CN103045635A - Expression process of coat protein gene of prunus necrotic ring spot virus (PNRSV), antiserum and kit - Google Patents
Expression process of coat protein gene of prunus necrotic ring spot virus (PNRSV), antiserum and kit Download PDFInfo
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- CN103045635A CN103045635A CN2012102458513A CN201210245851A CN103045635A CN 103045635 A CN103045635 A CN 103045635A CN 2012102458513 A CN2012102458513 A CN 2012102458513A CN 201210245851 A CN201210245851 A CN 201210245851A CN 103045635 A CN103045635 A CN 103045635A
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- 239000012137 tryptone Substances 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an expression process of a coat protein gene of prunus necrotic ring spot virus (PNRSV), an antiserum and a kit. The expression method comprises the following steps of: extracting a ribose nucleic acid (RNA) of the PNRSV in an affected material; adopting a transcription-polymerase chain reaction (RT-PCR) amplification process to clone the coat protein gene of the PNRSV; connecting the obtained coat protein gene to a pGEM-T vector, obtaining a pGEM-T-PNRSV recombinant vector, and using sequence analysis to determine the sequence accuracy of the coat protein gene of the PNRSV; building an eukaryotic expression vector of the coat protein gene of the PNRSV; transforming a pichia pastoris GS115 strain, and inducing a pichia pastoris expression coat protein; and using Western-blot to detect the size accuracy of an expression proteine. The invention aims to provide the process for using the pichia pastoris to express the PNRSV coat protein gene, the antiserum, and a PNRSV ELISA detection kit.
Description
Technical field
The present invention relates to a kind of method with Pichia anomala expression rose mosaic virus coat protein gene, the invention still further relates to a kind of antiserum(antisera) that adopts this expression coat protein gene preparation; The invention still further relates to a kind of rose mosaic virus ELISA detection kit.
Background technology
Rose mosaic virus (Plum necrosis ringspot virus, PNRSV) be the quarantine pathogen of the inward plant of the up-to-date promulgation of China, one of dangerous maximum virus of tone fruit trees, fruit tree is infected by it in a single day, can cause that immature fruit comes off in a large number, the output degradation, and fruit quality is significantly descended.And this virus disseminating speed is fast, namely may give at short notice bringing on a disaster property of the fruit growing development loss of whole country.Only there is the report that partial area have this virus to occur in China at present.Because China follows the state basic policy of reform and opening-up; exchange more and more frequent with other country's germ plasm resource; in order to prevent that this virus from entering China along with the introduction of the reproductive materials such as seed, nursery stock, with health and the Sustainable development of protection China agricultural, must strengthen this viral inspection and quarantine.
Enzyme linked immunosorbent assay analysis method (ELISA) is the present detection method of sensitive higher, more stable and a kind of maturation that flux is higher, is the conventional sense of port plant seedling virus and to the prefered method of the rapid detection of a plurality of samples.But because this virus is very low at the fruit tree in-vivo content, the breeding difficulty, therefore this virus of will purifying in a large number and the antiserum(antisera) for preparing high-titer just become very difficult, and the front row that belong to quarantine virus in China of this virales, not being preferably each detection department provides this virus as positive control, with regard to having limited to a great extent this virus is carried out the serology detection like this.
Summary of the invention
The objective of the invention is in order to overcome weak point of the prior art, a kind of method with Pichia anomala expression rose mosaic virus coat protein gene is provided;
Another object of the present invention provides a kind of antiserum(antisera) that adopts this expressing gene preparation;
Another object of the present invention provides a kind of rose mosaic virus ELISA detection kit.
In order to achieve the above object, the present invention adopts following scheme:
A kind of rose mosaic virus coat protein gene expression method is characterized in that may further comprise the steps:
A, from the susceptible material of PNRSV, extract the RNA of rose mosaic virus;
B, the method that adopts RT-PCR to increase are cloned the coat protein gene of Prunus necrotic ring spot virus; The coat protein gene that obtains is connected on the pGEM-T carrier, obtains the pGEM-T-PNRSV recombinant vectors, and determine the sequence exactness of this virus coat protein gene with sequential analysis;
C, structure PNRSV coat protein gene carrier for expression of eukaryon;
D, conversion Pichia pastoris GS115 bacterial strain are induced the Pichia anomala expression coat protein;
E, detect the exactness of expressing protein size with Western-blot.
Aforesaid a kind of rose mosaic virus coat protein gene expression method is characterized in that the RNA that extracts rose mosaic virus in the steps A specifically may further comprise the steps:
(1) get the tender tissue of the susceptible material of PNRSV some, liquid nitrogen grinding, be distributed into＜the 100mg aliquot is to precooling E.P pipe, and get the 100mg sample and add the 1mlTriZOL extracting;
(2) 12000rpm, 2-8 ℃ of centrifugal 10min;
(3) draw supernatant liquor, be transferred in the new 1.5mlE.P pipe, place 5min for 15-30 ℃;
(4) add the 0.2ml chloroform, fully extracting 8min places 2-3min for 15-30 ℃;
(5) 12000rpm, 2-8 ℃ of centrifugal 15min;
(6) draw supernatant liquor, be transferred in the new 1.5mlE.P. pipe, add the 0.5ml Virahol, place 10min for 15-30 ℃;
(7) 12000rpm, 2-8 ℃ of centrifugal 10min;
(8) abandoning supernatant gets colloidal RNA, adds 1-1.5ml75% ethanol, the vortex mixing
(9) 7500rpm, 2-8 ℃ of centrifugal 5min, abandoning supernatant stays colloidal RNA;
(10) dry 5-10min;
(11) be dissolved in the Rnase.free water, the rifle head is beaten even, and 55-60 ℃ of temperature bathed 10min;
(12) 5%Agarose glue detects, and measures concentration and transfers to 2-3 μ g/ μ L, and-70 ℃ store for future use.
Aforesaid a kind of rose mosaic virus coat protein gene expression method is characterized in that step B concrete grammar comprises:
A, RT-PCR amplifying target genes:
(1) primer
Upstream primer: PNRSV-F:5 '-GT
GGATCCTACGTAATGGTTTGCCGAATTTG-3 ', wherein
GGATCCBe the BamHI restriction enzyme site, TAC GTA is SnaB I restriction enzyme site;
Downstream primer: PNRSV-R:5 '-GG
AAGCTTGCGGCCGCGATCTCAAGCAGGTC-3 ', wherein
AAGCTTBe Hind III restriction enzyme site, GCGGCCGC is Not I restriction enzyme site;
Namely 5 ' and 3 ' SnaB I and NotI restriction enzyme site primer have been added respectively at the primer two ends;
(2) amplification system: template ribonucleic acid 1 μ L; 5 μ mol/L upstream primers, 1 μ L; 5
μ mol/L downstream primer 1 μ L; Amplification enzyme mixation 1 μ L; 2 * buffer, 12.5 μ L; RNase-free ddH
2O is to cumulative volume 25 μ L;
(3) amplification procedure: 50 ℃ of 30min, 94 ℃ of 3min, 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 1min, 72 ℃ of 10min, 4 ℃; 94 ℃ of 1min wherein, 60 ℃ of 30s, 2 ℃ of 1min, 72 ℃ of 10min circulations 35 times;
B, gene clone and sequence order-checking:
(1) recovery of PNRSV CP product
Get amplified production electrophoresis on the 1%TBE sepharose, electrophoresis is complete, downcuts the purpose band, adopts the PCR product to reclaim test kit and reclaims;
(2) structure of recombinant plasmid
Adopt the pGEM-T carrier to carry out the TA clone, construction recombination plasmid, concrete steps are: electrophoresis determines to reclaim purity and the concentration of product; In the PCR pipe, add successively approximately 4 μ L recovery product, 1 μ LpGEM-T carrier, 5 μ L connecting fluids are replenished ddH
2O is to cumulative volume 10 μ L; 16 ℃ of lower reactions are spent the night;
(3) competent cell preparation
Activation DH5 α bacterial strain on the LB culture medium flat plate, and picking list colony inoculation is in the 5mlLB liquid nutrient medium, 37 ℃ of lower wave and culture spend the night, and form kind of a daughter bacteria liquid; Get this 5ml bacterium liquid, in 100ml LB liquid nutrient medium, 37 ℃ of wave and culture 2～2.5hr are to OD
600=0.6～0.8 o'clock, draw 1.5ml bacterium liquid in the 1.5ml centrifuge tube, ice bath 5min, 4 ℃ of centrifugal 10min of lower 4000g abandon supernatant liquor, repeat to draw bacterium liquid once, and precipitation exhausts raffinate with sterilization filter paper, adds the 100mmol/L CaCl of 300 μ L ice precooling
