CN103543264B - The polyclonal antibody preparation of Chinese yam rhizome X virus, detection and application thereof - Google Patents

The polyclonal antibody preparation of Chinese yam rhizome X virus, detection and application thereof Download PDF

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CN103543264B
CN103543264B CN201310541274.7A CN201310541274A CN103543264B CN 103543264 B CN103543264 B CN 103543264B CN 201310541274 A CN201310541274 A CN 201310541274A CN 103543264 B CN103543264 B CN 103543264B
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陈保善
邹承武
蒙姣荣
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Guangxi University
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Abstract

The invention discloses a kind of potexvirus virus infecting Chinese yam rhizome---the polyclonal antibody preparation of Chinese yam rhizome X virus (Yam virus X, YVX), detection and application thereof.Based on YVX genome, inventor is by coat protein (the Coat protein of Prokaryotic expression, purification YVX, and prepare polyclonal antibody for immunizing rabbit CP), the coat protein of the YVX of this Anti-TNF-α physical efficiency specific recognition prokaryotic expression and infect the coat protein of YVX virus of Chinese yam rhizome, and produce specific immune response.Based on this, inventor also establishes a set of efficient, highly sensitive and IC-RT-PCR, ELISA and Western blot method accurately, can detect YVX specifically from the Chinese yam rhizome plant infecting YVX.Application the present invention, can be the quick detection of YVX and host's repercussion study and this virus, somatotype and molecular biology research provides material base and technical support, for solid foundation is laid in the Epidemiology monitor of this virus and control.

Description

The polyclonal antibody preparation of Chinese yam rhizome X virus, detection and application thereof
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of potexvirus virus infecting Chinese yam rhizome---the polyclonal antibody preparation of Chinese yam rhizome X virus, detection and application thereof.
Background technology
Chinese yam rhizome is also known as Chinese yam, Huai Shan, for Dioscoreaceae plant, its block root is rich in starch, protein, vitamin, amino acid and multiple medicinal ingredient as choline, saponin, mucopolysaccharide etc., also containing several mineral materials such as Fe, Zn, Cu, Ca, the difference of foundation kind and kind, can do medicinal or edible, there is bright market prospects and industry development potentiality.Virosis has a strong impact on the yield and quality of Chinese yam rhizome, on Chinese yam rhizome is produced, cause heavy losses.Document shows, in global range, the virus infecting Chinese yam rhizome under natural conditions has the ginseng potato baculoviral of Badnavirus (Dioscorea alata bacilliform virus, DaBV), Chinese yam rhizome mosaic virus (Yam mosaic virus, YMV), gentle mosaic virus (the Yam mild mosaicvirus of Chinese yam rhizome, YMMV), downright bad mosaic virus (the Chinese yam necrotic mosaic virus of China's Chinese yam rhizome, CYNMV), Japan Huaihe River mountain flower mosaic virus (Japanese yam mosaic virus, JYMV), broad bean wilt virus 2(Broad bean wiltvirus-2, BBWV-2).China has found that DaBV, YMMV, JYMV, CYNMV and BBWV-2 infect Chinese yam rhizome, and the symptom of initiation comprises floral leaf, mottled, veinclearing, chlorisis, growth retardation and leaf curling etc.Different Chinese yam rhizome virus causes symptom different, also can cause similar symptoms; Its vector is different, has different routes of transmission.In order to normally carrying out of ensureing that Chinese yam rhizome produces, first will ensure that potato seed does not carry virus, be secondly at production land for growing field crops Timeliness coverage and identifying virus, to formulate prophylactico-therapeutic measures targetedly, comprises and control vector to cut off the propagation of virosis.Therefore, require highly sensitive, that specificity is good virus detection techniques to detect the potato seed of Chinese yam rhizome and field sample.At present, the technical way for Plant RNA viral Pathogen test has RT-PCR, IC-RT-PCR, qRT-PCR, LAMP-PCR, ELISA, Western blot etc., and maximum in the application of virus causing disease quarantine context of detection is ELISA and qRT-PCR.
In investigation in recent years to each main Huai Shanchanqu of China, find the new virus infecting Chinese yam rhizome---classification belongs to potexvirus (Potexvirus) virus, tentative Chinese yam rhizome X virus (Yam virus X, YVX) by name.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of polyclonal antibody preparation of Chinese yam rhizome X virus, detection and application thereof, for use in detecting the Chinese yam rhizome X virus infecting Chinese yam rhizome fast.
For solving the problems of the technologies described above, the present invention by the following technical solutions: the preparation method of polyclonal antibody of Chinese yam rhizome X virus, the CP albumen (Coat Protein) of Prokaryotic expression, purification Chinese yam rhizome X virus also prepares polyclonal antibody for immunizing rabbit.
The CP albumen of Chinese yam rhizome X virus has the amino acid sequence of sequence table SEQ .ID.No.1, or by having the coded by said gene of base sequence of sequence table SEQ .ID.No.2.
Above-mentioned preparation method of polyclonal antibody, gets the recombinant C P albumen after purifying, adopts subcutaneous multi-point injection method immunize New Zealand White Rabbit, and later every 2 weeks booster immunizations once, are total to immunity 4 times; First immunisation adds the Freund's complete adjuvant with albumen equivalent, adds the incomplete Freund's adjuvant with albumen equivalent for 3 times afterwards; Final boost is after 7 days, Culling heart blood, separation of serum; The polyclonal antibody of this virus capsid protein CP is obtained again with antigen affinitive layer purification.
