A TGEV IgA antibody fluorescent microsphere immunochromatographic test strip and its preparation and application

Abstract

The present application relates to the technical field of antibody detection, in particular to a TGEV IgA antibody fluorescent microsphere immunochromatography test strip and preparation and application thereof.The TGEV IgA antibody fluorescent microsphere immunochromatography test strip comprises a base plate, and a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially and lapped and pasted on the base plate; the sample pad is a glass fiber membrane, and the glass fiber membrane is coated with a fluorescent microsphere labeled TGEV-S1 recombinant protein conjugate and a fluorescent microsphere labeled rabbit IgG antibody conjugate; the nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is coated with a goat anti-pig IgA antibody, and the quality control line is coated with a goat anti-rabbit IgG antibody.The test strip can accurately capture low-titer TGEV IgA antibodies, is suitable for high-throughput rapid on-site detection, and has the characteristics of low reagent cost and detection difficulty, high sensitivity and high specificity.

Description

Technical Field

[0001] This invention relates to the field of antibody detection technology, and in particular to a TGEV IgA antibody fluorescent microsphere immunochromatographic test strip and its preparation and application. Background Technology

[0002] In the prevention and control of porcine transmissible gastroenteritis virus (TGEV), rapid, sensitive, and field-operable detection methods are crucial. Currently, traditional laboratory detection techniques mainly include reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). While RT-PCR is highly sensitive, it requires expensive equipment and is complex to operate, making it difficult to meet the needs of rapid field testing. ELISA primarily detects IgG antibodies in serum, which is insufficient to reflect mucosal immune levels and lacks immediacy.

[0003] In recent years, immunochromatography (ICA) has been widely used in virus detection as a rapid on-site detection method due to its ease of operation and intuitive results. However, traditional colloidal gold immunochromatographic test strips have drawbacks such as low sensitivity and difficulty in achieving quantitative analysis. To address this issue, researchers have begun to explore labeling with higher sensitivity. For example, Du et al. used time-resolved fluorescent nanoparticles (TRFNPs) to label PEDV antibodies, establishing a fluorescent microsphere immunochromatographic test strip that successfully achieved rapid quantitative detection of PEDV antigen, with a sensitivity more than 10 times higher than that of traditional colloidal gold test strips. This study demonstrates that fluorescent microsphere labeling technology can significantly improve the sensitivity and specificity of immunochromatographic detection.

[0004] However, while fluorescent microsphere immunochromatography has made breakthroughs in the detection of viruses such as PEDV, research on TGEV has lagged behind. Currently, there is a lack of TGEV-specific fluorescent microsphere immunochromatographic test strips on the market. Considering the extremely high lethality of TGEV and its tendency to cause large-scale mortality in suckling piglets, there is an urgent need for an immunochromatographic tool that can rapidly detect TGEV on-site. Therefore, developing a TGEV-specific S1 protein-based fluorescent microsphere immunochromatographic test strip would not only fill a market gap but also provide strong technical support for TGEV prevention and control. Summary of the Invention

[0005] To overcome the shortcomings and drawbacks of existing fluorescent immunochromatographic detection methods for rapid on-site detection of porcine transmissible gastroenteritis virus (TGEV), such as insufficient sensitivity and complex operation, the primary objective of this invention is to provide a TGEV IgA antibody fluorescent microsphere immunochromatographic test strip that can be used for on-site detection of TGEV-specific IgA antibodies.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned test strip.

[0007] Another object of the present invention is to provide the application of the above-mentioned test strip.

[0008] The objective of this invention is achieved through the following technical solution: A TGEV IgA antibody fluorescent microsphere immunochromatographic test strip includes a base plate, and a sample pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped and pasted on the base plate; the sample pad is a glass fiber membrane, and the glass fiber membrane is coated with a TGEV-S1 recombinant protein conjugate labeled with fluorescent microspheres and a rabbit IgG antibody conjugate labeled with fluorescent microspheres; the nitrocellulose membrane is provided with a detection line and a control line, wherein the detection line is coated with goat anti-pig IgA antibody, and the control line is coated with goat anti-rabbit IgG antibody.

[0009] The preferred amount of TGEV-S1 recombinant protein conjugate coated with fluorescent microspheres on the glass fiber membrane is 0.55 μg / cm, and the preferred amount of rabbit IgG antibody conjugate coated with fluorescent microspheres is 0.30 μg / cm.

[0010] The preferred amount of goat anti-pig IgA antibody coated on the detection line is 0.01 mg / cm, and the preferred amount of goat anti-rabbit IgG antibody coated on the quality control line is 0.05 mg / cm.

[0011] The fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugate was prepared by the following method: the fluorescent microspheres were pre-washed, activated by EDC / NHS, acid-washed, resuspended, and then mixed with TGEV-S1 recombinant protein for conjugation. Finally, the conjugate was blocked to obtain the fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugate.

[0012] The specific pre-cleaning procedure is as follows: centrifuge the fluorescent microspheres and remove the supernatant; add sodium dihydrogen phosphate solution with pH=6.0, sonicate to resuspend, centrifuge, and remove the supernatant.

[0013] The specific operation for EDC / NHS activation is as follows: phosphate buffer containing EDC and phosphate buffer containing NHS are added to the pre-cleaned fluorescent microspheres and activated for 15-30 min; wherein the mass ratio of EDC to NHS is 1:1.

[0014] The ratio of fluorescent microspheres to recombinant TGEV-S1 protein is 10:1.

[0015] The coupling conditions are room temperature coupling for 2 hours.

[0016] The blocking reagent is preferably 2% (w / v) BSA, and the blocking condition is preferably room temperature blocking for 1 h.

[0017] The preferred nucleotide sequence encoding the TGEV-S1 recombinant protein is shown in SEQ ID NO. 1.

[0018] The method for preparing the TGEV-S1 recombinant protein includes the following steps: (1) The nucleotide sequence shown in SEQ ID NO. 1 of the gene was synthesized and then ligated with the expression vector to obtain the recombinant expression vector; (2) The recombinant expression vector obtained in step (1) was transformed into BL21(DE3) competent cells to obtain recombinant expression bacteria; (3) The recombinant expression bacteria obtained in step (2) were induced to express, the bacterial cells were collected and resuspended in PBS buffer, and sonicated. The precipitate after disruption was collected, which is the crude recombinant protein inclusion body. (4) The precipitate collected in step (3) is washed, dissolved, purified and refolded in sequence to obtain TGEV-S1 recombinant protein.

