Preparation and application of carrier for expressing triclocarban nano antibody in lettuce

Abstract

The invention discloses preparation and application of a vector for expressing a triclocarban nano-antibody in lettuce, the vector comprises a recombinant vector PBI121-EGFP-dTCC-9 of a dTCC-9-VHH triclocarban bivalent nano-antibody, a plant is used as an efficient platform for expressing recombinant protein, the triclocarban nano-antibody is expressed, and the expression amount of the triclocarban nano-antibody is increased. A protein immunoblotting method and a fluorescence microscope observation method are used for determining that the recombinant protein is successfully expressed in the lettuce, the time required for successful expression is short (3d), and an important foundation is laid for obtaining a large number of antibodies from the lettuce. According to the invention, plasmids containing a target protein are transferred into plant cells so as to obtain transient controllable expression of the gene in the plant cells. Compared with stable expression, homeopathic expression does not need a complex step of integrating an exogenous gene into a host chromosome, the required time is short, and an expected result can be obtained within 3-4 days.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and gene technology, and in particular to the preparation and application of a carrier for expressing triclocarban nano-antibody in lettuce. Background Art

[0002] Triclocarban is a white fine powder with poor water solubility and is easily soluble in organic solvents such as dimethyl sulfoxide, methanol, and ethanol. Triclocarban has antimicrobial activity against Gram-positive bacteria such as Staphylococcus and Streptococcus, as well as fungi. Therefore, it is used as a broad-spectrum antibacterial agent and is widely added to daily necessities such as laundry detergent, soap, and facial cleansers, as well as bactericides and disinfectants. It can also be used as an air freshener for sterilization and deodorization. After daily necessities containing triclocarban additives are used, triclocarban will be discharged into the environment through domestic wastewater. In the United States, Europe, and Asia, a fairly high content has been found in wastewater and sludge entering sewage treatment plants, and is continuously discharged into the environment. Studies have shown that triclocarban can be absorbed by fish in the water, and after metabolism, it remains in high levels in tissues with high fat content such as the liver and kidneys. Since triclocarban is easily absorbed by the human body, it is extremely harmful to humans. Expressing triclocarban nanoantibodies in plants may make plants resistant to and absorb triclocarban.

[0003] Immune response refers to the specific binding reaction between antigens and corresponding antibodies. Antibodies play an important role in immune response. Antibodies are mainly divided into three types: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. Genetically engineered antibodies are called "third-generation antibodies" and are obtained by combining cloning technology and recombinant antibody technology. Nano antibodies are more typical. The systems for expressing recombinant antibodies mainly include Escherichia coli expression system, yeast expression system, insect expression system, mammalian expression system and plant expression system.

[0004] Nanobodies are antibodies that naturally lack light chains in the peripheral blood of camels. Their heavy chain antibody variable region (VHH) is the smallest unit of known complete antigen-binding fragments, namely nanobodies (Nbs), which are genetically engineered antibodies. Nanobodies are superior to traditional antibodies in many aspects. The special structure of VHH single-domain antibodies based on alpaca heavy chain antibodies combines the advantages of traditional antibodies and almost overcomes the defects of traditional antibodies: long development cycle, low stability, harsh storage conditions, etc., and gradually becomes a new generation of biomedicine and clinical diagnostic reagents. Compared with conventional antibodies, the advantages of nanobodies are: nanobodies can recognize antigens through a single variable domain VHH, which is not recognized by traditional monoclonal antibodies; highly expressed in prokaryotic or eukaryotic systems; strong specificity and high affinity; weak immunogenicity to humans; easy to screen and purify, and relatively low production costs, making them more advantageous in disease diagnosis and treatment, and rapid detection of small molecule pollutants.

[0005] Various existing documents and patents show that plants can be used to produce recombinant proteins and antibodies, and this technology is cheap and fast. In 1989, Hiatt et al. successfully expressed functional antibodies in tobacco for the first time. Since then, different types of antibodies have been successfully expressed in plants. They have different purposes for expression in plants, such as regulating plant cell processes and producing biological drugs. Recombinant proteins such as antibodies, enzymes, and vaccines have been widely produced based on plant expression systems. At present, the commonly used technologies for expressing recombinant vectors in plants are Agrobacterium-mediated stable genetic transformation of plants and transient expression of Agrobacterium-infected plants. Plant transient expression technology is a technology that uses different technologies to transfer plasmids containing target proteins into plant cells when plants grow to a specific stage, establish an efficient and controllable expression system in plant cells, and obtain a short-term controllable expression of the target gene. Compared with stable expression, transient expression does not require the integration of exogenous genes into host plant chromosomes, and experimental results can be obtained in just a few days, which is shorter than the time required for stable expression. Compared with bacterial expression systems and animal expression systems, plant expression systems can correctly fold, process and modify the expressed proteins. The activity of the proteins produced is higher than that of bacterial expression systems. In addition, the cost of plant expression systems is very low and they are easy to express and produce on a large scale.

