CN110079511A - A kind of RNA polymerase preparation method and application that RNA is relied on - Google Patents
A kind of RNA polymerase preparation method and application that RNA is relied on Download PDFInfo
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- CN110079511A CN110079511A CN201910457310.9A CN201910457310A CN110079511A CN 110079511 A CN110079511 A CN 110079511A CN 201910457310 A CN201910457310 A CN 201910457310A CN 110079511 A CN110079511 A CN 110079511A
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- C12N9/1241—Nucleotidyltransferases (2.7.7)
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- 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 discloses a kind of RNA polymerase preparation method and application that RNA is relied on, the preparation method is using Carmovirus virus as raw material, viral replication protein using transmembrane region or cross-film region mutation containing polymerase domain and without the end N- is basic preparation and reorganization albumen, the RNA polymerase that there is RNA to rely on is obtained, specially chooses the Carmovirus virus of Tombusviridae first as raw material；Then using the transmembrane region for the end N- for truncating or being mutated removing viral RNA, template ribonucleic acid is obtained；Carry out the synthesis of RNA polymerase in the cell or extracellularly finally by prokaryotic expression or eukaryotic expression；Recombinant protein is obtained using method of the invention, RNA yield is greater than 1 mg/ml, is suitble to the synthesis and production of industrialization RNA.
Description
Technical field
The invention belongs to enzyme production technical fields, specifically, be related to a kind of RNA polymerase preparation method that RNA is relied on
Using.
Background technique
RNA is one of eukaryocyte and prokaryotic cell important component, and it is a variety of important that it participates in transcription, translation etc.
Function, transcription are genomic information transfer, and genomic information refers to DNA information, this information is transferred to cell by nucleus
Matter, translation, which refers to, is translated as amino acid or protein for transcriptional information, and RNA polymerase is using a DNA chain or RNA as mould
Plate, triphosphoric acid ribonucleotide be substrate, by phosphodiester bond polymerize synthesis RNA enzyme because in the cell with gene
It is related that the hereditary information of DNA is transcribed into RNA, so also referred to as transcriptase；
In the prior art, RNA can not be efficiently synthesized by RNA template, therefore causes the technology that can not be applied to industry
Metaplasia produces, and in order to solve this problem, the present invention provides following technical schemes.
Summary of the invention
The purpose of the present invention is to provide a kind of RNA RNA polymerase preparation method relied on and applications.
The technical problem to be solved in the invention are as follows:
In the prior art, it can not be efficiently synthesized RNA by template of RNA, led to not poly- suitable for industrialized RNA
Synthase synthesis and production.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of RNA polymerase preparation method that RNA is relied on, includes the following steps:
A, Tombusviridae (Tombusviridae) Carmovirus (Carmovirus) virus is chosen to make
For raw material；
B, using the transmembrane region for the end N- for truncating or being mutated removing viral RNA, to remove the cytotoxicity of virus protein
And enhance the solubility of recombinant protein；
C, the synthesis of RNA polymerase is carried out in the cell or extracellularly by prokaryotic expression or eukaryotic expression.
As further scheme of the invention, the specific side of the transmembrane region of the end N- is removed in the step b by mutation
Method is deletion mutation or point mutation；
As further scheme of the invention, in extracellularly preparation target RNA, RNA polymerase passes through protokaryon table first
Reach or eukaryotic expression after purify, become enzyme preparation, in extracellular buffered environment, utilize target template RNA synthesis complementary
Chain RNA, in one embodiment of the invention, buffer is by 100mM KCl, 5mM MgCl2With the 50mM Tris- of pH 7.5
HCl composition；
As further scheme of the invention, when preparing target RNA in the cell, polymerase template ribonucleic acid is constructed true
In nuclear expression carrier, after expressing in eukaryocyte, complementary strand RNA is synthesized using intracellular target template RNA；The eukaryon
Expression vector includes but is not limited to mammalian expression vector, insect expression vector, Yeast expression carrier；
Wherein, polymerase template ribonucleic acid is constructed into the method in carrier for expression of eukaryon are as follows:
The adhere-wall culture of S1, cell
Cell to be transfected is added in culture bottle, cell to be transfected, after cleaning, Xiang Qi are cleaned by PBS buffer solution
Middle addition pancreatin is digested by pancreatin and decomposes extracellular protein, so that the cell being bonded together be made to separate, passes through micro- sem observation
Cell separates situation, when cell is without large stretch of connection, serum is added into culture bottle, wherein serum is able to suppress and destroys pancreas