2The resuspension thalline, ice bath 30min, 4 ℃ of centrifugal 10min of lower 4000g, collecting precipitation adds the 100mmol/L CaCl that 100 μ L ice precooling
2The Eddy diffusion bacterial precipitation is competent cell, places 12～24hr for subsequent use in 4 ℃ of refrigerators; Or add the 100mmol/LCaCl of 70 μ L ice precooling
2Save backup in-70 ℃ with 30 μ L50% glycerine;
(4) conversion of recombinant plasmid and screening
From-70 ℃ of refrigerators, take out competent cell, place on ice and thaw, on Bechtop, get 10 μ L and connect product in 100 μ L competent cells, flick the tube wall mixing, ice bath 30min, centrifuge tube is placed 42 ℃ of water-bath heat shock 90sec, rapidly with centrifuge tube ice bath 3～5min.Add 800 μ LLB liquid nutrient mediums (not containing microbiotic) in every pipe, 37 ℃ of lower wave and culture 90min are beneficial to the recovery of bacterium and the expression of plasmid-encoded resistant gene;
Adopt microbiotic and α-complementary method screening recombinant plasmid; Get 100ml LB solid medium heating and melting, to be cooled to below 60 ℃ the time, add 100 μ LAmp, pour in separately 4 culture dish behind the mixing; After solidifying, every ware is got 4 μ LIPTG, even spread plate behind the filtration sterilization of the 200mg/ml aqueous solution and the 40 μ LX-gal mixings; The bacterium liquid of getting after liquid is absorbed after 200 μ l transform evenly is applied on the above-mentioned LB flat board, and forward is placed 30min and treated that liquid is absorbed, and 37 ℃ of lower inversions are cultivated 16～24hr; The picking diameter is about the white colony of 1～2mm size in 5ml LB liquid nutrient medium, 37 ℃ of lower wave and culture 12～16hr, after getting the sterile glycerol mixing of 0.7ml and 0.3ml 50% behind the mark, put under-80 ℃ and save backup, all the other bacterium liquid are used for recombinant plasmid extracting and evaluation;
(5) extracting of recombinant plasmid
Screen the bacterium colony culture to be checked precooling 5～10min in 4 ℃ of refrigerators that obtains through microbiotic and α-complementary method, adopt alkaline lysis extracting plasmid DNA; Concrete steps are as follows: the centrifuge tube label, get contain recombinant plasmid thalline liquid 1.5ml to centrifuge tube, 4 ℃, 12000g, centrifugal 1min, abandon supernatant, repeat to add thalline liquid once, pipe is inverted on the thieving paper after centrifugal, after flowing to end remaining liquid, add 150 μ L solution I, thermal agitation suspends thalline, and room temperature is placed 5～10min, add 250 μ L solution II, put upside down centrifuge tube for several times, gentle mixing, room temperature is placed 5min, add 180 μ L solution III, cover tightly centrifuge tube, gentleness is put upside down centrifuge tube for several times, makes the precipitation mixing, ice bath 6～7min, 4 ℃, the centrifugal 10min of 12000g, supernatant moves in the clean centrifuge tube, adds respectively the chloroform/primary isoamyl alcohol of 1/2 volume, the saturated phenol of Tris, mixing, 4 ℃, the centrifugal 5min of 12000g gets supernatant, and extracting is once again for chloroform/primary isoamyl alcohol; Supernatant water moves in the clean centrifuge tube mutually, adds 1/10 volume NaAc, 2 times of volume dehydrated alcohols, behind the mixing, 20min or spend the night in-20 ℃ of refrigerators, 4 ℃, the centrifugal 10min of 12000g abandon supernatant, the mouth of pipe opened be inverted on the thieving paper, blot remaining liquid, 70%, 100% ethanol is respectively washed once, and is air-dry under the room temperature, add 20 μ LTE, for subsequent use in-20 ℃ of refrigerators;
(6) evaluation of recombinant plasmid
1., specific primer PCR is identified
Utilize special primer to treat check weighing group plasmid and carry out pcr amplification, the reaction system program parameter is the same, and the PCR product is the electrophoresis evaluation on the 1%TBE sepharose, and all clones who contains the purpose amplified fragments further carry out enzyme and cut evaluation;
2., the recombinant plasmid enzyme is cut evaluation
Identify positive recombinant plasmid through PCR, carry out double digestion with following system: 10 * HBuffer1.5 μ L, BamH10.5 μ L, HindIII0.5 μ L, plasmid 4 μ L to be checked add ddH
2O is to cumulative volume 15 μ L, and 37 ℃ of lower reactions are spent the night, and get 10 μ L reaction solution electrophoresis detection enzymes and cut the result;
3., recombinant plasmid sequencing
Cut the clone who is accredited as the positive through above-mentioned PCR and enzyme, expand numerous thalline, behind extracting and the plasmid purification, utilize the universal sequencing primer thing that recombinant plasmid is carried out sequencing.
Aforesaid a kind of rose mosaic virus coat protein gene expression method is characterized in that the specific practice of step C:
Go out the PAP gene with pcr amplification, then use SnaB I and Not I double digestion after reclaiming this gene fragment, be connected with carrier pPIC9K with same double digestion again and be built into Expression vector pPIC9K-P, pPIC9K-P is transformed e. coli jm109 to be screened by screening culture medium, alkaline process extracts its recombinant plasmid, then cuts with enzyme and PCR identifies; Behind the positive recombinant that filters out with PCR and plasmid enzyme restriction authentication method, carry out again dna sequencing, with the exactness of checking reading frame.
Aforesaid a kind of rose mosaic virus coat protein gene expression method is characterized in that the specific practice of step D:
After the sequential analysis checking, use again enzyme BglII with Expression vector pPIC9K-P linearizing, then by the electrotransfer method it is imported the competent cell of yeast GS115 bacterial strain;
(1) preparation yeast competent cell:
Yeast strain P.pastoris GS115 in 500ml YPD 30 ℃ be cultured to A 600=1.5, the centrifugal collection thalline of 1500g, successively use the aseptic washing thalline of 500ml, 250ml precooling, the centrifugal supernatant of going, with the 1mol/L sorbyl alcohol suspension thalline of 20ml precooling, centrifugal rear thalline suspends rear for subsequent use with the sorbyl alcohol of 0.5ml precooling again;
(2) preparation transfering DNA: alkaline lysis is adopted in the extraction of plasmid DNA, then the DNA that extracts is cut with the BglII enzyme, makes it to be divided into two fragments, and one of them is the DNA section that contains the yeast expression box, and it is reclaimed after with the low melting-point agarose electrophoresis;
(3) conversion of yeast cell
1～5 μ gDNA that gets the above-mentioned steps preparation is added in the yeast cell of 40 μ l sorbyl alcohols suspension, ice bath 5min, and then with the electric shock instrument electric shock, parameter is: 0.8kV, 11.5 μ F; The 1mol/L sorbyl alcohol that adds immediately the 0.5ml precooling after electric shock finishes, getting 200 μ l is RDB:18.6% sorbyl alcohol, 2% glucose, 1.34%YNB, 0.00004% vitamin H, each 0.005% L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine, 2% agarose in solid RDB conversion substratum, upper coated plate is cultivated until transformant occurs for 30 ℃; Arrive MM:1.34%YNB, 0.00004% vitamin H, 0.5% methyl alcohol, 1.5% agarose with the corresponding dibbling of toothpick picking transformant, on flat board and MD:1.34%YNB, 0.00004% vitamin H, 2% glucose, 1.5% agarose, cultivate 2d for 30 ℃, normally be positive colony at MM growth transformant undesired or that do not grow in the MD growth;
(4) abduction delivering of PNRSV CP gene in yeast
Concrete operations are undertaken by Invitrogen company Pichia Expression Kit program.
Aforesaid a kind of rose mosaic virus coat protein gene expression method is characterized in that the specific practice of step e:
(1) mensuration of expressing protein molecular weight
Get the 1ml yeast fermentation broth, centrifugal 15 minutes of 13000rpm gets supernatant 10 μ l and carries out SDS-PAGE, dyes with Coomassie brilliant blue behind the electrophoresis;
(2) expressing protein Western-blot detects
Behind the SDS-PAGE electrophoresis with the expressing protein electrotransfer to nitrocellulose filter, then carry out immune response with the PNRSV antiserum(antisera) as the albumen on primary antibodie and the nitrocellulose filter, be that the goat-anti rabbit anti-serum of alkali phosphatase enzyme mark carries out specificity identification to expressing protein with the second antiserum(antisera) more afterwards, with the yeast culture liquid of not using methanol induction as negative control.