The polyclonal antibody of gained Chinese yam rhizome X virus is detecting the application in Chinese yam rhizome X virus.
Based on the ELISA detection method of the Chinese yam rhizome X virus of above-mentioned polyclonal antibody.
Above-mentioned ELISA detection method, comprises the following steps:
(1) with the fresh blade of 1mL antigen coated damping fluid grinding 0.1g Chinese yam rhizome, the centrifugal 5min of 9000rpm, gets supernatant and dilutes 20 times, adds elisa plate according to 100 μ l/ holes; The sick leaf of Chinese yam rhizome infecting YVX is positive control, and corresponding healthy leaf is negative control, hatches 2h for 37 DEG C;
(2), after washing twice with 200 μ l PBST, 30min is closed with 5% skimmed milk power;
(3), after washing twice with 200 μ l PBST, add the polyclonal antibody diluted according to 1:8000 with antibody dilution buffer in each hole, 100 μ l/ holes, hatch 1h or incubated at room 3h for 37 DEG C;
(4) after washing twice with 200 μ l PBST, the goat anti-rabbit igg bond that by specification dilutes the horseradish peroxidase-labeled of 3000 times in each hole, is added, 100 μ l/ holes, incubated at room 1h;
(5) after washing three times with 200 μ l PBST, develop the color with tmb substrate chromogenic reagent box (health is ShiJi Co., Ltd), room temperature lucifuge hatches 25-30min, and reaction product is in blue, and every hole adds 50 μ l0.5M H again 2sO 4color development stopping, now blue product becomes glassy yellow, reads OD immediately by microplate reader 450value, to be greater than 2.1 for positive with negative OD value ratio (P/N);
PBST is containing 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, 0.05%Tween20, pH7.4; Antigen coated damping fluid is carbonate buffer solution (30mM sodium carbonate-bicarbonate damping fluid, pH9.6); Antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.
Based on the Western blot detection method of the Chinese yam rhizome X virus of above-mentioned polyclonal antibody.
Above-mentioned Western blot detection method, comprises the following steps:
(1) extract kit with TCA/ acetone precipitation or employing phytoprotein and extract Chinese yam rhizome total protein sample, carry out quantification of protein by Bradford method, adjustment protein concentration is 2mg/mL;
(2) prepare protein electrophoresis gel, carry out SDS-PAGE;
(3) half dry type transfer: gel is cut to suitable size after electrophoresis terminates, by transferring film damping fluid balance, 5min × 3 time; Cut out the filter paper onesize with gel and pvdf membrane in advance, after soaking 1min with methyl alcohol, immerse 10min in transferring film damping fluid; Membrane-transferring device is put well by the order of carbon anode plate, 3 layers of 3mm filter paper, pvdf membrane, gel, 3 layers of 3mm filter paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, pvdf membrane Accurate align, each step removes bubble, switches on power after building negative electrode carbon plate, constant current 1mA/cm 2, transfer 1.5h, after transfer terminates, film takes out by deenergization;
(4) immune response: wash film with 0.01M PBS, 1min; Add confining liquid, room temperature closes 1 ~ 2h; Abandon confining liquid, wash film with 0.01M PBST, 5min × 3 time; Add the CP polyclonal antibody (primary antibodie) of the YVX diluted according to 1:5000 with antibody dilution buffer, place more than 12h for incubated at room 2h or 4 DEG C; Negative control, replace primary antibodie with 1%BSA, all the other steps are identical with experimental group; Abandon primary antibodie and 1%BSA, wash film respectively with 0.01M PBST, 5min × 4 time; Add the goat anti-rabbit igg bond (two resist) marked with the alkaline phosphatase (AP) that antibody dilution buffer dilutes 1000 times, steadily shake, room temperature 2h; Abandon two to resist, wash film with 0.01M PBST, 5min × 4 time; Add BCIP/NBT nitrite ion, colour developing puts into distilled water cessation reaction to when there is band;
Transferring film damping fluid is 25mM Tris, 192mM glycine, 0.01%SDS, 20%methanol, pH8.3; 0.01MPBS contains 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, pH7.4; Confining liquid is 5% skimmed milk power or 3%BSA.
Based on the IC-RT-PCR detection method of the Chinese yam rhizome X virus of above-mentioned polyclonal antibody.
