CN108486069B - Virus separation method for low-content sample of porcine epidemic diarrhea virus - Google Patents
Virus separation method for low-content sample of porcine epidemic diarrhea virus Download PDFInfo
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- CN108486069B CN108486069B CN201810570334.0A CN201810570334A CN108486069B CN 108486069 B CN108486069 B CN 108486069B CN 201810570334 A CN201810570334 A CN 201810570334A CN 108486069 B CN108486069 B CN 108486069B
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Abstract
The invention discloses a virus separation method for a low-content sample of Porcine Epidemic Diarrhea Virus (PEDV), and also discloses an immune nano magnetic bead for enrichment and separation of porcine epidemic diarrhea virus, wherein the immune nano magnetic bead has magnetic Fe2O3A core and a surface conjugated with a single domain antibody specific for a PEDV membrane protein. The traditional method for separating virus from multiple infection and low PEDV content samples needs virus purification and long-time blind transmission, has low separation efficiency, and limits research work of PEDV. According to the invention, the immune nano magnetic beads are prepared by using the nano magnetic beads and the PEDV specific single domain antibody, so that PEDV in various samples such as feces and tissues can be effectively captured and enriched. After the sample is processed, the immune nano magnetic beads are used for capturing and enriching viruses, and the immune nano magnetic beads combined with the viruses are directly inoculated to Vero cells, so that the virus separation can be realized. The method for separating the immune nano magnetic beads and the PEDV is suitable for quickly and effectively separating the PEDV in clinically complex samples with low virus content.
Description
Technical Field
The invention relates to an immune nano magnetic bead for separating Porcine Epidemic Diarrhea Virus (PEDV) and also relates to a high-efficiency virus separation method for a clinical sample with low content of PEDV. The invention belongs to the field of biotechnology.
Background
Porcine Epidemic Diarrheal Virus (PEDV) belongs to the order Nidovirales (Nidovirales), the family Coronaviridae (Coronaviridae), a member of the genus Coronavirus (Coronavirus), and is a pathogen that causes Porcine Epidemic Diarrhea (PED). PED is a highly-contact intestinal infectious disease which is mainly characterized by diarrhea, vomiting and dehydration of infected pigs, and pigs of various ages can be infected and diseased, and particularly has higher lethality to suckling piglets. The incidence area of the porcine epidemic diarrhea spreads all over the world, and great harm is brought to the pig raising industry. The rapid separation and identification of the virus in clinically ill PED pig samples is a necessary link for researching PEDV epidemiology, molecular biology and high-efficiency vaccine development. However, clinical pathogen infections are complex and diverse, clinical cases of single pathogen infections are rare, and this is very difficult to isolate using conventional methods. Meanwhile, for some samples with low pathogen content, the separation by using the conventional method is difficult to succeed. Therefore, the establishment of the method for effectively separating the target pathogen in clinical multiple infection and low pathogen content samples has important significance for the research of etiology, epidemiology, vaccine and the like.
The application of immunomagnetic beads in the field of life science is increasingly common, especially in the aspects of pathogen detection, separation and the like. The immunomagnetic bead separation technology takes antibody-coated magnetic beads as carriers, utilizes the specific combination of antigens and antibodies to form an antigen-antibody-magnetic bead compound, and the compound can move directionally under the action of a magnetic field, thereby realizing the specific separation of the antigens. The method combines the high efficiency of dispersed solid phase extraction, the specificity of immunoaffinity and the simplicity of magnetic recovery, and can effectively simplify the pretreatment process, shorten the pretreatment time and improve the enrichment and separation efficiency. With the continuous maturation and perfection of nano magnetic bead preparation and surface coating technology, various antibodies, specific affinity factors and the like can be used for magnetic bead coating, so that immunomagnetic beads are obtained, and the specificity of the immunomagnetic beads completely depends on the coated antibodies or affinity factors. Currently, the main antibodies commonly used include monoclonal antibodies (IgG), single chain antibodies, single domain antibodies (sdAb), and the like. The single domain antibody is an IgG antibody which naturally exists in blood serum of camelidae, cartilage shark and the like and only has a heavy chain, the antigen binding site of the antibody only consists of a single structural domain of a heavy chain variable region, and the IgG antibody is also called as a heavy chain antibody (VHH) or a nano antibody (Nanobody, Nb). The sdabs retain good, specific antigen binding capacity as do normal IgG antibody molecules. Meanwhile, the sdAb also has the unique properties of easy soluble expression in an escherichia coli expression system, high temperature resistance, extreme pH resistance and the like. These properties make sdabs ideal candidate antibodies for immunomagnetic bead preparation.
