CN104774873A - Grass carp reovirus in-vitro assembly preparation method - Google Patents
Grass carp reovirus in-vitro assembly preparation method Download PDFInfo
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Abstract
The invention relates to a grass carp reovirus in-vitro assembly preparation method, and specifically relates to an in-vitro assembly method of a grass carp reovirus (GCRV) core and baculovirus expression system expression outer capsid proteins VP5 and VP7. The method comprises the steps of GCRV873 proliferation, acquisition and centrifugal purification of virus core containing nucleic acid, outer capsid protein VP5 and VP7 expression, in-vitro assembly and centrifugal purification. The method is simple and is convenient to operate. The prepared purified in-vitro assembled virus virion R-GCRV assists in providing an effective tool for the researches on the mechanism that VP5 and VP7 mediated viruses infect host cells.
Description
Technical field
The present invention relates to field of virology, particularly a kind of GCRV of purifying (Grass carp reovirus, GCRV) core that adopts carries out with outer capsid proteins VP5 and VP7 adopting baculovirus expression system to express the method that vitro recombination forms complete virion.Virus strain used is GCRV Hunan Shaoyang strain GCRV873, and by China typical culture collection center 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, the easy infection GCRV when seedling, this virus can draw sudden and violent popular hemorrhagic disease of grass carp, and cause large quantities of Grass carps' fries dead, cause serious financial loss to freshwater aquaculture industry.Research for virus infection mechanism is conducive to the target spot finding antiviral effect, for the development of antiviral and the prevention of virus disease provide effective way.GCRV by double capsid form without togavirus, inner capsid comprises VP1-VP4 and VP6 five kinds of albumen, and outer capsid is made up of VP5 and VP7 heterotrimeric complex.Being different from togavirus adopts film to merge the approach entering cell, and the early infection of GCRV may be relevant with the membrane permeation that outer capsid proteins VP5 and VP7 mediates, but know little about it to the molecular mechanism that it enters cell.
At present, structure biology data have shown outer capsid proteins VP5 and VP7 has critical function in GCRV infection processs, in view of the double-stranded RNA of GCRV genome by 11 segmentations forms, the same with other double-strand segmentation RNA viruses, the method that there is no at present utilizes reverse genetics namely to realize the research to protein function by the change of gene level, limit the research to the membrane permeation mechanism that outer capsid proteins mediates in GCRV infection processs, therefore be necessary to provide a kind of method that effectively can disclose the GCRV infection mechanism that outer capsid proteins VP5 and VP7 mediates from molecular level.
Summary of the invention
Technical problem to be solved by this invention is: for prior art Problems existing, this research provides a kind of assembled in vitro GCRV preparation method, so that for the research and development that disclose the molecular mechanism that infects of GCRV and antiviral from molecular level and prevent and treat for hemorrhagic disease of grass carp and open up new effective way.
The present invention solves its technical problem and adopts following technical scheme:
Assembled in vitro GCRV preparation method provided by the invention, be a kind of method that GCRV GCRV core and baculovirus expression system express the assembled in vitro of outer capsid proteins VP5 and VP7, the method comprises the following steps:
(1) GCRV873 is bred:
Grass carp kidney cell line and CIK cell propagation GCRV873, GCRV873 is adopted to be Shaoyang strains of GCRV Hunan;
(2) acquisition of the nucleoid containing nucleic acid and centrifugal purification:
Adopt the virus of trypsin treatment purifying, with outer capsid proteins VP5 and VP7 that degrade, obtain the nucleoid containing nucleic acid, and adopt cesium chloride (CsCl) density gradient to carry out centrifugal purification;
(3) expression of outer capsid proteins VP5 and VP7:
Adopt recombinant baculovirus vAcGCRV-VP5/VP7 infected insect cell system and Sf9 cell, to carry out the great expression of outer capsid proteins VP5 and VP7;
(4) assembled in vitro:
The GCRV core of purifying is carried out mixing hatching according to suitable stoichiometric ratio with outer capsid proteins VP5 and VP7 at Sf9 cells, realizes the assembled in vitro of GCRV;
(5) centrifugal purification:
Adopt CsCl density gradient to carry out centrifugal purification to the virion after assembled in vitro, obtain the assembled in vitro virus particle (R-GCRV) of purifying, it is for having infective assembled in vitro GCRV particle.
