CN106906234A - One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector - Google Patents
One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector Download PDFInfo
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- CN106906234A CN106906234A CN201710091988.0A CN201710091988A CN106906234A CN 106906234 A CN106906234 A CN 106906234A CN 201710091988 A CN201710091988 A CN 201710091988A CN 106906234 A CN106906234 A CN 106906234A
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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
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- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
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Abstract
The present invention relates to phytovirology and molecular biology, there is provided one can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector, the entitled pMAL WYMV NIb of the plasmid vector, NIb albumen coded sequences containing WYMV Huangchuans separator, the plasmid can be utilized for the prokaryotic expression of NIb albumen, the replication in vitro system of NIb mediations is set up using the NIb albumen of purifying, can be used for the research of WYMV genome duplications regulation and control.
Description
Technical field
The present invention relates to phytovirology and molecular biology, there is provided one can prokaryotic expression WYMV pool
The plasmid vector of river separator NIb albumen.
Background technology
Wheat yellow mosaic reports that its cause of disease mainly includes WYMV (Wheat in nineteen twenty-seven first in Japan
Yellow mosaic virus, WYMV) and Chinese wheat mosaic virus (Chinese wheat yellow virus, CWMV),
The distributed areas of wherein WYMV are relatively broad.After 1980, China's Yangtze river basin each province area of wheat is also reported successively
Its harm of road, WYMV is widely distributed in China, and Yangtze river basin each province and Shandong, Henan, Shaanxi etc. save
Part is all distributed, and serious harm is constituted to wheat growth, development.With other Genus ymovirus (Bymovirus) virus
Equally, WYMV is that the Polymyxa Graminis (Polymyxa graminis) belonged to by many Acarasiales of Plasmodiophorales are propagated in the case of nature.This
Plant fungi and produce sporosorus, sporosorus resistance is extremely strong, and can at least survive 20-25 in soil.
WYMV particles are in bending, and length is divided into two kinds of 300 and 600nm.WYMV is diad genome, by two lines
Property single stranded positive-sense RNA composition.The total lengths of RNA 1 about 7.6kb, RNA 2 total length about 3.6kb, be separately encoded a 270kDa and
The polyprotein of 100kDa.There is covalently bound VPg at the 5 ' ends of WYMV RNA, and there is poly (A) tail at 3 ' ends.Wherein RNA1 is compiled
The NIb albumen of code has the activity of the RNA polymerase (RdRp) of RNA dependences, participates in the genome duplication of WYMV, and genome is multiple
Level processed directly determines that WYMV is pathogenic in plant.Accumulation of the RNA virus in cell body and genome duplication, albumen
Translation even virus particle package is relevant, and the regulation and control of independent studies genome duplication need to set up replication in vitro system.But
Research system there is presently no WYMV replication in vitro is, it is necessary to by prokaryotic expression and purify NIb albumen and set up replication in vitro
System, but homology of the coded sequence of NIb albumen in different WYMV separators is higher, and study at present only by homologous
Derivation knows that it should possess RNA polymerase (RdRp) activity of RNA dependences, but how to regulate and control WYMV genome duplications for it
Mechanism it is but still unclear, particularly possess RdRp activity albumen easily cause activity in prokaryotic expression, purge process
Lose, thus replication in vitro system cannot be set up according to prior art.
The content of the invention
The invention provides there is provided one can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid
Carrier, the entitled pMAL-WYMV-NIb of the plasmid vector, the NIb albumen coded sequences containing WYMV Huangchuans separator, the plasmid
The prokaryotic expression of NIb albumen is can be utilized for, the replication in vitro system of NIb mediations is set up using the NIb albumen of purifying, can be used for
The research of WYMV genome duplications regulation and control.
Inventor disclose first one can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid carry
Body, the entitled pMAL-WYMV-NIb of the plasmid vector, the coded sequence containing WYMV Huangchuans separator NIb albumen, its nucleotides
As shown in Seq ID No.1, the amino acid sequence of coding is as shown in Seq ID No.2 for sequence.
In order to obtain the plasmid, inventor is template with the plasmid containing NIb albumen coded sequences first with PCR method
The coded sequence of WYMV Huangchuans separator NIb albumen is obtained, then the coded sequence of NIb albumen is inserted using digestion and connection
To in pMAL-C2X (NEB companies) carrier, the prokaryotic expression plasmid vector containing WYMV NIb albumen is obtained,
The entitled pMAL-WYMV-NIb of the plasmid vector, wherein the coded sequence containing WYMV Huangchuans separator NIb albumen.
