Kit for detecting goat pox virus based on single-domain antibody 9-4-2 immunomagnetic beads and its application

Abstract

The present invention discloses a kit for direct amplification detection of goat pox virus based on single-domain antibody 9‑4‑2 immunomagnetic beads and its application. The kit includes single-domain antibody 9‑4‑2 immunomagnetic beads obtained by coupling the goat pox virus single-domain antibody 9‑4‑2 shown in SEQ ID NO.1 with streptavidin‑biotin and magnetic beads. The present invention uses the single-domain antibody 9‑4‑2 protein purified by gel cutting to couple with magnetic beads to obtain immunomagnetic beads. After the immunomagnetic beads are enriched with goat pox virus, the genomic RNA does not need to be extracted and can be directly amplified. The proposal of the present invention overcomes the phenomenon that goat pox virus is not easy to be found at the peak of virus replication due to the long time of forming the skin, the virus content of the collected diseased material is low, so the detection rate is low, and it is easy to miss the detection, etc.; as well as a series of problems such as time-consuming, costly, and laborious when extracting genomic DNA, and polluting the environment during operation, and it is efficient, sensitive, specific, stable, and practical. The proposal of the present invention provides an efficient technical means for the detection of goat pox virus disease.

Description

Technical Field

[0001] The invention relates to an immunomagnetic bead kit, in particular to a kit for directly amplifying goat pox virus based on single-domain antibody immunomagnetic beads and use thereof, and belongs to the field of biotechnology. Background Art

[0002] Sheeppox is an acute, febrile, highly contagious disease caused by goat or sheep pox virus, characterized by fever and systemic papules or nodules. It has high morbidity and mortality, and is the most serious of all animal pox diseases. The virus is mainly present in the papules, pustules, scabs of the skin and mucous membranes of sick sheep, and in the secretions of the nasal mucosa. Its typical change process generally forms red macules first, then changes from macules to white papules, and then forms blisters and scabs. The disease has a certain incubation period, and the virus content reaches a peak in 10-14 days, and will decrease after 3-6 weeks. When problems are discovered in farms, it is often in the late stage, and the virus content of the collected diseased materials is relatively low, which brings certain difficulties to the detection and isolation of the virus.

[0003] Single-domain antibodies are the smallest antigen-binding units of antibody molecules. They consist of only one variable domain or one engineered constant domain that only assists in target binding. They do not contain the Fc segment of ordinary antibodies. Therefore, single-domain antibodies can avoid complement reactions caused by the Fc segment. Therefore, they are increasingly used in biological detection, diagnosis, pharmaceuticals and other fields. Immunomagnetic beads (IMB) is a new diagnostic technology based on antigen-antibody reaction and magnetic carrier technology. After a certain treatment, the antibody is bound to the magnetic beads. After the magnetic beads as antibody carriers bind to the antigen substance, an immune complex is formed. Under the action of the magnetic force of the magnet, this complex undergoes mechanical movement, separating the antigen that specifically binds to the antibody on the magnetic special from other substances. This technology has been applied to immunoassays, cell separation, protein purification and other aspects. Due to its unique characteristics of rapidity, high efficiency, low toxicity, high specificity and simple operation, it provides a new idea for antigen detection of latent infection.

[0004] The present invention combines single-domain antibodies and immunomagnetic bead technology, does not require complex purification equipment and technology, and has no restrictions on sample clarity. It only requires a simple magnetic adsorption step to easily detect low-content capripox virus. Thus, an immunoassay method with fast detection speed, high specificity, high sensitivity and good repeatability is obtained. Summary of the invention

[0005] Aiming at a series of problems existing in the prior art, such as false negative results due to low virus content in the process of goat pox virus antigen detection, inability to meet the minimum requirements of PCR or Real-time-PCR detection, and time-consuming and labor-intensive RNA extraction, and expensive purchase of RNA extraction kits, the present invention, on the one hand, utilizes a gel excision purification method to prepare high-purity single-domain antibody protein, which has better protein purity and is more conducive to labeling than the common method, and the labeled immunomagnetic beads have stronger specificity in enriching goat pox pathogens; on the other hand, the key link of genomic DNA extraction is eliminated, and the enrichment product is directly amplified for real-time fluorescence quantitative detection, thereby saving time, manpower, material resources and financial resources, and improving economic benefits.