Enzymatic activity makes its failure, prevents pancreatin excessive decomposition extracellular protein, damages to cell itself；
Centrifugation obtains agglomerate cell, is centrifuged again after being cleaned by PBS buffer solution, PBS buffer solution is removed, into cell
MEM culture medium is added and dispels cell, cell culture medium is added in culture bottle, in the environment of 37 DEG C, 5% carbon dioxide
Stationary culture, keeps cell adherent and flanking cell does not bond mutually, and wherein the adherent area of cell accounts for culture bottle bottom of bottle area
60%-80%；
The case where cell of culture is separated first in the step, avoids the occurrence of multiple cell agglomerates, to cell phase
Mutually after dispersion, then the cell being dispersed is cultivated, makes cell adherent growth, be able to ascend in this way cell expose it is thin
The area of after birth, to promote the efficiency of subsequent processing；
The preparation of S2, transfection liquid
Polylea non-liposomal transfection reagent is added into physiological saline, ethyl alcohol is added thereto after concussion is uniformly dispersed
With A23187 carrier, it is stand-by that precursor is obtained after concussion dispersion；
The polymerase template ribonucleic acid for being 0.1-1g/L by MEM culture medium compound concentration, is then added dropwise in precursor,
Container is constantly shaken during being added dropwise, and is uniformly mixed the two；
S3, transfection
After the culture bottle that inner wall adherent growth obtained in step S1 has cell is taken out, culture solution is poured out, to culture
Transfection liquid is added dropwise in bottle, and transfection liquid slides into along bottle wall when dropwise addition, prevents cell and culture bottle of the droplet impact power by adherent growth
Separation；
By culture bottle in the environment of 37 DEG C, 5% carbon dioxide after stationary culture 10-15min, it is ultrasonically treated 20-
26min completes transfection after then cultivating 4-8h in the environment of 37 DEG C, 5% carbon dioxide.
During being ultrasonically treated, in order to guarantee that the uniformity of ultrasound, ultrasound occur point and culture bottle are arranged in
Underface, and the distance between point and culture bottle >=15cm occur for ultrasound；
In this step, first by cell in culture bottle in the environment of 37 DEG C, 5% carbon dioxide stationary culture 10-
15min, during this, calcium ion concentration is increased rapidly in A23187 carrier inducing cell, to promote the intake energy of cell
Power promotes the sponginess of cell membrane surface using ultrasound then by ultrasonic treatment, and polymerase template ribonucleic acid is accelerated to enter cell
It is interior, finally so that polymerase template ribonucleic acid is completed transfection, when whole process shortens transfection culture by ultrasound by stationary culture again
Between, while sonication treatment time is shortened by A23187 carrier function, that is, achieve the effect that shorten transfection time, and will not be big
It is big to extend ultrasonic time, guarantee cyto-architectural integrality.
As further scheme of the invention, to the albumen of viral source can by but be not limited to addition sequence label
Mode, in order to the purifying after protokaryon or eukaryotic expression；
As further scheme of the invention, the method for improving polymerase enzyme activity includes but is not limited to: improving intracellular
The copy number of gene, the expression for improving enzyme change the sequence of enzyme to improve stability, remove the amino acid for causing to inhibit
Residue and the transhipment and aggregation intracellular for changing protein；
It is double by will be formed with the short rna of target template RNA complementary pairing and template as further scheme of the invention
Chain structure can promote the synthesis of complementary strand RNA using the duplex structure as primer, improve RNA combined coefficient；
As further scheme of the invention, by being to be matched with self-complementary by the 3 ' tip designs of target template RNA,
And using the end RNA after pairing as primer, the synthesis of complementary strand RNA can be promoted.
In one embodiment of the invention, Tombusviridae (Tombusviridae) described in the step a is fragrant
It is specially turnip crinkle virus (Turnip crinkle virus) that China pink mottle virus, which belongs to virus, wherein turnip crinkle virus
In replication protein coding region sequence, 1-753 include transmembrane domains (having underscore part), and 754-2328 polymerize comprising RNA
Enzymatic activity is removed 1-753 in sequence transmembrane domains by truncating or being mutated, and 754-2328 using in sequence as RNA mould
Plate synthesizes complementary strand RNA, and RNA yield is 95% or more of template ribonucleic acid amount.
Beneficial effects of the present invention:
The present invention is sick using the xiangshizhubanbo of transmembrane region or cross-film region mutation containing polymerase domain and without the end N-
Poison belongs to virus for basic preparation and reorganization albumen, and RNA yield is greater than 1mg/mL, is suitble to the synthesis and production of industrialization RNA.
