CN108486069A - A kind of virus isolation procedure of Porcine epidemic diarrhea virus low content sample - Google Patents
A kind of virus isolation procedure of Porcine epidemic diarrhea virus low content sample Download PDFInfo
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- CN108486069A CN108486069A CN201810570334.0A CN201810570334A CN108486069A CN 108486069 A CN108486069 A CN 108486069A CN 201810570334 A CN201810570334 A CN 201810570334A CN 108486069 A CN108486069 A CN 108486069A
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- 229910052708 sodium Inorganic materials 0.000 description 1
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Abstract
The invention discloses a kind of virus isolation procedures of Porcine epidemic diarrhea virus (PEDV) low content sample, also disclose the immune nanometer magnetic bead for Porcine epidemic diarrhea virus enrichment and separation, which has magnetic Fe2O3Core and coupling have the surface of PEDV memebrane protein specificity single domain antibodies.Conventional method isolated viral from multiple infection and low PEDV contents sample, needs viral purification and long-time blind passage, low separation efficiency to limit the research work of PEDV.The present invention is prepared for immune nanometer magnetic bead using nanometer magnetic bead and PEDV specificity single domain antibodies, effectively can capture and be enriched with the PEDV in the various samples such as excrement, tissue.Sample carries out viral capture and enrichment using immune nanometer magnetic bead after processing, the immune nanometer magnetic bead of virus will be combined directly to be inoculated into Vero cells, you can realize to virus purification.Immune nanometer magnetic bead and PEDV separation methods prepared by the present invention, suitable for clinical complicated and low viral level sample PEDV it is quick, efficiently separate.
Description
Technical field
The present invention relates to a kind of immune nanometer magnetic beads for Porcine epidemic diarrhea virus (PEDV) separation, further relate to PEDV
The efficient virus separation method of low content clinical sample.The invention belongs to biotechnologies.
Background technology
Porcine epidemic diarrhea virus (poreine epidemic diarrhea virus, PEDV) belongs to the more virales of Buddhist nun
(Nidovirales), coronaviridae (Coronaviridae), coronavirus genus (Coronavirus) member, is to cause pig
The cause of disease of epidemic diarrhea (PED).PED is a kind of high degree in contact using the diarrhea of infected pigs, vomiting and dehydration as main feature
Property enteric infectious disease, the pig at various ages can infection morbidity, especially there is higher lethality to suckling pig.Pig epidemic
It rushes down diseased region to extend over the entire globe, significant damage is brought to pig breeding industry.To clinically suffering from quick separating mirror viral in PED pig samples
It is fixed, it is to study PEDV epidemiology, molecular biology and the necessary links of efficient vaccine development.However, current clinic cause of disease
Complicated various, the clinical case of rare single pathogen infection of infection, such case is using conventional method to single cause of disease point
It is difficult from very.Simultaneously for some low cause of disease content samples, it also is difficult to succeed using conventional method separation.Therefore, it builds
The vertical method that target cause of disease in clinical multiple infection and low cause of disease content sample is efficiently separated, to aetology, epidemiology
It is of great significance with researchs such as vaccines.
Immunomagnetic beads are increasingly universal in life science application, especially in Pathogen test and separation etc..Immune magnetic
Pearl isolation technics is to form Ag-Ab-magnetic using the specific binding of antigen, antibody using the coated magnetic bead of antibody as carrier
Pearl compound, which can be directed to movement under magnetic fields, to realize the specifically separation of antigen.This method combines
The simplicity of the high efficiency of dispersive solid-phase extraction, the specificity of immune affinity effect and magnetic recycling, can effectively simplify pre-treatment
Flow shortens the pre-treatment time, improves enrichment and separation efficiency.As nanometer magnetic bead is prepared and pan coating technology is not broken into
Ripe perfect, various antibody and the affine factor of specificity etc. can be used in magnetic bead coating, and to adaptive immune magnetic bead, these are immune
The specificity of magnetic bead then depends entirely on coated antibody or the affine factor.Currently, common main antibody includes that monoclonal is anti-
Body (IgG), single-chain antibody, single domain antibody (sdAb) etc..Single domain antibody is that the serum such as Camelidae and cartilage shark are naturally occurring
The antigen binding site of the only IgG antibody of heavy chain, such antibody is only made of the single structure domain of heavy chain variable region, also known as
Heavy chain antibody (VHH) or nano antibody (Nanobody, Nb).SdAb equally maintains good, special with common IgG antibody molecule
Different antigen binding capacity.Meanwhile sdAb also has and is easy in escherichia expression system solubility expression, high temperature resistant, resistance to
The peculiar properties such as extreme pH value.These characteristics make sdAb become ideal candidates antibody prepared by immunomagnetic beads.
