CN1322119C - Mandarin fish rhabdo virus toxic strain, its preparation method and application - Google Patents
Mandarin fish rhabdo virus toxic strain, its preparation method and application Download PDFInfo
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- CN1322119C CN1322119C CNB2003101162414A CN200310116241A CN1322119C CN 1322119 C CN1322119 C CN 1322119C CN B2003101162414 A CNB2003101162414 A CN B2003101162414A CN 200310116241 A CN200310116241 A CN 200310116241A CN 1322119 C CN1322119 C CN 1322119C
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Abstract
The present invention discloses a strain of a Siniperca chuatsi rhabdovirus, SCRV0208, with the preservation number of CCTCC No. V202008, a preparation method thereof and an application thereof. The preparation method comprises: GCF0208 cells are firstly cultured in a TC199 culture medium with bovine serum into monolayer cells, inoculated with virus liquid, sampled in different periods, sliced, and observed through an electronic microscope to determine the dot picking time; after dots are picked, the monolayer cells are inoculated with the cells to expend culture, and centrifugated to obtain purified virus. The simple and practical method can effectively separate rhabdovirus from accompanied infected virus to obtain high-purity virus antigens for the application in cell engineering vaccines and genetic engineering vaccines.
Description
Technical field
The present invention relates to separation, purifying and the technology of preparing of aquatic animal virus causing disease, be particularly related to the in-vitro multiplication of the famous-brand and high-quality fish virus of fresh water and the material source of detection technique, the pure antigenic technology of preparing of virus and development virocyte engineered vaccine and recombinant vaccine, more specifically relate to a kind of mandarin fish rhabdovirus strain, the preparation method who also relates to the strain of mandarin fish rhabdovirus also relates to the purposes of mandarin fish rhabdovirus strain in the detection of freshwater fish virus causing disease, evaluation and cell engineering vaccine and recombinant vaccine development.
Background technology
Mandarin fish (Siniperca chuatsi, mandarin fish) is a predacious fish, fine and tender taste, spinule between apleuria, ancient speech are said " peach blossom flowing water mandarin fish fertilizer ", as seen, not only body fertilizer is delicious for mandarin fish, and is full of poetic and pictorial splendor, is the cultured freshwater fish veriety of being favored especially.Mandarin fish has very big domestic and international demand, but along with the successful grasp and the breed amount of mandarin fish artificial propagation techniques constantly enlarges, its disease problem also becomes increasingly conspicuous and is serious, especially in middle and later periods nineteen nineties, large-area explosive prevailing disease has taken place in the mandarin fish that China's southern area is cultured, the mandarin fish mortality ratio surpasses 90% in the fishpond that causes, even reaches 100%.Only just cause average annual loss to surpass hundred million yuans heavy losses in the area, Guangdong.Confirm that the epiphytotics generation of mandarin fish is relevant with different virus infectiones.
Virus is the parasite in the host cell, still do not have active drug so far and can kill the intravital virus of invasion animal, preparation and use virus vaccines be still the popular and harm of prevention mandarin fish virus disease most promising by way of.Not only infectivity is strong for virus disease, rapid onset, can cause high mortality, and the virus causing disease of mandarin fish is complicated.Because virus disease causes the massive losses of culture fishery, caused the great attention of going up to the city father down to experts and scholars, relevant ministries and commissions have organized a plurality of R﹠D institutions to carry out the special project research of aquatic animal virus disease.
About the more existing reports of mandarin fish irido virus research, the ultra micro observation that comprises the mandarin fish irido virus with separate, fish body tieback infects and propagate analysis by way of, cell cultures, nucleic acid, PCR diagnostic method, and the mensuration [Wu Shuqin etc. of genome sequence, aquatic product journal, 1997,21 (supplementary issue): 56-60; 2000,24 (2): 165-169; 2001,25 (5): 460-463; He Jianguo etc., Zhongshan University's journal, 1998, (5): 25-31; He etc., Virology, 2001,291 (1): 126-39; Fang Qin etc., Chinese virusology, 2000,15 (3): 297-301].
Except irido virus, the ultrathin section(ing) of suffering from the epiphytotics mandarin fish tissue preparation of fulminant is carried out electron microscopic observation, show and have different mandarin fish virus, comprise that rhabdovirus SCRV, baculovirus SCBV virus and spherical virus SCSV[open strange Asia etc., Science Bulletin, 1999,44 (2): 192-195].This result also points out, though single vaccine is effective because of the prevention of damage by disease that a kind of virus causes to mandarin fish, the disease effect that anti-multiple virus causing disease causes still is limited.Therefore, have only to clone corresponding virus causing disease respectively, and development and use multiple vaccines, just may stop the propagation of mandarin fish virus disease and popular.
