CN110592128A - Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof - Google Patents
Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof Download PDFInfo
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Abstract
The invention discloses a plasmid vector capable of prokaryotic expression of cherry fruit number 1 virus RdRp protein, which is named as pMAL-LC-RdRp, wherein the plasmid vector contains a sequence shown by SEQ ID NO.1 and used for coding the cherry fruit number 1 virus RdRp protein, and the amino acid sequence of the coded cherry fruit number 1 virus RdRp protein is shown as SEQ ID NO. 2. The invention constructs a plasmid vector capable of prokaryotic expression of the RdRp protein of the cherry-fruitlet No.1 virus for the first time, the plasmid vector can be used for prokaryotic expression of the RdRp protein, and the purified RdRp protein is used for establishing an RdRp-mediated in-vitro replication system which can be used for research on replication regulation and control of LChV-1 genome.
Description
Technical Field
The invention relates to the technical field of plant virology and molecular biology, in particular to a plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof.
Background
In the last two decades, the cultivation area and the yield of the cherry planting industry are steadily increased. The main cherry producing areas in China mainly comprise Shandong, Liaonan, Jidong, Shaanxi, Beijing and Henan, and the Shandong is a province with the largest cultivation area and the largest yield of the sweet cherries in China. The sweet cherry production plays an important role in improving rural economic conditions and in the aspect of agricultural industrialization. However, the occurrence of sweet cherry virus disease seriously affects the yield and quality of cherries, and is one of important factors for restricting the continuous development of the cherry industry. At present, 14 virus diseases infecting sweet cherries are reported in China, wherein the cherry small fruit No.1 virus (LChV-1) is distributed in most cherry production areas in China, and the infection can cause symptoms of fruit reduction, sweetness reduction, late maturity and the like, thereby seriously affecting the yield of the cherries and bringing great economic loss. LChV-1 is a virus of the genus longilineans of the family Rhabdoviridae, LChV-1 is first reported in Poland 2004, LChV-1 is first reported to infect sweet cherries in 2015 in China, and the virus is mainly transmitted through sap, insect vectors and other ways. LChV-1 genome is a sense single-stranded RNA ((+) single-stranded RNA, (+) ssRNA), about 17,000nt in size, no poly (A) tail at 3' end, no tRNA-like structure, and about 75nt and 200nt in 5' end and 3' end untranslated region, respectively.
Plant RNA viruses complete a life cycle in a host cell including virion uncoating, viral protein translation, viral genome replication, virion assembly, which are performed almost simultaneously and cannot be strictly distinguished. Therefore, to study the RNA viral cycle in a single process, a separate in vitro study system was set up. Combining mutation analysis and RNA-out-of-structure analysis allows for the localization of core RNA regulatory elements in the template RNA and core amino acids in RdRp (RNA-dependent RNA polymerase). Currently, genomic sequences of multiple LChV-1 isolates from different regions of the world and different hosts are published at NCBI, and the software predicts that the coding patterns of each isolate are essentially identical, with large differences between the nucleotide sequences of the different isolates. The molecular biology research related to LChV-1 mainly focuses on aspects of virus detection, genome cloning, molecular evolution and the like, however, at present, no research system for LChV-1 in vitro replication exists, an RdRp protein needs to be expressed and purified through a prokaryotic system to establish an in vitro replication system, but the homology of a coding sequence of the RdRp protein in different LChV-1 isolates is high, and at present, the research only knows that the RdRp protein has RNA-dependent RNA polymerase (RdRp) activity through homology derivation, but a mechanism for regulating and controlling LChV-1 genome replication is still unclear, particularly, the protein with the RdRp activity easily causes activity loss in the prokaryotic expression and purification processes, so the in vitro replication system cannot be established according to the prior art.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a plasmid vector capable of prokaryotic expression of the RdRp protein of the cherry fruitlet No.1 virus. The plasmid vector constructed by the invention can be used for prokaryotic expression of the RdRp protein of the LChV-1 virus; the RdRp protein is used for establishing an RdRp mediated in-vitro replication system, and can be used for research on LChV-1 genome replication regulation.
In order to achieve the purpose, the invention adopts the following technical scheme:
in the first aspect of the invention, a plasmid vector capable of prokaryotic expression of the cherry fruit 1 virus RdRp protein is provided and named as pMAL-LC-RdRp, and the plasmid vector contains a sequence shown in SEQ ID NO.1 and used for coding the cherry fruit 1 virus RdRp protein.
Preferably, the amino acid sequence of the encoded virus RdRp protein of cherry fruit 1 is shown in SEQ ID NO. 2.
In a second aspect of the present invention, there is provided a process for preparing the above plasmid vector pMAL-LC-RdRp, comprising the steps of:
(1) amplifying a segment containing an RdRp coding sequence by using primers shown in SEQ ID NO.3 and SEQ ID NO.4 and an RT-PCR method;
(2) sequencing the amplified fragment in the step (1), designing a primer containing an enzyme cutting site according to a sequencing result, and accurately amplifying an RdRp coding sequence by using the primer containing the enzyme cutting site; the amplified product is subjected to double enzyme digestion by BamH I and Sal I, and then is connected with pMAL-C2X catalyzed by the same enzyme, and positive clone of pMAL-LC-RdRp is obtained after identification.