Described assembled in vitro GCRV particle, its core particle concentration used or granule number should reach 1.2mg/mL or 10
12individual/mL.
After adopting Sf9 cell great expression outer capsid proteins VP5 and VP7, its cell count should reach 10
7individual/mL.
Assembled in vitro process is: adopting 10mmol/L PBS (phosphate buffered saline buffer) nucleoid of purifying to be diluted to respectively concentration is that 0.8-1.2mg/mL or granule number are about 10
8-10
12individual/mL, it is 10 that the Sf9 cell that vAcGCRV-VP5/VP7 infects is diluted to cell count respectively
5-10
7individual/mL, the two, after freeze thawing, mixes according to different stoichiometric ratios, hatches 3h, complete assembled in vitro for 28 DEG C by the cell of its dilution.
During centrifugal purification, adopt 30-50%CsCl density gradient centrifugation to carry out the purifying of assembled in vitro virus, in 40%CsCl density gradient, obtain greatly the recombinant virus whole grain of a large amount of assembled in vitro.
The present invention can adopt the infectivity of plaque analyses method to R-GCRV to measure.
The present invention adopts 20mM NH
4cl carries out blocking experiment, analyzes the infectivity of R-GCRV and N-GCRV, to verify the route of infection of the two.
The present invention by the nucleoid of purifying with at Sf9 cells outer capsid proteins VP5 and VP7 at least according to 10
10: 10
6individual/mL ratio mixing is hatched.
The present invention compared with prior art, has following major advantage:
One. this method is simple, easy to operate, effectively overcomes the limitation that segmented genes papova is difficult to implement reverse genetics operative technique;
They are two years old. and this method packaging efficiency is high, the GCRV core of outer capsid proteins VP5 and VP7 and purifying is measured according to optimization and hatches than mixing, can obtain and have higher infective R-GCRV in a large number;
They are three years old. and experiment confirms that R-GCRV and N-GCRV has identical infection mechanism, and this assembled in vitro viral methods will become an effective tool of research GCRV infection mechanism.
Accompanying drawing explanation
Fig. 1 is the infectious schematic diagram that plaque assay measures vitro recombination virus.
In figure: A capable 1,2 is classified as the result that R-GCRV infects CIK cell, is experimental group; 3,4 being classified as the result that GCRV core infects CIK cell, is negative control group;
B capable 1,2 is classified as the result that N-GCRV infects CIK cell, is positive controls; 3,4 cell lysate supernatant being classified as VP5 and VP7 infect the result of CIK cell, are negative control group.
As can be seen from the figure, after R-GCRV and N-GCRV infects CIK attached cell, the plaque that is much significantly evenly distributed or cavity (figure A, B bottom culture dish, have been there is; 1,2 row), after showing that R-GCRV infects CIK cell, virus can copy in sensitive cells, causes lysis to produce plaque.This infection characteristic is the same with N-GCRV.And 3,4 cell lysate supernatant being classified as employing GCRV core and expression VP5 and VP7 that in figure, A is capable, B is capable infect CIK cell, without cavity generation, show that cell fails to be infected.
Embodiment
Assembled in vitro GCRV preparation method provided by the invention, carries out (the patent No.: 200710168642.2) achieving to utilize on baculovirus expression system great expression GCRV outer capsid proteins VP5 and VP7 basis.Adopt the GCRV core of outer capsid proteins VP5 and VP7 and the purifying of expressing to carry out assembled in vitro, obtain thus and there is infective recombinant virus particle.This method can be used for studying outer capsid proteins VP5 and VP7 and infects effect in host cell process at GCRV, thus discloses the molecular mechanism that GCRV infects.
