059] Example 1 Construction of phage display antibody library

[0060] 1.1 Animal immunization and extraction of total RNA from spleen cells

[0061] Benzonase was administered subcutaneously to 2-2.5 kg New Zealand white rabbits every two weeks. The primary immunization was performed with 1 mg of Benzonase emulsified in Freund's complete adjuvant, and the booster immunization was performed with 0.5 mg of Benzonase emulsified in incomplete adjuvant. Two months after immunization, 0.5 mg of protein was administered subcutaneously. Three days after the boost immunization, 30-50 mg of spleen tissue was obtained. Total RNA was extracted using the Ultrapure RNA Extraction Kit (DNase I). After thorough grinding in liquid nitrogen, 1 ml of TRIzon Reagent was added, or 1 ml of TRIzon Reagent was added to the spleen tissue and homogenized. After adding TRIzon Reagent, the sample was pipetted several times to fully lyse the sample. The sample was allowed to stand at room temperature for 5 minutes to completely dissociate the protein-nucleic acid complex. Chloroform was added at a ratio of 200 μl of chloroform per 1 ml of TRIzon Reagent. The tube was capped, shaken vigorously for 15 seconds, and allowed to stand at room temperature for 2 minutes. Centrifuge at 4°C, 12,000 rpm (~13,400 × g) for 10 min. The sample will separate into three layers: a red organic phase, an intermediate layer, and an upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube (self-prepared).

[0062] Add an equal volume of 70% ethanol (prepared with RNase-free water), approximately 600-700 μl, to the resulting aqueous solution and mix thoroughly by inversion. Add the entire solution from the previous step to the spin column (RM) placed in a collection tube. If the solution cannot be added all at once, add it in multiple additions. Centrifuge at 12,000 rpm for 20 seconds, discard the waste liquid from the collection tube, and return the spin column to the collection tube. Add 350 μl of Buffer RW1 to the spin column, centrifuge at 12,000 rpm for 20 seconds, discard the waste liquid from the collection tube, and return the spin column to the collection tube. Prepare the DNase I mixture: Add 8 μl of 10× Reaction Buffer and 20 μl of DNase I (1 U / μl) to 52 μl of RNase-Free Water. Mix thoroughly to a final reaction volume of 80 μl. Add 80 μl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes. Add 500 μl of Buffer RW2 to the adsorption column (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 20 seconds, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 minutes, pour out the waste liquid in the collection tube. Place the adsorption column at room temperature for 3 minutes and dry it thoroughly. Place the adsorption column in a new RNase-free centrifuge tube, add 30-50 μl of RNase-Free Water to the middle part of the adsorption column, place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, collect the RNA solution, take 1 μl of sample and run electrophoresis to detect the extraction effect. The results are as follows: Figure 1 The remaining RNA samples were stored in a -80°C refrigerator until use.

[0063] 1.2 scFv gene cloning and splicing

[0064] The present invention uses the reverse transcription kit of Yisheng Biotechnology Co., Ltd. to extract RNA from rabbit spleen cells as a template. According to the reverse transcription reaction system shown in Table 1-1, RNA template, RNase-free water, cDNA was obtained by incubating the reaction at 55°C for 15 minutes using III SuperMix, followed by reverse transcription at 55°C for 15 minutes and heating at 85°C for 5 minutes. The primers in Table 1-2 were synthesized by Shanghai Bioengineering. Both the light chain variable region gene and the heavy chain variable region gene used rabbit cDNA as a template. The light chain upstream primer and the downstream primer were mixed in pairs for PCR, and the heavy chain upstream primer and the downstream primer were mixed in pairs for PCR. The specific mixing method was as follows: heavy chain: VH-F1 was mixed with VH-R for PCR, VH-F2 was mixed with VH-R for PCR, VH-F3 was mixed with VH-R for PCR, and VH-F4 was mixed with VH-R for PCR; light chain: light chain VL-F1 was mixed with VL-R1 for PCR, VL-F1 was mixed with VL-R2 for PCR, VL-F1 was mixed with VL-R3 for PCR, VL-F2 was mixed with VL-R1 for PCR, VL-F2 was mixed with VL-R3 for PCR, and VLlamda-F was mixed with VLlamda-R for PCR; the components added to the PCR reaction were as shown in Table 1-3, and the PCR reaction was performed according to the reaction conditions in Table 1-4. The obtained PCR products of light chain VLK and heavy chain VH were loaded on 1.2% agarose gel and the amplification results were observed by electrophoresis. Figure 2 , use OMEGA's gel recovery kit according to the kit experimental steps to recover, and store the recovered product at -20℃.

[0065] The heavy chain and light chain genes recovered in the previous step were mixed in equal moles as templates, and PCR was performed using the corresponding heavy chain VH upstream primers and light chain downstream primers to splice into scFv. The PCR reaction system is shown in Table 1-5, and the reaction conditions are the same as the VH gene amplification conditions in Table 1-4. After the reaction is completed, the sample is loaded and tested by agarose gel electrophoresis. The results are as follows Figure 3 , glue recycling.