A kind of antiserum(antisera) of the present invention, it is characterized in that its preparation method may further comprise the steps: according to pre-expression result, choose the Pichia pastoris GS115 bacterial strain of high efficient expression, a large amount of abduction deliverings, through SDS-PAGE and coomassie brilliant blue staining analysis, cut the adhesive tape band and suitably after the dilution, add the emulsification of equal-volume freund 's incomplete adjuvant with physiological saline; Protein liquid 1mg/mL directly and freund 's incomplete adjuvant carry out emulsification; The immunity White Rabbit adopts intramuscular injection in conjunction with hypodermic mode, after for the first time immunity, respectively at the 21st day, the 35th day and the 49th day booster immunization 3 times; Begin a small amount of venous blood collection behind the last injecting immune in 7 days and measure and tire, measure greatly blood according to the situation gradation of tiring; The antiserum(antisera) of preparation adds 0.02% sodiumazide ,-20 ℃ of preservations.
Antiserum titre and specific assay:
After antiserum(antisera) carried out a series of dilutions, measure sero-fast tiring and specificity with indirect ELISA method.
(1). with coating buffer antigen diluent is become 2ug/mL, the 100ul/ hole is added in the removable enzyme plate in 96 holes, and 4 ℃ are spent the night.(2) .PBS-T washes three inferior to patting dry on the thieving paper, adds 200ul/ hole 1%BSA, 37 ℃ of thermostat containers 2 hours.(3). get antiserum(antisera) according to 1: 1000,1: 2000,1: 4000,1: 8000,1; 16000,1: 32000,1: 64000,1: 128000 make gradient dilution, and each sample is not less than 200ul.(4). take out the antigen plate in 37 ℃ of thermostat containers, PBS-T washes three inferior to patting dry on the thieving paper, adds the good antiserum(antisera) sample 100ul/ hole of dilution in the step 3, and each extent of dilution adds side by side two holes.(5). get negative serum 1: 1000 dilution and be added in order behind the antiserum(antisera) Cmin sample as negative control.Put 37 ℃ of thermostat containers 1 hour.(also will repeat 2 times) (6) .PBS-T washes three inferior to patting dry on the thieving paper, and goat-anti rabbit enzyme mark (horseradish peroxidase) two is anti-to be diluted according to operation instruction, and the 100ul/ hole is added in the enzyme plate, puts 37 ℃ of thermostat containers 1 hour.(7) .PBS-T washes five inferior to patting dry on the thieving paper, and TMB nitrite ion 100ul/ hole is added in the enzyme plate, puts 37 ℃ of thermostat containers 10 minutes.Microplate reader 450nm surveys OD value and print result.(8). usually regard 2.5 times of negative control OD values as the positive, according to judgement sample titre as a result.(9). select optimal working concentration, carry out immune response with viruses such as TMV, PVX, PVY respectively, to test the specificity of this serum.
A kind of test kit of the present invention is characterized in that comprising in this test kit: 1) .PNRSV antibody; 2). enzyme labelled antibody: alkaline phosphate ester enzyme labelling; 3). substrate: 4-NPP (ρ NPP); 4) .PNRSV positive control; 5) .PNRSV negative control.
Material, main agents, instrument and solution etc. among the present invention:
1, the susceptible material of PNRSV is provided by biotechnology center Molecular Biology Lab of Spain country, preserves in-80 ℃ of Ultralow Temperature Freezers.
2, main agents: Trizol reagent, Taq enzyme, M-MLV ThermoScript II, T4 ligase enzyme and methyl alcohol, sorbyl alcohol, yeast nitrogen base etc. are all available from Guangzhou Pu Bo biotech company; Restriction enzyme is all available from the precious biotech firm in Dalian; PPIC9K carrier, Pichia pastoris GS115 bacterial strain are available from Invitrogen company.
3, key instrument
BIOTEK enzyme connection detector, Japanese grads PCR instrument, the freezing high speed centrifugation of Labofuge400R type, high speed tabletop centrifuge, DYY-7 type electrophoretic blotting instrument, Pharmacia EPS601 type electrophoresis apparatus; France UVP gel imaging system; The Japan Shimadzu UV-120-02 of company type is visible-ultraviolet spectrophotometer; HARRIS ultralow temperature ice Sanyo board, the gloomy board pipettor of French gill; Single type Bechtop; Growth cabinet LRH-250-GS, water isolation type electro-heating standing-temperature cultivator PYX-DHS, HZQ-C airbath vibrator, electric-heated thermostatic water bath DK-S22 etc.
4, the preparation of main solution: (1) PBS damping fluid: among every 1000mL, contain 2.2gNa
2HP0
4.12H
2O, 0.2g KH
2P0
4, 8.0g NaCl, 0.2g KCl, 0.2g NaN
3, regulate pH to 7.3; (2) PBST: add 0.5mL Tween-20 among every 1000mL PBS.(3) frictional inoculation damping fluid: the α-benzoinoxime of adding 1% among the PBS.(4) the 0.1%DEPC aqueous solution: after adding 1mLDEPC in the 1000mL water, 37 ℃ of lower 24hr, autoclaving, Seal and preservations processed.(5) RNA Extraction buffer: the 4mol/L guanidinium isothiocyanate, the 25mmol/L Trisodium Citrate, 0.5% lauryl creatine acid is received (w/v), 0.1mol/L mercaptoethanol, 2mol/LNaAc, water-saturated phenol (pH3.5).(6)10×MOPS：0.2mol/L?MOPS(pH7.0)，10mmol/L?EDTA(pH8.0)，80mmol/L?NaAc。(7) LB substratum: Tryptones (Tryptone) 10g/L, yeast leach liquor (Yeast Extract) 5g/L, sodium-chlor (NaCl) 10g/L, pH 7.0～7.2, and solid medium adds 1.5% (w/v) agar powder.(8) plasmid extraction damping fluid: solution I: 50mmol/L glucose, 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA (pH8.0), autoclaving after the packing is in 4 ℃ of lower preservations; Solution II: 0.2mol/L NaOH, 1%SDS (w/v), now with the current with 4mol/L NaOH and 20% (w/v) SDS stock solution; Solution III: add 11.5mL glacial acetic acid and 28.5mL water in the potassium acetate solution of 60mL 5mol/L, being made into concentration is 3mol/L (pH5.3), and potassium acetate concentration is the solution of 5mol/L.(9) 10% (w/v) SDS solution (100mL): SDS 10g adds ddH
2O is to final volume 100mL.Room temperature preservation (guaranteeing not precipitation of SDS before the use).(10) the antigen coated damping fluid of related reagent A. of enzyme linked immunosorbent assay (ELISA) (0.05mol/L pH 9.6 carbonate buffer solutions): contain 1.59g Na among every 1000mL
2CO
3, 2.93gNaHCO
3, 0.2g NaN
3, regulate pH value to 9.6; B. lavation buffer solution (0.02mol/L pH7.3PBST damping fluid): get 8.0g NaCl, 0.2g KCl, 2.9g Na
2HPO
4, 0.2gKH
2P0
4, 0.5mL tween 20 (Tween-20), distilled water is settled to 1000mL, regulates pH to 7.3; C.IgG dilution buffer liquid (0.02mol/L PBST bovine serum albumin damping fluid): 0.1g bovine serum albumin (BSA) is dissolved in the 100mL washings; D. phosphate-citrate salts damping fluid (pH5.0): 0.2mol/L Na
2HPO
425.7mL, 0.1mol/L citric acid solution 24.3mL, adding distil water is settled to 100mL; E. substrate solution (OPD-H
2O
2): this solution matching while using, get phosphoric acid salt-citrate buffer solution 100mL, add 40mg (can tune to 16mg with the temperature rising) O-Phenylene Diamine (OPD), after lucifuge is fully dissolved, add the H of 0.15mL (can tune to 0.07mL with the temperature rising) 30%
2O
2F. stop buffer: 2mol/LH
2SO
4(11) penbritin (Amp): with the distilled water dissolving, be diluted to and store concentration 100mg/mL ,-20 ℃ of filtration sterilization postposition are for subsequent use; (12) chloroform: primary isoamyl alcohol (24: 1): get chloroform 48mL, primary isoamyl alcohol 2mL, fully behind the mixing, preserve in the brown bottle.(13) TE: contain 10mmol/LTris-C1,1mmol/L EDTA, adjust pH to 8.0; (14) EB mother liquor (10mg/mL): take by weighing 10mg EB and be dissolved in the 1mL distilled water, keep in Dark Place, working concentration is 0.5 μ g/mL.(15) 5 * TBE mother liquors: take by weighing 54g Tris base, 27.5g boric acid, 0.5mol/LEDTA (PH 8.0) 20mL, distilled water is settled to 1000mL.Diluting 10 times is 0.5 * TBE working fluid.