Preferably, in the step (2), the primer sequence containing the enzyme cutting site is shown as SEQ ID NO.5 and SEQ ID NO. 6.
In a third aspect of the invention, the application of the plasmid vector pMAL-LC-RdRp in prokaryotic expression of RdRp protein is provided.
The fourth aspect of the invention provides a method for prokaryotic expression of soluble RdRp protein of cherry small fruit No.1 virus, which comprises the following steps:
(1) transforming a plasmid vector pMAL-LC-RdRp into an escherichia coli competent cell, and selecting an escherichia coli strain containing a recombinant plasmid to be placed in an LB liquid culture medium containing IPTG for induction culture;
(2) carrying out ultrasonic crushing and centrifugation on the bacteria liquid after induction culture, and collecting supernatant;
(3) and (3) carrying out affinity chromatography on the supernatant to obtain purified soluble RdRp protein.
Preferably, in step (1), the final concentration of IPTG is 0.4 mmol/L.
Preferably, in step (1), the temperature of the induction culture is 18 ℃.
The fifth aspect of the invention provides the application of the plasmid vector pMAL-LC-RdRp or the RdRp protein expressed by the pronucleus thereof in the research of LChV-1 genome replication regulation;
further, in the application, the replication regulation of the LChV-1 genome is researched by establishing an RdRp mediated in vitro replication system by utilizing the RdRp protein.
The invention has the beneficial effects that:
the invention constructs a plasmid vector capable of prokaryotic expression of the RdRp protein of the cherry-fruitlet No.1 virus for the first time, the plasmid vector can be used for prokaryotic expression of the RdRp protein, and the purified RdRp protein is used for establishing an RdRp-mediated in-vitro replication system which can be used for research on replication regulation and control of LChV-1 genome.
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FIG. 1: schematic diagram of the electrophoresis result of the PCR product of the RdRp protein coding sequence of LChV-1; wherein M is DNA Ladder 2000 from SinoBio.
FIG. 2: the electrophoresis diagram of the restriction enzyme identification result of the plasmid pMAL-LC-RdRp; wherein M is DNA Ladder 5000 of SinoBio company, and a vector and an RdRp coding fragment are obtained after enzyme digestion respectively.
FIG. 3: a schematic representation of the pre-expression results for prokaryotic expression of plasmid pMAL-LC-RdRp; wherein M is Blue Plus II Protein Marker of Transgen company; IPTG induction was followed by expression of a specific protein of approximately 90kDa, with a molecular weight consistent with the sum of the molecular weights of RdRp (50kD) and MBP tag (40 kD).
FIG. 4: schematic diagram of the results of solubility analysis of the RdRp protein fused to MBP; wherein M is Blue Plus II Protein Marker of Transgen company, IPTG induced specific Protein expression of about 90kDa, ultrasonic treatment analysis shows the proportion of target Protein (MBP-RdRp) in supernatant and precipitate, wherein 18 ℃ induction can reach about 35%, and 37 ℃ induction is less than 5%.
FIG. 5: a schematic diagram of the chromatographic results of the RdRp protein fused to MBP; wherein M is Blue Plus II Protein Marker of Transgen company, and after IPTG induction, the specific Protein expression of about 90kDa is obtained, wherein the effect of the second tube after column chromatography is hung is the best.
"mock" in FIGS. 4 and 5 represents a bacterial solution without IPTG inducer added; "ck +" is the induced protein sample in FIG. 3.
FIG. 6: results of in vitro replication mediated by the RdRp fusion protein of LChV-1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background, RdRp is an essential central enzyme for virus replication and plays an extremely important role in the survival, propagation and evolution of viruses. However, the difference of different RdRp in amino acid sequence is huge, and no research system for RdRp-mediated LChV-1 in vitro replication exists at present.
Based on the technical scheme, the invention aims to provide a plasmid vector capable of prokaryotic expression of the RdRp protein of the cherry-fruitlet No.1 virus, and an in-vitro replication system is established by prokaryotic expression and purification of the RdRp protein through the plasmid vector.
The technical difficulty in constructing cloning plasmids for viruses is mainly that many viruses are unstable in E.coli. Their expression products in E.coli can affect E.coli growth. Therefore, there are often problems that a clone colony cannot be obtained after Escherichia coli is transformed with a vector containing a viral genome in a clone plasmid for constructing a virus, or that the obtained clone colony undergoes genetic mutation (base mutation, fragment deletion), which causes a great obstacle to the construction of a virus-infected clone.
