Infectious pancreatic necrosis vaccine and method for amplifying virus thereof on salmon embryo cells
阅读说明：本技术 传染性胰脏坏死病疫苗及其病毒在大鳞大麻哈鱼胚胎细胞上扩增的方法 (Infectious pancreatic necrosis vaccine and method for amplifying virus thereof on salmon embryo cells ) 是由 徐黎明 段凯越 邵轶智 赵景壮 任广明 卢彤岩 于 2021-07-27 设计创作，主要内容包括：本发明公开了传染性胰脏坏死病疫苗及其病毒在大鳞大麻哈鱼胚胎细胞上扩增的方法。本发明提供的体外增殖传染性胰脏坏死病毒的方法,包括如下步骤：将传染性胰脏坏死病毒按照剂量100～10000TCID-(50)接种于8.0×10～(5)个大鳞大麻哈鱼胚胎细胞,培养,实现体外增殖传染性胰脏坏死病毒。该方法所需收毒时间短、病毒滴度高且稳定。(The invention discloses an infectious pancreas necrosis vaccine and a method for amplifying virus thereof on embryo cells of salmon macroscales. The method for propagating the infectious pancreatic necrosis virus in vitro comprises the following steps: the infectious pancreatic necrosis virus is subjected to 100-10000 TCID (contact induced by infectious disease) according to the dosage 50 Inoculating to 8.0X 10 5 Culturing the embryo cells of the salmon to realize the in vitro multiplication of the infectious pancreatic necrosis virus. The method has the advantages of short virus recovery time, high virus titer and stability.)
1. A method for propagating infectious pancreatic necrosis virus in vitro, comprising the steps of: the infectious pancreatic necrosis virus is subjected to 100-10000 TCID (contact induced by infectious disease) according to the dosage50Inoculating to 8.0X 105Culturing the embryo cells of the salmon to realize the in vitro multiplication of the infectious pancreatic necrosis virus.
2. The method of claim 1, wherein: the culture time is 48-84 hours.
3. The method according to claim 1 or 2, characterized in that: the temperature of the culture was 15 ℃.
4. A method according to any one of claims 1-3, characterized in that: the culture time is 72 hours, the culture temperature is 15 ℃, and the inoculation dose is 100TCID50。
5. A method for preparing an infectious pancreatic necrosis disease vaccine, comprising the steps of:
1) the infectious pancreatic necrosis virus is subjected to 100-10000 TCID (contact induced by infectious disease) according to the dosage50Inoculating to 8.0X 105Culturing embryo cells of the salmon, and collecting supernatant to obtain in vitro propagated infectious pancreatic necrosis virus;
2) preparing the infectious pancreatic necrosis vaccine by using the infectious pancreatic necrosis virus propagated in vitro.
6. The method of claim 5, wherein: the culture time is 48-84 hours.
7. The method according to claim 4 or 5, characterized in that: the temperature of the culture was 15 ℃.
8. The method according to any one of claims 5-7, wherein: the culture time is 72 hours, the culture temperature is 15 ℃, and the dosage is 100TCID50。
9. The method according to any one of claims 5-8, wherein:
the preparation of the infectious pancreatic necrosis vaccine by using the in vitro propagated infectious pancreatic necrosis virus is to inactivate the in vitro propagated infectious pancreatic necrosis virus to obtain the infectious pancreatic necrosis inactivated vaccine.
10. An infectious pancreatic necrosis vaccine produced by the method of any one of claims 5 to 9;
or, an inactivated vaccine against infectious pancreatic necrosis, which is obtained by inactivating the in vitro propagated infectious pancreatic necrosis virus prepared by the method of any one of claims 1 to 4.
Technical Field
The invention relates to the technical field of biology, in particular to an infectious pancreas necrosis vaccine and a method for amplifying virus thereof on embryo cells of salmon pink fish.
Background
Infectious Pancreatic Necrosis Virus (IPNV) belongs to the aquatic birnaviridae family, Aquabirnavirus, which is the etiological agent of Infectious pancreatic necrosis disease (IPN). IPNs have started to become popular in the united states in the 50 th generation of the last century and have been spread to date to france, turkey, chile, mexico, finland, norway, spain, japan, korea, iran. The IPN epidemic situation is outbreak in 80 years in the first century in China, which causes huge economic loss and becomes one of important epidemic diseases restricting the development of the salmon and trout industry in China.