[0006] Therefore, in view of the deficiencies in the prior art, the present invention provides a preparation and application of a vector for expressing triclocarban nanobodies in lettuce. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation and application of a vector for expressing triclocarban nanobody in lettuce, so as to fill the gap in the current technology.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] Preparation of a vector for expressing triclocarban nanobody in lettuce, wherein the vector comprises a recombinant vector PBI121-EGFP-dTCC-9 of a dTCC-9-VHH triclocarban bivalent nanobody;

[0010] The preparation method of the carrier comprises the following steps:

[0011] S1. Codon optimization of the dTCC-9-VHH gene fragment was performed to adapt it to the lettuce expression system. The gene sequence was synthesized by Sangon Biotech Co., Ltd. The sequence of dTCC-9-VHH was obtained by immunizing camels and establishing a phage nanoantibody library, thereby screening the nanoantibody gene that is efficiently expressed in prokaryotes;

[0012] S2. Use the software SnapGene to design the required primers: dTCC-9F and dTCC-9R, and use the dTCC-9-VHH plasmid as a template to perform PCR to obtain the target fragment;

[0013] S3, PBI121-EGFP plasmid acquisition:

[0014] ① Take the original strain of PBI121-EGFP and spread it on LB solid medium with a concentration of 50 μg / mL kanamycin resistance, and culture it in an incubator;

[0015] ② Pick a single colony, inoculate it into LB liquid medium supplemented with 10 mg / mL kanamycin, and culture it in a shaker;

[0016] ③ The PBI121-EGFP plasmid was extracted using a plasmid extraction kit (Aidlab), and after measuring the concentration, it was stored in a refrigerator at -20 to -18°C for later use;

[0017] S4. Enzyme digestion and enzyme ligation: (1) Design an enzyme digestion system using restriction endonucleases HindIII and EcoRI;

[0018] (2) Agarose gel electrophoresis: Add DNA dye to the agarose gel and wait for it to solidify before use. Mix the above PCR amplification product with Loading Buffer and load it. Use DNA marker to indicate the band size. Perform electrophoresis at 120V and image it.

[0019] (3) Gel excision to recover target bands: Observe under ultraviolet light, cut the target band of the correct size, put the excised gel into a sterile centrifuge tube, and use a DNA gel recovery kit (Aidlab) to recover the target DNA: ① Weigh the mass of the excised gel block, add binding buffer at a mass to volume ratio of 1:1 (add 100 mL for every 100 mg), place the centrifuge tube in a metal bath and heat until the gel block is completely dissolved, and cool to room temperature; if the target fragment is less than 400 bp or greater than 10 kb, add one volume of isopropanol to the gel solution to improve the recovery efficiency;

[0020] ② Transfer the above gel solution to the purification column, place the purification column in the collection tube for centrifugation; in order to improve the recovery efficiency, pass the effluent after centrifugation through the purification column again;

[0021] ③ Add solution to the purification column (make sure to add ethanol before use), centrifuge, discard the effluent, repeat the operation once, add the purification column back to the collection tube, and perform the centrifugation step at the same speed;

[0022] ④ Place the purification column in a new centrifuge tube and heat it in a metal bath. At the same time, you can preheat the eluent in the metal bath;

[0023] ⑤ Add the eluent to the center of the purification column, place it at room temperature for 2 to 3 minutes, then centrifuge it, add the effluent back to the purification column, and repeat the elution; in order to improve the recovery efficiency;

[0024] ⑥ Take the recovered product for agarose gel electrophoresis to detect the concentration of the recovered target DNA for subsequent experiments;

[0025] (4) Linking the vector and the target fragment: Use homologous recombination to construct the PBI121-EGFP-dTCC-9 recombinant vector, and store the enzyme-linked product and the homologous recombination product in a refrigerator at -20 to -18°C for later use;

[0026] S5. Transformation of E. coli competent cells: Take out Top10 competent cells from -80℃ refrigerator;

[0027] S6. Identification of positive clones by colony PCR: Select several single colonies on the plate and mark them, prepare PCR reaction solution, add them to reaction tubes respectively, and pick up a small amount of bacteria with a sterilized pipette tip and place them in the corresponding PCR reaction tubes respectively, gently pipette several times to ensure that the bacteria are added to the reaction system, and then perform PCR reaction; after the PCR reaction is completed, perform agarose gel electrophoresis, pick up the colonies with the correct band size, clear and bright, culture them, extract the plasmid, send them for sequencing, and save the strains with correct sequencing results; (add 80% glycerol (1:1) to the bacterial solution and store it in a -80℃ refrigerator);

[0028] S7. Construction of plants transformed with nanoantibody genes:

[0029] (1) Freeze-thaw method for transformation of Agrobacterium: ① Thaw the competent cells of Agrobacterium stored at -80°C on ice, add the expression vector plasmid to the competent cells, mix gently, place the mixture in an ice bath, freeze it in liquid nitrogen, and quickly heat it in a water bath; add YEB medium without antibiotics and perform shaking culture;