[0066] Table 1-1 Reverse transcription reaction system

[0067]

[0068] Table 1-2 Primers used

[0069]

[0070] Table 1-3 PCR reaction system

[0071]

[0072]

[0073] Table 1-4 PCR amplification conditions

[0074] Destination fragment transsexual annealing extend End of reaction Number of cycles VL 98℃10s 55℃15s 68℃42s 4℃ 30 VH 98℃10s 60℃15s 68℃42s 4℃ 30

[0075] Table 1-5 PCR reaction system

[0076] Components Usage Prime STAR GXL Premix (2×) 25 μl Primer F (10 μM) 1 μl Primer R (10 μM) 1 μl Template(VH+VL) 2 μl sterile water 21 μl Total 50 μl

[0077] 1.3 Construction of pCANTAB5E-scFv recombinant plasmid

[0078] pCANTAB5E was transformed into DH5α for amplification and plasmid was prepared. According to the double enzyme digestion system of NEB Company in the UK, pCANTAB5E plasmid and scFv fragment were digested with Sfi1 and Not1 respectively. The digestion reaction is shown in Table 1-6 below. After digestion in a 37℃ water bath for 1 hour, 2μl Sfi1 was added, mixed thoroughly, and placed in a 50℃ water bath and digestion was continued for 1 hour. After the digestion was completed, the digestion results were observed by 1.2% agarose gel electrophoresis. The results are as follows. Figure 4 The product was recovered using an OMEGA gel recovery kit. Using NEB's electroporation-specific ligase, the digested scFv gene fragment scFv(S+N) and the plasmid pCANTAB5E(S+N) were ligated at 4°C overnight using the ligation reaction system in Table 1-7 at low temperature.

[0079] Table 1-6 Enzyme Digestion Reaction System

[0080]

[0081]

[0082] Table 1-7 Enzyme-linked system

[0083] Reaction components volume 10×Ligase buffer 2 μl pCANTAB5E(S+N) 100ng scFV(S+N) 50ng sterile water Make up to 20 μl

[0084] 1.4 Transformation of ligation product into competent TG1 and determination of library capacity and recombination rate

[0085] Thaw TG1 competent cells on ice, then add 1 μl of the pCANTAB5E-scFv ligation product and gently mix. Pour the cells along the tube wall into an ice-bathed electroporation cuvette. Let stand for 5-10 minutes before placing them in an electroporator. Set the voltage to 2.0 kV and hold the voltage constant for approximately 5.0 ms. Immediately after electroporation, add 1 ml of preheated SOB liquid medium and incubate on a shaker at 37°C for 1 hour. For this experiment, perform 20 transformations in parallel, electroporating 1 μl of the product once for a total of 20 transformations, yielding 20 ml of transformed competent cells.

[0086] After transformation and recovery culture, 100 μl of the bacterial solution was mixed and diluted by 10-fold gradient dilution method. 100 μl was spread on 2×YT-A (containing 50 mg / ml AMP) plates and incubated inverted at 37°C overnight to calculate the storage capacity. The ligation product transformation results are shown in Figure 2. Figure 5The next day, 26 single colonies were picked from the dilution plate with well-dispersed single colonies and placed in 2×YT-A (containing 50 mg / ml AMP) liquid culture medium, and cultured overnight at 37°C and 200 rpm / min. 1 μl of bacterial solution was taken from each of the 26 expanded single colonies as a template, and bacterial solution PCR was performed according to the scFv gene splicing PCR system. The PCR products were run on electrophoresis and the results were analyzed on a gel imager to calculate the recombination rate of the antibody library. The PCR identification results were as follows: Figure 6 .

[0087] Calculation formula: Storage capacity = number of single colonies × dilution factor × total volume (Formula 1-1)

[0088] Recombination rate (%) = positive clones / number of single clones picked (Formula 1-2).

[0089] 1.5 Construction of phage scFv antibody library

[0090] 1.5.1 Preparation of helper phage virus stocks

[0091] Inoculate 5 ml of SOB medium at a 1:100 ratio from an overnight TG1 culture. Cultivate at 37°C, 200 rpm / min, with shaking until the OD600 reaches 0.4-0.6. Add 5 μl of M13k07 helper phage and incubate at 37°C, 200 rpm / min, with shaking for 1 hour. Then, add 5 μl of kanamycin (50 mg / ml) and incubate at 37°C, 200 rpm / min, with shaking for 1 hour. Transfer the culture 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, 200 rpm / min. The next day, the overnight cultured bacterial solution was divided into 50 ml centrifuge tubes, centrifuged at 4°C, 10,000 rpm / min for 20 min, and the supernatant was transferred to a high-temperature sterilized 500 ml conical flask. PEG / NaCl with a volume of 1 / 5 of the initial bacterial solution was added, and the volume added for this operation was 20 ml. After mixing, the solution was divided into new 50 ml centrifuge tubes. After precipitation on ice for 2 h, the solution was centrifuged at 4°C, 10,000 rpm / min for 10 min, and the supernatant was discarded. The pellet was resuspended with 2 ml (1 / 50 volume) of 1× PBS, transferred to a sterile centrifuge tube, and centrifuged again at 10,000 rpm / min, 4°C for 5 min. The supernatant was carefully removed, sterilized by filtration with a 0.22 μM filter membrane, and glycerol was added to a final concentration of 50%. The solution was divided into sterile centrifuge tubes and stored at -20°C.