[0019] The preparation method of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip includes the following steps: The sample pad, NC membrane, and absorbent pad are then overlapped and pasted onto the base plate in sequence.

[0020] The application of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip in the preparation of products for detecting porcine TGEV IgA antibody or porcine TGEV.

[0021] A method for detecting TGEV IgA antibodies includes the following steps: Take a sample of sow's milk to be tested, defatt it, and add it to the sample pad of the above-mentioned TGEV IgA antibody fluorescent microsphere immunochromatographic test strip. After the chromatographic reaction, observe the fluorescence of the test line and the control line. If both the test line and the control line show fluorescence, the milk sample to be tested contains TGEV IgA antibody; if the test line does not show fluorescence but the control line does, the milk sample to be tested does not contain TGEV IgA antibody.

[0022] The preferred loading volume for the defatted sow milk sample is 50 μL.

[0023] The preferred time for the chromatography reaction is 5 to 10 minutes.

[0024] The above applications or methods are not intended for the treatment or diagnosis of diseases.

[0025] The principle of this invention: After the sample to be tested (such as skim emulsion) is dropped onto the sample pad, the sample liquid redissolves and carries the two fluorescent labels forward for chromatography: When TGEV IgA antibodies are present in the sample, they bind to fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugates, forming a fluorescent microsphere-TGEV-S1 recombinant protein-TGEV IgA antibody complex. This complex undergoes chromatography, and upon migrating to the T-line, it is captured by immobilized goat anti-porcine IgA antibody, resulting in its enrichment at the T-line. Simultaneously, when the fluorescent microsphere-labeled rabbit IgG antibody conjugate crosses the T-line chromatography and reaches the C-line, it is captured by immobilized goat anti-rabbit IgG antibody, resulting in its enrichment at the C-line. Under 365 nm ultraviolet light irradiation, the fluorescent microspheres captured on the T-line and C-line emit bright fluorescent signals. Analysis of these signals using a detector determines whether TGEV IgA antibodies are present in the sow's milk.

[0026] When TGEV IgA antibody is not present in the sample, the TGEV-S1 recombinant protein conjugate labeled with optical microspheres cannot be captured by the T line, and there is no signal on the T line.

[0027] The present invention has the following advantages and effects compared with the prior art. (1) Improving the sensitivity and specificity of on-site detection: Existing ELISA and some immunochromatographic techniques have insufficient sensitivity when detecting TGEVIgA antibodies, making it difficult to accurately detect them in the early or low-concentration infection situations. This invention utilizes the high fluorescence intensity and excellent optical properties of fluorescent microspheres to significantly improve the sensitivity and specificity of detection, achieving accurate capture of low-titer TGEVIgA antibodies.

[0028] (2) Achieving high-throughput rapid on-site detection: Traditional laboratory testing methods (such as RT-PCR) are costly and time-consuming, failing to meet the needs of pig farms for rapid decision-making. This invention develops a portable and easy-to-use immunochromatographic test strip, which, combined with the luminescent properties of fluorescent microspheres, enables rapid on-site detection without relying on large instruments, shortening the detection time and improving prevention and control efficiency.

[0029] (3) Reduce detection costs and operational barriers: In response to the problems of interference from live vaccine and the strong dependence of traditional detection methods on equipment, this invention reduces reagent costs and detection difficulty by optimizing the test strip process and fluorescent microsphere labeling system, so that grassroots farms or front-line epidemic prevention personnel can easily operate it. Attached Figure Description

[0030] Figure 1 This is a graph showing the hydrophilicity and hydrophobicity results of the TGEV-S1 protein.

[0031] Figure 2 This is a graph showing the prediction results of the TGEV-S1 protein signal peptide.

[0032] Figure 3 This is an analysis of the predicted transmembrane domains of the TGEV-S1 protein.

[0033] Figure 4 This is a spectrum of the TEGV-S1-28a plasmid.

[0034] Figure 5 This is a graph showing the preliminary induction and solubility analysis results of TGEV-S1 recombinant protein; where M: protein marker, 1: bacterial supernatant before induction, 2: bacterial supernatant after induction, 3: supernatant after ultrasonic disruption, and 4: precipitate after ultrasonic disruption.

[0035] Figure 6 The figures show the SDS-PAGE and Western blot results for different induction times of TGEV-S1 recombinant protein; where a is the SDS-PAGE analysis, M: protein marker, 1-4: 2, 4, 6, 8 h respectively; b is the Western blot analysis, 1-4: 2, 4, 6, 8 h respectively.

[0036] Figure 7 The figures show the SDS-PAGE and Western blot results for different concentrations of inducer for TGEV-S1 recombinant protein. In figure a, SDS-PAGE analysis is shown, M: protein marker, 1-4: 0.4, 0.6, 0.8, and 1.0 mmol / L IPTG, respectively; in figure b, Western blot analysis is shown, 1-4: 0.4, 0.6, 0.8, and 1.0 mmol / L IPTG, respectively.

[0037] Figure 8 The images show the SDS-PAGE and Western blot results for different induction temperatures of the TGEV-S1 recombinant protein. Figure a shows the SDS-PAGE analysis, where M is the protein marker and 1-4 represent 37, 28, 22, and 16℃ respectively. Figure b shows the Western blot analysis, where 1-4 represent 37, 28, 22, and 16℃ respectively.

[0038] Figure 9 This is an analysis diagram of the purification results of TGEV-S1 recombinant protein; where M: protein marker, 1: inclusion body precipitation (precipitate after ultrasonic disruption), 2: bacterial cell precipitation after induction, 3: bacterial supernatant before induction, 4: bacterial supernatant after induction, and 5: purified protein.

[0039] Figure 10 This is a chromatogram of the reactivity analysis of TGEV-S1 recombinant protein; where 1 and 2 represent the purified TGEV-S1 recombinant protein.

[0040] Figure 11 This is a schematic diagram of the structure of a fluorescent microsphere immunochromatographic test strip.

[0041] Figure 12 This is an analysis of the results of screening for the optimal antigen concentration in the assembly of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips.