Summary of the invention
The object of the present invention is to provide mandarin fish rhabdovirus strain, this mandarin fish rhabdovirus strain is at GCF
0208Breed in a large number in the cell, it is tired and can reach 10
7TCID
50/ ml.
Another object of the present invention has provided a kind of preparation method of mandarin fish rhabdovirus strain, and method is simple, this method effectively with rhabdovirus with it other virus of concomitant infections separate, with the amplification homogeneous SCRV
0208,, obtain high purity virus antigen by purifying.
The invention still further relates to the application of a kind of mandarin fish rhabdovirus strain in cell engineering vaccine and recombinant vaccine.
To achieve these goals, the present invention is by the following technical solutions:
The virus clone technology be exactly from virus cell or host's culture, obtain viral colony by viral offspring's propagation.The viral cultures of this biological character unanimity that is obtained is called the crohn strain, thereby can reach the purpose of purifying screening virus strain.The monolayer cell that the viral suspension inoculation of suitably dilution is responsive covers one deck solid dielectric again on cell, as agarose, then when virus after the initial cell internal breeding of infecting because the restriction of solid dielectric, can only and then infect and destroy the cell of vicinity.Through several such proliferating cycles, just will form visible degenerating cell district under limitation, naked eyes or the light microscopic, this is commonly called " plaque ".Theoretically, a virus particle can form a plaque.Simultaneously, plaque technique also is a kind of method accurately of measuring infective virus content in the viral suspension.The present invention cultivates by the picking plaque exactly again, and obtains clone's mandarin fish rhabdovirus strain.In suffering from epiphytotics mandarin fish tissue, observe the virus of 3 kinds of different shapes by electron microscope.Carry out ultra micro in conjunction with Electronic Speculum and observe and choose the spot clone, different mandarin fish viruses are separated, obtain clone mandarin fish rhabdovirus strain (Siniperca chuatsi rhabdovirus, SCRV recently
0208), CCTCC NO:V202008.Solid foundation has been established in this development for hydrocoles rhabdovirus cell engineering vaccine and recombinant vaccine.It is characterized in that: GCF
0208Cell adds 10% new-born calf serum through the TC199 substratum, cultivates 12-14 hour, grows up to monolayer cell, uses 0.1ml SCRV
0208(tire is 10 to virus liquid
7TCID
50/ ml) after the inoculation, the layer overlay agarose continues to cultivate in 25 ℃ of constant incubators.Behind virus inoculation, cultivated 12-36 hour simultaneously, and cultivate the period sampling in difference, the preparation ultrathin section(ing), and carry out electron microscopic observation, determine at SCRV
0208Be inoculated into GCF
0208Cultivate after 20 hours in the monolayer cell and choose spot.Through carrying out such spot process of choosing repeatedly 7 times, obtain clone's mandarin fish rhabdovirus strain SCRV
0208, continue enlarged culturing, again through differential centrifugation, sucrose gradient centrifugation, and, confirm to have obtained the SCRV of large-scale purification through negative staining and electron microscope observation
0208
In separation and purification to the basis of mandarin fish virus, utilize known different Rhabdovirus (vesicular stomatitis virus belongs to and Nola's Rhabdovirus) N gene and glycoprotein gene particular sequence for referencial use, and synthetic primer, with mandarin fish rhabdovirus genome is template, carry out RT-PCR amplification and comparison, recognizing that mandarin fish rhabdovirus and known other kinds of fish Rhabdovirus genomic constitutions there are differences, is a new strain.
The present invention compared with prior art has the following advantages and effect:
1. cell cultures is combined with electron microscopy, obtain the result directly perceived that the mandarin fish rhabdovirus breeds in host cell.
2. behind the window of opportunity of determining rhabdovirus a large amount of amplifications in host cell, can accurately choose the spot purifying.
3. obtain the strain of clone mandarin fish rhabdovirus.
4. obtain the rhabdovirus of purifying.
Description of drawings
Fig. 1, before choosing the spot pure culture, infect the cell electromicroscopic photograph of mandarin fish virus, show SCRV
0208, also the spherical virus of following with it arranged.x30000
Fig. 2, through after choosing spot for 7 times and separating, at GCF
0208The SCRV of middle pure culture
0208Electromicroscopic photograph.Be presented at and only deposit a large amount of SCRV in the host cell
0208, and shape virus if unsighted.x30000
Fig. 3, purifying SCRV
0208The negative staining electromicroscopic photograph, show the virion of a large amount of similar sub warhead samples.x60000
Embodiment
Collect the mandarin fish that typical illness is arranged in the popular pathogenetic epidemic-stricken area of mandarin fish, the symptom of sick mandarin fish mainly shows as kiss end, skin of head, dorsal fin and the hyperemia of tail fin base portion or the blutpunkte is arranged, and the flake ball that has is outstanding, and the cheek turns white because of ischemic.According to observations, in case these illnesss occur, the mandarin fish in the fishpond can mass mortality in 3-7 days.Take out the viscera tissue (comprising liver,spleen,kidney etc.) of disease fish, the phosphoric acid buffer (PBS) that shreds, adds 1-2 times of volume carries out homogenate, add again two anti-after, put refrigerator-20 ℃ freezing spending the night, take out next day treat that it melts after, with 1000-2000 rev/min speed centrifugal 10 minutes, obtain SCRV
0208The virus crude extract.With the SCRV for preparing
0208The virus crude extract after filtration, and at grass carp fin cell GCF
0208Middle propagation.At SCRV
0208The ultrathin section(ing) of virus crude extract cells infected through electron microscopic observation, promptly has spherical virus in the visible cell, rhabdovirus (Fig. 1) is also arranged.