Detailed description of the invention
Present invention is further described in detail in the following with reference to the drawings and specific embodiments.
Fig. 1 is the replication protein coding region sequence of turnip crinkle virus.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of RNA polymerase preparation method that RNA is relied on, includes the following steps:
A, turnip crinkle virus is chosen as raw material；
B, using the transmembrane region for the end N- for truncating or being mutated removing viral RNA, to remove the cytotoxicity of virus protein
And enhance the solubility of recombinant protein；
C, the synthesis of RNA polymerase is carried out in the cell or extracellularly by prokaryotic expression or eukaryotic expression.
The specific method for removing the transmembrane region of the end N- in the step b by mutation is deletion mutation；
Specifically, when preparing target RNA in the cell, by the building of polymerase template ribonucleic acid in carrier for expression of eukaryon, true
In nucleus after expression, complementary strand RNA is synthesized using intracellular target template RNA；The carrier for expression of eukaryon is dynamic for lactation
Object expression vector；
Polymerase template ribonucleic acid is wherein constructed into the method in carrier for expression of eukaryon are as follows:
The adhere-wall culture of S1, cell
Cell to be transfected is added in culture bottle, cell to be transfected, after cleaning, Xiang Qi are cleaned by PBS buffer solution
Middle addition pancreatin is digested by pancreatin and decomposes extracellular protein, so that the cell being bonded together be made to separate, passes through micro- sem observation
Cell separates situation, when cell is without large stretch of connection, serum is added into culture bottle, wherein serum is able to suppress and destroys pancreas
Enzymatic activity makes its failure, prevents pancreatin excessive decomposition extracellular protein, damages to cell itself；
Centrifugation obtains agglomerate cell, is centrifuged again after being cleaned by PBS buffer solution, PBS buffer solution is removed, into cell
MEM culture medium is added and dispels cell, cell culture medium is added in culture bottle, in the environment of 37 DEG C, 5% carbon dioxide
Stationary culture, keeps cell adherent and flanking cell does not bond mutually, and wherein the adherent area of cell accounts for culture bottle bottom of bottle area
63%；
The case where cell of culture is separated first in the step, avoids the occurrence of multiple cell agglomerates, to cell phase
Mutually after dispersion, then the cell being dispersed is cultivated, makes cell adherent growth, be able to ascend in this way cell expose it is thin
The area of after birth；
The preparation of S2, transfection liquid
Polylea non-liposomal transfection reagent is added into physiological saline, ethyl alcohol is added thereto after concussion is uniformly dispersed
With A23187 carrier, it is stand-by that precursor is obtained after concussion dispersion；
The polymerase template ribonucleic acid for being 0.1-1g/L by MEM culture medium compound concentration, is then added dropwise in precursor,
Container is constantly shaken during being added dropwise, and is uniformly mixed the two；
S3, transfection
After the culture bottle that inner wall adherent growth obtained in step S1 has cell is taken out, culture solution is poured out, to culture
Transfection liquid is added dropwise in bottle, and transfection liquid slides into along bottle wall when dropwise addition, prevents cell and culture bottle of the droplet impact power by adherent growth
Separation；
By culture bottle in the environment of 37 DEG C, 5% carbon dioxide after stationary culture 15min, it is ultrasonically treated 20min, then
Transfection is completed after cultivating 6h in the environment of 37 DEG C, 5% carbon dioxide.
During being ultrasonically treated, in order to guarantee that the uniformity of ultrasound, ultrasound occur point and culture bottle are arranged in
Underface；
By the way that duplex structure will be formed with the short rna of target template RNA complementary pairing and template, made using the duplex structure
For primer, the synthesis of complementary strand RNA can be promoted, improve RNA combined coefficient；
As shown in Figure 1, in the replication protein coding region sequence of the turnip crinkle virus, 1-753 comprising transmembrane domains (i.e.
Have underscore part), 754-2328 include rna polymerase activity, wear film for 1-753 in sequence by truncating or being mutated
Area removes, and 754-2328 using in sequence synthesize complementary strand RNA as RNA template, and RNA yield is template ribonucleic acid amount
95% or more.
