Kit for detecting peste des petits ruminants virus 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 bead direct spread detection small ruminant pestivirus kit and its application.The kit includes the single-domain antibody 9-4-1 immunomagnetic bead obtained by the single-domain antibody 9-4-1 shown in SEQ ID NO.1 small ruminant pestivirus through streptavidin-biotin and magnetic bead coupling.The single-domain antibody 9-4-1 protein purified by gel cutting is coupled with magnetic bead to obtain immunomagnetic bead, after small ruminant pestivirus is enriched by immunomagnetic bead, it can be directly amplified without extracting genomic RNA.The application overcomes the problems of low virus content, low detection rate, easy to miss detection, etc.;And a series of problems such as time-consuming, costly, laborious, environmental pollution during operation when extracting genomic RNA, by directly amplifying enrichment product, not only save time, save resources, but also efficient, sensitive, specific, stable, the application provides a kind of efficient technical means for small ruminant pestivirus antigen detection.

Description

TECHNICAL FIELD

[0001] The application relates to an immunomagnetic bead kit, in particular to a kit for detecting peste des petits ruminants virus based on single-domain antibody immunomagnetic beads and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Peste des petits ruminants (PPR) is also known as sheep plague, small ruminant pseudocow plague, enteropneumonia, etc., which is caused by peste des petits ruminants virus (PPRV) and has main clinical symptoms of fever, pneumonia, diarrhea, dyspnea, etc. The morbidity and mortality can be as high as 100% and 90%, respectively. The use of vaccines effectively controls the large-scale outbreak of the epidemic, but in recent years, PPRV has almost not been interrupted. The important epidemiological investigation of sheep diseases found that the persistent virus carrying in the wild Procapra przewalskii population in the north is a key factor for causing the current PPRV frequent outbreak or regional prevalence (Lingxia Li, et al. Epidemic and Evolutionary Characteristics of Peste des Petits Ruminants Virus Infecting Procapra przewalskii in Western China, Infection, Genetics and Evolution, 2019). This result brings new challenges to detection. The difficulty of continuous virus carrying detection lies in that the virus content in the body is low and the interference factors are complex, which can easily cause missed detection, thereby bringing certain difficulty to accurate diagnosis and isolation of the pathogen.

[0003] A single-domain antibody is the smallest antigen-binding unit of an antibody molecule, which is composed of only one variable domain or one engineered constant domain that only assists target binding and does not contain the Fc segment of a common antibody. Therefore, the single-domain antibody can avoid the complement reaction caused by the Fc segment, and is more and more applied to the fields of biological detection, diagnosis, medicine, etc. The immunomagnetic bead technology (IMB) is a new type of diagnostic technology based on antigen-antibody reaction and using magnetic carrier technology as a medium. After certain treatment, the antibody is combined with the magnetic bead as an antibody carrier. After the magnetic bead combined with the antigen material, an immune complex is formed. Under the action of the magnetic force of the magnet, the complex mechanically moves, separates the antigen specifically combined with the antibody on the magnetic bead from other substances. This technology has been applied to immunodetection, cell separation, protein purification, etc. Due to its unique characteristics of rapidness, high efficiency, low toxicity, high specificity, simple operation, etc., it provides a new idea for antigen examination of latent infection.

[0004] This invention combines optimized single-domain antibodies with immunomagnetic bead technology, eliminating the need for complex purification equipment and techniques and imposing no restrictions on sample clarity. It allows for convenient detection of low levels of peste des petits ruminants virus with just a simple magnetic adsorption step, thus establishing an immunoassay technique that is fast, specific, sensitive, and reproducible. Summary of the Invention

[0005] To address the challenges of existing technologies in detecting peste des petits ruminants (PPR) virus antigens, 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 and the high cost of RNA extraction kits, this invention offers two solutions. Firstly, it utilizes a gel extraction method to prepare high-purity single-domain antibody proteins, resulting in better protein purity and easier labeling compared to conventional methods. Labeled immunomagnetic beads then exhibit stronger specificity for enriching PPR pathogens. Secondly, it eliminates the crucial step of genomic RNA extraction, allowing direct amplification of the enriched products for real-time quantitative fluorescence detection, saving time, manpower, resources, and money, and improving economic efficiency.