With SCRV
0208The GCF of virus crude extract inoculation
0208Enchylema is collected, and is prepared into SCRV-20 ℃ of following freeze thawing twice
0208Enchylema is with SCRV
0208Enchylema is inoculated into 8 bottles of grass carp fin cell GCF that grown up to individual layer respectively
0208In, put under 25 ℃ of conditions and cultivate.From inoculating back 12 hours, got sample one time in every interval 2-4 hour, fix with glutaraldehyde, the preparation ultrathin section(ing), JEM-1230 type transmission electron microscope is observed down.Observations shows, at SCRV
0208In the cell ultrathin section(ing) after the inoculation between 12-18 hour, spherical virus is bigger than the rhabdovirus amount; But at SCRV
0208Inoculation GCF
0208In the cell ultrathin section(ing) of 20 hours or longer time, the amount of rhabdovirus increases greatly in the cell behind the cell, and spherical virus almost is difficult to find.Therefore determine, with virus inoculation to the GCF that grows up to individual layer
0208Behind the cell, cover agar, continue to cultivate after 20 hours and choose spot.Through 7 such spots of choosing, obtain clone mandarin fish rhabdovirus strain SCRV finally
0208At SCRV
0208The GCF that infects
0208In the cell ultrathin section(ing), the morphology of virus feature is that size is the rhabdovirus (Fig. 2) of an end tip, the other end tack for 70-110x30-45nm, form.
With the SCRV that obtains
0208Virus strain is at GCF
0208Amplification cultivation in the cell, collect virus after differential centrifugation, again through sucrose gradient centrifugation and the ultracentrifugation affination purifying of 20-60%.The viral sample of getting purification is through 2% phospho-wolframic acid negative staining and electron microscopic observation, and as seen the result has the virion (Fig. 3) of a large amount of similar sub warhead samples.
The purposes of mandarin fish rhabdovirus in the fresh-water fishes virus causing disease detects:
To inoculate mandarin fish rhabdovirus SCRV
0208Strain also the GCF of pathology occurs
0208Cell pyrolysis liquid is made antigen behind the differential centrifugation, weekly with hypodermic mode immune rabbit, and immunity is six times altogether.Then, through etherization, from the blood sampling of rabbit carotid artery, the centrifugal anti-SCRV of rabbit that obtains
0208Serum.Use this serum,, just can examine the survey fish and whether infect the virus causing disease that the serology dependency is arranged with the mandarin fish rhabdovirus by the immunodiffusion(ID) method.
Specific operation process is: get 60ul through GCF
0208Cell pyrolysis liquid absorption and the anti-SCRV of rabbit that does not adsorb
0208Serum is added in the quincuncial medium pore of agar plate, in 6 holes, adds the tissue homogenate (or a small amount of serum of fish) of different sample fishes to be checked respectively on every side.Then flat board is put into wet box, placed three days in 37 ℃ of incubators.Day by day observe its antigen antibody reaction precipitation line, the sample that precipitation line occurs is just for being confirmed as virus infection (positive).This method is easy and simple to handle, and expense is cheap, and suitable fish sample detection in enormous quantities when being particularly suitable for breeding nontoxic fry, is used for the screening of parent population.
The purposes of mandarin fish rhabdovirus in the recombinant vaccine development:
Mandarin fish rhabdovirus SCRV
0208Be to contain the genomic virus of not segmentation single stranded RNA.The rhabdovirus genome N that generally encodes, NS, M, G and L5 kind albumen are being analyzed SCRV
0208During gene structure, our increased N gene fragment of SCRV0208.Therefore, with 5 '-GAAGATCAGGYCRANTAYCC-3 ' and 3 '-TCTRACACGWCGTGABCGDTG-5 ' is primer, with mandarin fish rhabdovirus RNA is template, but the cDNA of the synthetic corresponding gene of reverse transcription, behind the electrophoresis purifying, insert on the pGEM-T carrier, and transformed into escherichia coli DH5 α, filter out positive colony after, enlarged culturing, propagation excretory virus antigen just is present in the nutrient solution, purifiedly just can be used for preparing the kinds of fish Rhabdovirus gene vaccine after adding adjuvant.The present invention not only provides good material and development approach for the development of fish recombinant vaccine, and more direct preparation manipulation method and the easy cell engineering vaccine of technology have been established sturdy basis.