[0030] ② Centrifuge and discard the supernatant, resuspend the remaining liquid, and culture the bacteria on solid YEB medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampicin;

[0031] ③ Detection of positive clones by PCR of bacterial solution;

[0032] (2) Vacuum infiltration method to express target protein: ① Pick a positive Agrobacterium single clone and place it in a test tube containing YEB liquid medium and kanamycin with a final concentration of 50ug / ml. After shaking culture, transfer it to YEB liquid medium at a ratio of 1:1000, incubate, and adjust OD600nm to 1.5;

[0033] ② Centrifuge to collect the cells, then resuspend them in infiltration buffer, adjust OD600nm to 0.6, and place them in the dark at 20-22℃ for 2-4h;

[0034] ③ Place the prepared Agrobacterium culture suspension in a container, cut off the first 10% of the lettuce, turn it upside down (core facing up) and gently rotate it in the bacterial suspension;

[0035] ④ Turn on the vacuum pump to evacuate the air, set the maximum pressure to -95kPa, maintain the pressure state for 30 to 60 seconds, then turn on the system to release the pressure, repeat this process 2 to 3 times, allow the permeate to penetrate into the space within the tissue, until it is clearly visible that the permeate has obvious diffusion in the lettuce tissue, then gently remove the lettuce tissue from the permeate and rinse it with distilled water, and the treated samples are cultured in the dark for 4 to 5 days;

[0036] S8. Identification of plants transfected with nanoantibody genes: (1) Observe plant leaves under a fluorescence microscope;

[0037] (2) Extraction of plant total protein: ① Grind the lettuce plant material into powder with liquid nitrogen, put it into a clean centrifuge tube, add an appropriate amount of NP40 lysis buffer (pre-cooled, added with PMSF and DTT, freshly prepared before use), repeatedly invert to dissolve, and incubate;

[0038] ② Centrifuge, the supernatant is the total plant protein, the protein concentration is measured, and it can be used for protein immunoblotting experiment (Western Blot experiment) or ELISA experiment;

[0039] (3) Western Blot experiment: ① After washing and drying the glass plate and the gel casting apparatus, assemble them; prepare 10% separation gel and add it to the middle of the assembled glass plate, then add distilled water to the surface of the separation gel, wait for it to solidify, pour out the upper layer of distilled water, and absorb the residual liquid with filter paper; prepare 5% concentrated gel and add it to the solidified separation gel, insert the comb vertically, and wait for it to solidify;

[0040] ② Add loading buffer to the protein sample, boil, and centrifuge; aspirate the loading solution, add it to the gel well, and start electrophoresis. Set the voltage of the concentrated gel to 90V and the voltage of the separation gel to 110V;

[0041] ③After the electrophoresis is completed, the protein gel is taken out, the upper concentrated gel is discarded, the lower separation gel is retained, the NC membrane is placed on the filter paper soaked in the transfer solution, the lower separation gel is covered on the membrane, and then the filter paper soaked in the transfer solution is covered on the separation gel. During this process, bubbles should be avoided; the protein separation gel faces the negative electrode, the NC membrane faces the positive electrode, and the electrophoresis is performed at a constant voltage of 130V for 120 minutes. During this process, the transfer tank needs to be ice-bathed;

[0042] ④ After the electrophoresis, take out the NC membrane, put it in 5% skim milk, block it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; add the diluted primary antibody, keep it at 4-5℃ overnight, take it out the next day and rewarm it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; add the diluted secondary antibody, keep it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; use a chemiluminescence instrument to detect and image, and use ImageJ software to analyze the band intensity.

[0043] Preferably, in step S3-①, the culture condition in the incubator is 37° C. constant temperature culture for 12 to 24 hours; in step S3-②, the culture condition in the shaker is 37° C., 220 rpm culture for 12 to 24 hours.

[0044] Preferably, in step S4-(3)-①, the centrifuge tube is heated in a metal bath at 65-68°C for 5-8 min; the centrifugal condition in step S4-(3)-② is centrifuged at 10000-12000 rpm for 1-2 min; the centrifugal condition in step S4-(3)-③ is centrifuged at 10000-12000 rpm for 1-2 min; and the centrifugal condition in step S4-(3)-④ is heated in a metal bath at 65-68°C for 5-8 min.

[0045] Preferably, in step S7-(1)-①, the mixture is placed in an ice bath for 5 to 6 minutes, frozen in liquid nitrogen for 5 to 6 minutes, and quickly placed in a water bath at 37°C for 5 to 6 minutes. After the culture medium is added, the mixture is shaken and cultured at 28°C and 200 rpm for 4 to 6 hours. In step S7-(1)-②, the conditions for centrifugation and discarding the supernatant are centrifugation at 4000 to 4200 rpm for 5 to 6 minutes, and the culture medium is cultured at 28°C for 2 to 4 days.