Technical solution of the present invention: the 1. total nucleic acid of extracting rose mosaic virus; 2. take total nucleic acid as template, be primer with Oligo (dT), reverse transcription becomes cDNA the first chain; 3. design the Auele Specific Primer of rose mosaic virus coat protein gene, take cDNA the first chain as template, the pcr amplification coat protein gene; With coat protein gene cloning to the pGEM-T carrier, then carry out sequencing, with the conclusive evidence gene order exactness; 5. from the pGEM-T carrier, coat protein gene is downcut, be cloned on the carrier for expression of eukaryon pPIC9K, transform the Pichia pastoris GS115 bacterial strain, then induce the Pichia anomala expression coat protein; 6. detect the exactness of expressing protein size with Western-blot; 7. then the great expression coat protein reclaims, immunizing rabbit; 8. extract blood in the immunizing rabbit body, detect it and whether produce antiserum(antisera) and specificity thereof; 9. measure sero-fast tiring, the preparation enzyme-linked immunologic detecting kit, and in actual testing, checked.
Modern molecular biology, Molecular Virology and immunology scheduling theory and technology have been merged in the present invention, traditional production antigen and the method for antibody have been avoided, adopt molecular biology method, utilize Eukaryotic breeding to come the coat protein that great expression should virus, this expressing protein can think that the serology detection provides positive control, can be used as again antigen and produce antiserum(antisera), offer the especially detection use of sanitary authority of detection department.Simultaneously since coat protein gene cloning that should virus on expression vector, therefore can produce continuously coat protein as the usefulness of positive control, avoid simultaneously this virus of direct usefulness or had the danger of viral escape by the host plant of this virus infection, thereby fully guaranteed sero-fast supply and biological safety problem in the testing.
Description of drawings
Fig. 1 is PNRSV recombinant plasmid PCR qualification result figure; Fig. 2 is the structure schematic diagram of eucaryon secreted expression carrier pPIC9K-PNRSV; Fig. 3 carrier for expression of eukaryon pPIC9K-PNRSV PCR and double digestion qualification result figure; Fig. 4 is yeast expression protein SDS-PAGE analytical results figure; Fig. 5 expressing protein Western-blotting specific detection is figure as a result; Fig. 6 reclaims the SDS-PAGE analytical results figure of PNRSVCP albumen.
Wherein 2,3,5, No. 6 transformants have PNRSV CP gene fragment to insert among Fig. 1." M " expression 100bp ladder DNA Marker. " → " is designated as PNRSV CP fragment (675bp).1. molecular weight contrast among Fig. 3,2. double digestion, 3.PCR identifies.1.Marker among Fig. 4 (Ku): 15,20,31,43,66.2; 2. expressing protein, 3. negative control; Among Fig. 51: the expression product of methanol induction; 2: negative control; 1:M among Fig. 6: protein molecular weight contrast (150kDa, 100kDa, 75kDa, 50kDa, 31kDa, 20kDa, 15kDa) 2:E1-E8: induction expression protein product
Embodiment
Below in conjunction with embodiment the present invention is described further:
1, the preservation of viral sample and evaluation
Preserve: mainly adopt two kinds of methods ,-be after choosing symptom typical blade sample accelerated freeze-drying, be stored in-80 ℃ of refrigerators; The 2nd, the total DNA of extracting diseased plant and RNA also are stored in-80 ℃ of refrigerators.Identify: the sample of collection adopts first serological method, mainly is that ELISA detects, and according to results of serological detection, proves conclusively with modern molecular biology method such as PCR detection method again.
2, the Serology test of PNRSV: adopt indirect ELISA method to carry out, step is as follows: (1) gets testing sample 0.1g, smashs to pieces with pestle, adds the antigen coated damping fluid of 1mL, changes in the 1.5mL centrifuge tube, and 2700g is centrifugal, gets supernatant liquid; (2) add 200 μ L supernatant liquid in the elisa plate sample well with envelope antigen, under 37 ℃, hatch 2hr; (3) outwell coating buffer, wash plate 3 times with PBST, each 3min; (4) add PNRSV specific IgG, every hole 150uL, 37 ℃, incubation 2hr; (5) outwell this solution, wash plate the same; (6) add enzyme mark goat anti-rabbit igg, every hole 100 μ L (working concentration 1: 3000), 37 ℃ of lower incubation 2hr; (7) outwell this solution, wash plate the same; (8) add substrate solution TMB, every hole 100 μ l, behind the room temperature 30min, every hole adds the H of 50 μ L 2mol/L
2SO
4Termination reaction; (9) microplate reader is measured OD
450NM, observational data colour developing situation, triplicate.Annotate: when doing the ELISA detection, each sample repeats 2 times at least, and positive control, negative control and blank need to be set; And developing time can not be long.
3, method for extracting total RNA:
(1) tender tissue is some, liquid nitrogen grinding, be distributed into＜the 100mg aliquot is to precooling E.P. pipe, and the 100mg sample adds 1mL TriZOL extracting several minutes (putting upside down mixing);
(2) 12000rpm, 2-8 ℃ of centrifugal 10min;
(3) draw supernatant (containing RNA), be transferred in the new 1.5mLE.P. pipe, place 5min (can place again extracting in January in-70 ℃ before this) for 15-30 ℃;
(4) (1/5 * VOl.TriZOL) chloroform, abundant extracting 8min place 2-3min for 15-30 ℃ to add 0.2mL;
(5) 12000rpm, 2-8 ℃ of centrifugal 15min (form the colourless water in upper strata, middle mutually with the lower floor organic phase, middlely can be used for DNA and protein extracting with organic phase mutually);
(6) draw supernatant, be transferred in the new 1.5mL E.P. pipe, (1/2 * VOl.TriZOL) Virahol is placed 10min for 15-30 ℃ to add 0.5mL;
(7) 12000rpm, 2-8 ℃ of centrifugal 10min;
(8) careful supernatant discarded gets colloidal RNA, adds 1-1.5mL (＞=1 * VOl.TriZOL) 75% ethanol, vortex mixing (in 75% ethanol, the RNA precipitation can be placed two weeks ,-70 ℃ in 2-8 ℃ and be placed 1 year);
(9) 7500rpm, 2-8 ℃ of centrifugal 5min, supernatant discarded stays colloidal RNA;
(10) dry 5-10min (overdrying can make RNA be difficult to dissolving);
(11) be dissolved in the RNase.free water, the rifle head is beaten even, and 55-60 ℃ of temperature bathed 10min;
(12) 5%Agarose glue detects, and measures concentration and transfers to 2-3 μ g/ μ L.-70 ℃ store for future use.
4, RT-PCR amplifying target genes:
(1) design of primers is synthetic
According to the PNRSV CP gene conserved sequence design of having measured, upstream primer: PNRSV-F:5 '-GT
GGATCCTACGTA ATGGTTTGCCGAATTTG-3 ', wherein
GGATCC isBamHI restriction enzyme site, TAC GTA are SnaB I restriction enzyme site;
Downstream primer: PNRSV-R:5 '-GG
AAGCTTGCGGCCGCGATCTCAAGCAGGTC-3 ', wherein
AAGCTTBe Hind III restriction enzyme site, GCGGCCGC is Not I restriction enzyme site; Namely 5 ' and 3 ' SnaB I and Not I restriction enzyme site primer have been added respectively at the primer two ends; Primer is synthetic by Beijing AudioCodes biotech firm.
(2) Amplification system
Adopt the test kit PrimeScript of the precious biotech firm in Dalian
TMOne-Step Ver.2.0 carries out single stage method RT-PCR amplification.5 μ L electrophoresis on 1% sepharose is got in 4 ℃ of preservations of PCR product, checks PCR result.
(2) amplification system: template ribonucleic acid 1 μ L; 5 μ mol/L upstream primers, 1 μ L; 5 μ mol/L downstream primers, 1 μ L; Amplification enzyme mixation 1 μ L; 2 * buffer, 12.5 μ L; RNase-free ddH
2O is to cumulative volume 25 μ L;
(3) amplification procedure: 50 ℃ of 30min, 94 ℃ of 3min, 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 1min, 72 ℃ of 10min, 4 ℃; 94 ℃ of 1min wherein, 60 ℃ of 30s, 2 ℃ of 1min, 72 ℃ of 10min circulations 35 times;
5, gene clone and sequence order-checking:
(1) recovery of PNRSV CP product
Get amplified production electrophoresis on the 1%TBE sepharose, electrophoresis is complete, downcuts the purpose band, adopts the PCR product to reclaim test kit and reclaims.Concrete steps reference reagent box explanation: get a 1.5mL centrifuge tube and weigh at electronic balance, downcut the as far as possible little gel piece of volume to centrifuge tube, weigh, add 300 μ L Buffer DE-A by every 100mg glue, behind the suspension mixing in 75 ℃ of heating in water bath, mix once every 2～3min, until gel piece melts fully, press 50% of Buffer DE-A volume and add Buffer DE-B, mix, the DNA-prep post places the 2mL centrifuge tube, and with in the above-mentioned mixed solution immigration DNA-prep post, the centrifugal 1min of 3600g, abandon filtrate, the DNA-prep post put get back in the former 2mL centrifuge tube, add 500 μ LBuffer W1, the centrifugal 1min of 3600g, abandon filtrate, the DNA-prep post put get back in the former 2mL centrifuge tube, add 700 μ L and added the Buffer W2 of dehydrated alcohol, the centrifugal 1min of 3600g, with same method again with 700 μ L Buffer W2 washing once, the DNA-prep post is placed the 1.5mL centrifuge tube, the centrifugal 1min of 12000g, the DNA-prep post is placed another clean 1.5mL centrifuge tube, add Eluent or the deionized water of 65 ℃ of preheatings of 25～30 μ L in silica film central authorities, room temperature leaves standstill 1min, the centrifugal 1min of 12000g, eluted dna ,-20 ℃ save backup.
