CN107513524B - Swine Saxifraga Valley virus strain and application thereof - Google Patents
Swine Saxifraga Valley virus strain and application thereof Download PDFInfo
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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Abstract
The invention discloses a suisaincai valley virus strain and application thereof. The Selaginella valley virus strain CH-FJZZ-2017 is separated from the disease material of pig idiopathic vesiculosis in pig farms of Fujian province, and the preservation number is CGMCC No. 12160. The separated porcine Selaginella Valvata virus CH-FJZZ-2017 strain is separated from a swinery which has newly developed the disease in China, represents a domestic epidemic dominant strain at present, has good strain background, can be used as an inactivated vaccine to produce a strain and a virus seed for inspection, provides a material for subsequent related experimental research, and lays a material foundation.
Description
Technical Field
The invention relates to the field of pathogenic microorganisms, belongs to the field of biological products for receiving, and particularly relates to an application of a suisaincaguagg virus strain in preparation of a suisaincaguagg inactivated vaccine.
Background
Senecavirus A (SVA), also known as Seneca Valley Virus (SVV), is a membrane-free single-stranded positive-strand RNA virus belonging to the genus Senecaviridae (Picornaviridae) Senecavirus. The SVA genome is about 7.3kb in total length and comprises an open reading frame and non-coding region sequences at two ends. SVA encodes a polyprotein of 2181aa in size that can be hydrolyzed by host cell and virus own proteases to form 12 proteins, L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D, respectively. Wherein, VP4, VP2, VP3 and VP1 are structural proteins of SVA, and VP2, VP3 and VP1 proteins are positioned on the surface of the capsid of the virus, and can induce the organism to generate good immune response.
SVA is a newly discovered acute pathogenic microorganism which can cause Swine Idiopathic Vesicular Disease (SIVD) and Epidemic Transient Neonatal Loss (ETNL), and has the advantages of high morbidity, fast disease course, fast propagation speed, low mortality rate and high piglet mortality rate. Clinically, the symptoms of blister, ulceration, lameness, lethargy, diarrhea, acute death and the like of the pig rhinoscope and the coronary area of the hooves are mainly shown. Clinical symptoms of SVA are similar to foot and mouth disease, swine vesicular disease, vesicular stomatitis, etc., and are difficult to distinguish by clinical diagnosis. The infection spectrum of the disease is narrow, and the disease has no reports of natural infection of livestock animals such as cattle, sheep, dogs, cats and the like by the pathogen. Porcine idiopathic vesiculopathy caused by SVA was first reported in American countries such as the United states, Brazil, Canada, and subsequently in China as well as Thailand, Columbia, and the like.
In 2015, the first in-plug valley epidemic situation occurs in Guangdong areas in China, in 2016 and 2017, the in-plug valley epidemic situation continues to spread, outbreaks occur in multiple provinces and markets in China, the epidemic range reaches provinces such as Guangdong, Fujian, Hunan, Henan and Heilongjiang, clinical symptoms are characterized by blister lesions, and occasionally, a large amount of piglets die in low days, so that huge economic loss is brought to the pig raising industry, and the public health safety and the breeding industry development in China are seriously threatened.
Currently, no biological vaccine preparation is available for the prevention and control of the disease. At present, the prevention and control of the Setaria inflata Valley disease of the pigs depend on the effective management of a pig farm, but the domestic pig raising modes are complex and various, the management of the pig farm is relatively laggard, and the development of corresponding biological products for preventing and controlling the outbreak and the epidemic of the epidemic disease is urgent in view of the pathogenic characteristics of the Setaria inflata Valley virus.
Disclosure of Invention
One of the purposes of the invention is to provide a new separated porcine Selaginella virus epidemic strain.
The invention also aims to provide an effective and safe Swine Selaginella vaccine and a preparation method of the inactivated vaccine aiming at the defects of the existing Selaginella virus biological product; the preparation method adopted by the inactivated vaccine is simple and safe, and the vaccine has a good immune effect.
The above object of the present invention is achieved by the following technical solutions:
the invention firstly provides a Selenaga valley virus, which is named as Senecavirus A CH-FJZZ-2017CGMCC No. 12160. The virus is isolated from pigs, also known as suisaincai valley virus; the international committee for virus classification classifies it as: picornavirus, Senecavirus; the porcine Senecavir Senevirus A (SVA) CH-FJZZ-2017 strain is separated from a pig farm in Fujian province where non-foot-and-mouth disease, swine vesicular stomatitis epidemic situation occurs. Has been preserved in China general microbiological culture Collection center (China academy of sciences, institute of microbiology, No. 3, West Lu No.1, Beijing, Chaoyang, North-West province) in 2017, 31.8.31.7.8.60, and the preservation number is CGMCC No. 12160.
