CN110343708A - A kind of pumpkin aphid passes the preparation of yellow viral movement protein purifying expression and its polyclonal antibody - Google Patents

A kind of pumpkin aphid passes the preparation of yellow viral movement protein purifying expression and its polyclonal antibody Download PDF

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CN110343708A
CN110343708A CN201910661457.XA CN201910661457A CN110343708A CN 110343708 A CN110343708 A CN 110343708A CN 201910661457 A CN201910661457 A CN 201910661457A CN 110343708 A CN110343708 A CN 110343708A
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cabyv
expression
albumen
protein
purifying
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韩成贵
张绍康
王颖
张宗英
李大伟
于嘉林
尚巧霞
王献兵
张永亮
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China Agricultural University
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China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00051—Methods of production or purification of viral material

Abstract

The present invention relates to a kind of pumpkin aphids to pass yellow virus (Cucurbit aphid-borne yellows virus, CABYV) the building of the preparation of motor protein (CABYV-MP) expression and purification method and CABYV-MP polyclonal antibody and detection kit, wherein CABYV-MP expression and purification step are as follows: (1) CABYV-MP gene being transferred and expanding: (2) CABYV-MP prokaryotic expression vector building；(3) prokaryotic expression of His-tag- (CABYV-MP) fusion protein；(4) purifying of His-tag- (CABYV-MP) fusion protein；(5) acquisition of CABYV-MP albumen.Not only accuracy rate is high, high sensitivity for the CABYV-MP protein polyclone antibody obtained using the method for the invention, but also specificity has great application value and market prospects well, it can be achieved that pumpkin aphid passes the accurate detection of yellow virus and its motor protein.

Description

A kind of pumpkin aphid passes yellow viral movement protein purifying expression and its Anti-TNF-α The preparation of body
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of detection pumpkin aphid passes yellow virus and its motor protein The preparation of serological method and its dedicated purifying expression and its polyclonal antibody.
Background technique
It is to infect cucurbit that pumpkin aphid, which passes yellow viral (Cucurbit aphid-borne yellows virus, CABYV), The important virus of section crop belongs to Lutoevirus section (Luteoviridae) Polerovirus (Polerovirus).It should Virus is confined to host plant phloem tissue, mainly passes through two kinds of aphids (Myzus persicae and Aphis gossypii) By persistently walk around to type and it is non-proliferative in a manner of propagate.In recent years, ground family crop virosis caused by being infected by CABYV is sent out in China Dough product constantly expands, and has seriously affected the yield and quality of ground family crop, has caused heavy economic losses, and CABYV both can be with It is single to infect, it can also be to cause the yellow of blade and thicken with Combined Infection, classical symptom, cause the field plant about underproduction 50%.
CABYV is sense single stranded rna (+ssRNA) virus, the regular dodecahedron spherical virus for being 25-30nm with diameter Particle is formed by T=3 by 180 protein protomers, is wrapped in geneome RNA, no coating.Virion is more stable, to chloroform and Non-ionic octoxynol detergent is insensitive, but long time treatment can be destroyed under high salt conditions.CABYV full-length genome about 5.7kb, altogether Containing 7 open reading frame (ORFs), 7 albumen are encoded, first three ORF is expressed by geneome RNA, and latter four pass through subunit Because of a group rna expression, wherein ORF4 encodes pumpkin aphid and passes yellow viral movement protein (abbreviation CABYV-MP albumen), is swept by infiltration Expression is retouched, can be positioned on plasmodesmus, plays important regulative in virus replication and moving process.
At present for the detection of CABYV, RT-PCR technology is mainly utilized, it was reported that serological technique mainly utilizes CABYV The antiserum of purified virus particle preparation is detected by Western blot or ELISA, however it is anti-to utilize MP albumen to prepare Serum detects CABYV there is not yet formal report.
Summary of the invention
Present invention firstly provides a kind of pumpkin aphids to pass yellow virus (Cucurbit aphid-borne yellows Virus, CABYV) motor protein (CABYV-MP) expression and purification method, specific steps are as follows:
(1) CABYV-MP gene being transferred and expands:
(2) CABYV-MP prokaryotic expression vector constructs；
(3) prokaryotic expression of His-tag- (CABYV-MP) fusion protein；
(4) purifying of His-tag- (CABYV-MP) fusion protein.