Above-mentioned assembled in vitro GCRV preparation method provided by the invention, comprises the following steps:
(1) adopt CIK cell (grass carp kidney cell line) to carry out virus multiplication, obtain a large amount of viral suspension infecting GCRV Hunan Shaoyang strain GCRV873 of CIK cell;
(2) by centrifuging, concentrated and purifying are carried out to the cell virus suspension having infected GCRV873;
(3) virus of trypsin treatment purifying is adopted, make intact virus outer capsid proteins VP5 and VP7 degradable, form the viral core particle containing nucleic acid, by 25-55% (w/v) CsCl density gradient centrifugation purifying, in 50%CsCl gradient solution, obtain a large amount of viral core particle;
(4) recombinant baculovirus vAcGCRV-VP5/VP7 is adopted to infect Sf9 cell, great expression outer capsid proteins VP5 and VP7;
(5) Sf9 cell warp-80 DEG C and 37 DEG C multigelations vAcGCRV-VP5/VP7 infected 3 times, the centrifugal 15min precipitate cell debris of 4000g;
(6) the outer capsid proteins VP5 of the nucleoid of purifying and baculovirus expression system great expression and VP7 cell lysate supernatant are measured according to different chemical hatch than carrying out mixing, hatch 3h, complete assembled in vitro for 28 DEG C;
(7) adopt 30-50% (w/v) CsCl density gradient centrifugation purifying, in about 40%CsCl gradient, obtain the R-GCRV particle of large-scale purification;
(8) adopt electron microscopy, SDS-PAGE and Plaque assay, carry out R-GCRV morphological structure, protein ingredient and analysis of infection.Result shows, R-GCRV and N-GCRV is very similar in morphological structure, protein ingredient and infectivity etc.
Below in conjunction with embodiment, above-mentioned preparation method is described further.
The propagation of one .GCRV873 and core preparation
CIK cell (grass carp kidney cell line) is adopted to carry out GCRV873 propagation.
1.CIK cell cultures:
Cell culture fluid is the Eagle's MEM substratum (MEM-10, Invitrogen Products) containing 10% foetal calf serum, PH 7.2.Get the CIK cell of preserving in liquid nitrogen, 37 DEG C of incubations melt.To be transferred to after the centrifugal 1min of 5000g in T25 Tissue Culture Flask (Corning Products) on desk centrifuge, the MEM-10 nutrient solution adding 5mL is cultivated.The suitableeest culture temperature of CIK cell is 28 DEG C, after at the bottom of the basic confluent culture bottle of cell, adopts the tryptic digestion attached cell of 0.25%, carries out Secondary Culture.
The propagation of 2.GCRV873:
Carry out connecing poison time at the bottom of the basic confluent culture bottle of the CIK cell gone down to posterity, first 10mmol/L phosphate buffered saline buffer (phosphate buffered salient is used, be called for short PBS) CIK cell of pre-vaccination is rinsed 2-3 time, to remove bovine serum albumin component; Then after the MEM of GCRV suspension serum-free being diluted, be about 1PFU/cell according to the infection multiplicity MOI (Multiplicity ofinfection) of virus and add the viral suspension of suitable volume of culture in monolayer cell culture bottle, supernatant liquor is outwelled after adherent cell 30min, clean cell 2-3 time with 10mmol/L PBS again, remove the virus particle of not adsorbing.Add MEM (MEM-2) maintenance medium containing 2% foetal calf serum that about 1/10 cell bottle amasss subsequently and carry out virus multiplication.After treating that the cell of about 85% produces cytopathy (cytopathic effect, CPE), collect viral suspension, save backup in 4 DEG C.
The purifying of 3.GCRV873:
By the viral suspension that 4 DEG C are preserved, with 3800g low-speed centrifugal 30min, the cell debris that removing is free.The viral suspension of removing cell debris is put ultracentrifuge (Beckman Products) with 100,000g ultracentrifugation 2h, precipitation GCRV873 particle.Abandon supernatant liquor, precipitate and be dissolved in 10mmol/L PBS by 1:250 times of volume.Subsequently on desk centrifuge again with 12,000g high speed centrifugation 5min, the cell debris that removing is remaining.