Inventor carries out prokaryotic expression and purifies NIb fusion proteins, profit by affinity chromatography method using the plasmid vector
Replication in vitro experiment is carried out with the NIb fusion proteins of purifying, genome 3'UTR can be successfully recognized, and is that template is produced mutually with it
Mend chain.Thus prove that the replication in vitro system of WYMV NIb mediation is successfully established, by for the duplication study on regulation of WYMV with
The internal pathogenic mechanism parsing offer system and data of WYMV are supported.
More specifically, inventor disclose this can prokaryotic expression WYMV Huangchuan separator NIb albumen
The construction method of plasmid vector pMAL-WYMV-NIb is as follows：
According to the sequence (accession number of WYMV Huangchuans separator (WYMV-HC) RNA1 announced in GenBank：AF067124)
With the sequence (accession number of RNA2：AF041041), design primer (primer information is shown in Table 1) amplification NIb coded sequence and other
Fragment (corresponds to the 5' ends of RNA1 and RNA2, intermediate sequence and 3' ends, for preparing corresponding RNA as replication in vitro
Template)；
With the plasmid pUWR1-1 (Dong Jiahong, 2004, thesis for the doctorate is recorded) containing WYMV Huangchuans separator RNA1 complete sequences
It is template, the NIb coded sequences that performing PCR amplification obtains WYMV Huangchuans separator is entered in the presence of Taq enzyme；This PCR primer is passed through
DNA QIAquick Gel Extraction Kits carry out BamH I and Sal I (Takara) double digestion, the pMAL-C2X carriers with same double digestion after reclaiming
(NEB companies) connects；Connection product converts bacillus coli DH 5 alpha, through bacterium colony PCR, plasmid enzyme restriction identification and DNA sequencing, is contained
There is the positive colony pMAL-WYMV-NIb of WYMV Huangchuans separator NIb albumen coded sequences.
The primer used in the present invention of table 1
| Primer name | Sequence(5'to 3') | PCR fragment | Seq ID No |
| WYM-R1-4921-BamHI-F | NIb | Seq ID No.3 | |
| WYM-R1-6504-SalI-R | NIb | Seq ID No.4 | |
| WYM-T7-R-1-F | ATtaatacgactcactataGGAAAATAAAACCACCACAAAC | RNA1 5'160nt | Seq ID No.5 |
| WYM-R1-162-R | ATCTCGAAGGGAGTGAAGAA | RNA1 5'160nt | Seq ID No.6 |
| WYM-R1-T7-390-F | ATtaatacgactcactataGCAGCCCCGCACATCTCATGC | RNA1int 160nt | Seq ID No.7 |
| WYM-R1-549-R | ATCTCGAGTGTGGTTGTTCCAAAGTG | RNA1int 160nt | Seq ID No.8 |
| WYM-T7-R1-7387-F | ATtaatacgactcactataGGACCATAACCCCCCTCCGC | RNA1 3'260nt | Seq ID No.9 |
| WYM-R1-7644-R | ATATTACCTTCTGGTACTCGTAAACGCAC | RNA1 3'260nt | Seq ID No.10 |
| WYM-T7-R-2-F | ATtaatacgactcactataGGAAAATAAAACCACCACAAAC | RNA2 5'170nt | Seq ID No.11 |
| WYM-R2-170-R | ATAGAAGCGGAAAGAACTAGG | RNA2 5'170nt | Seq ID No.12 |
| WYM-R2-T7-390-F | ATtaatacgactcactataGCTTGCTGCCAACCTTCGT | RNA2int 160nt | Seq ID No.13 |
| WYM-R2-549-R | ATGAAGGTTCGGACTGGTTGT | RNA2int 160nt | Seq ID No.14 |
| WYM-T7-R2-3460-F | ATtaatacgactcactataGGGCACCGCGCGTTGTGCCACG | RNA2 3'190nt | Seq ID No.15 |
| WYM-R2-3652-R | ATGTCACATTTCCTGTGTACAAAAGCTGG | RNA2 3'190nt | Seq ID No.16 |
Note：Restriction enzyme site represents that T7 promoter sequence small letter italics are represented with underscore；int：Represent intermediate sequence
nt：Nucleotides
After obtaining plasmid pMAL-WYMV-NIb, inventor carries out prokaryotic expression using the plasmid, and by affinity chromatography side
The NIb fusion proteins of method purification of soluble, and then the replication in vitro system of NIb mediations is set up, test result indicate that NIb albumen can
3 ' the terminal fragments of specific recognition WYMV RNA1 and RNA2, produce its complementary strand.Key step includes：
1) the pre- expression of the NIb albumen of fusion MBP：
By the competent cell of plasmid pMAL-WYMV-NIb conversion Escherichia coli Rossetta, using SDS-PAGE electrophoresis
Inducing action of the different IPTG concentration to destination protein (the NIb albumen with MBP labels, be named as MBP-NIb) is tested, is confirmed most
Optimization IPTG induced concentrations are 0.4mM.
