CN104774873B - A kind of assembled in vitro GCRV preparation method - Google Patents
A kind of assembled in vitro GCRV preparation method Download PDFInfo
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Abstract
The present invention is a kind of assembled in vitro GCRV preparation method, the method that specifically a kind of GCRV GCRV cores express outer capsid proteins VP5 and VP7 assembled in vitro with baculovirus expression system, this method includes propagation GCRV873, the acquisition of the virus core containing nucleic acid and centrifugal purification, outer capsid proteins VP5 and VP7 expression, assembled in vitro and steps by centrifugation.This method is simple and convenient to operate, and the assembled in vitro virion R GCRV of its purifying prepared provide effective tool for the mechanism of the virus infection host cell of research VP5 and VP7 mediations.
Description
Technical field
The present invention relates to field of virology, particularly a kind of GCRV (Grass carp using purifying
Reovirus, GCRV) outer capsid proteins VP5 and VP7 progress vitro recombination of the core with using baculovirus expression system expression
The method for forming complete virion.Strain used is GCRV Hunan Shaoyang strain GCRV873, by Chinese allusion quotation
Type culture collection preservation, preservation date is on July 23rd, 2004, and deposit number is CCTCC NO：V200414.
Background technology
Grass carp (Ctenopharyngodon idellus) is one of important cultured freshwater fish of China, easy in seedling
GCRV is infected, the virus can draw sudden and violent popular hemorrhagic disease of grass carp, and cause large quantities of Grass carps' fries dead, to fresh water
Aquaculture causes serious economic loss.Research for virus infection mechanism is conducive to finding the target of antiviral drugs effect
Point, the prevention of development and viral disease for antiviral drugs provides effective way.The nothing that GCRV is made up of double capsid
Togavirus, inner capsid includes five kinds of albumen of VP1-VP4 and VP6, and outer capsid is by VP5 and VP7 heterotrimeric complex groups
Into.Merge the approach for entering cell using film different from togavirus, GCRV early infection may with outer capsid proteins VP5 and
The film infiltration of VP7 mediations is relevant, but its molecular mechanism for entering cell is known little about it.
At present, structure biology data have shown that outer capsid proteins VP5 and VP7 has important in GCRV infection processs
Function, in view of GCRV genomes are made up of the double-stranded RNA of 11 segmentations, as other double-strands segmentation RNA virus, at present still
The research to protein function can not be realized using reverse genetics is the change by gene level, limited to outer capsid egg
The white film mediated in GCRV infection processs permeates the research of mechanism, therefore is necessary that provide one kind effectively can take off from molecular level
Show the method for the GCRV infection mechanisms that outer capsid proteins VP5 and VP7 are mediated.
The content of the invention
The technical problems to be solved by the invention are：The problem of existing for prior art, this research provides a kind of external
GCRV preparation method is assembled, so as to the molecular mechanism and antiviral drugs to be infected from molecular level announcement GCRV
Research and development and for hemorrhagic disease of grass carp preventing and treating open up new effective way.
The present invention solves its technical problem and uses following technical scheme：
The assembled in vitro GCRV preparation method that the present invention is provided, is a kind of GCRV GCRV cores
The method that the heart expresses outer capsid proteins VP5 and VP7 assembled in vitro with baculovirus expression system, this method includes following step
Suddenly：
(1) GCRV873 is bred：
It is CIK cell propagation GCRV873 using grass carp kidney cell line, GCRV873 is GCRV Hunan Shaoyang
Strain；
(2) acquisition of the virus core containing nucleic acid and centrifugal purification：
The virus purified using trypsin treatment, with the outer capsid proteins VP5 and VP7 of degrading, obtains the disease containing nucleic acid
Malicious core, and centrifugal purification is carried out using cesium chloride (CsCl) density gradient；
(3) outer capsid proteins VP5 and VP7 expression：
Using recombinant baculovirus vAcGCRV-VP5/VP7 infected insect cells, system is Sf9 cells, to carry out outer capsid egg
White VP5 and VP7 great expression；
(4) assembled in vitro：
The GCRV cores of purifying and the outer capsid proteins VP5 and VP7 expressed in Sf9 cells are counted according to suitable chemistry
Amount realizes GCRV assembled in vitro than carrying out mixing incubation；
(5) centrifugal purification：
Centrifugal purification, the assembled in vitro purified are carried out to the virion after assembled in vitro using CsCl density gradients
Virion (R-GCRV), it is with infective assembled in vitro GCRV particle.