Disclosure of Invention
A first object of the present invention is to provide immunomagnetic beads capable of binding and efficiently enriching Porcine Epidemic Diarrheic Virus (PEDV); it is another object of the present invention to provide a method for efficiently isolating viruses from samples with low PEDV content.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to an immunomagnetic bead capable of capturing and enriching Porcine Epidemic Diarrhea Virus (PEDV), which has magnetic Fe2O3The core and the surface coupled with the PEDV membrane protein specific single domain antibody, wherein the amino acid sequence of the PEDV membrane protein specific single domain antibody is shown as SEQ ID NO. 2.
Furthermore, the invention also provides a method for preparing the immune nano magnetic bead, which comprises the following steps:
(1) activation of Nano magnetic beads
Taking Fe with carboxyl surface2O3Adding nano magnetic beads into a centrifuge tube, washing for 3 times by using sterile PBS buffer solution, collecting the magnetic beads by using a magnetic frame, then adding 0.4mol/L EDC and 0.2mol/L NHS into the centrifuge tube with the magnetic beads, activating the surfaces of the magnetic beads, and washing for 5 times by using PBS buffer solution for later use;
(2) buffer replacement of coating antibody
Taking a PEDV membrane protein specific single domain antibody solution, and replacing a dissolving buffer solution with a PBS buffer solution by using an ultrafiltration tube for later use;
(3) preparation of immune nano magnetic bead
Adding a PEDV membrane protein specific single domain antibody solution with a replaced buffer solution into a centrifuge tube with activated magnetic beads, placing the centrifuge tube at 37 ℃, shaking at 120rpm, incubating for 2h, washing for 5 times by using PBS buffer solution, collecting the magnetic beads, then placing bovine serum albumin with the mass volume ratio of 0.1 percent at 37 ℃, sealing for 30min, collecting the magnetic beads, and resuspending by using PBS buffer solution containing 0.01 percent of sodium azide and 10 percent of glycerol in volume ratio, thus obtaining the PEDV membrane protein specific single domain antibody solution, and storing the PEDV membrane protein specific single domain antibody solution at 4 ℃ for later use.
Among them, preferably, Fe having a carboxyl surface2O3The diameter of the nanometer magnetic bead is 200 nm.
Among them, it is preferable that the volume ratio of 0.4mol/L EDC and 0.2mol/L NHS added is 1: 1.
Wherein, the pH value of the PBS buffer solution in the steps (1) to (3) is preferably 7.4.
Furthermore, the invention also provides a method for effectively separating PEDV low-content sample viruses, namely the method is used for capturing and enriching the PEDV in a low-virus-content clinical sample by using the immune nano magnetic beads, and virus separation is realized through cell separation and passage.
Wherein, preferably, the method comprises the following specific steps:
a. co-incubation of immune nano magnetic beads and a sample: soaking clinical suspected PEDV infected pig manure and secretion cotton swab type samples or tissue samples in sterile PBS buffer solution or homogenizing tissues, incubating the samples with immune nano magnetic beads for 1h at room temperature and 80rpm, washing the samples for 5 times by using sterile PBST, and collecting the immune nano magnetic beads by using a magnetic frame;
b. inoculating the immune nano magnetic beads and the virus compound with cells: the immunomagnetic beads were washed 5 times with DMEM containing 100IU/mL penicillin and 100. mu.g/mL streptomycin, collected on a magnetic rack, mixed with 1mL DMEM containing 5. mu.g/mL pancreatin, 100IU/mL penicillin and 100. mu.g/mL streptomycin, and added to the cell surfaceFlour, 37 deg.C, 5% CO2And incubating for 1 h;
c. replacing the cell culture solution: adding DMEM medium containing 5. mu.g/mL pancreatin, 100IU/mL penicillin and 100. mu.g/mL streptomycin at 37 deg.C with 5% CO2The culture was continued for 24h, and cytopathic effect (CPE) was observed and virus passaging was carried out to achieve virus isolation.
Wherein, preferably, the pH value of the PBS buffer solution in the step a is 7.4, and the cells in the step b are vero cells.