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

[0007] The present invention discloses a kit for detecting goatpox virus based on single-domain antibody 9-4-2 immunomagnetic beads direct amplification. The kit comprises single-domain antibody 9-4-2 immunomagnetic beads obtained by antibody coupling of goatpox virus single-domain antibody 9-4-2 with magnetic beads through streptavidin-biotin (SA-Biotin), and the amino acid sequence of the goatpox virus single-domain antibody 9-4-2 is shown in SEQ ID NO.1.

[0008] Among them, preferably, the goatpox virus single-domain antibody 9-4-2 is produced by a strain with a deposit number of CCTCC NO.M2019743 and a deposit name of Escherichia coli 9-4-2 / GTPV, and is obtained by a gel purification method.

[0009] Wherein, preferably, the kit further comprises an upstream primer, a downstream primer and a probe for direct amplification detection of goatpox virus, and the sequences of the upstream primer, the downstream primer and the probe are respectively:

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

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

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

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

[0014] Wherein, preferably, when the kit is used to detect goatpox virus antigen, the following steps are followed:

[0015] 1) Virus enrichment: Take 10 μL of single-domain antibody 9-4-2 immunomagnetic beads in a 1.5 mL EP tube, add 100 μL of the supernatant of the sample to be tested after centrifugation, shake and mix, place in a 4°C refrigerator, and enrich for 12 hours or overnight;

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

[0017] 3) Establish a 50 μL direct amplification reaction system: 40 μL direct amplification reaction solution, 5 μL upstream and downstream primers and probe mixture, 5 μL suspension; amplification program: 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 5 cycles of pre-amplification; 95°C for 10 s, 54°C for 30 s for a total of 40 cycles and collect fluorescence;

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

[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 probes are all 10 μM.

[0023] Furthermore, the present invention also proposes the use of the kit in preparing a reagent for detecting goatpox virus antigens.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The invention overcomes a series of problems such as low virus content in the process of goat pox virus antigen detection, which cannot meet the minimum requirements of RT-PCR detection and produce false negative results; time-consuming and labor-intensive DNA extraction; expensive DNA extraction kits; low concentration and purity of single-domain antibodies after expression and purification. The application uses optimized single-domain antibodies with high expression levels and good reaction activity. Its immunomagnetic beads are more economical to use when enriching pathogens. The kit obtained by directly amplifying the enriched product not only saves time and resources, but is also efficient, sensitive, specific, stable and practical, providing an efficient technical means for the detection of goat pox virus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE result of gel-cleaved purified 9-4-2 protein;

[0027] Left: pre-stained protein marker; Right: 9-4-2 protein target band;

[0028] Figure 2 This is the best binding result diagram of magnetic bead protein 9-4-2;

[0029] Figure 3 This is the result diagram of the enrichment sensitivity of magnetic bead protein 9-4-2;

[0030] Figure 4 This is a graph showing the specificity results of magnetic bead protein 9-4-2 amplification.

[0031] Collection information:

[0032] Nomenclature: Escherichia coli 9-4-2 / GTPV

[0033] Classification name: Escherichia coli 9-4-2 / GTPV

[0034] Deposit number: CCTCC NO.M 2019743

[0035] Collection time: September 23, 2019

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

[0037] Deposit address: Wuhan University, Wuhan, China DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are only used to illustrate the present invention and do not constitute any limitation to the scope of protection of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0039] Example 1 Screening of goat poxvirus single-domain antibodies and construction of strains expressing the single-domain antibodies

[0040] 1.1 Materials

[0041] The goat pox virus single domain antibody library is prepared according to the preparation method disclosed in the patent application with application number CN20161028158.3 and invention name "An anti-goat pox virus Bactrian camel VHH heavy chain single domain antibody cDNA library and preparation method thereof".

