Screening and rapid detection method of shrimp enterocytozoon hepatopenaei spore wall protein interaction polypeptide

Abstract

The invention discloses a screening and rapid detection method of shrimp enterocytozoon hepatopenaei spore wall protein interaction polypeptide. The screening and rapid detection method comprises the following steps: S1, constructing and extracting Pmal-EhSW12 recombinant plasmids; s2, expression and purification of recombinant protein; s3, screening of the EhSW12 protein interaction polypeptide; s4, polypeptide affinity identification; and S5, carrying out rapid detection on the EHP spore wall protein EhSW12. The method provided by the invention has the advantage of rapid detection of prawn enterocytozoon hepatopenaei, and solves the common problems of complex operation, dependence on precise instruments and long detection period in the use process of the current prawn enterocytozoon hepatopenaei detection method, and the problem that the demand of a grass-roots farm on rapid field detection is difficult to meet.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interacting polypeptides. Background Art

[0002] As one of the most important commercial shrimp species in the global aquaculture industry, Penaeus vannamei possesses extremely high economic value due to its rapid growth, strong adaptability, and low aquaculture costs. However, the recent prevalence of Enterocytozoa has become a major constraint on the development of this industry. Enterocytozoa is a microsporidian pathogen that specifically parasitizes the hepatopancreatic epithelial cells of shrimp. It primarily causes lesions in the hepatopancreas, impairing shrimp digestion and nutrient absorption, leading to growth retardation and even reduced production, resulting in severe economic losses for the shrimp aquaculture industry.

[0003] Shrimp hepatocellular parasites are mainly transmitted through oral ingestion of infectious spores. They can also spread through contaminated water sources, feed, aquaculture environments, and infected shrimp fry. Therefore, early and rapid detection is key to controlling the spread of the disease. Current detection methods for shrimp hepatocellular parasites mainly include molecular biology techniques such as loop-mediated isothermal amplification, fluorescence quantification, and recombinase polymerase amplification. Although these methods perform well in terms of sensitivity and specificity, they generally have problems such as complex operation, dependence on precision instruments, and long detection cycles, making it difficult to meet the needs of grassroots farms for rapid on-site detection. Therefore, there is an urgent need to develop a new detection method that is simple, rapid, and can be used without complex equipment to achieve immediate diagnosis and effective prevention and control of shrimp hepatocellular parasites. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for screening and rapid detection of shrimp hepato-enteric cytozoa spore wall protein interacting polypeptides, which has the advantage of rapid detection of shrimp hepato-enteric cytozoa and solves the problems of current shrimp hepato-enteric cytozoa detection methods during use, such as complex operation, reliance on precision instruments and long detection cycles, which makes it difficult to meet the needs of grassroots farms for on-site rapid detection.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interacting polypeptides, comprising the following steps:

[0006] S1, construction and extraction of Pmal-EhSW12 recombinant plasmid;

[0007] S2, expression and purification of recombinant proteins;

[0008] Screening of peptides interacting with S3 and EhSW12 proteins;

[0009] S4, peptide affinity identification;

[0010] S5. Rapid detection of EHP spore wall protein EhSW12.

[0011] As a preferred method for screening and rapid detection of a shrimp hepatoenteric cytozoan spore wall protein interacting polypeptide of the present invention, when constructing and extracting the recombinant plasmid in S1, first, according to the target gene EhSW12 sequence, a BamH1 amplification primer is used to specifically amplify the EhSW12 gene fragment in the subsequent PCR amplification process, and total DNA is extracted from a biological sample containing the target gene, and DNA fragments of the desired size are separated by agarose gel electrophoresis, and then the fragments are further purified using a gel purification kit to ensure that high-quality DNA samples are obtained for subsequent operations, and the prokaryotic vector Pmal-c5X(+) is selected, and BamH1 is used to amplify the EhSW12 gene fragment. The mH1 restriction endonuclease is used to cut it, preparing it to accept the target gene fragment so that the target gene can be inserted. The purified EhSW12 gene fragment and the enzyme-cut vector are ligated using a seamless cloning kit to generate a recombinant plasmid. The recombinant plasmid is introduced into competent bacterial cells TransT1 and cultured on LB plates containing ampicillin. Bacterial colonies that have successfully accepted the recombinant plasmid are screened, and single colonies are picked for PCR verification to confirm whether the target gene has been successfully inserted. Finally, the recombinant plasmid is extracted from the screened positive clones and sequenced to ensure that the inserted target gene sequence is accurate.

[0012] As a preferred method for screening and rapid detection of a shrimp hepato-enterocystis spore wall protein-interacting polypeptide of the present invention, when expressing and purifying the recombinant protein in S2, the constructed recombinant plasmid containing the target gene EhSW12 is transformed into BL21 (DE3) Escherichia coli to efficiently express the exogenous protein, a positive single colony cultured overnight is picked from the LB plate, inoculated into a fresh liquid culture medium for expansion culture, and when the bacteria grow to an appropriate density, isopropyl β-D-thiogalactoside is added to induce the expression of the target protein, the bacterial cells are collected by centrifugation, and then ultrasonic treatment is used to lyse the bacteria to release the internal protein, and then high-speed culture is performed again. After low-temperature centrifugation, a supernatant containing soluble protein and a precipitate of insoluble protein were obtained. These two parts were subjected to SDS-PAGE analysis to determine the main form of the recombinant protein. The recombinant protein mainly present in the supernatant was purified by affinity chromatography with the addition of starch resin. The purified recombinant protein was quantified using a Bradford kit. At the same time, an empty vector without an insert was treated according to the same expression and purification steps to prepare a protein with only an MBP tag, providing a control sample for subsequent reverse screening experiments. Finally, all obtained protein samples were stored at -80°C to maintain their activity and stability.

[0013] As a preferred method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interactive polypeptides of the present invention, when screening EhSW12 protein interactive polypeptides in S3, the recombinant protein MBP-EhSW12 is used as a substrate, and PH.D. TM The phage display peptide library in PHage Display Peptide Library Kits v2 was screened. First, MBP-EhSW12 was fixed on a solid support, and then the phage display peptide library was added to allow the polypeptides therein to contact and bind to the substrate. Phages that failed to bind to MBP-EhSW12 were washed away with TBST buffer, and only those phage-peptide complexes that successfully bound were retained. The bound phages were eluted by protein competition, that is, excess free MBP-EhSW12 or other related substances were used to competitively replace the phages bound to the substrate, thereby releasing phages with stronger binding force. The eluted phages were added to fresh Escherichia coli culture medium for infection and amplification. Subsequently, the supernatant of the bacterial culture medium was collected by low-temperature high-speed centrifugation, and the phages were concentrated using PEG / NaCl solution to obtain the phage collection after the first round of screening. The phage collection obtained in the previous round of screening was added to the phage collection obtained in the previous round of screening. The collaboration was used as the starting library for the next round of screening, and the above steps were repeated for a total of four rounds. In each round, phages with stronger binding to MBP-EhSW12 were enriched. Since the MBP tag itself is large, in order to exclude nonspecific binding caused by the MBP tag rather than the EhSW12 protein itself, a reverse screening step was introduced in the third round of screening. During the reverse screening, the expressed protein with only the MBP tag was used as a substrate, and the eluate at the end of the second round was used to bind to it, and the unbound phage was eluted and used for subsequent screening to reduce the impact of nonspecific binding. After the last round of screening, 20 phage plaques were randomly selected from the double-layer plate and sent to a biological company for DNA sequencing to determine the polypeptide sequence encoded by it. The screening effect was evaluated by comparing the changes in phage titers between different rounds, and sequence analysis was used to confirm which polypeptides were truly specifically bound to MBP-EhSW12.