(2) structure of recombinant plasmid
Adopt the pGEM-T carrier to carry out TA clone, construction recombination plasmid.The ligation method is undertaken by the method that the manufacturer recommends, and concrete steps are: electrophoresis determines to reclaim purity and the concentration of product; In the PCR pipe, add successively approximately 4 μ L recovery product, 1 μ LpGEM-T carrier, 5 μ L connecting fluids are replenished ddH
2O is to cumulative volume 10 μ L; 16 ℃ of lower reactions are spent the night.
(3) competent cell preparation
The colibacillary preparation of competence is with reference to the Calcium Chloride Method of (1989) such as Sambrook.
Concrete steps are: activation DH5 α bacterial strain on the LB culture medium flat plate, and picking list colony inoculation is in 5mL LB liquid nutrient medium, 37 ℃ of lower wave and culture spend the night (approximately 12～16hr), form kind of a daughter bacteria liquid.Get this 5mL bacterium liquid, in the 100mLLB liquid nutrient medium, 37 ℃ of wave and culture 2～2.5hr are to OD
600During=0.6～0.8 left and right sides, draw 1.5mL bacterium liquid in the 1.5mL centrifuge tube, ice bath 5min, 4 ℃ of centrifugal 10min of lower 4000g abandon supernatant liquor, repeat to draw bacterium liquid once, and precipitation exhausts raffinate with sterilization filter paper, adds the 100mmol/L CaCl of 300 μ L ice precooling
2The resuspension thalline, ice bath 30min, 4 ℃ of centrifugal 10min of lower 4000g, collecting precipitation adds the 100mmol/L CaCl that 100 μ L ice precooling
2The Eddy diffusion bacterial precipitation is competent cell, places 12～24hr for subsequent use in 4 ℃ of refrigerators; Or add the 100mmol/L CaCl of 70 μ L ice precooling
2Save backup in one 70 ℃ with 30 μ L50% glycerine.
(4) conversion of recombinant plasmid and screening
From-70 ℃ of refrigerators, take out competent cell, place on ice and thaw, on Bechtop, get 10 μ L and connect product in 100 μ L competent cells, flick the tube wall mixing, ice bath 30min, centrifuge tube is placed 42 ℃ of water-bath heat shock 90sec, rapidly with centrifuge tube ice bath 3～5min.Add 800 μ L LB liquid nutrient mediums (not containing microbiotic) in every pipe, 37 ℃ of lower wave and culture 90min are beneficial to the recovery of bacterium and the expression of plasmid-encoded resistant gene.
Adopt microbiotic and α-complementary method (Sambrook, et al., 1989) screening recombinant plasmid.Get 100mL LB solid medium heating and melting, to be cooled to below 60 ℃ the time, add 100 μ LAmp, pour in separately 4 culture dish behind the mixing; After solidifying, every ware is got behind 4 μ LIPTG (filtration sterilization of the 200mg/mL aqueous solution) and the 40 μ LX-gal mixings evenly spread plate; The bacterium liquid of getting after liquid is absorbed after 200 μ L transform evenly is applied on the above-mentioned LB flat board, and forward is placed 30min and treated that liquid is absorbed, and 37 ℃ of lower inversions are cultivated 16～24hr.The white colony that the picking diameter is about 1～2mm size (contains 100 μ g/mLAmp) in 5mL LB liquid nutrient medium 37 ℃ of lower wave and culture 12～16hr, after getting the sterile glycerol mixing of 0.7mL and 0.3mL 50% (v/v) behind the mark, put under-80 ℃ and save backup, all the other bacterium liquid are used for recombinant plasmid extracting and evaluation.
(5) extracting of recombinant plasmid
Screen the bacterium colony culture to be checked precooling 5～10min in 4 ℃ of refrigerators that obtains through microbiotic and α-complementary method, adopt alkaline lysis extracting plasmid DNA (Sambrook, et al., 1989).Concrete steps are as follows: the centrifuge tube label, get contain recombinant plasmid thalline liquid 1.5mL to centrifuge tube, 4 ℃, 12000g, centrifugal 1min, abandon supernatant, repeat to add thalline liquid once, pipe is inverted on the thieving paper after centrifugal, after flowing to end remaining liquid, add 150 μ L solution I, thermal agitation suspends thalline, and room temperature is placed 5～10min, add 250 μ L solution II, put upside down centrifuge tube for several times, gentle mixing, room temperature is placed 5min, add 180 μ L solution III, cover tightly centrifuge tube, gentleness is put upside down centrifuge tube for several times, makes the precipitation mixing, ice bath 6～7min, 4 ℃, the centrifugal 10min of 12000g, supernatant moves in the clean centrifuge tube, adds respectively the chloroform/primary isoamyl alcohol of 1/2 volume, the saturated phenol of Tris, mixing, 4 ℃, the centrifugal 5min of 12000g gets supernatant, and extracting is once again for chloroform/primary isoamyl alcohol.Supernatant water moves in the clean centrifuge tube mutually, adds 1/10 volume NaAc, 2 times of volume dehydrated alcohols, behind the mixing, 20min or spend the night in-20 ℃ of refrigerators, 4 ℃, the centrifugal 10min of 12000g abandon supernatant, the mouth of pipe opened be inverted on the thieving paper, blot remaining liquid, 70%, 100% ethanol is respectively washed once, and is air-dry under the room temperature, add 20 μ L TE, for subsequent use in-20 ℃ of refrigerators.
(6) evaluation of recombinant plasmid
A, specific primer PCR are identified
Utilize special primer to treat check weighing group plasmid and carry out pcr amplification.The reaction system program parameter is the same, and PCR product electrophoresis on the 1%TBE sepharose is identified.All clones who contains the purpose amplified fragments further carry out enzyme and cut evaluation.
B, recombinant plasmid enzyme are cut evaluation
Identify positive recombinant plasmid through PCR, carry out double digestion with following system: 10 * HBuffer1.5 μ L, BamH10.5 μ L (14U/ μ L), HindIII0.5 μ L (14U/ μ L), plasmid 4 μ L to be checked (approximately 400ng) add ddH
2O is to cumulative volume 15 μ L, and 37 ℃ of lower reactions are spent the night, and get 10 μ L reaction solution electrophoresis detection enzymes and cut the result.
C, recombinant plasmid sequencing
Cut the clone who is accredited as the positive through above-mentioned PCR and enzyme, expand numerous thalline, behind extracting and the plasmid purification, utilize the universal sequencing primer thing that recombinant plasmid is carried out sequencing, company limited carries out determined dna sequence by precious biotechnology (Dalian).
6, the structure of PNRSV CP gene eukaryotic expression vector:
Go out the PAP gene with pcr amplification, then use SnaB I and Not I double digestion after reclaiming this gene fragment, be connected with carrier pPIC9K with same double digestion again and be built into Expression vector pPIC9K-P, pPIC9K-P is transformed e. coli jm109 to be screened by screening culture medium, alkaline process extracts its recombinant plasmid, then cuts with enzyme and PCR identifies.Behind the positive recombinant that filters out with PCR and plasmid enzyme restriction authentication method, deliver to again precious biotechnology (Dalian) company limited and carry out dna sequencing, with the exactness of checking reading frame.
7, the conversion of the sequential analysis of expression vector and GS115
After the sequential analysis checking, use again enzyme BglII with Expression vector pPIC9K-P linearizing, then by the electrotransfer method it is imported the competent cell of yeast GS115 bacterial strain.Specified operational procedure is as follows:
7.1 the preparation of yeast competent cell: yeast strain P.pastoris GS115 in 500mlYPD (1% yeast extract, 2% peptone, 2% glucose) 30 ℃ be cultured to A600=1.5, the centrifugal collection thalline of 1500g, successively use the aseptic washing thalline of 500ml, 250ml precooling, the centrifugal supernatant of going, with the 1mol/L sorbyl alcohol suspension thalline of 20ml precooling, centrifugal rear thalline suspends rear for subsequent use with the sorbyl alcohol of 0.5ml precooling again.
7.2 the preparation of transfering DNA: alkaline lysis is adopted in the extraction of plasmid DNA, then the DNA that extracts is cut with the BglII enzyme, makes it to be divided into two fragments, and one of them is the DNA section that contains the yeast expression box, and it is reclaimed after with the low melting-point agarose electrophoresis.