Invention content
The first object of the present invention is to provide one kind can be in conjunction with simultaneously efficiently concentrating Porcine epidemic diarrhea virus (poreine
Epidemic diarrhea virus, PEDV) immune nanometer magnetic bead；It is a further object of the present invention to provide a kind of PEDV is low
The method that content sample virus efficiently separates.
In order to achieve the above object, present invention employs following technological means：
One kind of the present invention can capture and be enriched with Porcine epidemic diarrhea virus (poreine epidemic diarrhea
Virus, PEDV) immune nanometer magnetic bead, which has magnetic Fe2O3Core and coupling have PEDV memebrane proteins
The surface of specific single domain antibody, the amino acid sequence of the PEDV memebrane protein specificity single domain antibodies is as shown in SEQ ID NO.2.
Further, the invention also provides a kind of method preparing the immune nanometer magnetic bead, include the following steps：
(1) activation of nanometer magnetic bead
Take the Fe with carboxyl surface2O3Nanometer magnetic bead is added centrifuge tube, is washed 3 times with sterile PBS buffer, use magnetic frame
Collect magnetic bead, then into the centrifuge tube for have magnetic bead be added 0.4mol/L EDC and 0.2mol/L NHS, activated magnetic beads surface,
It is washed 5 times with PBS buffer solution, it is spare；
(2) coated antibody buffer exchange
PEDV memebrane protein specificity single domain antibody solution is taken, it is PBS buffer solution to replace dissolving buffer solution using super filter tube, standby
With；
(3) prepared by immune nanometer magnetic bead
The PEDV memebrane protein specificity single domain antibody solution for having replaced buffer solution is taken, the centrifuge tube for there are activated magnetic beads is added,
37 DEG C are set, 120rpm shakes, and is incubated 2h, is washed 5 times with PBS buffer solution, collects magnetic bead, and it is 0.1% then to use mass volume ratio
Bovine serum albumin(BSA) sets 37 DEG C, closes 30min, collects magnetic bead, is 0.01% Sodium azide, the PBS of 10% glycerine with containing volume ratio
Buffer solution is resuspended to get 4 DEG C store for future use.
Wherein, it is preferred that the Fe with carboxyl surface2O3A diameter of 200nm of nanometer magnetic bead.
Wherein, it is preferred that the volume ratio of the NHS of the EDC and 0.2mol/L of the 0.4mol/L of addition are 1:1.
Wherein, it is preferred that the pH value of the PBS buffer solution described in step (1)-(3) is 7.4.
Further, it the invention also provides a kind of method that PEDV low contents sample virus efficiently separates, that is, utilizes
Immune nanometer magnetic bead of the present invention is captured and is enriched with to the PEDV in low viral level clinical sample, and cell is being passed through
Separation passage, realizes virus purification.
Wherein, it is preferred that the method the specific steps are：
A. immune nanometer magnetic bead is incubated altogether with sample：By clinical doubtful PEDV infection swine excrements and secretion cotton swab subclass
Pattern product or tissue sample are impregnated with sterile PBS buffer or organize to be homogenized, and with immune nanometer magnetic bead, room temperature, 80rpm is incubated altogether
1h is educated, is washed 5 times with sterile PBST, magnetic frame collects immune nanometer magnetic bead；
B. immune nanometer magnetic bead and antiviral compound inoculating cell：With penicillin containing 100IU/mL and 100 μ g/mL streptomysins
DMEM, wash immune nanometer magnetic bead 5 times, magnetic frame collect, with 1mL contain 5 μ g/mL pancreatin, 100IU/mL penicillin and 100 μ g/
The DMEM of mL streptomysins is mixed, and is added to cell surface, 37 DEG C, 5%CO2, it is incubated 1h；
C. cell culture fluid is replaced：It is added and contains 5 μ g/mL pancreatin, 100IU/mL penicillin and 100 μ g/mL streptomysins
DMEM culture mediums, 37 DEG C, 5%CO2Continue culture for 24 hours, starts to observe cytopathy (CPE), and carry out viral passages, realize disease
Poison separation.