Embodiment 2
A kind of RNA polymerase preparation method that RNA is relied on, includes the following steps:
A, turnip crinkle virus is chosen as raw material；
B, using the transmembrane region for the end N- for truncating or being mutated removing viral RNA, to remove the cytotoxicity of virus protein
And enhance the solubility of recombinant protein；
C, the synthesis of RNA polymerase is carried out in the cell or extracellularly by prokaryotic expression or eukaryotic expression.
By truncating the specific method for the transmembrane region for removing the end N- as deletion mutation in the step b；
When preparing target RNA in the cell, by the building of polymerase template ribonucleic acid in carrier for expression of eukaryon, in eukaryocyte
After interior expression, complementary strand RNA is synthesized using intracellular target template RNA；The carrier for expression of eukaryon is Yeast expression carrier；
Polymerase template ribonucleic acid is wherein constructed into the method in carrier for expression of eukaryon are as follows:
The adhere-wall culture of S1, cell
Cell to be transfected is added in culture bottle, cell to be transfected, after cleaning, Xiang Qi are cleaned by PBS buffer solution
Middle addition pancreatin is digested by pancreatin and decomposes extracellular protein, so that the cell being bonded together be made to separate, passes through micro- sem observation
Cell separates situation, when cell is without large stretch of connection, serum is added into culture bottle, wherein serum is able to suppress and destroys pancreas
Enzymatic activity makes its failure, prevents pancreatin excessive decomposition extracellular protein, damages to cell itself；
Centrifugation obtains agglomerate cell, is centrifuged again after being cleaned by PBS buffer solution, PBS buffer solution is removed, into cell
MEM culture medium is added and dispels cell, cell culture medium is added in culture bottle, in the environment of 37 DEG C, 5% carbon dioxide
Stationary culture, keeps cell adherent and flanking cell does not bond mutually, and wherein the adherent area of cell accounts for culture bottle bottom of bottle area
71%；
The case where cell of culture is separated first in the step, avoids the occurrence of multiple cell agglomerates, to cell phase
Mutually after dispersion, then the cell being dispersed is cultivated, makes cell adherent growth, be able to ascend in this way cell expose it is thin
The area of after birth；
The preparation of S2, transfection liquid
Polylea non-liposomal transfection reagent is added into physiological saline, ethyl alcohol is added thereto after concussion is uniformly dispersed
With A23187 carrier, it is stand-by that precursor is obtained after concussion dispersion；
The polymerase template ribonucleic acid for being 1g/L by MEM culture medium compound concentration, is then added dropwise in precursor, is added dropwise
Container is constantly shaken in the process, is uniformly mixed the two；
S3, transfection
After the culture bottle that inner wall adherent growth obtained in step S1 has cell is taken out, culture solution is poured out, to culture
Transfection liquid is added dropwise in bottle, and transfection liquid slides into along bottle wall when dropwise addition, prevents cell and culture bottle of the droplet impact power by adherent growth
Separation；
By culture bottle in the environment of 37 DEG C, 5% carbon dioxide after stationary culture 15min, it is ultrasonically treated 25min, then
Transfection is completed after cultivating 6.5h in the environment of 37 DEG C, 5% carbon dioxide.
During being ultrasonically treated, in order to guarantee that the uniformity of ultrasound, ultrasound occur point and culture bottle are arranged in
Underface；
By the way that duplex structure will be formed with the short rna of target template RNA complementary pairing and template, made using the duplex structure
For primer, the synthesis of complementary strand RNA can be promoted, improve RNA combined coefficient；
In the replication protein coding region sequence of the turnip crinkle virus, 1-753 (have underscore portion comprising transmembrane domains
Point), 754-2328 include rna polymerase activity, 1-753 in sequence transmembrane domains are removed by truncating or being mutated, with
754-2328 synthesize complementary strand RNA as RNA template in sequence, and RNA yield is 95% or more of template ribonucleic acid amount.
Above content is only to structure of the invention example and explanation, affiliated those skilled in the art couple
Described specific embodiment does various modifications or additions or is substituted in a similar manner, without departing from invention
Structure or beyond the scope defined by this claim, is within the scope of protection of the invention.
Claims (10)
1. the RNA polymerase preparation method that a kind of RNA is relied on, which comprises the steps of:
A, the Carmovirus virus of Tombusviridae is chosen as raw material；
B, using the transmembrane region for the end N- for truncating or being mutated removing viral RNA, template ribonucleic acid is obtained；
C, the synthesis of RNA polymerase is carried out in the cell or extracellularly by prokaryotic expression or eukaryotic expression.
2. the RNA polymerase preparation method that a kind of RNA according to claim 1 is relied on, which is characterized in that the step b
In by mutation remove the end N- transmembrane region specific method be deletion mutation or point mutation.