[0046] Preferably, in step S7-(2)-①, the shaking culture conditions are shaking culture at 28°C and 200 r / min for 36 to 40 h, and the incubation conditions are incubation at 28°C for 18 to 24 h. In step S7-(2)-②, the centrifugal collection conditions are centrifugation at 4000 to 4200 rpm for 10 to 12 min to collect the bacteria.

[0047] Preferably, in step S8-(2)-①, the incubation condition is 4-5°C for 15-30 min; in step S8-(2)-②, the centrifugation condition is 13000-14000 rpm and 4-5°C for 15-18 min.

[0048] Preferably, in step S8-(3)-②, boiling is performed for 10 to 12 minutes; and the centrifugation condition is 5000 to 5200 rpm for 3 to 4 minutes.

[0049] The plant transient expression technology of the present invention refers to a technology that uses a specific technical method to transfer a plasmid containing a target protein into plant cells when the plant has grown to a certain stage, so as to obtain a short-term controllable expression of the gene in the plant cells; compared with stable expression, homeostatic expression does not require the complex step of integrating exogenous genes into host chromosomes, and the required time is shorter, and the desired results can be obtained in 3-4 days; the present invention provides the use of plants as hosts in expressing nano antibodies, using lettuce as an efficient platform for expressing recombinant proteins, expressing triclocarban nano antibodies, and using protein immunoblotting and fluorescence microscopy to determine that the recombinant protein is successfully expressed in the lettuce; in addition, the present invention is also applicable to other plants other than lettuce, such as tobacco or Arabidopsis; the plant organs of the plant are selected from leaves or rhizomes.

[0050] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0051] The present invention utilizes a plant expression system to express nano antibodies with higher sensitivity, which is named plant transient expression nano antibody technology. The present invention uses lettuce as an expression platform for producing recombinant proteins, and adopts a simple and effective Agrobacterium-mediated vacuum infiltration method to express nano antibodies in lettuce. This expression method can detect exogenous recombinant proteins in lettuce leaves 3 days after Agrobacterium infects lettuce. The present invention has successfully expressed triclocarban nano antibodies in lettuce, and transgenic lettuce expressing recombinant proteins has great potential for producing nano antibodies and absorbing pollutants in water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0053] Figure 1 This is a schematic diagram of the recombinant vector of Example 1 of the present invention;

[0054] Figure 2 This is a fluorescence image of transient expression of lettuce in Example 1 of the present invention;

[0055] Figure 3 This is a Western Blot immunoblot of the lettuce fusion protein in Example 1 of the present invention. DETAILED DESCRIPTION

[0056] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0057] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0058] Example 1

[0059] See attached Figure 1-3 This embodiment provides a method for preparing a vector for expressing triclocarban nanobody in lettuce, wherein the vector comprises a recombinant vector PBI121-EGFP-dTCC-9 of a dTCC-9-VHH triclocarban bivalent nanobody, and a schematic diagram of the recombinant vector is shown in the attached figure. Figure 1 As shown, in the specific operation process, the triclocarban bivalent nanobody codon is optimized to be a codon preferred by lettuce, and the preparation method of the vector comprises the following steps:

[0060] S1. Codon optimization of the dTCC-9-VHH gene fragment was carried out to facilitate its better adaptation to the lettuce expression system. The gene sequence was synthesized by Sangon Biotech Co., Ltd. The sequence of dTCC-9-VHH was established by my research group through immunizing camels in the early stage, and the nanoantibody gene that was efficiently expressed in prokaryotes was screened;

[0061] S2. Use SnapGene software to design the required primers: dTCC-9F, dTCC-9R,

[0062] PCR was performed using the dTCC-9-VHH plasmid as a template to obtain the target fragment. The reaction system is shown in Table 1;

[0063] Table 1

[0064]

[0065] PCR amplification procedure:

[0066]

[0067] S3, PBI121-EGFP plasmid acquisition: ① Using the original strain of PBI121-EGFP, take 5 μL and spread it on the kanamycin-resistant LB solid medium with a final concentration of 50 μg / mL, and place it in a 37°C constant temperature incubator for overnight culture;

[0068] ② Pick a single colony from the plate, inoculate it into 5 mL LB liquid medium supplemented with 5 μL of 10 mg / mL kanamycin, and culture it in a shaker at 37°C and 220 rpm overnight;

[0069] ③ The PBI121-EGFP plasmid was extracted using a plasmid extraction kit (Aidlab), and after measuring the concentration, it was stored in a -20°C refrigerator for later use;

[0070] S4. Enzyme digestion and enzyme ligation: (1) Design an enzyme digestion system (50 μL) using restriction endonucleases HindIII and EcoRI. The plasmid digestion system is shown in Table 2; the 3dTCC-9 fragment digestion system is shown in Table 3;

[0071] Table 2

[0072] Components volume Plasmids 4μL HindIII 1μL EcoRI 1μL 10×buffer 5μL <![CDATA[ddH2O]]> 39μL

[0073] Table 3

[0074] Components volume dTCC-9 fragment 13μL HindIII 1μL EcoRI 1μL 10×buffer 5μL <![CDATA[ddH2O]]> 30μL

[0075] (2) Agarose gel electrophoresis: Add DNA dye to 0.8% (w / v) agarose gel and wait for it to solidify before use. Mix the above PCR amplification product with Loading Buffer and load it. Use DNA marker to indicate the band size. Perform electrophoresis at 120V for 30 minutes and then image it.