Wherein, it is preferred that the pH value of the PBS buffer solution described in step a is 7.4, and the cell described in step b is vero
Cell.
Above-described Porcine epidemic diarrhea virus (poreine epidemic diarrhea virus, PEDV) film egg
White specificity single domain antibody and its nucleotide sequence also within protection scope of the present invention, the Porcine epidemic diarrhea virus
The amino acid sequence of memebrane protein specificity single domain antibody is as shown in SEQ ID NO.2.Preferably, the nucleotide sequence is such as
Shown in SEQ ID NO.1.
Compared to the prior art, the beneficial effects of the invention are as follows：
The present invention is prepared for immune nanometer magnetic bead using nanometer magnetic bead and PEDV specificity single domain antibodies, can effectively capture
With the PEDV being enriched in the various samples such as excrement, tissue.Sample after processing using immune nanometer magnetic bead carry out virus capture and
Enrichment will combine the immune nanometer magnetic bead of virus to be directly inoculated into cell, you can to realize to virus purification.Prepared by the present invention exempts from
Epidemic disease nanometer magnetic bead and PEDV separation methods, suitable for clinical complicated and low viral level sample PEDV it is quick, efficiently separate.
Description of the drawings
Fig. 1 is sequence analysis, expression and the purifying of PEDV specificity sdAb；
A：Skeleton is carried out by The Kabat et al. (Kabat, 1991) mode and complementary determining region divides.
(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：Expression and purification swimming lanes 1, blank control；Swimming lane 2, expression；Swimming lane 3, purifying；M, egg
White molecular weight standard.C：ELISA experimental analyses sdAb-Mc40 and M protein binding activities.
Fig. 2 is that immune nanometer magnetic bead analyzes M protein binding activities；
Recombinant expression M albumen is incubated altogether with immune nanometer magnetic bead, combines situation by SDS-PAGE detections, and pass through
Western blot verifications are primary antibody using PEDV antibody positive Swine serums, and the rabbit-anti pig antibody of HRP labels is secondary antibody；Swimming lane
1, the M albumen that immune nanometer magnetic bead combines；Swimming lane 2, the total recombinant M protein used；Swimming lane 3, the supernatant after being incubated with magnetic bead；
Swimming lane 4, the supernatant after being incubated with magnetic bead；Swimming lane 5, the total recombinant M protein used；Swimming lane 6, the M that immune nanometer magnetic bead combines
Albumen.
Fig. 3 is capture activity analysis of the immune nanometer magnetic bead to PEDV strains CV777；
It is incubated altogether using magnetic bead and CV777 infection vero cell conditioned mediums, collects magnetic bead, a part is drawn with PEDV specificity
Object is detected by RT-PCR, it is seen that be consistent band (A) with expected size, and magnetic bead detects after swimming lane 1 is incubated altogether, swimming lane M, DNA
marker；Another part is by 3% sodium phosphotungstate negative staining, with transmission electron microscope observing, it is seen that magnetic bead surfaces are combined with size and are about
The virion (B) of 100nm.
Fig. 4 is that viral enrichment is analyzed；
By PEDV cell toxicant CV777, PBS doubling dilutions, PCR detections, the dilution disease for taking RT-PCR that can not detect are utilized
Poison is incubated jointly with immune nanometer magnetic bead, is then detected by RT-PCR, is analyzed the viral enrichment of immune nanometer magnetic bead.Swimming
Road 1, the control of blank magnetic bead；Swimming lane 2, the RT-PCR detections of dilution restrovirus；3-swimming lane of swimming lane 9, respectively 400,600,800,
1000, RT-PCR testing results after 1200,1400 and 2000 μ L dilution viruses are incubated with magnetic bead.