The core preparation of 4.GCRV873:
By trypsin Sigma Products) join in the viral suspension of purifying, its working concentration is 200 μ g/mL, in 28 DEG C of water-baths, hatch 2h, makes viral outer capsid proteins composition degradable.Then through the CsCl density gradient of 25-55% (w/v) with 200,000g ultracentrifugation 4h, in 50%CsCl gradient solution, take out the sample containing a large amount of GCRV873 core, obtain pure nucleoid particle through 10mmol/L PBS dialysed overnight.Spectrophotometer (U.S. Biotek Products) is adopted to measure nucleoid concentration or the population of purifying.After measured, the GCRV873 core particle concentration of purifying is about 1.2mg/mL or granule number is about 1 × 10
12individual/mL, its sample saves backup in-20 DEG C.
Two. baculovirus expression system great expression outer capsid proteins VP5 and VP7
Sf9 cell (insect cell line) is adopted to carry out the great expression of outer capsid proteins VP5 and VP7.
Sf9 cell culture fluid is the Grace substratum (Grace-10, Invitrogen Products) containing 10% foetal calf serum, pH6.5.
1.Sf9 cell cultures:
Get the Sf9 cell preserved in liquid nitrogen, 37 DEG C of incubations melt.To be transferred to (Corning Products) in T25 culturing bottle after the centrifugal 1min of 5000g on desk centrifuge, add 5mL Grace-10 nutrient solution and cultivate.The suitableeest culture temperature of Sf9 cell is 28 DEG C, after at the bottom of the basic confluent culture bottle of culturing cell, adopts glass bend pipe to blow and beat attached cell gently, carries out Secondary Culture.
2. the great expression of outer capsid proteins VP5 and VP7:
Go down to posterity after Sf9 cell confluent monolayers at the bottom of cell bottle, first with 10mmol/L PBS, the cell of pre-vaccination is rinsed 2-3 time, removing bovine serum albumin component; Then be about by the infection multiplicity MOI of virus the vAcGCRV-VP5/VP7 suspension that 5PFU/cell adds suitable volume of culture, after adherent cell 30min, outwell the recombinant virus suspension do not adsorbed, then clean cell 2-3 time, thoroughly to remove the virion do not adsorbed with 10mmol/LPBS.Then add Grace (Grace-2) maintenance medium containing 2% foetal calf serum that about 1/10 cell bottle amasss and carry out virus multiplication.Cultivate after 48h, adding Aprotinin (Trypsin inhibitor,Trasylol, Sigma Products) and Leupeptin (but bright proteolytic enzyme, Sigma Products) proteinase inhibitor to final concentration is respectively 0.5 μ g/ml.After virus infection 72h, culture supernatant collected together with cells infected in 50mL centrifuge tube, with the centrifugal 30min sedimentation cell of 3800g, the cell precipitated is dissolved in the PBS of appropriate volume.Cell counter (U.S. Bio-Rad Products) is adopted to measure the cell count expressing outer capsid proteins VP5 and VP7.After measured, the cell count expressing VP5 and VP7 is 10
7individual/mL, saves backup in-80 DEG C.Through freeze thawing, the cell pyrolysis liquid containing great expression outer capsid proteins VP5 and VP7 can be obtained.