Described assembled in vitro GCRV particle, its core granule concentration used or granule number should reach
1.2mg/mL or 1012Individual/mL.
After Sf9 cell great expression outer capsid proteins VP5 and VP7, its cell number should reach 107Individual/mL.
Assembled in vitro process is：The virus core of purifying is diluted respectively using 10mmol/L PBS (phosphate buffer)
Into concentration be 0.8-1.2mg/mL or granule number is about 108-1012Individual/mL, the Sf9 cells point that vAcGCRV-VP5/VP7 is infected
It is 10 not to be diluted to cell number5-107Individual/mL, the cell that it dilutes enters the two according to different stoichiometric proportions after freeze thawing
Row mixing, 28 DEG C of incubation 3h, completes assembled in vitro.
During centrifugal purification, the purifying of assembled in vitro virus is carried out using 30-50%CsCl density gradient centrifugations, is about existed
The recombinant virus whole grain of a large amount of assembled in vitro is obtained in 40%CsCl density gradients.
The present invention can be measured using plaque analytic approach to R-GCRV infectivity.
The present invention is using 20mM NH4Cl carries out blocking experiment, and the infectivity to R-GCRV and N-GCRV is analyzed, with
Verify the route of infection of the two.
The present invention is by the virus core of purifying with expressing outer capsid proteins VP5 and VP7 in Sf9 cells at least according to 1010:
106Individual/mL ratios mixing is incubated.
The present invention compared with prior art, with following major advantage：
One the method is simple and convenient to operate, and is effectively overcome Undivided genome virus and is difficult to implement reverse genetics behaviour
Make the limitation of technology；
Secondly the method packaging efficiency is high, by outer capsid proteins VP5 and VP7 and the GCRV cores of purifying according to optimization
Learn metering to be incubated than mixing, can obtain largely has higher infective R-GCRV；
Thirdly experiments confirm that R-GCRV and N-GCRV have identical infection mechanism, the assembled in vitro viral methods will be into
To study an effective tool of GCRV infection mechanisms.
Brief description of the drawings
Fig. 1 is the infectious schematic diagram that plaque assay determines vitro recombination virus.
In figure：A rows 1,2 are classified as the result that R-GCRV infects CIK cell, are experimental group；3rd, 4 it is classified as the infection of GCRV cores
The result of CIK cell, is negative control group；
B rows 1,2 are classified as the result that N-GCRV infects CIK cell, are positive controls；3rd, 4 cell for being classified as VP5 and VP7
The result of lysate Supernatant infection CIK cell, is negative control group.
As can be seen from the figure after R-GCRV and N-GCRV infection CIK attached cells, culture dish bottom occurs in that many bright
The aobvious plaque being evenly distributed or cavity (figure A, B；1st, 2 row), show after R-GCRV infection CIK cells, virus can be in sensitivity
Replicated in cell, cause cell cracking to produce plaque.This infection characteristic is as N-GCRV.And A rows, the 3 of B rows, 4 are arranged in figure
CIK cell is infected for the cell lysate supernatant using GCRV cores and expression VP5 and VP7, no cavity is produced, and shows cell not
It can be infected.
Embodiment
The assembled in vitro GCRV preparation method that the present invention is provided, is to have been achieved with utilizing baculoviral table
(the patent No. carried out on the basis of up to system great expression GCRV outer capsid proteins VP5 and VP7：200710168642.2).Using
The outer capsid proteins VP5 and VP7 of expression and the GCRV cores of purifying carry out assembled in vitro, are derived from infective restructuring
Virion.The method can be used for effects of the research outer capsid proteins VP5 and VP7 during GCRV infects host cell, from
And disclose the molecular mechanism that GCRV infects.