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

[0007] This invention discloses a kit for direct amplification detection of peste des petits ruminants virus (PPR) 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 PPR single-domain antibody 9-4-1 to magnetic beads via streptavidin-biotin (SA-Biotin). The amino acid sequence of the PPR single-domain antibody 9-4-1 is shown in SEQ ID NO.1.

[0008] Preferably, the small ruminant plague virus single-domain antibody 9-4-1 is produced by a strain with accession number CCTCC NO.M2019740 and accession name Escherichia coli 9-4-1 / PPRV, and obtained by gel purification.

[0009] Preferably, the kit further includes upstream primers, downstream primers, and a probe for direct amplification detection of peste des petits ruminants virus, wherein the sequences of the upstream primers, downstream primers, and probe are as follows:

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

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

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

[0013] Preferably, the concentration of the upstream primer, the downstream primer and the probe is 10 μM.

[0014] Preferably, the kit further comprises a direct amplification reaction solution.

[0015] Preferably, when the kit is used for detecting PPRV antigen, the following steps are performed:

[0016] 1) Virus enrichment: take 10 μl of single-domain antibody 9-4-1 immunomagnetic beads in a 1.5 mL EP tube, add 100 μl of supernatant of the sample to be detected after centrifugation, shake and mix, place in a 4℃ refrigerator, and enrich for 12 h or overnight;

[0017] 2) Magnetic bead washing: after the virus enrichment is completed, place the EP tube on a magnetic stand, discard the supernatant, wash with 1 ml of PBS buffer solution with a pH value of 7.6 and a concentration of 0.05 M for 3 times, finally resuspend with 50 μl of PBS buffer solution to obtain a suspension as the product of enriched virus;

[0018] 3) Establish a 50 μl direct amplification reaction system: direct amplification reaction solution 40 μl, upstream primer and probe mixture 5 μl, and suspension 5 μl; the amplification program is as follows: 50℃ for 15 min; 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, and 72℃ for 30 s, 5 cycles of pre-amplification; 95℃ for 10 s, 51℃ for 30 s, a total of 40 cycles and fluorescence collection;

[0019] The sequences of the primers and the probe are as follows:

[0020] Upstream primer: 5'-GAGGGGAGTAGAGATGAAGACGAG-3';

[0021] Downstream primer: 5'-ATAATTGGAAAGGCGGGCTAAGAC-3';

[0022] Probe: 5'-GAGCTTGCAAGTTTCCTAATGGATCGG-3'.

[0023] Further, the application also proposes the use of the kit in the preparation of a reagent for detecting PPRV antigen.

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

[0025] The application overcomes a series of problems in the detection of PPRV antigen, such as low virus content, false negative results due to failure to meet the minimum requirement of RT-PCR detection, time-consuming and laborious extraction of RNA, high cost of purchasing RNA extraction kit, and low concentration and purity of single-domain antibody after expression and purification. The single-domain antibody prepared by optimization has high expression amount and good reactivity, and the use of immunomagnetic beads for pathogen enrichment is more economical. The kit obtained by directly amplifying the enrichment product not only saves time and resources, but also is efficient, sensitive, specific, stable and practical, and provides an efficient technical means for the detection of PPRV. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 SDS-PAGE result diagram for cutting and purifying 9-4-1 protein;

[0027] Left: 9-4-1 protein target band; right: pre-stained protein marker;

[0028] Figure 2 Result diagram of the best combination of magnetic bead protein 9-4-1;

[0029] Figure 3 Result diagram of the enrichment sensitivity of magnetic bead protein 9-4-1;

[0030] Figure 4 Result diagram of the amplification specificity of magnetic bead protein 9-4-1.