Above-mentioned IC-RT-PCR detection method, comprises the following steps:
(1) dilute YVX polyclonal antibody with antibody dilution buffer according to 1:8000, add the antibody that 100 μ l have diluted in the centrifuge tube of each 1.5mL, hatch 1h or incubated at room 3h for 37 DEG C;
(2) by Chinese yam rhizome blade with containing the 0.02M PBS grinding buffer solution of 2%PVP by mass volume ratio 0.2%(w/v) put into mortar, grind to form the homogenate of leaf juice, in suction 1.5mL centrifuge tube, the centrifugal 10min of 9000rpm;
(3) bag in step (1) is washed twice by the 1.5mL centrifuge tube PBST of good antibody, add 200 μ l step (2) ready leaf juice supernatants, 37 DEG C of water-bath 3h, the virion now exposed is attached to (immunocapture) on centrifuge tube inwall specifically;
(4) remove leaf juice, PBST washes 3 times, and DEPC washes 1 time, wash finish do at the bottom of of short duration centrifugal segregation pipe more than liquid;
(5) RT-PCR reaction is carried out with SuperRT One step RT-PCR kit (health is ShiJi Co., Ltd), it is YVX-F:5 '-CCAGGTCCATCAGTCACTTCT-3 ' and YVX-R:5 '-CTAATCTACCAGCAGTCACTTCATA-3 ' that YVX detects primer, it is 55 DEG C that the PCR stage arranges annealing temperature, 35 circulations;
(6), after RT-PCR terminates, the PCR primer mass body volume concentrations 1.0%(w/v of 5 μ l is got) Ago-Gel carries out electrophoresis detection, and expection clip size is 692bp.
Antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.
For setting up the detection method of Chinese yam rhizome X virus (YVX), inventor have extensively studied the full-length gene group sequence of YVX Henan separator (being separated the iron rod yam (TG) from Wen County, Henan Province), the full-length gene group sequence (see sequence table SEQ .ID.No.3) of this virus has 6159 nucleotide, there are 73 bases 5 ' noncoding region, there is the length of 235 bases (not comprising poly(A) 3 ' noncoding region), to encode 5 protein, be respectively from 5 ' end to the albumen size of the 3 ' sequential encoding of holding: 1335aa, 232aa, 109aa, 68aa, 227aa.Based on YVX genome, inventor is by the coat protein (CP) of Prokaryotic expression, purification YVX and prepare polyclonal antibody for immunizing rabbit, the coat protein of the YVX of this Anti-TNF-α physical efficiency specific recognition prokaryotic expression and infecting the coat protein of YVX virus of Chinese yam rhizome, and producing specific immune response, its ELISA tires as 1:16000.Inventor further, based on the specific antibody for YVX coat protein, establish IC-RT-PCR method in conjunction with the genomic Auele Specific Primer for this viral YVX, YVX can be detected efficient, highly sensitive and exactly from the Chinese yam rhizome plant infecting YVX; ELISA and the Western blot method in like manner set up also can detect YVX specifically from the Chinese yam rhizome plant infecting YVX.Apply YVX whole genome sequence provided by the invention and polyclonal antibody, can be the quick detection of YVX and host's repercussion study and this virus, somatotype and molecular biology research provides material base and technical support, for solid foundation is laid in the Epidemiology monitor of this virus and control.
Accompanying drawing explanation
Fig. 1 is 5 '/3 ' the RACE electrophoretogram of YVX, in figure: M is DNA molecular amount standard (O ' GeneRuler100bpPlus DNA Ladder, Thermo Scientific company), and 1 is 5 ' RACE product, and 2 is 3 ' RACE product.
Fig. 2 is the segmented-PCR amplification electrophoretogram of YVX, and in figure: M is DNA molecular amount standard (O ' GeneRuler1kb DNA Ladder, Thermo Scientific company), 1 be YVX fragment 2,2 is YVX fragment 3.
Fig. 3 is the genome structure schematic diagram of YVX.
Fig. 4 is the rdrp gene of YVX and the evolutionary relationship figure of other potexviruses virus representative species.
Fig. 5 is the coat protein gene of YVX and the evolutionary relationship figure of other potexviruses virus representative species.
Fig. 6 is the coat protein gene pcr amplification electrophoretogram of YVX, in figure: M is DNA molecular amount standard (O ' GeneRuler1kb DNA Ladder, Thermo Scientific company), and swimming lane 1 ~ 3 is the PCR primer of the CP protein gene of the YVX that 3 are repeated.。
Fig. 7 is the digestion verification electrophoretogram of prokaryotic expression carrier pET30a-YVX-CP, in figure: M be DNA molecular amount standard (O ' GeneRuler1kb DNA Ladder, Thermo Scientific company), swimming lane 1 ~ 3 is the digestion products of the pET30a-YVX-CP plasmid of the CP protein gene of YVX in 3 connections repeated.
Fig. 8 is the SDS-PAGE figure of the expression of recombinant proteins form of the CP gene of checking YVX, in figure: M is Protein Marker (PageRuler Unstained Protein Ladder, Thermo Scientific company), 1 is the Supernatant samples of collected by centrifugation after ultrasonic disruption cell, and 2 is the deposit sample of collected by centrifugation after ultrasonic disruption cell.
Fig. 9 is the SDS-PAGE figure of the recombinant protein of the CP gene of the YVX of affinity purification, in figure: M is Protein Marker (PageRuler Unstained Protein Ladder, Thermo Scientific company), 1 is the CP recombinant protein of the YVX with 6 × His label of ni-sepharose purification.
Figure 10 is YVX polyclonal antibody titration figure, in figure: 1 is preimmune serum, and 2 is antibody purification, and 3 is detecting factor.
Figure 11 is ACP-ELISA figure, in figure: 1 is the Chinese yam rhizome total protein sample (three repetitions) infecting YVX, 2 is the healthy Chinese yam rhizome total protein sample (three repetitions) not infecting YVX, 3 is the blanks (three repetitions) only adding protein lysate, and 4 is YVX-CP protein samples (three repetitions) of Prokaryotic expression, purification.
