CN108165539A - A kind of pears S7The vivoexpression method of-RNase albumen and its preparation method of polyclonal antibody - Google Patents
A kind of pears S7The vivoexpression method of-RNase albumen and its preparation method of polyclonal antibody Download PDFInfo
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- CN108165539A CN108165539A CN201711323194.9A CN201711323194A CN108165539A CN 108165539 A CN108165539 A CN 108165539A CN 201711323194 A CN201711323194 A CN 201711323194A CN 108165539 A CN108165539 A CN 108165539A
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
Abstract
The invention discloses a kind of pears S7The vivoexpression of RNase albumen and its preparation method of polyclonal antibody.Pears S is cloned by designing primer7RNase genes；Build Recombinant protein expression carrier S7‑cutSignalP‑pCold‑TF；By the recombinant expression carrier S of structure7CutSignalP pCold TF are transformed into Escherichia coli Rosetta (DE3), structure expression pears S7The recombinant bacterial strain of RNase albumen；Recombinant bacterial strain is expressed, and purifying recombination pears S is sent out with affinity chromatography7RNase albumen；With the recombination pears S of purifying7RNase albumen is antigen, and pears S is prepared using conventional polyclonal antibody preparation method7RNase polyclonal antibodies.With the method for the invention it is achieved that pears S7The vivoexpression of RNase albumen has simultaneously prepared the pears S that potency is high, specificity is good7RNase polyclonal antibodies can meet the needs of related experiment completely.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of pears S7The vivoexpression method of-RNase albumen and
The preparation method of its polyclonal antibody.
Background technology
Pears self-incompatibility is one of self-fertile mechanism of limitation of generally existing in angiosperm, it prevents gene
The identical pollen tube of type is normally grown in gynoecium, completes fertilization.Pears self-incompatibility is the gametophyte based on S-RNase
Type self-incompatibility response, numerous fruit trees such as apple, apricot, fruit plum etc., all shows this phenomenon.Since pear tree self-pollination cannot
It is solid, pollinizers must be configured in production or progress artificial pollination just can guarantee fruit setting, increase the production cost of peasant.
Therefore, double meaning of the research of pears self-incompatibility mechanism with theory value and more practical value.In the past 30 years, both at home and abroad
Many researchs have been carried out for pears self-incompatibility mechanism, what is faced is exactly the extraction of S-RNase the problem of standing in the breach, and
The problems such as due to the source of material, preservation and cumbersome protein extraction process, brings very big resistance to research.So it utilizes
Technique for gene engineering Prepare restructuring pears S-RNase albumen is the effective way to solve the above problems.
It is a kind of technological means with bright prospects using technique for gene engineering Prepare restructuring albumen.Up to the present,
Have multiple proteins expression system, such as prokaryotic expression system, eukaryotic expression system, Cell free expression system.Escherichia coli
Expression system is a kind of most common prokaryotic expression system, the method for extracting albumen from living materials compared to tradition, the system
Understand with genetic background and biochemical characteristic, grow fast, at low cost, expression quantity is high, expression product isolate and purify it is relatively easy and
The advantages that convenient for large-scale production.For a long time, has the case of countless successful expression recombinant proteins.At the same time, S-RNase
As the great influence factor of pears self-incompatibility reaction, in the market there are no the specific aim antibody that potency is high, specificity is good,
Therefore in order to further study effects and other related biological functions of the S-RNase in the reaction of pears self-incompatibility, this is specially
Profit is drawn up for the anti-pears S in rabbit source7- RNase protein antibodies.
Invention content
The object of the present invention is to provide a kind of low cost, high yield, easy purification pears S7- RNase albumen vivoexpressions
Preparation method.
The another purpose of the present invention is to provide the pears S that a kind of potency is high, specificity is good7The system of-RNase protein polyclone antibodies
Preparation Method.
Technical problem solved by the invention is realized using following technical scheme：
A kind of pears S7The method of-RNase albumen vivoexpressions, includes the following steps：
A. Isolation from Pear Style total serum IgE is extracted, reverse transcription expands pears S into cDNA, design primer PCR7- RNase genes；
B. structure expression pears S7The pronuclear recombination expression vector of-RNase albumen；
C. the pronuclear recombination expression vector that step b is built is transformed into coli strain Rosetta (DE3), built
S7- RNase recombinant strains；
D. the S constructed by incubation step c7- RNase recombinant strains are to OD600Reach 0.4-0.6, and low-temperature treatment
Afterwards, derivant IPTG, induced expression pears S are added in7- RNase albumen；
E. the pears S of induced expression is purified7- RNase albumen.
As a kind of optimal technical scheme, in step a, for PCR amplification pears S7The primer pair sequence of-RNase genes is：
Forward primer is 5 '-CCGCTCGAGATGTACGATTATTTTCAATTTACGCAGC-3 ',
Reverse primer is 5 '-CTAGTCTAGAATACTTAACATCGGCCGGGC-3 '.
It is further preferred that in step a, PCR amplification pears S7The PCR reaction systems of-RNase genes are：ddH233 μ l of O,
5 μ l, 2mM dNTPs of each 1 μ l, 10 × KOD-Plus-PCR buffer of 1.5 μ l, cDNA of upstream and downstream primer 5 μ l, 25mM
MgSO42μl,KOD-Plus-DNA polymerase 1μl；PCR reaction conditions are：94 DEG C of pre-degeneration 2min, then carry out 94
DEG C 15s, 60 DEG C of 30s, 68 DEG C of 1min, totally 30 cycles, last 68 DEG C of extensions 10min.