The Porcine Epidemic Diarrhea Virus (PEDV) membrane protein specific single domain antibody and the nucleotide sequence thereof are also within the protection scope of the invention, and the amino acid sequence of the porcine epidemic diarrhea virus membrane protein specific single domain antibody is shown in SEQ ID NO. 2. Preferably, the nucleotide sequence is shown as SEQ ID NO. 1.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the immune nano magnetic beads are prepared by using the nano magnetic beads and the PEDV specific single domain antibody, so that PEDV in various samples such as feces and tissues can be effectively captured and enriched. After the sample is processed, the immune nano magnetic beads are used for capturing and enriching viruses, and the immune nano magnetic beads combined with the viruses are directly inoculated to cells, so that the virus separation can be realized. The method for separating the immune nano magnetic beads and the PEDV is suitable for quickly and effectively separating the PEDV in clinically complex samples with low virus content.
Drawings
FIG. 1 is sequence analysis, expression and purification of PEDV-specific sdabs;
a: skeletal and complementarity determining region partitioning was performed by The Kabat et al (Kabat,1991) approach. (Kabat, E.A.,1991.Sequences of Proteins of Immunological interest. US Department of Health and Human Services, Public Health Service, National Institutes of Health.). B: lane 1, blank control; lane 2, expression; lane 3, purification; m, protein molecular weight standard. C: the binding activity of sdAb-Mc40 to M protein was analyzed in an ELISA assay.
FIG. 2 is an analysis of binding activity of immunomagnetic beads to M protein;
co-incubating the recombinant expression M protein and immune nano magnetic beads, detecting the binding condition through SDS-PAGE, and verifying through western blot, wherein PEDV antibody positive pig serum is used as a primary antibody, and HRP-labeled rabbit anti-pig antibody is used as a secondary antibody; lane 1, immunomagnetic bead-bound M protein; lane 2, total recombinant M protein used; lane 3, supernatant after incubation with magnetic beads; lane 4, supernatant after incubation with magnetic beads; lane 5, total recombinant M protein used; lane 6, immunomagnetic bead bound M protein.
FIG. 3 shows the capture activity analysis of PEDV strain CV777 by immunomagnetic beads;
incubating magnetic beads and CV777 infected vero cell supernatant, collecting the magnetic beads, detecting a part of the magnetic beads by using a PEDV specific primer through RT-PCR (reverse transcription-polymerase chain reaction), wherein a band (A) with the expected size is visible, detecting the magnetic beads after incubating in a lane 1, detecting a lane M and a DNA marker; the other part is negatively stained by 3% sodium phosphotungstate, and the surface of the magnetic beads is observed by a transmission electron microscope to be combined with virus particles (B) with the size of about 100 nm.
FIG. 4 is a virus enrichment assay;
diluting PEDV cytotoxic CV777 by PBS (phosphate buffer solution) in a multiple ratio manner, carrying out PCR (polymerase chain reaction) detection, taking diluted viruses which cannot be detected by RT-PCR (reverse transcription-polymerase chain reaction) and incubating the diluted viruses with immune nano magnetic beads, and then analyzing the virus enrichment effect of the immune nano magnetic beads by RT-PCR detection. Lane 1, blank magnetic bead control; lane 2, viral RT-PCR detection after dilution; lanes 3-9, RT-PCR detection results after incubation of 400, 600, 800, 1000, 1200, 1400 and 2000. mu.L diluted virus with magnetic beads, respectively.
FIG. 5 is the isolation and identification of field strains;
co-incubating immune nano magnetic beads and PEDV infected piglet feces samples, collecting the magnetic beads, detecting the magnetic beads through RT-PCR (B), a lane 1, and directly detecting the treated feces samples (200 mu L of the samples are used for RNA extraction); lane 2, 1mL of the treated sample was incubated with immunomagnetic beads and then detected; lane 3, blank magnetic bead control; lane 4; incubating 2mL of the treated sample with immune nano magnetic beads and detecting; lane 5, 3mL of the treated sample was incubated with immunomagnetic beads and detected. Vero cells were seeded and significant cytopathic effects (a) were observed; by using an immunofluorescence technique, the obvious fluorescence (C) in the cells can be seen; when the ultrathin cell section is observed by a transmission electron microscope, the virus particles are obvious in the cell, and the arrow indicates part of the virus particles (D).
FIG. 6 is an S gene-based evolution analysis.
The S gene sequences of the isolate jxdv1801 and 10 PEDV strains with known subtypes published on NCBI are compared and analyzed by MEGA6.0 software, and a phylogenetic tree is constructed by a maximum likelihood Method (ML) and a Poisson model (poisson model). As a result, it was confirmed that the isolate was subtype G1.
Detailed Description
The advantages of the present invention are further described below by way of examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the invention.