Three. the GCRV873 core of purifying and the assembled in vitro of outer capsid proteins VP5 and VP7
The concentration of purifying is about 1.2mg/mL to employing 10mmol/L PBS or granule number is 1 × 10
12it is that 0.8-1.2mg/mL or granule number are about 10 that individual/mL nucleoid is diluted to concentration respectively
8-10
12individual/mL; In addition, the Sf9 cell that vAcGCRV-VP5/VP7 infects being diluted to cell count is respectively 10
5-10
7individual/mL, through-80 DEG C and 37 DEG C of multigelations 3 times, the centrifugal 15min precipitate cell debris of 4000g.Then the nucleoid of different extension rate is mixed according to different stoichiometric ratios from the cell lysate supernatant of different extension rate, hatch assembling at 28 DEG C, carry out manually slowly shaking up, until complete vitro recombination after 3h every half an hour.Adopt the separation and purification of 30-50% (w/v) CsCl density gradient centrifugation, obtain the recombinant virus particle (R-GCRV) of purifying.Plaque assay is adopted to measure different chemical metering than mixing the infectivity of assembling the R-GCRV obtained afterwards.Result shows, 10
10individual viral core particle number and 10
6the cell lysate supernatant incubation reaction of individual coexpression VP5 and VP7 albumen, namely at least according to 10
10: 10
6when/mL stoichiometric ratio carries out incubated in vitro assembling, can obtain that there is higher infective R-GCRV, with original virus particle (N-GCRV), there is almost identical virus titer.
The biological property analysis of four .R-GCRV
1. adopt electron microscopy to carry out identification of morphology to the R-GCRV of separation and purification.Getting 3 μ L drips on the copper mesh being sprayed with carbon film through the R-GCRV of CsCl density gradient centrifugation separation and purification, blots after absorption at room temperature virus 3-5min with filter paper bar.Then use the phospho-wolframic acid of 2% (pH6.9) negative staining to be about about 1min, then suck unnecessary dyestuff with filter paper, place air drying.Observe under Hitachi 7000-FA transmission electron microscope.Result shows that R-GCRV and N-GCRV form under transmission electron microscope is very similar, and be the double capsid virus particle of icosahedron, diameter is about 75nm.
2. adopt denaturing polyacrylamide gel electrophoresis (SDS-PAGE) technology of 10% to carry out the analysis of R-GCRV protein ingredient, target protein band can be shown after coomassie brilliant blue staining.Result shows, R-GCRV and N-GCRV contains identical protein ingredient, all containing complete 7 kinds of structural protein (VP1-VP7).
3. adopt plaque assay to measure the infectivity of R-GCRV, be provided with N-GCRV in experiment is positive control simultaneously, and the GCRV core of purifying and the cell lysate supernatant of expression VP5 and VP7 are as negative control.
Result is as shown in Figure 1, similar to N-GCRV, and R-GCRV infects CIK cell and produces typical plaque, and the cell that the cell lysate supernatant of nucleoid or expression VP5 and VP7 infects does not produce obvious plaque.
4. adopt 20mM NH
4cl carries out the infection blocking experiment of R-GCRV and N-GCRV, and result shows, and the ability of R-GCRV and N-GCRV cells infected is all adding weak base NH
4be greatly suppressed after Cl, show that the two have employed identical route of infection.
In above-described embodiment, adopt the recombinant baculovirus (vAcGCRV-VP5/VP7) of GCRV outer capsid proteins VP5 and VP7 of Sf9 insect cell line propagation restructuring, its construction process is see " expressing recombinant baculovirus and the structure thereof of the GCRV outer capsid proteins " patent documentation (patent No.: 200710168642.2), the method is mainly: increased by RT-PCR, obtains the specific fragment of 2 coding GCRV outer capsid proteins VP5 and VP7 genes respectively; The restructuring pFastBacdual dual-expression vector pFbDGCRV-VP5/VP7 inserting GCRV VP5 and VP7 gene is obtained by gene clone screening; Transforming DH10Bac cell by screening the restructuring dual-expression vector obtained, obtaining the AcGCRV-VP5/VP7Bacmid of swivel base; And by its transfection Sf 9 insect cell, in Sf9 insect cell, obtain recombinant virus vAcGCRV-VP5/VP7.