As a kind of optimal technical scheme, in step b, structure expression pears S7The pronuclear recombination expression vector of-RNase albumen
When, used prokaryotic expression carrier is coli expression carrier pCold-TF, clone's pears S7The insertion position of-RNase genes
Point is between Xho I and Xba I restriction enzyme sites.It is further preferred that PCR product is analyzed simultaneously through 1% agarose gel electrophoresis
Gel extraction is connected to the large intestine bar through similary double digestion after recovery product Xho I and Xba I restriction enzymes double zyme cuttings
On Xho I and the Xba I sites of bacterium expression vector pCold-TF.
As a kind of optimal technical scheme, in step d, the low-temperature treatment is is placed in 5-10min on ice, then 15-
18 DEG C stand 40-60 minutes, the final concentration of 0.5-1.0mmol/L of the derivant IPTG, and the condition of the induced expression is
15-18 DEG C induces 18-24 hours.
It is further preferred that induced expression pears S in step d7The detailed process of-RNase albumen is：By the S of structure7-
RNase recombinant strains are according to 1:50(v:V) ratio is inoculated into LB liquid of the 100ml containing 100 μ g/ml ampicillins and trains
Support base in, 37 DEG C, 200~250rpm shake cultures stay overnight, then by 1:50 ratio is transferred to 300ml containing 100 μ g/ml ammonia benzyls
In the LB fluid nutrient mediums of penicillin, 37 DEG C, 200~250rpm shake cultures to bacterium solution OD600Reach 0.4-0.6, be immediately placed in
5-10 minutes on ice, then stand 40-60 minutes for 15-18 DEG C；Then the derivant of final concentration of 0.5-1.0mmol/L is added in
IPTG, 15~18 DEG C, 200~250rpm shake cultures, induced expression 18-24 hours；
The pears S of induced expression is purified in step e7The detailed process of-RNase albumen is：After the completion of induced expression, in 4 DEG C
Supernatant is abandoned in lower 12000rpm centrifugations after ten minutes, collects precipitation, lysate is added in into precipitation, precipitation is resuspended；Bacterium solution makes after resuspension
With ultrasonication, after the completion of ultrasonication, 12000rpm is centrifuged 15 minutes, collects supernatant, supernatant after 0.45 μm of membrane filtration,
Using affinity chromatography method to pears S7- RNase albumen is purified, and before affinity chromatography medium purification albumen, is needed
Chromatography media is balanced with the equilibration buffer of 10 times of column volumes；Purifying protein is collected, uses the super filter tube that combined closure system is 30kDa
It is concentrated under 6000rpm rotating speeds at 4 DEG C, desalination, is finally putting into -80 DEG C and saves backup；
Using affinity chromatography method to pears S7The process that-RNase albumen is purified is：20ml after membrane filtration
Final proof passes through purification column, coutroi velocity 1ml/min on albumen；Cleaning solution (the 500mM chlorinations of 20 times of column volume imidazoles containing 30mM
Sodium, 20mM tri- (methylol) aminomethane, 30mM imidazoles, pH 7.3) rinse pillar, coutroi velocity 1ml/min；10 times of cylinders
The elution fliud flushing (500mM sodium chloride, 20mM tri- (methylol) aminomethane, 80mM imidazoles, pH 7.3) of product imidazoles containing 80mM is washed
De- purification column, coutroi velocity 1ml/min collect eluent and obtain purifying protein.
The formula of the lysate is：140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphates, 1.8mM di(2-ethylhexyl)phosphates
Hydrogen potassium, 50 × EDTA-free protease inhibitor cocktail III (Merck KGaA Mi Libo), pH 7.3；
The formula of the equilibration buffer is：500mM sodium chloride, 20mM tri- (methylol) aminomethane, 5mM imidazoles, pH
It is 7.3.
The pears S7- RNase albumen n ends are with the histidine tag (His-Tag) on 6 expression vectors.
Recombination pears S prepared by the above method7- RNase albumen is preparing pears S as antigen7In-RNase polyclonal antibodies
Using.
A kind of pears S7The preparation method of-RNase polyclonal antibodies will recombinate pears S made from the above method7- RNase albumen
Japan large ear rabbit is immunized as antigen, it is immune for the first time using recombination pears S7- RNase albumen is helped completely with isometric Freund
Experimental rabbit is injected after agent emulsification, with recombination pears S after 3 weeks7- RNase albumen and after isometric incomplete Freund's adjuvant emulsification the
Secondary immunity experimental rabbit carries out 1 booster immunization every two weeks later, in total 4 times it is immune, take a blood sample, receive after last time is 7 days immune
Collect antiserum.
To evaluate potency and specificity the blood sampling 0.5ml before immune of antibody, serum is collected as negative serum, with feminine gender
Serum is as negative control, with ELISA method to pears S7- RNase polyclonal antibodies carry out antibody titer evaluation, use Western
Blotting methods evaluate antibody specificity.
Preferably, it is immune for the first time using 500 μ g recombination pears S7- RNase albumen and isometric Freund's complete adjuvant breast
Experimental rabbit is injected after change；With 300 μ g recombination pears S after 3 weeks7- RNase albumen and after isometric incomplete Freund's adjuvant emulsification the
Secondary immunity experimental rabbit.
