CN108484736A - Expression of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application - Google Patents
Expression of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- C12N2770/00011—Details
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- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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Abstract
Then the present invention relates to a kind of expressions of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application to be recombinantly expressed by truncating non-structural protein NS2A, expression is easy after truncation, and expression quantity is high；Recombinant protein is set to be expressed in inclusion body by Optimal Expression condition in expression, convenient for purifying, the albumen of purifying has immunogenicity, the high polyvalent antibody of potency can be obtained after immune animal, and antibody high sensitivity obtained, can be used for tembusu virus non-structural protein NS2A detection and and detect tembusu virus in infection different times non-structural protein NS2A positioning situations of change in the cell, be of great significance to the research of tembusu virus.
Description
Technical field
The invention belongs to biotechnologies, are related to the expression side of tembusu virus non-structural protein NS2A truncated proteins
Method, the truncated protein for further relating to expression and the application in preparing polyclonal antibody.
Background technology
Tembusu virus disease (Tembusu virus disease) is by tembusu virus (Tembusu virus, TMUV)
Caused acute infectious disease, tembusu virus are the virus for belonging to flaviviridae Flavivirus, and main infection aquatic bird causes bleeding
Property oaritis and nervous symptoms, clinical symptoms are mainly loss of appetite or useless exhausted, and weight drastically declines and egg production decline.With
The continuous expansion of China's aviculture scale, tembusu virus cause huge as one of the main virus for threatening China's aviculture
Big economic loss.But there is presently no the suitable specific drugs and therapeutic scheme for treating tembusu virus infection.
Tembusu virus non-structural protein NS2A has important role in the duplication of the gene of virus and the assembly of virus,
But there are no detailed researchs for the function and positioning scenarios up to the present, in relation to non-structural protein NS2A, and use label anti-
Body either has great limitation in research positioning or its function, and overall length NS2A albumen is one and possesses eight transmembrane regions
Transmembrane protein and contain many rare codons, therefore we provide the expression that a kind of truncation scheme is convenient for albumen, makes simultaneously
Standby antibody is capable of the NS2A albumen of the identification overall length of high specific and high sensitivity, and the polyclonal antibody of NS2A detecting
The positioning scenarios of NS2A have important role when DTMUV infects.
Therefore, it is badly in need of a kind of easy expression and the NS2A truncated segments with immunogenicity, the prevention to tembusu virus
It is of great significance with treatment.
Invention content
In view of this, one of the objects of the present invention is to provide a kind of tembusu virus non-structural protein NS2A truncated proteins
Expression；The second object of the present invention is to provide by the tembusu virus non-structural protein of the expression acquisition
NS2A truncated proteins；The third object of the present invention is that providing the tembusu virus non-structural protein NS2A truncated proteins is making
Application in standby anti-tembusu virus non-structural protein NS2A polyclonal antibodies；The fourth object of the present invention, which is to provide, utilizes institute
State anti-tembusu virus non-structural protein NS2A polyclonal antibodies made from tembusu virus non-structural protein NS2A truncated proteins；
The fifth object of the present invention is to provide the anti-tembusu virus non-structural protein NS2A polyclonal antibodies in detection Tan Busu
Application in virus infection.
In order to achieve the above objectives, the present invention provides the following technical solutions：
1, the expression of tembusu virus non-structural protein NS2A truncated proteins, is as follows：
1) using infect tembusu virus duck embryo fibroblasts extract RNA as template, reverse transcription synthesize cDNA, then
PCR amplification is carried out by primer of sequence shown in SEQ ID NO.1 and SEQ ID NO.2, amplification obtains NS2A truncated genes；
2) the NS2A truncated genes that step 1) amplification obtains are connected into expression vector and obtain recombinant expression carrier, then will weighed
Group expression vector converts host expresses bacterium, obtains engineering bacteria, purified to obtain smooth cloth then by engineering bacteria IPTG induced expressions
Soviet Union's virus nonstructural protein NS2A truncated proteins.
Preferably, in step 2), the expression vector is pGEX-4T-1 plasmids.