The vaccine immunization is the most effective method for preventing and controlling IPN at present, and the inactivated vaccine has the advantages of simple operation, good immunization effect, high safety, low production cost and the like, so that the inactivated vaccine becomes the first choice for preventing and controlling viral diseases. The efficient amplification of the virus is the basis of the preparation of the inactivated vaccine, the effect of the efficient amplification is influenced by the virus inoculation dose, when the virus inoculation is carried out at a high dose, a large amount of defective viruses are generated, and the defective viruses cannot be normally replicated, so that the virus cannot be normally passaged, and the production of the vaccine is influenced. When the inoculation dose is too small, the propagation speed of the virus is remarkably reduced, and the time required for virus propagation is prolonged. Currently, there is no report on the in vitro proliferation protocol of IPNV.
Disclosure of Invention
An object of the present invention is to provide a method for propagating infectious pancreatic necrosis virus in vitro.
The invention provides a method for propagating infectious pancreatic necrosis virus in vitro, which comprises the following steps: the infectious pancreatic necrosis virus is subjected to 100-10000 TCID (contact induced by infectious disease) according to the dosage50Inoculating to 8.0X 105Culturing the embryo cells of the salmon to realize the in vitro multiplication of the infectious pancreatic necrosis virus.
Optionally, in the method, the culturing time is 48 to 84 hours.
Optionally, in the above method, the temperature of the culturing is 15 ℃.
Optionally, in the above method, the culturing time is 72 hours, the culturing temperature is 15 ℃, and the inoculation is performedIs 100TCID50。
Another object of the present invention is to provide a method for preparing the infectious pancreatic necrosis vaccine.
The invention provides a method for preparing an infectious pancreatic necrosis vaccine, which comprises the following steps:
1) the infectious pancreatic necrosis virus is subjected to 100-10000 TCID (contact induced by infectious disease) according to the dosage50Inoculating to 8.0X 105Culturing embryo cells of the salmon, and collecting supernatant to obtain in vitro propagated infectious pancreatic necrosis virus;
2) preparing the infectious pancreatic necrosis vaccine by using the infectious pancreatic necrosis virus propagated in vitro.
Optionally, in the method, the culturing time is 48 to 84 hours.
Optionally, in the above method, the temperature of the culturing is 15 ℃.
Optionally, in the above method, the culturing time is 72 hours, the culturing temperature is 15 ℃, and the dose is 100TCID50。
Optionally, in the above method, the preparing the infectious pancreatic necrosis vaccine using the in vitro propagated infectious pancreatic necrosis virus is inactivating the in vitro propagated infectious pancreatic necrosis virus to obtain the infectious pancreatic necrosis inactivated vaccine.
The infectious pancreatic necrosis vaccine prepared by the method also belongs to the protection scope of the invention.
The invention also provides an inactivated vaccine for the infectious pancreatic necrosis, which is obtained by inactivating the in vitro propagated infectious pancreatic necrosis virus prepared by the method.
The IPNV sensitive cell large-scale salmon embryonic cell (CHSE-214) is utilized, virus inoculation is carried out at different doses, subculture is carried out, and an IPNV in-vitro multiplication scheme with small inoculation dose, high virus titer, stability and short virus receiving time is screened out by measuring the titer of each generation of virus and combining the optimal virus receiving time. The scheme is utilized to carry out large-scale culture and inactivation on IPNV, and then the immune protection effect of the inactivated virus is evaluated, so that reference is provided for large-scale production and application of the IPN inactivated vaccine.
At 100TCID50Dose of (2) IPNV to 8.0X 105The required virus harvesting time of each CHSE-214 cell is short, the virus titer is high and stable, and the optimal virus harvesting time is 72 hours after virus inoculation.
The IPN inactivated vaccines prepared by inactivating IPNV at room temperature for 48h by 0.500% BPL have obvious immune protection effect on rainbow trout within 60 days after immunization.
Drawings
FIG. 1 is an example 1IPNV strain at three doses (100 TCID)50、1000 TCID50、10000 TCID50) Titer of virus of different generations after inoculation.
FIG. 2 shows the IPNV strain of example 1 at 100TCID50Gene content varied at different times after inoculation into CHSE-214 cells, with different letters indicating significant differences (p < 0.05).