Figure 12 is that Western blot schemes, in figure: M is Protein Marker (PageRuler Prestained ProteinLadder, Thermo Scientific company), 1 is YVX positive, 2 is YVX positive, and 3 is YVX positive, and 4 is YVX negative sample, 5 is YVX positive, and 6 is YVX negative sample.
Figure 13 is IC-RT-PCR figure, in figure: M be DNA molecular amount standard (O ' GeneRuler100bp DNA Ladder, Thermo Scientific company), 1 is blank, 2 is negative control (healthy sample), 3 is positive control (the susceptible sample of YVX), and 4-6 is the testing sample (wherein the sample of No. 6 swimming lanes is detected as the YVX positive through ELISA) of doubtful virosis.
Embodiment
Escherichia coli used (Escherichia coli) strain DH5 α and BL21 (DE3) pLysS bacterial strain is preserved by this laboratory below, 5 '/3 ' RACE kit applies Life Sciences (Roche) purchased from Roche, PCR primer cloning vector is purchased from Invitrogen company, and restriction enzyme, PCR Mix etc. are purchased from Thermo company.
One, the clone of the full-length gene group sequence of YVX
3 ' the terminal sequence clone of 1.1YVX
According to the sequence of YVX that the order-checking of the transcript profile degree of depth obtains, design complementary YVX3 ' poly (A) end, cDNA first chain synthetic primer N1T(5 '-GACCACGCGTATCGATGTCGAC (T) with oligo (dT) 17v-3 ', is shown in sequence table SEQ .ID.No.4, and kit provides), forward primer YVX-3 '-1F
(5 '-TTGCTCGTCGTTCTCTGGCTGC-3 ', see sequence table SEQ .ID.No.5) and YVX-3 '-2F
(5 '-CCTCCTGCTAACTGGTCTGCTCTC-3 ', see sequence table SEQ .ID.No.6), reverse primer N1
(5 '-GACCACGCGTATCGATGTCGAC-3 ', see sequence table SEQ .ID.No.7, kit provides).5 '/3 ' RACE kit (Roche) is utilized to synthesize genomic 3 ' the end cDNA of YVX.7 μ l ddH are added in the reaction system of 20 μ l 2o (RNase-Free), 5 × cDNA synthesis buffer of 4 μ l, 1 μ l N1T primer (10 μMs), 5 μ l iron rod yam total serum IgE (1 μ g) and 2 μ l dNTP(10mM), 1 μ l Transcriptor Reverse Transcriptase(Roche company), mixing, 55 DEG C are reacted 60 minutes, then within 5 minutes, make reverse transcriptase inactivation 85 DEG C of reactions.The the first chain cDNA obtained with reverse transcription is for template, forward primer YVX-3 '-1F and reverse primer N1 is used to carry out first time PCR, 100 times are diluted for template again with first time PCR primer, be that primer carries out second time PCR with YVX-3 '-2F and N1, obtaining specific, an expection size is the DNA fragmentation (Fig. 1, swimming lane 2) of 489bp.PCR2.1PCR product cloning kit (Invitrogen company) is utilized to clone 3 ' RACE PCR primer.Coupled reaction system is: 1 μ l PCR primer, 2 μ l5 × ligase Buffer, 1 μ l pCR2.1vector, 5 μ l ddH 2o and 1 μ l T4DNA ligase.Coupled reaction liquid is placed in room temperature, react after 30 minutes for Transformed E .coli DH5 α competent cell, be coated on the ampicillin, the 10 μ L IPTG(24mg/mL that are added with 100 μ g/L) and 20 μ L X-Gal(20mg/mL) LA nutrient culture media on, 37 DEG C cultivate 18 hours.Picking white colony extracts plasmid, and cuts qualification with EcoR I enzyme, and the positive colony selecting 3 restriction enzyme mappings correct carries out sequencing, by recombinant plasmid called after pCR-YVX-3.