Example 1 preparation of PEDV-specific immunomagnetic Nanomagnetic beads
1.1 screening and expression of PEDV Membrane protein (M) -specific sdabs
Panning M protein immune bactrian camel library by using phage display technology to obtain phage specifically combined with M protein, performing sequencing analysis to obtain an sdAb gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO.1, and performing sequence analysis; then cloning the protein into a PET-32a expression plasmid, expressing the plasmid by using escherichia coli (E.coli), and purifying the target protein by using Ni purification resin; the binding activity of sdAb-Mc40 to M protein was analyzed by enzyme-linked immunosorbent assay (ELISA).
Phage sequencing analysis of M protein binding to obtain specific single domain antibody sdAb-Mc40 (FIG. 1A); after being connected with the 32a vector, the protein is transformed into escherichia coli to obtain expression protein with the same size, and the expression protein is soluble expression, and the target protein (shown in figure 1B) can be obtained by using Ni purification resin, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2; the results of the ELISA assay confirmed that sdAb-Mc40 had M protein binding activity.
1.2 activation of Nano-magnetic beads
Taking 500 mu L of Fe with carboxyl surface and the concentration of 2mg/mL and the diameter of 200nm2O3The nanobead was added to a 1.5mL centrifuge tube, washed 3 times with 1mL sterile PBS (pH7.4), and the magnetic beads were collected by a magnetic holder. Then, 200. mu.L each of 0.4mol/L EDC and 0.2mol/L NHS was added to the centrifuge tube with magnetic beads, the surfaces of the magnetic beads were activated, and the mixture was washed 5 times with PBS (pH7.4) for further use.
1.3 buffer replacement of coated antibody
1mL of prokaryotic expressed and purified sdAb-Mc40 was taken at a concentration of 2.35mg/mL, and its lysis buffer was replaced with PBS (pH7.4) using an ultrafiltration tube to a final volume of 1mL at a concentration of 1.8mg/mL for use.
1.4 preparation of Immunomagnetic beads
1mL of buffer-exchanged sdAb-Mc40 was added to a centrifuge tube containing activated magnetic beads, incubated at 37 ℃ for 2h while shaking at 120rpm, washed 5 times with PBS (pH7.4), the magnetic beads were collected, then suspended with Bovine Serum Albumin (BSA) at 0.1% by mass/volume (at 37 ℃ for 30 min), the magnetic beads were collected, resuspended in 1mL of PBS buffer (pH7.4) containing 0.01% by volume of sodium azide and 10% glycerol, and stored at 4 ℃ for further use.
2 analysis of binding Activity of Immunomagnetic bead Virus
2.1 analysis of the Capture Activity of Immunomagnetic beads on PEDV Membrane protein (M)
The binding activity of the immunomagnetic beads and the M protein is analyzed by SDS-PAGE and Western blot. Adding 200 μ L PBS (pH7.4) into 1.5mL centrifuge tube, adding 10 μ L immunomagnetic beads with concentration of 2mg/mL and 50 μ L recombinant M protein with concentration of 1.23mg/mL, shaking at 37 deg.C and 80rpm, and incubating for 1 h; meanwhile, a blank magnetic bead control of unconjugated single-domain antibody is arranged. Collecting magnetic beads and the conjugate with a magnetic rack, discarding the supernatant, washing with PBST (pH7.4) for 5 times, 80rpm for 5 min/time, and collecting the magnetic beads; adding SDS-PAGE sample buffer, boiling in boiling water for 10 min; a portion of the samples were directly analyzed by 12% SDS-PAGE, resulting in a band corresponding to the size of M protein in the sample lane incubated with immunomagnetic beads and M protein (FIG. 2).
Another portion of the sample was separated by 12% SDS-PAGE and transferred to PVDF membrane. Then incubated with PEDV positive pig serum at room temperature at 80rpm for 1h, and washed 4 times with PBST. Incubate with horseradish peroxidase (HRP) -labeled rabbit anti-porcine secondary antibody at room temperature, 80rpm for 1h, wash 5 times with PBST, 80rpm, 5 min/time. Developing with ECL developing solution; as a result, a band corresponding to the size of M protein appeared in the lane of the sample incubated with the immunomagnetic beads and the M protein (FIG. 2). These results show that the prepared immune nano magnetic beads have the M protein capture function.
