Kit for detecting capripoxvirus based on single-domain antibody 9-4-1 immunomagnetic beads and application thereof

Abstract

The application discloses a kind of based on single-domain antibody 9-4-1 immunomagnetic beads direct spread detection goat poxvirus kit and its application.The kit includes the single-domain antibody 9-4-1 immunomagnetic beads obtained by the single-domain antibody 9-4-1 shown in SEQ ID NO.1 goat poxvirus is coupled with magnetic bead by streptavidin-biotin.The single-domain antibody 9-4-1 protein purified by gel cutting is coupled with magnetic bead to obtain immunomagnetic beads, and after goat poxvirus is enriched, it can be directly amplified without extracting genomic RNA.The application overcomes the phenomenon that goat poxvirus is not easy to be found at virus replication peak period due to long time of forming sandwich, virus content is low when collecting disease material, detection rate is low, and a series of problems such as easy to miss detection;And it is time-consuming, costly and laborious to extract genomic DNA, and a series of problems such as environmental pollution during operation, and efficient, sensitive, specific, stable and practical, and the application provides an efficient technical means for detection of goat poxvirus disease.

Description

Technical Field

[0001] This invention relates to an immunomagnetic bead kit, and more particularly to a kit for direct amplification detection of goatpox virus based on single-domain antibody immunomagnetic beads and its applications. This invention belongs to the field of biotechnology. Background Technology

[0002] Sheep pox is an acute, febrile, highly contagious disease caused by the goat or sheep poxvirus, characterized primarily by fever and systemic papules or nodules. It has a high morbidity and mortality rate, making it the most severe of all animal pox diseases. The virus is mainly found in the papules, pustules, and scabs on the skin and mucous membranes of infected sheep, as well as in nasal secretions. The typical progression involves the initial formation of red macules, which then transform into white papules, followed by vesicles and scabs. The disease has an incubation period, with viral load reaching its peak after 10-14 days and decreasing after 3-6 weeks. When problems are discovered in farms, it is often in the later stages, and the viral load in collected samples is relatively low, making virus detection and isolation difficult.

[0003] Single-domain antibodies are the smallest antigen-binding units of antibody molecules, consisting of only a variable domain or an engineered constant domain that assists only in target binding. They lack the Fc region of ordinary antibodies, thus avoiding complement reactions caused by the Fc region. Therefore, they are increasingly used in fields such as biodetection, diagnostics, and pharmaceuticals. Immunomagnetic beads (IMB) are a novel diagnostic technique based on antigen-antibody reactions and mediated by magnetic carrier technology. After certain treatments, antibodies are bound to magnetic beads. The magnetic beads, acting as antibody carriers, bind to antigens, forming immune complexes. Under the influence of magnetic force, these complexes undergo mechanical movement, separating the antigens specifically bound to the antibodies on the magnetic beads from other substances. This technology has been applied in immunoassay, cell separation, and protein purification. Its unique characteristics of being rapid, efficient, low-toxicity, highly specific, and simple to operate provide a new approach for antigen detection of latent infections.

[0004] This invention combines single-domain antibody and immunomagnetic bead technology, eliminating the need for complex purification equipment and techniques, and placing no restrictions on sample clarity. It allows for convenient detection of low-level sheep poxvirus with just a simple magnetic adsorption step. This results in an immunoassay method that is fast, highly specific, highly sensitive, and reproducible. Summary of the Invention

[0005] To address the challenges of existing technologies in goatpox virus antigen detection, such as false negatives due to low viral loads failing to meet the minimum requirements for PCR or Real-time PCR, and the time-consuming and labor-intensive process of RNA extraction, coupled with the high cost of RNA extraction kits, this invention offers two solutions. Firstly, it utilizes a gel purification method to prepare high-purity single-domain antibody proteins, resulting in better protein purity and easier labeling compared to conventional methods. The labeled immunomagnetic beads then exhibit stronger specificity for enriching the goatpox pathogen. Secondly, it eliminates the crucial step of genomic DNA extraction, directly amplifying the enriched products for real-time quantitative fluorescence detection, saving time, manpower, resources, and money, thus improving economic efficiency.