[0042] Figure 13 This is an analysis of the preliminary evaluation results of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip; left: TGEV IgA antibody positive colostrum; right: TGEV IgA antibody negative colostrum.

[0043] Figure 14 This is an analysis of the results of screening for the optimal reaction time of TGEV IgA fluorescent microsphere immunochromatographic test strips.

[0044] Figure 15 This is an analysis of the specificity evaluation results of the TGEV IgA fluorescent microsphere test strip; from left to right, they are: TGEV IgA antibody-positive colostrum, PEDV IgA antibody-positive colostrum, and rotavirus IgA antibody-positive colostrum.

[0045] Figure 16 This is an analysis chart of the sensitivity evaluation results of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip. The dilutions from left to right are: 1:2, 1:10, 1:50, 1:250, and 1:1250.

[0046] Figure 17 This is an analysis of the intra-batch repeatability test results of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strips; where, left: the test results of the same batch of test strips on the same TGEV IgA positive colostrum; right: the test results of the same batch of test strips on the same TGEV IgA negative colostrum.

[0047] Figure 18 This is an analysis of the batch-to-batch repeatability of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips; left: test results of batches 1, 2, and 3 on the same TEGV IgA-positive colostrum; right: test results of batches 1, 2, and 3 on the same TEGV IgA-negative colostrum. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0049] Biomaterials, reagents, etc. involved in the examples: The pET-28a vector was obtained from the Veterinary Microbiology and Immunology Laboratory of South China Agricultural University and is commercially available.

[0050] Mouse-derived anti-TGEV S1 protein monoclonal antibody was purchased from Guangzhou Qianxun Biotechnology Co., Ltd.; antibiotics Kan and IPTG were purchased from Sigma-Aldrich, USA; and color-stained pre-stained protein molecular weight standards were purchased from Shanghai Yamei Biotechnology Co., Ltd.

[0051] Colostrum from sows that were positive for TGEV IgA antibodies, negative for TGEV IgA antibodies, positive for PEDV IgA antibodies, and positive for rotavirus IgA antibodies were obtained from the Guangdong Academy of Agricultural Sciences. Rabbit IgG antibodies were purchased from Beyotime Biotechnology Co., Ltd., goat anti-pig IgA antibodies were purchased from Thermo Fisher Scientific, and goat anti-rabbit IgG antibodies were purchased from Sigma-Aldrich. The porcine transmissible gastroenteritis virus IgA (TGEV-IgA) antibody assay kit (ELISA) was purchased from Quanzhou Ruixin Biotechnology Co., Ltd.

[0052] Example 1

[0053] I. Experimental Methods 1. TGEV S1 protein structure prediction (1) Primary structure analysis: The hydrophilicity and hydrophobicity of the TGEV S1 protein were analyzed using the online tool Prot Scale (https: / / www.expasy.org / ) in ExPASy.

[0054] (2) Signal peptide analysis: The protein signal peptide of TGEV S1 was predicted using an online website (https: / / www.novopro.cn / tools / signal / ).

[0055] (3) Transmembrane domain analysis: Transmembrane domain prediction of TGEV S1 protein was performed using an online website (https: / / www.novopro.cn / tools / tmhmm.html / ).

[0056] 2. Construction of the recombinant TEGV-S1-28a transfer vector The full-length gene sequence of the S1 protein from TGEV Purdue strain was obtained from GenBank and optimized based on the codon bias of E. coli. The optimized gene information was sent to Shanghai Sangon Biotech Co., Ltd., which synthesized the gene sequence and introduced BamHI and XhoI restriction enzyme sites into the synthesized gene. The sequence was then ligated into the pET-28a vector through the BamHI and XhoI restriction sites. The ligation product was transformed into DH5α competent cells, screened by plate culture, and positive bacteria were selected for expansion culture. Plasmids were extracted from the positive bacterial cultures and identified to obtain the recombinant expression plasmid TEGV-S1-28a.

[0057] Optimized full-length gene sequence of TGEV S1 protein (SEQ ID NO.1): 3. Expression, purification, and identification of TEGV-S1 recombinant protein (1) Obtaining recombinant expression bacteria ① Remove BL21(DE3) competent cells from the -80℃ freezer, place them on ice, thaw them, add 10 μL of recombinant expression plasmid TEGV-S1-28a, gently mix, incubate on ice for 30 min, then heat shock in a 42℃ water bath for 90 s, and then quickly place them on ice for 2 min, without shaking the centrifuge tube. Then add 900 μL of sterile, antibiotic-free LB liquid medium to the centrifuge tube, mix well, and incubate at 37℃, 200 r / min for 1 h. Centrifuge at 2500 r / min for 3 min, discard the 900 μL supernatant, resuspend the bacterial cells in the remaining medium, and spread them evenly on a Kan LB solid culture plate containing the corresponding amount. Place the culture plate upright in a 37℃ incubator for 10 min until the bacterial solution is completely absorbed, then invert the culture plate and incubate for 16 h. Observe the results.

[0058] ② Pick a single colony and place it in 5 mL of liquid LB medium supplemented with Kans. Incubate at 37°C and 200 r / min for 15 h with constant temperature shaking. Send the culture to Qingke Biotechnology for sequencing. Compare the sequencing results with the synthesized plasmid and retain positive bacteria. Mix the positive bacteria with 50% glycerol at a volume ratio of 7:3 and store in a 1.5 mL centrifuge tube.

[0059] (2) Preliminary induction of TEGV-S1 recombinant protein expression and identification of solubility ① Add the positive recombinant expression bacterial solution prepared in step (1) to liquid LB medium containing Kan at a volume ratio of 1:100, and shake to obtain OD 450 The pH was adjusted to 0.6, IPTG was added, and induction was performed for 6 h, with no IPTG induction serving as a control. After induction, the cells were centrifuged at 5000 r / min for 10 min, and the supernatant and bacterial pellet were collected separately and resuspended in PBS buffer. The bacterial cells were then sonicated with the following parameters: power 400 W, sonication every 5 s, with a treatment interval of 10 s, for a total of 40 min. After sonication, the cells were centrifuged at 10000 r / min for 10 min, and the supernatant before and after disruption, as well as the disrupted pellet, were collected separately. Subsequently, 80 μL of each sample was mixed with 20 μL of 5% (w / v) SDS-PAGE Loading Buffer, boiled for 10 min, cooled, and stored at -20℃.