In order to obtain the plasmid, the invention firstly utilizes an RT-PCR method to design primers shown in SEQ ID NO.3 and SEQ ID NO.4 to amplify a segment containing an RdRp coding sequence so as to obtain a coding sequence containing an RdRp protein, the fragment obtained was then ligated to pGEM-T by T4 ligase to obtain a plasmid containing the coding sequence of the RdRp protein, then using the plasmid containing the RdRp protein coding sequence as a template by utilizing a PCR method, designing primers SEQ ID NO.5 and SEQ ID NO.6 containing enzyme cutting sites to obtain the coding sequence of the LChV-1RdRp protein, the coding sequence of the RdRp protein was then inserted into the pMAL-C2X (NEB) vector using restriction and ligation, obtains a prokaryotic expression plasmid vector containing the RdRp protein of the cherry small fruit No.1 virus, the plasmid vector is named pMAL-LC-RdRp and contains a coding sequence of LChV-1RdRp protein.
Because the whole genome sequence of the cherry small fruit virus No.1 is not obtained, the coding sequence of the RdRp of the cherry small fruit virus No.1 can be quickly obtained by the method, and the experiment progress is greatly accelerated. The primers shown in SEQ ID NO.3 and SEQ ID NO.4 are designed to ensure that the complete RdRp coding frame can be contained in the amplified fragment, and to ensure that the required sequence can be successfully amplified according to the sequence primer design principle. SEQ ID NO.5 and SEQ ID NO.6 were designed to contain restriction sites in order to ensure that the expression vector containing the correct RdRp sequence could be successfully ligated into pMAL-C2X.
The coding sequence for the RdRp protein ligated to the plasmid requires its complete protein coding cassette, the sequence length does not exceed the length of the expression vector pMAL-C2X (6Kbp), and the optimal expression length is 1-4 Kbp. The fragments containing the coding sequence of the RdRp protein obtained by amplifying the primers shown in SEQ ID NO.3 and SEQ ID NO.4 can not be directly connected to be used for constructing prokaryotic expression, because the RdRp protein expression reading frame is not purely complete, and other sequences exist. Therefore, further processing of this fragment design is required to obtain the exact RdRp protein coding sequence.
pMAL-C2X is used as the expression vector because the expression vector is used for expressing protein in a commercial way, and is relatively stable and has good expression effect.
The plasmid vector is used for prokaryotic expression, RdRp fusion protein is purified by an affinity chromatography method, and the purified RdRp fusion protein is used for in vitro replication experiments, so that 3' UTR of a genome can be successfully identified and a complementary chain is generated by taking the UTR as a template. Therefore, the successful establishment of the LChV-1RdRp mediated in-vitro replication system is proved, and a system and data support is provided for the replication regulation and control research of LChV-1 and the in-vivo pathogenicity mechanism analysis of LChV-1.
More specifically, the invention discloses a construction method of a plasmid vector pMAL-LC-RdRp capable of prokaryotic expression of cherry fruit No.1 virus RdRp protein, which comprises the following steps:
primers (primer information see table 1) were designed to amplify fragments containing the RdRp coding sequence, the exact RdRp coding sequence and other fragments (corresponding to the 5 'end, middle sequence and 3' end of the genome for preparing the corresponding RNA as in vitro replication templates) based on the sequence of different isolates of LChV-1 published in GenBank (GenBank. ku715989, KR080325, JX669615, NC001836 and KR 736335);
primers LCh1-8-6324-F and LCh1-8-8614-R were designed for the peripheral upstream and downstream conserved regions of RdRp of LChV-1 (Table 1). And (3) amplifying a fragment containing the RdRp coding sequence by using an RT-PCR method, and designing a primer containing a restriction enzyme site according to a sequencing result for accurately amplifying the RdRp coding sequence. The PCR product was recovered by a DNA recovery kit and ligated with T4 DNA ligase (Takara) for 8h at 16 ℃. The ligation product was transformed into E.coli DH5 alpha. The sequence containing the RdRp fragment is obtained by colony PCR, enzyme digestion identification and final DNA sequencing. And then amplifying the RdRp fragment by PCR by using a primer containing the enzyme cutting site, wherein the PCR reaction conditions are the same as the above. The PCR product was recovered by DNA recovery kit, double digested with BamH I and Sal I, ligated with pMAL-C2X (NEB) catalyzed by the same enzyme. Positive clones of pMAL-LC-RdRp were obtained after identification.
Table 1: primers used in the present invention
Note: the cleavage sites are underlined, and the T7 promoter sequence is in lower italics; int: represents an intermediate sequence; nt: nucleotide, its preparation and use
After obtaining the plasmid pMAL-LC-RdRp, the invention uses the plasmid to carry out prokaryotic expression, purifies soluble RdRp fusion protein by an affinity chromatography method, further establishes an RdRp-mediated in vitro replication system, and experimental results show that the RdRp protein can specifically identify the 3' terminal fragment of the LChV-1 genome to generate a complementary chain thereof. The method mainly comprises the following steps:
(1) pre-expression of the RdRp protein fused to MBP:
the plasmid pMAL-LC-RdRp is transformed into competent cells of Escherichia coli Rossetta, and SDS-PAGE electrophoresis is utilized to test the induction effect of different IPTG concentrations on a target protein (the RdRp protein with an MBP label is named as MBP-RdRp), so that the optimized IPTG induction concentration is confirmed to be 0.4 mmoL/L.