Wherein, the CABYV-MP gene to transfer and expand be with the pCass- containing CABYV full length cDNA clone CABYV plasmid is template, and using 5 '-CATATGCAGGGAGGCGGAGGCGAAG-3 ', (underscore is restriction enzyme Nde I's Recognition site) and 5 '-CTCGAGCCTATTTCGGGTTTTGGACCTGG-3 ' (underscore be restriction enzyme Xho I knowledge Other site) it is that primer carries out PCR amplification；
Wherein, CABYV-MP prokaryotic expression vector building be by pcr amplification product obtained in step (1) and Cloning vector pMD19-T is attached, and obtains recombinant plasmid pMD19-T-CABYV-MP, with restriction enzyme Nde I and Xho I carries out digestion, recycles endonuclease bamhi, takes carrier pDB.His.MBP, carry out digestion with restriction enzyme Nde I and Xho I, Carrier framework is recycled, the endonuclease bamhi and the carrier framework are attached, recombinant plasmid pDB.His.MBP- is obtained CABYV-MP；
Wherein, the prokaryotic expression of His-tag- (CABYV-MP) fusion protein is the protokaryon that will be obtained in step (2) Expression vector imports in Escherichia coli Rosseta, obtains recombinant bacterium Rosseta-CABYV-MP, takes Rosseta-CABYV-MP mono- Clone, inoculated and cultured to OD600nmValue is 0.6~0.8, and the IPTG of final concentration of 0.1mM, 18 DEG C, 180rpm oscillation induction is added 16h is cultivated, 4 DEG C, 5000rpm centrifugation 6min collect bacterial sediment, supernatant is collected in ultrasonication after resuspension；
Wherein, the purifying of His-tag- (CABYV-MP) fusion protein is the supernatant and Ni that will be obtained in step (3) Affinity column combines, then elutes albumen with imidazole elution；
The present invention also provides the CABYV-MP albumen that the expression and purification method obtains according to the present invention, it is characterised in that institute CABYV-MP albumen is stated with the amino acid sequence as shown in SEQ ID NO:4.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of the coding CABYV-MP albumen of the invention.Those have by manually modified The nucleotide sequence 52% of the isolated CABYV-MP protein gene or the nucleotide of higher identity with the present invention, It is derived from nucleotide sequence of the invention and to be equal to sequence of the invention as long as encoding the CABYV-MP albumen.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair In bright polynucleotide amino acid sequence shown in SEQ ID NO:4 form protein nucleotide sequence have 52% or The nucleotide sequence of higher identity.Identity can with the naked eye or computer software is evaluated.Using computer software, two Identity between a or multiple sequences can be indicated with percentage (%), can be used to evaluate same between correlated series Property.
The present invention further provides above-mentioned CABYV-MP albumen to prepare the application in polyclonal antibody.
Wherein, the polyclonal antibody is by 1 parts by volume fusion protein solution (containing 400 μ g fusion proteins) and 1 parts by volume Freund's complete adjuvant is mixed to get mixed liquor first and 1 parts by volume fusion protein solution (containing 200 μ g fusion proteins) and 1 parts by volume Incomplete Freund's adjuvant is mixed to get the acquisition of mixed liquor second co-immunization New Zealand White Rabbit.
The present invention finally provides a kind of for pumpkin aphid biography yellow virus (Cucurbit aphid-borne yellows Virus, CABYV) detection kit, the kit include: according to the present invention the method obtain CABYV-MP albumen Polyclonal antibody.
The method comprises the steps of firstly, preparing amino acid sequence CABYV-MP albumen as shown in SEQ ID NO:4, then will CABYV-MP albumen is immunized new zealand white rabbit as immunogene, obtains polyclonal antiserum (antibody).Using Western blot detects pumpkin aphid as primary antibody using the polyclonal antibody and passes yellow virus and its motor protein, the Anti-TNF-α Not only accuracy rate is high for body, high sensitivity, but also specificity is good.The present invention can detect whether sample to be tested contains the biography yellow of pumpkin aphid Virus passes yellow virus detection kit for the function of study movement albumen, and building pumpkin aphid, has great application Value and market prospects.
Detailed description of the invention
The expression and purifying of Fig. 1: CABYV-MP fusion protein；
The titration of Fig. 2: CABYV-MP polyclonal antibody, wherein (A) sero-fast titration, (B) antiserum is most Suitable use scope measurement；
The sensitivity analysis of Fig. 3: CABYV-MP polyclonal antibody；
The specificity analysis of Fig. 4: CABYV-MP polyclonal antibody；
Application of Fig. 5: the CABYV-MP polyclonal antibody on viral diagnosis, wherein (A) is polyclonal antibody detection infection This life Tobacco Leaves of CABYV, (B) are the polyclonal antibody detection for simulating natural host Cucumber Infected CABYV.
Specific embodiment:
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.
Escherichia coli MC1022 is given by Strasbourg, FRA university Salah professor Bouzoubaa, Escherichia coli Rosseta be purchased from BIOMED company, Agrobacterium GV3101 by Britain University of Cambridge David Professor Baulcombe give, and the public can obtain from China Agricultural University (i.e. applicant), the biomaterial only attach most importance to duplicate invention Related experiment used in, not can be used as other purposes and use.
This life cigarette (Nicotiana benthamiana) by Britain John Innes Centre David Baulcombe Professor give, and the public can obtain from China Agricultural University (i.e. applicant), the biomaterial only attach most importance to duplicate invention correlation it is real Used in testing, it not can be used as other purposes and use.
