CN1306959C - Process for preparing subunit vaccine of reovirus antigen for grass carp - Google Patents
Process for preparing subunit vaccine of reovirus antigen for grass carp Download PDFInfo
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- CN1306959C CN1306959C CNB2004100608812A CN200410060881A CN1306959C CN 1306959 C CN1306959 C CN 1306959C CN B2004100608812 A CNB2004100608812 A CN B2004100608812A CN 200410060881 A CN200410060881 A CN 200410060881A CN 1306959 C CN1306959 C CN 1306959C
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Abstract
The present invention relates to a preparation method for the reovirus antigen subunit vaccines of grass carp, which comprises the following steps: adopting the renal cell system 3 of the grass carp, i.e. CIK cell, to proliferate GCRV873 which is the reovirus strain of the grass carp in the Shaoyang city of Hunan province; purifying and concentrating the suspension of cell viruses infected with the GCRV873 by a centrifugation method; processing the purified viruses with surfactant to disassemble intact virus structures for forming the subunit components of viral nucleic acid and capsid proteins; digesting and degrading the dissociative virus genome dsRNA with nuclease; carrying out dialysis processing to obtain the subunit preparations of the reovirus proteantigen of the grass carp without ribonucleic acid; and diluting the preparations with physiological saline to obtain the reovirus antigen subunit vaccines of the grass carp. The present invention has the advantages of no RNA of viral genetic materials, simple preparation method, convenient operation, high preparation purity, small packaging bulk and convenient transportation and preservation, and contain intact viral proteantigen components.
Description
Technical field
The present invention relates to field of virology, particularly a kind of GCRV antigen subunit vaccine preparation method.The used Strain of the present invention is GCRV Hunan Shaoyang strain GCRV873 (Grass carp reovirus), claim GCHV Hunan Shaoyang strain GCHV873 (Grass carp hemorrhagic virus) again, by China's typical culture collection center preservation, preservation date is on July 23rd, 2004, and deposit number is CCTCC NO:V200414.
Background technology
Ctenopharyngodon idellus (Ctenopharyngodon idellus) is one of China's freshwater aquiculture four large Chinese carps.GCRV is the main pathogen that causes Ctenopharyngodon idellus death, falls ill when serious, can cause the Ctenopharyngodon idellus fry morbidity more than 85% dead, causes serious economy loss to freshwater aquaculture industry thus.For the control of viral disease, vaccine is effective measures the most.Rough tissue milk of GCRV and cell inactivated vaccine sequential use and have been obtained certain effect in production.But because there are many unfavorable factors in the aspects such as stability, purity and safety of inactivated vaccine, its application and popularization value is limited.Hemorrhagic disease of grass carp still happens occasionally in some area of China, and, still very important in the hidden danger that China's large tracts of land is broken out once again.
Summary of the invention
Technical problem to be solved by this invention is: in view of factors such as attenuated live vaccine and the existing residual infection of rough fire extinguishing vaccine and the instabilities of tiring, be necessary to provide a kind of GCRV antigen subunit vaccine preparation method, thereby open up new effective way with viral vaccine for developing fish.
The technical solution adopted in the present invention is: adopting the Ctenopharyngodon idellus kidney cell line is CIK cell proliferation GCRV873, and GCRV873 is Shaoyang strain of GCRV Hunan; By centrifuging, the cell virus suspension that has infected GCRV873 is carried out purification and concentrated; With the virus of surfactant NP40 processing purification, the intact virus structure is disintegrated, form viral nucleic acid and capsomere component; Adopt nuclease digestion, degrade free virus genome dsRNA; By dialysis treatment, do not contained the GCRV proteantigen subunit preparation of nucleic acid; With normal saline virus dilution proteantigen subunit preparation, be prepared into GCRV antigen subunit vaccine.
The present invention compared with prior art has following major advantage:
One. preparation method is simple, easy to operate, is suitable for scale and produces in batches and use.
They are two years old. and product purity is higher: do not contain other (as cell and tissue) proteantigen composition substantially, can be used for preparing the vaccine of a kind of safety, high-efficiency prevention and control hemorrhagic disease of grass carp.
They are three years old. and product does not contain viral nucleic acid, and subunit vaccine contains intact virus antigen.Not only fish body and water environment are had safety, got rid of problems such as potential gene contamination simultaneously the aquaculture thing.