(2) Solubility analysis of MBP-RdRp protein:
and analyzing the solubility of the target protein on the premise of confirming that IPTG can induce the expression of the target protein. And testing the influence of different induction temperatures on the proportion of the soluble target protein, confirming that the optimal induction temperature is 18 ℃, and ensuring that the proportion of the soluble target protein can meet the requirement of subsequent affinity chromatography purification.
(3) Purification by affinity column chromatography of soluble protein from Amylose Resin (MBP tag):
a chromatographic column is prepared by using Amylose Resin (NEB company, 0111207), and soluble MBP-RdRp protein is purified by steps of column loading, balancing, loading, eluting and the like and is used for a subsequent in vitro replication activity test.
(4) RdRp-mediated in vitro replication system:
different fragments of the LChV-1 genome (including the 5' end, the middle sequence and the 3' end) were first prepared by in vitro transcription, then the catalytic ability of the purified MBP-RdRp protein was tested for different RNA fragments, and finally in vitro replication experiments showed that the MBP-RdRp protein specifically recognizes the 3' end fragment of the LChV-1 genome and catalyzes the synthesis of its complementary strand.
In conclusion, the invention obtains a plasmid vector capable of prokaryotic expression of cherry-small fruit No.1 virus RdRp protein, the plasmid vector is named as pMAL-LC-RdRp and contains an RdRp protein coding sequence of LChV-1, the plasmid can be used for prokaryotic expression of the RdRp protein, an RdRp mediated in-vitro replication system is established by utilizing the purified RdRp protein, and the plasmid can be used for research on replication regulation of LChV-1 genome.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.
Example 1: the construction of the plasmid vector pMAL-LC-RdRp for prokaryotic expression of the cherry small fruit No.1 virus RdRp protein comprises the following specific steps:
primers LCh1-8-6324-F (shown in SEQ ID NO. 3) and LCh1-8-8614-R (shown in SEQ ID NO. 4) were designed for peripheral upstream and downstream conserved regions of RdRp of LChV-1 based on sequence alignment analysis of different isolates of LChV-1 (GenBank. KU715989, KR080325, JX669615, NC001836 and KR736335) published in GenBank. A fragment containing the RdRp coding sequence is amplified by using an RT-PCR method, and LCh1-BamHI-7070-F (shown in SEQ ID NO. 5) and LCh1-SalI-8497-R (shown in SEQ ID NO. 6) containing enzyme cutting site primers are designed according to a sequencing result and are used for accurately amplifying the RdRp coding sequence.
25 μ L RT reaction: plant Total RNA, 5.0. mu.L, LCh1-8-8614-R, 5.0. mu.L, 10mmoL/L dNTP, 4.0. mu.L, ddH2O, 5.0 μ L; at 80 deg.C for 3min, and ice for 5 min; 5 × Reverse Transcriptase M-MLV Buffer,5.0 μ L, Reverse Transcriptase M-MLV, 0.5 μ L, Recombinant RNase Inhibitor, 0.5 μ L; 42 ℃ for 90 min.
25 μ L PCR reaction: 2.5 μ L10 XPCR buffer (Mg)2+plus), 1. mu.L dNTP (2.5 mmoL/L each), 1. mu.L each of LCh1-8-6324-F and LCh1-8-8614-R, 1. mu.L of RT reaction product, 1. mu.L of DNA polymerase (5U. mu.L-1), and complement of ddH2O Final reaction volume was 25. mu.L. The PCR reaction process comprises pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 40s, annealing at 50 deg.C for 40s, extension at 72 deg.C for 2min, 30 cycles, and final extension at 72 deg.C for 10 min.
The PCR product was recovered by a DNA recovery kit and ligated with T4 DNA ligase (Takara) for 8h at 16 ℃. The ligation product was transformed into E.coli DH5 alpha. Obtaining the fragment sequence containing the RdRp (the fragment sequence comprises 600bp before the RdRp + 120bp after the RdRp) through colony PCR, enzyme digestion identification and final DNA sequencing.
The RdRp fragment was amplified by PCR using primers LCh1-BamHI-7070-F and LCh1-SalI-8497-R containing the cleavage sites, and the PCR reaction conditions were the same as above. The PCR product was recovered by DNA recovery kit, double digested with BamH I and Sal I, ligated with pMAL-C2X (NEB) catalyzed by the same enzyme. Positive clones of pMAL-LC-RdRp were obtained after identification. And (4) analyzing results: electrophoresis of the PCR reaction products showed that the PCR product was approximately 1.6kb (shown in FIG. 1) and substantially matched the length of the coding sequence (1584 nucleotides) of LChV-1 RdRp. The PCR product is cut by BamH I and Sal I and then inserted into pMAL-C2X cut by BamH I and Sal I, the converted product is screened by colony PCR, the plasmid is identified by digestion, the double digestion identification of BamH I and Sal I is selected, the digestion result shows about 1.5kb digestion band, the size of the rest digestion fragment is about 6.7kb, and the plasmid is basically confirmed to contain gene sequence of LChV-1-RdRp (shown in figure 2); and finally identified by DNA sequencing of the plasmid.