[0076] (3) Gel excision to recover target bands: Observe under ultraviolet light, cut the target bands of the correct size with a knife, put the excised gel into a sterile 1.5 mL centrifuge tube, and use a DNA gel recovery kit (Aidlab) to recover the target DNA: ① Weigh the mass of the excised gel block, add binding buffer at a mass to volume ratio of 1:1 (add 100 mL for every 100 mg), place the centrifuge tube in a 65°C metal bath and heat for 5 min until the gel block is completely dissolved, and cool to room temperature; if the target fragment is less than 400 bp or greater than 10 kb, add one volume of isopropanol to the gel solution to improve the recovery efficiency;

[0077] ② Transfer the above gel solution to the purification column, place the purification column in the collection tube, and centrifuge at 12000rpm for 1min. In order to improve the recovery efficiency, pass the effluent after centrifugation through the purification column again;

[0078] ③ Add 600 mL of solution (confirm that ethanol has been added before use) to the purification column, centrifuge at 12000 rpm for 1 min, discard the effluent, repeat the operation once, add the purification column back to the collection tube, and centrifuge at the same speed;

[0079] ④ Place the purification column in a new 2 mL centrifuge tube and heat it in a 65°C metal bath for 5 min. At the same time, the eluent can be preheated in a 65°C metal bath;

[0080] ⑤ Add 40 mL of eluent to the center of the purification column, place it at room temperature for 2 minutes, then centrifuge it, add the effluent back to the purification column, and repeat the elution to improve the recovery efficiency;

[0081] ⑥ Take 3 mL of the recovered product for agarose gel electrophoresis to detect the concentration of the recovered target DNA for subsequent experiments;

[0082] (4) Linking the vector and the target fragment: Use homologous recombination to construct the PBI121-EGFP-dTCC-9 recombinant vector, and store the enzyme-linked product and the homologous recombination product in a -20°C refrigerator for later use; the homologous recombination system is shown in Table 4;

[0083] Table 4

[0084] Components volume Plasmids 10.5μL dTCC-9 fragment 0.5μL 2 × ClonExpressMix 5μL ddH2O 4μL

[0085] S5. Transformation of E. coli competent cells: ① Take out Top10 competent cells from -80℃ refrigerator;

[0086] S6. Identification of positive clones by colony PCR: Select several single colonies on the plate and mark them, prepare PCR reaction solution, add them to reaction tubes respectively, and use a sterilized pipette to pick up a small amount of bacteria and place them in the corresponding PCR reaction tubes respectively. Pipet gently several times to ensure that the bacteria are added to the reaction system, and then perform PCR reaction. The TaqPCR reaction system (25 μL) is shown in Table 5;

[0087] Table 5

[0088] Components volume Primer f 0.5μL Primer 0.5μL DNA 1μL MixTakara 12.5μL <![CDATA[ddH2O]]> 10.5μL

[0089] PCR amplification procedure:

[0090]

[0091] After the PCR reaction is completed, perform agarose gel electrophoresis, pick the colonies with the correct band size, clear and bright, culture them, extract the plasmid, send them to the company for sequencing, and preserve the strains with correct sequencing results (add 80% glycerol (1:1) to the bacterial solution and store it in a -80℃ refrigerator);

[0092] S7. Construction of plants transformed with nanoantibody genes:

[0093] (1) Freeze-thaw method for transformation of Agrobacterium: ① Thaw the competent Agrobacterium stored at -80°C on ice, add 2 μg of expression vector plasmid to 100 μL competent culture, mix gently, place the mixture on ice for 5 min, freeze in liquid nitrogen for 5 min, and quickly place in a water bath at 37°C for 5 min; add 900 μL of YEB medium without antibiotics, and culture at 28°C, 200 rpm, with shaking for 4 h;

[0094] ② Centrifuge at 4000 rpm for 5 min, discard the supernatant, keep 100 μL of liquid for re-suspending, and spread the bacteria on solid YEB medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampicin, and culture at 28°C for 3 days;

[0095] ③ Detection of positive clones by PCR of bacterial solution;

[0096] (2) Vacuum infiltration method to express target protein: ① Pick a positive Agrobacterium single clone and place it in a test tube containing 5 mL YEB liquid medium and a final concentration of 50 μg / ml kanamycin. After shaking culture at 28°C and 200 rpm for 36 h, transfer it to 300 mL YEB liquid medium (added with a final concentration of 20 μmol / L acetosyringone, 10 mmol / L MES pH 5.6 and 50 μg / ml kanamycin) at a ratio of 1:1000, incubate at 220 rpm, 28°C for 18-24 h, and adjust the OD600 nm to 1.5.