[0060] ② Clean the glass plate with tap water, place it in a vertical rack, secure it, fill with ultrapure water, and check for leaks for 5 minutes. Prepare the protein gel according to the steps of the protein gel kit (Guangzhou Yamei Company). After the protein gel solidifies, fix it on the electrophoresis tank and remove the comb. Add 10 μL of each sample to the corresponding well, and add 5 μL of protein marker as a control. Pour electrophoresis buffer into the electrophoresis tank, connect the power supply, and set the sample voltage in the electrophoresis parameters: 80 V for concentrating the gel and 120 V for separating the gel. Stop electrophoresis after about 2 hours, remove the gel, and place it in Coomassie Brilliant Blue staining solution. Shake and stain at room temperature for about 30 minutes. After staining, recover the Coomassie Brilliant Blue staining solution and replace it with Coomassie destaining solution for overnight destaining.

[0061] (3) Optimization of induction conditions for TEGV-S1 recombinant protein ① Set four different induction times: 2 h, 4 h, 6 h, and 8 h, with an induction temperature of 37℃ and an IPTG concentration of 1.0 mmol / L. The remaining steps are the same as in step (2). Prepare and collect protein samples with different induction times and perform SDS-PAGE analysis.

[0062] ② Set up 4 different concentrations of IPTG: 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, and 1.0 mmol / L, with an induction temperature of 37℃ and an induction time of 6 h. The remaining steps are the same as in step (2). Prepare and collect protein samples with different inducing agent concentrations and perform SDS-PAGE analysis.

[0063] ③ Set four different induction temperatures: 37℃, 28℃, 22℃ and 16℃, induction time 6 h, IPTG concentration 1.0 mmol / L, and the rest of the steps are the same as step (2). Prepare and collect protein samples at different induction temperatures and perform SDS-PAGE analysis.

[0064] (4) Purification of TEGV-S1 recombinant protein Following the optimal induction conditions explored in step (3), the recombinant protein was induced extensively. The precipitate after ultrasonic disruption was collected according to step (2). After obtaining crude recombinant protein inclusion bodies, the protein was washed, dissolved, purified, and refolded in sequence. The specific steps are as follows: ① Inclusion body washing and dissolution: Resuspend the recombinant protein inclusion bodies in 15 mL of pre-chilled PBS washing buffer and mix at room temperature for 1 h at a speed of 80-100 r / min; then centrifuge at 4 °C and 10,000 r / min for 15 min and collect the precipitate. Second wash of the precipitate: Resuspend the precipitate in 15 mL of 50 mmol / L Tris-HCl, gently shake at room temperature for 1 h, then centrifuge at 4 °C and 10,000 r / min for 15 min and collect the precipitate. Third wash of the precipitate: Resuspend the precipitate in 10 mL of a solution containing 8 mol / L urea (50 μM Tris-HCl + 500 mM NaCl + 8 M urea, pH=7.4), place in a 4 °C refrigerator, shake every 30 min for 4 times, and let stand overnight. After the solution becomes clear and transparent, centrifuge at 4 °C and 10,000 r / min for 15 min and collect the supernatant.

[0065] ② Protein purification: Remove IDA (iminodiacetic acid) from the 4℃ freezer, invert and resuspend it. Transfer 2 mL to an affinity chromatography column (Tiandi Renhe, catalog number: SA004GC05). Allow the IDA to settle to the bottom, remove the stopper, and discard the supernatant. Replace the stopper, add 8 mL of ddH2O to the affinity chromatography column, invert and resuspend the IDA, allow it to settle naturally, and discard the supernatant. Slowly add 10 mL of Binding Buffer (50 mM Tris-HCl, 500 mM NaCl, 8 M urea, 10 mM imidazole, pH 7.4), invert again to resuspend the IDA, allow it to settle naturally, and discard the supernatant. Repeat twice. Slowly add 10 mL of the supernatant collected in step ① (i.e., the denatured protein) to the affinity chromatography column, remove the stopper, discard the liquid, and incubate at 4℃ overnight with shaking until all protein is bound. The following day, the column was washed with Wash Buffer (50 mM Tris-HCl, 500 mM NaCl, 8 M urea, 30 mM imidazole, pH 7.4), and the target protein was eluted with an appropriate amount of Elution Buffer (50 mM Tris-HCl, 500 mM NaCl, 8 M urea, 300 mM imidazole, pH 7.4). 80 μL of the purified protein sample was taken for SDS-PAGE analysis to determine the purification effect.

[0066] ③ Protein refolding: Cut a dialysis bag to a suitable size, boil for 10 min to remove surface grease, and pre-cool by immersing in ice water. After pre-cooling, clamp one end of the dialysis bag with dialysis clamps, and gently inject the purified protein into the other end to remove air bubbles from the membrane. Then, clamp the other end of the dialysis bag tightly with dialysis clamps to check for leakage. After confirming that there are no leaks, place it in a beaker containing 6 mol / L urea refolding dialysis buffer, place the beaker on a magnetic stirrer, set the speed to 60 r / min, and dialyze for 4-6 h. Gradually decrease the urea gradient according to the concentrations of 6 mol / L, 4 mol / L, 2 mol / L, and 0 mol / L. Finally, replace the urea dialysis buffer in the beaker with pre-cooled PBS buffer, and dialyze overnight at 4°C to obtain the dialysis-refolded TEGV-S1 recombinant protein.

[0067] (5) Reactivity identification of TEGV-S1 recombinant protein After SDS-PAGE electrophoresis in step (4) ②, remove the gel and soak it in pre-cooled electrospinning buffer. Briefly immerse the polyvinylidene fluoride (PVDF) membrane in methanol to activate it, and stack them layer by layer in the following order: sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad. Place them in the electrospinning clamp and clamp them tightly, then place them in the electrospinning tank. Pour pre-cooled electrospinning buffer into the electrospinning tank, and place sufficient ice cubes or ice packs around the instrument after installation. Set the working current and time: 200mA, 90 min.

[0068] After electroporation, the PVDF membrane was removed and blocked with 5% (w / v) skim milk powder at room temperature for 2 h; washed 3 times with PBST; the mouse anti-TGEV S1 protein monoclonal antibody diluted 1:1000 (v / v) was used as the primary antibody and incubated overnight at 4°C; washed 3 times with PBST the next day; the HRP-labeled goat anti-mouse secondary antibody was diluted 1:10000 (v / v) and incubated at room temperature for 1 h; after washing 3 times with PBST, the results could be observed on a chemiluminescence imaging system.