[0031] DEPOSIT INFORMATION

[0032] Name: Escherichia coli 9-4-1 / PPRV

[0033] Classification name: Escherichia coli 9-4-1 / PPRV

[0034] Deposit number: CCTCC NO.M 2019740

[0035] Deposit date: September 23, 2019

[0036] Deposit organization: China Center for Type Culture Collection (CCTCC).

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

[0038] The advantages and features of the present application will become more apparent with the description of specific embodiments. The embodiments are only used to illustrate the present application and do not constitute any limitation on the protection scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0039] Example 1 Screening of PPRV single domain antibody and construction of strain expressing the single domain antibody

[0040] 1.1 Materials PPRV single domain antibody library See the patent: A camel-derived PPRV cDNA library and its preparation method and application (Patent No. ZL201810501728.0)

[0041] 1.2 Screening and construction of PPRV single domain antibody 9-4-1

[0042] The antibody library in 1.1 is amplified by two-step method, first to amplify VH-CH1-CH2 hinge region, recover VH-CH1-CH2 band and directly amplify VHH gene band (omit the amplification part of VHH-CH2 hinge region), clone and transform VHH gene, pick several colonies into LB medium, shake culture for 12-14 h, extract small amount of plasmid from positive ones, sequence, analyze and predict the sequences one by one, select high-quality genes meeting the single domain antibody framework, construct in expression vector pMal-c2X respectively, and transform into E. coli to express respective proteins, respectively use antibody detection kit, classical virus neutralization experiment and indirect immunofluorescence to verify the effectiveness of the antibody, i.e. the reactivity, and select the PPRV single domain antibody with higher expression and better activity in a true sense.

[0043] 1.3 Results

[0044] After sequencing, several sequences are obtained, which are compared, analyzed, expressed in small amount, and verified for activity one by one, and finally a PPRV single domain antibody 9-4-1 is 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 is named E. coli 9-4-1 / PPRV, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO. M 2019740.

[0045] Preparation of Peste des petits ruminants virus single domain antibody coupled immunomagnetic beads (9-4-1 immunomagnetic beads) of Example 2

[0046] 2.1 Purification of Peste des petits ruminants virus single domain antibody

[0047] Take the strain with preservation number M 2019740, the amino acid sequence of the single domain antibody 9-4-1 protein expressed by the strain is as shown in SEQ ID NO. 1, the expression vector is pMal-c2X, the N-terminal is an MBP solubilization tag; and the C-terminal is a HIS purification tag. 100 μl of the strain is taken into 5 ml of LB culture medium containing ampicillin resistance, shaken and cultured, then transferred to 1000 ml of LB culture medium containing ampicillin resistance, induced for expression, the bacteria are collected, ultrasonically lysed, centrifuged, the supernatant is collected, subjected to nickel column affinity layer purification, and finally the proteins combined with the column are eluted with 200 mM imidazole.

[0048] Under normal circumstances, the eluted proteins are directly subjected to subsequent Elisa tests and the like. However, they often contain impure proteins, which affect the purity of the proteins. The proteins are further subjected to gel cutting purification according to the present application. The proteins are subjected to SDS-PAGE again, the target band is cut under clean conditions, placed in a high-pressure sterilization mortar, liquid nitrogen is added under safe conditions, ground, resuspended in 0.05 M, pH 7.6 PBS buffer, centrifuged at 4000 rpm for 20 min, the supernatant is collected, a small amount of liquid nitrogen is added to the precipitate, which is ground again, resuspended in PBS buffer, and the two resuspensions are concentrated by a 100,000 molecular weight (MW) ultrafiltration concentration system. The obtained product is the VHH 9-4-1 / PPRV protein (6.9 mg / ml) which is purified to high purity and concentration, as shown in Figure 1 The conventional antibody reaction titer is 1:320-1:640, and after purification, it is at least increased to about 1:1280-1500.