[0042] 1.2 Screening and construction of goat poxvirus single-domain antibody 9-4-2

[0043] The 1.1 antibody library was amplified using a two-step method. The VH-CH1-CH2 hinge region was first amplified, the VH-CH1-CH2 band was recovered and directly amplified to obtain the VHH gene band (excluding the amplified part of the VHH-CH2 hinge region), the VHH gene was cloned and transformed, and several colony plaques were picked and placed in LB culture medium respectively. They were shaken and cultured within 12-14 hours, and a small amount of plasmids were extracted and sequenced for positive results. The sequences were analyzed and predicted one by one, and the high-quality genes that met the single-domain antibody framework were selected and constructed in the expression vector p Mal-c2X respectively. The respective proteins were expressed in small amounts, and the antibody effectiveness, that is, the reaction activity, was verified by antibody detection kits, classic virus neutralization experiments and indirect immunofluorescence, so as to screen out goat pox virus single-domain antibodies with higher expression levels and better activity in a true sense.

[0044] 1.3 Results

[0045] Several sequences were obtained by sequencing, and after comparison, analysis, small-scale expression, activity verification and other screening, a goat pox virus single-domain antibody 9-4-2 was finally obtained, and the amino acid sequence of the single-domain antibody 9-4-2 protein is shown in SEQ ID NO. 1. The Escherichia coli expressing the single-domain antibody 9-4-2 protein was named Escherichia coli 9-4-2 / GTPV (Escherichia coli 9-4-2 / GTPV), and was deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO.M2019743.

[0046] Example 2 Preparation of goat poxvirus single domain antibody-coupled immunomagnetic beads (9-4-2 immunomagnetic beads)

[0047] 2.1 Purification of goatpox virus single domain antibody

[0048] Take the strain with the accession number of CCTCC NO.M 2019743. The amino acid sequence of the single-domain antibody 9-4-2 protein expressed by the strain is as shown in SEQ ID NO.1. The expression vector is pMal-c2X, N-terminal-MBP solubility-promoting tag; C-terminal-HIS purification tag, pipette 100 μL into 5 ml of LB medium containing ampicillin resistance of the strain, shake and culture, transfer to 1000 ml of LB medium containing ampicillin resistance, induce expression, harvest the bacteria, ultrasonically lyse, centrifuge, collect the supernatant, purify with a nickel column affinity layer, and finally elute the protein bound to the column with 200 mM imidazole.

[0049] Under normal circumstances, the eluted protein is directly subjected to subsequent Elisa and other tests. However, it often contains foreign proteins, which affect the purity of the protein. The present invention further performs gel cutting purification on the above protein, and the above protein is subjected to SDS-PAGE again, and the target band is cut under clean conditions, placed in a high-pressure sterilized mortar, liquid nitrogen is added under safe conditions, ground, resuspended in 0.05M, pH7.6 PBS buffer, centrifuged at 4000rpm for 20min, the supernatant is collected, a small amount of liquid nitrogen is added to the precipitate, and ground again, and resuspended in PBS buffer. The two resuspensions are concentrated using a 100,000 molecular weight cutoff (MW) ultrafiltration concentration system, and the resulting product is a VHH 9-4-2 / GTPV protein (8mg / ml) purified to a high purity and concentration, such as Figure 1 The conventional antibody titer is 1:1280, which can be increased to at least 1:1500 after purification.

[0050] 2.2 Magnetic bead labeling of 9-4-2 protein

[0051] Add 1 ml of carboxyl magnetic beads to a centrifuge tube, place it on a magnetic rack, and discard the magnetic bead storage solution; wash three times with pH 5.20.05M MES buffer, discard the buffer, add 1.6 times the amount of magnetic beads DEAC, and activate on a rotating mixer for 30 minutes; add 20 μL of 9-4-2 protein with a concentration of 3.4 mg / ml, vortex mix at room temperature for 20 minutes, place the vortexed centrifuge tube on a magnetic rack for adsorption, discard the supernatant, add 1 ml of 1xPBS buffer to resuspend, wash, repeat 3 times, finally resuspend with 4 ml1xPBS and add 1% BSA as a stabilizer for the marker, the resulting product is a 9-4-2 protein magnetic bead marker, which is used to perform a 9-4-2 protein magnetic bead marker enrichment goat pox virus attenuated vaccine experiment.