Example 2: prokaryotic expression of LChV-1RdRp protein:
the specific operation steps are as follows:
pre-expression of the RdRp protein fused to MBP:
firstly, the plasmid is transformed into competent cells of Escherichia coli Rosseta, and an LB plate with 100mg/mL ampicillin is coated; the Rosseta strain containing the recombinant plasmid is picked and placed in an LB liquid culture medium of 5ml, and cultured in an incubator at 37 ℃ and 200rpm for overnight; adding overnight cultured bacterial liquid into 30mL of fresh LB liquid culture medium at a ratio of 1:100, culturing at 37 deg.C and 200rpm to OD600To 0.4 to 0.6; the freshly cultured broth was placed on ice for 10min and dispensed into finger tubes, 3ml each, for a total of 6 tubes. IPTG was added to final concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0mmoL/L, respectively.
Culturing at 37 deg.C and 200rpm for 4 hr; 1.5mL of the induction culture was put into a 1.5mL centrifuge tube at 13000rpm and centrifuged for 30 seconds, and the cells were collected and the supernatant was removed.
Sample treatment: to the collected bacteria, 100. mu.L of ddH was added2O, shake and suspend, add equal volume of 2 xSDS protein loading buffer. Boiling water bath for 8min, and standing on ice for 5 min.
Glue running and detection: 10 μ L of each sample was spotted and subjected to 120V electrophoresis until the bromophenol blue indicator band completely exited. Dyeing: placing the separation gel in a culture dish, adding staining solution, sealing with plastic wrap, heating with high fire in a microwave oven for 15s, taking out, shaking with horizontal shaking table for 10min, pouring off staining solution, and recovering staining solution. And (3) decoloring: adding decolorization solution, heating with high fire in microwave oven for 15s, shaking in horizontal shaker for 10min, removing decolorization solution, adding ddH2And O, oscillating the horizontal shaking table until the color is completely removed.
And (4) analyzing results: SDS-polyacrylamide gel electrophoresis results showed that, when the final concentrations of IPTG were 0.2, 0.4, 0.6, 0.8 and 1.0mmoL/L, respectively, compared with the negative control (IPTG concentration of 0mmoL/L), expression of specific proteins having a molecular weight of about 90kDa was induced in the bacterial fluid (shown in FIG. 3), and the molecular weight of the proteins substantially coincided with the size of RdRp protein fused with MBP. The final concentration of IPTG in subsequent experiments was 0.4 mmoL/L.
Example 3: solubility analysis of MBP-fused RdRp protein:
according to the basic steps of prokaryotic expression, inducing 10mL of bacterial liquid under the concentration of 0.4mmoL/L IPTG, carrying out shaking culture at 18 ℃ and 200rpm overnight, and setting a control group; concentrating the induced bacteria liquid in a 1.5mL centrifuge tube, and collecting 1mL bacteria liquid respectively under different temperature induction conditions (treating samples for later use); the collection of cells was resuspended in 1.0mL of protein buffer and PMSF (final concentration 0.1mmoL/L) was added. Note that: from this step, all operations were performed on ice.
Ultrasonication (conditions: 200W, 100 times, 2s of work, 2s of interval) was carried out on ice.
13000rpm, 15min, 4 ℃, the supernatant and the pellet were separated.
Sample treatment 1mL of the induced collection of cells and 1mL of the uninduced collection of cells were added to 100. mu.L of water, and after vigorous shaking and suspension, an equal volume of 2 XSDS protein loading buffer was added. For the precipitation after ultrasonic treatment, the treatment method is the same as above; for the sonicated supernatant, an equal volume of 2x SDS protein loading buffer was added. The sample is boiled in water bath for 8min, and kept stand on ice for 5 min.
The specific processes of glue running and detection are shown in the operation in the embodiment 2; finally, the ratio of the target protein in the supernatant and the precipitate is detected.
And (4) analyzing results: SDS-polyacrylamide gel electrophoresis results show that the target protein induced and expressed at 37 ℃ basically exists in the precipitate, and the content proportion of the target protein in the supernatant is extremely low and is less than 1 percent; the relative proportion of the target protein induced and expressed at 18 ℃ in the supernatant is obviously improved to about 35 percent (shown in figure 4), and the subsequent affinity chromatography experiment can be satisfied.
Example 4: purification of soluble protein by affinity column chromatography (applicable to MBP tag):
according to the prokaryotic expression method, 0.4mmoL/L IPTG induces 1000mL of bacterial liquid at 18 ℃, after centrifugation, column balance buffer solution is used for resuspending the bacteria, and ultrasonic crushing is carried out; after centrifugation, the supernatant was filtered through a 0.45 μm aqueous membrane into a 50ml centrifuge tube, and placed on ice for use, waiting for passing through a chromatography column.