Preferably, in step 2), the host expresses bacterium is BL21 (DE3).
Preferably, in step 2), when the induced expression is by recombinant to A600=0.6, by final concentration of 0.4
IPTG, induced expression 6~8 hours is added in~0.5mM.
Preferably, the purifying is takes induced expression bacterium solution, and thalline were collected by centrifugation, is then added and is equivalent to bacterium solution volume 1/
Thalline is resuspended in the Tris-HCl that 10 a concentration of 20mM, pH is 8, and resuspended bacterium solution, addition is taken to be equivalent to resuspended bacterium solution volume 1/5
6 × loading buffer, 100 DEG C of heating water baths are denaturalized the PAGE gel electrophoresis with 12% after ten minutes, are used after electrophoresis
The KCL solution of 0.25mMol/L develops the color, gel extraction target fragment, and obtaining tembusu virus NS2A using bag filter truncates egg
In vain.
2, the tembusu virus non-structural protein NS2A truncated proteins obtained by the expression.
3, the tembusu virus non-structural protein NS2A truncated proteins are preparing anti-tembusu virus non-structural protein
Application in NS2A polyclonal antibodies.
4, anti-tembusu virus non-structural protein made from the tembusu virus non-structural protein NS2A truncated proteins is utilized
White NS2A polyclonal antibodies.
5, the anti-tembusu virus non-structural protein NS2A polyclonal antibodies answering in detection tembusu virus infection
With.
The beneficial effects of the present invention are：The invention discloses the tables of tembusu virus non-structural protein NS2A truncated proteins
It is set to be easy expression, and expression quantity is high by truncating non-structural protein NS2A up to method；Make weight by Optimal Expression condition
Histone is expressed in inclusion body, and convenient for purifying, the albumen of purifying has immunogenicity, and potency height can be obtained after immune animal
Polyvalent antibody, and antibody high sensitivity obtained can be used for the detection of tembusu virus non-structural protein NS2A, and examine
Tembusu virus is surveyed in infection different times, the positioning situations of change of non-structural protein NS2A in the cell, therefore to Tan Busu
The research of virus is of great significance.
Description of the drawings
In order to keep the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides following attached drawing and carries out
Explanation：
Fig. 1 is the verification of NS2A truncated proteins expression and time-optimized (A：Truncated expression figure；M is Marker, and 1 is sky
Load induction 6h, 2 be unloaded induction 8h, and 3 be unloaded induction 10h, and 4 is do not induce 6h, and 5 is do not induce 8h, and 6 be not induce 10h, 7
To induce 6h, 8 be induction 8h, and 9 be induction 10h；B is total length expressed figure：M is Marker, and 1 is unloaded induction 6h, and 2 lure for zero load
8h is led, 3 is do not induce 6h, and 4 is do not induce 8h, and 5 is do not induce 10h, and 6 be induction 6h, and 7 be induction 8h, and 8 be induction 10h).
For the sample after induced expression, through western blot verifications, (M marker, the moon compare Fig. 2 to be unloaded, and full bacterium is
The sample of 6h after induction).
Fig. 3 be verifying purpose protein expression position (the moon is unloaded, and full bacterium be the full bacterium after expression, supernatant be it is ultrasonic from
Supernatant after the heart, the precipitation being precipitated as after ultrasound centrifugation includes inclusion body).
Fig. 4 is the albumen (A of verification after purification：The SDS-PAGE figures of albumen after purification, verify the purity of albumen after purification,
B：Whether the WB figures of albumen after purification, be purpose albumen to verify albumen after purification).
Fig. 5 is the result figure of antiserum titre inspection, is the positive when the positive is more than 2 than negative value.
Fig. 6 is to explore positioning (the NS2A NS2A of NS2A in the cell when DTMUV infects using the polyclonal antibody prepared
The positioning of albumen in the cell, DAPI are the positioning of nucleus, and merge is the positioning scenarios after two figures merge).