[0097] ② Centrifuge at 4000 rpm for 10 min to collect the cells, then resuspend them in infiltration buffer (10 mmol / LMgCl2, 10 mmol / LMES, 100 umol / L acetosyringone), adjust the OD600nm to 0.6, and place them at 22℃ in the dark for 2 h;

[0098] ③ Place the prepared Agrobacterium culture suspension in a 2L beaker, cut off the first 10% of the lettuce with a knife, turn it upside down (core facing up) and gently rotate it in the bacterial suspension;

[0099] ④ Turn on the vacuum pump to evacuate the air, set the maximum pressure to -95 kPa, maintain the pressure state for 60 seconds, and then turn on the system to release the pressure. Repeat this process twice to allow the permeate to penetrate into the space within the tissue until it is clearly visible that the permeate has obvious diffusion in the lettuce tissue. Then, gently remove the lettuce tissue from the permeate and rinse it with distilled water. The treated samples are cultured in the dark for 4 days.

[0100] S8. Identification of plants transfected with nanoantibody genes: (1) Observe plant leaves under a fluorescence microscope;

[0101] (2) Extraction of total plant protein: ① Grind the lettuce plant material into powder with liquid nitrogen, put it into a clean 1.5 mL centrifuge tube, add an appropriate amount of NP40 lysis buffer (pre-cooled, added with PMSF and DTT, prepared immediately before use), repeatedly invert to dissolve, and incubate at 4°C for 25 min;

[0102] ② Centrifuge at 13000rpm and 4℃ for 15min. The supernatant is the total plant protein. The protein concentration can be used for Western Blot or ELISA experiments.

[0103] (3) Western Blot experiment: ① After washing and drying the glass plate and the gel casting apparatus, assemble them; prepare 7 mL of 10% separation gel and add it to the middle of the assembled glass plate, then add distilled water to the surface of the separation gel, wait for it to solidify, pour out the upper layer of distilled water, and absorb the residual liquid with filter paper; prepare 3 mL of 5% concentrated gel and add it to the solidified separation gel, insert the comb vertically, and wait for it to solidify;

[0104] ② Add loading buffer to the protein sample, boil for 10 minutes, centrifuge at 5000 rpm for 3 minutes; draw 10 μL of loading solution, add it to the gel well, start electrophoresis, set the voltage of the concentrated gel to 90 V, and the voltage of the separation gel to 110 V;

[0105] ③After the electrophoresis is completed, the protein gel is taken out, the upper concentrated gel is discarded, the lower separation gel is retained, the NC membrane is placed on the filter paper soaked in the transfer solution, the lower separation gel is covered on the membrane, and then the filter paper soaked in the transfer solution is covered on the separation gel. During this process, bubbles should be avoided; the protein separation gel faces the negative electrode, the NC membrane faces the positive electrode, and the electrophoresis is performed at a constant voltage of 130V for 120 minutes. During this process, the transfer tank needs to be ice-bathed;

[0106] ④After the electrophoresis, take out the NC membrane, put it in 5% skim milk, block it at room temperature for 2 hours, and wash the NC membrane with PBST three times; add the diluted primary antibody, keep it at 4℃ overnight, take it out the next day and rewarm it at room temperature for 1 hour, wash the NC membrane with PBST three times; add the diluted secondary antibody, keep it at room temperature for 1 hour, and wash the NC membrane with PBST three times; use a chemiluminescence instrument to detect and image, and use ImageJ software to analyze the band intensity;

[0107] The experimental results obtained through the above steps are as follows: After the lettuce leaves were transiently transformed and cultured in the dark for three days, the fluorescence signal was observed in the lettuce leaves, and the distribution of the fluorescence signal in the leaves could be seen. For comparison, this experiment also set up empty lettuce plants and ordinary lettuce plants as control groups; it was observed that the GFP fluorescence signal existed in the cell nucleus and cell membrane, as shown in the attached Figure 2 As shown; after immunoblotting experiments, the results confirmed that the recombinant protein was successfully expressed in lettuce leaves. The size of the fusion protein dTCC-9 was 57.8 kda, and the size of TCC-TCS was 58.4 kda. The corresponding size protein bands can be seen as shown in the attached Figure 3 shown.

[0108] In summary, the present invention utilizes a plant expression system to express nanobodies with higher sensitivity, which is named plant transient expression of nanobodies technology. The present invention uses lettuce as an expression platform for producing recombinant proteins, and adopts a simple and effective Agrobacterium-mediated vacuum infiltration method to express nanobodies in lettuce. This expression method can detect exogenous recombinant proteins in lettuce leaves 3 days after Agrobacterium infects lettuce. The present invention has successfully expressed triclocarban nanobodies in lettuce, and transgenic lettuce expressing recombinant proteins has great potential for producing nanobodies and absorbing pollutants in water bodies.