[0092] 1.5.2 Helper phage virus titration

[0093] The prepared phage solution was diluted 10-fold using 1% BSA (in 1×PBS) as diluent.-4 , 10 -6 , 10 -8 , 10 -10 , 10 -12 Add 10 μl of the bacterial solution to TG1 with an OD600 of 0.4-0.6. Use the dilution and TG1 as negative controls, incubate at 37°C for 30 minutes, and shake for 30 minutes. After the incubation period, spread the five dilution gradients of bacterial solution and the two negative controls on plates containing 50 mg / ml of kanamycin and incubate at 37°C. The next day, count the number of colonies on the plates and calculate the phage titer (pfu / ml) according to the following formula. Generally, a titer of 10 12 Only then can subsequent experiments be performed. Calculation formula: Phage titer = number of monoclonal clones × dilution factor × 10 (Formula 1-3).

[0094] 1.5.3 Construction of primary phage antibody library

[0095] Add 10 ml of the remaining culture from the transformation to 90 ml of 2×YT-A medium (25 μg / ml AMP) and incubate at 37°C, 200 rpm, and shake for 1 hour. Add AMP to a final concentration of 50 μg / ml and continue incubating for another hour. Add 10 μl of helper phage M13K07 to the culture at a multiplicity of infection (MOI) of 20:1. Gently shake the culture at 37°C, 60 rpm, for 30 minutes to facilitate phage infection, and then incubate on a shaker for 30 minutes. Transfer the culture to a sterile centrifuge tube in a clean hood and centrifuge at 5000 rpm for 15 minutes. Carefully remove the supernatant. Aseptically resuspend the bacterial pellet in 100 ml of 2×YT-AK medium (50 mg / ml AMP and 50 mg / ml Kan) and incubate overnight with shaking. The next day, transfer the entire overnight culture to a sterile 50 ml centrifuge tube and centrifuge at 10,000 rpm at 4°C for 20 minutes.

[0096] Transfer the supernatant to a new sterile Erlenmeyer flask and add PEG-NaCl (20 ml) at 1 / 5 the volume of the initial bacterial solution. Let it sit on ice for 1 hour to precipitate. Centrifuge at 10,000 rpm at 4°C for 20 minutes, then remove the supernatant. Invert the tube for 2-5 minutes to remove as much excess liquid as possible. Resuspend the pellet in 1% BSA (in 1× PBS) at 1 / 50 the volume of the initial bacterial solution, transfer it to a 1.5 ml sterile tube, centrifuge at 10,000 rpm for 5 minutes, remove the pellet, 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.

[0097] 1.6 Immuno-tube panning for specific antibodies

[0098] According to the desired concentration, dilute the appropriate amount of antigen to 2 ml with 1× PBS, mix well, and coat the tubes. Incubate at 37°C for 1 hour (or overnight at 4°C), wash with 0.1% PBST, let stand for 3 minutes, and then spin dry. Repeat three times. Block with 2 ml of blocking solution, incubate at 37°C for 1 hour (or overnight at 4°C), wash with 0.1% PBST, let stand for 3 minutes, and then spin dry. Repeat three times and place at 4°C. Add the appropriate amount of phage library, dilute to 2 ml with 2.5% BSA (in 0.05% PBST), mix well, and incubate at 37°C for 2 hours. After the incubation is complete, retain a small amount of supernatant for titering. Elution: Use 2 ml of pH 2.2 Gly-HCl, let stand at 37°C for 10 minutes, then collect by vigorous pipetting for 3 minutes. Add 1 / 10 volume of pH 8.5 Tris-HCl to the collection tube to neutralize the pH. Then add 5% BSA (in 1× PBS) to dilute to a final concentration of 1%.

[0099] Amplification of the eluted product: Add 1 ml of eluted product to 4 ml of TG1 bacterial culture (OD600: 0.4-0.6) and incubate at 37°C, 60 rpm for 30 minutes, then adjust the speed to 200 rpm for an additional 30 minutes. Add 10 μl of M13K07 helper phage and incubate at 37°C, 60 rpm for 30 minutes, then adjust the speed to 200 rpm for an additional 30 minutes. Centrifuge the culture at 10,000 rpm at 4°C for 20 minutes. Resuspend the pellet in 100 ml of 2×YT medium (containing 100 μg / ml AMP and 50 μg / ml Kana) and incubate overnight with shaking. The next day, the bacterial suspension was centrifuged at 10,000 rpm and 4°C for 20 minutes. The supernatant was transferred to a fresh centrifuge tube and 1 / 5 volume of PEG-NaCl (20 ml) was added. After precipitation on ice for 1 hour, the supernatant was removed by centrifugation at 10,000 rpm and 4°C for 20 minutes. The supernatant was then removed and the tube was inverted for 2-5 minutes to remove as much excess liquid as possible. The pellet was resuspended in 1% BSA (in 1× PBS) at 1 / 50 the starting volume of the bacterial suspension, transferred to a 1.5 ml EP tube, and centrifuged at 10,000 rpm for 5 minutes. The pellet was removed and the supernatant was the amplified enriched product, which was immediately used for a new round of panning. Gradually increasing the stringency of each round of panning resulted in an increase in both the eluted product and the amplified titer, indicating that truly avid phage were being enriched.

