CN108949700B - Goat parainfluenza virus 3 type JS14-2 strain and application thereof - Google Patents
Goat parainfluenza virus 3 type JS14-2 strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological products for livestock and discloses a goat parainfluenza virus 3JS14-2 strain and application thereof in preparation of inactivated vaccines. Separating and purifying to obtain the goat parainfluenza virus 3 type JS14-2 virus strain, centrifuging the virus liquid, inactivating formaldehyde, adding adjuvant and emulsifying to obtain the inactivated vaccine. The invention provides a virus strain for preparing a goat parainfluenza virus 3 inactivated vaccine, which belongs to a domestic epidemic strain, and has strong toxicity and good antigenicity; the strain has better growth characteristics on MDBK cells, and can obtain stable high titer (more than or equal to 10)7 TCID50Per mL, blood coagulation valence of 2 or more8) (ii) a The vaccine preparation method is complete, and the immunogenicity and the immune protection effect are good; the use of the vaccine has low immunization dose, high safety and long immunization duration, greatly saves the production and use cost, and is favorable for effectively preventing and controlling the disease in clinical practice.
Description
Technical Field
The invention belongs to the field of biological products for veterinary use, and relates to a goat parainfluenza virus 3JS14-2 strain and application thereof.
Background
Goat parainfluenza virus type 3 (Caprine parainfluenza virus 3, CPIV3) is a newly identified member of the genus pneumovirus of the family paramyxoviridae, a enveloped RNA virus, which primarily infects goats and sheep. The virus is mainly spread through respiratory tract to cause respiratory tract diseases, and can cause obvious clinical symptoms under the conditions of stress, mixed or secondary infection with other pathogens and the like, thereby causing serious respiratory tract diseases and causing higher morbidity and mortality. Epidemiological studies indicate that domestic flocks have a high rate of viral infection. The virus is a newly discovered new pathogen, and no effective prevention and control method for the virus is reported at home and abroad. There are no vaccines and diagnostic reagents that can prevent infection by goat parainfluenza virus type 3. Therefore, the development of inactivated vaccines aiming at the epidemic strains in China and corresponding combined vaccine and attenuated live vaccines is urgent.
Disclosure of Invention
In order to solve the technical problems, the invention provides a goat parainfluenza virus 3JS14-2 strain, which has stronger virulence and good immunogenicity. In addition, aiming at the fact that no relevant report aiming at the goat parainfluenza virus type 3 vaccine exists at present, a method for preparing the goat parainfluenza virus type 3 inactivated vaccine by using the bovine kidney cell MDBK (MDBK cell for short) propagated virus is provided.
The invention screens out goat parainfluenza virus 3JS14-2 strain (Camine parainfluenza virus 3, JS14-2) with strong pathogenicity and high immunogenicity from the goat parainfluenza virus 3 epidemic strain which is epidemic in China, the classification name of the virus is goat parainfluenza virus 3 (Camine parainfluenza virus 3, CPIV3), the Latin literature name is Camine parainfluenza virus 3, the virus is preserved in China center for type culture Collection, the address is university of Wuhan, postal code 430072, the preservation number: CCTCC NO: v201832, date of deposit: 26/6/2018. The virus strain is derived from a virus strain which is acquired by an inventor in Jiangsu province and has stronger virulence and high immunogenicity.
The invention also provides a separation and purification method of the goat parainfluenza virus 3JS14-2 strain, which comprises the steps of identifying diseased sheep, collecting a nose swab sample of the diseased sheep, adding antibiotics into the nose swab sample, incubating the sample and centrifuging, taking supernatant, filtering and sterilizing through a filter membrane, inoculating the supernatant to a bovine kidney cell monolayer cell for culture, and observing cytopathic effect until stable cytopathic effect appears.
Further, the invention provides a method for separating and purifying the goat parainfluenza virus type 3JS14-2 strain, wherein the antibiotic is effective to both gram-positive bacteria and gram-negative bacteria.
Further, the present invention provides a method for isolating and purifying goat parainfluenza virus type 3JS14-2 strain, wherein the virus is passaged a plurality of times during the observation of cytopathic effect.