3. the RNA polymerase preparation method that a kind of RNA according to claim 1 is relied on, which is characterized in that the tomato clump
The Carmovirus virus of dwarf virus section is specially turnip crinkle virus.
4. the RNA polymerase that the RNA polymerase preparation method that a kind of RNA according to claim 1 is relied on is prepared,
It is characterized in that, the RNA polymerase for preparing target RNA in the cell or extracellularly.
5. the application of RNA polymerase according to claim 4, which is characterized in that first in extracellularly preparation target RNA
First RNA polymerase purifies after passing through prokaryotic expression or eukaryotic expression, becomes enzyme preparation, in extracellular buffered environment, benefit
Complementary strand RNA is synthesized with target template RNA.
6. the application of RNA polymerase according to claim 4, which is characterized in that, will when preparing target RNA in the cell
Polymerase template ribonucleic acid constructs in carrier for expression of eukaryon, after expressing in eukaryocyte, utilizes intracellular target template RNA
Synthesize complementary strand RNA.
7. the application of RNA polymerase according to claim 6, which is characterized in that constructing polymerase template ribonucleic acid in eukaryon
Method in expression vector are as follows:
The adhere-wall culture of S1, cell
By cell to be transfected be added culture bottle in, cell to be transfected is cleaned by PBS buffer solution, after cleaning, thereto plus
Enter pancreatin, digested by pancreatin and decompose extracellular protein, so that the cell being bonded together be made to separate, then blood is added into culture bottle
Clearly, pancreatin is made to fail；
Centrifugation obtains agglomerate cell, is centrifuged again after being cleaned by PBS buffer solution, PBS buffer solution is removed, be added into cell
MEM culture medium simultaneously dispels cell, and cell culture medium is added in culture bottle, is stood in the environment of 37 DEG C, 5% carbon dioxide
Culture, keeps cell adherent and flanking cell does not bond mutually, wherein the adherent area of cell accounts for the 60%- of culture bottle bottom of bottle area
80%；
The preparation of S2, transfection liquid
Into physiological saline be added polylea non-liposomal transfection reagent, concussion be uniformly dispersed after, thereto be added ethyl alcohol with
It is stand-by to obtain precursor after concussion dispersion for A23187 carrier；
The polymerase template ribonucleic acid for being 0.1-1g/L by MEM culture medium compound concentration, is then added dropwise in precursor, is added dropwise
Container is constantly shaken in the process, is uniformly mixed the two；
S3, transfection
After the culture bottle that inner wall adherent growth obtained in step S1 has cell is taken out, culture solution is poured out, into culture bottle
Transfection liquid is added dropwise, transfection liquid slides into along bottle wall when dropwise addition；
By culture bottle in the environment of 37 DEG C, 5% carbon dioxide after stationary culture 10-15min, it is ultrasonically treated 20-26min, so
Transfection is completed after cultivating 4-8h in the environment of 37 DEG C, 5% carbon dioxide afterwards；
During being ultrasonically treated, the underface that culture bottle is arranged in point occurs for ultrasound, and point and culture occur for ultrasound
The distance between bottle >=15cm.
8. the application of RNA polymerase according to claim 6, which is characterized in that the carrier for expression of eukaryon includes lactation
Animal expression vector, insect expression vector, Yeast expression carrier.
9. the application of RNA polymerase according to claim 4, which is characterized in that by with target template RNA complementary pairing
Short rna and the template ribonucleic acid form duplex structure, and using the duplex structure as primer, synthesize complementary strand RNA.
10. the application of RNA polymerase according to claim 4, which is characterized in that set the 3 ' ends of target template RNA
It is calculated as matching with self-complementary, and using the end RNA after pairing as primer, synthesizes complementary strand RNA.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265821A1 (en) * | 2001-07-30 | 2004-12-30 | Volker Sandig | Rna amplication system using plant components in animal cells |
| CN101168781A (en) * | 2007-10-17 | 2008-04-30 | 李越希 | In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof |
-
2019
- 2019-05-29 CN CN201910457310.9A patent/CN110079511A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265821A1 (en) * | 2001-07-30 | 2004-12-30 | Volker Sandig | Rna amplication system using plant components in animal cells |
| CN101168781A (en) * | 2007-10-17 | 2008-04-30 | 李越希 | In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| K. S. RAJENDRAN等: "Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants", 《JOURNAL OF VIROLOGY》 * |
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