[0100] Table 1-8 5-round screening conditions

[0101]

[0102] Table 1-9 Results of 5 rounds of screening

[0103]

[0104] 1.7 Phage-ELISA detection of phage antibodies

[0105] From the TG1 plate infected with the eluted product, single colonies were selected and cultured overnight. The next day, the culture was diluted 50-100-fold to an OD600 of approximately 0.1. After shaking to an OD600 of 0.4-0.6, 1 μl of ten-fold diluted M13K07 was added. The culture was incubated at 37°C and 60 rpm for 30 minutes, then the speed was adjusted to 200 rpm for an additional 30 minutes. The culture was centrifuged at 10,000 rpm and 4°C for 20 minutes, and the supernatant was removed. The pellet was resuspended in 4 ml of 2×YT medium (containing 100 μg / ml AMP and 50 μg / ml Kana) and incubated with shaking overnight. The next day, the culture was centrifuged at 10,000 rpm and 4°C for 20 minutes, and the supernatant was retained for ELISA analysis.

[0106] Antigen coating was performed at a concentration of 1 μg / ml, with 100 μl per well added, incubated at 37°C for 1 hour (or overnight at 4°C), washed with 0.1% PBST, allowed to stand for 1 minute, and then spun dry. This was repeated three times. Blocking was then performed with 200 μl per well in blocking solution, incubated at 37°C for 1 hour (or overnight at 4°C), washed with 0.1% PBST, allowed to stand for 1 minute, spun dry, and patted dry. This was repeated twice, and the cells were allowed to stand at 4°C. A control was also performed by directly coating with blocking solution, with 200 μl per well in blocking solution, incubated at 37°C for 2 hours (or overnight at 4°C), washed with 0.1% PBST, allowed to stand for 1 minute, spun dry, and patted dry. This was repeated twice, and the cells were allowed to stand at 4°C. Samples were then added sequentially in a clean hood, with 100 μl per well added, incubated at 37°C for 1 hour, the solution discarded, patted dry, washed with 0.1% PBST, allowed to stand for 1 minute, spun dry, and patted dry. This was repeated five times. Add 100 μl of 5000-fold diluted HRP-avidin to each well and incubate at 37°C for 1 hour. Discard the liquid, pat dry, and wash with 0.1% PBST. Let stand for 1 minute, shake, and pat dry. Repeat five times. Add 100 μl of TMB colorimetric solution to each well and develop at 37°C. Add 50 μl of 1M HCl as a stop solution to each well. After development, read the OD450 value. A positive clone is identified if the sample OD450 differs by at least 100% from the OD450 value of the blocking solution alone control.

[0107] 1.8 scFv expression in E. coli and ELISA identification

[0108] The TG1 shaker infected with the positive phage clone was extracted and treated, and then sent for sequencing. After obtaining the scFv gene sequence, the Immunoglobulin BLAST and Nucleotide.nucleotide BLAST analysis software in NCBI BLAST were used to analyze the CDR1, CDR2, and CDR3 regions. Based on the scFv sequencing results, the pET-Duet1-scFv expression vector was constructed. The primer design sequence is shown in the table below. Using the plasmid extracted from the positive clone as a template, PCR amplification was performed to obtain the scFv fragment, and PCR amplification was performed to obtain the pET-Duet1 fragment. The PET-Duet1-scFv was constructed by one-step cloning. After positive colony PCR identification, the plasmid was extracted and sequencing was used to verify the correct construction.

[0109] The constructed PET-Duet1-scFv plasmid was transformed into Rosseta (DE3) cells. A single clone was selected and expanded to 1 L. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM and expression was induced overnight at 16°C. After induction, the cells were centrifuged and the supernatant removed. The cells per 1 L of culture were resuspended in 50 ml of PBS and lysed using a homogenizer on ice for 5 minutes. The cells were then transferred to a centrifuge tube and centrifuged at 12,000 rpm at 4°C for 30 minutes. The supernatant was then transferred to a fresh centrifuge tube. The supernatant of the bacterial lysate was purified using a nickel affinity column. After incubation with 1 ml of nickel affinity medium at 4°C for 1 hour, the supernatant was loaded onto a gravity chromatography column. The flow-through was washed with 20 ml of PBS containing 30 mM imidazole and eluted with 250 mM imidazole in PBS. The eluted product was concentrated using a 10 kD concentrator. The protein concentration was measured by 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. The purification results of scFv1 are shown in Figure 2. Figure 7 The scFv was diluted to 1 μg / ml and serially diluted two-fold. The plate was coated with Benzonase and blocked with BSA. The diluted scFv samples were detected using an HRP-labeled anti-flag-tag antibody as a secondary antibody. BSA coating and blocking were used as controls. The ELISA results are shown in the table below. All six scFvs bound to Benzonase, but not to the BSA control.