Further, the invention provides a separation and purification method of the goat parainfluenza virus type 3JS14-2 strain, wherein the antibiotic is streptomycin.
Further, the invention provides a method for separating and purifying the goat parainfluenza virus type 3JS14-2 strain, which further comprises plaque purification of the separated virus.
Further, the invention provides a method for separating and purifying the goat parainfluenza virus type 3JS14-2 strain, which further comprises at least one plaque purification of the separated virus.
Further, the invention provides a method for separating and purifying the goat parainfluenza virus type 3JS14-2 strain, which further comprises diluting and purifying the separated virus before plaque purification.
Further, the invention provides a separation and purification method of the goat parainfluenza virus type 3JS14-2 strain, and the method also comprises the steps of carrying out RT-PCR and sequence analysis on the purified virus RNA.
Furthermore, the invention provides a method for separating and purifying the goat parainfluenza virus type 3JS14-2 strain, which also comprises the step of carrying out purity test on the purified virus.
The invention also provides a method for preparing the goat parainfluenza virus 3 type JS14-2 vaccine, which comprises the steps of preparing the goat parainfluenza virus 3 type JS14-2 challenge strain, inactivating the challenge strain, uniformly mixing the inactivated virus with Tween-80 to prepare a water phase, uniformly mixing the white oil for injection with span-80 to prepare an oil phase, and mixing and emulsifying the oil phase and the water phase to prepare the goat parainfluenza virus 3 vaccine.
The invention also provides a method for preparing the goat parainfluenza virus 3JS14-2 vaccine, wherein the preparation of the goat parainfluenza virus 3JS14-2 challenge strain comprises the steps of inoculating the goat parainfluenza virus 3JS14-2 strain to bovine kidney cells, collecting cell suspension after obvious cytopathic effect occurs, repeatedly freezing and thawing for multiple times, centrifuging, and collecting supernatant, namely the goat parainfluenza virus 3JS14-2 challenge strain.
The invention also provides a method for preparing the goat parainfluenza virus 3JS14-2 vaccine, which comprises detecting the inactivated challenge strain to determine whether the inactivation is complete.
The invention also provides a method for preparing the goat parainfluenza virus 3JS14-2 vaccine, wherein the detection of whether the challenge strain is completely inactivated is realized by inoculating the inactivated challenge strain to bovine kidney cells, carrying out passage after repeated freeze thawing, and observing cytopathic effect.
A goat parainfluenza virus type 3JS14-2 vaccine prepared using the method described above.
A therapeutic agent of goat parainfluenza virus type 3JS14-2 comprises the vaccine as above and the drug composed of the vaccine and the pharmaceutically acceptable antigen, immunologic adjuvant, carrier or adjuvant, such as inactivated vaccine, live carrier vaccine, monovalent vaccine, polyvalent vaccine or combined vaccine.
The invention also provides application of the goat parainfluenza virus 3JS14-2 strain as an attack strain in inducing the production of goat parainfluenza.
The invention also provides application of the goat parainfluenza virus 3JS14-2 strain as an attack strain to the establishment of an animal model for evaluating the effect of the goat parainfluenza virus 3 vaccine.
The invention also provides an evaluation method of the goat parainfluenza virus 3JS14-2 strain as an attack strain in establishing an animal model for evaluating the effect of the goat parainfluenza virus 3 vaccine, wherein the method comprises the step of observing the lesion of the animal subjected to the attack of the goat parainfluenza virus 3JS14-2 strain.
The invention also provides an evaluation method of the goat parainfluenza virus 3JS14-2 strain as an attack strain in establishing an animal model for evaluating the effect of the goat parainfluenza virus 3 vaccine, wherein the method comprises the steps of carrying out RT-PCR detection on viremia and detoxification on animals subjected to the attack of the goat parainfluenza virus 3JS14-2 strain.
The invention also provides application of the goat parainfluenza virus 3JS14-2 strain in preparation of a reagent for diagnosing diseases caused by the goat parainfluenza virus 3.