Cloning vector pMD19-T (simple) is the product of TaKaRa company.Ni affinity column is QIAGEN company.It is double First expression vector pMDC32 (Curtis and Grossniklaus, 2003), contain CABYV-MP gene recombination plasmid pCass- CABYV, pumpkin aphid pass yellow viral (CABYV), muskmelon aphid passes yellow viral (MABYV), sponge gourd aphid passes yellow virus (SABYV), rape yellow viral (BrYV), corium solani (PLRV), sugarcane yellow leaf virus (ScYLV) have Flag The MP transient expression vector of label, is constructed by this laboratory, and the public can obtain from China Agricultural University (i.e. applicant), is used here To detect the specificity of prepared antibody.
Carrier pDB.His.MBP (annular) is purchased from DNASU plasmid library, SEQ ID NO in nucleotide sequence such as sequence table: Shown in 1.
TBST buffer in following embodiments is containing 0.05% (v/v) Tween-20,150mM NaCl, 20mM The buffer of Tris-HCl (pH7.5).
The building of embodiment 1, recombinant plasmid pDB.His.MBP-CABYV-MP
1, using the pCass-CABYV plasmid containing CABYV full length cDNA clone as template, with 5 '-CATATGCAGGGAGGCGGAGGCGAAG-3 ' (recognition site that underscore is restriction enzyme Nde I) and 5 '-CTCGAGCCTATTTCGGGTTTTGGACCTGG-3 ' (recognition site that underscore is restriction enzyme Xho I) be primer into Row PCR amplification obtains the pcr amplification product of about 595bp.
2, pcr amplification product and cloning vector pMD19-T (simple) that step 1 obtains are attached, are recombinated Plasmid pMD19-T-CABYV-MP.
3, recombinant plasmid pMD19-T-CABYV-MP is taken, carries out digestion with restriction enzyme Nde I and Xho I, is recycled About 570bp endonuclease bamhi.
4, carrier pDB.His.MBP is taken, carries out digestion with restriction enzyme Nde I and Xho I, recycling about 6.5kb's Carrier framework.
5, the endonuclease bamhi that step 3 obtains and the carrier framework that step 4 obtains are attached, obtain recombinant plasmid pDB.His.MBP-CABYV-MP。
Recombinant plasmid pDB.His.MBP-CABYV-MP is sequenced.According to sequencing result, to recombinant plasmid PDB.His.MBP-CABYV-MP carries out structure and is described as follows: by the restriction enzyme Nde I of carrier pDB.His.MBP and DNA small fragment between Xho I identification sequence replaces with double chain DNA molecule shown in SEQ ID NO:2.SEQ ID NO:2 and limit The core as shown in SEQ ID NO:3 of ATG composition CABYV-MP gene in property restriction endonuclease Nde I identification sequence (CATATG) processed Nucleotide sequence.CABYV-MP gene encodes the CABYV-MP albumen as shown in SEQ ID NO:4.
In recombinant plasmid pDB.His.MBP-CABYV-MP, on DNA molecular shown in SEQ ID NO:3 and carrier framework The coded sequence of His-tag label (being made of 6 histidine residues) merges, shown in the SEQ ID NO:5 of formation sequence table Fusion expresses the fusion protein as shown in SEQ ID NO:6 with His-tag label.
The expression and purifying of embodiment 2, fusion protein
1, recombinant plasmid pDB.His.MBP-CABYV-MP is imported into Escherichia coli Rosseta (with bacterial strain one in material Cause), recombinant bacterium is obtained, which is named as Rosseta-CABYV-MP.
2, after completing step 1, Rosseta-CABYV-MP monoclonal is taken, is seeded to 5mL LB liquid medium (containing 50 μ g/ ML kanamycins), 37 DEG C, 220rpm shaken cultivation 12h obtain culture bacterium solution.
3, after completing step 2, culture bacterium solution is taken, is seeded to LB liquid medium (containing 50 μ g/mL for 1:1000 by volume Kanamycins), 37 DEG C, 220rpm shaken cultivation to OD600nmValue is 0.6~0.8.It is added the IPTG of final concentration of 0.1mM, 18 DEG C, 180rpm vibrate Fiber differentiation 16h, 4 DEG C, 5000rpm be centrifuged 6min, collect bacterial sediment 1.
Culture bacterium solution is taken, is seeded to LB liquid medium (containing 50 μ g/mL kanamycins) for 1:1000 by volume, 37 DEG C, 220rpm shaken cultivation to OD600nmValue is 0.6~0.8,4 DEG C, 5000rpm is centrifuged 6min, collects bacterial sediment 2 (as right According to).
4, it after completing step 3, takes bacterial sediment (bacterial sediment 1 or bacterial sediment 2), 100mL NaCl containing 500mM is added PH 8.0,20mM Tris-HCl buffer, ultrasonication after resuspension (ultrasonic power 600W, cyclic program are as follows: broken 4s stops 6s, and totally 3 times.4 DEG C, 16000rpm centrifugation 40min, collect supernatant.Ni affinity column is added in supernatant, then is used The imidazole elution of various concentration (20mM, 40mM, 60mM, 100mM, 200mM, 500mM) elutes albumen, collects eluent.It will The eluent of each purification process carries out SDS-PAGE detection, as shown in Figure 1.The result shows that there are fusion proteins in supernatant Suitable protein eluate is concentrated, obtains pDB.His.MBP-CABYV-MP fusion protein.