[0014] As a preferred method for screening and rapid detection of a shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptide of the present invention, when performing polypeptide affinity identification in the S4, the three screened polypeptides, the recombinant protein MBP-EhSW12, the protein with only the MBP tag, and PBS as a negative control are respectively added dropwise to a PVDF membrane, and different binding recognition substances are added to each point on the PVDF membrane, including the recombinant protein MBP-EhSW12, the protein with only the MBP tag, and the PBS control. First, a monoclonal antibody against the MBP tag is added as a primary antibody, and then , add HRP-labeled goat anti-mouse antibody as a secondary antibody. This antibody can recognize and bind to the primary antibody, thereby indirectly marking the location of proteins bound to MBP-EhSW12 or containing only the MBP tag. Finally, HRP-catalyzed substrate reaction produces a visible color change through chemiluminescence and other methods, thereby intuitively showing which points have undergone specific binding, helping to confirm which peptides have specific interactions with EhSW12. Further compare the affinity of the three peptides for MBP-EhSW12, and repeat the above dot blot experiment after diluting each peptide at different ratios. By comparing the signal intensity at different concentrations, it is determined which peptide can effectively bind to MBP-EhSW12 even at low concentrations, thereby determining their relative affinities. Based on the dot blot results, the peptides showing stronger binding ability are selected for subsequent processing.

[0015] As a preferred method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interacting polypeptides of the present invention, in the rapid detection of EHP spore wall protein EhSW12 in S5, EHP is separated from shrimp hepatopancreas tissue using Percoll density gradient centrifugation, the separated EHP is treated by enzymatic hydrolysis or mechanochemical method to release the spore wall protein EhSW12, biotinylated polypeptide 3 is labeled with colloidal gold solution, the screened specific polypeptide 2 is sprayed on the test line of the test strip, streptavidin is sprayed on the quality control line of the test strip, and the above components are integrated together to form a complete rapid detection test strip, the enzymatically hydrolyzed EhSW12 protein is used as a substrate and spotted on a membrane, the biotin-labeled polypeptide 3 is used as a test substance and dropped onto the sample point on the membrane, HRP-labeled streptavidin is added, and this enzyme-labeled secondary antibody can recognize and bind to the biotin-labeled polypeptide 3, thereby indirectly marking the position of the polypeptide bound to the substrate, and finally, the results are observed by color development to determine whether a specific protein-polypeptide interaction exists.

[0016] As a preferred method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interacting polypeptides of the present invention, when using Percoll density gradient centrifugation to separate EHP from shrimp hepatopancreas tissue, first, 1.5 mol / L NaCl solution and Percoll stock solution are mixed in a volume ratio of 1:9 to prepare 100% Percoll mother solution, then, the mother solution is diluted with 0.15 mol / L NaCl to prepare 50% and 75% Percoll working solutions, Percoll solutions of different concentrations are used to construct density gradients, 2 mL of 100%, 75%, and 50% Percoll solutions are sequentially stacked in a centrifuge tube, and a gradient separation system is constructed by density differences between layers. The tissue homogenate is filtered through 4 layers of gauze to remove larger tissue fragments, and the microsporidia are preliminarily enriched by differential centrifugation combined with repeated washing with PBS buffer to obtain a crude extract. Differential centrifugation uses different centrifugal forces to remove impurities and gradually concentrate the target microorganisms. The crude microsporidian extract obtained above is carefully added to the prepared In the Percoll gradient system, avoid disrupting the gradient. Centrifuge at 15,000 × g for 30 minutes at 4°C to allow components of different densities to distribute along the gradient. Collect the precipitates at each gradient interface and wash repeatedly with normal saline to remove residual Percoll solution. Finally, resuspend the highly purified microsporidia in normal saline. Add 1 mg / mL trypsin and 2 mg / mL chitosanase to the purified EHP suspension and incubate at 37°C for 2-4 hours with gentle stirring during incubation. Gentle stirring promotes effective tissue lysis and ensures sufficient release of the target protein.

[0017] As a preferred method for screening and rapid detection of a shrimp hepatoenteric cytozoon spore wall protein interacting polypeptide of the present invention, when the separated EHP is treated by mechanochemical method, the purified EHP spores are suspended in a lysis buffer containing PBS, 50mMTris-HCl, 1% SDS and 1% Triton X-100, glass beads with a diameter of 0.1-0.5mm are added, and a high-speed shaker is used to treat the spores at a speed of 6000rpm for 3 times, each time for 1 minute, and ice bath cooling is performed between each time to prevent the sample from being overheated and causing protein denaturation or inactivation. 10mM DTT was added and incubated at 60°C for 10 minutes to destroy the disulfide bonds in the protein and help completely cleave the protein structure. According to the characteristics of the spore wall components, 2% β-mercaptoethanol was added to further promote protein denaturation to ensure the maximum release of the target protein. Coomassie brilliant blue staining was used to observe the quality and concentration of protein samples obtained by different cleavage methods, so as to select the best cleavage method. The protein samples treated with the selected cleavage method were separated by SDS-PAGE electrophoresis and then wet-transferred to PVDF membrane at a constant voltage of 100V for 50 minutes. The PVDF membrane was blocked with 5% skim milk for 2 hours to reduce non- For specific binding, incubate the biotin-labeled peptide 3 overnight at 4°C to give it a chance to bind to the target protein on the membrane. Use PBST buffer to shake and wash the membrane for 6 minutes, repeat 7 times to remove unbound peptides, and incubate the membrane with HRP-labeled streptavidin for 2 hours at room temperature. Streptavidin can bind tightly to biotin to form a complex. Wash the membrane thoroughly with PBST buffer again to remove excess HRP-labeled streptavidin. Finally, perform a color reaction on a PVDF membrane for 30 seconds, and then use an ECL gel imaging system to capture and analyze the results to confirm the binding of Bio-peptide 3 to the target protein.

[0018] As a preferred method for screening and rapid detection of a shrimp hepatoenteric cytozoan spore wall protein interacting polypeptide of the present invention, when using colloidal gold solution to label the biotinylated polypeptide 3, a series of 8 connected tubes are taken, 200 μL of colloidal gold solution is added to each tube, and different amounts of 0.1 mol / L K2CO3 are added to each tube in turn to adjust the pH value of the colloidal gold solution. After adjusting the pH value, an excess amount of biotinylated polypeptide 3 is added to each tube, 10 μL of 10% NaCl is added, mixed evenly and allowed to stand, the color change is observed and the absorbance at 520 nm is measured using a spectrophotometer to determine the optimal pH value, and another series of 8 connected tubes are taken, 200 μL of colloidal gold solution is added to each tube, and an appropriate amount of K2CO3 is added according to the optimal pH value previously determined, different amounts of 1 mg / mL biotinylated polypeptide 3 are added to each tube in turn, and 10 μL of 10% NaCl is also added. 10% NaCl, mix and let stand, determine the optimal amount of biotinylated polypeptide labeling by observing the color change and measuring the OD value at 520nm, take 800μL colloidal gold solution in a centrifuge tube, add the corresponding amount of K2CO3 according to the optimal pH value determined previously, mix thoroughly, add the optimal amount of biotinylated polypeptide 3, and then add 200μL 5% BSA after mixing. BSA is used as a stabilizer to prevent nonspecific adsorption. The mixture is centrifuged at 10000rpm for 30 minutes at 4°C, the supernatant is discarded, and the precipitate is retained. 100μL of 0.01mol / L PBS buffer containing 5% trehalose and 1% BSA is added to the precipitate as a gold labeling solution for resuspending to obtain the final colloidal gold labeled polypeptide solution. The prepared gold labeling polypeptide solution is sprayed onto the gold label pad. The whole process is operated on ice to maintain the stability of the colloidal gold particles.