Specific implementation mode
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
The preparation of embodiment 1, cDNA masterplates
The Multiplying culture of 1.TMUV
TMUV-CQW1 kind poison is inoculated in the fine and close single layer duck embryo fibroblasts (DEF) just grown up to, 37 DEG C of absorption 1h
After abandon virus liquid, then plus containing volume fraction be 2% calf serum and 100IU/mL dual anti-(penicillin, streptomysin) DMEM
Nutrient solution is maintained, is cultivated 24~48 hours for 37 DEG C later.
2. cell total rna extracts
1) DEF (100mL cell bottle) of the TMUV kind poison infected cell lesions (CPE) up to 70% is selected；
2) incline cell culture fluid, and 1ml RNAiso are addedTMCell, room temperature (18~25 DEG C) stoichiometric number point is resuspended in plus
Clock；
3) 100 μ l BCP, manual mixing 30s are added to each sample respectively, place 10min on ice, 4 DEG C, 12000g from
Heart 15min；
4) carefully draw in transparent supernatant to new RNase-free EP pipes, often pipe be added 500 μ l isopropanols, gently on
Under overturn to mixing, be placed at room temperature for 10min, 4 DEG C, 12000g centrifuge 10min；
5) supernatant (noticing that white precipitate is not poured out) is removed, (DEPC water is matched to the ethyl alcohol of former EP pipes addition 1ml 75%
System), turn upside down mixing, 4 DEG C, 7500g centrifugations 5min；
6) supernatant is removed, step 4) is repeated；
7) several minutes of drying at room temperature, until precipitate it is transparent, often pipe be added 20 μ l DEPC water, flick hydrotropy；
8) total serum IgE of 1 μ l extractings is taken to detect its RNA concentration, RNA with 2000 protein nucleic acid concentration detectors of Nanodrop
Quality is weighed with the value of OD260/OD280 between 1.8~2.0.
3. reverse transcription synthesizes the first chains of cDNA
Take the RNase-free PCR pipes of precooling that pre-reaction system is added：2 μ g RNA are (according to the concentration conversion body measured
Product), RNase-free ddH are added in 1 μ l Oligo (dT) 182O supplies 8 μ l, and pipette tips blow and beat mixing, wink from 65 in PCR instrument
DEG C it is incubated 5min, takes out chilling 2min on ice.Continue that 10 μ l, HisCript Enzyme Mix of 2RT Mix are added in former pipe
2 μ l, it is 20 μ l to make the final volume of every pipe, and mixing, wink are from reacting 5min, reacted under the conditions of 50 DEG C under the conditions of 25 DEG C in PCR instrument
45min reacts 5min under the conditions of last 85 DEG C, and the cDNA for taking out synthesis is placed in -80 DEG C of refrigerators and saves backup.
Embodiment 2, TMUV CQW1 truncate NS2A gene fragment amplifications
1. design of primers
According to the CQW1 plants of sequence (Genbank of TMUV for having uploaded GenBank：KM233707), carried in conjunction with pGEX-4T-1
Restriction enzyme site on body designs using Primer 5 and truncates NS2A genetic fragment primers：
NS2A-F：5’-ccgcgtggatccccggaattcCtagtggcagctgcattt-3 ' (SEQ ID NO.1) (under
Scribing line italic is EcoRI restriction enzyme sites)；
NS2A-R：5’-gtcacgatgcggccgctcgaggtggtggtggtggtggtg aaaggagctaaccgtccc-
3 ' (SEQ ID NO.2) (underscore italic is XhoI restriction enzyme sites)；
NS2A-F, NS2A-R truncate NS2A genetic fragments (60-150aa) respectively as upstream and downstream primer amplification, cover
NS2A genes are synthesized from the genetic fragment of 178bp-450bp, primer by Qing Ke Bioisystech Co., Ltd.