[0006] Specifically, in order to achieve the above objectives, the present invention employs the following technical means:

[0007] The present invention discloses a kit for the direct amplification detection of goatpox virus based on single-domain antibody 9-4-1 immunomagnetic beads. The kit comprises single-domain antibody 9-4-1 immunomagnetic beads obtained by antibody conjugation of goatpox virus single-domain antibody 9-4-1 to magnetic beads via streptavidin-biotin (SA-Biotin). The amino acid sequence of the goatpox virus single-domain antibody 9-4-1 is shown in SEQ ID NO.1.

[0008] Preferably, the goatpox virus single-domain antibody 9-4-1 is produced by a strain with accession number CCTCCNO.M2019742 and accession name Escherichia coli 9-4-1 / GTPV, and is obtained by gel purification.

[0009] Preferably, the kit further includes an upstream primer, a downstream primer, and a probe for direct amplification detection of goatpox virus, wherein the sequences of the upstream primer, the downstream primer, and the probe are as follows:

[0010] Upstream primer: 5'-TATTTTGTGCGGAAACGTGTTTGTA-3';

[0011] Downstream primer: 5'-ATTATAGGAATTGTTGAGAGAGAT-3';

[0012] Probe: 5'-TTTTATCAGATGATTGCGTAATCTTAAC-3'.

[0013] Preferably, the kit further includes a direct amplification reaction solution.

[0014] Preferably, when using the kit to detect goatpox virus antigen, the following steps are performed:

[0015] 1) Virus enrichment: Take 10 μl of single-domain antibody 9-4-1 immunomagnetic beads into a 1.5 mL EP tube, add 100 μl of the supernatant of the sample to be tested after centrifugation, vortex to mix, place in a 4℃ refrigerator, and enrich for 12 h or overnight.

[0016] 2) Washing of magnetic beads: After the virus enrichment is completed, place the EP tube on a magnetic rack, discard the supernatant, wash 3 times with 1 ml of 0.05 M PBS buffer (pH 7.6), and finally resuspend with 50 μl of PBS buffer to obtain the suspension as the product of virus enrichment.

[0017] 3) Establish a 50 μl direct amplification reaction system: 40 μl direct amplification reaction solution, 5 μl mixture of upstream and downstream primers and probes, and 5 μl suspension; the amplification program is as follows: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, 5 cycles of pre-amplification; 95℃ for 10 s, 54℃ for 30 s for a total of 40 cycles and fluorescence collection.

[0018] The sequences of the primers and probes are as follows:

[0019] Upstream primer: 5'-TATTTTGTGCGGAAACGTGTTTGTA-3';

[0020] Downstream primer: 5'-ATTATAGGAATTGTTGAGAGAGAT-3';

[0021] Probe: 5'-TTTTATCAGATGATTGCGTAATCTTAAC-3'.

[0022] Preferably, the concentrations of the upstream and downstream primers and the probe are all 10 μM.

[0023] Furthermore, the present invention also proposes the application of the kit in the preparation of reagents for detecting goatpox virus antigen.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention overcomes a series of challenges in goatpox virus antigen detection, including low viral loads that fail to meet the minimum requirements for RT-PCR detection, leading to false negatives; time-consuming and labor-intensive DNA extraction; expensive DNA extraction kits; and low concentrations and purity after single-domain antibody expression and purification. This application utilizes optimized single-domain antibodies with high expression levels and good reactivity. The immunomagnetic beads used for pathogen enrichment are more economical, and the kit obtained by directly amplifying the enriched products not only saves time and resources but is also highly efficient, sensitive, specific, stable, and practical, providing a highly efficient technical means for the detection of goatpox virus disease. Attached Figure Description

[0026] Figure 1 The SDS-PAGE results for gel purification of 9-4-1 protein;

[0027] Left: Target band of 9-4-1 protein; Right: Pre-stained protein marker;

[0028] Figure 2 The diagram shows the optimal binding result of magnetic bead protein 9-4-1;

[0029] Figure 3 Figure showing the enrichment sensitivity results of magnetic globin 9-4-1;

[0030] Figure 4 This is a diagram showing the specific amplification results of magnetic bead protein 9-4-1.

[0031] Preservation information:

[0032] Nomenclature: Escherichia coli 9-4-1 / GTPV

[0033] Classification and nomenclature: Escherichia coli 9-4-1 / GTPV

[0034] Accession number: CCTCC NO.M 2019742

[0035] Storage date: September 23, 2019

[0036] Preservation institution: China Center for Type Culture Collection (CCTCC).