[0019] As a preferred method for screening and rapid detection of a shrimp hepatoenteric cytozoan spore wall protein interacting polypeptide of the present invention, when assembling a rapid detection test strip, a gold label pad, a sample pad and a water-absorbing pad are cut into 9 mm, 15 mm and 17 mm widths respectively using a strip cutting machine, soaked in PBS buffer containing 2% Tween-20 and 5% trehalose for 30 minutes, and then soaked in PBS buffer containing 0.2% Triton The pad was soaked in X-100 PBS buffer for 30 minutes. The soaked pad was thoroughly dried and set aside to remove excess moisture and prevent mold growth or other contamination. A test line and a quality control line were accurately drawn on the nitrocellulose membrane using an XYZ three-dimensional film spray gold apparatus. The test line was sprayed with the screened specific peptide 2, and the quality control line was sprayed with streptavidin. The prepared colloidal gold-labeled peptide solution was added dropwise to the gold-labeled pad until saturated, and then dried again for later use. Hepatopancreatic tissue homogenate was extracted from positive shrimp samples infected with different viruses. The tissue homogenate was treated to release the target protein and applied to the assembled colloidal gold test strip. The results were observed. If the target protein was present, it would bind to the labeled peptide on the test line, forming a visible color change. Similarly, the enzymatically hydrolyzed target protein was used as a substrate, and the biotin-labeled peptide 3 was used as a detector. HRP-labeled streptavidin was added for color development, and the results were observed to confirm specific binding.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention focuses on the shrimp hepatocystis spore wall protein EhSW12, and from the perspective of "interacting polypeptides", screens polypeptide sequences that can specifically bind to EhSW12, and constructs a rapid detection test strip based on colloidal gold immunochromatography technology. This method has the advantages of convenient operation, rapid detection, no need for complex instruments, and suitability for on-site application. It can provide farms and testing institutions with an efficient and practical shrimp hepatocystis rapid diagnosis tool, thereby improving the disease prevention and control capabilities of the shrimp farming industry and promoting the healthy and sustainable development of the whiteleg shrimp farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the colloidal gold test strip of the present invention;

[0023] Figure 2 This is a diagram of the construction of the Pmal-EhSW12 recombinant plasmid of the present invention;

[0024] Figure 3 This is a diagram showing the induction expression and purification of the recombinant protein MBP-EhSW12 of the present invention;

[0025] Figure 4 The plaque images on the plates of the four rounds of screening of the present invention are shown;

[0026] Figure 5This is a graph showing the recovery rate of phage display of the present invention;

[0027] Figure 6 This is a diagram showing the sequence analysis of the polypeptide specifically bound by the EhSW12 protein of the present invention;

[0028] Figure 7 This is the Dot blot verification analysis diagram of the present invention;

[0029] Figure 8 This is the SDS-PAGE analysis diagram of the present invention;

[0030] Figure 9 This is the Western Blot verification diagram of the present invention;

[0031] Figure 10 This is a graph showing the changes in colloidal gold solution at different pH values according to the present invention;

[0032] Figure 11 This is a graph showing changes in colloidal gold solution at different protein concentrations according to the present invention;

[0033] Figure 12 This is a graph showing the colloidal gold test strip and Dot Blot test results of the present invention;

[0034] Figure 13 This is a graph showing the specificity test results of the colloidal gold test strip and Dot Blot of the present invention;

[0035] Figure 14 This is a graph showing the sensitivity test results of the colloidal gold test strip and Dot Blot of the present invention;

[0036] Figure 15 This is a colloidal gold test strip and Dot Blot clinical test diagram of the present invention;

[0037] Figure 16 This is a comparison chart between the colloidal gold test strip of the present invention and the PCR detection method. DETAILED DESCRIPTION

[0038] See also Figures 1-16 A method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein interacting polypeptides comprises the following steps:

[0039] S1, construction and extraction of Pmal-EhSW12 recombinant plasmid;

[0040] S2, expression and purification of recombinant proteins;

[0041] Screening of peptides interacting with S3 and EhSW12 proteins;

[0042] S4, peptide affinity identification;

[0043] S5. Rapid detection of EHP spore wall protein EhSW12.

[0044] Furthermore, when constructing and extracting the recombinant plasmid in S1, first, according to the target gene EhSW12 sequence, BamH1 amplification primers were used to specifically amplify the EhSW12 gene fragment in the subsequent PCR amplification process. Total DNA was extracted from the biological sample containing the target gene, and the DNA fragment of the required size was separated by agarose gel electrophoresis. The fragment was further purified using a gel purification kit to ensure that high-quality DNA samples were obtained for the subsequent operation. The prokaryotic vector Pmal-c5X(+) was selected and cut with the BamH1 restriction endonuclease. , prepare to accept the target gene fragment so that the target gene can be inserted, use the seamless cloning kit to connect the purified EhSW12 gene fragment and the enzyme-cut vector to generate a recombinant plasmid, introduce the recombinant plasmid into the competent bacterial cell TransT1, and culture it on an LB plate containing ampicillin, screen out the bacterial colonies that have successfully accepted the recombinant plasmid, pick a single colony for PCR verification to confirm whether the target gene is successfully inserted, finally, extract the recombinant plasmid from the screened positive clone, and perform sequencing analysis on it to ensure that the inserted target gene sequence is accurate.

[0045] Furthermore, when expressing and purifying the recombinant protein in S2, the constructed recombinant plasmid containing the target gene EhSW12 was transformed into BL21 (DE3) Escherichia coli to efficiently express the exogenous protein. A positive single colony cultured overnight was picked from the LB plate and inoculated into fresh liquid culture medium for expansion. When the bacteria grew to an appropriate density, isopropyl β-D-thiogalactoside was added to induce the expression of the target protein. The bacterial cells were collected by centrifugation, and then ultrasonic treatment was used to lyse the bacteria to release the internal protein. After high-speed low-temperature centrifugation again, the soluble protein was obtained. The supernatant and the insoluble protein precipitate were separated and SDS-PAGE analysis was performed on each of them to determine the main form of the recombinant protein. The recombinant protein mainly present in the supernatant was purified by affinity chromatography with the addition of starch resin. The purified recombinant protein was quantified using a Bradford kit. At the same time, an empty vector without an insert was treated according to the same expression and purification steps to prepare a protein with only an MBP tag, providing a control sample for subsequent reverse screening experiments. Finally, all the obtained protein samples were stored at -80°C to maintain their activity and stability.

[0046] Furthermore, when screening for EhSW12 protein-interacting peptides in S3, the recombinant protein MBP-EhSW12 was used as a substrate and PH.D. TMThe phage display peptide library in PHage Display Peptide Library Kits v2 was screened. First, MBP-EhSW12 was fixed on a solid support, and then the phage display peptide library was added to allow the polypeptides therein to contact and bind to the substrate. Phages that failed to bind to MBP-EhSW12 were washed away with TBST buffer, and only those phage-peptide complexes that successfully bound were retained. The bound phages were eluted by protein competition, that is, excess free MBP-EhSW12 or other related substances were used to competitively replace the phages bound to the substrate, thereby releasing phages with stronger binding force. The eluted phages were added to fresh Escherichia coli culture medium for infection and amplification. Subsequently, the supernatant of the bacterial culture medium was collected by low-temperature high-speed centrifugation, and the phages were concentrated using PEG / NaCl solution to obtain the phage collection after the first round of screening. The phage collection obtained in the previous round of screening was added to the phage collection obtained in the previous round of screening. The collaboration was used as the starting library for the next round of screening, and the above steps were repeated for a total of four rounds. In each round, phages with stronger binding to MBP-EhSW12 were enriched. Since the MBP tag itself is large, in order to exclude nonspecific binding caused by the MBP tag rather than the EhSW12 protein itself, a reverse screening step was introduced in the third round of screening. During the reverse screening, the expressed protein with only the MBP tag was used as a substrate, and the eluate at the end of the second round was used to bind to it, and the unbound phage was eluted and used for subsequent screening to reduce the impact of nonspecific binding. After the last round of screening, 20 phage plaques were randomly selected from the double-layer plate and sent to a biological company for DNA sequencing to determine the polypeptide sequence encoded by it. The screening effect was evaluated by comparing the changes in phage titers between different rounds, and sequence analysis was used to confirm which polypeptides were truly specifically bound to MBP-EhSW12.

[0047] Furthermore, when performing peptide affinity identification in S4, the three screened peptides, recombinant protein MBP-EhSW12, protein with only MBP tag, and PBS as negative control were respectively added to the PVDF membrane. Different binding recognition substances were added to each point on the PVDF membrane, including recombinant protein MBP-EhSW12, protein with only MBP tag, and PBS control. First, a monoclonal antibody against the MBP tag was added as the primary antibody. Then, an HRP-labeled goat anti-mouse antibody was added as the secondary antibody. This antibody can recognize and bind to the primary antibody, thereby indirectly marking the position of the protein bound to MBP-EhSW12 or only containing the MBP tag. Finally, the HRP-catalyzed substrate reaction was used to produce a visible color change through chemiluminescence and other methods, thereby intuitively showing which points had specific binding, helping to confirm which peptides had specific interactions with EhSW12, and further comparing the affinity of the three peptides for MBP-EhSW12. Each peptide was diluted at different proportions and the above dot was repeated. In the dot blot experiment, by comparing the signal intensities at different concentrations, we determined which peptide could effectively bind to MBP-EhSW12 even at low concentrations, thereby determining their relative affinities. Based on the dot blot results, the peptide that showed stronger binding ability was selected for subsequent processing.