2.2 Immunomagnetic beads for PEDV cytotoxic (strain CV777) capture assay
The binding activity of immunomagnetic beads to PEDV (strain CV777) was analyzed by transmission electron microscopy and RT-PCR. Take 1mL of TCID50Value of 107.20.1mL of CV777 cytotoxic, and 20 g immune nanometer magnetic beads mixed, 37 degrees, 80rpm, incubated for 1h, then use the magnetic frame separation immune nanometer magnetic beads, abandon the supernatant, PBST washing 5 times. Some of the immunomagnetic beads collected by the magnetic frame were negatively stained with 3% phosphotungstic acid, and observed by transmission electron microscopy, and as a result, virus particles having a size of about 100nm were observed around the beads, corresponding to a size of 90-180nm of PEDV (FIG. 3B).
The other part was used for RNA extraction and direct detection by RT-PCR using PEDV specific detection primers, resulting in the appearance of specific bands consistent with the expected size in the immunomagnetic bead samples incubated with CV777 (fig. 3A). These results confirm that the prepared immunomagnetic beads have the PEDV virus capture function.
3 analysis of enrichment capacity of immune nano magnetic beads on low PEDV content samples
Diluting the PEDV cell strain CV777 by 10 times until RT-PCR can not be detected, taking a sample with the dilution, incubating the virus after dilution with different volumes by using immune nano magnetic beads, and detecting by RT-PCR. As a result, when the virus content in the sample is low and cannot be detected by PCR, the virus can be detected by RT-PCR after being enriched by the immune nano magnetic beads (figure 4), which shows that the immune nano magnetic beads prepared by the invention are suitable for virus enrichment of the sample with low PEDV content.
EXAMPLE 2 Virus isolation of samples with Low PEDV content
1 clinical sample processing
Placing suspected PEDV infected pig feces, secretion and other cotton swab type samples in a 2mL centrifuge tube, adding 1mL sterile PBS (pH7.4), placing at room temperature for 2h, centrifuging at 2000rpm for 5min, and taking the supernatant; a suspected PEDV infected dead pig tissue sample is homogenized with sterile PBS (pH7.4) (a homogenizing instrument or quartz sand grinding), placed at room temperature for 4h or 4 ℃ overnight, centrifuged at 10000rpm for 10min, and the supernatant is taken.
2 capturing PEDV in sample by immune nano magnetic bead
20 mu L of immune nano magnetic beads of 1mg/mL are aseptically taken and added into a 2mL centrifuge tube, and the immune nano magnetic beads are washed for 3 times by sterile PBS (pH7.4) and collected by a magnetic frame. And (3) adding 1mL of the supernatant of the treated sample into a centrifuge tube containing immune nano magnetic beads, uniformly mixing, incubating at room temperature for 1h, repeatedly taking enough sample supernatant and incubating immune nano magnetic beads for a sample with low PEDV content (PCR cannot be directly detected), and collecting the magnetic bead virus compound by using a magnetic frame.
3 cell inoculation
Washing the paramagnetic beads and virus complex with serum-free cell culture medium (DMEM) for 5 times (5 min each time), collecting with magnetic frame, adding to vero cell surface, 37 deg.C, and 5% CO2Incubating for 1h, removing the cell incubation liquid, adding DMEM medium containing 5 μ g/mL pancreatin, 100IU/mL penicillin and 100 μ g/mL streptomycin, incubating at 37 deg.C and 5% CO2Culturing for 24h, observing cytopathic effect (CPE), freezing and thawing for 3 times after 80% of cells have CPE, and carrying out virus passage on vero cells.
Example 3 viral isolation of field PEDV infected samples
After a clinical suspected infection excrement sample is treated, immune nano magnetic beads are used for enrichment, RT-PCR detection is carried out, then the captured virus is inoculated to vero cells, CPE is observed, and passage is carried out. Meanwhile, the sequence is verified by immunofluorescence and a cell ultrathin section transmission electron microscope method, and S genes are cloned by PCR for sequence analysis. Results the suspected PEDV infected piglet feces samples were directly treated by RT-PCR without significant bands after electrophoresis, whereas small bands were expected after incubation of different amounts of samples with magnetic beads by RT-PCR detection (fig. 5B). Significant CPE appeared 24h after incubation of the magnetic beads with the sample with vero cells (fig. 5A); the presence of significant fluorescence within the cells was observed by IFA after virus passage (fig. 5C); after the cells are harvested and fixed, ultrathin sections are manufactured, and the existence of a large number of virus particles in the cells can be observed through a transmission electron microscope (FIG. 5D); the S gene clone is sequenced to be 4149bp in length, and is proved to be PEDV G1 subtype by comparison evolution analysis with published sequences (figure 6), and the results show that PEDV in the sample is successfully enriched and separated by immune nano magnetic beads, so that the magnetic beads can be used for virus separation of samples with low PEDV content.