8, the conversion of yeast cell
1～5 μ g DNA that gets step 2.2.7.2 preparation is added in the yeast cell of 40 μ l sorbyl alcohols suspension, ice bath 5min, and then with the electric shock instrument electric shock, parameter is: 0.8kV, 11.5 μ F.The 1mol/L sorbyl alcohol that adds immediately the 0.5ml precooling after electric shock finishes, getting 200 μ l is RDB:(18.6% sorbyl alcohol, 2% glucose, 1.34%YNB, 0.00004% vitamin H, each 0.005% L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine, 2% agarose in solid RDB conversion substratum) upper coated plate, cultivate until transformant occurs for 30 ℃.Arrive on MM (1.34%YNB, 0.00004% vitamin H, 0.5% methyl alcohol, 1.5% agarose) flat board and the MD (1.34%YNB, 0.00004% vitamin H, 2% glucose, 1.5% agarose) with the corresponding dibbling of toothpick picking transformant; cultivate 2d for 30 ℃, normally be positive colony at MM growth transformant undesired or that do not grow in the MD growth.
9, the abduction delivering of PNRSV CP gene in yeast:
Concrete operations are undertaken by Invitrogen company Pichia Expression Kit program.
10, the mensuration of expressing protein molecular weight: get the 1ml yeast fermentation broth, centrifugal 15 minutes of 13000rpm gets supernatant 10 μ l and carries out SDS-PAGE, dyes with Coomassie brilliant blue behind the electrophoresis.
11, expressing protein Western-blot detects
Behind the SDS-PAGE electrophoresis with the expressing protein electrotransfer to nitrocellulose filter (NC), then carry out immune response with the PNRSV antiserum(antisera) as the albumen on primary antibodie and the NC film, be that the goat-anti rabbit anti-serum of alkali phosphatase enzyme mark carries out specificity identification to expressing protein with the second antiserum(antisera) more afterwards, with the yeast culture liquid of not using methanol induction as negative control.
The purifying of expression product of the present invention and antiserum(antisera) preparation:
According to pre-expression result, choose efficient expression strain, a large amount of abduction deliverings through SDS-PAGE and coomassie brilliant blue staining analysis, are cut the adhesive tape band and suitably after the dilution, are added the emulsification of equal-volume freund 's incomplete adjuvant with physiological saline; Protein liquid (1mg/mL) directly and freund 's incomplete adjuvant carry out emulsification.The immunity White Rabbit adopts intramuscular injection in conjunction with hypodermic mode, after for the first time immunity, respectively at the 21st day, the 35th day and the 49th day booster immunization 3 times.Begin a small amount of venous blood collection behind the last injecting immune in 7 days and measure and tire, measure greatly blood according to the situation gradation of tiring.The antiserum(antisera) of preparation adds 0.02% sodiumazide ,-20 ℃ of preservations.
Antiserum titre and specific assay: after antiserum(antisera) carried out a series of dilutions, measure sero-fast tiring and specificity with indirect ELISA method.
(1). with coating buffer antigen diluent is become 2ug/mL, the 100ul/ hole is added in the removable enzyme plate in 96 holes, and 4 ℃ are spent the night.(2) .PBS-T washes three inferior to patting dry on the thieving paper, adds 200ul/ hole 1%BSA, 37 ℃ of thermostat containers 2 hours.(3). get antiserum(antisera) according to 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000, make gradient dilution at 1: 128000, each sample is not less than 200ul.(4). take out the antigen plate in 37 ℃ of thermostat containers, PBS-T washes three inferior to patting dry on the thieving paper, adds the good antiserum(antisera) sample 100ul/ hole of dilution in the step 3, and each extent of dilution adds side by side two holes.(5). get negative serum 1: 1000 dilution and be added in order behind the antiserum(antisera) Cmin sample as negative control.Put 37 ℃ of thermostat containers 1 hour.(also will repeat 2 times) (6) .PBS-T washes three inferior to patting dry on the thieving paper, and goat-anti rabbit enzyme mark (horseradish peroxidase) two is anti-to be diluted according to operation instruction, and the 100ul/ hole is added in the enzyme plate, puts 37 ℃ of thermostat containers 1 hour.(7) .PBS-T washes five inferior to patting dry on the thieving paper, and TMB nitrite ion 100ul/ hole is added in the enzyme plate, puts 37 ℃ of thermostat containers 10 minutes.Microplate reader 450nm surveys OD value and print result.(8). usually regard 2.5 times of negative control OD values as the positive, according to judgement sample titre as a result.(9). select optimal working concentration, carry out immune response with viruses such as TMV, PVX, PVY respectively, to test the specificity of this serum.
For further checking technical solution of the present invention, done to test and identify and analyze:
1, the clone who extracts the total RNA of plant and contain the CP gene fragment:
Take the total RNA of blade in spite of illness as template, through the RT-PCR amplification, detect through agarose gel electrophoresis, obtain the approximately DNA purpose fragment of 675bp of length.The purified rear clone of amplified production arrives
On the carrier, cut evaluation through blue hickie screening and enzyme, the positive colony that obtains is carried out sequencing, determined the nucleotide sequence of amplified production, wherein comprise the PNRSV CP sequence of 675bp, consistent with the CP sequence of GenBank report.
2, the enzyme that contains the CP gene recombined vector is cut evaluation:
The CP gene PCR product that application specific primer PPV-F and PPV-R obtain is connected on same the swelled carrier with BamH1 and HindIII double digestion behind double digestion, sets up into recombinant vectors.Through the Colony pcr amplification, positive colony can obtain the band of 675bp size, and the extracting plasmid proves that through BamH1 and HindIII double digestion the positive recombinant that filters out is correct, sees Fig. 1.
3, the evaluation of Expression vector pPIC9K-PNRSV
The construction strategy of Expression vector pPIC9K-PNRSV such as Fig. 2. expression vector pPIC9K-PNRSV is identified and pcr amplification with PCR and SnaB I/Not I double digestion respectively, the result shows that PNRSV CP gene has been cloned on the Expression vector pPIC9K, such as Fig. 3: expression vector is carried out sequential analysis, found that CP direction of insertion and position are entirely true, can carry out next step abduction delivering.
4, the SDS-PAGE of the abduction delivering of PNRSV CP gene and expressing protein analyzes
With the GS115 of expression vector under 28 ℃ of conditions, at BMMY substratum (1% yeast extract, 2% peptone, the 100mM potassiumphosphate, pH 6.0,1.34% yeast nitrogen base (yeast nitrogen base, YNB), 0.00004 vitamin H, 1% methyl alcohol) cultured continuously is after 96 hours, get the substratum supernatant through the SDS-PAGE electrophoretic analysis, discovery has a very significantly protein band in swimming lane, size is about 25Ku, and almost can't see any protein band in negative control, such as Fig. 4, its expressing quantity is about 50-60 μ g/ml supernatant after measured.
5, the evaluation of expressing protein
Analyzing discovery expressing protein band through Western-blotting can specific immune response occur with the PNRSV rabbit anti-serum, such as Fig. 5, illustrates that PNRSV CP gene has obtained correction in yeast.
6, the purifying of expression product: the bacterium liquid of inducing in a large number downcuts the expressing protein band of 25ku as the direct immunization source behind the SDS-PAGE electrophoresis dying, see Fig. 6.
7, sero-fast preparation and tiring and specific assay:
CP protein immunization White Rabbit with cutting-out obtains has obtained the PNRSV specific antisera.Measuring its antiserum titre through ELISA is 3.2 * 10
4, preparing enzyme-linked immunologic detecting kit, and in actual testing, checked.According to the existing market price, the 40mL antiserum(antisera) economic worth of preparing is above 100,000,000 yuan, but more than 1,000 ten thousand parts of test sample, through the ELISA detection validation, this antiserum(antisera) does not all have serological reaction with PVX, PVY, TMV and healthy Japanese plum plants such as peach, Japanese plum and sweet cherry leaves.According to the existing market price, the 40mL antiserum(antisera) economic worth of preparing surpasses 100,000,000 yuan, but more than 1,000 ten thousand parts of test sample.Rose mosaic virus of the present invention (PNRSV) ELISA detection kit,
1, the test kit content comprises:
1) .PNRSV antibody; 2). enzyme labelled antibody: alkaline phosphate ester enzyme labelling; 3). substrate: 4-NPP (pNPP); 4) .PNRSV positive control; 5) .PNRSV negative control.
The purposes of test kit of the present invention and detection number: mainly detect the PNRSV in the Prunus plant, be used for the detection of 100 enzyme linked plate holes.