Affinity chromatography (performed at 4 ℃) and column packing: 2mL of an Amylose Resin (NEB, 0111207) packing was applied to a column to elute the liquid; balancing: balancing the chromatographic column by using a balance buffer solution with 5-10 times of column volume; loading: passing the sample through a chromatography column; washing: washing the chromatographic column with 5-10 times of column volume of an equilibrium buffer solution, and collecting an effluent liquid; and (3) elution: the protein of interest was eluted with 3mL of eluent (500. mu.l of eluent was collected in a centrifuge tube, labeled, and placed on ice).
And (3) detection: the quality of the purified protein was determined by SDS-PAGE of 10. mu.l each of the supernatant, the wash and the eluate containing maltose at different concentrations (FIG. 5). Finally, the solution containing the protein of interest was stored at-80 ℃.
Equilibration buffer (Amylose Resin) used therein: 20mmoL/L Tris-Cl (pH 8.0), 25mmoL/L NaCl, 1mmoL/L EDTA (pH 8.0), 10mmoL/L beta-mercaptoethanol, 10% glycerol;
elution buffer (Amylose Resin): 0.5moL/L maltose mother liquor is added into the equilibrium buffer solution to make the final concentration of maltose reach 10 mmoL/L.
Example 5: preparing LChV-1 genome different RNA fragments by in vitro transcription:
PCR fragments corresponding to different positions of LChV-1 were obtained by PCR amplification using the endmost 300bp sequence fragment containing LChV-1 as template (primers and amplified fragments used are shown in Table 1), and gel cutting recovery was performed using DNA recovery kit (all-purpose gold, EG 101-02). Using the above PCR product as a template, an in vitro transcription reaction was performed under the action of T7 RNA polymerase (Promega, P2075) (reaction system: PCR product 4.0. mu.g, 100mmoL/L DTT 12.0. mu.l, 5mmoL/L rNTPs12.0. mu.l, 5 XT 7 Buffer 24.0. mu.l, RNase inhibitor 0.5. mu.l, T7 RNA polymerase 2.0. mu.l, H2075)2Supplementing O to 120 mu l), reacting at 37 ℃ for 2.5 h; the reaction product was subjected to 1.5% agarose gel electrophoresis and then gel-cut to purify the corresponding RNA fragment. The purified RNA fragment is used as a template for in vitro replication.
Example 6: LChV-1RdRp mediated in vitro replication system:
in vitro replication reaction was performed under the action of MBP-RdRp protein (reaction system: RNA template 1.0. mu.g, 1moL/L Tris-Cl (pH) 8.2)2.5μl，1moL/L MgCl2 0.5μl，1moL/L DTT 0.5μl，1moL/L KCl 5μl，10mg/ml yeast tRNA 1.75μl，20mmoL/L ATP 2.5μl，20mmoL/L GTP 2.5μl，20mmoL/L CTP 2.5μl，1mmoL/L UTP 0.5μl，α-32P-UTP 0.5. mu.l, RdRp fusion protein 12.5. mu.l, H2Supplementing O to 50 mu l), and incubating for 1-1.5 h at the reaction temperature of 20 ℃; after the reaction was complete, 70. mu.L of ddH was added2Mixing, adding 120 μ L phenol/chloroform (0.5mL phenol/0.5 mL chloroform/0.04 mL 0.5moL/L EDTA/0.01mL 10% SDS mixture) and extracting; centrifuging the supernatant to add 2.4 volumes of NH4Ac/isopropanol(5moL/L NH4Ac, isoproanol 1:5), uniformly mixing, precipitating at-20 ℃ overnight, centrifuging, washing and airing the precipitate; adding 10 μ L RNA loading buffer, and then performing 1500mA electrophoresis for 2h in 5% PAGE gel (containing 8moL/L urea); dry glue and press phosphor screen imaging.