SCRV has now been set up in this laboratory
0208The amplification in vitro system of virus strain finds its sensitive cells GCF
0208We confirm in addition, utilize virus vaccines can induce the fish cell of vitro culture to produce antiviral substance.Therefore, the immune effect of vaccine and safety experiment can be measured by this system.Implementation process is given an example: earlier with GCF
0208Cell passes in two 96 porocyte plates, treat that it grows up to individual layer after, be divided into three groups.Wherein one group is the normal cell contrast; One winding kind SCRV
0208Another organizes first vaccination, uses SCRV again
0208Attack poison.Through microscopic examination, the immune protective rate (seeing Table 1) of cytopathy situation and vaccine as can be known just.
Table 1: vaccine protection ratio test experiments
| The experimental group numbering | The experimental cell hole count | The sick cell hole count appears | The sick cell hole count does not appear | Protection ratio % |
| The 1st group (cell control group) the 2nd group (directly attacking the poison group) the 3rd group (vaccine protection group) | 48 48 96 | 0 47 1 | 48 1 95 | 2.12％ 98.95％ |
The security of vaccine also can be measured by the cell of vitro culture.Execute process for example: earlier with GCF
0208Cell passes in two 24 porocyte plates, treat that it grows up to individual layer after, be divided into two groups.Wherein one group is the normal cell contrast; One winding kind SCRV
0208Vaccine, whether the microscopic examination cell pathology occurs, and just whether safely vaccine (sees Table 2) as can be known.
Table 2: vaccine safety test experiments
| The experimental group numbering | The experimental cell hole count | The sick cell hole count appears | The sick cell hole count does not appear | Safety factor % |
| The 1st group (control group) the 2nd group (vaccine group) | 24 24 | 0 0 | 24 24 | 100 |
Claims (2)
1, a kind of mandarin fish rhabdovirus strain is characterized in that being mandarin fish rhabdovirus strain Siniperca chuatsirhabdovirus, SCRV
0208, CCTCC NO:V202008.
2, a kind of mandarin fish rhabdovirus strain according to claim 1 is characterized in that viral micromorphological features is: size is the rhabdovirus of an end tip, the other end tack for 70-110 * 30-45nm, form.
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|---|---|---|---|
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| CN 02139283 CN1410535A (en) | 2002-11-14 | 2002-11-14 | Mandarin fish rhabdo virus toxic strain, its preparation method and application |
| CNB2003101162414A CN1322119C (en) | 2002-11-14 | 2003-11-13 | Mandarin fish rhabdo virus toxic strain, its preparation method and application |
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| CN108251384B (en) * | 2017-12-13 | 2021-06-15 | 中国水产科学研究院珠江水产研究所 | Fish rhabdovirus attenuated vaccine strain |
| CN109762939A (en) * | 2019-02-02 | 2019-05-17 | 中国水产科学研究院珠江水产研究所 | For detecting the primer and probe and kit of mandarin fish rhabdovirus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001055330A2 (en) * | 2000-01-28 | 2001-08-02 | Thomas Jefferson University | Recombinant rhabdoviruses as live-viral vaccines for immunodeficiency viruses |
| CN1370831A (en) * | 2001-02-23 | 2002-09-25 | 国家海洋局第一海洋研究所 | Purificaltion process of sea fish lymphocysis virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001055330A2 (en) * | 2000-01-28 | 2001-08-02 | Thomas Jefferson University | Recombinant rhabdoviruses as live-viral vaccines for immunodeficiency viruses |
| CN1370831A (en) * | 2001-02-23 | 2002-09-25 | 国家海洋局第一海洋研究所 | Purificaltion process of sea fish lymphocysis virus |
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Assignee: Guangzhou City Haiwei Fertilizer Co., Ltd. Assignor: Institute of Hydrobiology, Chinese Academy of Sciences Contract fulfillment period: 2007.9.1 to 2012.8.31 contract change Contract record no.: 2008420000018 Denomination of invention: Mandarin fish rhabdo virus toxic strain, its preparation method and application Granted publication date: 20070620 License type: Exclusive license Record date: 2008.7.7 |
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Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2007.9.1 TO 2012.8.31 Name of requester: HAI WEI CO., LTD., GUANGZHOU CITY Effective date: 20080707 |
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Granted publication date: 20070620 Termination date: 20131113 |