[0048] Furthermore, in S5, when performing a rapid test for the EHP spore wall protein EhSW12, EHP is separated from the hepatopancreas tissue of shrimp using Percoll density gradient centrifugation. The separated EHP is treated with enzymatic or mechanochemical methods to release the spore wall protein EhSW12. A colloidal gold solution is used to label the biotinylated peptide 3. The screened specific peptide 2 is sprayed on the test line of the test strip. Streptavidin is sprayed on the quality control line of the test strip. The above components are integrated together to form a complete rapid test strip. The enzymatically hydrolyzed EhSW12 protein is used as a substrate and spotted on the membrane. The biotin-labeled peptide 3 is used as a detector and dropped onto the sample point on the membrane. HRP-labeled streptavidin is added. This enzyme-labeled secondary antibody can recognize and bind to the biotin-labeled peptide 3, thereby indirectly marking the position of the peptide bound to the substrate. Finally, the results are observed through a color reaction to determine whether a specific protein-peptide interaction exists.

[0049] Furthermore, when using the Percoll density gradient centrifugation method to separate EHP from the hepatopancreas tissue of shrimp, 1.5 mol / L NaCl solution was first mixed with the Percoll stock solution in a volume ratio of 1:9 to prepare a 100% Percoll stock solution. Then, the stock solution was diluted with 0.15 mol / L NaCl to prepare 50% and 75% Percoll working solutions. Percoll solutions of different concentrations were used to construct a density gradient. 2 mL of 100%, 75%, and 50% Percoll solutions were stacked in a centrifuge tube in sequence. A gradient separation system was constructed by the density difference between the layers. The tissue homogenate was filtered through 4 layers of gauze to remove larger tissue fragments. Microsporidia were initially enriched by differential centrifugation and repeated washing with PBS buffer to obtain a crude extract. Differential centrifugation uses different centrifugal forces to remove impurities and gradually concentrate the target microorganisms. The crude microsporidia extract obtained above was carefully added to the prepared In the Percoll gradient system, avoid disrupting the gradient. Centrifuge at 15,000 × g for 30 minutes at 4°C to allow components of different densities to distribute along the gradient. Collect the precipitates at each gradient interface and wash repeatedly with normal saline to remove residual Percoll solution. Finally, resuspend the highly purified microsporidia in normal saline. Add 1 mg / mL trypsin and 2 mg / mL chitosanase to the purified EHP suspension and incubate at 37°C for 2-4 hours with gentle stirring during incubation. Gentle stirring promotes effective tissue lysis and ensures sufficient release of the target protein.

[0050] Furthermore, when the isolated EHP was treated by mechanochemical method, the purified EHP spores were suspended in a lysis buffer containing PBS, 50 mM Tris-HCl, 1% SDS and 1% Triton X-100, and glass beads with a diameter of 0.1-0.5 mm were added. The spores were treated with a high-speed shaker at 6000 rpm for 3 times, each for 1 minute, and ice-bathed between each time to prevent the sample from overheating and causing protein denaturation or inactivation. 10 mM DTT was added and incubated at 60°C for 10 minutes to destroy the disulfide bonds in the protein and help completely cleave the protein structure. According to the characteristics of the spore wall components, 2% β-mercaptoethanol was added to further promote protein denaturation to ensure the maximum release of the target protein. Coomassie brilliant blue staining was used to observe the quality and concentration of protein samples obtained by different cleavage methods, so as to select the best cleavage method. The protein samples treated with the selected cleavage method were separated by SDS-PAGE electrophoresis and then wet-transferred to PVDF membrane at a constant voltage of 100V for 50 minutes. The PVDF membrane was blocked with 5% skim milk for 2 hours to reduce non- For specific binding, incubate the biotin-labeled peptide 3 overnight at 4°C to give it a chance to bind to the target protein on the membrane. Use PBST buffer to shake and wash the membrane for 6 minutes, repeat 7 times to remove unbound peptides, and incubate the membrane with HRP-labeled streptavidin for 2 hours at room temperature. Streptavidin can bind tightly to biotin to form a complex. Wash the membrane thoroughly with PBST buffer again to remove excess HRP-labeled streptavidin. Finally, perform a color reaction on a PVDF membrane for 30 seconds, and then use an ECL gel imaging system to capture and analyze the results to confirm the binding of Bio-peptide 3 to the target protein.

[0051] Furthermore, when using colloidal gold solution to label biotinylated peptide 3, a series of 8 tubes were taken, 200 μL of colloidal gold solution was added to each tube, and different amounts of 0.1 mol / L K2CO3 were added to each tube in turn to adjust the pH value of the colloidal gold solution. After the pH value was adjusted, an excess amount of biotinylated peptide 3 was added to each tube, 10 μL of 10% NaCl was added, mixed well and allowed to stand, the color change was observed and the absorbance at 520 nm was measured using a spectrophotometer to determine the optimal pH value. Another series of 8 tubes were taken, 200 μL of colloidal gold solution was added to each tube, and an appropriate amount of K2CO3 was added according to the optimal pH value determined previously. Different amounts of 1 mg / mL biotinylated peptide 3 were added to each tube in turn, and 10 μL of 10% NaCl was also added. 10% NaCl, mix and let stand, determine the optimal amount of biotinylated peptide labeling by observing the color change and measuring the OD value at 520nm, take 800μL colloidal gold solution in a centrifuge tube, add the corresponding amount of K2CO3 according to the optimal pH value determined previously, mix thoroughly, add the optimal amount of biotinylated peptide 3, and then add 200μL 5% BSA after mixing. BSA is used as a stabilizer to prevent nonspecific adsorption. The mixture is centrifuged at 10000rpm for 30 minutes at 4℃, the supernatant is discarded, and the precipitate is retained. 100μL of 0.01mol / L PBS buffer containing 5% trehalose and 1% BSA is added to the precipitate as a gold labeling solution for resuspending to obtain the final colloidal gold labeled peptide solution. The prepared gold label peptide solution is sprayed onto the gold label pad. The whole process is operated on ice to maintain the stability of the colloidal gold particles.

[0052] Furthermore, when assembling the rapid test strips, the gold label pad, sample pad, and absorbent pad were cut into 9 mm, 15 mm, and 17 mm widths, respectively, using a strip cutting machine, and soaked in PBS buffer containing 2% Tween-20 and 5% trehalose for 30 minutes, and then soaked in PBS buffer containing 0.2% Triton X-ray dilution. The pad was soaked in X-100 PBS buffer for 30 minutes. The soaked pad was thoroughly dried and set aside to remove excess moisture and prevent mold growth or other contamination. A test line and a quality control line were accurately drawn on the nitrocellulose membrane using an XYZ three-dimensional film spray gold apparatus. The test line was sprayed with the screened specific peptide 2, and the quality control line was sprayed with streptavidin. The prepared colloidal gold-labeled peptide solution was added dropwise to the gold-labeled pad until saturated, and then dried again for later use. Hepatopancreatic tissue homogenate was extracted from positive shrimp samples infected with different viruses. The tissue homogenate was treated to release the target protein and applied to the assembled colloidal gold test strip. The results were observed. If the target protein was present, it would bind to the labeled peptide on the test line, forming a visible color change. Similarly, the enzymatically hydrolyzed target protein was used as a substrate, and the biotin-labeled peptide 3 was used as a detector. HRP-labeled streptavidin was added for color development, and the results were observed to confirm specific binding.

[0053] Furthermore, when verifying the sensitivity of the colloidal gold test strip, tissue DNA was extracted from 400 μL of tissue homogenate, ultimately obtaining 50 μL of DNA solution. 1 μL of DNA sample was taken and qPCR detection was performed using a quantitative method to determine the EHP content in the sample. Another 500 μL of tissue homogenate was taken, from which the spore wall protein of EHP was extracted and purified, ultimately obtaining 200 μL of protein solution. 20 μL of this solution was taken and treated by enzymatic hydrolysis for colloidal gold test strip detection.

[0054] The purified spore wall protein solution was serially diluted and then detected using colloidal gold test strips and dot blot methods, respectively. The relationship between the detection results at different dilutions and the qPCR results was compared to evaluate the sensitivity of the colloidal gold test strips and dot blot method relative to qPCR.