Claims (10)
1. an assembled in vitro GCRV preparation method, it is characterized in that a kind of GCRV GCRV core and baculovirus expression system express the method for the assembled in vitro of outer capsid proteins VP5 and VP7, the method comprises the following steps:
(1) GCRV873 is bred:
Grass carp kidney cell line and CIK cell propagation GCRV873, GCRV873 is adopted to be Shaoyang strains of GCRV Hunan;
(2) acquisition of the nucleoid containing nucleic acid and centrifugal purification:
Adopt the virus of trypsin treatment purifying, with outer capsid proteins VP5 and VP7 that degrade, obtain the nucleoid containing nucleic acid, and adopt cesium chloride CsCl density gradient to carry out centrifugal purification;
(3) expression of outer capsid proteins VP5 and VP7:
Recombinant baculovirus vAcGCRV-VP5/VP7 infected insect cell system and Sf9 cell is adopted to carry out outer capsid proteins VP5 and VP7 great expression;
(4) smudge cells:
The Sf9 cell of expression outer capsid proteins VP5 and VP7 of collection is carried out freeze thawing treatment, with smudge cells;
(5) assembled in vitro:
The GCRV core of purifying is carried out mixing hatching according to suitable stoichiometric ratio with outer capsid proteins VP5 and VP7 at Sf9 cells, realizes the assembled in vitro of GCRV;
(6) centrifugal purification:
Adopt CsCl density gradient to carry out centrifugal purification to the virion after assembled in vitro, obtain the assembled in vitro virus particle R-GCRV of purifying, it is for having infective assembled in vitro GCRV particle.
2. assembled in vitro GCRV preparation method according to claim 1, is characterized in that assembled in vitro GCRV particle, and its core particle concentration used or granule number should reach 1.2mg/mL or 10
12individual/mL.
3. assembled in vitro GCRV preparation method according to claim 1, after it is characterized in that adopting Sf9 cell great expression outer capsid proteins VP5 and VP7, its cell count should reach 10
7individual/mL.
4. assembled in vitro GCRV preparation method according to claim 1, is characterized in that assembled in vitro process is: the nucleoid of purifying is diluted to concentration 1mg/mL to employing 10mmol/L phosphate buffered saline buffer PBS or granule number is about 10
10individual/mL, becomes cell count to be 10 the Sf9 cell dilution that vAcGCRV-VP5/VP7 infects
6individual/mL.
5. assembled in vitro GCRV preparation method according to claim 1, is characterized in that the Sf9 cell that infected by vAcGCRV-VP5/VP7 is through-80 DEG C and 37 DEG C of multigelations 3 times, the centrifugal 15min precipitate cell debris of 4000g.
6. assembled in vitro GCRV preparation method according to claim 1, is characterized in that getting the supernatant liquor containing VP5/VP7 albumen mixes with the nucleoid of purifying, hatches 3h, completes assembled in vitro for 28 DEG C.
7. assembled in vitro GCRV preparation method according to claim 1, when it is characterized in that centrifugal purification, adopt 30-50%CsCl density gradient centrifugation to carry out the purifying of assembled in vitro virus, in 40%CsCl density gradient, obtain greatly the recombinant virus whole grain R-GCRV of a large amount of assembled in vitro.
8. assembled in vitro GCRV preparation method according to claim 1, is characterized in that adopting the infectivity of plaque analyses method to R-GCRV to measure.
9. assembled in vitro GCRV preparation method according to claim 1, is characterized in that adopting 20mM NH
4cl carries out blocking experiment, analyzes the infectious difference of R-GCRV and protovirus N-GCRV, to verify the route of infection of the two.
10. assembled in vitro GCRV preparation method according to claim 1, it is characterized in that the nucleoid of purifying to hatch with mixing according to the suitableeest stoichiometric ratio at Sf9 cells outer capsid proteins VP5 and VP7, ratio both it can not lower than 10
10: 10
6/ mL.
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| CN115850397A (en) * | 2022-07-22 | 2023-03-28 | 南阳师范学院 | II-type grass carp reovirus-like particle and preparation method and application thereof |
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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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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| FANG QIN ET AL.: "3D reconstruction and capsid protein characterization of grass carp reovirus", 《SCIENCE IN CHINA SER. C LIFE SCIENCES》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115850397A (en) * | 2022-07-22 | 2023-03-28 | 南阳师范学院 | II-type grass carp reovirus-like particle and preparation method and application thereof |
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