[0069] II. Test Results 1. Results of TGEV-S1 protein structure analysis The primary structure of the TGEV-S1 protein was analyzed using the Prot Scale online tool in the ExPASy database. The results showed that the protein size was 46.48 kDa, the theoretical isoelectric point was 5.03, the maximum and minimum hydrophobic values ​​of the amino acids were 1.811 and 1.922, respectively, and the average hydrophilicity was -0.111. Figure 1This suggests that the TGEV-S1 protein is likely a hydrophilic protein. Hydrophilicity implies strong solubility and affinity in water. This indicates that the TGEV-S1 protein can interact with water molecules and dissolve easily in water. This hydrophilic property makes the TGEV-S1 protein play an important role in cells and likely participate in many biological processes.

[0070] Signal peptide prediction of TGEV-S1 protein showed that ( Figure 2 The probability of the TGEV-S1 protein containing a signal peptide is 0.222%, suggesting that this protein likely does not contain a signal peptide. Signal peptides are small molecules that play a guiding, localizing, and regulatory role in protein synthesis. They help proteins accurately locate specific sites within the cell or be secreted extracellularly. However, based on the prediction results, the probability of the TGEV-S1 protein containing a signal peptide is very low, meaning that it may not require a signal peptide for localization or secretion.

[0071] Prediction of the transmembrane domains of the TGEV-S1 protein showed that ( Figure 3 This protein does not have a transmembrane domain, so the full gene sequence of the protein can be used for prokaryotic expression.

[0072] 2. Protein expression and purification (1) Attempt to induce expression of TGEV-S1 recombinant protein The recombinant expression plasmid TEGV-S1-28a was transformed into BL21(DE3) competent cells, and single clones were selected for sequencing identification after culture. The experimental results showed that the recombinant expression strain TGEV-S1-28a was successfully constructed. Figure 4 ).

[0073] The recombinant expression bacteria were induced with IPTG for 6 h, centrifuged, and the supernatant (as sample 2) and bacterial pellet were collected. The bacterial pellet was disrupted by sonication, and the supernatant and pellet were collected as samples 3 and 4. The supernatant of the pre-induction *E. coli* bacterial culture was used as sample 1. All samples were subjected to 12.5% ​​SDS-PAGE gel electrophoresis and stained with Coomassie brilliant blue. SDS-PAGE results showed a distinct protein band of pET-28a-TGEV-S1 at approximately 45 kDa, with a size consistent with the target protein. This indicates successful expression of the TGEV-S1 recombinant protein, and the protein was mainly present in the pellet after sonication, suggesting that it primarily exists in inclusion body form. Figure 5 ).

[0074] (2) Exploration of the optimal induction time for TEGV-S1 recombinant protein Following step (3) of the experimental method, four induction times were set, with other conditions remaining unchanged (temperature 37℃, inducer concentration 1.0 mmol / L). The induction time was optimized, and the results are as follows: Figure 6 The results showed that the TGEV-S1 recombinant protein was fully expressed after 6 h of induction. Although the expression level after 8 h of induction was slightly higher than that after 6 h, 6 h was chosen as the optimal induction time for the TGEV-S1 recombinant protein in order to save time.

[0075] (3) Exploration of the optimal inducer concentration for TEGV-S1 recombinant protein Following step (3) of the experimental method, four inducer concentrations were set, with other conditions remaining unchanged (temperature 37℃, induction time 6 h), to optimize the inducer concentrations. The results are as follows: Figure 7 As shown, the expression level of TGEV-S1 recombinant protein was most significant at an inducer concentration of 1.0 mmol / L. The protein expression levels induced by the other three different inducer concentrations were comparable. Therefore, 1.0 mmol / L was selected for the induction of TGEV-S1 recombinant protein expression.

[0076] (4) Exploration of the optimal induction temperature for TEGV-S1 recombinant protein expression Following step (3) of the experimental method, four induction temperatures were set, with other conditions remaining unchanged (inducer concentration 1.0 mmol / L, induction time 6 h), to optimize the induction temperature, such as... Figure 8 As shown, the induction temperature of 16℃ had the most significant effect on the expression level of the TGEV-S1 recombinant protein, indicating that the protein can be fully expressed after low-temperature induction, and that high-temperature induction may be detrimental to the induction and expression of the protein. Therefore, the lowest induction temperature, 16℃, was selected as the optimal induction temperature for the TGEV-S1 recombinant protein.

[0077] (5) Purification and reaction-promoting identification of TEGV-S1 recombinant protein To obtain a purer protein and reduce its impact on the specificity of the detection method, this invention purifies the target protein. First, after denaturation, the purified protein is obtained by nickel column chromatography. SDS-PAGE analysis of the purified protein shows that all TGEV-S1 recombinant protein samples exhibit a single protein band with a molecular weight of approximately 46 kDa, consistent with the molecular size of the TGEV-S1 recombinant protein. Figure 9 The concentration of the purified and refolded protein was then measured using a spectrophotometer, and the results showed that the concentration of the TGEV-S1 recombinant protein was 0.76 g / mL.

[0078] To confirm whether the purified protein possessed antigenicity, Western blot analysis was performed. The purified protein was reacted with a mouse-derived anti-TGEV S1 protein monoclonal antibody. The results are as follows: Figure 10 As shown, the purified protein exhibits good antigenicity.

[0079] Example 2. Development and application of TGEV fluorescent microsphere immunochromatographic test strips I. Experimental Methods 1. The specific method for coupling fluorescent microspheres with antigens is as follows: (1) Take 50 µL of fluorescent microspheres (Thermo Fisher Scientific, F8810) and add them to a centrifuge tube. Centrifuge and discard the supernatant.

[0080] (2) Add 1 mL of sodium dihydrogen phosphate solution (0.01 mol / L, pH 6.0), resuspend the precipitate using an ultrasonic cell disruptor; centrifuge and discard the supernatant.