Protein eluate 1: the Tris-HCl buffer of the pH 8.0 of NaCl containing 150mM and 200mM imidazoles, 20mM.
Protein eluate 2: the Tris-HCl buffer of the pH 8.0 of NaCl containing 150mM and 500mM imidazoles, 20mM.
5, after completing step 4, solution after column was taken, is concentrated into 1.5mL with ultra-filtration centrifuge tube, obtains concentrate egg white liquor.
It is detected, it is about 1mg/mL that fusion protein concentration in the CABYV-MP concentrate egg white liquor that IPTG induction obtains, which is added,. The concentrate egg white liquor (hereinafter referred to as concentrate egg white liquor) that addition IPTG induction obtains is all made of below to be tested.
The preparation of embodiment 3, polyclonal antibody
1, concentrate egg white liquor is taken, normal saline dilution is added to 200-500 μ L, obtains fusion protein solution.
2,1 parts by volume fusion protein solution (containing 400 μ g fusion proteins) and 1 parts by volume Freund's complete adjuvant are mixed, cream Change, obtains mixed liquor first.By 1 parts by volume fusion protein solution (containing 200 μ g fusion proteins) and 1 parts by volume incomplete Freund's adjuvant Mixing, emulsification, obtains mixed liquor second.
3, taking weight is New Zealand White Rabbit 1 of 2kg or so, through dorsal sc injection (8-10 is selected) mixed liquor first.
4, the 14d for completing step 3, through dorsal sc injection (8-10 is selected) mixed liquor second.
5, the 24d for completing step 3, through dorsal sc injection (8-10 is selected) mixed liquor second.
6, the 34d for completing step 3, through dorsal sc injection (8-10 is selected) mixed liquor second.7, the of step 3 is completed 35d takes a blood sample to the arteria carotis of New Zealand White Rabbit, separates serum；The serum is the polyclonal antibody prepared.Specifically: with fiber crops Liquor-saturated dose of anesthesia New Zealand White Rabbit (yellow Jackets, 30mg/kg, vein, abdominal cavity, intramuscular injection), fixed frame is fixed, and hand is used Art instrument cuts off neck outer layer fur, and arteria carotis is found below tracheae side, and haemostatic clamp clamps arterial and cuts bloodletting, receives Collection；5,000rpm centrifugation 10min twice, collect serum.
Above-mentioned steps are operated by BeiJing HuaDa protein Research Center Co., Ltd.
Embodiment 4, polyclonal antibody detect CABYV-MP
This life Tobacco Leaves total protein of transient expression CABYV-MP albumen is subjected to SDS-PAGE and Western blot inspection It surveys, CABYV is detected as primary antibody using polyclonal antibody prepared by step 3 in embodiment 1.Specific step is as follows:
1, this life Tobacco Leaves total protein of transient expression CABYV-MP albumen is extracted.
2, after completing step 1, the leaves total protein is subjected to SDS-PAGE, then uses the method for electrotransfer by protein (200mA, 90min) is transferred on nitrocellulose filter.
3, after completing step 2, the nitrocellulose filter is placed in the TBST buffer containing 5% (w/v) skimmed milk power, 37 DEG C of closing 1h.
4, after completing step 3, (1:1000 is dilute for the dilution of the polyclonal antibody of step 4 preparation in addition embodiment 1 Release), 37 DEG C of incubation 1h, TBST buffer washes film 3 times, each 10min.
5, complete step 4 after, by the nitrocellulose filter be placed in AP label goat anti-rabbit igg secondary antibody working solution (1: 20000 dilutions), 37 DEG C of incubation 1h, TBST buffer washes film 3 times, each 10min.
6, after completing step 5, under the conditions of being protected from light, the nitrocellulose filter is placed in containing 330 μ g/mL NBT and 165 μ g/ It is clear to band to develop the color in the colorbuffer of mL BCIP, is terminated and is reacted with distilled water.
The result shows that the polyclonal antibody that in embodiment 1 prepared by step 3 can successfully detect CABYV.
The titration of embodiment 5, polyclonal antibody
1. extracting this life Tobacco Leaves of transient expression CABYV-MP albumen and this life Tobacco Leaves of injection pMDC32 empty carrier The total protein of (as negative control).
2, the CABYV-MP polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains To dilution.The extension rate of dilution is respectively 1000,2000,4000,8000,16000,32000,64000,100000, 128000,200000,256000,400000,500000,512000,800000,1000000 and 1600000.
3, the total protein for extracting step 1 carries out SDS-PAGE and Western blot, the dilution obtained using step 2 CABYV is detected as primary antibody.
