Preparation and evaluation method of scolospora toxin gene engineering antibody

Abstract

The preparation and evaluation method comprises the following steps: 1, extracting a heavy-chain DNA fragment and a light-chain DNA fragment, carrying out recombinant transformation by using an overlapping PCR method to construct an scFv gene, 2, constructing an expression vector, culturing the expression vector, carrying out sequencing identification to obtain an scFv bacterial solution, recombining the scFv bacterial solution with a mouse Fc fragment, and identifying a sequence to obtain an MN8 bacterial solution, the method comprises the following steps: 1, preparing an MN8 bacterial liquid, 2, extracting the MN8 bacterial liquid, 3, carrying out inoculated culture on the MN8 bacterial liquid to obtain an MN8 genetically engineered antibody bacterial liquid, 4, extracting MN8 genetically engineered antibody bacterial liquid plasmids and carrying out transfection expression purification, and 5, carrying out antibody titer and thermal stability determination on the MN8 genetically engineered antibody. The scFv single-chain antibody obtained by recombination according to the method has the advantages of small molecular weight, high penetrability and the like, the stability and the sensitivity of an immunoassay method of the scolosporins can be improved, and a certain basis is provided for rapid determination of the scolosporins.

Description

Technical Field

[0001] This invention relates to the field of biological research technology, and in particular to a method for preparing and evaluating a genetically engineered antibody against *Cyclophorus* toxin. Background Technology

[0002] Cylindrospermopsin (CYN) is a type of cyanobacterial toxin that has attracted widespread attention after microcystin. It poses potential hazards to humans, animals, and plants. Antibody-based immunoassay is one of the commonly used techniques for risk assessment of cyanobacterial toxins. The foundation of immunoassays lies in the successful preparation of monoclonal or polyclonal antibodies. Antibody preparation for microcystins began in the 1980s, and to date, numerous studies have been conducted on the preparation of antibodies against different isoforms and epitopes of microcystins, as well as the development of various immunoassay methods. In comparison, reports on antibody preparation for CYN are extremely rare. Research and application of genetically engineered antibodies in the determination of cyanobacterial toxins mainly focus on antibody modification of microcystins. Compared with the monitoring methods for microcystins, the monitoring methods for Cyclopsacylus toxins are still extremely limited. Therefore, this invention proposes a method for the preparation and evaluation of genetically engineered antibodies against Cyclopsacylus toxins to overcome the problems existing in the prior art. Summary of the Invention

[0003] To address the aforementioned problems, the present invention aims to provide a method for preparing and evaluating a genetically engineered antibody against *Cyclopyralidus*. This method aims to improve the detection sensitivity of the direct competitive ELISA method and provide a foundation for the rapid and accurate determination of *Cyclopyralidus*.

[0004] To achieve the objectives of this invention, the invention is implemented through the following technical solution: a method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.*, comprising the following steps: Step 1: cDNA of the CYN-M010 hybridoma cell line that produces N8 monoclonal antibody was obtained by RT-PCR, and the heavy chain DNA fragment and light chain DNA fragment of the variable region gene were extracted by degenerate primers. The fragments were then recombined and transformed by overlapping PCR to construct the scFv gene. Step 2: The scFv gene and mouse Fc were ligated and recombined into the enzyme-digested linear peaSKY203 vector using overlapping PCR to obtain the expression vector. The expression vector was transformed into competent E. coli and plated. Single colonies were picked and sequenced to identify the MN8 bacterial culture with correct sequencing. Step 3: Inoculate and culture the correctly sequenced MN8 bacterial culture to obtain MN8 genetically engineered antibody bacterial culture, extract plasmids and determine their concentration, and identify them using ELISA. Step 4: Inoculate and culture the MN8 genetically engineered antibody bacterial culture, extract plasmids for transfection and expression, purify using a proteinG purification column, and finally identify using a direct competitive ELISA method. Step 5: Determine the antibody titer and thermal stability of the MN8 genetically engineered antibody.