And (4) analyzing results: in vitro replication results As shown in FIG. 6, MBP-RdRp protein specifically recognizes the 3' terminal sequence of LChV-1 genome, producing its complementary strand. The MBP-RdRp protein which is expressed and purified by the plasmid pMAL-LC-RdRp pronucleus can mediate an in vitro replication system aiming at LChV-1.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof
<130> 2019
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1428
<212> DNA
<213> Artificial sequence
<400> 1
atggacttga ctttcaatgg tttgtcttct gttgattttc tgaccaggac tcttcgattc 60
gagtattctg atattgatct gccttgtctt gaagatcttc tgttgacccc atctaaatct 120
aaaccctatc aagcagttaa ttgtaagata cctattctta taggcaaagg tgagcgaagt 180
agaccagata cttggaaaca agttatttta tccttatcca aacgaaattt tgcagcaccc 240
agactgaatg aagatctttc cattgatgaa actgctatta ggttatttaa tagtttgatg 300
agatgtatgg agcctagtcg tttggcagag tattatgatg tcgttgaacc cgatgttaat 360
aaaatcaatt cttggttact atccagagac aggaggaagt atggcaacgt tttgaggggg 420
tttgatggga atgattggac tacgaaactt tttaatttga agctaatggt taagggtgat 480
atgaaaccga aacttgatat gtctggtgtc tccgaatatt caccaacttc aaacatcatt 540
tactatcaaa ctatgataaa tatgtttttc agccccatct ttcttgaaat ttttggtaga 600
attaaatact gtttaggtga taaagttatt ttattttctg gtatgaacct tgatgaaatg 660
ggtgaacttc ttgaaggtcg tttgtgttat cctttaaatt cttacaattt tgtggaagtt 720
gactttagca aatttgataa atctcaaggt catgtgatca agctctacga agagttggta 780
tacaaaattt ttaaattcag cccaaatatt tacgacaatt tcaaattaag tgaatatttt 840
tgtcgtgctt ctgcaacttg tggagttaat ttagatttgt attgccagcg aaggaccggt 900
tcacccaata cttggttatc taactccttg gcgactctag ccatgctggc aagtgtgtac 960
aatttagatg aaatagatct aattattgta tctggagacg attctttgat catcagtcgt 1020
aatgtaattg agaataaatg ttttgaaatt aataatgact tcggaatgga tgctaaattc 1080
cttgctaacc ctgtccctta tttttgttcg aaattcataa ttcaagtgga caatagaatt 1140
agattggtac ctgatcctgt tagattcctt gaaaaattga gtactcctgt gactttagtt 1200
caattggagc atcccacttt actccgtgag cgtttcactt cttatcgtga cctcatggga 1260
gcttattttg atgaaaatgt tattatagca gttgataggt ttgtttcttt gaagtataac 1320
acaccaattg gtagtggtta cgccgctttt tgctttattc attgtcttct gtctagctat 1380
aaaaattttt taacgatctt tgataacgat attagtattc agttgtaa 1428
<210> 2
<211> 475
<212> PRT
<213> Artificial sequence
<400> 2
Met Asp Leu Thr Phe Asn Gly Leu Ser Ser Val Asp Phe Leu Thr Arg
1 5 10 15
Thr Leu Arg Phe Glu Tyr Ser Asp Ile Asp Leu Pro Cys Leu Glu Asp
20 25 30
Leu Leu Leu Thr Pro Ser Lys Ser Lys Pro Tyr Gln Ala Val Asn Cys
35 40 45
Lys Ile Pro Ile Leu Ile Gly Lys Gly Glu Arg Ser Arg Pro Asp Thr
50 55 60
Trp Lys Gln Val Ile Leu Ser Leu Ser Lys Arg Asn Phe Ala Ala Pro
65 70 75 80
Arg Leu Asn Glu Asp Leu Ser Ile Asp Glu Thr Ala Ile Arg Leu Phe
85 90 95
Asn Ser Leu Met Arg Cys Met Glu Pro Ser Arg Leu Ala Glu Tyr Tyr
100 105 110
Asp Val Val Glu Pro Asp Val Asn Lys Ile Asn Ser Trp Leu Leu Ser
115 120 125
Arg Asp Arg Arg Lys Tyr Gly Asn Val Leu Arg Gly Phe Asp Gly Asn
130 135 140
Asp Trp Thr Thr Lys Leu Phe Asn Leu Lys Leu Met Val Lys Gly Asp
145 150 155 160
Met Lys Pro Lys Leu Asp Met Ser Gly Val Ser Glu Tyr Ser Pro Thr
165 170 175
Ser Asn Ile Ile Tyr Tyr Gln Thr Met Ile Asn Met Phe Phe Ser Pro
180 185 190
Ile Phe Leu Glu Ile Phe Gly Arg Ile Lys Tyr Cys Leu Gly Asp Lys
195 200 205
Val Ile Leu Phe Ser Gly Met Asn Leu Asp Glu Met Gly Glu Leu Leu
210 215 220
Glu Gly Arg Leu Cys Tyr Pro Leu Asn Ser Tyr Asn Phe Val Glu Val
225 230 235 240
Asp Phe Ser Lys Phe Asp Lys Ser Gln Gly His Val Ile Lys Leu Tyr
245 250 255
Glu Glu Leu Val Tyr Lys Ile Phe Lys Phe Ser Pro Asn Ile Tyr Asp
260 265 270
Asn Phe Lys Leu Ser Glu Tyr Phe Cys Arg Ala Ser Ala Thr Cys Gly
275 280 285
Val Asn Leu Asp Leu Tyr Cys Gln Arg Arg Thr Gly Ser Pro Asn Thr
290 295 300
Trp Leu Ser Asn Ser Leu Ala Thr Leu Ala Met Leu Ala Ser Val Tyr
305 310 315 320
Asn Leu Asp Glu Ile Asp Leu Ile Ile Val Ser Gly Asp Asp Ser Leu
325 330 335
Ile Ile Ser Arg Asn Val Ile Glu Asn Lys Cys Phe Glu Ile Asn Asn
340 345 350
Asp Phe Gly Met Asp Ala Lys Phe Leu Ala Asn Pro Val Pro Tyr Phe
355 360 365
Cys Ser Lys Phe Ile Ile Gln Val Asp Asn Arg Ile Arg Leu Val Pro
370 375 380
Asp Pro Val Arg Phe Leu Glu Lys Leu Ser Thr Pro Val Thr Leu Val
385 390 395 400
Gln Leu Glu His Pro Thr Leu Leu Arg Glu Arg Phe Thr Ser Tyr Arg
405 410 415
Asp Leu Met Gly Ala Tyr Phe Asp Glu Asn Val Ile Ile Ala Val Asp
420 425 430
Arg Phe Val Ser Leu Lys Tyr Asn Thr Pro Ile Gly Ser Gly Tyr Ala
435 440 445
Ala Phe Cys Phe Ile His Cys Leu Leu Ser Ser Tyr Lys Asn Phe Leu