[0055] The specific steps for comparing colloidal gold test strips, dot blot, and qPCR clinical test results are as follows:

[0056] Sample collection: 20 diseased shrimp samples were collected from a farm in Guangdong;

[0057] Sample processing and testing: The hepatopancreas tissue of each shrimp was enzymatically hydrolyzed and prepared for colloidal gold strips and dot blot testing;

[0058] At the same time, hepatopancreatic tissue DNA was extracted from these samples for qPCR detection;

[0059] Comparison and analysis of results: The detection results of colloidal gold test strips and dot blot were compared with the results of qPCR.

[0060] Kappa analysis was used to evaluate the agreement between two rapid detection methods (colloidal gold test strips and dot blot) and qPCR.

[0061] The target gene (EhSW12) was amplified by PCR using designed primers with specific restriction sites.

[0062] The pmal vector universal primers were used to perform PCR verification on the empty vector and ligated products.

[0063] Electrophoresis observation confirmed that the size of the recombinant plasmid was approximately 1016 bp, which was consistent with the expected total of 260 bp of the empty vector plus 756 bp of the target fragment.

[0064] The recombinant plasmid was extracted and sent to a biological company for sequencing to ensure that the sequence completely matched the target gene, indicating that the recombinant plasmid was successfully constructed.

[0065] For further reference, Figure 2 The recombinant plasmid containing the target gene EhSW12 fused with the MBP tag was transformed into Escherichia coli BL21 (DE3), and isopropyl β-D-thiogalactopyranoside (IPTG) was used to induce the bacteria to express the recombinant protein MBP-EhSW12. After induction, a clear band was observed at 73KD on the SDS-PAGE electrophoresis graph, which was consistent with the expected size of the recombinant protein (EhSW12 protein is approximately 28.7KD and MBP tag is approximately 45KD).

[0066] After induction, the bacterial culture medium was treated with ultrasound to disrupt the cells, causing the bacteria to lyse and release the internal proteins. The lysate was then centrifuged and divided into a supernatant and a precipitate. SDS-PAGE analysis revealed that the recombinant protein was expressed at high levels in both the supernatant and the precipitate. Since the precipitate contained a large amount of impurities and required denaturants such as urea for further purification, affinity chromatography purification was performed from the supernatant.

[0067] Utilizing the characteristics of the MBP tag, the recombinant protein MBP-EhSW12 in the supernatant was purified by affinity chromatography. The purified protein sample showed a single major band by SDS-PAGE analysis, indicating a high purity.

[0068] The purified protein solution was concentrated using PEG20000 to facilitate subsequent experimental operations. The concentration of the purified recombinant protein (MBP-EhSW12) was determined to be 0.83 mg / mL using the Bradford method. The concentration of the control protein with only the MBP tag was also determined to be 2 mg / mL.

[0069] refer to Figure 2 and Figure 3 IPTG (isopropyl-β-D-thiogalactopyranoside) was used to induce bacteria to express a specific recombinant protein, which consisted of a 28.7KD EhSW12 protein and a 45KD MBP (maltose binding protein) tag, with a total molecular weight of approximately 73KD, which was consistent with the expected results.

[0070] After induction of expression, the bacterial culture was ultrasonically treated to lyse the cells, and the supernatant and precipitate were separated by centrifugation. The two parts were then subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. The results showed that the target recombinant protein could be detected in both the supernatant and the precipitate. However, since the precipitate contained a large amount of impurities and required purification using denaturants such as urea, further protein purification was performed from the supernatant. The recombinant protein in the supernatant was purified using affinity chromatography, and a relatively single band was obtained, indicating that a higher purity was obtained. The purified protein was concentrated with PEG20000 (polyethylene glycol 20000) and set aside. Finally, the concentration of the recombinant protein was determined by the Bradford method to be 0.83 mg / ml, while the concentration of the MBP-tagged protein alone was 2 mg / ml.

[0071] refer to Figure 4-Figure 6 , used PH.D. TM PHage Display Peptide Library Kits v2 kit, after each round of screening, the phage titer in the eluate and amplified product was measured to ensure the effectiveness and accuracy of the screening process. The phage titer refers to the number of active phages per unit volume. This step is crucial for evaluating the screening effect. After four rounds of screening and amplification, phage plaques were observed, and it was shown that the phage titer showed a positive correlation growth trend with the increase in the number of selection rounds. This means that after each round of screening, those phages that can specifically bind to EhSW12 are continuously enriched. After the fourth round of screening, 20 phage plaques were selected from the obtained phage population, and these phages were sent to a biological company for DNA sequencing to determine the encoded polypeptide sequence. Based on the sequencing results, the three polypeptides with the highest recurrence frequency were selected as the final candidates.

[0072] refer to Figure 7 , three peptides (peptide 1, peptide 2, peptide 3) were synthesized, and dot-blot experiments were performed to evaluate the specific binding and affinity between these peptides and the target protein (EhSW12). Peptide 1, peptide 2, peptide 3 at a concentration of 1 mg / ml and PBS (phosphate buffer) control were spotted at different positions on the membrane (in the second, third, fourth, and fifth columns of wells). Then, recombinant protein (including EhSW12), MBP-tagged protein, and PBS control were added to different wells in each row as binders to detect whether these peptides can specifically bind to EhSW12 but not to MBP. The first row of the first column was used as a positive control for recombinant protein to ensure that the experimental conditions enable the recombinant protein to be correctly recognized. The second row was used as a positive control for MBP-tagged protein to verify the experimental settings.

[0073] The results showed that all three peptides could specifically bind to the EhSW12 protein, but not to the MBP-tagged protein. This indicates that the selected peptides have good specificity. To further compare the binding affinity between the three peptides and the EhSW12 protein, the three peptides were diluted proportionally and the dot-blot experiment was performed again.

[0074] The experimental results showed that the third polypeptide had the strongest specific binding affinity to the EhSW12 protein, which means that it can effectively bind to EhSW12 even at lower concentrations.

[0075] refer to Figure 8-Figure 9 , choose the most suitable lysis method to fully release the target protein and provide high-quality samples for subsequent interaction experiments, use proteases to gently degrade cell walls or cell membranes to release proteins, and destroy cell structures through grinding, ultrasonic disruption or chemical lysis solutions.

[0076] Enzymatic hydrolysis has a more complete cleavage effect, while mechanical chemical methods may cause protein degradation or impurity interference due to excessive fragmentation. Enzymatic hydrolysis is suitable for processing complex or hard samples, and can preserve the natural conformation and activity of proteins, avoiding the damage to proteins caused by heat generated by mechanical methods or strong denaturants by chemical methods.

[0077] To verify the specific binding of the synthesized Bio-Peptide 3 to the spore wall protein and determine its reactivity, EHP from the purified enzymatic lysate was used as a substrate, and Bio-Peptide 3 was incubated with the substrate as a probe. The proteins were separated by SDS-PAGE and transferred to a PVDF / NC membrane. Binding events were detected using a specific antibody or labeled probe. A clear band appeared at approximately 25 kD, consistent with the expected molecular weight of the spore wall protein, indicating successful binding of Bio-Peptide 3 to the spore wall protein. The band appeared only at the target location and was not bound to unrelated proteins. The high band intensity indicated strong binding affinity. Western blotting is typically used to detect protein expression, but this method, combined with Far-Western blotting, demonstrated that Bio-Peptide 3 can be used as a specific binding tool for spore wall proteins. The enzymatic method is superior to mechanical / chemical methods due to its mildness and efficiency, ensuring the complete release of spore wall proteins.

[0078] refer to Figure 10 Colloidal gold is often used in colorimetric detection technology for biomolecule labeling. The labeling effect is significantly affected by the pH value of the solution. By adjusting the pH value, the surface charge of colloidal gold can be matched with the charge characteristics of the polypeptide, thereby promoting stable binding and avoiding particle aggregation.

[0079] Reagents and conditions:

[0080] The fixed volume of colloidal gold solution was 200 μL.

[0081] K2CO3 solution: 0.1 mol / L, used to adjust the pH value (K2CO3 is an alkaline reagent, increasing its volume will increase the pH).

[0082] Well plate setting: 8 wells in total, add different volumes of K2CO3 solution (0μL to 14μL) to each well, corresponding to different pH values.