The isolation culture method of the suijianeigao virus comprises the following specific steps:
collecting a sample of the porcine vesicular skin lesion infected by the Valley virus in the stopple, shearing the sample into pieces, preparing the pieces into 1:5 suspension by using sterile PBS containing 100U/mL penicillin and 100ug/mL streptomycin, grinding the suspension, uniformly mixing the suspension by vortex oscillation, repeatedly freezing and thawing for 3 times, centrifuging the suspension for 10min at 10000 Xg, filtering and sterilizing the supernatant by using a 0.22 mu m filter, and subpackaging the sterilized suspension at-70 ℃ for later use;
will be 1 × 106Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; 1mL of the filtered Sabavirus treatment solution was inoculated into PK-15 cells while negative control wells were placed at 37 ℃ with 5% CO2Adsorbing for 2 hours in an incubator, removing the inoculum, adding 2mL of maintenance liquid, continuing culturing for 4-5 days, and observing the cell state every day; when obvious cytopathic effect occurs, freezing and thawing the cells for 3 times, collecting the cells and culture solution, continuously subculturing, and simultaneously carrying out RT-PCR identification on each generation of cell culture; when the virus PK-15 group to be inoculated has stable and obvious cytopathic effect, a stable passage porcine Sailurus valley virus strain is obtained by separation.
The plaque purification method of the suijianei valley virus comprises the following specific steps:
1) preparing a single-layer PK-15 cell, adding a stable passage Saikoujia virus solution, and adsorbing for 2 hours;
2) washing the adsorbed cells twice with PBS, covering a layer of solid culture medium for culturing mammalian cells on the surface, solidifying the solid culture medium, and inversely culturing until virus plaques appear;
3) selecting 8-10 virus plaques, inoculating a monolayer PK-15 cell, and harvesting a virus culture when the cell is diseased;
4) and (3) carrying out the next round of cloning and purifying test on the harvested virus culture, and carrying out 3 rounds of plaque purifying test to obtain the cloned and purified strain of the Seneca valley virus.
The porcine Seneca Valley virus CH-FJZZ-2017CGMCC No.12160 provided by the invention can be used for preparing vaccines for preventing diseases caused by the porcine Seneca Valley virus, and can also be used for preparing diagnostic reagents and therapeutic drugs related to the porcine Seneca Valley virus.
The invention provides a vaccine for preventing diseases caused by suis Seagagu virus, the active component of which is inactivated Seagagu virus CH-FJZZ-2017CGMCC No. 12160; the vaccine specifically contains inactivated Selaginella virus CH-FJZZ-2017CGMCC No.12160 and pharmaceutically acceptable adjuvant.
The invention also discloses a preparation method of the vaccine, which comprises the following steps:
1) and (3) virus culture: inoculating the porcine epididymis Valley virus CH-FJZZ-2017CGMCC No.12160 to PK-15 cells for culture, and harvesting a virus culture;
2) inactivating the virus culture, repeatedly freezing and thawing the virus culture for three times, centrifuging, collecting supernatant, adding an inactivating agent, and inactivating the virus to obtain an inactivated virus solution;
3) and mixing the inactivated porcine epinastine added with the valley virus liquid and an adjuvant according to a proportion, emulsifying, and preparing the porcine epinastine added with the valley virus inactivated vaccine.
The preparation method of the vaccine composition comprises the following specific steps of step 1): using low sugar DMEM medium containing 10% fetal calf serum, 100U/mL penicillin, 100ug/mL streptomycin, at 37 deg.C and 5% CO2Culturing PK-15 cells in an adherent manner by using an incubator; when the fusion degree reaches about 80%, the culture solution is discarded, the porcine epizoon is inoculated with the valley virus CH-FJZZ-2017 strain according to the infection complex number of 1, then a low-sugar DMEM culture medium containing 2% fetal calf serum, 100U/mL penicillin and 100g/mL streptomycin is used for adherent culture at 37 ℃ for about 48 hours in a 5% CO2 culture box, and after the cells are completely diseased, the cell culture is obtained.