[0005] A further improvement is made in that step one specifically includes: S1. RNA was extracted from the preserved cell lines after reviving using the trizol method, and the RNA was stored at -80℃. S2. Using the RT-PCR method, the extracted RNA was processed using a kit to obtain the N8 cDNA fragment. S3. Using degenerate primers, PCR amplification was performed with cDNA as a template. After amplification, the reaction products were identified by 1.5% agarose gel electrophoresis and the DNA gel was extracted using a DNA gel extraction kit. S4. Take 1 part of the recovered heavy chain DNA fragment and 1 part of the recovered light chain DNA fragment. Recombination and ligation were performed separately with the T vector, and the ligation products were converted to 10... To Escherichia coli DH5 The competent cells were plated and incubated overnight at 37°C. S5. Select single colonies of recombinant bacteria for sequencing. Analyze the results using SnapGene software. Add the bacterial solution with the correct sequence to 50% glycerol at a 1:1 ratio and store at -80℃ for later use. S6. Using bacterial culture with the correct sequence, extract heavy chain DNA fragments and light chain DNA fragment plasmids, then use overlap PCR to synthesize scFv fragments via Linker-(GGGGS)n, perform sequence identification as above, and preserve them to obtain the scFv gene.

[0006] A further improvement is made in the following: the transformed competent cells in S4 are subjected to an ice bath for 15 minutes, followed by heat shock at 42°C for 60 seconds, placed on ice for 2 minutes, and then 500 cells are taken from each cell. Coated on Incubate on plate.

[0007] A further improvement is that the primers used in the overlap PCR in step two are 203MN8SCLF, MN8SCHR, 2AFCF and 2AFCR, and the primer sequences are shown in SEQ ID No. 1 to SEQ ID No. 4, respectively.

[0008] A further improvement is made in step three, which involves adding 6 ml of sterile 2YT culture medium to a sterile test tube and adding 1:1000 ampicillin. Then, the correctly sequenced MN8 bacterial culture is inoculated into a glass shaker tube with an inoculation volume of 1 ml. The plasmids were cultured overnight on a shaker, and then extracted using a small-scale plasmid extraction kit to determine their concentration.

[0009] A further improvement is made in step three, where the ELISA method is used for identification. Specifically, 293F cells are pre-cultured in 12-well plates and transfected the following day; 7 cells are then collected. plasmid and 75 Mix the cell culture medium and let it stand for 3 minutes. Then add 4.5... Transfection reagent PEI and 75 Mix the cell culture medium and let it stand for 3 minutes. Finally, mix the two mediums together, shake well, and let it stand for 15-20 minutes. Then, suspend the medium and add it evenly to a 12-well plate and incubate for 48 hours. Collect the cell supernatant and perform ELISA identification.

[0010] A further improvement is made in step four, where the MN8 genetically engineered antibody bacterial culture is inoculated by adding 400ml of sterilized 2YT culture medium to a 1000ml Erlenmeyer flask, adding 1:1000 ampicillin in a clean bench, and inoculating for 10... The bacterial culture was preserved and cultured overnight at 37°C with shaking. The precipitate was collected by centrifugation the next day, and plasmids were extracted and endotoxins removed using a large-scale plasmid extraction kit. The concentration of the obtained plasmids was measured, and then transfection expression was performed.

[0011] A further improvement is made in that: the transfection expression specifically involves pre-resuscitating and culturing 293F cells in a 250ml cell culture flask until their density reaches 1.2-2.0× Transfection can be performed at a rate of cells / ml. Add plasmid to 300 ml. Mix with serum-free cell culture medium in a test tube and let stand for 3 minutes. In another test tube, add 900 ml of serum-free cell culture medium. Mix the transfection reagent PEI with serum-free culture medium and let stand for 3 minutes. Then mix the solutions in the two test tubes and let stand for 15-20 minutes before adding them to the cell culture flask to complete the transfection.

[0012] A further improvement is made in step four: before purification, the cultured cell solution is centrifuged at 6000 rpm for 10 minutes to remove the precipitate and retain the supernatant, which is then used for purification at 0.22... The solution is filtered through a membrane before purification; during purification, all bacteria in the solution pass through a 0.45 μL filter. Samples are injected after membrane filtration.

[0013] A further improvement is made in step five, where the antibody titer determination is performed using an indirect competitive ELISA. The HRP-CYN concentration is 1:5000, and the MN8 genetically engineered antibody is diluted sequentially in 10-fold increments using a microplate reader. Absorbance at nm was measured; thermal stability was determined by heating the antibody at different temperatures for 5 minutes, then restoring it to room temperature for direct competitive ELISA assay. HRP-CYN plus antibodies treated at different temperatures 50 The activity of residual antibody binding to antigen was determined. Three parallel groups were set up for each group, with the binding value after treatment at 25℃ as the 100% binding control. The average absorbance value of the samples measured repeatedly in each group was calculated, and the relative residual binding activity (RT Binding) of each antibody was calculated according to the following formula.