[0081] (3) Add 1 mL of activation solution, resuspend the precipitate using an ultrasonic cell disruptor, and then shake with a rotary mixer at room temperature for 15 min; centrifuge and discard the supernatant. The activation solution is prepared by mixing 10 mM phosphate buffer (PB, pH 6.0) containing 20 mg / mL EDC and 10 mM phosphate buffer (PB, pH 6.0) containing 20 mg / mL NHS at a volume ratio of 1:1, and using immediately.

[0082] (4) Add 1 mL of hydrochloric acid solution (0.002 mol / L), resuspend the precipitate with an ultrasonic fine electrolysis device; centrifuge and remove the supernatant.

[0083] (5) Add 500 µL of PBS buffer (0.01 mol / L, pH 7.4) and resuspend the precipitate using an ultrasonic cell disruptor to ensure uniform dispersion of the fluorescent microspheres.

[0084] (6) Immediately add 1 mL of the purified TGEV-S1 recombinant protein from Example 1, wherein the mass ratio of fluorescent microspheres to TGEV-S1 recombinant protein is 10:1, mix well, and shake with a rotary mixer at room temperature for 2 h; centrifuge and remove the supernatant.

[0085] (7) Add 500 µL of blocking solution (2% (w / v) BSA), mix well, and shake with a rotary mixer at room temperature for 1 h; centrifuge and discard the supernatant.

[0086] (8) Add 200 µL of preservation solution (20 mM boric acid), mix well, and obtain fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugate. At the same time, prepare fluorescent microsphere-labeled rabbit IgG antibody conjugate according to the above method.

[0087] 2. Preliminary assembly and antigen coating concentration screening of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips The fluorescent microsphere immunochromatographic test strip includes a base plate, a sample pad, a detection membrane (NC membrane), and an absorbent pad. The detection membrane has a detection line (T line) and a control line (C line). The sample pad, NC membrane, and absorbent pad are sequentially overlapped and adhered to the base plate according to the chromatography direction. Figure 11 ),in: (1) The sample pad was prepared by immersing the glass fiber membrane in a 0.01 mol / L PBS solution (pH 7.4) containing 5% (m / v) D-(+) trehalose, 1% (m / v) BSA and 0.1% (v / v) Tween20, and then drying it in a 37℃ oven for 3h to obtain the pretreated sample pad. The fluorescent microsphere-labeled anti-TGEV-S1 recombinant protein conjugate obtained in step 1 was diluted to different concentrations, and then each concentration of conjugate was sprayed onto the upper end of the pretreated sample pad using an XYZ3050 spraying system at a spraying amount of 10 µL / cm, and then dried in a 37℃ oven for 3h.

[0088] (2) The NC membrane was prepared as follows: using an XYZ3050 spraying system, goat anti-pig IgA antibody was diluted to 1 mg / mL and sprayed onto the T-line of the NC membrane at a spraying rate of 10 μL / cm. Goat anti-rabbit IgG antibody was diluted to 0.5 mg / mL and sprayed onto the C-line of the NC membrane at a spraying rate of 10 μL / cm. The detection line was placed near the sample pad end, and the control line was placed near the absorbent pad end. After spraying, the membrane was dried at 37℃ for 3 h and then dried and stored at 4℃.

[0089] (3) Assemble and paste the sample pad, NC membrane and absorbent pad prepared above onto the base plate in sequence, and cut them into 3.5 mm wide strips using the CM4000 cutting system to obtain fluorescent microsphere immunochromatographic test strips.

[0090] (4) After defatting, equal amounts of the same sample of TGEV IgA antibody-positive colostrum from sows were added to the central area of ​​the sample pad of the above-mentioned fluorescent microsphere immunochromatographic test strip, and the antigen coating concentration was tested three times. By comparing the differences in T / C results of the test strips, the protein concentration with the largest difference was selected as the optimal coating concentration. Wherein, T / C value = fluorescence intensity of the detection line (T) / fluorescence intensity of the control line (C).

[0091] 3. Final assembly of TGEV IgA antibody fluorescent microsphere immunochromatographic test strip The fluorescent microsphere immunochromatographic test strip includes a base plate, a sample pad, a detection membrane (NC membrane), and an absorbent pad. The detection membrane has a detection line (T line) and a control line (C line). The sample pad, NC membrane, and absorbent pad are sequentially overlapped and adhered to the base plate according to the chromatography direction. Figure 11 ),in: (1) The sample pad was prepared by immersing the glass fiber membrane in a 0.01 mol / L PBS solution (pH 7.4) containing 5% (m / v) D-(+) trehalose, 1% (m / v) BSA and 0.1% (v / v) Tween20, and then drying it in a 37℃ oven for 3 h to obtain the pretreated sample pad. The fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugate obtained in step 1 was mixed with the fluorescent microsphere-labeled rabbit IgG antibody conjugate to obtain a mixed probe. The final protein concentrations of the two in the mixed probe were 55 μg / mL and 30 μg / mL, respectively. The mixed probe solution was sprayed onto the upper end of the treated sample pad using an XYZ3050 spraying system at a spraying rate of 10 μL / cm, and then dried in a 37℃ oven for 3 h.

[0092] (2) The NC membrane was prepared as follows: using an XYZ3050 spraying system, goat anti-pig IgA antibody at a concentration of 1.0 mg / mL was sprayed onto the T-line of the NC membrane at a spraying rate of 10 μL / cm, and goat anti-rabbit IgG antibody at a concentration of 0.5 mg / mL was sprayed onto the C-line of the NC membrane at a spraying rate of 10 μL / cm. The detection line was placed near the sample pad end, and the control line was placed near the absorbent pad end. After spraying, the nitrocellulose membrane was dried in a 37℃ oven for 3 h for later use.

[0093] (3) Assemble and paste the sample pad, NC membrane and absorbent pad prepared above onto the base plate in sequence, and cut them into 3.5 mm wide strips using the CM4000 cutting system to obtain the fluorescent microsphere immunochromatographic test strip for detecting TGEV IgA antibody in milk.

