CN1239697C

CN1239697C - Large brill rhabdo virus toxic strain and its preparation method and application

Info

Publication number: CN1239697C
Application number: CN 200310116250
Authority: CN
Inventors: 张奇亚; 李正秋; 桂建芳
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2002-11-14
Filing date: 2003-11-13
Publication date: 2006-02-01
Anticipated expiration: 2023-11-13
Also published as: CN1500866A

Abstract

The present invention discloses a toxic strain of scophthalmus maximus rhabdovirus SMV0207 with the preservation number of CCTCC No. V202006, preparation method thereof and application thereof. The preparation method comprises the following steps: taking the liver, kidney, heart and spleen tissue of affected scophthalmus maximus, crushing, adding PBS for homogenation, adding amphitolerant, freezing in a refrigerator, thawing, centrifugating, filtering coarse virus extracting liquid, respectively inoculating to CLC0207 cells for further culture at constant temperature. With the easy method, the virus strain can be rapidly proliferated in the CLC0207 cells in potency of 10<8>TCID50/ml, and can be used for preparing vaccines for cell engineering of sea water fish rhabdovirus.

Description

Turbot rhabdovirus strain and its production and application

Technical field

The present invention relates to aquatic animal disease cause of disease and separation, vitro culture and authenticate technology, particularly the in-vitro multiplication of sea farming fish virus, detection diagnostic techniques.More specifically relate to a kind of Da Ling Maximus rhabdovirus strain, the preparation method who also relates to a kind of turbot rhabdovirus strain also relates to a kind of biological method simultaneously and is identifying seawater fish rhabdovirus and the sea water fish rhabdovirus purposes in the cell engineering vaccine preparation.

Background technology

Big water chestnut separates that (Scophthalmus maximus is the Pleuronectiformes seawater fish of dwelling at the bottom of a kind of cold water that originates in Europe turbour), also is local famous sea farming breeding.Because the smooth delicacy of turbot mouthfeel and deliciousness, spur are few, no off-flavors, fin limit and subcutaneous abundant colloid arranged can be compared U.S. with soft-shelled turtle with sea cucumber, be that ideal keeps healthy and beauty food.Turbot personality docility, there is not mutual residual phenomenon, and growth is rapidly, acceptant mixed feed, and the food efficiency of conversion was up to 1: 1, be easy to characteristics such as intensive culture, be subjected to the great attention of international mariculture industry, and the peculiar figure of flatfish is arranged, in water body, can build colourful, the appreciation effect that light butterfly is danced exquisitely becomes the upstart of whole world culture fishery rapidly.China introduced a fine variety the beginning of the nineties, and under the batch production condition, the fry of 5cm was cultured 1 year at present, and body weight can reach 800-1000g.But with the expansion of the scale of breed, the disease of turbot also becomes increasingly conspicuous, especially viral disease [Angulo etc., 1994, the J.Fish Dis.17:163-169 that causes; Breton etc., 1997, J.Fish Dis, 20:145-152], become the restraining factors of turbot aquaculture development.

The turbot virus disease is by the bait throwing in horizontal transmission a bit, as viral nervous necrosis [Breton etc., 1997] and infectious pancreatic necrosis [Mortensen etc., 1995, J World Aqacul.Soci.26:217-219].Some disease directly contacts by water body or with ill fish and propagates.Also have a kind of spherical virus particle, the hemocyte of directtissima turbot causes fish body anoxic [Lamas etc., 1995, J.Fish Dis, 18:425-433] by influencing erythrocytic function.Cultivation density increases, and propagating for the turbot virus disease provides more opportunity.

Known hydrocoles rhabdovirus majority is the strong pathogenic agent of fish, is the pathogenic agent of fish septicemia as viral septicemia virus (VHSV), and they can cause the fatal disease that host animal is serious, and morbidity host mortality ratio in 1 week can reach 100%.Also have barracuda rhabdovirus (pike fry rhabdovirus), snakehead fish rhabdovirus (snakehead rhabdovirus), SVCV (spring viremia of carp), the beautiful fish rhabdovirus of Bluepoint (Rio grande cichlid rhabdovirus) and perch rhabdovirus (perch rhabdovirus) multiple in addition.These rhabdoviruses can both cause fish morbidity and dead, cause heavy economic big loss.Therefore, set up corresponding method, the turbot virus causing disease is separated, especially the fulminant disease that causes of rhabdovirus is identified, can set up corresponding early diagnosis technology, and understand its fashion trend, in conjunction with the corresponding virus vaccines of development, thereby can play effective monitoring and prophylactic effect to its propagation.

Summary of the invention

The object of the present invention is to provide a kind of turbot rhabdovirus strain, turbot rhabdovirus strain is at CLC ₀₂₀₇High efficiently multiplying in the cell, propagation is tired and can be reached 10 ⁸TCID ₅₀/ ml.

Another object of the present invention also relates to a kind of preparation method who prepares turbot rhabdovirus strain, this method is simple, and can be external a large amount of amplification flounder rhabdoviruses, and the fish body infection experiment condition that has solved turbot virus is difficult to control, the cost height, the problem of poor repeatability.