Pears S of the present invention7The most preferred technique scheme of the method for-RNase albumen vivoexpressions includes the following steps：
(1) pears S7The sequence analysis of-RNase genes and clone
(1.1) a kind of pears S7- RNase (BAA32416.1) gene, nucleotides sequence are classified as SEQ ID NO:1, amino acid
Sequence is SEQ ID NO:2.Utilize sequence analysis website SignalP 4.1Serve (http://www.cbs.dtu.dk/
Services/SignalP/ it) analyzes and removes signal peptide sequence.
(1.2) Dangshan pear style RNA is extracted according to plant total RNA extraction reagent box specification, uses reverse transcription reagent box
Reverse transcription is into cDNA.
(1.3) design of primers and PCR reactions：Designing forward primer (S7-pCold-cutSignalP-Xho I-Lp) is
5’-CCGCTCGAG(underscore is Xho I restriction enzyme sites to ATGTACGATTATTTTCAATTTACGCAGC-3 ', and thickened portion is
Initiation codon), reverse primer (S7-pCold-cutSignalP-Xba I-Rp) for 5 '-
CTAGTCTAGAATACTTAACATCGGCCGGGC-3 ' (underscore is Xba I restriction enzyme sites)；
PCR reaction systems are：ddH233 μ l of O, upstream and downstream primer each 1.5 μ l, cDNA 1 μ l, 10 × KOD-Plus-PCR
buffer 5μl,2mM dNTPs 5μl,25mM MgSO42μl,KOD-Plus-DNA polymerase 1μl；PCR reacts item
Part is：Then 94 DEG C of pre-degeneration 2min carry out 94 DEG C of 15s, 60 DEG C of 30s, 68 DEG C of 1min, totally 30 cycles, last 68 DEG C of extensions
10min。
(1.4) above-mentioned PCR product is through the analysis of 1% agarose gel electrophoresis and gel extraction, if attached drawing 2 is as it can be seen that 500
~750bp has a clear bright band, meets with expected size.After recovery product Xho I and Xba I restriction enzymes double zyme cuttings
It is connected on Xho I and the Xba I sites of the coli expression carrier pCold-TF through similary double digestion (referring to TAKARA public affairs
Take charge of Vector map).
(1.5) recombinant expression carrier S is built7- cutSignalP-pCold-TF, after digestion identification is correct, then through Suzhou
Jin Wei intelligence bio tech ltd DNA sequencing verifies its correctness.
(2) structure of bacterial strain is expressed
It will verify correct recombinant expression carrier S7- cutSignalP-pCold-TF is transformed into Escherichia coli through chemical method
In strain Rosetta (DE3), in the upper 37 DEG C of cultures of LB solid plates (containing 100 μ g/ml ampicillins), picking single bacterium falls on 1ml
In LB (contain 100 μ g/ml ampicillins) fluid nutrient medium, 37 DEG C, 250rpm shake cultures stay overnight, draw 1 μ l bacterium solutions and do mould
Plate is added in 50% (v/v) glycerine of 300 μ l sterilizings by 700 μ l bacterium solutions after PCR verifications are correct, after mixing liquid nitrogen flash freezer and in-
80 DEG C of refrigerators preserve, and the bacterial strain of preservation is recombinant strains.
(3) pears S7The induced expression of-RNase and purifying
(3.1) pears S7The induced expression of-RNase
By the recombinant strains preserved in step (2) according to 1:50 volume ratio is inoculated into 100ml LB (containing 100 μ g/
Ml ammonia benzyls mycin) in fluid nutrient medium, 37 DEG C, 200-250rpm shake cultures stay overnight, by 1:50 are transferred to 300ml LB again (contains
100 μ g/ml ampicillins) in fluid nutrient medium, 37 DEG C, 200-250rpm to bacterium solution OD600Reach 0.4-0.6, be immediately placed in
It 5-10 minutes on ice, is then placed in 15-18 DEG C of shaking table and stands 40-60 minutes, in 0.5- under 15-18 DEG C, 200-250rpm
1.0mmol/L IPTG concentration shake cultures, induced expression 18-24 hours.