Fig. 5 is that the separation of field strain is identified；
It is incubated altogether with immune nanometer magnetic bead with PEDV infection piglet fecal specimens are suspected to be, collects magnetic bead, examined by RT-PCR
It surveys (B), swimming lane 1, fecal specimens directly detect (200 μ L samples are extracted for RNA) after processing；Swimming lane 2, sample after 1mL processing
It is detected after being incubated with immune nanometer magnetic bead；Swimming lane 3, the control of blank magnetic bead；Swimming lane 4；Sample and immune nanometer magnetic bead after 2mL processing
It is detected after incubation；Swimming lane 5, sample detects after being incubated with immune nanometer magnetic bead after 3mL processing.Vero cells are inoculated with, are observed bright
Aobvious cytopathy (A)；Utilize immunofluorescence technique, it is seen that intracellular apparent fluorescence (C)；It is ultra-thin with transmission electron microscope side's observation cell
Slice, it is seen that intracellular apparent virion, fractionated viral particle shown in arrow (D).
Fig. 6 is the evolutionary analysis based on S genes.
With MEGA6.0 softwares, the S gene sequences of 10 plants of PEDV of known hypotype will be announced on separation strains jxdv1801 and NCBI
Row are compared, and maximum likelihood method (ML), Poisson model (poisson model) phylogenetic tree construction is used in combination.As a result
Confirmation separation strains are G1 hypotypes.
Specific implementation mode
Further describe beneficial effects of the present invention by the following examples, it should be understood that these embodiments are only
For illustration purposes, it is never limited in protection scope of the present invention.
The preparation of 1 PEDV specific immunity nanometer magnetic beads of embodiment
The screening and expression of 1.1 PEDV memebrane proteins (M) specificity sdAb
Using display technique of bacteriophage, elutriation M protein immunization two-humped camels library obtains the phagocytosis with M albumen specific bonds
Body carries out sequencing analysis, obtains sdAb gene orders, and nucleotide sequence carries out sequence analysis as shown in SEQ ID NO.1；
PET-32a expression plasmids are then cloned into, are expressed with Escherichia coli (E.coli), mesh is carried out using Ni Purification Resins
Protein purification；By enzyme-linked immunosorbent assay (ELISA), the combination activity of sdAb-Mc40 and M albumen is analyzed.
To the protein bound bacteriophage sequencing analysis of M, specificity single domain antibody sdAb-Mc40 (Figure 1A) is obtained；Connect 32a
After carrier, Escherichia coli are transformed into, the expression albumen that size is consistent is obtained, and be solubility expression, utilizes Ni Purification Resins
Destination protein (Figure 1B) can be obtained, amino acid sequence is as shown in SEQ ID NO.2；Elisa assay result confirms sdAb-
Mc40 has M protein binding activities.
The activation of 1.2 nanometer magnetic beads
500 μ L, a concentration of 2mg/mL, diameter 200nm are taken, the Fe on carboxyl surface is carried2O3Nanometer magnetic bead, be added 1.5mL from
Heart pipe is washed 3 times with the sterile PBS of 1mL (pH7.4), and magnetic bead is collected with magnetic frame.Then the EDC and 0.2mol/ of 0.4mol/L is added
Each 200 μ L of NHS of L, to having in the centrifuge tube of magnetic bead, activated magnetic beads surface is washed 5 times with PBS (pH 7.4), spare.
1.3 coated antibody buffer exchanges
A concentration of 2.35mg/mL prokaryotic expressions of 1mL and the sdAb-Mc40 of purifying are taken, it is slow to replace its dissolving using super filter tube
Fliud flushing be PBS (pH 7.4), and reach final volume be 1mL, a concentration of 1.8mg/mL, it is spare.
It is prepared by 1.4 immune nanometer magnetic beads
It takes 1mL to replace the sdAb-Mc40 of buffer solution, the centrifuge tube for having activated magnetic beads is added, sets 37 DEG C, 120rpm shakes
It is dynamic, it is incubated 2h, is washed 5 times with PBS (pH 7.4), magnetic bead is collected, it is 0.1% bovine serum albumin(BSA) then to use mass volume ratio,
37 DEG C are set, 30min is closed, collects magnetic bead, it is 0.01% Sodium azide, the PBS buffer solution (pH of 10% glycerine to contain volume ratio with 1mL
7.4) it is resuspended, 4 DEG C store for future use.