[0049] 2.2 9-4-1 protein magnetic bead labeling

[0050] Carboxyl magnetic beads 1 ml are added to a centrifuge tube, which is placed on a magnetic stand, and the magnetic bead storage solution is discarded; washed with 0.05 M MES buffer solution with pH 5.2 for 3 times, the buffer solution is discarded, 1.6 times the amount of DEAC is added to the magnetic beads, and activated on a rotary mixing instrument for 30 min; 20 μL of 9-4-1 protein with a concentration of 6.9 mg / ml is added, vortexed at room temperature for 20 min, the vortexed centrifuge tube is placed on a magnetic stand for adsorption, the supernatant is discarded, 1 ml of 1xPBS buffer solution is added for resuspension, washed repeatedly for 3 times, finally resuspended with 9 ml of 1xPBS and added with 1% BSA as a stabilizer of the labeled product, and the obtained product is the 9-4-1 protein magnetic bead label, which is used for the enrichment of Peste des petits ruminants virus attenuated vaccine experiment of the 9-4-1 protein magnetic bead label.

[0051] Example 3 Optimal binding test of PPRV single domain antibody conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0052] PPRV attenuated vaccine powder was purchased and diluted to about 10 3 TCID 50 / mL of virus solution, 100 μl of each was taken, 5 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl of PPRV single domain antibody magnetic beads marker (prepared in Example 2) was added in turn for enrichment, then washed with 1xPBS, finally resuspended with 50 μl of 1xPBS, and real-time fluorescence amplification was performed. The sequences of the upstream primer, downstream primer and probe used were as follows:

[0053] Upstream primer: 5'-GAGGGGAGTAGAGATGAAGACGAG-3';

[0054] Downstream primer: 5'-ATAATTGGAAAGGCGGGCTAAGAC-3';

[0055] Probe: 5'-GAGCTTGCAAGTTTCCTAATGGATCGG-3'.

[0056] A 50 μl direct amplification reaction system was established: direct amplification reaction solution 40 μl, primer probe mixture 5 μl, resuspension solution 5 μl; the amplification program was: 50°C for 15 min; 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, 5 cycles of pre-amplification; 95°C for 10 s, 51°C for 30 s, 40 cycles of amplification and fluorescence collection, and the enrichment of magnetic beads was observed.

[0057] Results: From Figure 2 It can be seen that 10 μl of magnetic beads is saved and the adsorption capacity is optimal.

[0058] Example 4 Optimal adsorption time of PPRV single domain antibody conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0059] To determine the optimal adsorption time of 9-4-1 protein immunomagnetic beads for PPRV, two 1.5 ml ep tubes were taken, each containing 100 μl of PPRV attenuated vaccine diluent as in Example 3, 10 μl of 9-4-1 immunomagnetic beads was added, one was placed at 4°C overnight, and the other was placed at 37°C for 2 h, and quantitative amplification was performed as in Example 3.

[0060] Results: The CT value of the virus enriched at 4°C overnight was 15.69, and the CT value of the virus enriched at 37°C for 2 h was 17.33, which fully demonstrated that 4°C overnight was the optimal reaction time for 9-4-1 immunomagnetic beads to enrich PPRV.

[0061] Sensitivity of PPRV single domain antibody conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0062] Take 1 mL of PPRV attenuated vaccine diluent as in Example 3 (CT value is 16.12), and dilute with 1xPBS by ten times to obtain virus diluents of 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , respectively. Take 100 μl of each and conduct 4°C overnight enrichment with 10 μl of 9-4-1 immunomagnetic beads, and detect the sensitivity of 9-4-1 immunomagnetic beads the next day.

[0063] According to the results as in Figure 3 , according to the fluorescence quantitative PCR determination standard, CT value greater than 35 needs to be re-determined, so from Table 1, it can be seen that after enrichment, 10 -4 diluted virus solution can be detected, and 10 -3 diluted virus solution can only be detected without magnetic bead enrichment. In addition, from the overall detection results, the virus copy number content determined after enrichment is generally higher than that without enrichment. It is shown that after enrichment of 9-4-1 immunomagnetic beads, the detection rate is obviously increased, and the sensitivity is improved by at least 10 times.