(3.2) pears S7The purifying of-RNase albumen
12000rpm is centrifuged after the completion of induced expression, at 4 DEG C abandons supernatant after ten minutes, collects precipitation, is added in into precipitation
Lysate (140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphates, 1.8mM potassium dihydrogen phosphates, 50 × EDTA-free
Protease inhibitor cocktail III (Merck KGaA Mi Libo), pH 7.3) precipitation is resuspended.Bacterium solution makes after resuspension
With ultrasonication, power 240W, condition is：3s is opened, stops 7s, totally 27 minutes.After the completion of ultrasonication, 12000rpm centrifugations 15
Minute, supernatant is collected, supernatant is after 0.45 μm of membrane filtration, using affinity chromatography method to pears S7- RNase albumen carries out pure
Change, before affinity chromatography medium purification albumen, equilibration buffer (500mM sodium chloride, the 20mM of 10 times of column volumes need to be used
Three (methylol) aminomethanes, 5mM imidazoles, pH 7.3) balance chromatography media；Final proof on 20ml albumen after membrane filtration
By purification column, coutroi velocity 1ml/min；Cleaning solution (500mM sodium chloride, the 20mM tri- of 20 times of column volume imidazoles containing 30mM
(methylol) aminomethane, 30mM imidazoles, pH 7.3) rinse pillar, coutroi velocity 1ml/min；10 times of column volumes contain 80mM
The elution fliud flushing (500mM sodium chloride, 20mM tri- (methylol) aminomethane, 80mM imidazoles, pH 7.3) of imidazoles is purified by flash column,
Coutroi velocity is 1ml/min, collects eluent and obtains purifying protein.Using super filter tube (combined closure system 30kDa) at 4 DEG C
It is concentrated under 6000rpm rotating speeds, desalination.50 μ l samples after purification are taken later, add in 50 μ l 2 × SDS-PAGE loading buffers
Liquid takes 10 μ l to carry out SDS-PAGE electrophoresis, and pears S is determined after coomassie brilliant blue staining7- RNase purification conditions collect purifying egg
In vain, it saves backup for -80 DEG C；
(4) pears S7The preparation of-RNase protein polyclone antibodies
According to Shanghai Sheng Gong bio-engineering corporations BCA determination of protein concentration kit to recombinating pears S after purification7- RNase eggs
White protein concentration is measured.By recombination pears S after purification7Japan large ear rabbit is immunized as antigen in-RNase albumen.It is real
Immune preceding blood sampling 0.5ml is tested, collects serum as negative serum.The immune recombination pears S purified with 500 μ g for the first time7-RNase
Albumen is with immunization experiment rabbit after isometric Freund's complete adjuvant emulsification, the recombination pears S purified after 3 weeks with 300 μ g7- RNase eggs
In vain with second of immunization experiment rabbit after isometric incomplete Freund's adjuvant emulsification, the recombination purified every two weeks with 300 μ g later
Pears S7- RNase albumen carries out 1 booster immunization, in total 4 times it is immune, last time booster immunization is taken a blood sample after a week, collects anti-blood
Clearly；For negative serum as negative control, ELISA method measures antibody titer, and Western blotting measure antibody specificity.
Beneficial effects of the present invention
(1) method that the present invention uses molecular biology vivoexpression and has purified pears S for the first time7- RNase albumen, also for
Antibody is further prepared to lay the foundation.
It (2), can be according to certainly suitable for all pears S genes using the vivoexpression and the method for purifying protein in the present invention
Oneself research purpose is with needing flexible.
(3) pears S in the present invention7- RNase protein antibodies can be with Isolation from Pear Style histone and the pears S of purifying7- RNase albumen
Specific binding, specificity is good, is widely used in various detection pears S7The immune detection tool of-RNase albumen.
(4) the pears S that the present invention expresses7- RNase albumen can directly cause oneself of Dangshan pear pollen under in vitro conditions
Not compatible reaction is handed over, there is theory directive significance and using reference value to the research of pears self-incompatibility mechanism.
Description of the drawings
Fig. 1 is pears S7- RNase gene order signal peptide prediction diagrams.
Fig. 2 is PCR amplification pears S7The agarose gel electrophoresis diagram of-RNase genes；
Wherein, M is DNA standard molecule quality；1 is pcr amplification product.
Fig. 3 is recombination pears S7The induced expression of-RNase albumen and after purification SDS-PAGE diagrams；
Wherein, M is protein standard marker；1 is to induce bacterial protein without IPTG；2 be 0.5mmol/L IPTG
Bacterial protein after induction；3 be recombination pears S after purification7- RNase albumen.
Fig. 4 is anti-His (C-term) monoclonal antibody to recombinate pears S after primary antibody purification Identification7- RNase albumen
Western blotting diagrams；
Wherein, M is protein standard marker；1 is recombination pears S after purification7- RNase albumen.
Fig. 5 is ELISA method evaluation antibody titer diagram；
Wherein, abscissa is different dilution ratios；Ordinate is OD490 absorption values.
Fig. 6 is the polyclonal antibody and recombination pears S prepared7The Western blot diagrams of-RNase albumen；
Wherein, M is protein standard marker；1 is recombination pears S after purification7- RNase albumen.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below with reference to the accompanying drawings are exemplary, only
For explaining the present invention, and it is not considered as limiting the invention.
Embodiment 1：S7The structure of-cutSignalP-pCold-TF recombinant plasmids and identification
Fresh Dangshan pear pear flower style is taken, plant total RNA extraction reagent box extraction style total serum IgE uses Reverse Transcription
Box reverse transcription obtains total cDNA；As shown in Figure 1, it analyzes and removes pears S7The signal peptide sequence of-RNase genes designs primer, just
To primer (S7-pCold-cutSignalP-Xho I-Lp) for 5 '-
CCGCTCGAG(underscore is Xho I restriction enzyme sites to ATGTACGATTATTTTCAATTTACGCAGC-3 ', and thickened portion is starting
Codon), reverse primer (S7-pCold-cutSignalP-Xba I-Rp) for 5 '-
CTAGTCTAGAATACTTAACATCGGCCGGGC-3 ' (underscore is Xba I restriction enzyme sites)；
PCR amplification is carried out by template of cDNA, PCR reaction systems (50 μ l of overall reaction system) are：ddH233 μ l of O, up and down
Swim 5 μ l, 2mM dNTPs of primer (10 μm) each 1 μ l, 10 × KOD-Plus-PCR buffer of 1.5 μ l, cDNA 5 μ l, 25mM
MgSO42μl,KOD-Plus-DNA polymerase 1μl.PCR reaction conditions are：94 DEG C of pre-degeneration 2min, then carry out 94
DEG C 15s, 60 DEG C of 30s, 68 DEG C of 1min, totally 30 cycles, finally 68 DEG C of extension 10min again.PCR product is coagulated through 1% agarose
Gel electrophoresis is identified.From Figure 2 it can be seen that there is a clearly bright band between 500~750bp, it coincide with expected size.