[0110] Table 1-10 ELISA results

[0111]

[0112]

[0113] 1.9 Six strains of scFv E. coli expressed antibodies paired to form an immune kit

[0114] From the six successfully expressed and identified scFv antibodies, scFv1, scFv4, and scFv5 were selected for biotin labeling. The remaining three scFv antibodies were randomly paired with the labeled biotin antibody and tested for Benzonase using a double-antibody sandwich ELISA. 0.5 mg of each scFv1, scFv4, and scFv5 antibody was dissolved in 0.01 M PBS buffer at pH 7.4. (Proteins in Tris or other amine-containing buffers must be converted to the appropriate buffer.) An appropriate amount of 10 mM biotin reagent solution (2.0 mg NHS-Biotin reagent dissolved in 590 μl dimethyl sulfoxide (DMSO)) was added to the protein solution. The solution was incubated at room temperature for 30 minutes to complete scFv antibody labeling. The labeled proteins were then purified by dialysis to achieve optimal performance and stability. Preliminary testing of the labeled proteins using ELISA confirmed the proper function and labeling of the labeled antibodies. The steps and results of the preliminary ELISA pairing test of the labeled scFv antibodies are as follows:

[0115] scFv2, scFv3, and scFv6 were diluted to 8 μg / ml and coated overnight at 4°C using a two-fold serial dilution series. After blocking with BSA, the Benzonase sample was tested, and PBS was added as a blank control. The detection antibodies (labeled scFv antibodies) corresponding to scFv1, scFv4, and scFv5 were diluted to 16 μg / ml and then tested using a two-fold serial dilution series. HRP-labeled anti-biotin streptavidin was used as the secondary antibody. The ELISA results are shown in Tables 1-11 below. Among the six paired scFvs, the results of the scFv2-coated antibody paired with the scFv1 antibody labeled with biotin and the scFv3-coated antibody paired with the scFv4 antibody labeled with biotin met expectations. The blank control was positive, and further testing was performed to confirm sensitivity. The scFv6-coated antibody paired with the scFv5 antibody labeled with biotin showed high background, so this pairing was eliminated.

[0116] Table 1-11 ELISA results

[0117]

[0118]

[0119]

[0120]

[0121] Based on the previous experiment, the optimal working concentrations of the coating antibody and detection antibody were selected. Further debugging was performed, maintaining the coating and detection antibody concentrations constant. A gradient of standards with varying concentrations was set up, and the reaction values ​​were measured using a double-antibody sandwich ELISA to determine the highest sensitivity of the kit. The experimental results are shown in Table 1-12. The scFv2-coated antibody paired with the scFv1 antibody labeled with biotin as the detection antibody achieved the highest sensitivity, reaching 4 pg / ml. Three replicate wells were set up for each standard concentration in this experiment. The OD450 values ​​in the table are average values. The specific experimental steps are as follows:

[0122] Coating: Dilute scFv2 or scFv3 antibodies to the optimal working concentration using coating solution (0.05 M NaHCO3, pH 9.6), add 100 μl / well, and coat overnight at 4°C. Remove the coating solution and wash three times with PBST solution.

[0123] Blocking: Remove the coating solution, wash with PBS solution three times, add 0.3 ml of blocking solution (5% BSA + PBS) to each well and incubate at 37°C for 2 hours.

[0124] Standard addition: Dilute Benzonase samples to 9 ng / ml, 6 ng / ml, 3 ng / ml, 1.5 ng / ml, 0.5 ng / ml, 0.1 ng / ml, 0.037 ng / ml, 0.012 ng / ml, and 0.004 ng / ml using PBS containing 2% BSA as dilution buffer. Add 100 μl of diluted sample to each well. Wells containing dilution buffer alone serve as negative controls. Incubate at 37°C for 1 hour, remove the sample solution, and wash twice with PBS.

[0125] Biotin-labeled scFv4 and scFv5 detection antibody addition: add 100ul of biotin-labeled antibody of optimal concentration to each well, incubate at 37℃ for 0.5h, then remove the biotin-labeled antibody and wash with PBST solution three times.

[0126] Addition of marker HRP-conjugated streptavidin: add 100 μl of HRP-conjugated anti-biotin protein diluted 1:5000 with dilution buffer to each well, incubate at 37°C for 0.5 h, remove the enzyme label solution, and wash with PBS solution three times.