The clone of 5 ' the end cDNA of 1.2YVX
According to the sequence of the YVX that high-flux sequence obtains, design reverse primer YVX-5 '-1R(5 '-TGATGGGTGTTTGAAGTAAGCCTCTG-3 ', see sequence table SEQ .ID.No.8) and YVX-5 '-2R(5 '-TCTGATATGTATGCCACGGGTGTTTG-3 ', see sequence table SEQ .ID.No.9).The cDNA utilizing 5 '/3 ' RACE kit (Roche) to synthesize 5 ' of YVX to hold: add 7 μ l ddH in the reaction system of 20 μ l 2o, the cDNA synthesis buffer of 4 μ l, 2 μ l dNTP(10mM), 1 μ l YVX-5 '-1R (10 μMs), 5 μ l iron rod yam total serum IgE (1 μ g) and 1 μ l Transcriptor Reverse Transcriptase, mixing, 55 DEG C are reacted 60 minutes, then within 5 minutes, make transcriptase inactivation 85 DEG C of reactions.After reaction terminates, with PCR primer purification kit (Roche trade name) purifying first chain cDNA product.Get the first chain cDNA product of 19 μ l purifying, add 2.5 μ l10 × Reaction Buffer, 2mM dATP, soft mixing is also of short duration centrifugal, in 94 DEG C of reactions 3 minutes, is placed in immediately on ice.Of short duration centrifugal, add 1 μ l terminal enzyme (DNA) Terminal Transferase(Roche company), softly mix and be placed in 37 DEG C of reactions 20 minutes, then reacting 10 minutes to make terminal enzyme (DNA) inactivation at 70 DEG C.Get 5 μ l and add the cDNA of A tail as template, add 1 μ l primer N1T(10 μM), 1 μ l YVX-5 '-1R(10 μM), 1 μ l dNTP(10mM), 5 μ l10 × Reaction Buffer, 0.5 μ l Taq DNA Polymerase(5U/ μ l) and 36.5 μ l ddH 2o, carries out first time PCR, then dilutes 100 times for template, with YVX-5 '-2R(10 μM with first time PCR primer) and N1(10 μM) for primer carries out second time PCR, amplify the band (Fig. 1, swimming lane 1) that a specificity expection size is 541bp.PCR2.1PCR product cloning kit (Invitrogen company) is utilized to clone 5 ' RACE PCR primer.Coupled reaction system is: the fresh PCR primer of 1 μ l, 2 μ l5 × ligase Buffer, 1 μ l pCR2.1vector, 5 μ l ddH2O and 1 μ l T4DNA ligase.Coupled reaction liquid is placed in room temperature, react after 30 minutes for the competent cell of Transformed E .coli DH5 α, be coated on the ampicillin, the 10 μ L IPTG(24mg/mL that are added with 100 μ g/L) and 20 μ L X-Gal(20mg/mL) LA nutrient culture media on, 37 DEG C cultivate 18 hours.Picking white colony extracts plasmid, and cuts qualification with EcoR I enzyme, selects 3 correct positive colonies of qualification and carries out sequencing, by recombinant plasmid called after pCR-YVX-5.
1.3 cloning and sequencing checking high-flux sequence results
3 clones of picking 3 '-cDNA fragment and 5 '-cDNA fragment carry out sequencing respectively, obtain viral genome Partial Fragment sequence (Fig. 1).Sequences Design two pairs of primers according to obtaining: forward primer YVX-268F(5 '-ACATACCCATGCCGCCTGTAAAG-3 ', see sequence table SEQ .ID.No.10) and reverse primer YVX-3446R(5 '-GCATTGCTCCGTCCTGAGATTGAT-3 ', see sequence table SEQ .ID.No.11); Forward primer YVX-3277F(5 '-CGTCCGCCGTCGAAGTTCAA-3 ', is shown in sequence table SEQ .ID.No.12) and reverse primer YVX-5824R(5 '-CACGCTCAATCTCTGTAGGCTCTC-3 ', see sequence table SEQ .ID.No.13).Expection pcr amplified fragment size is respectively 3179bp and 2771bp.With the cDNA of 3 ' RACE for template, carry out with these two pairs of primers the object fragment (Fig. 2) that pcr amplification obtains expection size respectively, be cloned into pCR2.1vector respectively and picking 3 clones check order.
By 3 ' RACE, 5 ' RACE and the final splicing of cDNA clones order-checking obtain the full-length gene group sequence (see sequence table SEQ .ID.No.3) comprising the YVX of 6139nt, through domain prediction and homologous sequence compare of analysis, find its coding 5 virus proteins, be respectively replicase polyprotein (replicase polyprotein, see sequence table SEQ .ID.No.18), triple gene block albumen 1(triple gene block protein1, see sequence table SEQ .ID.No.19), triple gene district albumen 2(triplegene block protein2, see sequence table SEQ .ID.No.20), triple gene district albumen 3(triple gene block protein3, see sequence table SEQ .ID.No.21) and coat protein (coat protein, see sequence table SEQ .ID.No.1) (Fig. 3).
Choose the amino acid sequence of replicase polyprotein and coat protein, with the adjacent method constructing system chadogram of MEGA4.0 software.Two chadograms all show, and this virus and the relation of Nerine virus X on evolving are recently (Fig. 4 and Fig. 5).Two, the prokaryotic expression of YVX virus capsid protein and polyclonal antibody preparation
The clone of 2.1YVX coat protein gene
CP gene order according to acquired YVX designs a pair Auele Specific Primer: forward primer YVX-CP-F(3 '-CCC gAATTCaTGATTGAAAGTATGTCTACACC-5 ', is shown in sequence table SEQ .ID.No.14, and its 5 ' end adds EcoR I restriction enzyme site), reverse primer YVX-CP-R(3 '-TTT gTCGACtTAAGGTTCAGGCAAGTTGAG-5 ', see sequence table SEQ .ID.No.15, its 5 ' end adds Sal I restriction enzyme site), expection product is 771bp.Through RT-PCR, obtain the CP genetic fragment (Fig. 6) that size is about the YVX of 750bp.By this product cloning to pUC19, build pUC19-YVX-CP, Transformed E .coli DH5 α competent cell.Picking 3 positive colony order-checkings, result shows that pUC19-YVX-CP comprises the complete sequence of the CP gene of YVX.BLASTX comparison result shows, with the CP homology of YVX the highest be the CP(46% of Nerine virus X), be secondly the CP(44% of White clover mosaic virus).