PCR product is the quick Ago-Gel DNA QIAquick Gel Extraction Kits of ShiJi Co., Ltd Gel extraction kit with reference to health
Specification is recycled.PCR product after recycling after Xho I and Xba I double digestions by T4-DNA ligases be inserted into through
In the pCold-TF expression vectors of similary double digestion, 4 DEG C connect 18 hours, build S7- cutSignalP-pCold-TF recombinates matter
Grain, by recombinant plasmid transformed to coli strain DH5 α, in 37 on LB solid plates (containing 100 μ g/ml ampicillins)
DEG C culture, picking individual colonies are after digestion identification is correct, then pass through Suzhou Jin Weizhi bio tech ltd DNA sequencing and verify
Its correct sequence.Sequencing result shows obtained pears S7- RNase coding sequences fulfill the expectation.Illustrate to recombinate
Plasmid construction success is simultaneously named as S7-cutSignalP-pCold-TF.Pears S7- RNase coding sequences with
PCold-TF connection cloning and sequencings result is SEQ ID NO:3, amino acid sequence is SEQ ID NO:4.
Embodiment 2：Pears S7Expression of-the RNase in Escherichia coli
1. obtain expression pears S7The recombinant strains of-RNase
The successful bacterium of sequencing is chosen to be inoculated into the LB fluid nutrient mediums that 4ml contains ampicillin (100 μ g/ml), 37 DEG C,
250rpm shaken cultivations are stayed overnight, according to the small extraction of QuickPure Plasmid Mini kit rapid plasmids that health is ShiJi Co., Ltd
Kit is by S7- cutSignalP-pCold-TF recombinant expression carriers extract, and take 1ng recombinant expression carriers S7-
CutSignalP-pCold-TF conversion Escherichia coli Rosetta (DE3), are coated on the ampicillin plate containing 100 μ g/mL
Upper screening recon, cultivation temperature are 37 DEG C, and incubation time obtains recon as expression pears S to be incubated overnight7The base of-RNase
It because of engineering bacteria, randomly selects 1 single bacterium colony and carries out scribing line culture, the scribing line grown on a small quantity culture bacterium is taken to be inoculated in 1ml LB and (is contained
100 μ g/ml ampicillins) in fluid nutrient medium, 37 DEG C, 250rpm shaken cultivations stay overnight, then added in by 700 μ l bacterium solutions
300 μ l sterilize 20% (v/v) glycerine, are stored in 80 DEG C of refrigerators of ﹣ with liquid nitrogen flash freezer after mixing, obtain expression pears S7- RNase is recombinated
Express bacterial strain.
2. pears S7The expression of-RNase recombinant proteins
By pears S obtained above7- RNase recombinant strains press 1:50 are inoculated into 100ml LB (containing 100 μ g/ml ammonia benzyls
Penicillin) in fluid nutrient medium, 37 DEG C, 250rpm shaken cultivations stay overnight, activate recombinant strains.By the recombinant expression of activation
Bacterial strain presses 1 again:50 go to culture, condition of culture 37 in 300ml LB fluid nutrient mediums (the 100 μ g/mL containing ampicillin)
DEG C, 200rpm, shaken cultivation to OD6005ml bacterium solutions are taken after during for 0.4-0.6, and as negative control, remaining bacterium solution is immediately placed in ice
It was then placed in 15 DEG C of shaking tables and stands 40 minutes upper 5 minute, and be eventually adding the IPTG of final concentration of 0.5mmol/L, 15 DEG C,
240rpm shake cultures, induced expression 24 hours.Expression takes 5ml bacterium solutions to centrifuge after the completion, collects bacterial sediment, precipitation and feminine gender
Compare 200 μ l 10%SDS (10g/100ml) of each addition, 100 DEG C of boiling water water-baths 10 minutes, are placed in ice and cool down 2 points after mixing
Clock, centrifuged 10 minutes after 12000rpm at 4 DEG C, respectively take 50 μ l of supernatant, taken after adding in 50 μ l 2 × albumen sample-loading buffers
10 μ l carry out 12% routine SDS-PAGE electrophoresis, detect recombinant protein expression after coomassie brilliant blue staining, decoloration, as a result such as
The the 1st, 2 article of swimming lane of Fig. 3 is shown.
Embodiment 3：Recombinate pears S7- RNase albumen isolating and purifying and identifying
By pears S7- RNase recombinant strains press 1:50 inoculation 100ml LB (containing 100 μ g/ml ampicillins) liquid
In culture medium, 37 DEG C, 250rpm shaken cultivations stay overnight, activate recombinant strains.The recombinant strains of activation are pressed 1 again:
50 go to culture in 300ml LB culture mediums (the 100 μ g/mL containing ampicillin), and condition of culture is 37 DEG C, 200rpm, oscillation training
It supports to OD600It after during for 0.4-0.6, is immediately placed in 5 minutes on ice, is then placed in 15 DEG C of shaking tables and stands 40 minutes, be eventually adding
The IPTG of final concentration of 0.5mmol/L, 15 DEG C, 240rpm shake cultures induced expression 24 hours.4 DEG C after the completion of expression,
Supernatant is abandoned in 12000rpm centrifugations after ten minutes, collects bacterial sediment, with 20ml lysates (140mM sodium chloride, 2.7mM potassium chloride,
10mM disodium hydrogen phosphates, 1.8mM potassium dihydrogen phosphates, 50 × EDTA-free protease inhibitor cocktail III
(Merck KGaA Mi Libo), pH 7.3) ultrasonic disruption after thalline, power 240W is resuspended, condition is：3s is opened, stops 7s, altogether
27 minutes, it is crushed to solution clarification.After the completion of ultrasonication, 12000rpm is centrifuged 15 minutes at 4 DEG C, collects supernatant, supernatant
Impurity is removed through 0.45 μm of membrane filtration.It is purified using the Ni-NTA agarose affinity chromatographies filler of Merck KGaA Millipore Corp.