The above-mentioned assembled in vitro GCRV preparation method that the present invention is provided, comprises the following steps：
(1) virus multiplication is carried out using CIK cell (grass carp kidney cell line), the grass carp for obtaining a large amount of infection CIK cells exhales
The lonely viral Hunan Shaoyang strain GCRV873 of intestines viral suspension；
(2) by centrifugal process, the cell virus suspension for having infected GCRV873 is concentrated and purified；
(3) virus purified using trypsin treatment, makes intact virus outer capsid proteins VP5 and VP7 degradable, shape
Into the viral core particle containing nucleic acid, purified by 25-55% (w/v) CsCl density gradient centrifugations, in 50%CsCl gradients
Substantial amounts of viral core particle is obtained in solution；
(4) using recombinant baculovirus vAcGCRV-VP5/VP7 infection Sf9 cells, great expression outer capsid proteins VP5 and
VP7；
(5) -80 DEG C and 37 DEG C of the Sf9 cells warp multigelation 3 times for infecting vAcGCRV-VP5/VP7,4000g centrifugations
15min sedimentation cell fragments；
(6) by the virus core of purifying and outer capsid proteins VP5 and the VP7 cell of baculovirus expression system great expression
Lysate supernatant carries out mixing incubation according to different stoichiometric proportions, and 28 DEG C of incubation 3h complete assembled in vitro；
(7) using the purifying of 30-50% (w/v) CsCl density gradient centrifugations, obtain a large amount of in about 40%CsCl gradients
The R-GCRV particles of purifying；
(8) electron microscopy, SDS-PAGE and Plaque assay are used, R-GCRV morphosis, protein component and infection is carried out
Property analysis.As a result show, R-GCRV and N-GCRV is very much like in terms of morphosis, protein component and infectivity.
Above-mentioned preparation method is described further with reference to embodiment.
It is prepared by one .GCRV873 propagation and core
GCRV873 propagation is carried out using CIK cell (grass carp kidney cell line).
1.CIK cell culture：
For the Eagle's MEM culture mediums containing 10% hyclone, (MEM-10, Invitrogen company produce cell culture fluid
Product), PH 7.2.The CIK cell preserved in liquid nitrogen is taken, 37 DEG C incubate thawing.Centrifuged on desk centrifuge with 5000g after 1min
It is transferred in T25 Tissue Culture Flasks (Corning Products), plus 5mL MEM-10 nutrient solutions are cultivated.CIK cell is most
Suitable cultivation temperature is 28 DEG C, after after the basic confluent cultures bottom of bottle of cell, using 0.25% Trypsin Induced attached cell, is entered
Row Secondary Culture.
2.GCRV873 propagation：
Carry out connecing poison during the basic confluent cultures bottom of bottle of the CIK cell of passage, 10mmol/L phosphate buffers are used first
(phosphate buffered salient, abbreviation PBS) rinses the CIK cell of pre-vaccination 2-3 times, to remove cow's serum egg
Bai Zufen；Then after the MEM of GCRV suspension serum-frees is diluted, according to the infection multiplicity MOI (Multiplicity of virus
Of infection) it is about that 1PFU/cell adds the viral suspension of appropriate volume of culture into monolayer cell culture bottle, absorption is thin
Outwell supernatant after born of the same parents 30min, then with 10mmol/L PBSs cell 2-3 times, remove unadsorbed virion.Then add
MEM (MEM-2) maintaining liquid containing 2% hyclone for entering about 1/10 cell bottle volume carries out virus multiplication.Treat 85% or so
Cell is produced after cytopathy (cytopathic effect, CPE), is collected viral suspension, is saved backup in 4 DEG C.
3.GCRV873 purifying：
By the viral suspension of 4 DEG C of preservations, with 3800g low-speed centrifugal 30min, free cell fragment is removed.It will remove thin
The viral suspension of born of the same parents' fragment puts ultracentrifuge (Beckman Products) with 100,000g ultracentrifugation 2h, precipitates GCRV873
Particle.Supernatant is abandoned, precipitation presses 1:250 times of volumes are dissolved in 10mmol/L PBS.Then again with 12 on desk centrifuge,
000g high speed centrifugation 5min, remove remaining cell fragment.