They are four years old. and use GCRV873 subunit preparation, soaked 3～5cm Ctenopharyngodon idellus fry 30 minutes, can reach 80%～85% immune protective effect.
They are five years old. and the subunit preparation is a concentrating virus antigen subunit product, has characteristics such as the little and high valency of packaging volume, is convenient to transportation and preserves.
They are six years old. and have the commercialization promotional value, use simultaneously, can further improve and improve fish body immune protective rate with immunological adjuvant.
The specific embodiment
GCRV antigen subunit vaccine provided by the invention, its preparation process comprises:
(1) adopts CIK cell (Ctenopharyngodon idellus kidney cell line) to carry out virus multiplication, obtain the viral suspension of GCRV Hunan Shaoyang strain GCRV873 of infection CIK cell.
(2), the cell virus suspension that has infected GCRV873 is carried out purification and concentrated by centrifuging.
(3) surfactant is handled the virus of purification, and the complete virion structure is disintegrated, and forms viral nucleic acid and capsomere component.Surfactant can adopt NP40 (Fluka company product); Its concentration is 0.5%～1%, can make viral capsid proteins become loose like this and is unlikely to degrade fully.
(4) adopt nuclease to digest, free virus genome dsRNA in the degraded GCRV873 subunit preparation.
(5), do not contained the GCRV antigen subunit protein preparation of nucleic acid by dialysis treatment.
(6) with normal saline virus antigen subunit protein preparation is diluted, obtain GCRV antigen subunit vaccine.
Below in conjunction with embodiment above-mentioned preparation method is described further.
One .GCRV873 virus multiplication
1. adopt CIK cell (Ctenopharyngodon idellus kidney cell line) to carry out GCRV873 propagation.
1) cell culture fluid is the Eagle ' s MEM culture medium (MEM-10) that contains 10% super pure calf serum, pH7.2.
2) CIK cell culture: get the CIK cell of preserving in the liquid nitrogen, 37 ℃ of incubation 2～3min.Directly behind centrifugal 1min on the desk centrifuge, add an amount of MEM-10 culture fluid, be transferred in the culture bottle and cultivate.The suitableeest cultivation temperature of CIK cell is 28 ℃, cell culture 2～3 days, treat at the bottom of the basic confluent cultures bottle after, adopt 0.1%～0.25% trypsinization attached cell, the cultivation of going down to posterity.
3) GCRV873 virus preparation: passage cell is in exponential phase or compiles at about 80% o'clock at the bottom of bottle, with the CIK cell of inoculating in advance, at first use 10mmol/L phosphate buffer (phosphate buffered salient is called for short PBS) flushing 3 times, remove the bovine serum albumin component; The viral suspension that adds infection then by 1/10 volume of culture, the infection multiplicity MOI (Multiplicity of infection) of virus is 5～10PFU/cell, behind viral suspension adherent cell 30～60min, outwell the not viral suspension of absorption, and then, remove the not virion of absorption with 10mmol/L PBS cleaning 3 times.The MEM (MEM-2) that employing contains 2% calf serum keeps liquid and carries out virus multiplication.At viral infection 48～72h, treat the basic cracking of most of cell after, collect viral suspension, standby in 4 ℃ of preservations.
2. dripping valency measures: viral suspension to be measured is carried out a series of 10 times of dilutions (10
-1～10
-15), infect the CIK cell by above-mentioned virus multiplication method.At viral infection 48～72h, observation of cell pathological changes situation.Press the Reed-Muech method and calculate 50% a neutralization valency.A valency of measuring virus-culturing fluid is 10
11TCID
50/ ml.
Two .GCRV873 viral purifications
Collect to infect the GCRV viral suspension of CIK cell,, remove the free cell fragment with 8000r/min (6000g) low-speed centrifugal 30 minutes.With the viral suspension of removing cell debris with 26,000r/min (80,000g) ultracentrifugation 2.5hrs, precipitation GCRV granule.Abandon supernatant, precipitation is dissolved among the 10mmol/L PBS by 1: 100 times of volume.Again with 8000r/min (6000g) low-speed centrifugal 20 minutes, remove the residual cells fragment subsequently.Supernatant adds 0.5%～1%Nonidet p40 (Flaka company product is called for short NP40) or other kind surfactant such as Triton-X-100 (being dissolved in 10mmol/L PBS) by 1/10 volume ratio, handles 2～4h, makes the viral capsid composition become loose for 28 ℃.In above-mentioned solution, add 50-100mg/ml nuclease (RNase), 28 ℃ of digestion 2～4h, degraded free virus nucleic acid.To distilled water flowing water dialysis 48h, remove the nucleic acid of degraded at last.The sample of purification is standby in-20 ℃ of preservations.