2) soluble analysis of MBP-NIb albumen：
On the premise of confirming that the inducible destination proteins of IPTG are expressed, the solubility of analysis purpose albumen.And test difference
Inducing temperature confirms that optimal inducing temperature is 20 DEG C, it is ensured that soluble purpose for the influence of soluble destination protein ratio
The ratio of albumen can meet follow-up affinitive layer purification.
3) Amylose Resin affinity column chromatographies purifying (MBP labels) of soluble protein：
Using Amylose Resin (NEB companies, 0111207) preparative chromatography post, through filling post, balance, loading, wash-out etc.
Step purification of soluble MBP-NIb albumen, for follow-up replication in vitro active testing.
4) the replication in vitro system of NIb mediations：
Different fragments (including 5' ends, intermediate sequence and the 3' of WYMV RNA1 and RNA2 are prepared by in-vitro transcription first
End), the catalytic capability of the MBP-NIb albumen for different RNA fragments of purifying is then tested, final replication in vitro experiment shows
The 3' terminal fragments of MBP-NIb protein-specifics identification WYMV RNA1 and RNA2, and catalyze and synthesize its complementary strand.
In sum, can prokaryotic expression WYMV Huangchuan separator NIb albumen present invention obtains one
Plasmid vector, the entitled pMAL-WYMV-NIb of the plasmid vector, the NIb albumen coded sequences containing WYMV Huangchuans separator should
Plasmid can be utilized for the prokaryotic expression of NIb albumen, and the replication in vitro system of NIb mediations is set up using the NIb albumen of purifying, can
For the research of WYMV genome duplications regulation and control.
Brief description of the drawings
Fig. 1 is the PCR primer electrophoresis result schematic diagram of WYMV Huangchuans separator NIb albumen coded sequences,
Wherein M is the DNA Ladder 5000 of SinoBio companies, and PCR primer should be 1600bp；
Fig. 2 is the digestion qualification result electrophoresis schematic diagram of plasmid pMAL-WYMV-NIb,
Wherein M is the DNA Ladder 5000 of SinoBio companies, and carrier and NIb coding fragments are respectively obtained after digestion；
Fig. 3 is the pre- expression result schematic diagram of plasmid pMAL-WYMV-NIb prokaryotic expressions,
Wherein M is the Blue Plus II Protein Marker of Transgen companies.There is about 100kDa after IPTG inductions
Differential protein expression, its molecular weight is consistent with the molecular weight sum of NIb (58kD) and MBP labels (40kD)；
Fig. 4 is the soluble analysis result schematic diagram of the NIb albumen for merging MBP,
Wherein M has about 100kDa for Blue Plus II the Protein Marker, IPTG of Transgen companies after inducing
Differential protein expression, the ratio of destination protein (MBP-NIb) in ultrasonication post analysis visible supernatant precipitation, wherein 20
DEG C induction can reach 20% or so, and 37 DEG C of inductions are lower is less than 1%；
Fig. 5 is the fusion protein mediated replication in vitro result schematic diagrams of WYMV Huangchuans separator NIb,
Replication in vitro experiment is that analysis purifying obtains whether NIb fusion proteins have the specificity of catalysis activity and its identification
Template,
As seen from the figure, MBP-NIb can specific recognition RNA1 3' fragments and the 3' fragments of RNA2, catalysis produces respective
Complementary strand；The strong and weak also explanation MBP-NIb of simultaneously visible wherein reaction band is for the different identification fragment (3 ' fragments of RNA1
With the 3' fragments of RNA2) catalytic efficiency it is different.