Western blot result is shown in A in Fig. 2.
4, this life Tobacco Leaves of transient expression CABYV-MP albumen are extracted and inject this life Tobacco Leaves of pMDC32 empty carrier The total protein of (as negative control).
5, the CABYV-MP polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains To dilution.The extension rate of dilution is respectively 10000,20000,40000,60000,80000,100000,120000, 140000,160000 and 180000.
6, the total protein for extracting step 4 carries out SDS-PAGE and Western blot, the dilution obtained using step 5 CABYV is detected as primary antibody.
Western blot result is shown in B in Fig. 2.
The result shows that polyclonal antibody is dilute when detecting polyclonal antibody potency with this life Tobacco Leaves of transient expression CABYV It releases and still can detecte faint protein band when multiple reaches 1000000, polyclonal antibody extension rate reaches 1600000 It cannot detect purpose band, therefore, the potency for the CABYV-MP polyclonal antibody that in embodiment 1 prepared by step 4 is about 1: 1000000。
The best scope of application that polyclonal antibody is detected with this life Tobacco Leaves of transient expression CABYV, in 1:10000~1: Color developing effect is all more obvious in the range of 100000.When extension rate reaches 100000, from color developing effect and economic angle, This life cigarette detection infected to CABYV is more satisfactory.
The sensitivity analysis of embodiment 6, polyclonal antibody
1,300 μ L 2 × SDS eggs are added in this life Tobacco Leaves 0.1g for taking transient expression CABYV-MP albumen after being fully ground White sample-loading buffer obtains protein sample 1 (1:4).
2 × SDS albumen sample-loading buffer: containing 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, The buffer of 100mM Tris-HCl (pH6.8).
2, after completing step 1,1,2 times of doubling dilutions of protein sample are taken, obtain extension rate be respectively 8,16,32,64, 128,256,512 and 1024 protein sample.
3, protein sample 1 separately is taken, the 1 parts by volume 2 × SDS albumen sample-loading buffer of parts by volume protein sample 1 and 9 is mixed, Obtain protein sample 2 (i.e. extension rate 10,1:40)
4. taking 2,2 times of doubling dilutions of protein sample, obtaining extension rate is respectively 80,160,320 and after completing step 3 640 protein sample.
5, the CABYV-MP polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains To dilution.The extension rate of dilution is 1000,10000 and 20000.
6, the protein sample for obtaining step 2 and step 4 carries out SDS-PAGE and Western blot, is obtained using step 5 The dilution arrived detects CABYV as primary antibody.
The Western blot result of CABYV-MP is shown in Fig. 3.
The result shows that the total protein of this life Tobacco Leaves of 0.1g transient expression CABYV-MP is laid eggs in 1:1000 concentration conditions It reacts and is positive after white 128 times of dilution, albumen dilutes 80 times and is positive under the conditions of 1:10000, albumen under the conditions of 1:20000 64 times of dilution is positive.
The specificity analysis of embodiment 7, polyclonal antibody
Blade to be measured be transient expression CABYV-MP, MABYV-MP, SABYV-3Flag-MP, BrYV-3Flag-MP, This life Tobacco Leaves of PLRV-3Flag-MP, ScYLV-3Flag-MP and pMDC32 empty carrier.
1, the total protein of blade to be measured is extracted.
2, the CABYV polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains Dilution.The extension rate of dilution is 1000,10000 and 20000.
3, the total protein for obtaining step 1 carries out SDS-PAGE and Western blot, the dilution obtained using step 2 CABYV is detected as primary antibody.Western blot result is shown in Fig. 4, the only total protein of this life Tobacco Leaves of transient expression CABYV It reacts and is positive under its corresponding serum condition.The result shows that the polyclonal antibody that in embodiment 1 prepared by step 3 is to CABYV Specificity with higher, including with can mixed infection melon MABYV, SABYV serological cross reaction is also not present.
The application of embodiment 8, polyclonal antibody on viral diagnosis
One, this life Tobacco Leaves of polyclonal antibody detection infection CABYV
Blade to be measured is that this life Tobacco Leaves for being inoculated with CABYV infectious cDNA clone and inoculation pCass-RZ empty carrier (are made For negative control) this life Tobacco Leaves.
1, the total protein of blade to be measured is extracted.
2, the CABYV polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains Dilution.The extension rate of dilution is 10000.
3, the total protein for obtaining step 1 carries out SDS-PAGE and Western blot, the dilution obtained using step 2 CABYV is detected as primary antibody.Western blot result is shown in Fig. 5 A, the results showed that CABYV-MP polyclonal antiserum can be special Property detects the CABYV in this life Tobacco Leaves, and consistent with the testing result of RT-PCR.Illustrate that CABYV-MP antiserum is applicable in The detection of CABYV, application value with higher in model plant this life cigarette being widely used at present.