[0014] in This represents the average absorbance value of the heat-treated group. This represents the average absorbance value of the room temperature treatment group.

[0015] The beneficial effects of this invention are as follows: Based on the monoclonal antibody N8 against Cyclostoma toxin, this invention successfully obtained the VH and VL fragment sequences of the antibody variable region using RT-PCR, degenerate primers, overlap PCR, seamless cloning and other techniques, and recombined them into scFv single-chain antibody. Then, the mouse Fc fragment was ligated to it by overlap PCR to obtain the MN8 (scFv-Fc) antibody, which can improve the stability and sensitivity of the immunoassay method for Cyclostoma toxin, and provide a certain foundation for the rapid and accurate determination of Cyclostoma toxin. Attached Figure Description

[0016] Figure 1 This is an electrophoresis diagram of the variable region fragment of the antibody of this invention.

[0017] Figure 2 This is an electrophoresis image of the mouse-derived Fc segment of this invention.

[0018] Figure 3 This is a colony PCR electrophoresis image of the antibody scFv-Fc fragment of this invention.

[0019] Figure 4 This is a diagram illustrating the activity of MN8 in this invention.

[0020] Figure 5 This is an SDS-PAGE image of the purified MN8 antibody of this invention.

[0021] Figure 6 This is a graph showing the antibody titer determination of the present invention.

[0022] Figure 7This is a comparison graph showing the temperature stability of different antibodies in this invention.

[0023] Figure 8 This is a standard curve for the detection of *Cyclophorus toxin* by a direct competitor ELISA of this invention. Detailed Implementation

[0024] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0025] The CYN-M010 hybridoma cell line that produced the N8 monoclonal antibody in this invention is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C2019253, deposit date November 14, 2019; plasmid vector peaSKY203 (laboratory-constructed); Escherichia coli (DH5α) The following reagents were purchased from Kangti Life Insurance Co., Ltd.: CYN standard from ALEXIS; horseradish peroxidase (HRP) from Sigma-Aldrich; DEME medium from Thermo Fisher Scientific; 293 serum-free medium from Beijing Yiqiao Shenzhou Technology Co., Ltd.; HEK293F cells (laboratory-preserved); cell transfection reagent (PEI) from Shanghai Liji Biotechnology Co., Ltd.; seamless cloning reagent from Wuhan Aibote Technology Co., Ltd.; rapid PCR polymerase from Beijing Baori Medical Co., Ltd.; ampicillin from Beijing Solarbio Co., Ltd.; plasmid mini-extraction kit, DNA gel recovery kit, endotoxin-free plasmid extraction kit, and TA cloning kit were all purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.; GLDNA marker 1000 and GLDNA marker 2000 were purchased from Aikerui Biotechnology Co., Ltd.; mouse secondary antibody plate, chromogenic solutions A and B, and stop solution were provided by Guangzhou Darui Biotechnology Co., Ltd.; other reagents, unless otherwise specified, were from Sinopharm Chemical Reagent Co., Ltd.

[0026] Example according to Figures 1-8 As shown in the figure, this embodiment provides a method for preparing and evaluating a genetically engineered antibody against *Cyclophorus spp.*, comprising the following steps: Step 1: cDNA of the CYN-M010 hybridoma cell line that produces N8 monoclonal antibody was obtained by RT-PCR, and the heavy chain DNA fragment and light chain DNA fragment of the variable region gene were extracted by degenerate primers. The fragments were then recombined and transformed by overlapping PCR to construct the scFv gene. Specifically, it includes: S1. After reviving the preserved cell line using the trizol method, RNA was extracted. The entire process must be sterile and enzyme-free, and the RNA was stored at -80°C to prevent degradation and contamination. S2. Using the RT-PCR method, the extracted RNA was processed using a kit to obtain the N8 cDNA fragment. S3. Using degenerate primers, PCR amplification was performed with cDNA as a template. After amplification, the reaction products were identified by 1.5% agarose gel electrophoresis and the DNA gel was extracted and recovered using a DNA gel extraction kit. The gel was then stored at -20℃ for later use. S4. Take 1 part of the recovered heavy chain DNA fragment and 1 part of the recovered light chain DNA fragment. Recombination and ligation were performed separately with the T vector, and the ligation products were converted to 10... To Escherichia coli DH5 In competent cells, gently mix, incubate on ice for 15 min, then heat shock at 42°C for 60 s, place on ice for 2 min, and then take 500 from each sample. Coated on The plates were incubated overnight in an incubator at 37°C, with the plates inverted. S5. Select single colonies of recombinant bacteria for sequencing. Analyze the results using SnapGene software. Add the bacterial solution with the correct sequence to 50% glycerol at a 1:1 ratio and store at -80℃ for later use. S6. Using bacterial culture with the correct sequence, extract heavy chain DNA fragments and light chain DNA fragment plasmids, then use overlap PCR to synthesize scFv fragments via Linker-(GGGGS)n, perform sequence identification as above, and preserve them to obtain the scFv gene.

