CN111202029A - Establishment method and application of Seneca virus rabbit animal model - Google Patents
Establishment method and application of Seneca virus rabbit animal model Download PDFInfo
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Abstract
The invention discloses a method for establishing a Seneca virus rabbit animal model and application thereof, belonging to the field of establishment of animal morbidity models. The establishment method of the Seneca virus rabbit animal model comprises inoculating the rabbit with a virus amount of not less than 108.5TCID50The Seneca virus solution can cause lip onset symptoms which are highly similar to those of Seneca virus infected pigs. The rabbit animal model established by the method provided by the invention can replace an experimental pig animal model to be applied to screening the Seneca virus vaccine or the anti-Seneca virus medicine.
Description
Technical Field
The invention belongs to the field of animal morbidity model establishment, and particularly relates to an establishment method and application of a seneca virus rabbit animal model, in particular to application in a vaccine or anti-infective drug screening process.
Background
Seneca Virus A (SVA) is a virus that primarily infects pigs and belongs to the genus Seneca virus of the family Picornaviridae. The virus is mainly transmitted by contact, can cause the rhinoceros and hoof coronary vesicular lesions of pigs, is extremely similar to diseases such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis in clinical symptoms, and is difficult to distinguish. The virus is expressed as subclinical infection and reproduction for adult pigs, and the pigs have pathological changes on hoof crowns and noses or have recessive infection, so that the pigs on the market have huge risks, and the death rate of newborn piglets within 7 days can reach 30-70%. China researches the disease later, no safe and efficient vaccine for preventing and controlling the virus exists in the market at present, and the vaccine is in the research and development stage. Animal experiments are particularly critical in the research and development stage, however, due to the influence of swine infectious diseases such as African swine fever, experimental swine is very difficult to purchase and is no longer safe and reliable, and the experimental progress and the development of vaccines or anti-infective drugs are greatly restricted. Therefore, the construction of SVA-related animal models is of great importance for the development of vaccines or anti-infective drugs, and the research and development costs can be greatly saved.
Disclosure of Invention
In view of one or more of the problems of the prior art, one aspect of the present invention provides a method for establishing a rabbit animal model of seneca virus, comprising inoculating a seneca virus solution to rabbits, wherein the inoculation toxicity of the seneca virus solution is not less than 108.5TCID50。
The toxic value of the above-mentioned saineika virus liquor is not less than 107.5TCID500.1ml, the inoculation amount is more than or equal to 1.0ml per seed.
The Seneca virus liquid is SVV/CH/ZZ/2016CGMCC No.14886 virus liquid.
The inoculation is carried out in a nasal drip mode.
The weight of the rabbit is 2-3 kg.
The method for establishing the senany virus rabbit animal model further comprises the step of determining that the establishment of the senany virus rabbit animal model is successful, and specifically comprises the following steps: observing the lips of the rabbits after inoculation, and determining that the establishment of the seneca virus rabbit animal model is successful if a white powder thorn-like bulge appears.
The method for establishing the senany virus rabbit animal model further comprises the step of determining that the establishment of the senany virus rabbit animal model is successful, and specifically comprises the following steps: collecting the white powder prick-like bulges, collecting the saintpaka virus liquid, respectively extracting RNA, carrying out PCR detection, and determining that the establishment of the saintpaka virus rabbit animal model is successful if a target strip is amplified and the amplified target strip of the white powder prick-like bulges is consistent with the amplified target strip of the saintpaka virus liquid.
The primers detected by the PCR are shown as SEQ ID NO 1 and SEQ ID NO 2 in the sequence table, and the size of the target band is 129 bp.
The invention also provides a seneca virus rabbit animal model which is obtained by the establishment method of the seneca virus rabbit animal model, and lips of rabbits inoculated with seneca virus have white acne-like bulges.
The establishing method of the seneca virus rabbit animal model or the application of the seneca virus rabbit animal model in screening seneca virus vaccines or anti-seneca virus medicines also belong to the content of the invention.
The establishment method of the senany virus rabbit animal model based on the technical scheme can effectively use rabbits to replace experimental pigs to establish the senany virus animal model, and eliminates the influence caused by swine infectious diseases such as African swine fever and the like from the source; meanwhile, the animal model establishing method provided by the invention adopts the rabbits as experimental animals, and compared with experimental pigs, the rabbits are relatively low in feeding cost and purchasing cost, so that the cost can be effectively reduced. The clinical symptoms of the SVA rabbit animal model established by the invention are highly similar to those of the SVA model taking pigs as experimental animals, and in the established SVA rabbit animal model, acne-like bulges are the disease symptoms of rabbits caused by Seneca virus, so that the rabbits can be used as experimental animals for researching and developing Seneca virus vaccines and medicaments. The establishment method of the SVA rabbit animal model is simple and convenient to operate, provides an optimized evaluation scheme and indexes for development of novel Seneca virus vaccines or anti-Seneca virus medicines, and has very important theoretical significance and application value.