FIG. 3 shows the 100TCID used in example 150CHSE-214 cells at various times after inoculation of IPNV.
FIG. 4 is the titer of the virus of example 2 in rainbow trout tissues with different letters indicating significant differences (p < 0.05).
FIG. 5 is the viral load in tissues after challenge with the immune rainbow trout of example 3, with different letters indicating significant differences (p < 0.05).
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Data were processed using GraphpadPrism8 statistical software and results were expressed as mean ± standard deviation using One-way ANOVA test.
M199 cell culture medium (Gibco, cat # C11150500 BT).
Salmon embryo cells (CHES-214 cells) were maintained in the laboratory and were described in the following references: xu L M, Zhao J Z, Liu M, et al. Bivalent DNA vaccine antigens infection infected heterologous biochemical secretion virus and infection pathological secretion virus in raw stomach [ J ]. Scientific reports, 2017,7 (1): 5700..
IPNV strains were isolated and stored by the laboratory and are described in the following references: duan K Y, ZHao J Z, Ren G M, et al molecular Evolution of Infectious coronary thrombosis in China [ J ]. Virus, 2021, 13 (3): 488..
Example 1 preparation of IPNV Virus solution
First, recovery and culture of CHSE-214 cells
Taking out the frozen CHSE-214 cells from the liquid nitrogen tank, quickly putting the cells into a constant-temperature water bath kettle at 30 ℃, after the cells are melted, transferring the cells into a 15m1 centrifuge tube under the aseptic condition, centrifuging the cells at 1000g/min for 2min, discarding the cell frozen stock, adding 1ml of M199 cell culture solution (containing 10% fetal calf serum and 1% double antibody) and uniformly mixing. And transferring the uniformly blown cells into a T-25 cell culture bottle, supplementing 4ml of cell culture solution, uniformly blowing, and placing into a carbon dioxide biochemical incubator at 20 ℃ for standing culture. After the cells were collected into a monolayer, they were digested with 0.25% trypsin and passaged to 6-well or 96-well cell culture plates, etc., as required for the experiment.
Second, study of the optimal proliferation protocol of IPNV on CHSE-214 cells
1. Different concentrations of IPNV infected CHSE-214 cells
Taking 6-well plate, the amount of single layer of CHSE-214 cells in each well is 8.0 × 105Separately, IPNV strain (1.00X 10) isolated in this laboratory6.00 TCID500.1ml) was diluted with PBS buffer, each using 100TCID50、1000 TCID50、10000 TCID50A monolayer of CHSE-214 cells was inoculated at a dose of 1ml virus fluid per well. After incubation for 1h in a carbon dioxide constant temperature incubator at 15 ℃, the culture solution containing the virus is discarded, each well is replaced by 2mL of cell maintenance solution (2% fetal calf serum in M199 culture medium), and the cells are kept still for culture at 15 ℃ and observed for cytopathic conditions every day.
(please supplement the adjustment according to the actual situation)
And (3) when 80% of cells have pathological changes, harvesting the virus, repeatedly freezing and thawing twice in a refrigerator at-80 ℃, centrifuging at 12000g/min at 4 ℃ for 15min, and collecting virus supernatant (namely the primary virus).
Primary viruses were inoculated in 6-well cell culture plates at the three doses described above and serially subcultured in the same manner until passage 10. And subpackaging the virus supernatant harvested by each passage and storing in a refrigerator at-80 ℃ for later use.
2. IPNV titer determination
The harvested 10-generation virus supernatant was subjected to a 10-fold dilution gradient (10) using a cell maintenance medium in a 1.5mL sterile centrifuge tube-1～10-8) Monolayer of CHSE-214 cells seeded in 96-well cell culture plates (4.0X 10 cells per well)48 wells per dilution, 0.1ml of diluted virus supernatant per well is put into a carbon dioxide biochemical incubator at 15 ℃ for culture for 7 days, then the virus titer is calculated according to the Reed-Muench method, and the optimal virus inoculation dose with high and stable virus titer is screened out. Cell maintenance solution inoculated monolayer of CHSE-214 cells was a negative control.