It is prepared by 4.GCRV873 core：
Trypsase (Sigma Products) is added in the viral suspension of purifying, its working concentration is 200 μ g/mL,
2h is incubated in 28 DEG C of water-baths, makes viral outer capsid proteins composition degradable.Then the CsCl through 25-55% (w/v) is close
Gradient is spent with 200,000g ultracentrifugation 4h, and the sample containing a large amount of GCRV873 cores is taken out in 50%CsCl gradient solutions,
Stayed overnight through 10mmol/L PBSs and obtain pure virus core particle.Using spectrophotometer, (Biotek companies of the U.S. produce
Product) determine the virus core concentration or population purified.After measured, the GCRV873 core granule concentration of purifying is about 1.2mg/
ML or granule number are about 1 × 1012Individual/mL, its sample is saved backup in -20 DEG C.
Two, baculovirus expression system great expression outer capsid proteins VP5 and VP7
Outer capsid proteins VP5 and VP7 great expression are carried out using Sf9 cells (insect cell line).
For the Grace culture mediums containing 10% hyclone, (Grace-10, Invitrogen company produce Sf9 cell culture fluids
Product), pH6.5.
1.Sf9 cell culture：
The Sf9 cells preserved in liquid nitrogen are taken, 37 DEG C incubate thawing.With after 5000g centrifugations 1min turns on desk centrifuge
Move in T25 blake bottles (Corning Products), add 5mL Grace-10 nutrient solutions and cultivated.The most suitable training of Sf9 cells
It is 28 DEG C to support temperature, after after the culture basic confluent cultures bottom of bottle of cell, gently blows and beats attached cell using glass bend pipe, is passed
It is commissioned to train foster.
2. outer capsid proteins VP5 and VP7 great expression：
Sf9 cells are passed on after the confluent monolayers of cell bottle bottom, first the cell of pre-vaccination are rinsed with 10mmol/L PBS
2-3 times, remove bovine serum albumin component；Then it is about that 5PFU/cell adds appropriate volume of culture by the infection multiplicity MOI of virus
VAcGCRV-VP5/VP7 suspensions, after adherent cell 30min, outwell unadsorbed recombinant virus suspension, then use 10mmol/L
PBS cell 2-3 times, thoroughly to remove unadsorbed virion.Then add about 1/10 cell bottle volume contains 2% tire
Grace (Grace-2) maintaining liquid of cow's serum carries out virus multiplication.Cultivate after 48h, add Aprotinin (Aprotinin, Sigma
Products) and Leupeptin (bright suppression protease, Sigma Products) protease inhibitors to final concentration respectively be 0.5 μ g/
ml.After virus infection 72h, culture supernatant is collected into 50mL centrifuge tubes together with infection cell, centrifuged with 3800g
30min sedimentation cells, the cell precipitated is dissolved in the PBS of appropriate volume.Using cell counter, (U.S. Bio-Rad is public
Take charge of product) determine the cell number for expressing outer capsid proteins VP5 and VP7.After measured, expression VP5 and VP7 cell number is 107Individual/
ML, is saved backup in -80 DEG C.Through freeze thawing, the cell pyrolysis liquid containing great expression outer capsid proteins VP5 and VP7 can be obtained.