According to the preparation method of the vaccine composition, the inactivator in the step 2) is diethylene imine, the final concentration of the added diethylene imine in the virus liquid is 1mmol/L, and the inactivation condition is 30 ℃ for 28 h; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml. The inactivation effect is inoculated to PK-15 blind passage 3, and no lesion appears, so that the inactivation is qualified.
According to the preparation method of the vaccine composition, the adjuvant in the step 3) is ISA206 or ISA201, and the volume ratio of the inactivated virus solution to the adjuvant is 1: 1.
The invention has the following positive beneficial effects:
the separated Selaginella Valvata virus CH-FJZZ-2017CGMCC No.12160 is separated from a swinery which has newly developed the epidemic disease in China, represents the current domestic epidemic dominant strain, has good strain background, can be used as an inactivated vaccine production strain and a virus seed for detection, provides materials for subsequent related experimental research, and lays a material foundation.
The invention discloses a method for separating, culturing and plaque purifying porcine Saxifraga Valley virus, which has the advantages of stability and easy operation, and provides a simple and feasible method for separating and purifying porcine Saxifraga Valley virus.
The vaccine composition has good immunogenicity, and can induce animals to generate stronger neutralizing antibodies after immunization. As the market at home and abroad does not have biological products related to the vaccine of the pathogen, the strain used by the vaccine composition is a strain which is newly separated in recent years, and compared with an early strain, the strain has a closer genetic relationship with a recently epidemic strain, has stronger product competitive advantage, can effectively prevent the prevalence and spread of SVA in swinery, reduces the economic loss caused by the disease, and has wide application prospect.
The preparation method adopted by the vaccine is quick, simple and safe.
Drawings
FIG. 1 is a SVA-PCR assay in tissue disease.
FIG. 2 shows that SVA-infected PK-15 cells induced cytopathic effects.
FIG. 3 shows the nucleic acid detection of SVA at PK-15 cell passage, wherein P1, P2, P3, P4 and P5 respectively show the detection results of SVA at passage 1-5.
FIG. 4 is a plaque assay of SVA-infected PK-15 cells.
FIG. 5 shows that SVA-infected PK-15 cells induced cytopathic effects.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to specific examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the invention, an SVA virus epidemic strain is separated from a sick pig body with the symptoms of the porcine vesicular lesion in a pig farm with non-foot-and-mouth disease, porcine vesicular disease and vesicular stomatitis epidemic situation in southeast province of China, and is subjected to separation culture, plaque-removing cloning and purification. And a method for preparing the inactivated vaccine based on the epidemic strain is further established, the immunogenicity of the inactivated vaccine is evaluated, and a foundation is laid for timely, rapid, safe and efficient porcine epididymis valley virus inactivated vaccine development. As detailed in the examples below.
Example 1: separation and identification of Selaginella virus CH-FJZZ-2017CGMCC No.12160
1.1 Experimental materials
The disease sample used in the experiment is from a pig farm in Fujian province where non-foot-and-mouth disease, swine vesicular disease and vesicular stomatitis epidemic situation occur, and the collection time is 2017 and 6 months; porcine Kidney cell line (Porcine Kidney, PK-15) was purchased from American type culture center ATCC.
1.2 primer design
1 pair of specific primers synthesized by Biotechnology engineering (Shanghai) GmbH were designed using Primier 5 with reference to SVA CH-HN-2017(KY747511) gene series published in GenBank. The nucleotide sequence of the specific primer is as follows: SVA F: 5'-GCCCTCATGCCCAGTCCTTC-3'; SVA R: 5'-GTTCAGTGATCCGAGGTGG-3'.
1.3 Collection and treatment of pathological Material
Collecting a pig trotter crown part blister sample with idiopathic vesiculosis, shearing, preparing into 1:5 suspension with sterile PBS containing 100U/mL penicillin and 100g/mL streptomycin, grinding with a mortar to homogenate, vortex oscillating, mixing, repeatedly freezing and thawing for 3 times, centrifuging at 10000 Xg for 10min, collecting supernatant, filtering with a 0.22 μm filter for sterilization, and subpackaging at-70 deg.C for later use;
1.4 RT-PCR detection of SVA RNA in tissue specimens
Extracting total RNA of the pathological material according to a Trizol method; taking the extracted RNA as a template, configuring a reverse transcription reaction system according to RevertAID First Strand cDNA Synthesis Kit (Invitrogen) to carry out reverse transcription reaction; carrying out PCR amplification by taking cDNA obtained by reverse transcription as a template, wherein an amplification reaction system (50 mu L) comprises: 2 XTaq PCR StarMix 25. mu.L, upstream and downstream primers (10 pmol/. mu.L) 1.5. mu. L, cDNA template 2. mu.L each, supplemented with sterile double distilled water to 50. mu.L. The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 10 min. 10 μ of the amplification product was detected by electrophoresis on a 1% agarose gel (see FIG. 1). As can be seen from FIG. 1, a band of about 372bp was amplified, which is consistent with the expected size of the amplified fragment.