[0052] Example 3 Optimal binding test of goat poxvirus single domain antibody-coupled immunomagnetic beads (9-4-2 immunomagnetic beads)

[0053] Buy goat pox virus attenuated vaccine powder (Dou Bingying), dilute to about 10 3100 μL of TCID50 / mL virus solution was aspirated, and 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL of goat pox virus single domain antibody magnetic bead markers were added in sequence for enrichment, then washed with 1xPBS, and finally resuspended with 50 μL 1xPBS for real-time fluorescence amplification. The upper primer, lower primer, and probe sequences are:

[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: 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, and 5 cycles of pre-amplification; 95°C for 10 s, 54°C for 30 s, for a total of 40 cycles and fluorescence was collected to observe the enrichment of magnetic beads.

[0058] Results: From Figure 2 It can be seen that 10 μL magnetic beads are economical in adsorption and the adsorption capacity is optimal.

[0059] Example 4 Optimal adsorption time of goat poxvirus single domain antibody-coupled immunomagnetic beads (9-4-2 immunomagnetic beads)

[0060] In order to clarify the optimal time for 9-4-2 protein immunomagnetic beads to adsorb goatpox virus, two 1.5 ml eppendorf tubes were taken, 100 μL of the goatpox virus attenuated vaccine dilution solution as in Example 3 was taken into each tube, 10 μL of magnetic beads were added, one tube was placed at 4°C overnight, and the other tube was placed at 37°C for 2 hours, and quantitative amplification was performed as in Example 3.

[0061] Results: The CT value after enrichment at 4℃ overnight was 15.22, and the CT value after enrichment at 37℃ for 2h was 16.23, which fully demonstrated that overnight at 4℃ was the optimal reaction time for enrichment of goatpox virus by 9-4-2 protein immunomagnetic beads.

[0062] Example 5 Sensitivity of goat poxvirus single domain antibody-coupled immunomagnetic beads (9-4-2 immunomagnetic beads)

[0063] Take 1 mL of the goatpox virus attenuated vaccine dilution solution (CT value is 16.75) as in Example 3, and dilute it tenfold with 1xPBS to obtain 10 0 , 10 -1 , 10-2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Take 100 μL of the virus dilution and enrich with 10 μL of 9-4-2 protein immunomagnetic beads at 4°C overnight. The sensitivity of 9-4-2 protein immunomagnetic beads is tested the next day.

[0064] The results are as follows Figure 3 According to the fluorescent quantitative PCR judgment standard, a CT value greater than 35 needs to be re-measured. Therefore, it can be seen from Table 1 that after enrichment, 10- 4 The diluted virus solution without magnetic bead enrichment can only detect 10 -3 In addition, from the overall test results, the viral copy number content measured after enrichment is generally higher than that before enrichment. This shows that after magnetic bead enrichment, the detection rate is significantly increased, and the sensitivity is increased by at least 10 times.

[0065] Example 6 Specificity of goat poxvirus single domain antibody-coupled immunomagnetic beads (9-4-2 immunomagnetic beads)

[0066] The immunomagnetic beads coupled with the goatpox virus single domain antibody in Example 2 were taken, and the steps were the same as in Example 3 to enrich the sheeppox, sheep ulcer and peste des petits ruminants attenuated vaccines for specific detection.

[0067] The results are as follows Figure 4 The positive control and sheep pox showed peaks, while the negative control, sheep sores and peste des petits ruminants attenuated vaccine did not show peaks, indicating that the 9-4-2 protein magnetic beads are specific to sheep pox virus and have strong specificity.