Two, the polyclonal antibody detection of natural host Cucumber Infected CABYV is simulated
Blade to be measured be inject empty carrier pMDC32 this life Tobacco Leaves, this life Tobacco Leaves of transient expression CABYV-MP, The cucumber leaves of health.
1, the total protein of blade to be measured is extracted.
2, the analog detection that CABYV infects is carried out with the natural host cucumber of health.It extracts according to the ratio of 1:4 in this life The CABYV-MP albumen of Tobacco Leaves transient expression, the total protein with the healthy cucumber leaves extracted respectively according to 1:4 and 1:8 ratio Mixed diluting is carried out to 6 gradients: 1:8,1:16,1:32,1:64,1:128,1:256.To extract injection empty carrier pMDC32's This life Tobacco Leaves and the cucumber leaves albumen of health are negative control, with this life Tobacco Leaves of the transient expression CABYV-MP of extraction Albumen is positive control.
3, the CABYV polyclonal antibody that in Example 1 prepared by step 3 is added TBST buffer and is diluted, obtains Dilution.The extension rate of dilution is 10000.
4, the total protein for obtaining step 1 carries out SDS-PAGE and Western blot, the dilution obtained using step 2 CABYV is detected as primary antibody.Western blot result is shown in B in Fig. 5.
The result shows that under the cucumber leaves total protein diluted concentration background extracted with 1:4, CABYV-MP Anti-TNF-α blood It is able to detect that clearly with the CABYV-MP albumen of 1:256 ratio mixed diluting, in the cucumber leaves total protein dilution extracted with 1:8 Under concentration background, CABYV-MP polyclonal antiserum is able to detect that with the CABYV-MP albumen of 1:128 ratio mixed diluting, and And with the antiserum serological reaction does not occur for the total protein of healthy cucumber leaves, and does not influence antiserum and CABYV-MP albumen Specific reaction occurs, for the sero-fast Fields detection application and the kit of preparation detection CABYV and its motor protein Theoretical foundation is provided.
This embodiment is merely preferred embodiments of the present invention, but scope of protection of the present invention is not limited thereto, In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art, It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with scope of protection of the claims Subject to.
Sequence table
<110>China Agricultural University
<120>a kind of pumpkin aphid passes the preparation of yellow viral movement protein purifying expression and its polyclonal antibody
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6494
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
ggtaccaaaa ctgaagaagg taaactggta atctggatta acggcgataa aggctataac 5160
ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 5220
catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 5280
attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 5340
atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 5400
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 5460
aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 5520
ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 5580
ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 5640
aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 5700
attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 5760
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 5820
aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 5880
gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag 5940
ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 6000
ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 6060
gccaccatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 6120
ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 6180
gaagccctga aagacgcgca gactggtacc gattacgata tcccaacgac cgaaaacctt 6240
tacttccagg gccatatggc tagcatgact ggtggacagc aaatgggtcg cggatccgaa 6300
ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga 6360
tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 6420
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 6480
aactatatcc ggat 6494
<210> 2
<211> 570
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cagggaggcg gaggcgaaga aatcagcgct ctatacggcg cgaccgcgtg gttgtggtca 60
acccctctgg gggaccatca cgcggaagac gacaacgaag aaaccgccga cgccctaatc 120