The specific primer sequence of primer for designing amplification NS2A full length genes simultaneously is as follows：
NS2Afull-F：5’-ccgcgtggatccccggaattctttcaagggggtggcatgg-3’(SEQ ID
NO.3) (underscore italic is EcoRI restriction enzyme sites)；
NS2Afull-R：5’-gtcacgatgcggccgctcgaggtggtggtggtggtggtgtctccgtgtcactggc
Ttc-3 ' (SEQ ID NO.4) (underscore italic is XhoI restriction enzyme sites)；
2.PCR reacts
PCR reaction systems are as follows：
PCR response procedures are as follows：
Step 1：98 DEG C, 2min；
Step 2：98 DEG C, 10s；59 DEG C, 15s；72 DEG C, 5s；(30 cycles)
Step 3：72 DEG C, 5min.
PCR amplification result is observed with 2% agarose gel electrophoresis；Special purpose band is according to TIANGEN DNA
The specification operating procedure for purifying QIAquick Gel Extraction Kit recycles pcr amplification product, and -20 DEG C save backup.
Embodiment 3, pGEX-4T-NS2A60-150aaRecombined pronucleus expression plasmid construction
Double digestion pGEX-4T-1 empty plasmids, digestion system are as follows：
At 37 DEG C react 15min after, with TIANGEN DNA purify QIAquick Gel Extraction Kit recycling linear DNA fragment.
It usesII One Step Cloning Kit kits, by truncation NS2A genes after purification
Segment is connected on the pGEX-4T-1 empty plasmids after double digestion.Reaction system is as follows：
Note：Most suitable cloning vector usage amount=[0.02 cloning vector base logarithm] ng (0.03pmol)
Most suitable Insert Fragment usage amount=[0.04 Insert Fragment base logarithm] ng (0.06pmol)
It after reaction system reacts 30min with 37 DEG C, is placed on ice, 10 μ l connection products is taken to be transformed into 100 μ l expression bacterial strains
BL21 (DE3) competent cell, picking positive colony bacterium is enlarged culture on Amp plates, and extracting plasmid is sequenced, is recombinated
Plasmid is named as pGEX-4T-1-NS2A.
NS2A full length sequences are connected into pGEX-4T-1 according to above-mentioned same procedure, the recombinant plasmid of acquisition is named as：
pGEX-4T-1-NS2A’。
Embodiment 4, TMUV cut the induced expression of NS2A genetic recombination prokaryotic expression proteins
It will identify correct recombinant plasmid pGEX-4T-1-NS2A and pGEX-4T-1-NS2A ' conversion BL21 (DE3) hosts
Express bacterium, picking monoclonal is inoculated into overnight incubation in the LB liquid medium containing 50 μ g/mL Amp, next day take shake it is overnight
Bacterium solution, which is inoculated in the new LB culture mediums containing 50 μ g/mLAmp, makes the culture solution A after addition bacterium solution600=0.05, continue to train
It supports to A600When ≈ 0.6, isopropyl-B-D- thio-pyrylium galactolipins sweet (IPTG) are added by final concentration of 0.4mM and 0.5mM and lure
Expression is led, respectively in expression 6h, 8h and thalline were collected by centrifugation in 10 hours.It is added 20mM's by 1/10th of original LB amounts
Thalline is resuspended in Tris-HCL (pH=8).The re-suspension liquid of 100 μ L is taken to be added the 6 × albumen loading buffer of 20 μ L, 100 DEG C
Heating water bath, which is denaturalized, carries out 12%SDS-PAGE gel electrophoresises for 10 minutes, and coomassie brilliant blue staining observes expression of results.As a result it weighs
Group plasmid expresses the fusion protein of about 35kDa after induction, and empty carrier group and the group of full length gene containing NS2A do not have respective strap
(Fig. 1).
Embodiment 5, pGEX-4T-NS2A60-150aaThe identification of recombined pronucleus expression albumen
It is analyzed using Western-blotting, steps are as follows：
1) expression albumen is transferred to after SDS-PAGE is separated by electrophoresis on pvdf membrane, with containing 5% (w/v) skimmed milk power
37 DEG C of TBST slowly shakes closing 2 hours；
2) after TBST washs film 3 times (5min/ times), the anti-His of the diluted mouse of TBST containing 5% (w/v) skimmed milk power is added
Antibody, 37 DEG C of incubation 2h；
3) after TBST washs film 3 times (5min/ times), the diluted HRP marks of TBST containing 5% (w/v) skimmed milk power are added
The sheep anti-mouse igg secondary antibody of note, 37 DEG C of incubation 1h；
4) after TBST washs film 3 times (5min/ time), ECL colour reagent boxes develop the color, as a result it is observed that specific
React band (Fig. 2).