The results show that when IPNV is 100TCID50When the virus is inoculated, the titer gradually rises along with the increase of the number of passages, and becomes stable when the virus is transmitted to the 5 th passage, and the average titer is 1.00 multiplied by 107.50 TCID500.1 ml. At 1000TCID50Upon inoculation, IPNV titers rose rapidly and still did not level off when the 10 th passage was reached. In 10000TCID50Upon inoculation, IPNV titers increased rapidly in the first 4 passages and slowed down when transmitted to passage 5, but still did not reach steady state. In view of the 100TCID50IPNV titres were high and stable after virus inoculation, thus determined for 8.0X 1051ml of 100TCID for one CHSE-214 cell50Of virusesThe amount of inoculation was the optimal inoculation dose (fig. 1).
3. Determination of IPNV virus recovery time
The experiment was divided into a virus inoculation group and a control group.
The receiving group is used for receiving the optimal receiving dose (100 TCID)50) IPNV was seeded onto a monolayer of CHSE-214 cells (8.0X 10 per well) in 6-well cell culture plates5Individual cell), incubating in a carbon dioxide constant-temperature incubator at 15 ℃ for 1h, discarding culture solution containing virus, replacing each hole with 2mL of cell maintenance solution, standing and culturing at 15 ℃, photographing the cell by using an inverted microscope every day, and recording the pathological change condition of the cell. Harvest cell cultures at 12, 24, 48, 60, 72 and 84h post-inoculation culture total RNA was extracted using Trizol reagent and 2 using RT-qPCR-ΔΔCTThe method detects changes in IPNV gene content in cells to determine the optimal time for harvesting the virus.
The control group was normal CHSE-214 cells cultured in 6-well cell culture plates using cell maintenance media at 8.0X 10/well5The cells, 2ml of cell maintenance liquid, were cultured by standing in a carbon dioxide biochemical incubator at 15 ℃. Cell cultures were harvested at the same time as the vaccinated group and total RNA of the cultures was extracted using Trizol reagent as a negative control.
Uses IPNV A chain gene as target gene and beta-acin as reference gene.
A chain gene upstream primer F: GCATT CAACT ACGGG AGAC
A chain gene downstream primer R: CATCA GGCT GTTGTA GGTTAG
Beta-acin upstream primer F: GCCGGCCGCGACCTCACAGACTAC
A beta-acin downstream primer R: CGGCCGTGGTGGTGAAGCTGTAAC
Each 20. mu.l RT-qPCR system included: 10 μ 12 Xone Step TB Green RT-PCR Buffer 4, 1.2 μ l TaKaRa Ex Taq HS Mix, 0.4 μ l PrimeScript PLUS RTase Mix, 2 μ l RNA template, 0.8 μ l upstream and downstream primers, and 4.8 μ l RNase Free dH2And O. The reaction procedure is as follows: 42 ℃ 5min (1 cycle), 95 ℃ 10s (1 cycle), 95 ℃ 5s (40 cycles), 60 ℃ 34s (40 cycles), 95 ℃ 15s (1 cycle), 60 ℃ 1min (1 cycle), 95 ℃ 30s (1 cycle), 60 ℃ 15s (1 cycle)Cycling).
The gene content multiple is the IPNV gene content of the virus inoculation group/the IPNV gene content of the control group.
RT-qPCR results showed that IPNV gene content in CHSE-214 cells increased first and then decreased. The virus nucleic acid content is lower within 24 hours after inoculation; the gene content begins to increase 48-72 hours after inoculation, which is 19.8 times, 29.4 times and 120.0 times of that of a control group respectively, and the difference of the gene content of IPNV among time points is obvious; at 84h after the inoculation, the gene expression level begins to decrease, which is 89.7 times that of the control group, and is significantly lower than 72h after the inoculation, but still significantly higher than other time points (p < 0.05) (FIG. 2).
The observation result of cytopathic effect shows that the cells do not change within 48h after virus inoculation, and the CHSE-214 cells begin to have pathological changes 60h after virus inoculation, which is represented as cell thinning, cell ablation and circular membrane structure; after 72h of inoculation, cells begin to have large-area lesions and are disintegrated; most of the cells disintegrated and fell off 84h after inoculation (FIG. 3).
This time point was chosen as the optimal time for virus harvest, given that IPNV is the highest gene content and cytopathic effects are evident at 72h post-inoculation.
Taken together, the optimal proliferation protocol for IPNV on CHSE-214 cells was: the inoculation dose was 100TCID50And culturing at 15 ℃ for 72h to obtain the virus.