Specific embodiment
Other use prior art unless otherwise specified in the embodiment of the present invention；
Embodiment 1
Can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector pMAL-WYMV-NIb structure
Construction method, comprises the following steps that：
According to the sequence (accession number of WYMV Huangchuans separator (WYMV-HC) RNA1 announced in GenBank：
AF067124), primer WYM-R1-4921-BamHI-F and WYM-R1-6504-SalI-R are designed, this PCR amplification to primer is produced
Thing is the coded sequence of WYMV Huangchuans separator NIb albumen.
With the plasmid pUWR1-1 (Dong Jiahong, 2004, thesis for the doctorate is recorded) containing WYMV Huangchuans separator RNA1 complete sequences
Be template, enter performing PCR in the presence of Taq enzyme (Quan Shijin, AP221-01), primer pair be WYM-R1-4921-BamH1-F and
WYM-R1-6504-Sal1-R；
PCR conditions：94 DEG C of predegeneration 5min；94 DEG C of 40s denaturation, 51 DEG C of annealing 40s, 72 DEG C of extension 2min, 30 circulations；
72 DEG C of extension 10min；4 DEG C of preservations；Amplification obtains the NIb coded sequences of WYMV Huangchuans separator；
This PCR primer carries out BamH I and Sal I after being reclaimed through DNA QIAquick Gel Extraction Kits (Quan Shijin, EG101-02)
(Takara) double digestion, with the pMAL-C2X of same double digestion in the presence of T4DNA ligases (Takara, 2011A) 16
Overnight connected at DEG C；Connection product converts the competent cell of bacillus coli DH 5 alpha, applies with 100mg/mL ampicillins
LB flat boards, confirm to obtain positive colony pMAL-WYMV-NIb through bacterium colony PCR screenings, digestion identification and DNA sequencing.
Interpretation of result：The electrophoresis result of PCR product shows that PCR primer is about 1.6kb (shown in Fig. 1), with WYMV
Coded sequence (1584 nucleotides) length of NIb is coincide substantially.This PCR primer is inserted together after BamH I and Sal I digestions
In pMAL-C2X of the sample through BamH I and Sal I digestions, after bacterium colony PCR primary dcreening operations, upgrading grain carries out digestion identification to converted product,
From the identification of BamH I and Sal I double digestions, digestion result shows the digestion band of about 1.6kb, and remaining digestion piece
Duan great little is for about 6.7kb, the gene order containing WYMV-NIb in the basic confirmation plasmid (shown in Fig. 2)；And by plasmid
DNA sequencing is finally identified.
Embodiment 2
The prokaryotic expression of WYMV Huangchuans separator NIb albumen：Concrete operation step is as follows：
Merge the pre- expression of the NIb albumen of MBP：
The competent cell of plasmid conversion Escherichia coli Rossetta, applies with 100mg/mL ampicillins first
LB flat boards；Rossetta bacterial strain of the picking containing recombinant plasmid is placed in the LB fluid nutrient mediums of 5ml, 37 DEG C, 200rpm incubators
Middle overnight incubation；The bacterium solution of incubated overnight is taken by 1:100 ratio is added in the fresh LB fluid nutrient mediums of 30mL, 37 DEG C,
200rpm, culture to OD600To between 0.4-0.6；The bacterium solution of fresh cultured is put on ice for 10min, dactylethrae is dispensed into
In, often pipe 3ml, 6 manages totally.IPTG is added to be respectively 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM to final concentration.
37 DEG C, 200rpm cultures 4h；1.5ml Induced cultures are taken in 1.5mL centrifuge tubes, 13000rpm is centrifuged 30s,
Collects thalline, removes supernatant.
Sample treatment：100 μ L ddH are added in bacterium to collection2O, vibration suspends, and adds isometric 2 × SDS albumen
Sample-loading buffer.Boiling water bath 5min, stands 5min on ice.
Run glue and detection：Above-mentioned sample is respectively taken into 10 μ L point samples, 120V electrophoresis is run out of completely to bromophenol blue index strip.Dye
Color：Separation gel is placed in culture dish, adds dyeing liquor, preservative film to seal, middle fire heating 15s high, takes out, water in micro-wave oven
Yawing bed shake 10min, outwells dyeing liquor, and dyeing liquor is reclaimed.Decolourize：Destainer is added, the middle fire heating high in micro-wave oven
15s, horizontal shaker shake 10min, removes destainer, adds ddH2O, horizontal shaker vibrates clean to decolourizing.