[0027] Step 2: The scFv gene and mouse Fc were ligated and recombined into the enzyme-digested linear peaSKY203 vector using overlapping PCR to obtain the expression vector. The expression vector was transformed into competent E. coli and plated. Single colonies were picked and sequenced to identify the MN8 bacterial culture with correct sequencing. The primers used for overlap PCR were 203MN8SCLF, MN8SCHR, 2AFCF and 2AFCR, and the primer sequences are shown in SEQ ID No. 1 to SEQ ID No. 4, respectively. See Table 1 below for details.

[0028] Table 1 Primer sequence listing

[0029] Step 3: Inoculate and culture the correctly sequenced MN8 bacterial culture to obtain MN8 genetically engineered antibody bacterial culture, extract plasmids and determine their concentration, and identify them using ELISA. Specifically, 6 ml of sterile 2YT culture medium was added to a sterile test tube, and ampicillin was added at a dilution of 1:1000. Then, the correctly sequenced MN8 bacterial culture was inoculated into a glass shaker tube with an inoculation volume of 1. The plasmids were cultured overnight on a shaker, and then extracted using a small-scale plasmid extraction kit and their concentration was determined. The ELISA method was used for identification. Specifically, 293F cells were pre-cultured in 12-well plates and transfected the following day; 7 cells were then collected. plasmid and 75 Mix the cell culture medium and let it stand for 3 minutes. Then add 4.5... Transfection reagent PEI and 75 Mix the cell culture medium and let stand for 3 minutes. Then, mix the two solutions thoroughly and let stand for another 15-20 minutes before uniformly adding the suspension to 12-well plates and culturing for 48 hours. Collect the cell supernatant and perform ELISA identification. Beforehand, remove the purchased mouse secondary antibody plate from -20°C and let it stand at room temperature for about 30 minutes. Add 50 ml of the mouse secondary antibody solution... Add 50 to the pre-connected HRP-CYN (1:1000) Incubate the cell supernatant at 37°C for 1 hour, wash the plate 5 times and spin dry the washing buffer, then add 100 ml of chromogenic solution A and B (1:1 mixture). Incubate in the dark for 30 minutes to allow color development; a positive reaction will show a blue color. Finally, add 50 ml of stop solution (2M sulfuric acid). The color will change from blue to yellow.

[0030] Step 4: Inoculate and culture the MN8 genetically engineered antibody bacterial culture and transfect it for expression. Purify the cultured cells using a protein G purification column. Before purification, centrifuge the cultured cell solution at 6000 rpm for 10 min to remove the precipitate and retain the supernatant. Then, use 0.22... The solution is filtered through a membrane before purification; all solutions required for purification are passed through a 0.45 μm filter. After membrane filtration, the purified antibody was added to the neutralization solution and its concentration was determined. It was then stored at -20℃ for later use and finally identified using the direct competitive ELISA method. A certain amount of purified protein sample was denatured at high temperature and subjected to SDS-PAGE electrophoresis. After electrophoresis, Coomassie brilliant blue staining was performed to observe whether the band size was correct. The ELISA method was used to determine whether it bound to CYN and produced color, and the range of the genetically engineered antibody and the enzyme-labeled antigen HRP-CYN was determined by checkerboard titration. The MN8 genetically engineered antibody bacterial culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80℃. The inoculation and culture process involved adding 400ml of sterilized 2YT medium to a 1000ml Erlenmeyer flask, and then adding 1:1000 ampicillin (original concentration 50mg / ml) in a clean bench. After inoculation for 10 days... The bacterial culture was preserved and cultured overnight at 37°C with shaking. The precipitate was collected by centrifugation the next day, and plasmids were extracted and endotoxins were removed using a large-scale plasmid extraction kit. The concentration of the obtained plasmids was determined, and then transfection and expression were performed. Transfection expression was specifically performed by pre-resuscitating and culturing 293F cells in 250ml cell culture flasks until their density reached 1.2-2.0× Transfection can be performed at a rate of cells / ml. Add plasmid to 300 ml. Mix with serum-free cell culture medium in a test tube and let stand for 3 minutes. In another test tube, add 900 ml of serum-free cell culture medium. Mix the transfection reagent PEI with serum-free culture medium and let stand for 3 minutes. Then mix the solutions in the two test tubes and let stand for 15-20 minutes before adding them to the cell culture flask to complete the transfection.