Pears S7- RNase recombinant proteins, concrete operations are：Equilibration buffer (500mM sodium chloride, (the hydroxyl first of 20mM tri- of 10 times of column volumes
Base) aminomethane, 5mM imidazoles, pH 7.3) balance pillar, coutroi velocity 1ml/min；20ml albumen after membrane filtration
Upper final proof passes through purification column, coutroi velocity 1ml/min；20 times of column volume imidazoles containing 30mM cleaning solution (500mM sodium chloride,
20mM tri- (methylol) aminomethane, 30mM imidazoles, pH 7.3) rinse pillar, coutroi velocity 1ml/min；10 times of column volumes
Elution fliud flushing (500mM sodium chloride, 20mM tri- (methylol) aminomethane, 80mM imidazoles, the pH 7.3) elution of the imidazoles containing 80mM
Purification column, coutroi velocity 1ml/min collect eluent and obtain purifying protein, using super filter tube (combined closure system 30kDa) in 4 DEG C
Lower 6000rpm is concentrated, desalination, last 80 DEG C of preservations of ﹣.Take a small amount of albumen carry out the analysis of SDS-PAGE electrophoresis purities and
Western Blotting are identified, as a result as shown in the 3rd article of swimming lane of Fig. 3.Utilize the Western of anti-His monoclonal antibodies
Blotting qualification results determine that it is pears S as shown in the 1st article of swimming lane of Fig. 47- RNase recombinant proteins.
Embodiment 4：Pears S7The preparation of the polyclonal antibody of-RNase albumen and antibody titer and specificity are evaluated
According to BCA determination of protein concentration kit specification to recombinating pears S after purification7The protein concentration of-RNase albumen into
Row measures.By recombination pears S after purification7Japan large ear rabbit is immunized as antigen in-RNase albumen.Ear is quiet before experiment immunization
Arteries and veins blood sampling 0.5ml, collects the negative control that serum is detected as follow-up ELISA.First immunisation is dissolved in 0.5ml PBS with 500 μ g
The recombination pears S of purifying7- RNase albumen and immunization experiment rabbit after isometric Freund's complete adjuvant emulsification, it is molten with 300 μ g after 3 weeks
In the recombination pears S of 0.3ml PBS purifying7- RNase albumen is immunized real with second after isometric incomplete Freund's adjuvant emulsification
Rabbit is tested, the recombination pears S purified every two weeks with 300 μ g later7- RNase albumen carries out 1 booster immunization, in total 4 times it is immune, most
A booster immunization is taken a blood sample after a week afterwards, collects antiserum；Negative serum measures antibody titer as negative control, ELISA method
(negative serum is as negative control), Western blotting measure antibody specificity.Antiserum after dilution is inhaled in 490nm
Luminosity be more than it is immune before 2.1 times of (negative control) serum when highest extension rate be defined as the potency of antibody, ELISA results are such as
Shown in Fig. 5, show the pears S through preparation7- RNase polyclonal antibody antiserum titres are about 1:1024000；The pears S of preparation7-
The antibody specificity of RNase polyclonal antibodies show that its specificity is good through Western blotting analyses, the 1st article of swimming of Fig. 6
Shown in road.
Sequence table
<110>Agricultural University Of Nanjing
<120>A kind of vivoexpression method of pears S7-RNase albumen and its preparation method of polyclonal antibody
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 681
<212> DNA
<213>Pears (Pyrus spp)
<400> 1
atggggatta cggggatgat atatatagtt acgatggtat tttcattaat tgtattgata 60
ttgtcttcct ctacggtggg atacgattat tttcaattta cgcagcaata tcagccagct 120
gtctgcaact ccaaacctac tccttgtaag gatcctcctg acaagttgtt cacggttcac 180
ggtttgtggc cttcaaactt gaatggacct cacccagaaa attgcactaa tgcaaccgtg 240
aatcctcaca ggataaaaaa tatccaagcc cagttgaaaa ttatttggcc gaatgtactc 300
gatcgaacca atcatgtagg cttctggaat aaacagtgga taaaacatgg cagctgtggg 360
tatcccgcaa taatgaacga cacgcattac tttcaaacag taatcaacat gtacataacc 420
cagaaacaaa acgtctctga aatactctca aaggcgaaga ttgaaccgtt gggaatacaa 480