[0094] 4. Preliminary application of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip detection method Take 2 mL of colostrum from sows that are positive for TGEV IgA antibodies or negative for IgA antibodies, centrifuge at 6000 r / min for 5 min, and collect the lower layer of skimmed emulsion as the test sample. Add 50 µL of the skimmed emulsion to the sample end of the test strip for detection. When TGEV IgA antibodies are present in the milk, they can bind to the fluorescent microsphere-labeled anti-TGEV-S1 recombinant protein conjugate. When the conjugate undergoes chromatography at the T-line, it is captured by goat anti-porcine IgA antibody, resulting in its enrichment at the T-line. Simultaneously, when the fluorescent microsphere-labeled rabbit IgG antibody conjugate crosses the T-line chromatography and reaches the C-line, it is captured by the immobilized goat anti-rabbit IgG antibody, resulting in its enrichment at the C-line. Under 365 nm ultraviolet light, the fluorescent microspheres captured at the T-line and C-line emit bright fluorescent signals. Analyzing these signals with a detector determines whether the sow's milk contains TGEV IgA antibodies.

[0095] (1) Determination of reaction time Observe the test strip's reaction every 3 minutes. Determine the optimal reaction time based on the test strip's reaction speed and the clarity of the results. Observe the test strip's results immediately after each time point. Note the appearance of any lines, color changes, or other relevant indicators. Compare the results with those from different time points. Based on the observations and comparisons, select the optimal reaction time.

[0096] (2) Establishment of critical values ​​for negative and positive determination of visual instruments This study used a developed TGEV IgA antibody fluorescent microsphere immunochromatographic test strip to detect 100 clinical breast milk samples that were positive for TGEV IgA antibodies, preserved in our laboratory. A visual detection instrument (Shenzhen Sanfangyuan Biotechnology Co., Ltd.) was used to detect fluorescence intensity, obtaining the T-line detection values ​​for all samples. The minimum value displayed on the instrument at the T-line position was considered the positive detection limit, while the maximum value displayed on the instrument at the T-line position was considered the negative detection limit. The visual detection instrument measures 35 mm (length) × 20 mm (width) × 5 mm (height), using a light-emitting diode (LED) with a peak wavelength of 365 nm as the excitation light source and equipped with a bandpass filter system with a center wavelength of 498 nm and a bandwidth of 7 nm. This effectively excites the fluorescence signal of the fluorescent microspheres on the test strip and converts it into an electrical signal, which is then displayed on the instrument screen as a fluorescence intensity value. The test results can also be connected to a printer for output.

[0097] (3) Specific detection of TGEV IgA antibody fluorescent microsphere immunochromatographic test strip The established immunochromatographic method was used to detect TGEV IgA antibody-positive colostrum, PEDV IgA antibody-positive colostrum, and rotavirus IgA antibody-positive colostrum from sows, respectively. The assembled test strips were used for detection, and the samples were placed in a 37°C oven for 10 min to observe the results in order to evaluate the specificity of the test strips.

[0098] (4) Sensitivity detection of TGEV IgA antibody fluorescent microsphere immunochromatographic test strip The TGEV IgA antibody-positive colostrum was serially diluted 5-fold starting at a 1:2 ratio with PBS buffer. 50 µL of sample was taken from each dilution and tested using the assembled test strip. The strips were placed in a 37°C oven for 10 min to observe the results and evaluate their sensitivity.

[0099] (5) Repeatability evaluation of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips Three different batches of test strips were used to apply samples to three known TGEV IgA antibody-negative and positive colostrum samples to determine the results of different batches. Simultaneously, one batch of test strips was randomly selected, and the same milk sample was tested three times to evaluate the repeatability of the test strips.

[0100] (6) Detection of clinical samples using TGEV IgA antibody fluorescent microsphere immunochromatographic test strips One hundred TGEV IgA antibody-positive colostrum samples were collected from pregnant sows and tested using IgA antibody fluorescent microsphere immunochromatographic strips and commercial ELISA kits, respectively, to evaluate the clinical application efficacy of this method.

[0101] II. Evaluation of the Experimental Results of the Fluorescent Microsphere Immunochromatographic Test Strip 1. Determination of the concentration of the assembled antigen Data obtained from Shanghai Kundao Biotechnology Co., Ltd. shows that when the TGEV-S1 concentration on the NC membrane is 55 μg / mL, the T / C value difference in positive milk detected by the test strip is the largest, which is the optimal coating concentration. Figure 12 ).

[0102] 2. Preliminary application of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip detection method The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip was used to detect TGEV IgA antibody-positive colostrum and TGEV IgA antibody-negative colostrum (identified by an ELISA kit) from pregnant sows immunized with a TGEV attenuated live vaccine. After a 10-minute reaction, the fluorescence values ​​were read using a detector. The results showed ( Figure 13This method yielded positive results for colostrum from pregnant sows that were immunized with a live attenuated TGEV vaccine and positive results for colostrum from sows that were TGEV IgA antibody-positive, while the results for colostrum from sows that were TGEV IgA antibody-negative were negative. These results can preliminarily determine whether sow milk contains TGEV IgA antibodies.

[0103] (1) Determination of the optimal reaction time The observation results show that ( Figure 14 The optimal reaction time is 5-10 minutes. Too short a time results in insufficient antigen-antibody reaction and unclear color development; too long a time causes the chromatography membrane to dry out, also leading to unclear color development. Both of these situations can easily lead to misinterpretation of the results.

[0104] Table 1. Optimal reaction time analysis of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips

[0105] (2) Determination of diagnostic criteria for TGEV negative and positive One hundred TGEV antibody-positive milk samples were tested using TGEV IgA antibody fluorescent microsphere immunochromatographic test strips, and the fluorescence value was measured using a visualization instrument. Based on the sample test results in Tables 2-5 and the sensitivity test results in Table 6, the positive judgment criteria for the fluorescent microsphere test strips were determined as follows: instrument detection value ≥ 1.01; instrument detection value < 1.00 was judged as negative.

[0106] Table 2. Clinical sample detection data of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips (1-25)

[0107] Table 3. Clinical sample detection data of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips (26-50)

[0108] Table 4. Clinical sample detection data of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips (51-75)

[0109] Table 5. Clinical sample detection data of TGEV IgA antibody fluorescent microsphere immunochromatographic test strips (76-100)

[0110] (3) Specificity evaluation The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip was used to detect TGEV IgA antibody-positive colostrum, PEDV IgA antibody-positive colostrum, and rotavirus IgA antibody-positive colostrum (control group) from pregnant sows immunized with attenuated TGEV vaccine. Fluorescence values ​​were read using a detector after 10 minutes of reaction. Results showed ( Figure 15 This method only yields positive results for TGEV IgA antibody-positive colostrum from pregnant sows that have been immunized with a live attenuated TGEV vaccine, while the results for other colostrum are negative. This indicates that the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip has good specificity.