2 immune nanometer magnetic bead virus combination activity analysis
2.1 immune nanometer magnetic beads capture activity analysis to PEDV memebrane proteins (M)
The combination activity of immune nanometer magnetic bead and M albumen, is analyzed by SDS-PAGE and Western blot.It takes
1.5mL centrifuge tubes are added in 200 μ L PBS (pH7.4), and the immune nanometer magnetic bead and 50 μ L of 10 a concentration of 2mg/mL of μ L is then added
A concentration of 1.23mg/mL recombinant M proteins, 37 DEG C, 80rpm shakes, and is incubated 1h；Setting is not coupled the blank magnetic of single domain antibody simultaneously
Pearl compares.Magnetic bead and conjugate are collected with magnetic frame, supernatant is discarded, is washed 5 times, 80rpm, 5min/ times with PBST (PH7.4), is received
Collect magnetic bead；SDS-PAGE sample-loading buffers, boiling water boiling 10min is added；A part of sample is directly divided by 12% SDS-PAGE
Analysis, as a result immune nanometer magnetic bead and M albumen are total to samples of incubation swimming lane and occur and M albumen band in the same size (Fig. 2).
After another part sample is by 12% SDS-PAGE separation, pvdf membrane is gone to.Then with PEDV positive Swine serums,
Room temperature, 80rpm are incubated 1h, and PBST is washed 4 times.In the rabbit-anti pig secondary antibody marked with horseradish peroxidase (HRP), room temperature,
80rpm is incubated 1h and is washed 5 times, 5min/ times with PBST, 80rpm.It is developed the color with ECL developing solutions；As a result in immune nanometer magnetic bead and M
Albumen, which is total to samples of incubation swimming lane, to be occurred and M albumen band in the same size (Fig. 2).These are the result shows that the immune nanometer magnetic bead prepared
With M albumen capturing functions.
2.2 immune nanometer magnetic beads capture PEDV cell toxicants (strain CV777) and analyze
The combination activity of immune nanometer magnetic bead and PEDV (strain CV777) are analyzed by transmission electron microscope and RT-PCR.
Take 1mL TCID50Value is 107.2The CV777 cell toxicants of/0.1mL mix, 37 DEG C, 80rpm with 20 μ g immune nanometer magnetic beads, are incubated
Then 1h uses magnetic frame separating immune nanometer magnetic bead, discards supernatant, washed 5 times with PBST.The immune nano magnetic collected with magnetic frame
Pearl, a part of 3% phosphotungstic acid negative staining, with transmission electron microscope observing, it is about 100nm's that size as a result, which can be observed, in periphery of magnetic beads
Virion is consistent (Fig. 3 B) with PEDV sizes 90-180nm.
Another part using PEDV specific detection primers, is directly detected by RT-PCR for extracting RNA, as a result with
CV777 is incubated immune nanometer magnetic bead sample and the specific band (Fig. 3 A) that is consistent with expected size occurs altogether.These results confirm system
Standby immune nanometer magnetic bead has PEDV virus capturing functions.
3 immune nanometer magnetic beads are to low PEDV contents example enrichment capability analysis
By PEDV cell strains CV777,10 times of doubling dilutions take this dilution sample until RT-PCR cannot be detected
Product are incubated using immune nanometer magnetic bead and different volumes dilution restrovirus, are detected by RT-PCR.As a result when in sample virus contain
When amount can not be detected as low as PCR, (figure after being enriched with to virus using immune nanometer magnetic bead, can be detected by RT-PCR
4), show that the immune nanometer magnetic bead of invention preparation is suitable for the virus enrichment of low PEDV contents sample.
The virus isolation procedure of 2 PEDV low content samples of embodiment
The processing of 1 clinical sample
Doubtful PEDV infects the cotton swabs type of sample such as swine excrement and secretion, is placed in 2mL centrifuge tubes, be added 1mL without
Bacterium PBS (pH 7.4) is placed at room temperature for 2h, and 2000rpm centrifuges 5min, takes supernatant；Doubtful PEDV infects sick dead pig tissue sample, uses
Sterile PBS (pH 7.4) tissue homogenate (homogenate instrument or quartzite sand grind), is placed at room temperature for 4h or 4 DEG C overnight, 10000rpm from
Heart 10min, takes supernatant.