aggccactgg tgcatattga aaatgccata cggaatagta ccaacaataa gaaaccaaaa 540
ttcaagtgcc aaaagaattc tggggtgact gaattagttg aggtcggtct ttgcagcgat 600
ggcagcttaa cgcagttcag aaattgcccc cacccaccac caggatcacc atatctctgc 660
ccggccgatg ttaagtatta a 681
<210> 2
<211> 226
<212> PRT
<213>Pears (Pyrus spp)
<400> 2
Met Gly Ile Thr Gly Met Ile Tyr Ile Val Thr Met Val Phe Ser Leu
1 5 10 15
Ile Val Leu Ile Leu Ser Ser Ser Thr Val Gly Tyr Asp Tyr Phe Gln
20 25 30
Phe Thr Gln Gln Tyr Gln Pro Ala Val Cys Asn Ser Lys Pro Thr Pro
35 40 45
Cys Lys Asp Pro Pro Asp Lys Leu Phe Thr Val His Gly Leu Trp Pro
50 55 60
Ser Asn Leu Asn Gly Pro His Pro Glu Asn Cys Thr Asn Ala Thr Val
65 70 75 80
Asn Pro His Arg Ile Lys Asn Ile Gln Ala Gln Leu Lys Ile Ile Trp
85 90 95
Pro Asn Val Leu Asp Arg Thr Asn His Val Gly Phe Trp Asn Lys Gln
100 105 110
Trp Ile Lys His Gly Ser Cys Gly Tyr Pro Ala Ile Met Asn Asp Thr
115 120 125
His Tyr Phe Gln Thr Val Ile Asn Met Tyr Ile Thr Gln Lys Gln Asn
130 135 140
Val Ser Glu Ile Leu Ser Lys Ala Lys Ile Glu Pro Leu Gly Ile Gln
145 150 155 160
Arg Pro Leu Val His Ile Glu Asn Ala Ile Arg Asn Ser Thr Asn Asn
165 170 175
Lys Lys Pro Lys Phe Lys Cys Gln Lys Asn Ser Gly Val Thr Glu Leu
180 185 190
Val Glu Val Gly Leu Cys Ser Asp Gly Ser Leu Thr Gln Phe Arg Asn
195 200 205
Cys Pro His Pro Pro Pro Gly Ser Pro Tyr Leu Cys Pro Ala Asp Val
210 215 220
Lys Tyr
225
<210> 3
<211> 603
<212> DNA
<213>Pears (Pyrus spp)
<400> 3
atgtacgatt attttcaatt tacgcagcaa tatcagccag ctgtctgcaa ctccaaacct 60
actccttgta aggatcctcc tgacaagttg ttcacggttc acggtttgtg gccttcaaac 120
ttgaatggac ctcacccaga aaattgcact aatgcaaccg tgaatcctca caggataaaa 180
aatatccaag cccagttgaa aattatttgg ccgaatgtac tcgatcgaac caatcatgta 240
ggcttctgga ataaacagtg gataaaacat ggcagctgtg ggtatcccgc aataatgaac 300
gacacgcatt actttcaaac agtaatcaac atgtacataa cccagaaaca aaacgtctct 360
gaaatactct caaaggcgaa gattgaaccg ttgggaatac aaaggccact ggtgcatatt 420
gaaaatgcca tacggaatag taccaacaat aagaaaccaa aattcaagtg ccaaaagaat 480
tctggggtga ctgaattagt tgaggtcggt ctttgcagcg atggcagctt aacgcagttc 540
agaaattgcc cccacccacc accaggatca ccatatctct gcccggccga tgttaagtat 600
taa 603
<210> 4
<211> 200
<212> PRT
<213>Pears (Pyrus spp)
<400> 4
Met Tyr Asp Tyr Phe Gln Phe Thr Gln Gln Tyr Gln Pro Ala Val Cys
1 5 10 15
Asn Ser Lys Pro Thr Pro Cys Lys Asp Pro Pro Asp Lys Leu Phe Thr
20 25 30
Val His Gly Leu Trp Pro Ser Asn Leu Asn Gly Pro His Pro Glu Asn
35 40 45
Cys Thr Asn Ala Thr Val Asn Pro His Arg Ile Lys Asn Ile Gln Ala
50 55 60
Gln Leu Lys Ile Ile Trp Pro Asn Val Leu Asp Arg Thr Asn His Val
65 70 75 80
Gly Phe Trp Asn Lys Gln Trp Ile Lys His Gly Ser Cys Gly Tyr Pro
85 90 95
Ala Ile Met Asn Asp Thr His Tyr Phe Gln Thr Val Ile Asn Met Tyr
100 105 110
Ile Thr Gln Lys Gln Asn Val Ser Glu Ile Leu Ser Lys Ala Lys Ile
115 120 125
Glu Pro Leu Gly Ile Gln Arg Pro Leu Val His Ile Glu Asn Ala Ile
130 135 140
Arg Asn Ser Thr Asn Asn Lys Lys Pro Lys Phe Lys Cys Gln Lys Asn
145 150 155 160
Ser Gly Val Thr Glu Leu Val Glu Val Gly Leu Cys Ser Asp Gly Ser
165 170 175
Leu Thr Gln Phe Arg Asn Cys Pro His Pro Pro Pro Gly Ser Pro Tyr
180 185 190
Leu Cys Pro Ala Asp Val Lys Tyr
195 200
Claims (10)
1. a kind of pears S7The method of-RNase albumen vivoexpressions, which is characterized in that include the following steps：
A. Isolation from Pear Style total serum IgE is extracted, reverse transcription expands pears S into cDNA, design primer PCR7- RNase genes；
B. structure expression pears S7The pronuclear recombination expression vector of-RNase albumen；
C. the pronuclear recombination expression vector that step b is built is transformed into coli strain Rosetta (DE3), builds S7-
RNase recombinant strains；
D. the S constructed by incubation step c7- RNase recombinant strains are to OD600Reach 0.4-0.6, and after low-temperature treatment, add in
Derivant IPTG, induced expression pears S7- RNase albumen；
E. the pears S of induced expression is purified7- RNase albumen.
2. pears S according to claim 17The method of-RNase albumen vivoexpressions, which is characterized in that in step a, be used for
PCR amplification pears S7The forward primer of-RNase genes is 5 '-CCGCTCGAGATGTACGATTATTTTCAATTTACGCAGC-3 ',
Reverse primer is 5 '-CTAGTCTAGAATACTTAACATCGGCCGGGC-3 '.