gaggaggcag agctagagga aggagtccag gcgaaacatt cgtattttca aaggacaatc 180
tcacgggcag ttcctcagga agtatcacct tcgggccgtc tctatcagag agcccagcat 240
tcagctctgg aatactcaag gcctaccatg aatataagat catcatggtc cagctggagt 300
tcatctccga ggcctcttcc acctcctcgg gctccatctc ttatgagttg gacccccact 360
gcaagcttag ctccctccaa tccacgatta ataaatttgg aatcaccaag agtggattac 420
gacgttgggc agccaggcag atcaacggga tggaatggca tgacgcaact gaggaccaat 480
tcaagatcct ctataaaggg aacggatctt cctcggttgc aggcagcttc aggatcacca 540
tcaagtgcca ggtccaaaac ccgaaatagg 570
<210> 3
<211> 573
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcagggag gcggaggcga agaaatcagc gctctatacg gcgcgaccgc gtggttgtgg 60
tcaacccctc tgggggacca tcacgcggaa gacgacaacg aagaaaccgc cgacgcccta 120
atcgaggagg cagagctaga ggaaggagtc caggcgaaac attcgtattt tcaaaggaca 180
atctcacggg cagttcctca ggaagtatca ccttcgggcc gtctctatca gagagcccag 240
cattcagctc tggaatactc aaggcctacc atgaatataa gatcatcatg gtccagctgg 300
agttcatctc cgaggcctct tccacctcct cgggctccat ctcttatgag ttggaccccc 360
actgcaagct tagctccctc caatccacga ttaataaatt tggaatcacc aagagtggat 420
tacgacgttg ggcagccagg cagatcaacg ggatggaatg gcatgacgca actgaggacc 480
aattcaagat cctctataaa gggaacggat cttcctcggt tgcaggcagc ttcaggatca 540
ccatcaagtg ccaggtccaa aacccgaaat agg 573
<210> 4
<211> 191
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Gln Gly Gly Gly Gly Glu Glu Ile Ser Ala Leu Tyr Gly Ala Thr
1 5 10 15
Ala Trp Leu Trp Ser Thr Pro Leu Gly Asp His His Ala Glu Asp Asp
20 25 30
Asn Glu Glu Thr Ala Asp Ala Leu Ile Glu Glu Ala Glu Leu Glu Glu
35 40 45
Gly Val Gln Ala Lys His Ser Tyr Phe Gln Arg Thr Ile Ser Arg Ala
50 55 60
Val Pro Gln Glu Val Ser Pro Ser Gly Arg Leu Tyr Gln Arg Ala Gln
65 70 75 80
His Ser Ala Leu Glu Tyr Ser Arg Pro Thr Met Asn Ile Arg Ser Ser
85 90 95
Trp Ser Ser Trp Ser Ser Ser Pro Arg Pro Leu Pro Pro Pro Arg Ala
100 105 110
Pro Ser Leu Met Ser Trp Thr Pro Thr Ala Ser Leu Ala Pro Ser Asn
115 120 125
Pro Arg Leu Ile Asn Leu Glu Ser Pro Arg Val Asp Tyr Asp Val Gly
130 135 140
Gln Pro Gly Arg Ser Thr Gly Trp Asn Gly Met Thr Gln Leu Arg Thr
145 150 155 160
Asn Ser Arg Ser Ser Ile Lys Gly Thr Asp Leu Pro Arg Leu Gln Ala
165 170 175
Ala Ser Gly Ser Pro Ser Ser Ala Arg Ser Lys Thr Arg Asn Arg
180 185 190
<210> 5
<211> 600
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atgcagggag gcggaggcga agaaatcagc gctctatacg gcgcgaccgc gtggttgtgg 60
tcaacccctc tgggggacca tcacgcggaa gacgacaacg aagaaaccgc cgacgcccta 120
atcgaggagg cagagctaga ggaaggagtc caggcgaaac attcgtattt tcaaaggaca 180
atctcacggg cagttcctca ggaagtatca ccttcgggcc gtctctatca gagagcccag 240
cattcagctc tggaatactc aaggcctacc atgaatataa gatcatcatg gtccagctgg 300
agttcatctc cgaggcctct tccacctcct cgggctccat ctcttatgag ttggaccccc 360
actgcaagct tagctccctc caatccacga ttaataaatt tggaatcacc aagagtggat 420
tacgacgttg ggcagccagg cagatcaacg ggatggaatg gcatgacgca actgaggacc 480
aattcaagat cctctataaa gggaacggat cttcctcggt tgcaggcagc ttcaggatca 540
ccatcaagtg ccaggtccaa aacccgaaat aggctcgagc accaccacca ccaccactag 600
<210> 6
<211> 199
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Met Gln Gly Gly Gly Gly Glu Glu Ile Ser Ala Leu Tyr Gly Ala Thr
1 5 10 15
Ala Trp Leu Trp Ser Thr Pro Leu Gly Asp His His Ala Glu Asp Asp
20 25 30
Asn Glu Glu Thr Ala Asp Ala Leu Ile Glu Glu Ala Glu Leu Glu Glu
35 40 45
Gly Val Gln Ala Lys His Ser Tyr Phe Gln Arg Thr Ile Ser Arg Ala
50 55 60
Val Pro Gln Glu Val Ser Pro Ser Gly Arg Leu Tyr Gln Arg Ala Gln
65 70 75 80
His Ser Ala Leu Glu Tyr Ser Arg Pro Thr Met Asn Ile Arg Ser Ser
85 90 95
Trp Ser Ser Trp Ser Ser Ser Pro Arg Pro Leu Pro Pro Pro Arg Ala
100 105 110
Pro Ser Leu Met Ser Trp Thr Pro Thr Ala Ser Leu Ala Pro Ser Asn
115 120 125
Pro Arg Leu Ile Asn Leu Glu Ser Pro Arg Val Asp Tyr Asp Val Gly
130 135 140
Gln Pro Gly Arg Ser Thr Gly Trp Asn Gly Met Thr Gln Leu Arg Thr
145 150 155 160
Asn Ser Arg Ser Ser Ile Lys Gly Thr Asp Leu Pro Arg Leu Gln Ala
165 170 175
Ala Ser Gly Ser Pro Ser Ser Ala Arg Ser Lys Thr Arg Asn Arg Leu
180 185 190
Glu His His His His His His
195

Claims (8)

1. a kind of pumpkin aphid passes viral (Cucurbit aphid-borne yellows virus, the CABYV) motor protein of yellow (CABYV-MP) expression and purification method, specific steps are as follows:
(1) CABYV-MP gene being transferred and expands:
(2) CABYV-MP prokaryotic expression vector constructs；
(3) prokaryotic expression of His-tag- (CABYV-MP) fusion protein；
(4) purifying of His-tag- (CABYV-MP) fusion protein.