Has the advantages that: the goat parainfluenza virus 3JS14-2 strain has stable biological characteristics and strong pathogenicity, and can be used for establishing a virus attack pathogenesis model. The goat parainfluenza virus 3JS14-2 strain has good immunogenicity, and can be used for establishing an antibody detection method. The oil adjuvant inactivated vaccine prepared by the goat parainfluenza virus 3JS14-2 strain is safe and reliable, can generate a high-level antibody after the immunization of a goat, has the duration of 12 months, has good protection effect on homologous virus challenge, obviously reduces the symptoms and toxin expulsion of an immunized goat flock, and can effectively prevent the epidemic of the goat parainfluenza virus 3.
Drawings
For a better illustration of the invention, the following figures are provided, which are provided for illustration of the invention only and are not limiting of the invention:
FIG. 1 is an electron microscope observation of goat parainfluenza virus type 3JS14-2 strain virions;
FIG. 2 shows the variation of the antibody titer of each group of HI antibody in the goat parainfluenza virus type 3JS14-2 inactivated vaccine immunity efficacy test;
FIG. 3 shows the detection of goat parainfluenza virus type 3JS14-2 inactivated vaccine immune antibody for a sustained period.
Detailed Description
The invention will be further described with reference to specific embodiments and drawings attached to the description, but the invention is not limited thereto.
Example 1 isolation, purification and characterization of goat parainfluenza Virus type 3JS14-2 Strain
1 isolation of viruses
In 2013, diseases mainly with respiratory symptoms appear in large-scale goat farms in many places such as Jiangsu Anhui, China, and the like, and the diseases are manifested as mental depression, cough, serous fluid or thick nasal fluid. A nasal swab sample of a diseased goat is collected and detected to be identified as the goat parainfluenza virus type 3. And then, continuously carrying out etiological detection on a plurality of similar cases in Jiangsu, collecting a nasal swab sample of a diseased sheep farm, and storing at-80 ℃.
Adding streptomycin into nasal swab sample of diseased goat detected as positive parainfluenza virus, incubating at 37 deg.C for 30min, centrifuging at 12000r/m for 20min, collecting supernatant, filtering with 0.22 μm filter membrane for sterilization, inoculating to bovine kidney cells (MDBK) (purchased from Chinese veterinary medicine inspection institute), culturing in single layer cell, culturing at 37 deg.C with 5% CO2The cells were incubated in the incubator protected from light and observed daily for cytopathic effect (CPE). Blindly passed to passage 2, significant cytopathic effect (CPE) began to develop 48h after vaccination, and stable cytopathic effect appeared after passage 3.
2 Virus purification
The isolated 3 rd generation virus was diluted in nutrient solution in 10-fold gradient. Then, the cells were individually plated in 24-well cell culture plates full of MDBK cell monolayers, with 2 wells at each dilution, and 100. mu.L per well. While a negative control without virus inoculation was set. Adsorbing at 37 ℃ for 1h, discarding virus liquid, adding 0.5mL of prepared agar into each hole, culturing at 37 ℃ for 5-7 days, staining neutral red for 2h, selecting hollow spots in the holes, inoculating the hollow spots into MDBK cells on a 96-hole plate, culturing and passaging for 2 generations, collecting the culture with cytopathic effect, performing plaque purification once again, and subculturing and amplifying purified strains.
3RT-PCR and sequence analysis
Extracting the purified virus RNA, performing RT-PCR amplification on a virus M gene by adopting a one-step RT-PCR detection kit of Beijing Quanjin company, wherein the total volume of the RT-PCR is 20 mu L, 2 XR-Mix Buffer is 10 mu L, upstream and downstream primers are respectively 0.5 mu L (F: 5'-AGTGATCTAGATGATGATCCA-3', R: 5'-GTTATTGATCCAATTGCTGT-3'), E-Mix is 0.4 mu L, an RNA template is 4 mu L, and RNase-free water is added to 20 mu L. And (3) amplification procedure: reverse transcription is carried out for 40min at the temperature of 45 ℃; 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃. The recovered PCR product was sequenced by Biometrics, and it was confirmed to be CPIV3 by BLAST alignment analysis, and this strain was designated as goat parainfluenza virus type 3JS14-2 strain.