Drawings
FIG. 1 shows the result of PCR detection of pimple-like projections;
fig. 2 is a photograph showing the morphology of pimple-like bumps.
Detailed Description
The invention aims to establish a rabbit animal model of the Seneca virus to replace a model taking pigs as experimental animals, and provides an evaluation scheme and indexes for the development of novel Seneca virus vaccines or anti-Seneca virus drugs.
The present invention will be described in detail with reference to the following specific embodiments.
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.
Example 1: preparation of epinakava virus liquid
This example selects SVV/CH/ZZ/2016CGMCC No.14886(CN110551694A) as the seed virus for the preparation of Selcaard virus solution, comprising the following steps:
(1) inoculating Seneca virus seed virus into full monolayer PK-15 cells (porcine kidney cells, provided by Jinyubao Ling biopharmaceutical Co., Ltd.), culturing in a CO2 incubator at 37 deg.C for 5 days, repeatedly freezing and thawing for 3 times to obtain virus culture solution, recording as F1 generation, and storing at-20 deg.C. Collecting harvested virus culture fluid F1 generation, inoculating full monolayer PK-15 cells according to content of 10% (V/V), and placing in CO at 37 deg.C2The cells were incubated in an incubator and observed for cytopathic effect (CPE) daily, and the virus broth was harvested 48 hours after inoculation and recorded as F2 generation. Passage was continued as described above until the appearance of typical lesions (manifested by cell rounding, aggregation, voiding, or even shedding), which occurred in this example when cultured for F3. Continuing to passage F7, collecting virus liquid as the most typical lowest passage of the lesion, and determining the titer of virus to 107.5TCID500.1 ml. The process is completed by national engineering laboratory of Jinyubingling biopharmaceutical GmbH.
(2) RNA extraction (using RNA extraction kit, Biotechnology, Inc.): adding 200 mul of virus liquid into a 1.5ml centrifuge tube, adding 200 mul of Buffer V-L, mixing uniformly by vortex oscillation, and standing for 5 minutes; adding 75 mul Buffer V-N, mixing evenly by vortex oscillation, and centrifuging for 5 minutes at 12000 rpm; transferring the supernatant into a new 2ml centrifuge tube, adding 300 mu l of isopropanol (1% glacial acetic acid), inverting for 6-8 times, mixing uniformly, transferring the mixture into a preparation tube, placing the preparation tube into the 2ml centrifuge tube, and centrifuging at 6000rpm/min for 1 minute; discarding the filtrate, placing the preparation tube back into a 2ml centrifuge tube, adding 500. mu.l buffer W1A, placing the tube in a greenhouse for standing for 1 minute, and centrifuging at 12000rpm for 1 minute; discarding the filtrate, placing the preparation tube back into a 2ml centrifuge tube, adding 800. mu.l Buffer W2, and centrifuging at 12000rpm for 1 minute; discarding the filtrate, putting the preparation tube back into a 2ml centrifuge tube, and centrifuging at 12000rpm for 1 minute; the prepared tube is placed in a clean 1.5ml centrifuge tube, 40 mul of enzyme-free water is added in the center of the prepared tube membrane, the tube membrane is kept still for 1 minute at room temperature, 12000g of the tube membrane is centrifuged for 1 minute to elute RNA, and the RNA is frozen and stored for standby at the temperature of minus 20 ℃.
(3) PCR detection of the harvested virus fluid: identifying SVA by PCR method from the virus liquid RNA extracted in the step (2), the method is as follows: the upstream primer SVA-F: 5'-TATCTCAGATCCCTGGCTGTC-3' (SEQ ID NO: 1); the downstream primer SVA-R: 5'-CCTGATGATCACATTGTTGAGC-3' (SEQ ID NO: 2). Reaction system: 2 × One Step RT-PCRbuffer III 12.5 μ l, TaKaRa Ex Taq HS 0.5 μ l, PrimeScript RT Enzyme Mix II 0.5 μ l, SVA-F (20 μ M)0.5 μ l, SVA-R (20 μ M)0.5 μ l, RNA template 2.0 μ l, RNA-free H2O5.8. mu.l. The PCR reaction conditions are as follows: firstly, reverse transcription is carried out for 15 minutes at 42 ℃, and pre-denaturation is carried out for 2 minutes at 95 ℃; then denatured at 94 ℃ for 10 seconds, annealed at 59 ℃ for 30 seconds, and subjected to 45 cycles (PCR amplification). And (3) carrying out 1.5% agarose gel electrophoresis on the PCR amplification product, observing the result under a gel imager, and determining that the virus solution is seneca virus solution, wherein a bright single band appears near 129bp and is consistent with the size of the amplified target fragment of the PCR primer.