[0109] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. Preparation of a vector for expressing triclocarban nanobody in lettuce, characterized in that: The vector comprises the recombinant vector PBI121-EGFP-dTCC-9 of the dTCC-9-VHH triclocarban bivalent nanobody; The preparation method of the carrier comprises the following steps: S1. Codon optimization of the dTCC-9-VHH gene fragment was performed to adapt it to the lettuce expression system. The gene sequence was synthesized by Sangon Biotech Co., Ltd. The sequence of dTCC-9-VHH was obtained by immunizing camels and establishing a phage nanoantibody library, thereby screening the nanoantibody gene that is efficiently expressed in prokaryotes; S2. Use the software SnapGene to design the required primers: dTCC-9F and dTCC-9R, and use the dTCC-9-VHH plasmid as a template to perform PCR to obtain the target fragment; S3, PBI121-EGFP plasmid acquisition: ① Take the original strain of PBI121-EGFP and spread it on LB solid medium with a concentration of 50 μg / mL kanamycin resistance, and culture it in an incubator; ② Pick a single colony, inoculate it into LB liquid medium supplemented with 10 mg / mL kanamycin, and culture it in a shaker; ③ Use a plasmid extraction kit to extract the PBI121-EGFP plasmid, measure the concentration and store it in a -20 to -18℃ refrigerator for later use; S4. Enzyme digestion and enzyme ligation: (1) Design an enzyme digestion system using restriction endonucleases HindIII and EcoRI; (2) Agarose gel electrophoresis: Add DNA dye to the agarose gel and wait for it to solidify before use. Mix the above PCR amplification product with Loading Buffer and load it. Use DNA marker to indicate the band size. Perform electrophoresis at 120V and image it. (3) Gel excision to recover target bands: Observe under ultraviolet light, cut the target band of the correct size, put the excised gel into a sterile centrifuge tube, and use a DNA gel recovery kit to recover the target DNA: ① Weigh the mass of the excised gel block, add binding buffer at a mass to volume ratio of 1:1, place the centrifuge tube in a metal bath and heat until the gel block is completely dissolved, and cool to room temperature; ② Transfer the above gel solution to the purification column, place the purification column in the collection tube for centrifugation; in order to improve the recovery efficiency, pass the effluent after centrifugation through the purification column again; ③ Add the solution to the purification column, centrifuge, discard the effluent, repeat the operation once, add the purification column back to the collection tube, and perform the centrifugation step at the same speed; ④ Place the purification column in a new centrifuge tube and heat it in a metal bath. At the same time, you can preheat the eluent in the metal bath; ⑤ Add the eluent to the center of the purification column, place it at room temperature for 2 to 3 minutes, then centrifuge it, add the effluent back to the purification column, and repeat the elution; ⑥ Take the recovered product for agarose gel electrophoresis to detect the concentration of the recovered target DNA for subsequent experiments; (4) Linking the vector and the target fragment: Use homologous recombination to construct the PBI121-EGFP-dTCC-9 recombinant vector, and store the enzyme-linked product and the homologous recombination product in a refrigerator at -20 to -18°C for later use; S5. Transformation of E. coli competent cells: Take out Top10 competent cells from -80℃ refrigerator; S6. Identification of positive clones by colony PCR: Select several single colonies on the plate and mark them, prepare PCR reaction solution, add them to reaction tubes respectively, and use a sterilized pipette to pick up a small amount of bacteria and place them in the corresponding PCR reaction tubes respectively. Gently pipette several times to ensure that the bacteria are added to the reaction system, and then perform PCR reaction; after the PCR reaction is completed, perform agarose gel electrophoresis, select colonies with the correct band size, clear and bright, culture them, extract plasmids, send them for sequencing, and save the strains with correct sequencing results; S7. Construction of plants transformed with nanoantibody genes: (1) Freeze-thaw method for transformation of Agrobacterium: ① Thaw the competent cells of Agrobacterium stored at -80°C on ice, add the expression vector plasmid to the competent cells, mix gently, place the mixture in an ice bath, freeze it in liquid nitrogen, and quickly heat it in a water bath; add YEB medium without antibiotics and perform shaking culture; ② Centrifuge and discard the supernatant, resuspend the remaining liquid, and culture the bacteria on solid YEB medium supplemented with 50 mg / L kanamycin and 100 mg / L rifampicin; ③ Detection of positive clones by PCR of bacterial solution; (2) Vacuum infiltration method to express target protein: ① Pick a positive Agrobacterium single clone and place it in a test tube containing YEB liquid medium and kanamycin with a final concentration of 50ug / ml. After shaking culture, transfer it to YEB liquid medium at a ratio of 1:1000, incubate, and adjust OD600nm to 1.5; ② Centrifuge to collect the cells, then resuspend them in infiltration buffer, adjust OD600nm to 0.6, and place them in the dark at 20-22℃ for 2-4h; ③ Place the prepared Agrobacterium culture suspension in a container, cut off the first 10% of the lettuce, turn it upside down and gently rotate it in the bacterial suspension; ④ Turn on the vacuum pump to evacuate the air, set the maximum pressure to -95kPa, maintain the pressure state for 30 to 60 seconds, then turn on the system to release the pressure, repeat this process 2 to 3 times, allow the permeate to penetrate into the space within the tissue, until it is clearly visible that the permeate has obvious diffusion in the lettuce tissue, then gently remove the lettuce tissue from the permeate and rinse it with distilled water, and the treated samples are cultured in the dark for 4 to 5 days; S8. Identification of plants transfected with nanoantibody genes: (1) Observe plant leaves under a fluorescence microscope; (2) Extraction of total plant protein: ① Grind the lettuce plant material into powder with liquid nitrogen, put it into a clean centrifuge tube, add an appropriate amount of NP40 lysis buffer, repeatedly invert to dissolve it, and incubate; ② Centrifuge, the supernatant is the total plant protein, the protein concentration is measured, and it can be used for protein immunoblotting or ELISA experiments; (3) Protein immunoblotting experiment: ① Wash and dry the glass plate and gel casting apparatus, and assemble them; prepare 10% separation gel and add it to the middle of the assembled glass plate, then add distilled water to the surface of the separation gel, wait for it to solidify, pour out the upper layer of distilled water, and absorb the residual liquid with filter paper; prepare 5% concentrated gel and add it to the solidified separation gel, insert the comb vertically, and wait for it to solidify; ② Add loading buffer to the protein sample, boil, and centrifuge; aspirate the loading solution, add it to the gel well, and start electrophoresis. Set the voltage of the concentrated gel to 90V and the voltage of the separation gel to 110V; ③After the electrophoresis is completed, the protein gel is taken out, the upper concentrated gel is discarded, the lower separation gel is retained, the NC membrane is placed on the filter paper soaked in the transfer solution, the lower separation gel is covered on the membrane, and then the filter paper soaked in the transfer solution is covered on the separation gel. During this process, bubbles should be avoided; the protein separation gel faces the negative electrode, the NC membrane faces the positive electrode, and the electrophoresis is performed at a constant voltage of 130V for 120 minutes. During this process, the transfer tank needs to be ice-bathed; ④ After the electrophoresis, take out the NC membrane, put it in 5% skim milk, block it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; add the diluted primary antibody, keep it at 4-5℃ overnight, take it out the next day and rewarm it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; add the diluted secondary antibody, keep it at room temperature for 1-2 hours, and wash the NC membrane with PBST for 3-4 times; use a chemiluminescence instrument to detect and image, and use ImageJ software to analyze the band intensity.

2. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In step S3-①, the culture condition in the incubator is 37° C. constant temperature culture for 12 to 24 hours; in step S3-②, the culture condition in the shaker is 37° C., 220 rpm culture for 12 to 24 hours.

3. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In step S4-(3)-①, the centrifuge tube is heated in a metal bath at 65-68°C for 5-8 min; the centrifugal condition in step S4-(3)-② is centrifuged at 10000-12000 rpm for 1-2 min; the centrifugal condition in step S4-(3)-③ is centrifuged at 10000-12000 rpm for 1-2 min; and in step S4-(3)-④, the centrifuge tube is heated in a metal bath at 65-68°C for 5-8 min.

4. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In step S7-(1)-①, the mixture is placed in an ice bath for 5 to 6 minutes, frozen in liquid nitrogen for 5 to 6 minutes, and quickly placed in a water bath at 37°C for 5 to 6 minutes. After the culture medium is added, the mixture is shaken and cultured at 28°C and 200 rpm for 4 to 6 hours. In step S7-(1)-②, the conditions for centrifugation and discarding the supernatant are centrifugation at 4000 to 4200 rpm for 5 to 6 minutes, and the culture medium is cultured at 28°C for 2 to 4 days.

5. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In step S7-(2)-①, the shaking culture conditions are 28°C 200r / min shaking culture for 36 to 40h, and the incubation conditions are 28°C incubation for 18 to 24h. In step S7-(2)-②, the centrifugal collection conditions are 4000 to 4200rpm centrifugation for 10 to 12min to collect the bacteria.

6. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In step S8-(2)-①, the incubation condition is 4-5°C for 15-30 min; in step S8-(2)-②, the centrifugation condition is 13000-14000 rpm and 4-5°C for 15-18 min.

7. The preparation of a vector for expressing triclocarban nanobody in lettuce according to claim 1, characterized in that: In the step S8-(3)-②, the mixture is boiled for 10 to 12 minutes and centrifuged at 5000 to 5200 rpm for 3 to 4 minutes.

8. A vector obtained by preparing the vector for expressing triclocarban nanobody in lettuce as claimed in any one of claims 1 to 7.

9. A vector prepared by the vector for expressing triclocarban nanobody in lettuce as claimed in any one of claims 1 to 7 or use of the vector as claimed in claim 8.

Images