The abduction delivering of 2.2 albumen and purifying
With EcoR I and Sal I double digestion pUC19-YVX-CP plasmid, be separated through gel electrophoresis, reclaim the CP fragment (Fig. 7) of YVX and be cloned into prokaryotic expression carrier pET30a, build prokaryotic expression plasmid pET30a-YVX-CP.With pET30a-YVX-CP Transformed E .coli BL21 (DE3) pLysS competent cell, with PCR and double digestion method screening positive clone.The mono-bacterium colony of BL21 (DE3) pLysS containing pET30a-YVX-CP of picking fresh cultured, be inoculated in l0mL containing in the LB nutrient culture media of 50 μ g/L kanamycins, 37 DEG C of shaken cultivation are spent the night.In the ratio of l:100, the bacterium liquid of incubated overnight is joined fresh the containing in the LB nutrient culture media of 25 μ g/L kanamycins of 200mL, 37 DEG C of joltings are cultured to OD 600=0.4 ~ 0.6.Add the IPTG of final concentration 0.5mM, in 20 DEG C, 160rpm shaken cultivation abduction delivering 5h.Collected by centrifugation thalline, suspends by the cell lysis buffer solution (50mM NaH2PO4,300mM NaCl, 10mM Imidazole, 10mM β-ME, pH8.0) of 1/20 volume of culture, then carries out ultrasonic disruption.The upper cleer and peaceful precipitation that takes a morsel after centrifugal carries out SDS-PAGE analysis respectively, determines that this albumen is solubility expression (Fig. 8).The affinity purification of YVX-CP albumen is carried out with Ni-NTA resin (QIAgen company) the cell centrifugation supernatant to ultrasonic disruption.SDS-PAGE detection display, the YVX-CP albumen after affinity purification is single band, and molecular weight is about 35kDa(Fig. 9).
2.3 polyclonal antibody preparation and titration
Immunity is new zealand white rabbit (2) with rabbit.Normal serum is gathered as negative control before immunity.Getting the recombinant protein 2mL(protein concentration after purifying is 1mg/mL), adopt subcutaneous multi-point injection method immunize New Zealand White Rabbit, later every 2 weeks booster immunizations once, are total to immunity 4 times.First immunisation adds the Freund's complete adjuvant with albumen equivalent, adds the incomplete Freund's adjuvant with albumen equivalent for 3 times afterwards.Final boost is after 7 days, Culling heart blood, separation of serum, then uses antigen affinity chromatography, obtains the polyclonal antibody of purifying.With the YVX CP of prokaryotic expression for antigen, measure through ELISA method, the polyclonal antibody after purifying is tired as 1:16000(Figure 10).
Three, based on the detection method of YVX coat protein polyclonal antibody
3.1 indirect antigen coated ELISA(ACP-ELISA) method detection virus
The operation steps of ACP-ELISA method:
(1) with the fresh blade of 1mL antigen coated damping fluid grinding 0.1g Chinese yam rhizome, the centrifugal 5min of 9000rpm, gets supernatant and dilutes 20 times, adds elisa plate according to 100 μ l/ holes; The sick leaf of Chinese yam rhizome infecting YVX is positive control, and corresponding healthy leaf is negative control, hatches 2h for 37 DEG C;
(2) with 200 μ l PBST(3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, 0.05%Tween20, pH7.4) wash twice after, close 30min with 5% skimmed milk power;
(3), after washing twice with 200 μ l PBST, add the YVX polyclonal antibody diluted according to 1:8000 with antibody dilution buffer in each hole, 100 μ l/ holes, hatch 1h or incubated at room 3h for 37 DEG C;
(4), after washing twice with 200 μ l PBST, the goat anti-rabbit igg bond (Abmart company) that horseradish peroxidase (HRP) that by specification dilutes 3000 times marks is added in each hole, 100 μ l/ holes, incubated at room 1h;
(5) after washing three times with 200 μ l PBST, carry out develop the color (Figure 11) with tmb substrate chromogenic reagent box (health is ShiJi Co., Ltd), room temperature lucifuge hatches 25-30min, and reaction product is in blue, and every hole adds 50 μ l0.5M H again 2sO 4color development stopping, now blue product becomes glassy yellow, reads OD immediately by microplate reader 450value, to be greater than 2.1 for positive with negative OD value ratio (P/N);
Wherein, PBST is containing 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, 0.05%Tween20, pH7.4; Antigen coated damping fluid is carbonate buffer solution (30mM sodium carbonate-bicarbonate damping fluid, pH9.6); Antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.