[0068] Example 7 Kit for direct amplification detection of goatpox virus based on immunomagnetic beads coupled to goatpox virus single domain antibody

[0069] The kit includes 10 μL of immunomagnetic beads coupled with the goat pox virus single domain antibody 9-4-2 prepared in Example 2, 40 μL of direct amplification reaction solution, and 5 μL of primer-probe mixture. The concentrations of the upstream and downstream primers and probes are all 10 μM, and the sequences are:

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

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

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

[0073] The amplification reaction conditions are as follows: 95° C. for 10 min; 95° C. for 10 s, 60° C. for 30 s, 5 cycles of pre-amplification; 95° C. for 10 s, 54° C. for 30 s, a total of 40 cycles and fluorescence collection. Sequence Listing <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> Kit for detecting goat pox virus based on single-domain antibody 9-4-2 immunomagnetic beads and its application <141> 2022-05-12 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 131 <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 Val Val Asn Gly Asn Thr Tyr Asn Asn Tyr 20 25 30 Cys Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Gly Val Gly Arg Thr Arg Tyr Ala Asp Ser Val Asn 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Lys Arg Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Arg Trp Ser Leu Thr Thr Arg Pro Leu Ile Asn Arg Cys Glu 100 105 110 Ala Val Pro Gly Leu Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr 115 120 125 Val Ser Ser 130

Claims

1. A kit for direct amplification detection of goat pox virus based on single domain antibody 9-4-2 immunomagnetic beads, characterized in that: The kit comprises single-domain antibody 9-4-2 immunomagnetic beads obtained by coupling goatpox virus single-domain antibody 9-4-2 with magnetic beads through streptavidin-biotin (SA-Biotin), and upstream primers, downstream primers and probes for direct amplification detection of peste des petits ruminants virus; the amino acid sequence of the goatpox virus single-domain antibody 9-4-2 is shown in SEQ ID NO.1; The sequences of the upstream primer, downstream primer and probe are respectively: Upstream primer: 5′-TATTTTGTGCGGAAACGTGTTTGTA-3′; Downstream primer: 5′-ATTATAGGAATTGTTGAGAGAGAT-3′; Probe: 5′-TTTTATCAGATGATTGCGTAATCTTAAC-3′.

2. The kit according to claim 1, characterized in that The goat poxvirus single-domain antibody 9-4-2 is produced by a strain with a deposit number of CCTCC NO.M 2019743 and a deposit name of Escherichia coli 9-4-2 / GTPV, and is obtained by a gel excision purification method.

3. The kit according to claim 1, characterized in that The kit also includes a direct amplification reaction solution.

4. The kit according to claim 1, characterized in that When using the kit for detecting goat pox virus antigen, follow the steps below: 1) Virus enrichment: Take 10 μL of single-domain antibody 9-4-2 immunomagnetic beads in a 1.5 mL EP tube, add 100 μL of the supernatant of the sample to be tested after centrifugation, shake and mix, place in a 4°C refrigerator, and enrich for 12 hours or overnight; 2) Washing of magnetic beads: After the virus enrichment is completed, place the EP tube on a magnetic stand, discard the supernatant, wash three times with 1 ml of pH 7.6, 0.05 M PBS buffer, and finally resuspend with 50 μL of PBS buffer to obtain a suspension as the virus enrichment product; 3) Establish a 50 μL direct amplification reaction system: 40 μL direct amplification reaction solution, 5 μL upstream and downstream primers and probe mixture, 5 μL suspension; amplification program: 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 5 cycles of pre-amplification; 95°C for 10 s, 54°C for 30 s, a total of 40 cycles and collect fluorescence; The sequences of the primers and probes are: Upstream primer: 5′-TATTTTGTGCGGAAACGTGTTTGTA-3′; Downstream primer: 5′-ATTATAGGAATTGTTGAGAGAGAT-3′; Probe: 5′-TTTTATCAGATGATTGCGTAATCTTAAC-3′.

5. The kit according to claim 4, characterized in that The concentrations of the upstream and downstream primers and probes are all 10 μM.

6. Use of the kit according to any one of claims 1 to 5 in preparing a reagent for detecting goatpox virus antigen.

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