2 immune nanometer magnetic beads capture PEDV in sample
The sterile 20 μ L of immune nanometer magnetic bead for taking 1mg/mL are added 2mL centrifuge tubes, are washed 3 times with sterile PBS (pH 7.4),
Magnetic frame collects immune nanometer magnetic bead.Processing Sample supernatants 1mL is taken, the centrifuge tube containing immune nanometer magnetic bead, mixing, room are added to
Temperature is incubated 1h, for low PEDV contents sample (PCR is not directly detected), can take fully repeatedly enough sample supernatants with
Immune nanometer magnetic bead is incubated, and magnetic bead antiviral compound is collected with magnetic frame.
3 cell inoculations
Magnetic bead magnetic bead antiviral compound is washed with serum-free cell culture medium (DMEM) 5 times, washes 5min every time, and magnetic frame is received
Collection, is then added to vero cell surfaces, 37 DEG C, 5%CO2, it is incubated 1h, removes and contains 5 μ g/mL with cell incubation liquid, addition
Pancreatin, the DMEM culture mediums of 100IU/mL penicillin and 100 μ g/mL streptomysins, 37 DEG C, 5%CO2Continue culture for 24 hours, starts to see
Cytopathy (CPE) is examined, after CPE occurs in 80% cell, receives cell freeze thawing 3 times, viral passages are carried out in vero cells.
The virus purification of embodiment 3 field PEDV infection samples
After the processing of clinical suspected infection fecal specimens, it is enriched with using immune nanometer magnetic bead, is detected by RT-PCR, then
By the virus inoculation of capture to vero cells, observes CPE and passed on.It is saturating by immunofluorescence and cell ultrathin sections simultaneously
Radio mirror method validation, and S genes are cloned by PCR, carry out sequence analysis.As a result at doubtful PEDV infection piglet fecal specimens
Directly by RT-PCR after reason, apparent band is had no after electrophoresis, and after being incubated by different amounts of sample and magnetic bead, RT-PCR inspections
It surveys, occurs anticipating small band (Fig. 5 B).It is inoculated with after vero cells for 24 hours with the magnetic bead after sample incubation, apparent CPE occurs
(Fig. 5 A)；It is able to observe that into the cell there is apparent fluorescence (Fig. 5 C) by IFA after viral passages；After harvesting cell fixation, system
Make ultra-thin section, is able to observe that into the cell there are a large amount of virion (Fig. 5 D) by transmission electron microscope；To S gene clonings, survey
The length of 4149bp of sequence is confirmed that it is PEDV G1 hypotypes (Fig. 6), these result tables with sequence alignment evolutionary analysis has been announced
It is bright that enrichment and separation is successfully made to PEDV in sample by immune nanometer magnetic bead, it was demonstrated that the magnetic bead can be used for low PEDV contents
The virus purification of sample.
Virus purification culture is the essential links such as aetology, epidemiology and the vaccine of research virus.This hair
The immune nanometer magnetic bead of bright preparation can detach PEDV in complex sample, and what the solution single cause of disease of mixed infection was difficult to detach asks
Topic, while immune nanometer magnetic bead can also be enriched with low viral level sample, to realize in low viral level sample
Efficient virus is detected and is detached.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of virus isolation procedure of Porcine epidemic diarrhea virus low content sample
<160>2
<170>Patent-In 3.5
<210>1
<211>378
<212>DNA
<213> poreine epidemic diarrhea virus
<400>1
gatgtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt ctcctttact agcaacgtca tgggctggtt ccgccaggct 120
ccaggaaagg aacgcgaggg ggtcgcagct atttcggtag gtagtggtag cacatggtat 180
ggcgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacactgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gactgcgaga 300
cgtggagtta ttcttacact aagcccagag acctatgaca tttactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210>2
<211>126
<212>PRT
<213> poreine epidemic diarrhea virus
<400>2
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Thr 30
Ser Asn Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 45
Glu Gly Val Ala Ala Ile Ser Val Gly Ser Gly Ser Thr Trp Tyr 60
Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Leu Asp Ser Ala 75
Asn Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 90
Thr Ala Met Tyr Tyr Cys Ala Thr Ala Arg Arg Gly Val Ile Leu 105
Thr Leu Ser Pro Glu Thr Tyr Asp Ile Tyr Trp Gly Gln Gly Thr 120
Gln Val Thr Val Ser Ser 126
Claims (10)
1. one kind can capture and be enriched with Porcine epidemic diarrhea virus (poreine epidemic diarrhea virus,
PEDV immune nanometer magnetic bead), it is characterised in that the immune nanometer magnetic bead has magnetic Fe2O3Core and coupling have PEDV films
The surface of protein-specific single domain antibody, the amino acid sequence such as SEQ ID NO.2 of the PEDV memebrane protein specificity single domain antibodies
It is shown.