The 3.2 Western Immuno markings (Western blot) method detects virus
Operation steps:
(1) extract kit (health is ShiJi Co., Ltd) with TCA/ acetone precipitation or employing phytoprotein and extract Chinese yam rhizome total protein sample, carry out quantification of protein by Bradford method, adjustment protein concentration is 2mg/mL;
(2) prepare protein electrophoresis gel, carry out SDS-PAGE;
(3) half dry type transfer: gel is cut to suitable size after electrophoresis terminates, by transferring film damping fluid balance, 5min × 3 time; Cut out the filter paper onesize with gel and pvdf membrane in advance, after soaking 1min with methyl alcohol, immerse 10min in transferring film damping fluid; Membrane-transferring device is put well by the order of carbon anode plate, 3 layers of 3mm filter paper, pvdf membrane, gel, 3 layers of 3mm filter paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, pvdf membrane Accurate align, each step removes bubble, switches on power after building negative electrode carbon plate, constant current 1mA/cm 2, transfer 1.5h, after transfer terminates, film takes out by deenergization;
(4) immune response: with 0.01M PBS(3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, pH7.4) and wash film, 1min; Add confining liquid (5% skimmed milk power or 3%BSA), room temperature closes 1 ~ 2h; Abandon confining liquid, wash film with 0.01M PBST, 5min × 3 time; Add the CP polyclonal antibody (primary antibodie) of the YVX diluted according to 1:5000 with antibody dilution buffer, place more than 12h for incubated at room 2h or 4 DEG C; Negative control, replace primary antibodie with 1%BSA, all the other steps are identical with experimental group; Abandon primary antibodie and 1%BSA, wash film respectively with 0.01M PBST, 5min × 4 time; Add the goat anti-rabbit igg bond (two resist) marked with the alkaline phosphatase (AP) that antibody dilution buffer dilutes 1000 times, steadily shake, room temperature 2h; Abandon two to resist, wash film with 0.01M PBST, 5min × 4 time; Add BCIP/NBT nitrite ion, colour developing is to putting into distilled water cessation reaction (Figure 12) when there is band;
Wherein, transferring film damping fluid is 25mM Tris, 192mM glycine, 0.01%SDS, 20%methanol, pH8.3; 0.01M PBS contains 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, pH7.4; Confining liquid is 5% skimmed milk power or 3%BSA.
3.3 detect virus by immunocapture inverse transcription polymerase chain reaction (IC-RT-PCR) method
The operation steps of IC-RT-PCR method:
(1) dilute YVX polyclonal antibody with antibody dilution buffer according to 1:8000, add the antibody that 100 μ l have diluted in the centrifuge tube of each 1.5mL, hatch 1h or incubated at room 3h for 37 DEG C;
(2) by Chinese yam rhizome blade with containing the 0.02M PBS grinding buffer solution of 2%PVP by mass volume ratio 0.2%(w/v) put into mortar, grind to form the homogenate of leaf juice, in suction 1.5mL centrifuge tube, the centrifugal 10min of 9000rpm;
(3) bag in step (1) is washed twice by the 1.5mL centrifuge tube PBST of good antibody, add 200 μ l step (2) ready leaf juice supernatants, 37 DEG C of water-bath 3h, the virion now exposed is attached to (immunocapture) on centrifuge tube inwall specifically;
(4) remove leaf juice, PBST washes 3 times, and DEPC washes 1 time, wash finish do at the bottom of of short duration centrifugal segregation pipe more than liquid;
(5) RT-PCR reaction is carried out to specifications with SuperRT One step RT-PCR kit (health is ShiJi Co., Ltd), it is YVX-F:5 '-CCAGGTCCATCAGTCACTTCT-3 ' (see sequence table SEQ .ID.No.16) and YVX-R:5 '-CTAATCTACCAGCAGTCACTTCATA-3 ' (see sequence table SEQ .ID.No.17) that YVX detects primer, it is 55 DEG C that the PCR stage arranges annealing temperature, 35 circulations;
(6), after RT-PCR terminates, the PCR primer mass body volume concentrations 1.0%(w/v of 5 μ l is got) Ago-Gel carries out electrophoresis detection, expection clip size is 692bp(Figure 13).
Wherein, antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.

Claims (9)

1. a preparation method of polyclonal antibody for Chinese yam rhizome X virus (YVX), is characterized in that: the CP albumen of Prokaryotic expression, purification Chinese yam rhizome X virus also prepares polyclonal antibody for immunizing rabbit; The CP albumen of described Chinese yam rhizome X virus has the amino acid sequence of sequence table SEQ .ID.No.1, or by having the coded by said gene of base sequence of sequence table SEQ .ID.No.2.
2. the preparation method of polyclonal antibody of Chinese yam rhizome X virus (YVX) according to claim 1, it is characterized in that: get the recombinant C P albumen after purifying, adopt subcutaneous multi-point injection method immunize New Zealand White Rabbit, later every 2 weeks booster immunizations once, are total to immunity 4 times; First immunisation adds the Freund's complete adjuvant with albumen equivalent, adds the incomplete Freund's adjuvant with albumen equivalent for 3 times afterwards; Final boost is after 7 days, Culling heart blood, separation of serum; The polyclonal antibody of this virus capsid protein CP is obtained again with antigen affinitive layer purification.
3. the polyclonal antibody of claim 1 or 2 gained Chinese yam rhizome X virus (YVX) is detecting the application in Chinese yam rhizome X virus.
4. based on the ELISA detection method of the Chinese yam rhizome X virus (YVX) of polyclonal antibody described in claim 1.