And recovering the target fragment of the PCR product by using a glue recovery and purification kit, cloning the target fragment to a pMD-18T vector, and sequencing the target fragment by using a biological engineering (Shanghai) corporation. The sequencing result is subjected to manual proofreading and sequence splicing, and similarity comparison is carried out in an NCBI database by using a Blast program, so that the highest homology of the sequence gene and the known sequence of the SVA is found, the similarity of 93-98% exists, and the result proves that the SVA nucleic acid in the pathological material is positive.
1.5 isolation and culture of the Virus
Will be 1 × 106Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; inoculating PK-15 cells with the frozen and reserved Valley virus filtrate obtained in the step 1.3, wherein the inoculation amount is 1mL, and setting a negative control hole at 37 ℃ and 5% CO2Adsorbing for 2 hours in an incubator, removing the inoculum, adding 2mL of maintenance liquid, continuing culturing for 4-5 days, and observing the cell state every day; if no obvious cytopathic effect appears in the inoculated cells at the 5 th day, freezing and thawing the cells for 3 times, and collecting the cells and culture solution for continuous subculture; when 4 serial passages were made on PK-15 cells, significant cytopathic effects occurred. Simultaneously extracting sample RNA from each generation of cell culture, and performing RT-PCR detection on cells according to step 1.4Adding valley virus nucleic acid into the middle plug.
It was observed that during subculture, distinct cytopathic effects appeared in each passage from passage 5, as evidenced by the aggregation of cells into "grape-like" lesions, followed by rounding, shedding, and the appearance of plaques (fig. 2). The porcine epididymis added with an oryzavirus specific primer SVA F/SVAR to perform RT-PCR amplification on cell culture RNA, so that an obvious SVA specific target fragment can be detected (see figure 3), and a non-inoculated PK-15 cell negative control group is used for amplifying a corresponding nucleic acid fragment. When the virus is transmitted to the 5 th generation blindly, stable cytopathy appears in 48h of inoculation, cells become round and fall off, the virus is proved to be adapted to PK-15 cells and can be stably propagated on the cells, and an SVA strain capable of being passaged stably is obtained through separation.
Example 2: method for purifying porcine epinastine plus valley virus plaque
2.1 preparation of cell monolayers
Mixing T75cm2PK-15 cells with good growth and a confluency of 98% or more in a cell culture flask were washed with PBS 1 time, digested with 3mL of 0.25% trypsin (Gibco), suspended in 70mL of a low-sugar DMEM medium containing 10% fetal calf serum, 100U/mL of penicillin and 100g/mL of streptomycin to prepare a suspension, inoculated into a six-well cell culture plate at 3 mL/well, placed at 37 ℃ in a 5% CO atmosphere2Culturing in an incubator, and performing subsequent plaque purification experiments when the cells grow into a monolayer.
2.2 dilution and infection of the Virus
Diluting SVA virus solution stably propagated in step 1.5 to 10-fold serial dilution by using low-sugar DMEM medium containing 2% fetal calf serum, 100U/mL penicillin and 100g/mL streptomycin-8And placing for later use. The original cell culture medium in the six-well plate was discarded, the cells in each well were washed 1 time with PBS, and 1mL of the above dilution 10 was added-4～10-8The virus diluent of (1) is subjected to 2-time repeated hole every dilution, meanwhile, a negative control group which is not inoculated with the virus is arranged, and the virus diluent is placed in a 5% CO2 incubator at 37 ℃ for adsorption for 2 hours.