Interpretation of result：SDS- polyacrylamide gel electrophoresis results show, with negative control (IPTG concentration is 0mM) phase
Than when IPTG final concentrations are respectively 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, having molecular weight about in induction bacterium solution
The differential protein of 100kDa is expressed (shown in Fig. 3), and this molecular weight of albumen coincide substantially with the size of the NIb albumen for merging MBP.Afterwards
The final concentration of 0.4mM of IPTG in use in continuous experiment.
Embodiment 3
Merge the soluble analysis of the NIb albumen of MBP：
According to the basic step of prokaryotic expression, under 0.4mM IPTG concentration, 10mL bacterium solutions are induced, 20 DEG C, 200rpm shakes
Overnight incubation is swung, and control group is set；The bacterium solution that will have been induced is concentrated in 1.5mL centrifuge tubes, different temperatures inductive condition point
Shou Ji not 1mL bacterium solutions (sample treatment is standby)；Microorganism collection thing is suspended again with 1.0mL albumen buffer solutions, and adds PMSF
(final concentration of 0.1mM).Note：Since this step, all operations are carried out on ice.
Ultrasonic disruption (condition：400W, 100 times, work 4s, is spaced 4s) carried out on ice.
13000rpm, 15min, 4 DEG C, supernatant and precipitation are separated.
The microorganism collection thing that sample treatment induces 1mL and the microorganism collection thing that 1ml is not induced are separately added into 100 μ L water,
Acutely vibration adds isometric 2 × SDS albumen sample-loading buffers after suspending.For the precipitation after ultrasonication, treatment side
Method is ibid；For the supernatant after ultrasonication, isometric 2 × SDS albumen sample-loading buffers are added.By the boiling of above-mentioned sample
Water-bath 5min, stands 5min on ice.
Run the operation that glue and detection detailed process are shown in embodiment 2；Final testing goal albumen is in supernatant precipitation
Ratio.
Interpretation of result：SDS- polyacrylamide gel electrophoresis results show, the destination protein base of induced expression at 37 DEG C
This is all present in the form of the inclusion body in precipitation, and destination protein content ratio is extremely low in supernatant, less than 1%；And at 20 DEG C
Relative scale of the destination protein of induced expression in supernatant is significantly improved, and 20% or so (shown in Fig. 4) is can reach, after can meeting
Continuous affinity chromatography experiment.
Embodiment 4
The Amylose Resin affinity column chromatographies purifying (being applied to MBP labels) of soluble protein：
According to the method for above-mentioned prokaryotic expression, 0.4mM IPTG induce 500mL bacterium solutions in 20 DEG C, slow with column equilibration after centrifugation
The resuspended thalline of fliud flushing, and carry out ultrasonic disruption；Supernatant is centrifuged with 0.45 μm of water system membrane filtration to 50ml after centrifugation
Guan Zhong, puts standby on ice, waits chromatographic column.
Affinity chromatography (is carried out) in 4 DEG C of environment, fills post：Take 2mL Amylose Resin (NEB companies, 0111207) filler
Add in chromatographic column, let the liquid out；Balance：With the 5-10 times of equilibration buffer chromatographic column of column volume；Loading：By sample
By chromatographic column；Washing：With the 5-10 times of equilibration buffer solution chromatographic column of column volume, efflux is collected；Wash-out：Washed with 3mL
De- liquid wash-out destination protein (collect eluent with centrifuge tube, often the μ l of pipe 500, and make marks, be positioned on ice).
Detection：The eluent of supernatant, cleaning solution and various concentrations maltose respectively takes 10 μ l and carries out SDS-PAGE electrophoresis,
Detect the quality of purifying protein.Finally, by the solution storage containing destination protein at -80 DEG C.
Level pad (Amylose Resin) employed in it：20mM Tris-Cl (pH 8.0), 25mM NaCl,
1mM EDTA (pH 8.0), 10mM β-mercaptoethanol, 10%glycerol；
Elution buffer (Amylose Resin)：0.5M maltose mother liquors are added in level pad, is made
Maltose final concentrations reach 10mM.