[0037] Location of collection: Wuhan University, Wuhan, China Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are only for illustrating the present invention and do not constitute any limitation on the scope of protection of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the scope of protection of the present invention.

[0039] Example 1: Screening of goatpox virus single-domain antibodies and construction of strains expressing such single-domain antibodies

[0040] 1.1 Materials

[0041] The goat poxvirus single-domain antibody library was prepared using the method disclosed in patent application CN20161028158.3, entitled "An anti-goat poxvirus Bactrian camel VHH heavy chain single-domain antibody cDNA library and its preparation method".

[0042] 1.2 Screening and Construction of Goatpox Virus Single-Domain Antibody 9-4-1

[0043] The antibody library was amplified using a two-step method. First, the VH-CH1-CH2 hinge region was amplified. The VH-CH1-CH2 band was recovered and directly amplified to obtain the VHH gene band (omitting the amplification of the VHH-CH2 hinge region). The VHH gene was cloned and transformed. Several colonies were picked and placed in LB medium. They were shake-cultured for 12-14 hours. Positive colonies underwent small-scale plasmid extraction and sequencing. The obtained sequences were analyzed and predicted one by one. Genes that conformed to the single-domain antibody framework were selected and constructed into expression vector pMal-c2X. Their respective proteins were expressed in small quantities. The effectiveness of the antibodies, i.e., their reactivity, was verified by antibody detection kits, classical virus neutralization experiments, and indirect immunofluorescence. From these, single-domain antibodies with high expression levels and good activity of goatpox virus were screened out.

[0044] 1.3 Results

[0045] Several sequences were obtained through sequencing. After comparison, analysis, and screening through small-scale expression and activity verification, a single-domain antibody 9-4-1 for goat pox virus was finally obtained. The amino acid sequence of the single-domain antibody 9-4-1 protein is shown in SEQ ID NO.1. The *E. coli* expressing the single-domain antibody 9-4-1 protein was named *Escherichia coli* 9-4-1 / GTPV and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M2019742.

[0046] Example 2: Preparation of goatpox virus single-domain antibody-conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0047] 2.1 Purification of Goatpox Virus Single-Domain Antibody

[0048] The strain with accession number CCTCC NO.M 2019742 was used. The amino acid sequence of the single-domain antibody 9-4-1 protein expressed by this strain is shown in SEQ ID NO.1. The expression vector was pMal-c2X, with an N-terminal MBP solubilization tag and a C-terminal HIS purification tag. 100 μl of the strain was added to 5 ml of ampicillin-resistant LB medium, shaken, and then transferred to 1000 ml of ampicillin-resistant LB medium for induction of expression. The strain was harvested, sonicated, centrifuged, and the supernatant was collected for purification by nickel column affinity chromatography. Finally, the protein bound to the column was eluted with 200 mM imidazole.

[0049] Under normal circumstances, the eluted proteins are directly used for subsequent ELISA and other experiments. However, they often contain contaminating proteins, affecting protein purity. This invention further purifies the above proteins by gel extraction. The proteins are then subjected to another SDS-PAGE. Under clean conditions, the target band is excised, placed in an autoclaved mortar, and liquid nitrogen is added under safe conditions. The mixture is ground, resuspended in 0.05M, pH 7.6 PBS buffer, centrifuged at 4000 rpm for 20 min, and the supernatant is collected. A small amount of liquid nitrogen is added to the precipitate, and the mixture is ground again and resuspended in PBS buffer. The two resuspended solutions are concentrated using a 100,000 molecular weight cutoff (MW) ultrafiltration system. The resulting product is VHH 9-4-1 / GTPV protein (3.4 mg / ml) purified to a high purity and concentration. Figure 1 When the titer of a conventional antibody reaction is 1:640, purification can increase it to at least 1:1280.

[0050] 2.2 Magnetic bead labeling of 9-4-1 protein

[0051] Add 1 ml of carboxyl magnetic beads to a centrifuge tube and place it on a magnetic rack. Discard the magnetic bead storage solution. Wash three times with pH 5.2 0.05M MES buffer, discard the buffer, add 1.6 times the amount of DEAC magnetic beads, and activate on a vortex mixer for 30 min. Add 20 μL of 3.4 mg / ml 9-4-1 protein, vortex at room temperature for 20 min, place the vortexed centrifuge tube on a magnetic rack for adsorption, discard the supernatant, resuspend in 1 ml of 1xPBS buffer, wash, repeat three times, and finally resuspend in 4 ml of 1xPBS and add 1% BSA as a stabilizer for the label. The resulting product is the 9-4-1 protein magnetic bead label, which is used to enrich goatpox virus attenuated vaccine with the 9-4-1 protein magnetic bead label.