[0031] Step 5: Determine the antibody titer and thermal stability of the MN8 genetically engineered antibody; Antibody titer determination was specifically performed using an indirect competitive ELISA. The HRP-CYN was diluted 1:5000, and the MN8 genetically engineered antibody was diluted sequentially in 10-fold increments (1:10~1:10000). The titers were then measured using an ELISA reader. Measurement of absorbance value in nm; The thermal stability assay specifically involves heating the antibody at different temperatures for 5 minutes each, then restoring it to room temperature for direct competitive ELISA assay. HRP-CYN (1:5000) plus antibodies treated at different temperatures 50 (Original concentration 0.3 mg / ml) (1:100), the activity of the remaining antibody binding to the antigen was determined. Three parallel groups were set up for each group. The binding value after treatment at room temperature of 25℃ was used as the 100% binding control. The average absorbance value of the repeated measurements of each group was calculated, and the relative residual binding activity RT Binding of each antibody was calculated according to the following formula.

[0032] in This represents the average absorbance value of the heat-treated group. This represents the average absorbance value of the room temperature treatment group.

[0033] result: 1. Obtaining the N8 variable region gene of monoclonal antibody After PCR amplification, the variable regions VH and VL fragments were obtained, and the band size was identified by agarose gel electrophoresis, as per the instruction manual. Figure 1As shown in the figure, M is the DNA marker DL1000; 1, 3, 5, and 6 are PCR products amplified from the heavy chain fragment; 2 and 4 are PCR products amplified from the light chain fragment. Bands 1, 2, and 6 were successfully amplified and their sizes met expectations, and can be used for subsequent recombination construction.

[0034] 2. Construction and expression of recombinant N8-scFv-mFc (MN8) fusion protein Single-chain antibodies were synthesized using a linker via overlap PCR, and an Fc fragment was ligated into them to improve their stability. (See attached instruction manual.) Figure 2 Electrophoresis image of the amplified mouse Fc fragment, as shown in the instruction manual. Figure 3 The image shows an electrophoresis result of the antibody scFv-Fc(MN8) fragment amplified by colony PCR. The correct-sized band was recovered by gel cutting.

[0035] 3. MN8 Expression and Identification After antibody transfection and expression, the results were verified using an indirect competitive ELISA. The colorimetric results are shown in the attached instructions. Figure 4 Compared with the positive control, this indicates that antibody MN8 still has the ability to bind to CYN.

[0036] 4. Large-scale expression and identification of genetically engineered antibodies The constructed genetically engineered antibody MN8 was extracted in large quantities and transfected into HEK293F cells. The plasmid was purified using protein G affinity chromatography, and the results were identified by SDS-PAGE electrophoresis as shown in the instruction manual. Figure 5 As shown in the figure, M1 and M2 are 10-150kDa protein markers, band 1 is the stock solution before MN8 elution, band 2 is the solution after MN8 elution, and bands 3-4 are the purified MN8 antibody solution. From band 1, it can be seen that the antibody concentration in the cell supernatant was low before purification. After enrichment and elution, the concentration increased in bands 3 and 4, and the antibody was enriched in the packing material. The eluted solution contained virtually no antibody (as shown in band 2). Purification increases the concentration and removes impurities such as cell debris, improving the specificity of the antibody in later stages.

[0037] 5. Antibody titer determination As per the instruction manual Figure 6 The comparison of the titers of two antibodies is shown. N8 is a monoclonal antibody of the CYN-M010 hybridoma cell line previously preserved in the laboratory. It was determined by indirect ELISA. The antibody was serially diluted with HRP-CYN at a fixed amount (1:5000). As shown in the figure, when the dilution gradient reaches 1:10000 (the original concentration is 0.3 mg / ml), the color development drops to the minimum and the OD value tends to flatten. Therefore, it can be seen that the titer of the genetically engineered antibody MN8 is very similar to that of the monoclonal antibody N8.