Example 2: establishment and identification of Seneca virus rabbit animal model
In this example, the inoculation of rabbits with the seneca virus solution obtained in example 1 includes the following steps:
(1) selecting experimental animals: 28 rabbits (purchased from Dallas permanent farming and animal husbandry, Inc. of Ordorsi) with the weight of 2-3 kg are selected, the male and female are not limited, and the SVA serum neutralizing antibody is less than 1: 2. The experimental group had 24 rabbits, and the control group had 4 rabbits.
(2) The cell maintenance solution (containing 1.5% (by mass) NaHCO) for the Seneca virus solution obtained in example 13MEM medium) to 106.5TCID50/0.1ml、105.5TCID50/0.1ml、104.5TCID50/0.1ml、103.5TCID50/0.1ml、102.5TCID50/0.1ml。
(3) The rabbits of the experimental group and the rabbits of the control group were inoculated, respectively, according to the inoculation method shown in table 1 below, wherein the inoculation route was nasal drip, and the inoculation amount was 1 ml/rabbit.
Table 1: SVA virus inoculation method
(4) Observing lip changes of the rabbits of each group: carefully observing lips of all groups of rabbits after inoculation, and finding that the inoculation toxicity is 10 days after SVA virus liquid is inoculated for 5-8 days3.5TCID50(i.e., inoculation titer of 10)2.5TCID500.1ml of virus solution 1ml) of rabbit 0/5; the inoculation toxicity is 104.5TCID50(i.e., inoculation titer of 10)3.5TCID500.1ml of virus solution 1ml) of rabbit 0/5; the inoculation toxicity is 105.5TCID50(i.e., inoculation titer of 10)4.5TCID500.1ml of viral solution 1ml) rabbit 1/5 exhibited pimple-like bulges on the lips, as shown in panels (a) and (B) of fig. 2; the inoculation toxicity is 106.5TCID50(i.e., inoculation titer of 10)5.5TCID500.1ml of viral solution 1ml) rabbit 2/5 developed comedo-like bulges in the lips; the inoculation toxicity is 107.5TCID50(i.e., inoculation titer of 10)6.5TCID500.1ml of viral solution 1ml) rabbit 3/5 developed comedo-like bulges in the lips; the inoculation toxicity is 108.5TCID50(i.e., inoculation titer of 10)7.5TCID500.1ml of viral solution 1ml) rabbit 5/5 developed pimple-like bumps on the lips. No lip abnormality occurred in the control rabbit. It was confirmed (described in detail below) that the pimple-like bulge is a disease symptom caused by Seneca virus, and therefore it was confirmed that the virus amount of the Seneca virus solution when inoculated was not less than 108.5TCID50In time, can ensure the rabbit of the virus of the epikayavirus to moveThe model was successfully established, and the lips of the rabbits inoculated with the virus fluid all showed pimple-like bulges.
(5) Collecting raised parts of the powder prick sample, and performing separation, detection and identification: taking off the white bulge part by using sterile ophthalmological forceps, putting the white bulge part into a sterile small tube containing PBS liquid, keeping the low temperature state and returning the white bulge part to a laboratory. Crushing the mixture by using a tissue dispersion instrument to prepare a tissue suspension, repeatedly freezing and thawing the tissue suspension for 2 times in a refrigerator at the temperature of-70 ℃, centrifuging the tissue suspension for 10 minutes at 3500rpm/min, and taking a supernatant as a sample to be detected for later use. Then, RNA extraction and PCR detection are performed on the sample to be detected according to the methods of the step (2) and the step (3) in the above example 1, and the detection result is shown in FIG. 1, wherein M represents Marker, lane F represents the sample to be detected, lane N represents negative control, lane P represents positive control, a bright single band appears near 129bp, the size of the amplified fragment is consistent with that of the PCR primer, and the detection result is consistent with the PCR detection result of the virus solution of the Seneca virus, so that the sample to be detected contains Seneca virus, that is, the acne-like bulge is the disease symptom caused by the Seneca virus, the establishment success of the Seneca virus rabbit animal model with the acne-like bulge appearing on the lips is confirmed, and finally, the inoculation virus amount of the Seneca virus is determined to be 108.5TCID50(i.e., inoculation titer of 10)7.5TCID500.1ml virus solution 1ml) to establish a Seneca virus rabbit animal model.