The GCRV873 cores and outer capsid proteins VP5 and VP7 assembled in vitro of three, purifying
Use that the concentration of purifying is about 1.2mg/mL by 10mmol/L PBS or granule number is 1 × 1012Individual/mL virus cores
It is about 10 that concentration is diluted to respectively for 0.8-1.2mg/mL or granule number8-1012Individual/mL；In addition, vAcGCRV-VP5/VP7 is felt
It is 10 that the Sf9 cells of dye are diluted to cell number respectively5-107Individual/mL, through -80 DEG C and 37 DEG C of multigelations 3 times, 4000g centrifugations
15min sedimentation cell fragments.Then by cell lysate supernatant of the virus core of different extension rates from different extension rates
Mixed according to different stoichiometric proportions, assembling is incubated at 28 DEG C, carry out slowly shaking up manually every half an hour, until 3h
After complete vitro recombination.Isolated and purified using 30-50% (w/v) CsCl density gradient centrifugations, obtain the recombinant virus grain of purifying
Sub (R-GCRV).The infectivity that obtained R-GCRV is assembled after different stoichiometric proportion mixing is determined using plaque assay.As a result
Show, 1010Individual viral core particle number and 106The cell lysate supernatant incubation reaction of individual coexpression VP5 and VP7 albumen, i.e.,
At least according to 1010:106When/mL stoichiometric proportions carry out incubated in vitro assembling, it can obtain that there is higher infective R-GCRV,
There is almost identical virus titer with original virion (N-GCRV).
Four .R-GCRV biological property analysis
1. Morphological Identification is carried out to the R-GCRV isolated and purified using electron microscopy.3 μ L are taken through CsCl density gradient centrifugations
The R-GCRV drops isolated and purified are blotted after viral adsorption 3-5min with filter paper bar at room temperature in being sprayed with the copper mesh of carbon film.Then
Unnecessary dyestuff is sucked with 2% phosphotungstic acid (pH6.9) negative staining about 1min or so, then with filter paper, air drying is placed.
Observed under Hitachi 7000-FA transmission electron microscopes.As a result R-GCRV and N-GCRV form poles under transmission electron microscope are shown
It is the double capsid virion of regular dodecahedron, diameter is about 75nm to be similar.
2. R-GCRV protein components point are carried out using 10% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) technology
Analysis, can show destination protein band after coomassie brilliant blue staining.As a result show, R-GCRV and N-GCRV contains identical
Protein component, containing 7 kinds of complete structural proteins (VP1-VP7).
3. determining R-GCRV infectivity using plaque assay, it is positive control to be provided with N-GCRV in experiment simultaneously, pure
The GCRV cores of change and expression VP5 and VP7 cell lysate supernatant are used as negative control.
As a result as shown in figure 1, similar to N-GCRV, R-GCRV infection CIK cells produce typical plaque, and virus core
Or the cell of expression VP5 and VP7 cell lysate supernatant infection does not produce obvious plaque.
4. using 20mM NH4Cl carries out R-GCRV and N-GCRV infection blocking experiment, as a result shows, R-GCRV and N-
The ability of GCRV infection cells is all adding weak base NH4It is greatly suppressed after Cl, shows that the two employs identical infection way
Footpath.
In above-described embodiment, breed GCRV the outer capsid proteins VP5 and VP7 of restructuring restructuring using Sf9 insect cell lines
Baculoviral (vAcGCRV-VP5/VP7), its construction method is referring to " the restructuring bar of expression GCRV outer capsid proteins
The shape virus and its structure " patent document (patent No.：200710168642.2), this method is mainly：Expanded by RT-PCR,
Respectively obtain the specific fragment of 2 coding GCRV outer capsid proteins VP5 and VP7 genes；Inserted by gene cloning screening
GCRV VP5 and VP7 genes restructuring pFastBacdual dual-expression vectors pFbDGCRV-VP5/VP7；It will screen what is obtained
Dual-expression vector conversion DH10Bac cells are recombinated, the AcGCRV-VP5/VP7Bacmid of swivel base is obtained；And transfected Sf9 elder brothers
Worm cell, obtains recombinant virus vAcGCRV-VP5/VP7 in Sf9 insect cells.