[0083] Detection indicators:

[0084] OD value (optical density): The absorbance of the colloidal gold solution at 520 nm (OD520) was measured by a spectrophotometer. The higher the OD value, the more stable the colloidal gold (the better the particle dispersion).

[0085] Color change: Observe the color of the solution with the naked eye (red → blue-purple indicates aggregation).

[0086] Result Analysis

[0087] The relationship between OD value and pH:

[0088] The OD value of well 4 (6 μL K2CO3) was the highest, indicating that colloidal gold was the most stable at this time.

[0089] However: The solution in well 4 showed aggregation (the color became darker or blue-purple), indicating that the colloidal gold particles began to precipitate, which may affect the subsequent labeling efficiency.

[0090] Relationship between color change and pH:

[0091] The OD value of well 3 (4 μL K2CO3) was the second highest, and the color showed no obvious aggregation (still remained red), indicating that the colloidal gold was stable and had not aggregated.

[0092] Final selection:

[0093] Well 3 (4 μL K2CO3) was selected as the optimal pH condition because:

[0094] The OD value is high enough (close to the maximum value) to ensure the stability of colloidal gold;

[0095] No aggregation, ensuring even dispersion of particles after labeling, suitable for subsequent applications.

[0096] refer to Figure 11 When colloidal gold is used to label biological molecules, it is necessary to adjust the amount of protein or peptide added to balance the charge with the surface charge of the colloidal gold, thereby stabilizing the colloidal gold particles and avoiding aggregation.

[0097] The amount of protein or peptide added directly affects the stability of colloidal gold. If too little is added, the colloidal gold cannot be stabilized; if too much is added, it may cause excessive coverage or interfere with the labeling effect.

[0098] Reagents and conditions:

[0099] Colloidal gold solution: fixed volume.

[0100] MCP peptide: The concentration is 1 mg / mL, and the amount added ranges from 0 to 14 μL (8 wells in total).

[0101] Well plate setting: add 0, 2, 4, 6, 8, 10, 12, and 14 μL of peptide solution to wells 1-8, respectively.

[0102] Detection indicators:

[0103] Color change: Observe the color of the colloidal gold solution with the naked eye (red → blue-purple indicates aggregation).

[0104] OD value: Measure the absorbance of colloidal gold at 520 nm (OD520) using a spectrophotometer. The higher the OD value, the more stable the colloidal gold (the better the particle dispersion).

[0105] Result Analysis

[0106] The relationship between color change and protein amount:

[0107] Well 1 (0 μL peptide): No protein was added, and the colloidal gold solution turned blue-purple, indicating severe aggregation (poor stability).

[0108] Well 2 (2 μL peptide): The amount of protein is too small, and slight aggregation can still be seen (the color is bluish-purple), indicating that the protein is not sufficient to stabilize the colloidal gold.

[0109] Well 5 (8 μL polypeptide): The color remains red and there is no aggregation, indicating that the protein amount is moderate and the colloidal gold is stable.

[0110] The relationship between OD value and protein amount:

[0111] The OD value of well 5 (8 μL of polypeptide) was the highest, indicating that the colloidal gold was the most stable (the particle dispersion was the best) at this time.

[0112] The OD values of other wells (such as wells 1 and 2) decreased significantly, indicating that the aggregation of colloidal gold led to a decrease in absorbance.

[0113] in conclusion:

[0114] Well 5 (8 μL peptide) was selected as the optimal labeling amount because:

[0115] The OD value is the highest, and the colloidal gold is the most stable;

[0116] No aggregation, color remains red;

[0117] This indicates that the binding efficiency between MCP polypeptide and colloidal gold is optimal under these conditions.

[0118] refer to Figure 12 , colloidal gold immunochromatography technology, the test strip consists of a sample pad, a colloidal gold pad, a nitrocellulose membrane (NC membrane) and a water absorbent pad.

[0119] T line (test line): immobilized with antibodies or antigens that can bind to target analytes (such as specific proteins or peptides).

[0120] Line C (quality control line): A second antibody that can bind to the colloidal gold-labeled antibody is fixed on it and is used to verify whether the test is effective.

[0121] When the sample contains the target analyte, the colloidal gold-labeled antibody binds to the target, forming a visible color band at the T line; regardless of whether the target is detected, the C line will show color to confirm that the test process is normal.

[0122] Experimental results analysis

[0123] Positive sample detection

[0124] Operation: Add a positive sample containing the target analyte (such as a specific protein or peptide) onto the test strip.

[0125] result:

[0126] Within 5-10 minutes, the T line and C line will appear simultaneously (usually red or purple stripes).

[0127] Conclusion: T line coloration indicates that the target analyte is detected (positive), and C line coloration indicates that the test process is effective.

[0128] Advantages: The detection time is extremely short (only a few minutes), suitable for rapid screening.

[0129] Negative control test

[0130] Procedure: Add a negative control (such as purified water) that does not contain the target analyte onto the test strip.

[0131] result:

[0132] Within 5-10 minutes, only the C line will show color, while the T line will not show color.

[0133] Conclusion: The target analyte was not detected (negative), and the C line color development indicated that the test process was effective.

[0134] Significance: Verify the specificity of the test strip (responds only to the target analyte).

[0135] Comparison with PCR testing

[0136] PCR test:

[0137] Principle: Amplify the target DNA fragment through polymerase chain reaction (PCR) and detect its presence or absence.

[0138] step:

[0139] Sample processing (DNA extraction);

[0140] PCR amplification (denaturation, annealing, and extension cycles);

[0141] Result detection (gel electrophoresis or fluorescent probe).

[0142] Time required: Approximately 1 hour (including sample processing and detection steps).

[0143] Test strips vs. PCR:

[0144] speed:

[0145] Test strips: Rapid results in 5-10 minutes, suitable for on-site instant testing.

[0146] PCR: More than 1 hour, requires laboratory equipment and professional operation.

[0147] accuracy:

[0148] Test strips: Depends on the specificity of antigen-antibody binding, and may have false positives / false negatives (needs to be verified in combination with other methods).

[0149] PCR: High specificity (based on DNA sequence) and higher sensitivity, but the steps are complex.

[0150] Application scenarios:

[0151] Test strips: rapid screening, on-site initial screening (such as rapid disease diagnosis, food safety testing).

[0152] PCR: Confirmation, quantitative analysis, scientific research-grade testing.

[0153] 4. Advantages of test strips

[0154] Speed: The test can be completed in 5-10 minutes without the need for complex equipment.

[0155] Simplicity: easy to operate, readable by naked eyes, suitable for non-professionals.

[0156] Low cost: The production cost of test strips is low, making them suitable for large-scale screening.

[0157] Portability: It can be used anytime and anywhere, especially suitable for resource-limited environments.

[0158] refer to Figure 13 , the test strips have the ability to accurately detect target pathogens without nonspecific reactions with other pathogens. Poor specificity may lead to false positive results and affect diagnostic accuracy.

[0159] This test strip is designed to detect target pathogens, while WSSV, DIV1, and HLV are other pathogens that may be confused with the target pathogens.

[0160] Experimental design

[0161] Test sample:

[0162] Positive control: target pathogen.

[0163] Non-target pathogens: White spot virus (WSSV), Decapoda iridescent virus (DIV1), and highly pathogenic Vibrio (HLV).

[0164] Operation: Add the extracts of the above pathogens to the test strips respectively and observe the color development of the T line (test line) and C line (quality control line).

[0165] Result Analysis

[0166] Target pathogen detection:

[0167] Both the T line and the C line are colored: indicating that the test strip successfully detects the target pathogen (positive result).

[0168] Non-target pathogen detection:

[0169] The T line does not show color, and only the C line shows color: This indicates that the test strip does not bind to non-target pathogens such as WSSV, DIV1, and HLV, and there is no cross-reaction.

[0170] in conclusion:

[0171] The test strip has good specificity and only reacts to the target pathogen without being interfered with by other related pathogens.

[0172] Key Principles

[0173] Antigen-antibody specific binding:

[0174] The test line (T line) of the test strip is immobilized with antibodies against specific antigens of the target pathogen.

[0175] When the target pathogen is present in the sample, its antigen binds to the colloidal gold-labeled antibody and forms a visible band at the T line.

[0176] Antigens from non-target pathogens have no binding ability to T-line antibodies and therefore do not trigger color development.