2, adopt the detection method of test kit of the present invention:
1). sample preparation: with testing sample and normal healthy controls sample by 1: 10 (weight: volume) add coated damping fluid, grind pulping with mortar, centrifugal 10 minutes of 2000r/min, supernatant is the test sample for preparing.Negative control, positive control are correspondingly processed;
2). application of sample: add the test sample, negative control, the positive control that prepare, add a cover in 100 μ L/ holes, and 4 ℃ of refrigerator overnight incubation are then with PBST washing 3 times, each 3 minutes;
3). add antibody: with the enzyme labelled antibody damping fluid PNRSV antibody is pressed the working concentration dilution, add in the hole of elisa plate, add a cover in 100 μ L/ holes, hatched 2 hours for 37 ℃, empties solution in the hole, PBST washing 3 times, each 3 minutes;
4). add enzyme labelled antibody:, add in the elisa plate on request multiple dilution of antibody with the enzyme labelled antibody damping fluid, add a cover in 100 μ L/ holes, hatched 4 hours for 37 ℃, then with PBST washing 3 times, each 3 minutes;
5). add substrate: substrate ρ NPP is joined to make final concentration in the substrate buffer solution be 1mg/mL (now with the current), by 100 μ L/ holes, join in the elisa plate incubated at room;
6). reading: within the different time as in 30 minutes, 1 hour, read the OD value with enzyme connection instrument at the 405nm place.
3. data processing and result judge
1). the OD of control wells
405Value (damping fluid hole, negative control and positive control hole) should be in quality control clearance.That is: the OD of damping fluid hole and negative control hole
405Value＜0.15.OD when negative control hole
405Be worth＜0.05 o'clock, and calculated by 0.05; Positive control OD
405Value/negative control OD
405Value＞5-10; Hole repeated basically identical.
2). after having satisfied above-mentioned specification of quality, the result can judge as follows in principle: sample OD
405Value/negative control OD
405Value obviously＞2 is judged to the positive; Sample OD
405Value/negative control OD
405Value is judged to suspicious specimen at Near Threshold, need again do once, or use the additive method proved; Sample OD
405Value/negative control OD
405Value obviously＜2 is judged to feminine gender; If do not satisfy above-mentioned specification of quality, then can not carry out the result and judge.
4. the preparation of damping fluid
1). coated damping fluid (pH9.6): Na
2CO
31.59g; NaHCO
32.93g; NaN
30.2g; Be dissolved in distilled water, adjust pH to 9.6, distilled water is settled to 1000mL, 4 ℃ of storages.
2) .PBST damping fluid (lavation buffer solution) adds in 1000mL distilled water:
8.0g NaCl, 1.15g Na
2HPO
4, 0.2g KH
2PO
4, 0.2g KCl, 0.5mL Tween-20, adjust pH to 7.4.
3). the enzyme labelled antibody damping fluid:
Add to 1000mLPBST: 2.0g BSA (bovine serum albumin) or skim-milk, 20.0gPVP (MW24,000-40,000), 0.2g NaN
3, adjust pH to 7.4,4 ℃ of storages.
4). substrate buffer solution:
In 800mL distilled water, add: 0.1g MgCl
20.2g NaN
3The 97mL diethanolamine; With HCl adjust pH to 9.8, distilled water is dissolved to 1000mL, 4 ℃ of storages.
Claims (8)
1. rose mosaic virus coat protein gene expression method is characterized in that may further comprise the steps:
A, from the susceptible material of PNRSV, extract the RNA of rose mosaic virus;
B, the method that adopts RT-PCR to increase are cloned the coat protein gene of Prunus necrotic ring spot virus; The coat protein gene that obtains is connected on the pGEM-T carrier, obtains the pGEM-T-PNRSV recombinant vectors, and determine the sequence exactness of this virus coat protein gene with sequential analysis;
C, structure PNRSV coat protein gene carrier for expression of eukaryon;
D, conversion Pichia pastoris GS115 bacterial strain are induced the Pichia anomala expression coat protein;
E, detect the exactness of expressing protein size with Western-blot.
2. a kind of rose mosaic virus coat protein gene expression method according to claim 1 is characterized in that the RNA that extracts rose mosaic virus in the steps A specifically may further comprise the steps:
(1) get the tender tissue of the susceptible material of PNRSV some, liquid nitrogen grinding, be distributed into＜the 100mg aliquot is to precooling E.P pipe, and get the 100mg sample and add the 1mlTriZOL extracting;
(2) 12000rpm, 2-8 ℃ of centrifugal 10min;
(3) draw supernatant liquor, be transferred in the new 1.5mlE.P pipe, place 5min for 15-30 ℃;
(4) add the 0.2ml chloroform, fully extracting 8min places 2-3min for 15-30 ℃;
(5) 12000rpm, 2-8 ℃ of centrifugal 15min;
(6) draw supernatant liquor, be transferred in the new 1.5mlE.P. pipe, add the 0.5ml Virahol, place 10min for 15-30 ℃;
(7) 12000rpm, 2-8 ℃ of centrifugal 10min;
(8) abandoning supernatant gets colloidal RNA, adds 1-1.5ml75% ethanol, the vortex mixing
(9) 7500rpm, 2-8 ℃ of centrifugal 5min, abandoning supernatant stays colloidal RNA;
(10) dry 5-10min;
(11) be dissolved in the Rnase.free water, the rifle head is beaten even, and 55-60 ℃ of temperature bathed 10min;
(12) 5%Agarose glue detects, and measures concentration and transfers to 2-3 μ g/ μ L, and-70 ℃ store for future use.
3. a kind of rose mosaic virus coat protein gene expression method according to claim 1 is characterized in that step B concrete grammar comprises:
A, RT-PCR amplifying target genes:
(1) primer
Upstream primer: PNRSV-F:5 '-GT
GGATCCTACGTAATGGTTTGCCGAATTTG-3 ', wherein
GGATCCBe the BamHI restriction enzyme site, TAC GTA is SnaB I restriction enzyme site;
Downstream primer: PNRSV-R:5 '-GG
AAGCTTGCGGCCGCGATCTCAAGCAGGTC-3 ', wherein
AAGCTTBe Hind III restriction enzyme site, GCGGCCGC is Not I restriction enzyme site;
Namely 5 ' and 3 ' SnaB I and Not I restriction enzyme site primer have been added respectively at the primer two ends;
(2) amplification system
(3) amplification procedure:
50 ℃ of 30min, 94 ℃ of 3min, 94 ℃ of 1min, 60 ℃ of 30s, 72 ℃ of 1min, 72 ℃ of 10min, 4 ℃; 94 ℃ of 1min wherein, 60 ℃ of 30s, 2 ℃ of 1min, 72 ℃ of 10min circulations 35 times;
B, gene clone and sequence order-checking:
(1) recovery of PNRSV CP product
Get amplified production electrophoresis on the 1%TBE sepharose, electrophoresis is complete, downcuts the purpose band, adopts the PCR product to reclaim test kit and reclaims;
(2) structure of recombinant plasmid
Adopt the pGEM-T carrier to carry out the TA clone, construction recombination plasmid, concrete steps are: electrophoresis determines to reclaim purity and the concentration of product; In the PCR pipe, add successively approximately 4 μ L recovery product, 1 μ LpGEM-T carrier, 5 μ L connecting fluids are replenished ddH
2O is to cumulative volume 10 μ L; 16 ℃ of lower reactions are spent the night;
(3) competent cell preparation
Activation DH5 α bacterial strain on the LB culture medium flat plate, and in the sub-5mlLB liquid nutrient medium of picking list colony inoculation, 37 ℃ of lower wave and culture spend the night, and form kind of a daughter bacteria liquid; Get this 5ml bacterium liquid, in 100ml LB liquid nutrient medium, 37 ℃ of wave and culture 2～2.5hr to OD600=0.6～0.8 o'clock, draw 1.5ml bacterium liquid in the 1.5ml centrifuge tube, ice bath 5min, 4 ℃ of centrifugal 10min of lower 4000g abandon supernatant liquor, repeat to draw bacterium liquid once, precipitation exhausts raffinate with sterilization filter paper, adds the 100mmol/L CaCl of 300 μ L ice precooling
2The resuspension thalline, ice bath 30min, 4 ℃ of centrifugal 10min of lower 4000g, collecting precipitation adds the 100mmol/L CaCl that 100 μ L ice precooling
2The Eddy diffusion bacterial precipitation is competent cell, places 12～24hr for subsequent use in 4 ℃ of refrigerators; Or add the 100mmol/L of 70 μ L ice precooling
CaCl
2Save backup in-70 ℃ with 30 μ L50% glycerine;
(4) conversion of recombinant plasmid and screening
From-70 ℃ of refrigerators, take out competent cell, place on ice and thaw, on Bechtop, get 10 μ L and connect product in 100 μ L competent cells, flick the tube wall mixing, ice bath 30min, centrifuge tube is placed 42 ℃ of water-bath heat shock 90sec, rapidly with centrifuge tube ice bath 3～5min.Add 800 μ LLB liquid nutrient mediums (not containing microbiotic) in every pipe, 37 ℃ of lower wave and culture 90min are beneficial to the recovery of bacterium and the expression of plasmid-encoded resistant gene;