[0064] Specificity of PPRV single domain antibody conjugated immunomagnetic beads (9-4-1 immunomagnetic beads)

[0065] Take the magnetic beads in this example 2, and conduct enrichment of sheep pox, orf and PPRV attenuated vaccine according to the steps in Example 3 for specificity detection.

[0066] The results are as in Figure 4 , the positive control and PPRV sample appear peak values, and the negative control, sheep pox and orf attenuated vaccine do not appear peak values, which shows that the 9-4-1 immunomagnetic beads are specific to PPRV, and have strong specificity.

[0067] Kit for PPRV direct amplification detection based on PPRV single domain antibody conjugated immunomagnetic beads

[0068] The kit comprises PPRV single domain antibody 9-4-1 conjugated immunomagnetic beads prepared in Example 2, direct amplification reaction solution, and primer probe mixture. The concentrations of the used upstream primer, downstream primer and probe are all 10 μM, and the sequences are as follows:

[0069] Upstream primer: 5'-GAGGGGAGTAGAGATGAAGACGAG-3';

[0070] Downstream primer: 5'-ATAATTGGAAAGGCGGGCTAAGAC-3';

[0071] Probe: 5'-GAGCTTGCAAGTTTCCTAATGGATCGG-3'.

[0072] A 50 μl direct amplification reaction system was established: direct amplification reaction solution 40 μl, primer probe mixture solution 5 μl, resuspension solution 5 μl; the amplification reaction conditions: 50℃ 15 min; 95℃ 10 min; 95℃ 10 s, 60℃ 30 s, 72℃ 30 s, 5 cycles of pre-amplification; 95℃ 10 s, 51℃ 30 s, a total of 40 cycles and fluorescence collection. SEQUENCE LISTING <110> Lanzhou Institute of Animal Health, Chinese Academy of Agricultural Sciences <120> Reagent kit for direct amplification detection of peste des petits ruminants virus based on single-domain antibody 9-4-1 immunomagnetic beads and application thereof <141> 12 May 2022 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 125 <212> PRT <213> Peste des petits ruminants virus antibody <400> 1 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr lie Asn Ser Gly Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ser Arg Leu Thr lie Gin Ala Leu Ala Thr Tyr Leu Val Val Tyr 100 105 110 Asn Tyr Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 115 120 125

Claims

1. A kit for detecting Peste des petits ruminants 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 PPRV 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 direct extension detection of PPRV, wherein the amino acid sequence of the PPRV 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: The upstream primer is 5'-GAGGGGAGTAGAGATGAAGACGAG-3'; The downstream primer is 5'-ATAATTGGAAAGGCGGGCTAAGAC-3'; The probe is 5'-GAGCTTGCAAGTTTCCTAATGGATCGG-3'.

2. The kit of claim 1, wherein The PPRV single-domain antibody 9-4-1 is produced by a strain with a preservation number of CCTCC NO.M 2019740 and a preservation name of Escherichia coli 9-4-1 / PPRV, and is obtained by a gel cutting and purifying method.

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 PPRV 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 hours 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 mixed solution 5 μl, and suspension 5 μl; the amplification program is as follows: 50℃ for 15 min; 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, and 72℃ for 30 s, 5 cycles of pre-amplification; 95℃ for 10 s, 51℃ for 30 s, a total of 40 cycles and collection of fluorescence; The sequences of the primers and the probe are respectively: The upstream primer is 5'-GAGGGGAGTAGAGATGAAGACGAG-3'; The downstream primer is 5'-ATAATTGGAAAGGCGGGCTAAGAC-3'; The probe is 5'-GAGCTTGCAAGTTTCCTAATGGATCGG-3'.

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 PPRV antigens.

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