The main antigen gene HN of the goat parainfluenza virus 3JS14-2 strain is amplified by RT-PCR and sequenced, compared with the reported isolated strain of goat parainfluenza virus 3, the gene sequence and the coded amino acid sequence have a plurality of variations, and the coded HN protein has enhanced antigenicity and better immunogenicity through software prediction analysis.
Example 2 hemagglutination assay
Hemagglutination of isolated and purified 3 rd generation goat parainfluenza virus type 3 virus was determined by hemagglutination assay. Different virus solutions were diluted 2-fold in 96-well hemagglutination plates, and then 1% guinea pig erythrocytes were added and incubated at 37 ℃ for 45min, so that the highest dilution that allowed complete agglutination of erythrocytes was taken as the hemagglutination value of the virus. The results show that: the hemagglutination test shows that the hemagglutination price can reach 256.
Example 3 morphological Observation of Virus particles
200mL of goat parainfluenza virus 3JS14-2 strain cell culture is taken, centrifuged at 12000 r/min for 5min to remove cell debris, the supernatant is ultracentrifuged at 40000 r/min for 2h, and the precipitate is taken and dissolved with PBS overnight to obtain virus suspension. The viral suspension was loaded onto a copper mesh and the virion morphology was observed under a transmission electron microscope after negative staining with 2% phosphotungstic acid. As shown in FIG. 1, a large number of enveloped virus particles were observed, and the virus exhibited an irregular morphology with a size of about 100-150 nm.
Example 4 pathogenicity test and infection pathogenesis model of goat parainfluenza virus type 3JS14-2 Strain
1. Preparation of virus seeds for counteracting toxic pathogen
Inoculating goat parainfluenza virus type 3 virus strain JS14-2 to MDBK cells at MOI of 0.1, and sucking at 37 deg.CAnd (3) after 2h, removing the culture solution, adding a DMEM culture medium containing 2% fetal calf serum, culturing for 5 days at 37 ℃ until obvious cytopathic effect appears on the cells, collecting cell suspension, repeatedly freezing and thawing for 3 times, centrifuging at 10000r/min, and obtaining the supernatant which is the virus seed of the goat parainfluenza virus 3 type JS14-2 strain. Hemagglutination test for determining goat parainfluenza virus type 3JS14-2 strain hemagglutination value is 256, and TCID is calculated according to Reed-Muench method50Is 10-7and/mL. Purity test is carried out according to the appendix of the current pharmacopoeia of the people's republic of China, and the virus liquid should grow without mixed bacteria. The goat parainfluenza virus type 3JS14-2 strain has good growth characteristics in MDBK cells, and can obtain stable and high-titer virus solution more quickly compared with the prior isolated strain of goat parainfluenza virus type 3.
Animal grouping and viral infection procedure
Selecting 10 CPIV3 healthy goats of 2-3 months of age with negative antigen and antibody detection, and randomly dividing the goats into a challenge group (CC) and a control group (NC), wherein each group comprises 5 goats; the purified goat parainfluenza virus 3JS14-2 strain (10) is inoculated in the challenge group6TCID50Per mL), 2mL for nasal drip. The control group was inoculated with MDBK cell culture supernatant at the same dose and method. After the challenge, the goats were housed in two groups and observed for 14 days, and clinical symptoms (cough, nasal secretion, etc.) of the goats in each group were observed and recorded every day. Nasal swabs and serum samples of the goats were collected on days 1, 3, 5, 7, 9, 11 and 14 after challenge and stored at-80 ℃. RT-PCR is used for detecting viremia and expelling toxin. Gross anatomical lesions were observed 14 days after challenge.