The invention still further relates to the application of a kind of turbot rhabdovirus strain in biological method evaluation seawater fish rhabdovirus.

The invention still further relates to the application of a kind of turbot rhabdovirus strain in kinds of fish Rhabdovirus detection accurately and effectively and diagnosis.

The invention still further relates to the application of a kind of turbot rhabdovirus strain in the preparation of sea water fish rhabdovirus cell engineering vaccine.

In order to achieve the above object, the present invention by the following technical solutions;

From the Da Ling Maximus that suffers from fatal disease, be separated to a strain rhabdovirus, be called turbot rhabdovirus (Scophthalmus maximus rahbdovirus, SMRV ₀₂₀₇), CCTCC NO:V202006 proves that through the inoculation experiments to multiple fish cell this virus can cause that host cell produces the cracking performance pathology.This is the wider kinds of fish Rhabdovirus cause of disease of a kind of host range.Virus disease with other animal is the same, does not still have medicine so far and can effectively control them, have only by virus vaccines carry out immunity by way of, just for the prevention virus disease, keep the turbot healthy aquaculture to show hope.This laboratory utilizes the superiority condition with fish cell storehouse, and find when whether turbot is carried out conventional sense by virus infection culturing by cultivating fish cell: the turbot of coastal certain plant of China is infected by a kind of rhabdovirus.Carry out the fish body and attack poison experiment and cultured cell in vitro infection experiment, and in conjunction with biochemical method analyses such as electron microscopic observation, Flow Cytometry detection and electrophoresis, show that this virus can cause Apoptosis of Host Cells, confirmed the lethality cause of disease that this strain rhabdovirus is a turbot.For this reason, we are through carrying out amplification in vitro and cultivate, separate repeatedly and screen, measure virus structural protein and carry out series of studies such as pcr amplification to this virus, and (tire is 10 by force to have obtained virulence ⁸TCID ₅₀/ ml), biological heredity stable performance, the kinds of fish Rhabdovirus strain SMRV that can carry out pure culture ₀₂₀₇

The illness of ill turbot is to have occurred many vesicles and blutpunkte on body surface, skin, and liver has the irregular piebald of the depth, the nephridial tissue erosion, and body surface diseases occurs 24 hours, and sick fish will be dead.Get the organization material of disease fish, be prepared into tissue homogenate, be inoculated in the sensitive cells, carry out the separation and Culture of virus; Or the organization material of getting the disease fish directly with glutaraldehyde fixing after, the preparation ultrathin section(ing) is used for electron microscopic observation.

To SMRV ₀₂₀₇Infect CLC ₀₂₀₇The ultrathin section(ing) of cell carries out electron microscopic observation, show consistently with viewed situation in ill turbot cell, turbot rhabdovirus morphological specificity is: it is that 110-150 * 40-60nm, form are the rhabdoviruses of an end tip, the other end tack that a lot of sizes are arranged in the cell.

As material, vitro culture and evaluation have been carried out with ill or dying big water chestnut Maximus tissue from different aspects to wherein rhabdovirus.Comprise:

After A, ill turbot tissue preparation become homogenate, infect different fish cells, clear and definite CLC ₀₂₀₇Cell is to SMRV ₀₂₀₇Sensitivity can produce the pathology spot behind the virus inoculation, and SMRV ₀₂₀₇Virus strain is at CLC ₀₂₀₇Propagation in the cell is tired and can be reached 10 ⁸TCID ₅₀/ ml.

The directly fixing fresh sick fish tissue sample of B carries out ultramicroscopic view and examines after ultrathin section(ing), dyeing, size, the form of clear and definite virus and cause the ultra micro pathological change of host cell.

Description of drawings

Fig. 1, turbot rhabdovirus SMRV ₀₂₀₇The ultra micro form, showing a large amount of sizes among the figure is that 110-150 * 40-60nm, form are the rhabdoviruses of an end tip, the other end tack.×30 000

Fig. 2, (A), normal CLC ₀₂₀₇Cell and (B), infect SMRV ₀₂₀₇, and the CLC of pathology appears ₀₂₀₇The cell Photomicrograph.×40

Fig. 3, SMRV ₀₂₀₇Infect CLC ₀₂₀₇Cavity appears in the electromicroscopic photograph of cell in the cells infected.×4 000

Embodiment

Vessel equipment, substratum, the reagent of the cell cultures that is useful on and virus amplification all require in advance to keep aseptic through processing such as autoclaving or filtration sterilizations.The compound method of part common agents is as follows:

Phosphate buffered saline buffer (PBS, no Ca ⁺⁺, Mg ⁺⁺):

NaCl 8.00g

KCl 0.20g

Na ₂HPO ₄ 2.31g

KH ₂PO ₄ 0.31g

Be dissolved in the 500ml distilled water, add 5ml 0.4% phenol red liquid, add distilled water to 1000ml after stirring.10 pounds of sterilizations in 15 minutes are standby.