5. ELISA detection method according to claim 4, is characterized in that comprising the following steps:
(1) with the fresh blade of 1mL antigen coated damping fluid grinding 0.1g Chinese yam rhizome, the centrifugal 5min of 9000rpm, gets supernatant and dilutes 20 times, adds elisa plate according to 100 μ l/ holes; The sick leaf of Chinese yam rhizome infecting YVX is positive control, and corresponding healthy leaf is negative control, hatches 2h for 37 DEG C;
(2), after washing twice with 200 μ l PBST, 30min is closed with 5% skimmed milk power;
(3), after washing twice with 200 μ l PBST, add the polyclonal antibody diluted according to 1:8000 with antibody dilution buffer in each hole, 100 μ l/ holes, hatch 1h or incubated at room 3h for 37 DEG C;
(4) after washing twice with 200 μ l PBST, the goat anti-rabbit igg bond that by specification dilutes the horseradish peroxidase-labeled of 3000 times in each hole, is added, 100 μ l/ holes, incubated at room 1h;
(5) after washing three times with 200 μ l PBST, develop the color with tmb substrate chromogenic reagent box, room temperature lucifuge hatches 25-30min, and reaction product is in blue, and every hole adds 50 μ l 0.5M H again 2sO 4color development stopping, now blue product becomes glassy yellow, reads OD immediately by microplate reader 450value, to be greater than 2.1 for positive with negative OD value ratio;
Described PBST is containing 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, 0.05%Tween 20, pH 7.4; Described antigen coated damping fluid is carbonate buffer solution, 30mM sodium carbonate-bicarbonate damping fluid, pH9.6; Described antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.
6. based on the Western blot detection method of the Chinese yam rhizome X virus (YVX) of polyclonal antibody described in claim 1.
7. Western blot detection method according to claim 6, is characterized in that comprising the following steps:
(1) extract kit with TCA/ acetone precipitation or employing phytoprotein and extract Chinese yam rhizome total protein sample, carry out quantification of protein by Bradford method, adjustment protein concentration is 2mg/mL;
(2) prepare protein electrophoresis gel, carry out SDS-PAGE;
(3) half dry type transfer: gel is cut to suitable size after electrophoresis terminates, by transferring film damping fluid balance, 5min × 3 time; Cut out the filter paper onesize with gel and pvdf membrane in advance, after soaking 1min with methyl alcohol, immerse 10min in transferring film damping fluid; Membrane-transferring device is put well by the order of carbon anode plate, 3 layers of 3mm filter paper, pvdf membrane, gel, 3 layers of 3mm filter paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, pvdf membrane Accurate align, each step removes bubble, switches on power after building negative electrode carbon plate, constant current 1mA/cm 2, transfer 1.5h, after transfer terminates, film takes out by deenergization;
(4) immune response: wash film with 0.01M PBS, 1min; Add confining liquid, room temperature closes 1 ~ 2h; Abandon confining liquid, wash film with 0.01M PBST, 5min × 3 time; Add the CP protein polyclone antibody of the YVX diluted according to 1:5000 with antibody dilution buffer, place more than 12h for incubated at room 2h or 4 DEG C; Negative control, replace primary antibodie with 1%BSA, all the other steps are identical with experimental group; Abandon primary antibodie and 1%BSA, wash film respectively with 0.01M PBST, 5min × 4 time; Add the goat anti-rabbit igg bond diluting the alkali phosphatase enzyme mark of 1000 times with 0.01M PBS antibody dilution buffer, steadily shake, room temperature 2h; Abandon two to resist, wash film with 0.01M PBST, 5min × 4 time; Add BCIP/NBT nitrite ion, colour developing puts into distilled water cessation reaction to when there is band;
Described transferring film damping fluid is 25mM Tris, 192mM glycine, 0.01%SDS, 20%methanol, pH 8.3; Described 0.01M PBS contains 3.2mM Na 2hPO 4, 0.5mM KH 2pO 4, 1.3mM KCl, 135mM NaCl, pH 7.4; Described confining liquid is 5% skimmed milk power or 3%BSA.
8. based on the IC-RT-PCR detection method of the Chinese yam rhizome X virus (YVX) of polyclonal antibody described in claim 1.
9. IC-RT-PCR detection method according to claim 8, is characterized in that comprising the following steps:
(1) dilute YVX polyclonal antibody with antibody dilution buffer according to 1:8000, add the antibody that 100 μ l have diluted in the centrifuge tube of each 1.5mL, hatch 1h or incubated at room 3h for 37 DEG C;
(2) Chinese yam rhizome blade and the 0.02M PBS grinding buffer solution containing 2%PVP are put into mortar by mass volume ratio 0.2%, grind to form the homogenate of leaf juice, suck in 1.5mL centrifuge tube, the centrifugal 10min of 9000rpm;
(3) bag in step (1) is washed twice by the 1.5mL centrifuge tube PBST of good antibody, add 200 μ l step (2) ready leaf juice supernatants, 37 DEG C of water-bath 3h;
(4) remove leaf juice, PBST washes 3 times, and DEPC washes 1 time, wash finish do at the bottom of of short duration centrifugal segregation pipe more than liquid;
(5) RT-PCR reaction is carried out with SuperRT One step RT-PCR kit, detecting primer is YVX-F:5 '-CCAGGTCCATCAGTCACTTCT-3 ' and YVX-R:5 '-CTAATCTACCAGCAGTCACTTCATA-3 ', it is 55 DEG C that the PCR stage arranges annealing temperature, 35 circulations;
(6), after RT-PCR terminates, PCR primer mass body volume concentrations 1.0% Ago-Gel getting 5 μ l carries out electrophoresis detection;
Described antibody dilution buffer is 1%BSA, 0.01M PBS pH7.2.
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