The virus isolation culture is an essential link for researching the etiology, epidemiology, vaccine and the like of the virus. The immune nano magnetic beads prepared by the invention can separate PEDV from a complex sample, solve the problem that mixed infection single pathogen is difficult to separate, and simultaneously can enrich a low-virus-content sample, thereby realizing efficient detection and separation of viruses in the low-virus-content sample.
Sequence listing
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Claims (8)
1. An immune nano magnetic bead capable of capturing and enriching Porcine Epidemic Diarrhea Virus (PEDV), which is characterized in that the immune nano magnetic bead has magnetic Fe2O3The core and the surface coupled with the PEDV membrane protein specific single domain antibody, wherein the amino acid sequence of the PEDV membrane protein specific single domain antibody is shown as SEQ ID NO. 2.
2. A method for preparing the immunomagnetic beads of claim 1, comprising the steps of:
(1) activation of Nano magnetic beads
Taking Fe with carboxyl surface2O3Adding nano magnetic beads into a centrifuge tube, washing for 3 times by using sterile PBS buffer solution, collecting the magnetic beads by using a magnetic frame, then adding 0.4mol/L EDC and 0.2mol/L NHS into the centrifuge tube with the magnetic beads, activating the surfaces of the magnetic beads, and washing for 5 times by using PBS buffer solution for later use;
(2) buffer replacement of coating antibody
Taking a PEDV membrane protein specific single domain antibody solution, and replacing a dissolving buffer solution with a PBS buffer solution by using an ultrafiltration tube for later use;
(3) preparation of immune nano magnetic bead
Adding a PEDV membrane protein specific single domain antibody solution with a replaced buffer solution into a centrifuge tube with activated magnetic beads, shaking at 120rpm at 37 ℃, incubating for 2h, washing for 5 times by using PBS buffer solution, collecting the magnetic beads, then placing bovine serum albumin with the mass-volume ratio of 0.1% at 37 ℃, sealing for 30min, collecting the magnetic beads, and resuspending by using PBS buffer solution containing 0.01% of sodium azide and 10% of glycerol in volume ratio, thus obtaining the PEDV membrane protein specific single domain antibody, and storing at 4 ℃ for later use.
3. The method of claim 2, wherein said Fe has a carboxyl surface2O3The diameter of the nanometer magnetic bead is 200 nm.
4. The method of claim 2, wherein the volume ratio of 0.4mol/L EDC and 0.2mol/L NHS added is 1: 1.
5. The method of claim 2, wherein the PBS buffer of steps (1) - (3) has a pH of 7.4.
6. A PEDV (porcine epidemic diarrheum virus) membrane protein specific single-domain antibody is characterized in that the amino acid sequence of the PEDV membrane protein specific single-domain antibody is shown as SEQ ID NO. 2.
7. A polynucleotide encoding a PEDV membrane protein specific single domain antibody of claim 6.
8. The polynucleotide of claim 7, wherein the sequence of said polynucleotide is set forth in SEQ ID No. 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104804082A (en) * | 2015-04-01 | 2015-07-29 | 广东海大畜牧兽医研究院有限公司 | Immunofluorescence detection test strip and preparation method thereof for rapid quantitative detection of porcine epidemic diarrhea viruses |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
| CN105861746A (en) * | 2016-04-14 | 2016-08-17 | 西北农林科技大学 | Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology |
| CN106435023A (en) * | 2016-09-23 | 2017-02-22 | 湖南新南方养殖服务有限公司 | Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
| CN104804082A (en) * | 2015-04-01 | 2015-07-29 | 广东海大畜牧兽医研究院有限公司 | Immunofluorescence detection test strip and preparation method thereof for rapid quantitative detection of porcine epidemic diarrhea viruses |
| CN105861746A (en) * | 2016-04-14 | 2016-08-17 | 西北农林科技大学 | Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology |
| CN106435023A (en) * | 2016-09-23 | 2017-02-22 | 湖南新南方养殖服务有限公司 | Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Ultrasensitive Detection of Porcine Epidemic Diarrhea Virus from Fecal Samples Using Functionalized Nanoparticles;Na Xing等;《PLoS One》;20161231;第11卷(第12期);第1-16页 * |
| 快速检测TGEV和PEDV早期感染的UNDP-PCR方法的建立;邢娜;《中国优秀硕士学位论文全文数据库 农业科技辑》;20161115(第11期);第D050-140页 * |
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