2. a kind of method preparing immune nanometer magnetic bead described in claim 1, it is characterised in that include the following steps：
(1) activation of nanometer magnetic bead
Take the Fe with carboxyl surface2O3Nanometer magnetic bead is added centrifuge tube, is washed 3 times with sterile PBS buffer, collected with magnetic frame
Magnetic bead, is then added the NHS of the EDC and 0.2mol/L of 0.4mol/L into the centrifuge tube for have magnetic bead, and activated magnetic beads surface is used
PBS buffer solution is washed 5 times, spare；
(2) coated antibody buffer exchange
PEDV memebrane protein specificity single domain antibody solution is taken, it is PBS buffer solution to replace dissolving buffer solution using super filter tube, spare；
(3) prepared by immune nanometer magnetic bead
The PEDV memebrane protein specificity single domain antibody solution for having replaced buffer solution is taken, the centrifuge tube for there are activated magnetic beads is added, sets 37
DEG C, 120rpm shakes, and is incubated 2h, is washed 5 times with PBS buffer solution, collects magnetic bead, and it is 0.1% ox blood then to use mass volume ratio
Pure albumen sets 37 DEG C, closes 30min, collects magnetic bead, and with being 0.01% Sodium azide containing volume ratio, the PBS of 10% glycerine is buffered
Liquid is resuspended to get 4 DEG C store for future use.
3. method as claimed in claim 2, it is characterised in that the Fe with carboxyl surface2O3The diameter of nanometer magnetic bead
For 200nm.
4. method as claimed in claim 2, it is characterised in that the body of the NHS of the EDC and 0.2mol/L of the 0.4mol/L of addition
Product is than being 1:1.
5. method as claimed in claim 2, it is characterised in that the pH value of the PBS buffer solution described in step (1)-(3) is
7.4。
6. a kind of Porcine epidemic diarrhea virus (poreine epidemic diarrhea virus, PEDV) low content sample disease
The method that poison efficiently separates, it is characterised in that using immune nanometer magnetic bead described in claim 1 to low viral level clinic sample
PEDV in product is captured and is enriched with, and is passed on being detached by cell, realizes virus purification.
7. method as claimed in claim 6, which is characterized in that the specific steps are：
A. immune nanometer magnetic bead is incubated altogether with sample：By clinical doubtful PEDV infection swine excrements and secretion cotton swab subtype sample
Product or tissue sample are impregnated with sterile PBS buffer or organize to be homogenized, and with immune nanometer magnetic bead, room temperature, 80rpm is incubated altogether
1h is washed 5 times with sterile PBST, and magnetic frame collects immune nanometer magnetic bead；
B. immune nanometer magnetic bead and antiviral compound inoculating cell：With penicillin containing 100IU/mL and 100 μ g/mL streptomysins
DMEM, washes immune nanometer magnetic bead 5 times, and magnetic frame is collected, and contains 5 μ g/mL pancreatin, 100IU/mL penicillin and 100 μ g/mL with 1mL
The DMEM of streptomysin is mixed, and is added to cell surface, 37 DEG C, 5%CO2, it is incubated 1h；
C. cell culture fluid is replaced：It is added and contains 5 μ g/mL pancreatin, the DMEM trainings of 100IU/mL penicillin and 100 μ g/mL streptomysins
Foster base, 37 DEG C, 5%CO2Continue culture for 24 hours, starts to observe cytopathy (CPE), and carry out viral passages, realize virus point
From.
8. the method for claim 7, it is characterised in that the pH value of the PBS buffer solution described in step a is 7.4, step b
Described in cell be vero cells.
9. a kind of Porcine epidemic diarrhea virus (poreine epidemic diarrhea virus, PEDV) memebrane protein specificity
Single domain antibody, which is characterized in that the amino acid sequence such as SEQ ID NO.2 institutes of the PEDV memebrane protein specificity single domain antibodies
Show.
10. encoding the nucleotide sequence of the PEDV memebrane protein specificity single domain antibodies described in claim 9, it is preferred that described
Nucleotide sequence is as shown in SEQ ID NO.1.
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