[0127] Colorimetry and result calculation: Add 100 μl TMB colorimetric solution to each well, incubate in the dark at room temperature for 15 minutes, then add 0.05 ml 2M H2SO4 stop solution to terminate the reaction, and measure the OD450 value using an enzyme-labeled colorimeter.

[0128] Table 1-12

[0129]

[0130] Based on the previous two experimental steps, we selected the most sensitive coating antibody (scFv2) and biotinylated scFv1 detection antibody, along with the standard concentration range they could detect. While maintaining the coating and detection antibody concentrations constant, we set up a standard gradient with seven different concentrations. Three replicates were run for each concentration to minimize experimental manipulation errors. ELISA reactions were measured on the same plate using the same protocol as described above, and a standard curve was constructed based on these reactions. The standard concentrations and corresponding reaction values ​​are shown in Tables 1-13.

[0131] Table 1-13 Standard curve data

[0132]

[0133]

[0134] From the data in the table, we found that the data has a clear gradient and the difference between the replicate wells is very small, which can meet the experimental requirements of ELISA. The standard curve is drawn using the linear fitting method, such as Figure 8 As shown. Figure 8 It can be seen that the standard curve R 2 The value is 0.9991, which is highly reliable. Sequence Listing <110> Yisheng Biotechnology (Shanghai) Co., Ltd. <120> Benzonase immune phage display antibody library construction method, monoclonal antibodies, encoding genes and kits <141> 2022-03-17 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 244 <212> PRT <213> Artificial Sequence <400> 1 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Thr Pro Gly Thr Pro Leu 1 5 10 15 Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Trp Met 20 25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Val 35 40 45 Ile Ser Ser Ser Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr Ser 65 70 75 80 Leu Thr Thr Glu Asp Thr Ala Ile Tyr Phe Cys Ala Arg Tyr Ser Ser 85 90 95 Asp Trp Asn Ala Tyr Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Ile 100 105 110 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly Gly 130 135 140 Thr Val Thr Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr Gly Asn Asn 145 150 155 160 Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 165 170 175 Ile Tyr Gln Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Thr Ile Ser Gly Val Gln 195 200 205 Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Trp Ser Gly Ser Gly 210 215 220 Thr Asp Ala Val Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Leu Gly 225 230 235 240 Arg Ser Gly Pro <210> 2 <211> 244 <212> PRT <213> Artifical Sequence <400> 2 Gln Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Asn Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asn Trp Gly Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly His 100 105 110 Gly His Arg Leu Leu Ser Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala 130 135 140 Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala 145 150 155 160 Ser Ser Ser Ile Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Ser 165 170 175 Gly Ala Ser Pro Lys Pro Leu Ile His Arg Thr Ser Asn Leu Ala Ser 180 185 190 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 195 200 205 Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Trp Ser Gly Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys Arg <210> 3 <211> 241 <212> PRT <213> Artifical Sequence <400> 3 Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro Leu 1 5 10 15 Thr Leu Thr Cys Thr Ala Ser Gly Phe Ala Leu Ser Gly Tyr Tyr Met 20 25 30 Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ile 35 40 45 Thr His Pro Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Ala Ser Thr Thr Val Asp Leu Arg Ile Thr Ser 65 70 75 80 Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val Trp Thr 85 90 95 His Tyr Ser Ser Gly Gly Gly Asp Leu Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Ile Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Asp Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val 130 135 140 Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Glu Ser Val Tyr Lys 145 150 155 160 Asn Asn Asp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 165 170 175 Leu Leu Ile Ser Ala Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg 180 185 190 Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly 195 200 205 Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser 210 215 220 Gly Asp Asn Val Val Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val 225 230 235 240 Lys <210> 4 <211> 244 <212> PRT <213> Artifical Sequence <400> 4 Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ala Ser Leu 1 5 10 15 Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ser Asn Tyr Trp 20 25 30 Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Cys Ile Tyr Thr Gly Thr Ser Gly Thr Thr Tyr Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Glu Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ser 85 90 95 Lys Asn Thr Tyr Gly Tyr Ala Leu Ala Ala Arg Leu Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Ile Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Met Thr Gln Thr Pro Ala Ser 130 135 140 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 145 150 155 160 Gln Ser Ile Gly Ser Asp Leu Ala Trp Tyr Gln Gln Lys Leu Gly Gln 165 170 175 Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Glu Ser Gly Ala 180 185 190 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr 195 200 205 Ile Thr Asp Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly 210 215 220 Asp Tyr Ser Ser Gly Asn Asp Asn Gly Phe Gly Gly Gly Thr Glu Val 225 230 235 240 Val Val Lys Ala <210> 5 <211> 241 <212> PRT <213> Artifical Sequence <400> 5 Gln Leu Met Glu Ser Gly Gly Gly Leu Val Thr Pro Gly Gly Thr Leu 1 5 10 15 Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr Trp Met 20 25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Ile 35 40 45 Ile Ser Asn Tyr Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile Thr Ser 65 70 75 80 Pro Thr Ile Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Phe Ala 85 90 95 Gly Val Phe Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly Gly Thr Val 130 135 140 Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn Lys Asn Leu 145 150 155 160 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr 165 170 175 Lys Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly Ser 180 185 190 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala 195 200 205 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly His Asp Thr Asn Val Gln 210 215 220 Asn Leu Phe Gly Gly Gly Thr Glu Val Val Val Lys Ala Ala Ala Gly 225 230 235 240 Ala <210> 6 <211> 243 <212> PRT <213> Artifical Sequence <400> 6 Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser Leu 1 5 10 15 Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Leu Ser Ser Tyr Trp Met 20 25 30 Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Cys 35 40 45 Ile Asp Thr Gly Ser Ser Asp Ser Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val Thr Leu Gln 65 70 75 80 Met Thr Ser Val Thr Pro Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg 85 90 95 His Leu Arg Tyr Ser Thr Asn Asn Asn Asp Trp Asp Leu Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Leu Thr Gln Thr Pro Ala Ser Val 130 135 140 Glu Ala Ala Val Gly Asp Thr Val Thr Ile Lys Cys Gln Ala Ser Gln 145 150 155 160 Ser Ile Tyr Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro 165 170 175 Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Ala Ser Gly Val Pro 180 185 190 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile 195 200 205 Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Ala 210 215 220 Tyr Ser Thr Tyr Gly Asp Asn Gly Phe Gly Gly Gly Thr Glu Val Val 225 230 235 240 Val Lys Ala <210> 7 <211> 732 <212> DNA <213> Artifical Sequence <400> 7 cagctggtgg agtccggagg aggcctggta acgcctggga cacccctgac actcacctgc 60 120. acagcctctg gattctccct cagtagctac tggatgagct gggtccgcca ggctccaggg aaggggctgg aatggatcgg ggtcattagt agtaggta gcacggacta cgcgagctgg 180 gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgaa aatcaccagt ctgacaaccg aggacacggc catctatttc tgtgccagat atagtagtga ctggaatgcc tatgatttgt ggggccaagg caccctggtc actatctcct caggaggtgg aggttctgga 360 ggaggtggta gtggtggtgg aggatctgtg ctgacccaga ctccatcccc cgtgtctgcg 420 gctgttggag gcacagtcac catcagttgc caggccagtc aggtgttta tggtaacaac 540. tatttatcct ggtttcaaca gaaaccaggg cagcctccca agctcctgat ctaccaggca tccactctgg catctggggt cccatcgcgg ttcagcggca gtggatctgg gacacagttc 600 tctctcacca tcagcggcgt gcagtgtgac gatgctgcca cttactactg tgcaggatgg 660 agtggtagtg gtactgatgc tgttgctttc ggcggaggga ccgagctgga gatcctaggc 720 cgatctggcc cg 732 <210> 8 <211> 732 <212> DNA <213> Artificial Sequence <400> 8 caggtgaagc tgcagcagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acatgcaccg tctcagggtt ttcattaacc agctatggtg tacactgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagta atatgggctg gtggaagcac aaactataat 180 tcagctctca aatccagact gaacatcagc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctccaaac tgatgacaca gccatgtact actgtgccag aaactggggc 300 agctactggt acttcgatgt ctggggccaa ggccacggtc accgtctcct cagtggaggc 360 ggttcaggcg gaggtggcgg aggtggctct ggcggtggcg gatcggacat tgagctcacc 420 cagtctccag caatcatgtc tgcatctcca ggggaaaagg tcaccatgac ctgcagggcc 480 agctcaagta taagttccag ttacttgcac tggtaccagc agaagtcagg cgcttccccc 540 aaacccttga ttcataggac atccaacctg gcttctggag tcccagctcg cttcagtggc 600 agtgggtctg ggacctctta ctctctcaca atcagcagcg tggaggctga agatgatgca 660 acttattact gccagcagtg gagtggttac ccattcacgt