3. pears S according to claim 1 or 27The method of-RNase albumen vivoexpressions, which is characterized in that in step a,
PCR amplification pears S7The PCR reaction systems of-RNase genes are：ddH233 μ l of O, upstream and downstream primer each 1.5 μ l, cDNA 1 μ l, 10
×KOD-Plus-PCR buffer 5μl,2mM dNTPs 5μl,25mM MgSO4 2μl,KOD-Plus-DNA
polymerase 1μl；PCR reaction conditions are：Then 94 DEG C of pre-degeneration 2min carry out 94 DEG C of 15s, 60 DEG C of 30s, 68 DEG C of 1min,
Totally 30 cycles, last 68 DEG C of extensions 10min.
4. pears S according to claim 17The method of-RNase albumen vivoexpressions, which is characterized in that in step b, structure
Express pears S7During the pronuclear recombination expression vector of-RNase albumen, used prokaryotic expression carrier is coli expression carrier
PCold-TF, clone's pears S7The insertion point of-RNase genes is between Xho I and Xba I restriction enzyme sites.
5. pears S according to claim 47The method of-RNase albumen vivoexpressions, which is characterized in that PCR product is through 1%
Agarose gel electrophoresis analysis and gel extraction, connect after recovery product Xho I and Xba I restriction enzymes double zyme cuttings
Onto Xho I and the Xba I sites of the coli expression carrier pCold-TF through similary double digestion.
6. pears S according to claim 17The method of-RNase albumen vivoexpressions, which is characterized in that described in step d
Low-temperature treatment to be placed in 5-10min on ice, then stand 40-60 minutes for 15-18 DEG C, the derivant IPTG's is final concentration of
0.5-1.0mmol/L, the condition of the induced expression induce 18-24 hours for 15-18 DEG C.
7. the pears S according to claim 1 or 67The method of-RNase albumen vivoexpressions, which is characterized in that lured in step d
Lead expression pears S7The detailed process of-RNase albumen is：By the S of structure7- RNase recombinant strains are according to 1:50 ratio connects
Kind to 100ml containing 100 μ g/ml ampicillins LB fluid nutrient mediums in, 37 DEG C, 200~250rpm shake cultures stay overnight, so
1 is pressed afterwards:50 ratio is transferred in LB fluid nutrient mediums of the 300ml containing 100 μ g/ml ampicillins, 37 DEG C, 200~
250rpm shake cultures are to bacterium solution OD600Reach 0.4-0.6, be immediately placed in 5-10 minutes on ice, then stand 40-60 for 15-18 DEG C
Minute；Then the derivant IPTG of final concentration of 0.5-1.0mmol/L is added in, 15~18 DEG C, 200~250rpm shake cultures,
Induced expression 18-24 hours；
The pears S of induced expression is purified in step e7The detailed process of-RNase albumen is：After the completion of induced expression, at 4 DEG C
Supernatant is abandoned in 12000rpm centrifugations after ten minutes, collects precipitation, lysate is added in into precipitation, precipitation is resuspended；Bacterium solution uses after resuspension
Ultrasonication, after the completion of ultrasonication, 12000rpm is centrifuged 15 minutes, collects supernatant, and supernatant makes after 0.45 μm of membrane filtration
With affinity chromatography method to pears S7- RNase albumen is purified, and before affinity chromatography medium purification albumen, needs to use
The equilibration buffers of 10 times of column volumes balances chromatography media；Purifying protein is collected, is existed using the super filter tube that combined closure system is 30kDa
It is concentrated under 6000rpm rotating speeds at 4 DEG C, desalination, is finally putting into -80 DEG C and saves backup；
The formula of the lysate is：140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphates, 1.8mM biphosphates
Potassium, 50 × EDTA-free protease inhibitor cocktail III, pH 7.3；
The formula of the equilibration buffer is：500mM sodium chloride, 20mM tri- (methylol) aminomethane, 5mM imidazoles, pH are
7.3。
8. pears S according to claim 17The method of-RNase albumen vivoexpressions, which is characterized in that the pears S7-
RNase albumen n ends carry 6 histidine tags.
9. recombination pears S prepared by the method described in claim 1-87- RNase albumen is preparing pears S as antigen7- RNase is more
Application in clonal antibody.
10. a kind of pears S7The preparation method of-RNase polyclonal antibodies, it is characterised in that：By the method system described in claim 1-8
The recombination pears S obtained7Japan large ear rabbit is immunized as antigen in-RNase albumen, immune for the first time using recombination pears S7- RNase eggs
In vain with injecting experimental rabbit after isometric Freund's complete adjuvant emulsification, with recombination pears S after 3 weeks7- RNase albumen with it is isometric
Second of immunization experiment rabbit after incomplete Freund's adjuvant emulsification, carries out 1 booster immunization every two weeks later, in total 4 times it is immune, most
Primary immunization is taken a blood sample after 7 days afterwards, collects antiserum.
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