The GCRV873 virion is the symmetric spheroidal particle of icosahedron, and ripe viral diameter is about 75nm, has double capsid, no cyst membrane.This virus is very stable in the pH3-11 scope, and is insensitive to organic solvents such as chloroform and ether, and its genome is made up of 11 dsRNA.
The mensuration that the neutralization of three .GCRV873 subunits is tired
Virus antigen subunit behind the purification is measured through ultraviolet spectrophotometer, adopts 10mmol/L PBS that its antigen subunit is made final concentration 0.5～1mg/ml preparation.Get GCRV subunit 100-150 μ g (the about 0.5 μ g/ μ l of protein content) immunizing rabbit, get the neutralization of antiserum mensuration and tire.Adopt fixed virus amount and doubling dilution serum method carry out viral in and titration.Experiment is provided with 3 groups of repetitions, carries out in 24 hole Cast plates.Establish each one group of viral infection and normal cell contrast simultaneously.Press the Reed-Muech method and calculate 50% a neutralization valency mensuration.Recording that antibody neutralization tires is 1: 1025.
Four .GCRV873 viral sub-units preparation detection of nucleic acids
Adopt conventional chloroform: the GCRV subunit preparation that the phenol extraction process is handled the RNA enzyme carries out nucleic acid extraction, gets 15 μ l nucleic acid extractives, through the polyacrylamide electrophoresis, does not see GCRV nucleic acid band, and the contrast without the RNA enzyme is handled presents tangible 11 nucleic acid bands.In addition,, adopt the GCRV cDNA probe of digoxigenin labeled, do not detect remaining RNA for further improving detection sensitivity.The positive control that is provided with can detect the GCRVRNA of 10pg.
Five .GCRV873 viral sub-units vaccines are to the protection recruitment evaluation of fry
The GCRV subunit vaccine is diluted to 10 with normal saline (0.85%NaCl)
-2～10
-6A series of gradients adopt infusion method that 3～4cm hatching was soaked 30 minutes, obtain immune protective effect preferably.10
-3The subunit preparation protective rate of diluent can reach 85%, 10
-4Dilution protective rate is more than 75%.Show that adopting dilution factor is 10
-3Or 10
-4GCRV subunit preparation carries out immersion treatment to the Ctenopharyngodon idellus fry, can prevent and treat hemorrhagic disease of grass carp effectively.
Claims (5)
1. GCRV antigen subunit vaccine preparation method is characterized in that comprising following concrete steps:
(1) adopting the Ctenopharyngodon idellus kidney cell line is CIK cell proliferation GCRV873, and GCRV873 is Shaoyang strain of GCRV Hunan,
(2) by centrifuging, the cell virus suspension that has infected GCRV873 is carried out purification and concentrated,
(3) with the virus of surfactant NP40 processing purification, the intact virus structure is disintegrated, forms viral nucleic acid and capsomere component,
(4) adopt nuclease digestion, degrade free virus genome dsRNA,
(5) by dialysis treatment, do not contained the GCRV proteantigen subunit preparation of nucleic acid,
(6) with normal saline virus dilution proteantigen subunit preparation, be prepared into GCRV antigen subunit vaccine.
2. GCRV antigen subunit vaccine preparation method according to claim 1 is characterized in that the preparation of GCRV873 suspension may further comprise the steps:
(1) the CIK cell that will inoculate in advance is phosphate buffer flushing 3 times with PBS, removes the bovine serum albumin component, and PBS concentration is 10mmol/L,
(2) volume of culture of pressing cell culture fluid 1/10 adds the virus stock solution used of GCRV873, and the virus stock solution used infection multiplicity is 5～10PFU/cell,
(3) virus absorption onto cell was outwelled the not virus stock solution used of absorption after 30～60 minutes, cleaned 3 times with PBS then, removed the not virion of absorption,
(4) adopt the MEM contain 2% calf serum or MEM-2 to keep liquid and carry out virus multiplication, behind viral infection 48～72h, collect viral suspension, standby in 4 ℃ of preservations.