Embodiment 5
In-vitro transcription prepares the different RNA fragments of WYMV RNA1 and RNA2：
With containing WYMV Huangchuans separator RNA1 and RNA2 complete sequence plasmid pUWR1-1 and pUWR2-1 (Dong Jiahong,
2004, thesis for the doctorate is recorded) it is template, the PCR fragment (the primer obtained corresponding to WYMV diverse locations is expanded by PCR
It is shown in Table 1) with amplified fragments, and gel extraction is carried out using DNA QIAquick Gel Extraction Kits (Quan Shijin, EG101-02).Using above PCR
Product is template, and in-vitro transcription reaction (reaction system is carried out in the presence of t7 rna polymerase (Pu Luomaige, P2075)：PCR
4.0 μ g, 100mM DTT of product, 12.0 μ l, 5mM rNTPs 12.0,24.0 μ l, Rnase enzyme inhibitors of μ l, 5 × T7Buffer
0.5 μ l, t7 rna polymerase 2.0 μ l, H2O is mended to 120 μ l), 37 DEG C of reaction temperature, reaction time 2.5h；Product is passed through
The corresponding RNA fragments of glue purification are cut after 1.5% agarose gel electrophoresis.After purification RNA fragments as replication in vitro template
It is standby.
Embodiment 6
The replication in vitro system of WYMV NIb mediations：
Replication in vitro reaction (reaction system is carried out in the presence of MBP-NIb albumen：RNA templates 1.0 μ g, 1M Tris-
Cl (pH 8.2) 2.5 μ l, 1M MgCl2μ l, the 10mg/ml yeast tRNA of 0.5 μ l, 1M DTT, 0.5 μ l, 1M KCl 5
1.75 μ l, 20mM ATP, 2.5 μ l, 20mM GTP, 2.5 μ l, 20mM CTP, 2.5 μ l, 1mM UTP 0.5 μ l, α-32P-UTP
0.5 μ l, NIb fusion protein 12.5 μ l, H2O is mended to 50 μ l), 20 DEG C of reaction temperature is incubated 1h-1.5h；Reaction is added after terminating
70μL ddH2O is mixed, and adds 120 μ L phenol/imitative (0.5mL phenol/0.5mL chloroforms/0.04mL 0.5M EDTA/0.01mL10%
SDS mixtures) extracting；Centrifuged supernatant adds 2.4 times of NH of volume4Ac/isopropanol(5M NH4Ac:isopropanol
=1:5), mix, -20 DEG C of overnight precipitations, precipitate through being centrifuged, washing, dry；10 μ L RNA loading buffer are added, so
Afterwards in 5%PAGE glue (urea containing 8M), 1500mA electrophoresis 1-2h；Dry glue, the screen imaging of pressure phosphorus.
Interpretation of result：Replication in vitro result such as Fig. 5 shows that MBP-NIb albumen can specific recognition WYMV RNA1 and RNA2
3' end sequences, produce its complementary strand.Wherein the strong and weak also explanation MBP-NIb of reaction band is for different specific recognition pieces
The catalytic efficiency of section (3 ' fragments of RNA1 and the 3' fragments of RNA2) is different.Illustrate that this passes through plasmid pMAL-WYMV-NIb protokaryons
The MBP-NIb albumen expressed and purify can mediate the replication in vitro system for WYMV.
<110>Shandong Agricultural University
<120>One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector
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tat gcg aca aac ctg aaa gct cca cac acg gta gga atc aac aaa ttc 768
Tyr Ala Thr Asn Leu Lys Ala Pro His Thr Val Gly Ile Asn Lys Phe
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agc aga ggc tgg gaa atg ctc cac gac aag ctc aac cgc cca ggt tgg 816
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ctt cac ggc agt ggc gat ggg tca cga ttc gat agt tca att gac cct 864
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290 295 300
gag cac cat cgc gca atc gac cta ata tac gac gag att ctc aac acc 960
Glu His His Arg Ala Ile Asp Leu Ile Tyr Asp Glu Ile Leu Asn Thr
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aac agc ggg cag cca agc acg gtc gta gac aac aca ctt gtt ctc atg 1056
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aaa ttt gcc att tcc cca gaa ttc gac gcc gag ttc ggt cac gac ttt 1200
Lys Phe Ala Ile Ser Pro Glu Phe Asp Ala Glu Phe Gly His Asp Phe
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tca ccg gag ctc aca gag cta ggt ttg acg tac gag ttt gac gac ata 1248
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aca gat gac atc tgt gca aac ccc tac atg tcc ctg acc atg gtc cgc 1296