Embodiment 6, pGEX-4T-NS2A60-150aaThe purifying of recombined pronucleus expression albumen
It is identified, pGEX-4T-NS2A60-150aaRecombined pronucleus expression albumen expresses (Fig. 3) in bacterial inclusion bodies, therefore will
After inclusion body containing target gene albumen is used for PAGE gel electrophoresis, the KCl solution of the 0.25mMol/L of precooling is used
Colour developing, gel extraction target fragment obtain tembusu virus after purification using bag filter and truncate NS2A genetic recombination protokaryon tables
Up to albumen.Concrete operations are as follows：
1) match glue：Separation gel 4mL, is flattened with absolute ethyl alcohol, and concentration glue 0.8mL is flattened with absolute ethyl alcohol；
2) electrophoresis：The sample of 0.6-1mL is added in every piece of glue；
3) bag filter is handled：Prepare solution：2% NaHCO3, the EDTA of 1mM, constant volume to 1L is put into bag filter after boiling
10min is boiled, is put into after cooked in RO water and is placed in 4 DEG C for use；
4) glue is cut：The KCl solution of 0.25mM is prepared, precooling is used as developing solution, is put into developing solution after glue is peeled, in egg
White place sees a white band, is fitted into after the glue at this is cut in bag filter and Tris- glycine is added to bag filter and delayed
Fliud flushing (one piece of glue 0.5mL) ensures no leakage；
5) electroelution：Tris- glycine buffers are filled into electrophoresis tank, and the bag filter equipped with blob of viscose is put into electrophoresis
Slot keeps bag filter direction vertical with current direction, two hours of 120V, 120V races 2min after reversion electrode, will after electrophoresis
Liquid in bag filter, which is drawn into, to be put -80 DEG C in EP pipes and saves backup.
TMUV after purification is truncated into NS2A prokaryotic expression proteins after SDS-PAGE and Western Blot analyses, as a result
As shown in Figure 4.As a result display successfully obtains destination protein.
It is prepared by the polyclonal antibody that embodiment 7, TMUV truncate NS2A genes
1. zoopery prepares
6~8 week old Kunming mouse 6 is bought, a progress eye frame blood sampling is randomly selected.After (18~25 DEG C) of room temperature places 1h
It moves into 4 DEG C to stand overnight, 4 DEG C, 3000rmp centrifugation 10min detach negative serum, remaining 5 Kunming mouse is as experimental group.
2. purifying protein concentration mensuration
Using the Pierce of Thermo Scientific companiesTMBCA Protein Assay Kit kits are to purifying
The concentration of the truncation NS2A prokaryotic expression proteins of acquisition is measured, and concrete operations are as follows：
1) standard items BSA is diluted to final concentration of 2000 μ g/ml, 1500 μ respectively with elution buffer (being free of urea)
The liquid of g/ml, 1000 μ g/ml, 750 μ g/ml, 500 μ g/ml, 250 μ g/ml and 125 μ g/ml, the volume of each concentration are
40μl；
2) according to required determination sample number and various concentration standard items quantity summation, by A liquid：Liquid=50 B：1 ratio is matched
Working solution processed；
3) 10 μ l are taken to be added in EP pipes respectively the good standard items of above-mentioned dilution and sample to be tested, often pipe adds 200 μ l
Prepared working solution；
4) 37 DEG C of incubation 30min, after cooling, with 2000 protein nucleic acid concentration detectors of Nanodrop in 562nm wavelength
Lower its light absorption value of survey first detects the standard concentration diluted, establishes standard curve.According to standard curve, measuring samples are obtained
Concentration, a concentration of 1mg/mL.