Two anti-(penicillin, Streptomycin sulphate) solution

With 10 ⁶Unit penicillin G and 10 ⁶μ g Streptomycin sulphate is dissolved in the tri-distilled water of 100ml sterilization, makes every ml penicillin 10 ⁴Unit, Streptomycin sulphate 10 ⁴μ g.

The TC119 substratum

To purchase next TC199 substratum pulvis, the amount that by specification requires adds with fresh tri-distilled water dilution, fully the dissolving back adds serum (10% new-born calf serum), two anti-, and adds the light sodium solution of carbonic acid and regulate pH to 7.2-7.4, use the tri-distilled water constant volume again after, filter packing through filter.

The operation steps of virus inoculation and amplification:

After the liver of ill turbot, kidney, the heart, spleen tissue taken out, the PBS that shreds, adds 1-2 times of volume carried out homogenate, adds two anti-again, 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 SMRV ₀₂₀₇The virus crude extract.With the SMRV for preparing ₀₂₀₇The virus crude extract is inoculated into the CLC that has grown up to individual layer respectively after filtering ₀₂₀₇In the cell.Be provided with the contrast of corresponding normal cell simultaneously, and to put temperature be to continue to cultivate in 20 ℃ the constant incubator.Utilize opticmicroscope, regularly observe having inoculated viral active somatic cell, and make comparisons with corresponding normal cell.Behind the virus inoculation 12-24 hour, under inverted microscope, can see: at fine and close originally CLC ₀₂₀₇In the cell monolayer, the pathology spot successively occurred, beginning is the dispersive small amounts of cells formed little cavity that comes off, and the cavity increases (Fig. 2) gradually.To at this moment the section got, SMRV ₀₂₀₇The electron microscopic sample of cells infected preparation is observed, and show that the mandarin fish rhabdovirus breeds in a large number in host cell, and tangible pathology (Fig. 3) has appearred in cell.The cell that does not come off as yet in the cell monolayer forms netted subsequently.

The authentication method of a kind of sea water fish rhabdovirus:

The tissue homogenate of fish to be checked is inoculated into the CLC that grows up to individual layer ₀₂₀₇In the cell (being adopted cell), cultivated about 12 hours down for 25 ℃-30 ℃,, the phenomenon of attached cell local shedding as seen whether occurs through om observation by the present invention.The collection viral cell that increased carries out electron microscopyc sample preparation and observation again, just can be easier to determine that whether fish to be checked is by rhabdovirus infection.

A kind of preparation method of sea water fish rhabdovirus cell engineering vaccine:

At first obtained virus strain SMRV ₀₂₀₇Pure growth.

Secondly, screen and be suitable for virus strain SMRV ₀₂₀₇The sensitive cells CLC that height is tired and bred ₀₂₀₇Cell.

Moreover, by aftermentioned method and step, can prepare the rhabdovirus cell engineering vaccine of fish.CLC ₀₂₀₇Cell is put in 25 ℃ of-30 ℃ of cell culture incubators and is cultivated with 199 substratum (commercial product culture medium)+10% calf serum, grow up to individual layer after, the inoculation 0.1ml (contain 10 ⁵TCID ₅₀/ ml) SMRV ₀₂₀₇Viral suspension continue to be cultivated 12-20 hour, collected the enchylema that contains amplicon virus, with the formaldehyde of 0.1-0.5%, handled 4-6 hour down at 30 ℃-56 ℃, and is aseptic subpackaged, frozen, just prepare the cell engineering rhabdovirus vaccine of the many antigen components of sea water fish.This vaccine is safe in utilization, and can produce in batches.

Owing to set up viral amplification in vitro system, screen responsive host cell, therefore, the immune effect of vaccine and safety experiment can be measured by this system.Implementation process is given an example: earlier with CLC ₀₂₀₇Cell passes in the 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 SMRV ₀₂₀₇Virus; Another organizes first vaccination, uses SMRV again ₀₂₀₇Virus is attacked 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 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)	32 32 32	0 32 1	32 0 31	0 97％

The cell of vitro culture is more more responsive to toxicity and Effect of Environmental than animal body, so the safety experiment of vaccine also can be measured by cell.Execute process for example: earlier with CLC ₀₂₀₇Cell passes in the 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 SMRV ₀₂₀₇Vaccine, 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 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 (cell control group) the 2nd group (vaccination group)	12 12	0 0	12 12	100

Claims

1, a kind of turbot rhabdovirus strain is characterized in that turbot rhabdovirus strain Scophthalmusmaximus rahbdovirus, SMRV ₀₂₀₇, CCTCC NO:V202006.

2, a kind of turbot rhabdovirus strain according to claim 1 is characterized in that Da Ling Maximus rhabdovirus is: size is the rhabdovirus of an end tip, the other end tack for 110-150 * 40-60nm, form.

3, the application of the described a kind of turbot rhabdovirus strain of claim 1 in the preparation of sea water fish rhabdovirus cell engineering vaccine.