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act ccc ttt gga att ggg ttc tct ttg ccc gtg gaa cgc att gta gca 1344
Thr Pro Phe Gly Ile Gly Phe Ser Leu Pro Val Glu Arg Ile Val Ala
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caa tag 1590
Gln
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Met Ala Ser Asp Thr Leu Ser Lys Ile Asn Asn Ser Ile Leu Gly Phe
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Gly Ser Asp Leu Lys Gly Gln Leu Val Gln Pro Val Thr Pro Ala Leu
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Arg Thr Arg Phe Glu Ala Leu Phe Gly Gly Gly Ser Phe Glu Leu Val
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Gly Thr Met Asn Lys Gly Leu Ile Asp Lys His Ile Ile Val Gly Glu
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Asn Asp Asn Val His Asp Phe Met Arg Glu His Pro Thr Phe Ala Trp
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Leu Gln Gly Phe Met Asp Glu Tyr Ala Pro Ser Val Leu Asn Tyr Ser
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Ala Tyr Tyr Lys Asp Leu Cys Lys Tyr Asn Arg Lys Lys His Gln Leu
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Ser Phe Asn Pro His Glu Leu Arg Ser Ala Thr Ala Gly Met Ile Arg
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Met Leu Glu Asp Ala Gly Leu Thr Gln Gly Asp Val Arg Thr Pro Gln
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Gln Val Val Ser Asp Met Gln Trp Asn Thr Ser Ala Gly Pro Ser Tyr
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Gln Gly Lys Lys Arg Asp Val Cys Ala His Leu Ser Glu Gln Glu Val
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Ser Leu Phe Ala Met Lys Phe Tyr Val Asp Asp Phe Asn Lys Lys Phe
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Tyr Ala Thr Asn Leu Lys Ala Pro His Thr Val Gly Ile Asn Lys Phe
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Ser Arg Gly Trp Glu Met Leu His Asp Lys Leu Asn Arg Pro Gly Trp
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Leu His Gly Ser Gly Asp Gly Ser Arg Phe Asp Ser Ser Ile Asp Pro
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Phe Phe Phe Asp Ile Ile Lys Glu Ile Arg Lys His Phe Leu Pro Val
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Glu His His Arg Ala Ile Asp Leu Ile Tyr Asp Glu Ile Leu Asn Thr
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Asn Ile Cys Leu Ala Asn Gly Met Val Ile Arg Lys Asn Val Gly Asn
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Asn Ser Gly Gln Pro Ser Thr Val Val Asp Asn Thr Leu Val Leu Met
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Val Ser Phe Leu Tyr Ala Tyr Ile His Lys Thr Gly Asp His Met Leu
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Lys Lys Leu Asn Asp Arg Phe Val Phe Val Cys Asn Gly Asp Asp Asn
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Ser Pro Glu Leu Thr Glu Leu Gly Leu Thr Tyr Glu Phe Asp Asp Ile
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Thr Asp Asp Ile Cys Ala Asn Pro Tyr Met Ser Leu Thr Met Val Arg
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Thr Pro Phe Gly Ile Gly Phe Ser Leu Pro Val Glu Arg Ile Val Ala
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Gly Ile Ser Ala Ile Tyr Glu Ser Phe Asn Thr Pro Lys Leu Phe Lys
465 470 475 480
Ser Val Tyr Ala Tyr Leu Leu Trp Leu Thr Glu Glu His Gly Ser Asp
485 490 495
Ile Leu Ala Ala Met Thr Gly Thr Ala Thr Ala Leu Pro Ile Pro Ser
500 505 510
Met Leu Asp Val Tyr Arg Leu His Tyr Gly Asp Ser Ser Ile Glu Leu
515 520 525
Gln
529
<210>3
<211>29
<212>DNA
<213>Artificial sequence
<400>3
atggatccat ggcctccgac actctcagc 29
<210>4
<211>32
<212>DNA
<213>Artificial sequence
<400>4
atgtcgacga tagtttggag ctcaatgctg ct 32
<210>5
<211>41
<212>DNA
<213>Artificial sequence
<400>5
attaatacga ctcactatag gaaaataaaa ccaccacaaa c 41
<210>6
<211>20
<212>DNA
<213>Artificial sequence
<400>6
atctcgaagg gagtgaagaa 20
<210>7
<211>40
<212>DNA
<213>Artificial sequence
<400>7
attaatacga ctcactatag cagccccgca catctcatgc 40
<210>8
<211>26
<212>DNA
<213>Artificial sequence
<400>8
atctcgagtg tggttgttcc aaagtg 26
<210>9
<211>39