2.3 plaque formation by overlay culture
When the virus adsorption step in the step 2.2 is left for 40min, 10mL of 2g/L low-melting-point agarose sterile gellan gum solution is placed in a 70 ℃ water bath for preheating and melting; after they were completely thawed, they were incubated with 10mL of 3% fetal bovine serum, 100U/mL of penicillin, and 100g/mL of streptomycin in 2 Xlow sugar DMEM medium in a 50 ℃ water bath. And absorbing the virus for 2h, removing the virus liquid in the six-hole plate, washing PK-15 cells for 2 times by using PBS, quickly and uniformly mixing the 2g/L low-melting-point agarose gel solution and the 2 Xlow-sugar DMEM culture solution, adding the uniformly mixed culture medium into each cell hole before solidification, wherein each hole is 3mL, and cooling at room temperature for 30 min. The cooled six-well plate was placed upside down in a 5% CO2 incubator at 37 ℃. Cytopathic phenomena were observed and recorded day by day.
It was observed that the monolayer of cells covered with the low melting agarose gel showed significant viral plaques after 48h, with the extent of viral plaque formation gradually expanding but with no significant difference over time up to 96h (fig. 4).
2.4 Spot-picking purified Virus
When obvious plaques are formed in the step 2.3, the cell holes with fewer virus plaques (less than 10) and better shapes are selected for carrying out the plaque picking under the observation of a microscope. The method for picking the single plaque comprises the following steps: firstly, marking the plaque on the bottom layer of a cell culture plate by using a marking pen; sucking the covering layer at the marked plaque position by using a sterile 200-mu L micropipettor head, adding 1mL serum-free low-sugar DMEM medium, and blowing and beating for several times to ensure that the covering layer medium is fused in the medium; then sucking 50 mu L of serum-free low-sugar DMEM culture medium to repeatedly blow the plaque part, completely blowing up the virus single plaque, adding the sucked liquid into 1mL of serum-free low-sugar DMEM culture medium again, repeatedly freezing and thawing for 2 times, and performing amplification and propagation on the virus liquid by using PK-15 cells for 2 generations.
And (3) preparing plaques by repeating the plaque purification 2 times after the virus liquid of the 2 generation of monoclonal amplification is prepared again, thus obtaining the cloned and purified SVA virus strain which is named as CH-FJZZ-2017 strain. The CH-FJZZ-2017 strain is subjected to RT-PCR amplification by using the following primers to obtain a sequence shown in a sequence 1: SVA F: 5'-GCCCTCATGCCCAGTCCTTC-3'; SVA R: 5'-GTTCAGTGATCCGAGGTGG-3'. Comparing the strain sequence (sequence 1 in the sequence table) of the Selaginella virus CH-FJZZ-2017 obtained by the RT-PCR amplification with other 53 Selaginella viruses in GenBank, wherein the homology is between 93.7% and 98.5%. Analysis shows that the Selaginella virus CH-FJZZ-2017 strain has a different sequence from the previously separated Selaginella virus strain, and is a new separated strain.
The swine Sai interior valley virus CH-FJZZ-2017 strain is a single-stranded positive-strand RNA virus, and virus particles are in a typical icosahedral symmetrical structure, have the diameter of about 27nm and have the molecular weight of about 30 KD. The outer layer of the virus particle has no envelope and has certain resistance to lipid solvent. The viral genome is approximately 7.3kb in length and includes an open reading frame and non-coding sequences at both ends, ending with a poly (A) at the 3' end of the genome. The non-coding regions at both ends of the genome can form complex RNA structures, such as stem-loop structures and prosthesis structures, and play an important role in the replication and translation processes of viruses. The Selaginella Valeriana RNA encodes a polyprotein, 2181aa in size, which is hydrolyzed by the host cell and virus own proteases to form 12 proteins, L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D, respectively. Wherein, VP4, VP2, VP3 and VP1 are structural proteins of SVA, and VP2, VP3 and VP1 proteins are positioned on the surface of the capsid of the virus, and can induce the organism to generate good immune response.
Up to now, senega virus contains only one serotype. The infection spectrum of the disease is narrow, and the disease has no reports of natural infection of livestock animals such as cattle, sheep, dogs, cats and the like by the pathogen.
The CH-FJZZ-2017 strain is preserved in China general microbiological culture Collection center (the address: the microbial research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, respectively) in 31.8.7.2017, and the preservation number is CGMCC No. 12160.