[0177] Avoidance of cross-reactivity:

[0178] By selecting highly specific antibodies (which only recognize unique antigenic epitopes of the target pathogen), cross-reactions with other pathogens can be effectively avoided.

[0179] refer to Figure 14, evaluate the detection sensitivity of colloidal gold test strips and Dot Blot technology for EHP and determine its minimum detection limit.

[0180] The sensitivity of the test strip was calculated according to the national standard qPCR. The copy number of EHP in 1 μL DNA solution was 8.93×106 copies, and the copy number of EHP after 20 μL enzymatic hydrolysis was 5.6×107. The 20 μL enzymatic hydrolysis EHP was diluted in the proportions of 1:2, 1:4, 1:6, 1:8, and 1:10. As shown in the figure, the highest dilution factor was 1:8, from which the lowest detection line was 7×106 EHP copies. 1.5 μL was spotted onto the DotBlot plate and diluted in a certain proportion to calculate the lowest detection line. As shown in the figure, the highest dilution factor was 1:80, from which the lowest detection line of the DotBlot detection method was 5.2×104 copies.

[0181] refer to Figure 15 The test strip conformity test results showed that 20 samples of whiteleg shrimp were tested using three detection methods: colloidal gold test strips, Dot Blot and national standard PCR. The Kappa values of colloidal gold test strips and Dot Blot compared with national standard PCR were 0.78 and 0.886 respectively. The results showed that the accuracy of the two methods was good.

[0182] refer to Figure 16 , Figure 16 The results are compared between colloidal gold test strips and PCR detection methods.

[0183] Furthermore, the test materials are as follows:

[0184] The pmal-c5X plasmid was preserved in the laboratory, the colloidal gold solution was prepared in the laboratory, the positive samples of shrimp hepatocellular carcinoma and common shrimp diseases were provided by a farm in Guangdong, and the polypeptide sequence was synthesized by Shanghai Bioengineering.

[0185] Furthermore, the test reagents are as follows:

[0186] DL2000 DNA Marker and PrimeSTAR Max DNA Polymerase were purchased from TaKaRa, competent cells were purchased from TransGen, seamless cloning kit and Bradford protein concentration assay kit were purchased from Shanghai Beyotime, BamHⅠ rapid endonuclease, purification, plasmid extraction, and nucleic acid extraction kits were purchased from TianGen, starch resin was purchased from Sangon Biotechnology, PH.D. TMPHage Display Peptide Library Kits v2 were purchased from NEB, Goat anti-Mouse IgG (H+L) Secondary Antibody and HRP were purchased from Invitrogen, HRP-streptavidin was purchased from SolarBio, and gold reagent was purchased from Sigma-Aldrich. Test strip consumables were purchased from Shanghai Jiening Biotechnology.

[0187] Furthermore, the main instruments of the test are as follows:

[0188] The blotting substrate gel imaging system and electrophoresis instrument were purchased from Shanghai Tianneng Company, the PCR instrument was purchased from Bio-Rad Company, the bacterial incubator was purchased from Hualida Company, the constant temperature shaker (THZ-92A) was purchased from Shanghai Shenxian Equipment Factory, the ultra-clean workbench (VD-850) was purchased from LICHEN Company, and the test strip equipment were all purchased from Shanghai Jinbiao Biological.

[0189] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for screening and rapid detection of peptides interacting with shrimp hepatoenteric cytozoan spore wall proteins, characterized in that: The following steps are involved: S1, construction and extraction of Pmal-EhSW12 recombinant plasmid; S2, expression and purification of recombinant proteins; Screening of peptides interacting with S3 and EhSW12 proteins; S4, peptide affinity identification; S5. Rapid detection of EHP spore wall protein EhSW12.

2. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 1, characterized in that: When constructing and extracting the recombinant plasmid in S1, first, according to the target gene EhSW12 sequence, BamH1 amplification primers are used to specifically amplify the EhSW12 gene fragment in the subsequent PCR amplification process, total DNA is extracted from the biological sample containing the target gene, and the DNA fragment of the required size is separated by agarose gel electrophoresis, and then the fragment is further purified using a gel purification kit to ensure that a high-quality DNA sample is obtained for subsequent operations. The prokaryotic vector Pmal-c5X(+) is selected and cut with BamH1 restriction endonuclease to prepare to receive the target gene fragment so that the target gene can be inserted. The purified EhSW12 gene fragment and the enzyme-cut vector are ligated using a seamless cloning kit to generate a recombinant plasmid. The recombinant plasmid is introduced into competent bacterial cells TransT1 and cultured on an LB plate containing ampicillin. Bacterial colonies that successfully received the recombinant plasmid are screened, and a single colony is picked for PCR verification to confirm whether the target gene is successfully inserted. Finally, the recombinant plasmid is extracted from the screened positive clones and sequenced to ensure that the inserted target gene sequence is accurate.

3. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 2, characterized in that: When expressing and purifying the recombinant protein in S2, the constructed recombinant plasmid containing the target gene EhSW12 is transformed into BL21 (DE3) Escherichia coli to efficiently express the exogenous protein. A positive single colony cultured overnight is picked from the LB plate and inoculated into a fresh liquid culture medium for expansion. When the bacteria grow to an appropriate density, isopropyl β-D-thiogalactoside is added to induce the expression of the target protein. The bacterial cells are collected by centrifugation, and then ultrasonic treatment is used to lyse the bacteria to release the internal protein. After high-speed low-temperature centrifugation again, a soluble protein containing the protein is obtained. The supernatant and the insoluble protein precipitate were subjected to SDS-PAGE analysis to determine the main form of the recombinant protein. The recombinant protein mainly present in the supernatant was purified by affinity chromatography with the addition of starch resin. The purified recombinant protein was quantified using a Bradford kit. At the same time, an empty vector without an insert was treated according to the same expression and purification steps to prepare a protein with only an MBP tag, providing a control sample for subsequent reverse screening experiments. Finally, all obtained protein samples were stored at -80°C to maintain their activity and stability.

4. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 3, characterized in that: When screening for EhSW12 protein-interacting polypeptides in S3, the recombinant protein MBP-EhSW12 was used as a substrate and PH.D. TM The phage display peptide library in PHage Display Peptide Library Kits v2 was screened. First, MBP-EhSW12 was fixed on a solid support, and then the phage display peptide library was added to allow the polypeptides therein to contact and bind to the substrate. Phages that failed to bind to MBP-EhSW12 were washed away with TBST buffer, and only those phage-peptide complexes that successfully bound were retained. The bound phages were eluted by protein competition, that is, excess free MBP-EhSW12 or other related substances were used to competitively replace the phages bound to the substrate, thereby releasing phages with stronger binding force. The eluted phages were added to fresh Escherichia coli culture medium for infection and amplification. Subsequently, the supernatant of the bacterial culture medium was collected by low-temperature high-speed centrifugation, and the phages were concentrated using PEG / NaCl solution to obtain the phage collection after the first round of screening. The phage collection obtained in the previous round of screening was added to the phage collection obtained in the previous round of screening. The collaboration was used as the starting library for the next round of screening, and the above steps were repeated for a total of four rounds. In each round, phages with stronger binding to MBP-EhSW12 were enriched. Since the MBP tag itself is large, in order to exclude nonspecific binding caused by the MBP tag rather than the EhSW12 protein itself, a reverse screening step was introduced in the third round of screening. During the reverse screening, the expressed protein with only the MBP tag was used as a substrate, and the eluate at the end of the second round was used to bind to it, and the unbound phage was eluted and used for subsequent screening to reduce the impact of nonspecific binding. After the last round of screening, 20 phage plaques were randomly selected from the double-layer plate and sent to a biological company for DNA sequencing to determine the polypeptide sequence encoded by it. The screening effect was evaluated by comparing the changes in phage titers between different rounds, and sequence analysis was used to confirm which polypeptides were truly specifically bound to MBP-EhSW12.

5. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 4, characterized in that: When performing peptide affinity identification in the S4, the three screened peptides, the recombinant protein MBP-EhSW12, the protein with only the MBP tag, and PBS as a negative control are respectively added dropwise to the PVDF membrane. Different binding recognition substances are added to each point on the PVDF membrane, including the recombinant protein MBP-EhSW12, the protein with only the MBP tag, and the PBS control. First, a monoclonal antibody against the MBP tag is added as a primary antibody, and then an HRP-labeled goat anti-mouse antibody is added as a secondary antibody. This antibody can recognize and bind to the primary antibody, thereby indirectly marking the peptide bound to MBP-EhSW12. W12 or the position of the protein containing only the MBP tag. Finally, the HRP-catalyzed substrate reaction produces a visible color change through chemiluminescence and other methods, thereby intuitively showing which points have undergone specific binding, helping to confirm which peptides have specific interactions with EhSW12. Further compare the affinities of the three peptides for MBP-EhSW12, and repeat the above dotblot experiment after diluting each peptide at different ratios. By comparing the signal intensity at different concentrations, it is determined which peptide can effectively bind to MBP-EhSW12 even at low concentrations, thereby determining their relative affinities. Based on the dot blot results, the peptide showing stronger binding ability is selected for subsequent processing.

6. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 5, characterized in that: In the S5, when performing rapid detection of the EHP spore wall protein EhSW12, EHP is separated from shrimp hepatopancreas tissue using Percoll density gradient centrifugation, the separated EHP is treated by enzymatic hydrolysis or mechanochemical method to release the spore wall protein EhSW12, a colloidal gold solution is used to label a biotinylated polypeptide 3, the screened specific polypeptide 2 is sprayed on the test line of the test strip, and streptavidin is sprayed on the quality control line of the test strip. The above components are integrated together to form a complete rapid detection test strip, the enzymatically hydrolyzed EhSW12 protein is used as a substrate and spotted on a membrane, the biotin-labeled polypeptide 3 is used as a test substance and dropped onto the sample point on the membrane, and HRP-labeled streptavidin is added. This enzyme-labeled secondary antibody can recognize and bind to the biotin-labeled polypeptide 3, thereby indirectly marking the position of the polypeptide bound to the substrate. Finally, the results are observed by color development to determine whether a specific protein-polypeptide interaction exists.

7. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 6, characterized in that: When using the Percoll density gradient centrifugation method to separate EHP from the hepatopancreas tissue of shrimp, first use 1.5mol / L NaCl solution and Percoll stock solution in a volume ratio of 1:9 to prepare 100% Percoll stock solution. Then, the stock solution is diluted with 0.15mol / L NaCl to prepare 50% and 75% Percoll working solutions. Percoll solutions of different concentrations are used to construct density gradients. 2mL of 100%, 75%, and 50% Percoll solutions are stacked in the centrifuge tube in sequence. The gradient separation system is constructed by the density difference between the layers. The tissue homogenate is filtered through 4 layers of gauze to remove larger tissue fragments. The microsporidia are initially enriched by differential centrifugation and repeated washing with PBS buffer to obtain a crude extract. Differential centrifugation uses different centrifugal forces to remove impurities and gradually concentrate the target microsporidia. Carefully add the crude microsporidium extract obtained above to the prepared Percoll gradient system to avoid disrupting the gradient. Centrifuge at 15,000 × g at 4°C for 30 minutes to allow components of different densities to distribute along the gradient. Collect the precipitates at each gradient interface and wash repeatedly by centrifugation with normal saline to remove residual Percoll solution. Finally, resuspend the highly purified microsporidia in normal saline. Add 1 mg / mL trypsin and 2 mg / mL chitosanase to the purified EHP suspension and incubate at 37°C for 2-4 hours with gentle stirring during incubation. Gentle stirring promotes effective tissue lysis and ensures sufficient release of the target protein.

8. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 7, characterized in that: When using the mechanochemical method to treat the isolated EHP, the purified EHP spores were suspended in a lysis buffer containing PBS, 50mMTris-HCl, 1% SDS and 1% Triton X-100, and glass beads with a diameter of 0.1-0.5mm were added. The spores were treated at a high-speed shaker at 6000rpm for 3 times, each time for 1 minute, and ice bath cooling was performed between each time to prevent the sample from overheating and causing protein denaturation or inactivation. 10mM DTT was added to the solution and incubated at 60℃ for 10 minutes to destroy the disulfide bonds in the protein and help completely cleave the protein structure. According to the characteristics of the spore wall components, 2% β-mercaptoethanol was added to further promote protein denaturation to ensure the maximum release of the target protein. Coomassie brilliant blue staining was used to observe the quality and concentration of protein samples obtained by different lysis methods, so as to select the best lysis method. The protein samples treated with the selected lysis method were separated by SDS-PAGE electrophoresis and then wet-transferred to PVDF membrane under a constant voltage of 100V for 50 minutes. The PVDF membrane was blocked with 5% skim milk for 2 hours to reduce non- For specific binding, incubate the biotin-labeled peptide 3 overnight at 4°C to give it a chance to bind to the target protein on the membrane. Use PBST buffer to shake and wash the membrane for 6 minutes, repeat 7 times to remove unbound peptides, and incubate the membrane with HRP-labeled streptavidin for 2 hours at room temperature. Streptavidin can bind tightly to biotin to form a complex. Wash the membrane thoroughly with PBST buffer again to remove excess HRP-labeled streptavidin. Finally, perform a color reaction on a PVDF membrane for 30 seconds, and then use an ECL gel imaging system to capture and analyze the results to confirm the binding of Bio-peptide 3 to the target protein.

9. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 8, characterized in that: When using colloidal gold solution to label biotinylated peptide 3, take a series of 8 tubes, add 200 μL of colloidal gold solution to each tube, and add different amounts of 0.1 mol / L K2CO3 to each tube to adjust the pH of the colloidal gold solution. After adjusting the pH, add an excess of biotinylated peptide 3 to each tube, add 10 μL of 10% NaCl, mix well, and let it stand. Observe the color change and measure the absorbance at 520 nm using a spectrophotometer to determine the optimal pH. Take another series of 8 tubes, add 200 μL of colloidal gold solution to each tube, and add an appropriate amount of K2CO3 according to the previously determined optimal pH. Add different amounts of 1 mg / mL biotinylated peptide 3 to each tube, and also add 10 μL 10% NaCl, mix well and let it stand. The optimal amount of biotinylated peptide labeling was determined by observing the color change and measuring the OD value at 520nm. 800μL of colloidal gold solution was placed in a centrifuge tube. The corresponding amount of K2CO3 was added according to the optimal pH value determined previously. The mixture was thoroughly mixed and the optimal amount of biotinylated peptide 3 was added. After mixing, 200μL of 5% BSA was added. BSA was used as a stabilizer to prevent nonspecific adsorption. The mixture was centrifuged at 10000rpm for 30 minutes at 4°C. The supernatant was discarded and the precipitate was retained. 100μL of 0.01mol / L PBS buffer containing 5% trehalose and 1% BSA was added to the precipitate as a gold labeling solution for resuspending to obtain the final colloidal gold labeled peptide solution. The prepared gold label peptide solution was sprayed onto the gold label pad. The entire process was performed on ice to maintain the stability of the colloidal gold particles.

10. The method for screening and rapid detection of shrimp hepatoenteric cytozoan spore wall protein-interacting polypeptides according to claim 9, characterized in that: When assembling the rapid test strips, use a strip cutter to cut the gold label pad, sample pad and absorbent pad into 9mm, 15mm and 17mm widths respectively, soak them in PBS buffer containing 2% Tween-20 and 5% trehalose for 30 minutes, and then soak them in PBS buffer containing 0.2% Triton. The pad was soaked in X-100 PBS buffer for 30 minutes. The soaked pad was thoroughly dried and set aside to remove excess moisture and prevent mold growth or other contamination. A test line and a quality control line were accurately drawn on the nitrocellulose membrane using an XYZ three-dimensional film spray gold apparatus. The test line was sprayed with the screened specific peptide 2, and the quality control line was sprayed with streptavidin. The prepared colloidal gold-labeled peptide solution was added dropwise to the gold-labeled pad until saturated, and then dried again for later use. Hepatopancreatic tissue homogenate was extracted from positive shrimp samples infected with different viruses. The tissue homogenate was treated to release the target protein and applied to the assembled colloidal gold test strip. The results were observed. If the target protein was present, it would bind to the labeled peptide on the test line, forming a visible color change. Similarly, the enzymatically hydrolyzed target protein was used as a substrate, and the biotin-labeled peptide 3 was used as a detector. HRP-labeled streptavidin was added for color development, and the results were observed to confirm specific binding.

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