tcggtgctgg gaccaagctc 720 gagatcaaac gg 732 <210> 9 <211> 723 <212> DNA <213> Artifical Sequence <400> 9 cagctggtgg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 60 acagcctctg gattcgccct cagtggctac tacatgacct gggtccgcca ggctccaggg 120 aaggggctgg aatggatcgg gatcacacat cctagtggta gcacatacta cgcgagctgg 180 gtgaaaggcc gattcaccat ctccaaggcc tcgaccacgg tggatctgag aatcaccagt 240 ccgacaaccg aggacacggc cacctatttc tgtgccagag tatggactca ttatagtagt 300 ggtggtgggg acttgtgggg cccaggcacc ctggtcacca tctcttcagg aggtggaggt 360 tctggaggag gtggtagtgg tggtggagga tctgatctga cccagactcc atcctccgtg 420 tctgcagctg tgggaggcac agtcaccatc aattgccagt ccagtgagag tgtttataag 480 aacaacgact tatcctggta tcagcagaaa ccagggcagc ctcccaagct cctaatcagt 540 gctgcatcca ctctggcatc tggggtccca tcccggttca aaggcagtgg atctgggaca 600 cagttcactc tcaccatcag cggcgtgcag tgtgacgatg ctgccactta ctactgtcaa 660 cagggttata gtggtgataa tgttgtgaat actttcggcg gagggaccga ggtggtggtc aaa 723 <210> 10 <211> 732 <212> DNA <213> Artificial Sequence <400> 10 cagctgatgg agtccggggg aggcctggtc cagcctgggg catccctgac actcacctgc 120. acagcctctg gattctcctt cagtagcaac tactggatat gctgggtccg ccaggctcca gggaaggggc tggagtggat cggatgcatt tatactggta ctagtggtac cacttactac 240. gcgagctggg cgaaaggccg attcaccatc tccgaaacct cgtcgaccac ggtgactctg 300. caaatgacca gtctgacagc cgcggacacg gccacctatt tctgttcaaa gaatacttat ggttatgctt tggctgctcg gttggatctc tggggccagg gcaccctggt caccatctct 360 tcaggaggtg gaggttctgg aggaggtggt agtggtggtg gaggatctga tatgacccag 420 <h2 style=";text-align:left;direction:ltr">actccagcct ctgtggaggt agctgtggga ggcacagtca ccatcaagtg ccaggccagt 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagagcattg gtagtgactt agcctggtat cagcagaaac taggccagcc tcccaaactc 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgatctatt atgcatccac tctggaatct ggggccccag cgcggttcag cggcagtgga 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tctgggacag agtacactct caccatcacc gacgtgcagt gtgacgatgc tgccacttac 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tactgtgcag gcgattatag tagtggtaat gataatggtt tcggcggagg gaccgaggtg 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtggtcaaag cg 732<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 723<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagctgatgg agtccggagg aggcctggta acgcctggag gaaccctgac actcacctgc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acagcctctg gattctccct cagtaactac tggatgagct gggtccgcca ggctccaggg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaggggctgg aatggatcgg catcattagt aattatggta gtacatatta cgcgagctgg 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgag aatcaccagt 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccgacaatcg aggacacggc cacctatttc tgtgccagag aatttgctgg tgtctttaac 300<h2 style=";text-align:left;direction:ltr"> ttgtggggcc caggcaccct ggtcaccgtc tcctcaggag gtggaggttc tggaggaggt 360 ggtagtggtg gtggaggatc tgatatgacc cagactccag cctccgtgtc tgaacctgtg 420 ggaggcacag tcaccatcaa ctgccaggcc agtcagagtg tttataataa caaaaatta 480 gcctggtatc agcagaaacc ggggcagcct cccaaggtcc tgatctacaa ggcatccact 540 ctggcatctg gggtctcatc gcggttcaaa ggcagtggat ctgggacaga gttcactctc 600 accatcagcg acctggagtg tgccgatgct gccacttact actgtcaaca gggtcatgat 660 accaacgtcc aaaatctttt cggcggaggg accgaggtgg tcgtcaaagc ggccgcaggt 720 gcg 723 <210> 12 <211> 729 <212> DNA <213> Artificial Sequence <400> 12 cagctggtgg agtccggggg agacctggtc aagcctgggg catccctgac actcacctgc 60 acagcctctg gattcaccct cagtagctac tggatgtgct gggtccgcca ggctccaggg 120 aaggggctgg agtggatcgg atgcattgat actggtagta gtgatagcac ttactacgcg 180 aactgggcga aaggccgatt caccatctcc aaagcctcgt cgaccacggt gactctgcaa 240 atgaccagtg tgacacccgc ggacacggcc acctatttct gtgcgagaca tctccgatat 300 agtactaata ataatgattg ggacttgtgg ggcccaggca ccctggtcac cgtctcctca 360 ggaggtggag gttctggagg aggtggtagt ggtggtggag gatctgatct gacccagact 420 ccagcctccg tggaggcagc tgtgggagac acagtcacca tcaagtgcca ggccagtcag 480 agcatttaca actacttagc ctggtatcag cagaaaccag ggcagcctcc caaggtcctg 540 atctatgctg catccaatct ggcatctggg gtcccatcgc ggttcagcgg cagtggatct 600 gggacacagt tcactctcac catcagcggc gtgcagtgtg acgatgctgc cacttactac 660 tgtgcaggcg cttatagtac gtatggtgat aatggtttcg gcggagggac cgaggtggtg 720 gtcaaagcg 729

Claims

1. A Benzonase monoclonal antibody scFv, characterized in that The sequence thereof is any one of SEQ ID No. 1 to 6.

2. A Benzonase monoclonal antibody scFv encoding gene, characterized in that Its sequence is any one of SEQ ID No. 7 to 12.

3. A Benzonase detection kit, characterized in that: Contains the Benzonase monoclonal antibody scFv according to claim 1.

4. Benzonase detection kit according to claim 3, characterized in that: The antibody containing the sequence shown in SEQ ID No. 2 is used as a coating antibody, and the antibody containing the sequence shown in SEQ ID No. 1 which is labeled with biotin.

Images