Adopt microbiotic and α-complementary method screening recombinant plasmid; Get 100ml LB solid medium heating and melting, to be cooled to below 60 ℃ the time, add 100 μ LAmp, pour in separately 4 culture dish behind the mixing; After solidifying, every ware is got 4 μ LIPTG, even spread plate behind the filtration sterilization of the 200mg/ml aqueous solution and the 40 μ LX-gal mixings; The bacterium liquid of getting after liquid is absorbed after 200 μ l transform evenly is applied on the above-mentioned LB flat board, and forward is placed 30min and treated that liquid is absorbed, and 37 ℃ of lower inversions are cultivated 16～24hr; The picking diameter is about the white colony of 1～2mm size in 5ml LB liquid nutrient medium, 37 ℃ of lower wave and culture 12～16hr, after getting the sterile glycerol mixing of 0.7ml and 0.3ml 50% behind the mark, put under-80 ℃ and save backup, all the other bacterium liquid are used for recombinant plasmid extracting and evaluation;
(5) extracting of recombinant plasmid
Screen the bacterium colony culture to be checked precooling 5～10min in 4 ℃ of refrigerators that obtains through microbiotic and α-complementary method, adopt alkaline lysis extracting plasmid DNA; Concrete steps are as follows: the centrifuge tube label, get contain recombinant plasmid thalline liquid 1.5ml to centrifuge tube, 4 ℃, 12000g, centrifugal 1min, abandon supernatant, repeat to add thalline liquid once, pipe is inverted on the thieving paper after centrifugal, after flowing to end remaining liquid, add 150 μ L solution I, thermal agitation suspends thalline, and room temperature is placed 5～10min, add 250 μ L solution II, put upside down centrifuge tube for several times, gentle mixing, room temperature is placed 5min, add 180 μ L solution III, cover tightly centrifuge tube, gentleness is put upside down centrifuge tube for several times, makes the precipitation mixing, ice bath 6～7min, 4 ℃, the centrifugal 10min of 12000g, supernatant moves in the clean centrifuge tube, adds respectively the chloroform/primary isoamyl alcohol of 1/2 volume, the saturated phenol of Tris, mixing, 4 ℃, the centrifugal 5min of 12000g gets supernatant, and extracting is once again for chloroform/primary isoamyl alcohol; Supernatant water moves in the clean centrifuge tube mutually, adds 1/10 volume NaAc, 2 times of volume dehydrated alcohols, behind the mixing, 20min or spend the night in-20 ℃ of refrigerators, 4 ℃, the centrifugal 10min of 12000g abandon supernatant, the mouth of pipe opened be inverted on the thieving paper, blot remaining liquid, 70%, 100% ethanol is respectively washed once, and is air-dry under the room temperature, add 20 μ LTE, for subsequent use in-20 ℃ of refrigerators;
(6) evaluation of recombinant plasmid
1., specific primer PCR is identified
Utilize special primer to treat check weighing group plasmid and carry out pcr amplification, the reaction system program parameter is the same, and the PCR product is the electrophoresis evaluation on the 1%TBE sepharose, and all clones who contains the purpose amplified fragments further carry out enzyme and cut evaluation;
2., the recombinant plasmid enzyme is cut evaluation
Identify positive recombinant plasmid through PCR, carry out double digestion with following system: 10 * HBuffer1.5 μ L, BamH10.5 μ L, HindIII0.5 μ L, plasmid 4 μ L to be checked add ddH
2O is to cumulative volume 15 μ L, and 37 ℃ of lower reactions are spent the night, and get 10 μ L reaction solution electrophoresis detection enzymes and cut the result;
3., recombinant plasmid sequencing
Cut the clone who is accredited as the positive through above-mentioned PCR and enzyme, expand numerous thalline, behind extracting and the plasmid purification, utilize the universal sequencing primer thing that recombinant plasmid is carried out sequencing.
4. a kind of rose mosaic virus coat protein gene expression method according to claim 1 is characterized in that the specific practice of step C:
Go out the PAP gene with pcr amplification, then use SnaB I and Not I double digestion after reclaiming this gene fragment, be connected with carrier pPIC9K with same double digestion again and be built into Expression vector pPIC9K-P, pPIC9K-P is transformed e. coli jm109 to be screened by screening culture medium, alkaline process extracts its recombinant plasmid, then cuts with enzyme and PCR identifies; Behind the positive recombinant that filters out with PCR and plasmid enzyme restriction authentication method, carry out again dna sequencing, with the exactness of checking reading frame.
5. a kind of rose mosaic virus coat protein gene expression method according to claim 1 is characterized in that the specific practice of step D:
After the sequential analysis checking, use again enzyme BglII with Expression vector pPIC9K-P linearizing, then by the electrotransfer method it is imported the competent cell of yeast GS115 bacterial strain;
(1) preparation yeast competent cell:
Yeast strain P.pastoris GS 115 in 500ml YPD 30 ℃ be cultured to A 600=1.5, the centrifugal collection thalline of 1500g, successively use the aseptic washing thalline of 500ml, 250ml precooling, the centrifugal supernatant of going, with the 1mol/L sorbyl alcohol suspension thalline of 20ml precooling, centrifugal rear thalline suspends rear for subsequent use with the sorbyl alcohol of 0.5ml precooling again;
(2) preparation transfering DNA: alkaline lysis is adopted in the extraction of plasmid DNA, then the DNA that extracts is cut with Bgl II enzyme, makes it to be divided into two fragments, and one of them is the DNA section that contains the yeast expression box, and it is reclaimed after with the low melting-point agarose electrophoresis;
(3) conversion of yeast cell
1～5 μ gDNA that gets the above-mentioned steps preparation is added in the yeast cell of 40 μ l sorbyl alcohols suspension, ice bath 5min, and then with the electric shock instrument electric shock, parameter is: 0.8kV, 11.5 μ F; The 1mol/L sorbyl alcohol that adds immediately the 0.5ml precooling after electric shock finishes, getting 200 μ l is RDB:18.6% sorbyl alcohol, 2% glucose, 1.34%YNB, 0.00004% vitamin H, each 0.005% L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine, 2% agarose in solid RDB conversion substratum, upper coated plate is cultivated until transformant occurs for 30 ℃; Arrive MM:1.34%YNB, 0.00004% vitamin H, 0.5% methyl alcohol, 1.5% agarose with the corresponding dibbling of toothpick picking transformant, on flat board and MD:1.34%YNB, 0.00004% vitamin H, 2% glucose, 1.5% agarose, cultivate 2d for 30 ℃, normally be positive colony at MM growth transformant undesired or that do not grow in the MD growth;
(4) abduction delivering of PNRSV CP gene in yeast
Concrete operations are undertaken by Invitrogen company Pichia Expression Kit program.
6. a kind of rose mosaic virus coat protein gene expression method according to claim 1 is characterized in that the specific practice of step e:
(1) mensuration of expressing protein molecular weight
Get the 1ml yeast fermentation broth, centrifugal 15 minutes of 13000rpm gets supernatant 10 μ l and carries out SDS-PAGE, dyes with Coomassie brilliant blue behind the electrophoresis;
(2) expressing protein Western-blot detects
Behind the SDS-PAGE electrophoresis with the expressing protein electrotransfer to nitrocellulose filter, then carry out immune response with the PNRSV antiserum(antisera) as the albumen on primary antibodie and the nitrocellulose filter, be that the goat-anti rabbit anti-serum of alkali phosphatase enzyme mark carries out specificity identification to expressing protein with the second antiserum(antisera) more afterwards, with the yeast culture liquid of not using methanol induction as negative control.
7. antiserum(antisera), it is characterized in that its preparation method may further comprise the steps: according to pre-expression result, choose the Pichia pastoris GS115 bacterial strain of high efficient expression, a large amount of abduction deliverings, through SDS-PAGE and coomassie brilliant blue staining analysis, cut the adhesive tape band and suitably after the dilution, add the emulsification of equal-volume freund 's incomplete adjuvant with physiological saline; Protein liquid 1mg/mL directly and freund 's incomplete adjuvant carry out emulsification; The immunity White Rabbit adopts intramuscular injection in conjunction with hypodermic mode, after for the first time immunity, respectively at the 21st day, the 35th day and the 49th day booster immunization 3 times; Begin a small amount of venous blood collection behind the last injecting immune in 7 days and measure and tire, measure greatly blood according to the situation gradation of tiring; The antiserum(antisera) of preparation adds 0.02% sodiumazide ,-20 ℃ of preservations.
8. test kit is characterized in that comprising in this test kit:
1) .PNRSV antibody
2). enzyme labelled antibody: alkaline phosphate ester enzyme labelling
3). substrate: 4-NPP (pNPP)
4) .PNRSV positive control
5) .PNRSV negative control.
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