3. immune Kunming mouse prepares TMUV and truncates NS2A gene polyvalent antibodies
1) immune for the first time：According to the protein content of every 100 μ g of mouse, by the albumen of purifying and Freund's complete adjuvant 1:1
Ratio row emulsified, emulsification to Water-In-Oil state instills 1min in water and does not scatter, subcutaneous multi-point injection complete as emulsification
Kunming mouse (200 μ L/ are only)；
2) it is immunized for second：After one exempts from 2 weeks, also according to the protein content of every 100 μ g of mouse, the egg of equivalent emulsification purifying
In vain and Freund's incomplete adjuvant, after emulsification completely, subcutaneous multi-point injection Kunming mouse (200 μ L/ are only)；
3) third time is immune：One exempts from after three weeks, and immune programme is exempted from identical with two；
4) the 4th booster immunization：After one exempts from 4 weeks, immune programme is exempted from identical with two, the 10th day after immune, extracts eye
Ball is largely taken a blood sample, and serum is detached；
Embodiment 8, TMUV truncate the polyclonal antibody titration of NS2A genes
The polyclonal antibody potency that TMUV after purification truncates NS2A genes is measured using indirect ELISA, specific steps are such as
Under：
1) antigen coat：After using sodium carbonate-bicarbonate buffer solution people to be diluted to 10 μ g/mL in albumen after purification as
100 μ L are added per hole for coating buffer, and 4 DEG C, coating is overnight.
2) sealase mark reacting hole：Three times are washed with PBST, 5min/ times, use 5% BSA as confining liquid after patting dry again, often
100 μ L are added in hole, and 37 DEG C are closed 40min, are washed three times, are patted dry with PSBT after closing.
3) detected sample is added：100 μ L will be added per hole after antiserum and negative serum difference doubling dilution, 37 DEG C anti-
1h is answered, is washed three times, is patted dry with PBST after reaction.
4) enzyme labelled antibody is added：The sheep anti mouse secondary antibody by specification of HRP labels is illustrated 1:It is added per hole after 500 dilutions
The dilution of 100 μ L is washed three times with PBST after being placed in 37 DEG C of reaction 1h, is patted dry.
5) substrate reactions liquid is added：TMB- hydrogen peroxide ureas solution need to be used as reaction solution, 100 μ L are added per hole, set
It is protected from light 5min in 37 DEG C
6) reaction is terminated：The terminate liquid that 50 μ L are added in per hole terminates reaction.
7) absorbance is measured：Absorbance is measured under 450nm wavelength after terminating reaction, the absorbance and feminine gender of specimen hole are right
According to ratio be more than 2 potency as antibody, as a result such as Fig. 5.The results show that the polyclonal potency finally obtained is 1：
128000。
The positioning variation of NS2A albumen when embodiment 9, the infection of indirect immunofluorescene assay virus
1) virus infected cell：With 6 orifice plates culture duck embryo fibroblasts, it is all often put into cell climbing sheet in hole, cell is long
It when to suitable density, is infected with DTMUV, 12,24,36,48,60 and 72h after infection takes out creep plate respectively, is used in combination
4% paraformaldehyde is fixed.
2) permeabilization：Sample etc. all time points is all collected together, washed three times with PBST after fixed, then with being pre-chilled
0.22% triton x-100 permeabilization, 4 DEG C of permeabilization 20min are washed three times with PBST again after permeabilization.
3) it closes：30min is closed with BSA37 DEG C of 5%, is washed three times with PBST again after closing.
4) it is incubated primary antibody：With 1%BSA by the anti-NS2A polyclonal antibodies of the mouse of preparation 1:It was incubated at 4 DEG C after 500 dilutions
Night, then washed three times with PSBT.
5) it is incubated secondary antibody：With 1:1000 37 DEG C of diluted FITC- sheep anti mouses secondary antibodies use PBST tri- times after being incubated 2h.
6) core, mounting are contaminated：By DAPI1:It is incubated 10min after 1000 dilutions, mounting after washing three times with PBST.