[0052] Example 3: Optimal binding assay of goatpox virus single-domain antibody-conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0053] Purchase goat pox virus attenuated vaccine powder (Poxwin), and dilute it to approximately 10... 3100 μl of TCID50 / mL viral solution was pipetted into each container, and 5 μl, 10 μl, 20 μl, 30 μl, 40 μl, and 50 μl of goatpox virus single-domain antibody magnetic bead labeling were added sequentially for enrichment. The mixture was then washed with 1xPBS and resuspended in 50 μl of 1xPBS for real-time fluorescence amplification. The upstream primer, downstream primer, and probe sequences are as follows:

[0054] Upstream primer: 5'-TATTTTGTGCGGAAACGTGTTTGTA-3';

[0055] Downstream primer: 5'-ATTATAGGAATTGTTGAGAGAGAT-3';

[0056] Probe: 5'-TTTTATCAGATGATTGCGTAATCTTAAC-3'.

[0057] A 50 μl direct amplification reaction system was established: 40 μl direct amplification reaction solution, 5 μl primer probe, and 5 μl resuspension solution. The amplification program was as follows: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, 5 cycles of pre-amplification; 95℃ for 10 s, 54℃ for 30 s, for a total of 40 cycles, and fluorescence was collected to observe the enrichment of magnetic beads.

[0058] Result: From Figure 2 It can be seen that 10μl magnetic beads are economical in adsorption and achieve optimal adsorption capacity.

[0059] Example 4: Optimal adsorption time of goatpox virus single-domain antibody-conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0060] To determine the optimal time for the adsorption of goatpox virus by 9-4-1 protein immunomagnetic beads, two 1.5ml ep tubes were used. 100μl of the diluted goatpox virus attenuated vaccine solution as in Example 3 was taken from each tube, and 10μl of magnetic beads were added to each tube. One tube was incubated at 4°C overnight, and the other tube was incubated at 37°C for 2 hours. Quantitative amplification was performed as in Example 3.

[0061] Results: The CT value of the virus enriched overnight at 4℃ was 15.43, and the CT value of the virus enriched at 37℃ for 2 hours was 18.59, which fully demonstrates that overnight incubation at 4℃ is the optimal reaction time for enriching goatpox virus with 9-4-1 protein immunomagnetic beads.

[0062] Example 5: Sensitivity of Goatpox Virus Single-Domain Antibody-Conjugated Immunomagnetic Beads (9-4-1 Immunomagnetic Beads)

[0063] Take 1 mL of the goatpox virus attenuated vaccine dilution solution (CT value 16.75) as described in Example 3, and serially dilute it 10-fold with 1xPBS to obtain 100, 10... -1 10 -2 10-3 10 -4 10 -5 10 -6 10 -7 The virus dilution was diluted 100 μl and then mixed with 10 μl of 9-4-1 protein immunomagnetic beads for enrichment at 4°C overnight. The sensitivity of the VHH4-1 protein magnetic beads was then tested the next day.

[0064] The results are as follows Figure 3 According to the criteria for quantitative real-time PCR, a CT value greater than 35 requires retesting. Therefore, as shown in Table 1, after enrichment, 10 can be detected. -4 Diluted virus solution, without magnetic bead enrichment, can only be detected at 10. -3 Furthermore, overall, the viral copy number measured after enrichment was generally higher than that before enrichment. This indicates that the detection rate significantly increased and the sensitivity improved by at least 10 times after magnetic bead enrichment.

[0065] Example 6: Specificity of Goatpox Virus Single-Domain Antibody-Conjugated Immunomagnetic Beads (9-4-1 Immunomagnetic Beads)

[0066] Take the magnetic beads from Example 2 and follow the steps in Example 3 to enrich sheep pox, sheep oral thrush and small ruminant plague attenuated live vaccines for specific detection.

[0067] The results are as follows Figure 4 The positive control and sheep pox showed peak values, while the negative control, sheep oral thrush, and small ruminant attenuated vaccine did not show peak values, indicating that the 9-4-1 protein magnetic beads are specifically targeted at sheep pox virus and have strong specificity.