2. expression and purification method according to claim 1, it is characterised in that: the CABYV-MP gene being transferred and expanding It is using the pCass-CABYV plasmid containing CABYV full length cDNA clone as template, with 5 '-CATATGCAGGGAGGCGGAGGCGAAG-3 ' (recognition site that underscore is restriction enzyme Nde I) and 5 '-CTCGAGCCTATTTCGGGTTTTGGACCTGG-3 ' (recognition site that underscore is restriction enzyme Xho I) be primer into Row PCR amplification.
3. -2 described in any item expression and purification methods according to claim 1, it is characterised in that: the CABYV-MP gene protokaryon Expression vector establishment is to be attached pcr amplification product obtained in step (1) and cloning vector pMD19-T, is recombinated Plasmid pMD19-T-CABYV-MP carries out digestion with restriction enzyme Nde I and Xho I, recycles endonuclease bamhi, take carrier PDB.His.MBP carries out digestion with restriction enzyme Nde I and Xho I, recycles carrier framework, by the endonuclease bamhi and The carrier framework is attached, and obtains recombinant plasmid pDB.His.MBP-CABYV-MP.
4. purifying expression according to claim 1-3, which is characterized in that the His-tag- (CABYV- MP) prokaryotic expression of fusion protein is that the prokaryotic expression carrier that will be obtained in step (2) imports in Escherichia coli Rosseta, is obtained To recombinant bacterium Rosseta-CABYV-MP, Rosseta-CABYV-MP monoclonal, inoculated and cultured to OD are taken600nmValue for 0.6~ 0.8, the IPTG of final concentration of 0.1mM is added, 18 DEG C, 180rpm oscillation Fiber differentiation 16h, 4 DEG C, 5000rpm centrifugation 6min are received Collect bacterial sediment, supernatant is collected in ultrasonication after resuspension.
5. purifying expression according to claim 1-4, which is characterized in that the His-tag- (CABYV- MP) purifying of fusion protein is to elute the supernatant obtained in step (3) in conjunction with Ni affinity column, then with imidazole elution Albumen.
6. any one of -5 purifying expression CABYV-MP albumen obtained according to claim 1, which is characterized in that institute CABYV-MP albumen is stated with the amino acid sequence as shown in SEQ ID NO:4.
7. according to claim 1 any one of -5 purifying expression CABYV-MP albumen obtained prepare it is polyclonal Application in antibody, which is characterized in that the CABYV-MP albumen has the amino acid sequence as shown in SEQ ID NO:4.
8. one kind passes yellow viral (Cucurbit aphid-borne yellows virus, CABYV) detection for pumpkin aphid Kit, the kit includes: by any one of claim 1-5 purifying expression CABYV-MP egg obtained The white prepared polyclonal antibody obtained, the CABYV-MP albumen have the amino acid sequence as shown in SEQ ID NO:4.
CN201910661457.XA 2019-07-22 2019-07-22 A kind of pumpkin aphid passes the preparation of yellow viral movement protein purifying expression and its polyclonal antibody Pending CN110343708A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110885363A (en) * 2019-12-17 2020-03-17 中国农业大学 Expression and purification method of luffa aphid-borne yellowed virus motor protein and preparation of polyclonal antiserum thereof
CN110938645A (en) * 2019-12-17 2020-03-31 中国农业大学 Sugarcane yellow leaf virus motor protein expression and purification method and preparation of polyclonal antiserum thereof
CN115010793A (en) * 2022-06-17 2022-09-06 中国农业大学 Preparation method and application of rose-leaf malformation virus coat protein polyclonal antibody

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109164261A (en) * 2018-08-24 2019-01-08 中国农业大学 A kind of method and its special antibody detecting corium solani

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109164261A (en) * 2018-08-24 2019-01-08 中国农业大学 A kind of method and its special antibody detecting corium solani

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KWAK,H.-R.等: ""Cucurbit aphid-borne yellows virus isolate HS1, complete genome"", 《GENBANK DATABASE》 *
ZHANG SHAOKANG等: ""First report of Cucurbit aphid-borne yellows virus in passion fruit plants exhibiting mosaic and mottling in China."", 《PLANT DISEASE》 *
张绍康等: ""南瓜蚜传黄化病毒运动蛋白的原核表达、纯化和抗血清的制备"", 《中国植物病理学会2019年学术年会论文集》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110885363A (en) * 2019-12-17 2020-03-17 中国农业大学 Expression and purification method of luffa aphid-borne yellowed virus motor protein and preparation of polyclonal antiserum thereof
CN110938645A (en) * 2019-12-17 2020-03-31 中国农业大学 Sugarcane yellow leaf virus motor protein expression and purification method and preparation of polyclonal antiserum thereof
CN115010793A (en) * 2022-06-17 2022-09-06 中国农业大学 Preparation method and application of rose-leaf malformation virus coat protein polyclonal antibody

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