Example 3: determination of titer of porcine Selaginella Valeriana virus strain CH-FJZZ-2017 virus
Inoculating the digested and blown-off PK-15 cell suspension into a 96-well cell culture plate, wherein each well is 150 mu L, and culturing overnight; taking the cell culture of the CH-FJZZ-2017 strain cloned and purified for 3 times in the step 2.4, and diluting the cell culture by 10 times by using a cell maintenance solution, wherein the dilution is 10 times-1To 10-10(ii) a The cell culture medium in a 96-well cell culture plate is sucked and discarded, PK-15 cells are washed for 1 time by sterile PBS, and cell maintenance liquid is added, wherein each well is 100 mu L; then adding the CH-FJZZ-2017 strain virus diluent of each dilution into a 96-well plate, wherein each well is 100 mu L, each dilution is repeated for 8 times, and each cell culture plate is provided with a negative control group of the non-inoculated virus; culturing in a 5% CO2 incubator at 37 deg.C. And 4d, continuously observing and recording the pathological changes day by day. TCID calculation according to Reed-Muench method50. The result shows that the toxin value of the porcine Seneca Valley virus CH-FJZZ-2017 strain is 107.78TCID50/0.1mL
Example 4: preparation of porcine Sagnac Valley virus CH-FJZZ-2017 strain inactivated vaccine
4.1 Virus culture
Low-sugar DMEM medium containing 10% fetal calf serum, 100U/mL penicillin and 100g/mL streptomycin, T225cm2Cell culture flask, 37 deg.C, 5% CO2Culturing PK-15 cells in an adherent manner by using an incubator; when the fusion degree reaches about 80%, the culture solution is discarded, the porcine Sampus virus CH-FJZZ-2017 strain is inoculated according to the infection complex number of 1, and then a low-sugar DMEM culture medium containing 2% fetal calf serum, 100U/mL penicillin and 100ug/mL streptomycin is used at 37 ℃ and 5% CO2Culturing in an incubator for about 48h, repeatedly freezing and thawing for 3 times after the cells are completely diseased, centrifuging for 10min at 10000 Xg, removing cell debris, collecting supernatant to obtain Suaeda salsa added with valley virus liquid, and standing at-80 deg.C for use.
4.2 inactivation of viral fluid and inactivation test
In virus liquid with qualified virus content (preferably more than or equal to 10)7.0TCID50/0.1mL) is added with an inactivating agent which is divinyl imine, so that the final concentration of the divinyl imine is 1mmol/L, and the mixture is placed in a constant temperature shaking table at 30 ℃ and at 80rpm for inactivation for 28 hours; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml.
Will be 1 × 106Inoculating the PK-15 cells to a six-hole cell culture plate, and washing twice by using PBS when the fusion degree of the PK-15 cells reaches 80%; diluting the inactivated virus solution 10 times with a maintenance solution, wherein the inoculation amount is 500 μ L, and setting negativeControl wells at 37 ℃ 5% CO2Adsorbing for 2h in the incubator, discarding the inoculum, adding 2mL of maintenance liquid, continuing to culture for 4-5 d, and observing the cell state every day. Blind passage for 3 generations, no cytopathic effect; and extracting sample RNA from each cell culture generation, and detecting the Selenarctic valley virus nucleic acid in the cells by RT-PCR according to the step 1.4. If the results are negative, the virus liquid is completely inactivated.
4.3 preparation of vaccine composition containing Sagnac Valley Virus CH-FJZZ-2017 Strain
Mixing completely inactivated CH-FJZZ-2017 strain virus liquid with ISA206 adjuvant at a volume ratio of 1:1, and emulsifying oil phase and water phase under the conditions of constant temperature shaking table at 30 ℃ and 120rpm for 15 min. And (3) after the mixing is complete, obtaining the vaccine composition, wherein the emulsification quality of the vaccine can reach the quality standard specified by the state.
Example 5: establishment of indirect immunofluorescence method of porcine Saxiega virus
In order to screen negative pigs with the porcine Selaginella valley virus antibodies to evaluate the immunogenicity of the porcine Selaginella valley virus CH-FJZZ-2017 strain inactivated vaccine, an indirect immunofluorescence method applied to detection of the porcine Selaginella valley virus antibodies is established due to the lack of a proper antibody diagnosis kit and method.