Claims (8)
1. a kind of assembled in vitro GCRV preparation method, it is characterized in that a kind of GCRV GCRV cores with
The method of baculovirus expression system expression outer capsid proteins VP5 and VP7 assembled in vitro, this method comprises the following steps：
(1) GCRV873 is bred：
It is that CIK cell propagation GCRV873, GCRV873 are the strains of GCRV Hunan Shaoyang using grass carp kidney cell line；
(2) acquisition of the virus core containing nucleic acid and centrifugal purification：
The virus purified using trypsin treatment, with the outer capsid proteins VP5 and VP7 of degrading, obtains the viral core containing nucleic acid
The heart, and centrifugal purification is carried out using cesium chloride CsCl density gradients；
(3) outer capsid proteins VP5 and VP7 expression：
Using recombinant baculovirus vAcGCRV-VP5/VP7 infected insect cells system be Sf9 cells carry out outer capsid proteins VP5 and
VP7 great expressions；
(4) assembled in vitro：
Using 10mmol/L PBS to be diluted to virus core, concentration is 0.8-1.2mg/mL or granule number is 108-1012Individual/mL；
It is 10 that the vAcGCRV-VP5/VP7 Sf9 cells infected are diluted into cell number5-107Individual/mL, carries out freeze thawing treatment, with broken thin
Born of the same parents；By the cell lysate supernatant of the virus core of different extension rates and different extension rates according to different stoichiometric proportions
Mixed, the two ratio cannot be below 1010:106/mL；Assembling is incubated at 28 DEG C, carries out slowly shaking manually every half an hour
It is even, until completing vitro recombination；
(5) centrifugal purification：
Centrifugal purification is carried out to the virion after assembled in vitro using CsCl density gradients, the assembled in vitro virus purified
Particle R-GCRV, it is with infective assembled in vitro GCRV particle.
2. assembled in vitro GCRV preparation method according to claim 1, it is characterized in that assembled in vitro grass carp
Reovirus particle, its core granule concentration used or granule number are 1.2mg/mL or 1012Individual/mL.
3. assembled in vitro GCRV preparation method according to claim 1, it is characterized in that big using Sf9 cells
After amount expression outer capsid proteins VP5 and VP7, its cell number is 107Individual/mL.
4. assembled in vitro GCRV preparation method according to claim 1, it is characterised in that by vAcGCRV-
- 80 DEG C and 37 DEG C of the Sf9 cells warp multigelation 3 times of VP5/VP7 infection, 4000g centrifugation 15min sedimentation cell fragments.
5. assembled in vitro GCRV preparation method according to claim 1, it is characterised in that take containing VP5/
The supernatant of VP7 albumen is mixed with the virus core purified, 28 DEG C of incubation 3h, completes assembled in vitro.
6. assembled in vitro GCRV preparation method according to claim 1, it is characterised in that during centrifugal purification,
The purifying of assembled in vitro virus is carried out using 30-50%CsCl density gradient centrifugations, is about obtained in 40%CsCl density gradients
Obtain the recombinant virus whole grain R-GCRV of a large amount of assembled in vitro.
7. assembled in vitro GCRV preparation method according to claim 1, it is characterised in that using plaque point
Analysis method is measured to R-GCRV infectivity.
8. assembled in vitro GCRV preparation method according to claim 1, it is characterised in that use 20mM
NH4Cl carries out blocking experiment, R-GCRV and protovirus N-GCRV infection sex differernce is analyzed, to verify the sense of the two
Dye approach.
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| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
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|---|---|---|---|---|
| CN1616094A (en) * | 2004-09-21 | 2005-05-18 | 中国科学院武汉病毒研究所 | Process for preparing subunit vaccine of reovirus antigen for grass carp |
| CN101205545B (en) * | 2007-12-06 | 2010-06-23 | 中国科学院武汉病毒研究所 | Recombinant baculoviral for expressing grass carp reovirus external casing protein and construction thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616094A (en) * | 2004-09-21 | 2005-05-18 | 中国科学院武汉病毒研究所 | Process for preparing subunit vaccine of reovirus antigen for grass carp |
| CN101205545B (en) * | 2007-12-06 | 2010-06-23 | 中国科学院武汉病毒研究所 | Recombinant baculoviral for expressing grass carp reovirus external casing protein and construction thereof |
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| 利用噬菌体展示技术淘选草鱼呼肠孤病毒的单链抗体;张凤 等;《水生生物学报》;20140531;第38卷(第3期);全文 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881155A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP55 recombinant baculovirus and application |
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