3. GCRV antigen subunit vaccine preparation method according to claim 1 is characterized in that the GCRV873 viral suspension is carried out purification and spissated method is:
(1) collect the CIK cell virus suspension infected GCRV873,, remove the free cell fragment with 6000g low-speed centrifugal 25～35 minutes,
(2) viral suspension that will remove cell debris obtains GCRV873 solids precipitation thing with 80000g ultracentrifugation 2～3h,
(3) precipitate is dissolved in PBS by 1: 100 times of volume,
(4) with 6000g low-speed centrifugal 20～25 minutes, remove the residual cells fragment,
(5) supernatant is added 0.5%～1%NP40 surfactant in 1/10 ratio, handles 2～4h for 28 ℃, virus structure is disintegrated,
(6) in above-mentioned solution, add the 50-100mg/ml nuclease, 28 ℃ of digestion 2～4h, degraded free virus nucleic acid,
(7) through distilled water flowing water dialysis 40～50h, remove the nucleic acid of degrading in the preparation, obtain purer GCRV873 antigen protein subunit composition, standby in-20 ℃ of preservations.
4. GCRV antigen subunit vaccine preparation method according to claim 3, the virus protein that it is characterized in that the GCRV873 behind the purification is measured its content through ultraviolet spectrophotometer, is made into the antigen subunit preparation that final concentration is 0.5～1mg/ml with the PBS dilution.
5. GCRV antigen subunit vaccine preparation method according to claim 1, the concentration that it is characterized in that surfactant NP40 is 0.05～0.1%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104918913A (en) * | 2012-11-16 | 2015-09-16 | 特里纳普克公司 | Synthesis of tetrabutylammonium bis(fluorosulfonyl)imide and related salts |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1895667B (en) * | 2006-06-06 | 2010-11-03 | 瑞普（保定）生物药业有限公司 | Reovirus inactivated vaccine and its preparation |
| CN102218136B (en) * | 2011-04-12 | 2013-07-24 | 上海海洋大学 | Anti-reovirus oral gene engineering vaccine and its preparation method |
| CN102532310B (en) * | 2012-03-08 | 2013-10-30 | 中华人民共和国北京出入境检验检疫局 | Monoclonal antibody against grass carp reovirus VP5 protein and application of monoclonal antibody |
| CN103382221B (en) * | 2013-07-05 | 2015-03-11 | 中国科学院水生生物研究所 | Single chain antibody 19B7 for specific recognition and neutralization of grass carp reovirus |
| CN104774873B (en) * | 2015-03-31 | 2017-10-03 | 中国科学院武汉病毒研究所 | A kind of assembled in vitro GCRV preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001161355A (en) * | 1999-11-25 | 2001-06-19 | Natl Sci Council | Immortal cell line obtained from epinephelus coioides of family serranidae and application thereof |
| WO2003072811A2 (en) * | 2002-02-28 | 2003-09-04 | Oncolytics Biotech Inc. | The use of ribozymes in the detection of adventitious agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001161355A (en) * | 1999-11-25 | 2001-06-19 | Natl Sci Council | Immortal cell line obtained from epinephelus coioides of family serranidae and application thereof |
| WO2003072811A2 (en) * | 2002-02-28 | 2003-09-04 | Oncolytics Biotech Inc. | The use of ribozymes in the detection of adventitious agents |
Non-Patent Citations (5)
| Title |
|---|
| 水生呼肠孤病毒研究进展 方勤等，中国病毒学，第18卷第1期 2003 * |
| 草鱼出血病病毒多肽及其基因组的体外翻译 柯丽华，病毒学杂志，第8卷第2期 1992 * |
| 草鱼出血病病毒多肽及其基因组的体外翻译 柯丽华等，病毒学报，第8卷第2期 1992 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104918913A (en) * | 2012-11-16 | 2015-09-16 | 特里纳普克公司 | Synthesis of tetrabutylammonium bis(fluorosulfonyl)imide and related salts |
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