4. In vitro proliferation of IPNV
Dose is 100TCID50In 6-well cell culture plates (8.0X 10 cells per well)5Individual cell), standing and culturing for 72h in a carbon dioxide constant-temperature incubator at 15 ℃, harvesting the virus, repeatedly freezing and thawing for 2 times in a refrigerator at-80 ℃, centrifuging for 10min (4 ℃) at 12000g/min, and taking the supernatant as IPNV virus solution after amplification.
Example 2 establishment of IPNV challenge model
At 1.00X 105.00 TCID50Dose/tail IPNV (IPNV strain isolated in this laboratory (1.00X 10)6.00TCID50/0.1m1)) to perform intraperitoneal injection for counteracting toxic substance to rainbow trout (with the weight of 10 +/-2 g). 3, 7, 14 and 30 days after toxin attack, respectively taking visceral tissues (liver, spleen and head and kidney) of 3 fish to obtainThe supernatant was homogenized in PBS buffer at a ratio of 1: 10 (g: ml) and then centrifuged at 12000g/min4 ℃ for 15 min. The supernatant was sterile filtered and diluted 10-fold (10)-1～10-8) Then inoculated into 96-well cell culture plates, and after 7d, the virus titer was calculated by the Reed-Muench method to determine the time point at which the virus content in the tissue was the highest, which was repeated three times for each sample. Rainbow trout tissues injected intraperitoneally with an equal amount of PBS buffer were used as a control group.
The results showed that IPNV titers in rainbow trout tissues were highest 3d after challenge and then began to decline. At 3, 7, 15, 30d post-challenge, mean IPNV titres in tissues were 1.51X 10, respectively8.00、5.46×107.00、4.25×106.00、1.47×105.00 TCID50(ii) in terms of/g. This time point was chosen as the post challenge sampling time point, considering that IPNV had the highest viral titer at 3d post challenge (p < 0.05) (fig. 4).
In summary, the challenge model was the use of the IPNV strain isolated in example 1 (1.00X 10)6.00 TCID500.1ml), the rainbow trout is detoxified by intraperitoneal injection, and the dosage is 10 mul/tail. And taking the 3d after the challenge as a sampling time point after the challenge.
Example 3 preparation of an IPN inactivated vaccine and immunoprotection potency analysis
Preparation of IPN inactivated vaccine
0.153ml of beta-propiolactone (BPL) was added to 30ml of IPNV virus solution (virus titer of 1.00X 10) obtained by "4, in vitro propagation of IPNV" in example 17.50 TCID500.1ml) to ensure that the final concentration of the BPL is 0.500 percent, mixing uniformly, putting the mixture in a shaking table at room temperature (25 ℃), inactivating the mixture for 48 hours at 100r/min, and carrying out water bath at 37 ℃ for 2 hours to obtain the IPN inactivated vaccine.
Second, analysis of immunoprotection potency of IPN inactivated vaccine
Immunization groups were administered 0.1 ml/tail (1.00X 10) of the IPN inactivated vaccine prepared as described above7.50 TCID50) The dose of the immune rainbow trout was intraperitoneally injected, and the control group was intraperitoneally injected with the same amount of PBS buffer solution with pH of 6.5. The weight of the rainbow trout is 10 plus or minus 2g, and each group has 80 tails.
At different time points after immunization, virus challenge was performed according to the challenge model established in example 2, and the virus titer was determined 3 days after challenge as a sampling point. The virus titer was determined in the same manner as in example 2.
The degree of viral load reduction is expressed as the ratio of the viral titer of the control group to that of the immunized group.
The results showed that the rainbow trout virus titer in the immunized group was significantly lower than that in the control group (p < 0.05), with the titer decreased the most at 30d after immunization. The mean titers of IPNV in the tissues of the control rainbow trout at 3, 7, 14, 30, 45 and 60d after immunization were 4.7-fold, 13.5-fold, 9.3-fold, 60513.3-fold, 435.8-fold and 29.6-fold (p < 0.05) that of the immunized rainbow trout, respectively. The result shows that the IPN inactivated vaccine can effectively inhibit virus proliferation within 60 days after immunization and continuously generate immune protection effect on the rainbow trout, wherein the antiviral ability is strongest at 30d after immunization (figure 5).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种HSV减毒株及其制备方法与应用