450 455 460
Thr Ile Phe Asp Asn Asp Ile Ser Ile Gln Leu
465 470 475
<210> 3
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attcactcaa ggtgagaagg atg 23
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<212> DNA
<213> Artificial sequence
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atacagacat cagatcaacc aaa 23
<210> 5
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<212> DNA
<213> Artificial sequence
<400> 5
aaggatccat ggacttgact ttcaatgg 28
<210> 6
<211> 34
<212> DNA
<213> Artificial sequence
<400> 6
ttgtcgacct attacaactg aatactaata tcgt 34
<210> 7
<211> 59
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<213> Artificial sequence
<400> 7
attaatacga ctcactatag ggtttataat aagtttctat attataaata tattatcaa 59
<210> 8
<211> 25
<212> DNA
<213> Artificial sequence
<400> 8
gcacctttta ttttttatat atgca 25
<210> 9
<211> 41
<212> DNA
<213> Artificial sequence
<400> 9
attaatacga ctcactatag gcgtttttat ctcccagctt t 41
<210> 10
<211> 19
<212> DNA
<213> Artificial sequence
<400> 10
tactgaaagg aaagttgcg 19
<210> 11
<211> 44
<212> DNA
<213> Artificial sequence
<400> 11
attaatacga ctcactatag gcccgcagca atgaagacat tctc 44
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence
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tacagcaaca tcagcacttg aaa 23
Claims (10)
1. A plasmid vector capable of prokaryotic expression of cherry fruit number 1 virus RdRp protein is named as pMAL-LC-RdRp, and the plasmid vector contains a sequence which is shown in SEQ ID NO.1 and used for coding the cherry fruit number 1 virus RdRp protein.
2. The plasmid vector of claim 1, wherein the amino acid sequence of the encoded virus of cherry fruit number 1RdRp protein is shown in SEQ ID No. 2.
3. The method for producing a plasmid vector according to claim 1 or 2, comprising the steps of:
(1) amplifying a segment containing an RdRp coding sequence by using primers shown in SEQ ID NO.3 and SEQ ID NO.4 and an RT-PCR method;
(2) sequencing the amplified fragment in the step (1), designing a primer containing an enzyme cutting site according to a sequencing result, and accurately amplifying an RdRp coding sequence by using the primer containing the enzyme cutting site; the amplified product is subjected to double enzyme digestion by BamH I and Sal I, and then is connected with pMAL-C2X catalyzed by the same enzyme, and positive clone of pMAL-LC-RdRp is obtained after identification.
4. The method according to claim 3, wherein in the step (2), the primer sequence containing the cleavage site is shown as SEQ ID NO.5 and SEQ ID NO. 6.
5. Use of the plasmid vector pMAL-LC-RdRp of claim 1 or 2 for prokaryotic expression of the RdRp protein.
6. A method for prokaryotic expression of soluble RdRp protein of cherry fruit No.1 virus is characterized by comprising the following steps:
(1) transforming an escherichia coli competent cell by the plasmid vector pMAL-LC-RdRp of claim 1 or 2, and selecting an escherichia coli strain containing a recombinant plasmid to be placed in an LB liquid culture medium containing IPTG for induction culture;
(2) carrying out ultrasonic crushing and centrifugation on the bacteria liquid after induction culture, and collecting supernatant;
(3) and (3) carrying out affinity chromatography on the supernatant to obtain purified soluble RdRp protein.
7. The method of claim 6, wherein in step (1), the final concentration of IPTG is 0.4 mmol/L.
8. The method according to claim 6, wherein the temperature of the induction culture in the step (1) is 18 ℃.
9. Use of the plasmid vector pMAL-LC-RdRp of claim 1 or 2 or the RdRp protein prepared by the method of claim 6 in LChV-1 genome replication regulation studies.
10. Use according to claim 9, characterized in that the regulation of replication of the LChV-1 genome is studied by establishing an RdRp-mediated in vitro replication system using the RdRp protein.
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| CN201910811957.7A CN110592128B (en) | 2019-08-30 | 2019-08-30 | Plasmid vector capable of prokaryotic expression of cherry small fruit No.1 virus RdRp protein and application thereof |
| AU2020101483A AU2020101483A4 (en) | 2019-08-30 | 2020-07-24 | Plasmid vector capable of prokaryotically expressing RdRp protein of LChV-1 and its application |
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| CN110592128B (en) | 2022-02-11 |
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