7) it observes：With fluorescence microscope, green fluorescence indicates the positioning scenarios of NS2A in the cell, blue-fluorescence table
Show the position of core, and take pictures, the results are shown in Figure 6.As a result show that NS2A is first located in nucleus week in DTMUV infection cells
It encloses, as the time of infection increases, NS2A gradually starts being distributed in the cytoplasm of entire cell of dispersivity.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Sichuan Agricultural University
<120>Expression of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ccgcgtggat ccccggaatt cctagtggca gctgcattt 39
<210> 2
<211> 57
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gtcacgatgc ggccgctcga ggtggtggtg gtggtggtga aaggagctaa ccgtccc 57
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccgcgtggat ccccggaatt ctttcaaggg ggtggcatgg 40
<210> 4
<211> 58
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gtcacgatgc ggccgctcga ggtggtggtg gtggtggtgt ctccgtgtca ctggcttc 58
Claims (9)
1. the expression of tembusu virus non-structural protein NS2A truncated proteins, which is characterized in that be as follows：
1) using infect tembusu virus duck embryo fibroblasts extract RNA as template, reverse transcription synthesize cDNA, then with
Sequence shown in SEQ ID NO.1 and SEQ ID NO.2 is that primer carries out PCR amplification, and amplification obtains NS2A truncated genes；
2) the NS2A truncated genes that step 1) amplification obtains are connected into expression vector and obtain recombinant expression carrier, then will recombinate table
Host expresses bacterium is converted up to carrier, obtains engineering bacteria, then by engineering bacteria IPTG induced expressions, purified acquisition Tan Busu diseases
Malicious non-structural protein NS2A truncated proteins.
2. the expression of tembusu virus non-structural protein NS2A truncated proteins according to claim 1, it is characterised in that：
In step 2), the expression vector is pGEX-4T-1 plasmids.
3. the expression of tembusu virus non-structural protein NS2A truncated proteins according to claim 1, it is characterised in that：
In step 2), the host expresses bacterium is BL21 (DE3).
4. the expression of tembusu virus non-structural protein NS2A truncated proteins according to claim 1, it is characterised in that：
In step 2), when the induced expression is by recombinant to A600=0.6, it is added by final concentration of 0.4~0.5mM
IPTG, induced expression 6~8 hours.
5. the expression of tembusu virus non-structural protein NS2A truncated proteins according to claim 1, it is characterised in that：
The purifying is takes induced expression bacterium solution, and thalline were collected by centrifugation, be then added be equivalent to bacterium solution volume 1/10 a concentration of 20mM,
Thalline is resuspended in the Tris-HCl that pH is 8, takes resuspended bacterium solution, and the 6 × loading for being equivalent to resuspended bacterium solution volume 1/5 is added
Buffer, 100 DEG C of heating water baths are denaturalized the PAGE gel electrophoresis with 12% after ten minutes, use 0.25mMol/L's after electrophoresis
KCL solution develops the color, gel extraction target fragment, and tembusu virus NS2A truncated proteins are obtained using bag filter.
6. truncating egg by the tembusu virus non-structural protein NS2A that Claims 1 to 5 any one of them expression obtains
In vain.
7. tembusu virus non-structural protein NS2A truncated proteins described in claim 6 are preparing anti-tembusu virus non-structural protein
Application in white NS2A polyclonal antibodies.
8. utilizing the anti-non- knot of tembusu virus made from tembusu virus non-structural protein NS2A truncated proteins described in claim 6
Structure albumen NS2A polyclonal antibodies.
9. anti-tembusu virus non-structural protein NS2A polyclonal antibodies described in claim 8 are in detection tembusu virus infection
Application.
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| CN113736790A (en) * | 2021-10-14 | 2021-12-03 | 四川农业大学 | sgRNA and cell line with duck hnRNPA3 gene knocked out, and construction method and application thereof |
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| CN113736790A (en) * | 2021-10-14 | 2021-12-03 | 四川农业大学 | sgRNA and cell line with duck hnRNPA3 gene knocked out, and construction method and application thereof |
| CN113736790B (en) * | 2021-10-14 | 2023-05-02 | 四川农业大学 | sgRNA (ribonucleic acid) for knocking out duck hnRNPA3 gene, cell line, construction method and application thereof |
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