Spreading PK-15 cells on a 96-well cell culture plate, when the fusion degree of the PK-15 cells reaches 80%, absorbing and discarding a cell culture medium in the 96-well cell culture plate, washing the PK-15 cells for 1 time by using sterile PBS, diluting the CH-FJZZ-2017 strain virus by using a cell maintenance solution to a proper concentration, inoculating a porcine epididymis virus CH-FJZZ-2017 strain according to the infection multiplicity of 0.1, setting an unvoxic cell group at the same time, and supplementing 200 mu L of maintenance solution to each well; after infection for 30h, removing culture supernatant by suction, washing with PBS, fixing with cold acetone at-20 deg.C for 10min, removing fixing solution, washing with PBS for 3 times, each time for 5 min; add 100. mu.L of 1: 20 diluting the tested serum or SVA positive serum and negative serum, placing in a 37 deg.C wet box, acting for 1h, washing with PBS for 3 times, each time for 5 min; adding 100 μ L rabbit anti-pig fluorescent antibody (purchased from Sigma, diluted according to the instruction), performing wet box action at 37 deg.C for 45min, and washing with PBS for 5min for 3 times; 50 μ L of PBS was added to each well and observed under a fluorescent microscope. And (4) judging a result: typical specific bright green fluorescence was observed in the cytoplasm or nucleus of positive cells in wells with negative and blank controls (fig. 5).
Example 6: immunogenicity test of Selaginella virus CH-FJZZ-2017 strain inactivated vaccine
6.1 Sai Neigavirus CH-FJZZ-2017 strain inactivated vaccine immunization
Antigen-antibody negative screening is carried out on 6-week-old piglets by the method of the step 1.4 and the example 5, and 8 antigen-antibody negative piglets are selected to carry out immunogenicity evaluation test of the inactivated vaccine of the Sendai Valeriana virus CH-FJZZ-2017 strain. Grouping condition: randomly selecting 5 piglets to be immunized, adding a valley virus CH-FJZZ-2017 strain inactivated vaccine into the piglet immune stopper, wherein each piglet immune stopper contains 1 part of vaccine and each part contains 2mL of vaccine composition; in the control group, 2mL of cell maintenance solution was injected into the head and neck muscles of 3 subjects. Measuring the body temperature every day from 0 to 7 days of immunization; continuously observing the feeding condition of the swinery for 14 days; and observing and touching whether the local part of the injection part has the tumor or not.
After the piglets are immunized by the inactivated vaccine of the strain CH-FJZZ-2017 of the porcine epididymitis added with the valley virus, no obvious body temperature rise phenomenon occurs (Table 1), and the herds have normal ingestion and good mental state; the vaccine injection part has no pain and lumps after 5 days of immunization. The vaccine is proved to have good safety for the immunized piglets.
TABLE 1 animal body temperature monitoring watch after inactivated vaccine immunization
6.2 determination of Inula Valley virus CH-FJZZ-2017 strain inactivated vaccine induced antibody
In order to evaluate the immune effect of the CH-FJZZ-2017 strain inactivated vaccine, serum is collected at 14d and 28d after immunization, the generation state of anti-Swine Selaginella virus antibodies in the serum is evaluated by using indirect immunofluorescence and a neutralization test, and the immunogenicity of the CH-FJZZ-2017 strain inactivated vaccine is evaluated.
According to the step 5, the situation of the antibody generated after the inactivated vaccine of the CH-FJZZ-2017 strain is immunized is detected by an indirect immunofluorescence method, as can be seen from figure 2, the inactivated vaccine of the CH-FJZZ-2017 strain 14 and 28d can induce the immunized piglet to generate the Seleninella virus antibody, and the control group does not generate the corresponding antibody.
In order to further evaluate the titer of the antibody generated by the inactivated vaccine CH-FJZZ-2017 strain induced piglets, the level of the antibody generated by the inactivated vaccine induced immunized piglets is detected by utilizing a neutralization test technology. The method comprises the following specific steps:
1) putting all the pig serum to be detected in a 56 ℃ water bath for incubation for 30min, and performing inactivation treatment;
2) each serum was subjected to 1:5, 1:10, 1: 20. dilutions at 1:40, 1:80, 1:160 and 1:320 fold ratios, 8 replicates for each dilution, containing 100 μ L of diluted serum per well, were used in FMDV neutralizing antibody assay.
3) Diluting the CH-FJZZ-2017 strain virus solution to 200TCID by using a cell maintenance solution500.1mL, adding the diluted virus solution into the diluted serum obtained in the previous step, mixing, placing at 37 deg.C and 5% CO2Incubate in incubator for 2 h.