<212>DNA
<213>Artificial sequence
<400>9
attaatacga ctcactatag gaccataacc cccctccgc 39
<210>10
<211>29
<212>DNA
<213>Artificial sequence
<400>10
atattacctt ctggtactcg taaacgcac 29
<210>11
<211>41
<212>DNA
<213>Artificial sequence
<400>11
attaatacga ctcactatag gaaaataaaa ccaccacaaa c 41
<210>12
<211>21
<212>DNA
<213>Artificial sequence
<400>12
atagaagcgg aaagaactag g 21
<210>13
<211>38
<212>DNA
<213>Artificial sequence
<400>13
attaatacga ctcactatag cttgctgcca accttcgt 38
<210>14
<211>21
<212>DNA
<213>Artificial sequence
<400>14
atgaaggttc ggactggttg t 21
<210>15
<211>41
<212>DNA
<213>Artificial sequence
<400>15
attaatacga ctcactatag ggcaccgcgc gttgtgccac g 41
<210>16
<211>29
<212>DNA
<213>Artificial sequence
<400>16
atgtcacatt tcctgtgtac aaaagctgg 29
Claims (1)
1. one can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector, it is characterised in that：The matter
Grain container name be pMAL-WYMV-NIb, the coded sequence containing WYMV Huangchuans separator NIb albumen, its nucleotide sequence is such as
Shown in Seq ID No.1, the amino acid sequence of coding is as shown in Seq ID No.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710091988.0A CN106906234A (en) | 2017-02-21 | 2017-02-21 | One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710091988.0A CN106906234A (en) | 2017-02-21 | 2017-02-21 | One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106906234A true CN106906234A (en) | 2017-06-30 |
Family
ID=59207952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710091988.0A Pending CN106906234A (en) | 2017-02-21 | 2017-02-21 | One can prokaryotic expression WYMV Huangchuan separator NIb albumen plasmid vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106906234A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486148A (en) * | 2018-03-29 | 2018-09-04 | 山东农业大学 | The weak malicious mutant plasmids carriers of cucumber mosaic virus RNA2 of the genetic fragments of PDS containing tobacco and its application |
| CN110592128A (en) * | 2019-08-30 | 2019-12-20 | 山东农业大学 | Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof |
| CN114605504A (en) * | 2022-02-25 | 2022-06-10 | 宁波大学 | Wheat yellow mosaic virus 14K protein capable of inducing plant cell necrosis and application thereof in virus resistance |
-
2017
- 2017-02-21 CN CN201710091988.0A patent/CN106906234A/en active Pending
Non-Patent Citations (8)
| Title |
|---|
| ANINDYA R等: "Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase", 《VIROLOGY》 * |
| CHEN J等: "登录号：AJ239037.1", 《GENBANK》 * |
| GOVIND KUNDURI等: "Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication", 《FEBS OPEN BIO》 * |
| LI YI等: "Identification acid characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus", 《JOURNAL OF VIROLOGY》 * |
| RAJENDRAN KS等: "Comparison of turnip crinkle virus RNA-dependent RNA polymerase preparations expressed in Escherichia coli or derived from infected plants", 《JOURNAL OF VIROLOGY》 * |
| YU J等: "登录号：AF067124.2", 《GENBANK》 * |
| 唐伟: "小麦黄花叶病毒全序列分析及蛋白互作和亚细胞定位", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 董家红: "小麦黄花叶病毒侵染性cDNA克隆及细胞侵染体系的建立", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486148A (en) * | 2018-03-29 | 2018-09-04 | 山东农业大学 | The weak malicious mutant plasmids carriers of cucumber mosaic virus RNA2 of the genetic fragments of PDS containing tobacco and its application |
| CN110592128A (en) * | 2019-08-30 | 2019-12-20 | 山东农业大学 | Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof |
| CN110592128B (en) * | 2019-08-30 | 2022-02-11 | 山东农业大学 | Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof |
| CN114605504A (en) * | 2022-02-25 | 2022-06-10 | 宁波大学 | Wheat yellow mosaic virus 14K protein capable of inducing plant cell necrosis and application thereof in virus resistance |
| CN114605504B (en) * | 2022-02-25 | 2023-05-09 | 宁波大学 | Wheat yellow mosaic virus 14K protein capable of inducing plant cell necrosis and application thereof in antiviral |
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Application publication date: 20170630 |