Example 3: clinical symptoms of Seneca virus rabbit animal model
Option 10 in this embodiment8.5TCID50(i.e., inoculation titer of 10)7.5TCID500.1ml of virus solution 1ml) as an inoculation dose, and observing the clinical symptoms of the rabbits, specifically comprising the following steps:
(1) selecting experimental animals: 8 rabbits (purchased from Dallas permanent farming and animal husbandry, Inc. of Ordorsi) with the weight of 2-3 kg are selected, the male and female are not limited, and the SVA serum neutralizing antibody is less than 1: 2. The experimental group had 4 rabbits, and the control group had 4 rabbits.
(2) The in-stopper-Calla virus solution (with a titer of 10) used in example 2 was taken7.5TCID50/01ml) the rabbits of the experimental group were inoculated by nasal drip in an amount of 1 ml/rabbit (10 doses of inoculum)8.5TCID50). Another cell maintenance solution (containing 1.5% (by weight) NaHCO is taken3MEM medium of (d) was inoculated into control rabbits by nasal drip, in an amount of 1 ml/rabbit.
(3) Observation of clinical symptoms: after inoculation, the clinical symptom changes of the rabbit lips of the experimental group and the control group at the positions with little or no hair are carefully observed, the fact that the nose lip of the experimental group rabbit starts to appear a plurality of red and swollen parts with different degrees on the 5 th day after toxin attack is found, the red and swollen parts gradually bulge on the 6 th to 7 th days to form acne-like pathological changes, the rabbits of the experimental group all suffer from diseases on the 8 th day after toxin attack, and the nose lip of the diseased rabbit appears acne-like bulge; no similar symptoms were found in the control rabbits.
In the past, in the saincard virus model taking pigs as experimental animals, after the experimental pigs are infected with the saincard virus, the clinical symptoms are characterized by blister-like damage of nasal discs and lips, in the embodiment, rabbits are used as the experimental animals to establish the saincard virus pathogenesis model, and clinical manifestations are acne-like bulges which are all the pathogenesis symptoms caused by the saincard virus, so the saincard virus pathogenesis model is highly similar to the typical symptoms of the experimental pigs infected with the saincard virus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for establishing an animal model of a rabbit with Seneca virus is characterized by comprising inoculating Seneca virus liquid to the rabbit, wherein the inoculation toxicity of the Seneca virus liquid is not less than 108.5TCID50。
2. The method for establishing a seneca virus rabbit animal model according to claim 1, wherein the toxic valence of the seneca virus liquid is not less than 107.5TCID500.1ml, the inoculation amount is more than or equal to 1.0ml per seed.
3. The method for establishing a seneca virus rabbit animal model according to claim 1 or 2, wherein the seneca virus liquid is SVV/CH/ZZ/2016CGMCC No.14886 virus liquid.
4. The method of establishing according to any one of claims 1-3, wherein the inoculation is performed nasally.
5. The method for establishing a seneca virus rabbit animal model according to any one of claims 1-4, wherein the weight of the rabbit is 2-3 kg.
6. The method for establishing a senany rabbit animal model according to any one of claims 1 to 5, further comprising the step of determining that the establishment of the senany rabbit animal model is successful, specifically: observing the lips of the rabbits after inoculation, and determining that the establishment of the seneca virus rabbit animal model is successful if a white powder thorn-like bulge appears.
7. The method for establishing the senany rabbit animal model according to claim 6, further comprising the step of determining that the establishment of the senany rabbit animal model is successful, specifically: collecting the white powder prick-like bulges, collecting the saintpaka virus liquid, respectively extracting RNA, carrying out PCR detection, and determining that the establishment of the saintpaka virus rabbit animal model is successful if a target strip is amplified and the amplified target strip of the white powder prick-like bulges is consistent with the amplified target strip of the saintpaka virus liquid.
8. The method for establishing the seneca virus rabbit animal model according to claim 7, wherein the primers detected by PCR are shown by SEQ ID NO.1 and SEQ ID NO. 2 in the sequence table, and the size of the target band is 129 bp.
9. An animal model of a seneca virus rabbit obtained by the method for constructing an animal model of a seneca virus rabbit according to any one of claims 1 to 8, wherein the lips of the rabbit inoculated with seneca virus have pimple-like projections.
10. The method of establishing the seneca virus rabbit animal model according to any one of claims 1 to 8 or the use of the seneca virus rabbit animal model according to claim 9 for screening a seneca virus vaccine or an anti-seneca virus drug.
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