4) The PK-15 cells with good growth state are transferred into a 96-well plate in advance, when the fusion degree is 80-90%, PBS is used for washing for 1 time, and the virus liquid-serum mixed solution is added into the 96-well plate at 200 mu l/well. And simultaneously setting a positive control group and a negative control group of the virus liquid and the virus liquid which is not added. Placing at 37 ℃ and 5% CO2Culturing in an incubator, observing the pathological condition of cells every day, recording the pathological condition, and calculating the serum neutralizing antibody condition of each group according to a Reed-Muench method.
The data were collated after determination of the serum neutralizing antibody titres and the results are shown in table 2. As can be seen from Table 2, after the inactivated vaccine CH-FJZZ-2017 strain is immunized for 14d, the inactivated vaccine can induce immunized piglets to generate specific antibodies, the immunization requirements are basically met, and the highest antibody can reach 1: 128; the 28d antibody continued to rise, with the highest antibody being able to reach 1: 258. In contrast, no neutralizing antibodies against porcine Sendai Valley virus were detected in the control immunoreduced DMEM maintenance fluid group (Table 2).
TABLE 2 level of neutralizing antibodies produced by animals immunized with inactivated vaccine
The porcine Seneca Valley virus vaccine composition of the invention is proved to be capable of inducing immune animals to generate neutralizing antibodies with higher titer against porcine Seneca Valley virus. The data lays a certain theoretical and practical foundation for the clinical use of the inactivated vaccine.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (10)
1. A strain of Selaginella valley virusSenecavirus A ) A strain characterized by: the strain is named as CH-FJZZ-2017, and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC number 12160.
2. Use of the Seneca Valley virus CH-FJZZ-2017 as claimed in claim 1 in the preparation of a vaccine for the prevention and treatment of diseases caused by porcine Seneca Valley virus.
3. Use according to claim 2, characterized in that: the vaccine for preventing and treating diseases caused by the suijianeijiagu virus is an inactivated vaccine.
4. The active component of the vaccine for preventing and treating diseases caused by the porcine Selaginella virus is an inactivated Selaginella virus strain CH-FJZZ-2017, and the preservation number of the Selaginella virus strain CH-FJZZ-2017 is CGMCC number 12160.
5. The vaccine for the control of diseases caused by suis calavavirus according to claim 4, characterized in that: the vaccine for preventing and treating diseases caused by the suis Selaginella virus comprises inactivated suis Selaginella virus CH-FJZZ-2017 and a pharmaceutically acceptable adjuvant.
6. The method for preparing the vaccine for preventing and treating diseases caused by suis calva virus according to claim 4 or 5, comprising the steps of:
1) and (3) virus culture: inoculating the Selenmevirus CH-FJZZ-2017 to PK-15 cells for culture, and harvesting a virus culture;
2) inactivating the virus culture, repeatedly freezing and thawing the virus culture for three times, centrifuging, collecting supernatant, adding an inactivating agent, and inactivating the virus to obtain an inactivated virus solution;
3) and mixing the inactivated Sasa Veitchii virus CH-FJZZ-2017 virus liquid and an adjuvant in proportion, emulsifying, and preparing the inactivated Swine Sasa Veitchii virus vaccine.
7. The preparation method according to claim 6, wherein the inactivating agent is diethylene imine, the final concentration of the added diethylene imine in the virus solution is 1mmol/L, and the inactivating condition is 30 ℃ for 28 h; after inactivation, sodium thiosulfate solution was added to neutralize the divinyl imine, the final concentration of the added sodium thiosulfate solution being 0.02 g/ml.
8. The method according to claim 7, wherein the adjuvant is ISA206 or ISA201, and the volume ratio of the inactivated virus solution to the adjuvant is 1: 1.
9. Use of the Selaginella virus CH-FJZZ-2017 as claimed in claim 1 for establishing an animal model of Selaginella virus disease.
10. Use of Selaginella virus CH-FJZZ-2017 as claimed in claim 1 for the preparation of antibodies against Selaginella virus disease.
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| CN108103078B (en) * | 2018-01-03 | 2019-02-19 | 中国农业科学院兰州兽医研究所 | Seneca Valley virus vaccine and its preparation method and application |
| CN110551694B (en) * | 2018-06-01 | 2021-03-19 | 金宇保灵生物药品有限公司 | Selenka valley virus SVV/CH/ZZ/2016 |
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