[0038] 6. Antibody thermal stability assay As per the instruction manual Figure 7 The thermostability assays for the two antibodies are shown. RT Binding represents the relative residual binding activity (with the 25℃ treatment group as a control). A higher RT value indicates better thermostability. The graph shows that the stability of both antibodies gradually decreases after the temperature exceeds 60℃, indicating that their antibody performance is consistent. The typical incubation condition for ELISA is 37℃, at which point there is a highly significant difference between the two antibodies (P < 0.01), with MN8 exhibiting higher relative residual activity.

[0039] 7. Determine the optimal reaction dilution between the antibody and HRP-CYN. With a fixed HRP-CYN dilution, the OD value decreased as the antibody concentration decreased. When the antibody dilution was 1:400, the OD value dropped sharply. To ensure a high absorbance value, the MN8 dilution gradient for the direct competitive ELISA method was finally determined to be 1:200, and the HRP-CYN dilution gradient was determined to be 1:8000.

[0040] 8. Direct competition ELISA standard curve The standard curve for direct competition ELISA is attached to the instruction manual. Figure 8 Figure (a) shows the absorbance change curve of antibody MN8 in the range of 0–400 ng / mL, and (b) shows the logit-log linear standard curve of antibody MN8. Within the range of 0–400 ng / mL, the standard curve exhibits a typical S-shaped curve. Linear fitting using the logit-log method shows good linearity for the antibody in the range of 0–4 ng / mL. Furthermore, the coefficients of variation for repeated measurements of the three MN8 standards ranged from 0.09% to 2.45%, indicating that the ELISA standard curve established in this study has good reproducibility.

[0041] 9. Sensitivity and stability of the method When the CYN concentration is 0 ng / mL, it is in The concentration value corresponding to the average absorbance at nm minus twice the standard deviation, substituted into the standard curve, represents the sensitivity of this direct competitive ELISA method. The calculated value for MN8 antibody was 0.079 ng / mL, which is comparable to the sensitivity of N8 (0.069 ng / mL, Yang et al., 2023). The mean IC80 at three different time points was 3.43 ng / mL, with a SD less than 10%, demonstrating good stability. The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing and evaluating a genetically engineered antibody against *Cyclophorus* toxin, characterized in that, Includes the following steps: Step 1: cDNA of the CYN-M010 hybridoma cell line that produces N8 monoclonal antibody was obtained by RT-PCR, and the heavy chain DNA fragment and light chain DNA fragment of the variable region gene were extracted by degenerate primers. The fragments were then recombined and transformed by overlapping PCR to construct the scFv gene. Step 2: The scFv gene and mouse Fc were ligated and recombined into the enzyme-digested linear peaSKY203 vector using overlapping PCR to obtain the expression vector. The expression vector was transformed into competent E. coli and plated. Single colonies were picked and sequenced to identify the MN8 bacterial culture with correct sequencing. Step 3: Inoculate and culture the correctly sequenced MN8 bacterial culture to obtain MN8 genetically engineered antibody bacterial culture, extract plasmids and determine their concentration, and identify them using ELISA. Step 4: Inoculate and culture the MN8 genetically engineered antibody bacterial culture, extract plasmids for transfection and expression, purify using a proteinG purification column, and finally identify using a direct competitive ELISA method. Step 5: Determine the antibody titer and thermal stability of the MN8 genetically engineered antibody.

2. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: Step one specifically includes S1. RNA was extracted from the preserved cell lines after reviving using the trizol method, and the RNA was stored at -80℃. S2. Using the RT-PCR method, the extracted RNA was processed using a kit to obtain the N8 cDNA fragment. S3. Using degenerate primers, PCR amplification was performed with cDNA as a template. After amplification, the reaction products were identified by 1.5% agarose gel electrophoresis and the DNA gel was extracted using a DNA gel extraction kit. S4. Take 1 part of the recovered heavy chain DNA fragment and 1 part of the recovered light chain DNA fragment. Recombination and ligation were performed separately with the T vector, and the ligation products were converted to 10... To Escherichia coli DH5 The competent cells were plated and incubated overnight at 37°C. S5. Select single colonies of recombinant bacteria for sequencing. Analyze the results using SnapGene software. Add the bacterial solution with the correct sequence to 50% glycerol at a 1:1 ratio and store at -80℃ for later use. S6. Using bacterial culture with the correct sequence, extract heavy chain DNA fragments and light chain DNA fragment plasmids, then use overlap PCR to synthesize scFv fragments via Linker-(GGGGS)n, perform sequence identification as above, and preserve them to obtain the scFv gene.

3. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 2, characterized in that: The transformed competent cells in S4 were subjected to an ice bath for 15 minutes, followed by heat shock at 42°C for 60 seconds, and then placed on ice for 2 minutes. Subsequently, 500 cells were taken from each cell. Coated on Incubate on plate.

4. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: The primers used for overlap PCR in step two are 203MN8SCLF, MN8SCHR, 2AFCF and 2AFCR, and the primer sequences are shown in SEQ ID No. 1 to SEQ ID No. 4, respectively.

5. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: Step three specifically involves adding 6 ml of sterile 2YT culture medium to a sterile test tube and adding ampicillin at a dilution of 1:1000. Then, the correctly sequenced MN8 bacterial culture is inoculated into a glass shaker tube with an inoculation volume of 1 ml. The plasmids were cultured overnight on a shaker, and then extracted using a small-scale plasmid extraction kit to determine their concentration.

6. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: In step three, the ELISA method for identification specifically involves pre-coating 293F cells in 12-well plates and transfecting them the following day; then taking 7... plasmid and 75 Mix the cell culture medium and let it stand for 3 minutes. Then add 4.5... Transfection reagent PEI and 75 Mix the cell culture medium and let it stand for 3 minutes. Finally, mix the two mediums together, shake well, and let it stand for 15-20 minutes. Then, suspend the medium and add it evenly to a 12-well plate and incubate for 48 hours. Collect the cell supernatant and perform ELISA identification.

7. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: In step four, the inoculation and culture of the MN8 gene-engineered antibody bacterial suspension specifically involves adding 400ml of sterilized 2YT culture medium to a 1000ml Erlenmeyer flask, adding 1:1000 ampicillin in a clean bench, and inoculating for 10... The bacterial culture was preserved and cultured overnight at 37°C with shaking. The precipitate was collected by centrifugation the next day, and plasmids were extracted and endotoxins removed using a large-scale plasmid extraction kit. The concentration of the obtained plasmids was measured, and then transfection expression was performed.

8. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 7, characterized in that: The transfection expression specifically involves pre-resuscitating and culturing 293F cells in 250ml cell culture flasks until their density reaches 1.2-2.0× Transfection can be performed at a rate of cells / ml. Add plasmid to 300 ml. Mix with serum-free cell culture medium in a test tube and let stand for 3 minutes. In another test tube, add 900 ml of serum-free cell culture medium. Mix the transfection reagent PEI with serum-free culture medium and let stand for 3 minutes. Then mix the solutions in the two test tubes and let stand for 15-20 minutes before adding them to the cell culture flask to complete the transfection.

9. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: Before purification in step four, the cultured cell solution is centrifuged at 6000 rpm for 10 min to remove the precipitate and retain the supernatant, which is then used in 0.22... Purification is performed after filtration through a filter membrane; All solutions required for purification were passed through a 0.45 ohmmeter. Samples are injected after membrane filtration.

10. The method for preparing and evaluating a gene-engineered antibody against *Cyclophorus spp.* according to claim 1, characterized in that: Step five, the antibody titer determination, specifically involves using an indirect competitive ELISA to determine the antibody titer. The HRP-CYN concentration is 1:5000, and the MN8 genetically engineered antibody is diluted sequentially in 10-fold increments using a microplate reader. Absorbance at nm was measured; thermal stability was determined by heating the antibody at different temperatures for 5 minutes, then restoring it to room temperature for direct competitive ELISA assay. HRP-CYN plus antibodies treated at different temperatures 50 The activity of residual antibody binding to antigen was determined. Three parallel groups were set up for each group, with the binding value after treatment at 25℃ as the 100% binding control. The average absorbance value of the samples measured repeatedly in each group was calculated, and the relative residual binding activity (RT Binding) of each antibody was calculated according to the following formula. in This represents the average absorbance value of the heat-treated group. This represents the average absorbance value of the room temperature treatment group.

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