[0111] (4) Sensitivity assessment PBS buffer was used to perform serial dilutions (1:2, 1:10, 1:50, 1:250, and 1:1250) on TGEV IgA antibody-positive colostrum from pregnant sows immunized with a live attenuated TGEV vaccine. Subsequently, TGEV IgA antibody fluorescent microsphere immunochromatographic test strips were used to detect samples at each dilution. After a 10-minute reaction time, fluorescence values ​​were read using a detector. The experimental results are as follows: Figure 16 As shown, under 365 nm UV excitation, the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip exhibited two fluorescent bands, a C band and a T band, in samples at dilutions ranging from 1:2 to 1:50, with the fluorescence intensity gradually decreasing. Therefore, the limit of detection for colostrum from pregnant sows using the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip can be determined to be a 1:50 dilution.

[0112] Table 6 Sensitivity analysis of IgA antibody fluorescent microsphere immunochromatographic test strips

[0113] (5) Repeatability evaluation like Figure 17 As shown, the TGEV IgA antibody fluorescent microsphere immunochromatographic test strips from the same production batch yielded consistent results when the same positive and negative samples were tested three times, indicating that the prepared TGEV IgA antibody fluorescent microsphere immunochromatographic test strips have good intra-batch repeatability.

[0114] like Figure 18 As shown, the TGEV IgA antibody fluorescent microsphere immunochromatographic test strips from different production batches yielded consistent results when testing the same positive and negative samples, indicating that the prepared TGEV IgA antibody fluorescent microsphere immunochromatographic test strips have good batch-to-batch repeatability.

[0115] (6) Clinical sample testing The assembled TGEV IgA antibody fluorescent microsphere immunochromatographic test strip and a commercial ELISA kit were used to simultaneously detect the colostrum of 100 clinically TGEV attenuated live vaccine-immunized sows. The results are shown in the figure. The positive detection rates of the test strip and the commercial ELISA kit were 98% and 96%, respectively, and the concordance rate between the two was 95.9% (see Tables 2-5).

[0116] This invention utilizes fluorescent microspheres as a novel luminescent marker, replacing traditional colloidal gold or enzyme markers, to detect mucosal immune IgA antibodies in TGEVs. The fluorescence intensity of the fluorescent microspheres is read using a spectrometer, significantly improving detection sensitivity and specificity. Compared to conventional IgG detection, IgA antibody levels better reflect the maternal immune status of the pig herd and have a longer detection window. A portable fluorescence reader is used for quantitative or semi-quantitative readings, providing intuitive fluorescence intensity data for rapid on-site interpretation. The TGEV-S1 recombinant protein is used as a capture antigen, leveraging its strong immunogenicity and specific binding region to improve antibody capture efficiency.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A TGEV IgA antibody fluorescent microsphere immunochromatographic test strip, characterized in that... The device includes a base plate, and a sample pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and pasted onto the base plate. The sample pad is a glass fiber membrane coated with a TGEV-S1 recombinant protein conjugate labeled with fluorescent microspheres and a rabbit IgG antibody conjugate labeled with fluorescent microspheres. The nitrocellulose membrane has a detection line and a control line, wherein the detection line is coated with goat anti-pig IgA antibody, and the control line is coated with goat anti-rabbit IgG antibody. The nucleotide sequence encoding the TGEV-S1 recombinant protein is shown in SEQ ID NO.

1.

2. The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to claim 1, characterized in that: The amount of TGEV-S1 recombinant protein conjugate labeled with fluorescent microspheres on the glass fiber membrane is 0.55 μg / cm, and the amount of rabbit IgG antibody conjugate labeled with fluorescent microspheres is 0.30 μg / cm.

3. The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to claim 1, characterized in that: The amount of goat anti-pig IgA antibody used to coat the detection line is 0.01 mg / cm, and the amount of goat anti-rabbit IgG antibody used to coat the quality control line is 0.05 mg / cm.

4. The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to claim 1, characterized in that: The fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugate was prepared by the following method: After pre-washing the fluorescent microspheres, they were activated using EDC / NHS, acid-washed, resuspended, and then conjugated with TGEV-S1 recombinant protein. Finally, they were blocked to obtain fluorescent microsphere-labeled TGEV-S1 recombinant protein conjugates.

5. The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to claim 4, characterized in that: The ratio of fluorescent microspheres to recombinant TGEV-S1 protein is 10:

1.

6. The TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to claim 4, characterized in that: The method for preparing the TGEV-S1 recombinant protein includes the following steps: (1) The nucleotide sequence shown in SEQ ID NO. 1 of the gene was synthesized and then ligated with the expression vector to obtain the recombinant expression vector; (2) The recombinant expression vector obtained in step (1) was transformed into BL21(DE3) competent cells to obtain recombinant expression bacteria; (3) The recombinant expression bacteria obtained in step (2) were induced to express, the bacterial cells were collected and resuspended in PBS buffer, and sonicated. The precipitate after disruption was collected, which is the crude recombinant protein inclusion body. (4) The precipitate collected in step (3) is washed, dissolved, purified and refolded in sequence to obtain TGEV-S1 recombinant protein.

7. The method for preparing the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to any one of claims 1-6, characterized in that... It includes the following steps: The sample pad, NC membrane, and absorbent pad are then overlapped and pasted onto the base plate in sequence.

8. The use of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to any one of claims 1-6 in the preparation of products for detecting porcine TGEV IgA antibodies or porcine TGEV.

9. A method for detecting TGEV IgA antibodies, characterized in that... It includes the following steps: Take a sample of sow's milk to be tested, defatt it, and add it to the sample pad of the TGEV IgA antibody fluorescent microsphere immunochromatographic test strip according to any one of claims 1-6. After the chromatographic reaction, observe the fluorescence of the detection line and the control line. If both the detection line and the control line show fluorescence, the milk sample to be tested contains TGEV IgA antibody; if the detection line has no fluorescence and the control line has fluorescence, the milk sample to be tested does not contain TGEV IgA antibody.

10. The method for detecting TGEV IgA antibodies according to claim 9, characterized in that: The chromatography reaction time is 5-10 min.

Images