[0068] Example 7: Kit for direct amplification detection of goatpox virus based on immunomagnetic beads conjugated with single-domain antibodies against goatpox virus

[0069] The kit includes 10 μl of immunomagnetic beads conjugated with the goatpox virus single-domain antibody 9-4-1 prepared in Example 2, 40 μl of direct amplification reaction solution, and 5 μl of primer-probe mixture. The concentrations of the upstream primer, downstream primer, and probe are all 10 μM, and their sequences are as follows:

[0070] Upstream primer: 5'-TATTTTGTGCGGAAACGTGTTTGTA-3';

[0071] Downstream primer: 5'-ATTATAGGAATTGTTGAGAGAGAT-3';

[0072] Probe: 5'-TTTTATCAGATGATTGCGTAATCTTAAC-3'.

[0073] The amplification reaction conditions were as follows: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, for 5 cycles of pre-amplification; 95℃ for 10 s, 54℃ for 30 s, for a total of 40 cycles, during which fluorescence was collected. sequence list <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> A kit for direct amplification detection of goatpox virus based on single-domain antibody 9-4-1 immunomagnetic beads and its application. <141> 2022-05-12 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 123 <212> PRT <213> peste des petits ruminants virus antibody <400> 1 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Trp Thr Tyr Thr Gly Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Thr Ala Ile Glu Ser Asp Gly Thr Thr Thr Tyr Tyr Thr Asp Ala Val Lys 50 55 60 Glu Arg Phe Thr Ile Ser Arg Asp Lys Asn Val Asn Ile Ala Arg Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Val Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Thr Asp Leu Arg Gly Ser Cys Thr Lys Glu Arg Gly Lys Val Arg Tyr 100 105 110 Trp Gly Leu Gly Thr Gln Val Thr Val Ser Ser 115 120

Claims

1. A reagent kit for detecting goatpox virus based on single-domain antibody 9-4-1 immunomagnetic beads, characterized in that, The kit comprises single-domain antibody 9-4-1 immunomagnetic beads obtained by coupling goatpoxvirus single-domain antibody 9-4-1 with magnetic beads through streptavidin-biotin (SA-Biotin), and an upstream primer, a downstream primer and a probe for detecting goatpoxvirus by direct extension, wherein the amino acid sequence of the goatpoxvirus single-domain antibody 9-4-1 is shown as SEQ ID NO. 1, and the sequences of the upstream primer, the downstream primer and the probe are respectively: 。 2. The kit of claim 1, wherein The goatpox virus single-domain antibody 9-4-1 is produced by a strain with the preservation number CCTCC NO. M 2019742 and the preservation name Escherichia coli 9-4-1 / GTPV, and is obtained by a gel cutting purification method. Escherichia coli 9-4-1 / GTPV.

3. The kit of claim 1, wherein The kit further comprises a direct extension reaction solution.

4. The kit of claim 1, wherein When the kit is used for detecting goatpoxvirus antigens, the following steps are performed: 1) Virus enrichment: 10 μl of single-domain antibody 9-4-1 immunomagnetic beads are taken in a 1.5 mL EP tube, 100 μl of supernatant of a sample to be detected after centrifugation is added, and the mixture is shaken and mixed, and then placed in a 4℃ refrigerator for enrichment for 12 h or overnight; 2) Magnetic bead washing: after the virus enrichment is completed, the EP tube is placed on a magnetic stand, the supernatant is discarded, 1 ml of PBS buffer with a pH value of 7.6 and a concentration of 0.05 M is used for washing 3 times, and finally 50 μl of PBS buffer is used for resuspension to obtain a suspension as the product of enriched virus; 3) Establishment of a 50 μl direct extension reaction system: direct extension reaction solution 40 μl, upstream and downstream primer and probe mixture 5 μl, and suspension 5 μl; the amplification procedure is as follows: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, 5 cycles of pre-amplification; 95℃ for 10 s, 54℃ for 30 s, a total of 40 cycles and collection of fluorescence; The sequences of the primers and the probe are respectively: 。 5. The kit of claim 4, wherein The concentrations of the upstream and downstream primers and the probe are all 10 μM.

6. Use of the kit of any one of claims 1-5 in the preparation of a reagent for detecting goatpoxvirus antigens.

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