RT-PCR detection of viremia and detoxification
Extracting virus RNA in goat nose swab samples of the challenge group and the control group, carrying out RT-PCR amplification on M genes to detect the detoxification condition, wherein the total volume of RT-PCR is 20 muL, wherein 2 XR-Mix Buffer is 10 muL, the upstream primer and the downstream primer are respectively 0.5 muL (F: 5'-AGTGATCTAGATGATGATCCA-3', R: 5'-GTTATTGATCCAATTGCTGT-3'), E-Mix is 0.4 muL, the RNA template is 4 muL, and RNase-free water is added to 20 muL. And (3) amplification procedure: reverse transcription is carried out for 40min at the temperature of 45 ℃; 5min at 94 ℃; 30s at 94 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃. After the reaction is finished, taking the PCR product to carry out electrophoresis detection in 1.2% agarose gel, and photographing and recording. Selecting a sample with positive RT-PCR detection, carrying out virus separation on MDBK cells, and observing cytopathic effect. The results are shown in Table 1.
TABLE 1 viremia and detoxification test
Note: -, negative; positive, positive
4 clinical symptoms and necropsy lesions
The clinical symptom results are shown in table 2, the goat in the toxin attacking group starts to have nasal discharge and cough on the 3 rd day after toxin attacking, and has obvious respiratory symptoms on the 5 th to 9 th days, wherein the symptoms are manifested as poor spirit, head fluid or purulent nasal discharge and cough; the control group had no apparent clinical symptoms. The results of RT-PCR detection of viremia and detoxification are shown in Table 2, the goat in the toxin-attacking group can detect viremia 3-9 days after infection, and can detect nasal detoxification 3-11 days after infection. The clinical sample detection of the control group goat is negative. The band detected as positive is recovered and sent to a sequencing company for sequencing, and the result shows that the amplified strain is CPIV3JS14-2 strain.
The autopsy result shows that the goat lung in the offending group has obvious hyperplasia and solid change, and part of the surface has serous exudate which is adhered to the chest wall. No obvious lesions were observed in other organs. No significant pathological changes were observed in the lung tissue of the control group.
RT-PCR detection positive nasal swab samples can generate stable cytopathic effect after MDBK cell separation, and collected passage viruses are detected as CPIV3JS14-2 strain through RT-PCR detection.
TABLE 2 clinical symptoms of goat parainfluenza virus type 3JS14-2 strain after challenge
Note: -, normal; +, light; + +, medium; + + + + +, severe degree
Example 5CPIV 3JS14-2 Strain Hemagglutination Inhibition (HI) antibody assay
The HA titre of the virus was tested first (see example 4). The antigen was diluted to 4 HA units. 25. mu.L of physiological saline was added to each well of a 96-well V-type microplate. Adding 25 mu L of serum into the first hole, uniformly mixing, continuously diluting by 2 times, uniformly mixing 1 hole, sucking 25 mu L of serum, and discarding. At the same time, 4 units of antigen control and negative control with only guinea pig erythrocytes were set and left at 37 ℃ for 40 min. mu.L of 1% guinea pig erythrocyte solution was added to each well and left at 37 ℃ for 40 min. The highest dilution of antibody that completely inhibited agglutination was the hemagglutination-inhibiting antibody (HI) titer of serum. The antibody titer is more than or equal to 16 (2)4) Is positive and less than or equal to 8 (2)3) It was negative. The results showed that HI antibodies were produced after vaccine immunization (fig. 2, fig. 3).
Example 6 preparation of inactivated vaccine of CPIV3JS14-2 Strain
The goat parainfluenza virus 3JS14-2 strain seed virus prepared in the example 4 is inactivated by formaldehyde with the final concentration of 0.1% for 16h after centrifugation, and the inactivated virus liquid is stored at the temperature of 2-8 ℃ for standby. Diluting the inactivated virus liquid sample by 10 times with a cell maintenance liquid, inoculating the diluted virus liquid sample to MDBK cells with 90% confluency, adsorbing for 1h at 37 ℃, adding the cell maintenance liquid to culture for 5d, repeatedly freezing and thawing for 3 times, and then harvesting virus liquid, and performing blind passage on the MDBK cells for 2 generations according to the method. No cytopathic effect of MDBK cells was observed, indicating complete virus inactivation.
Mixing white oil for injection with span-80 at volume ratio of 94:6, mixing, and autoclaving to obtain oil phase. And uniformly mixing the inactivated virus solution and tween-80 according to the volume ratio of 96:4 to prepare a water phase. And mixing the oil phase and the water phase according to the volume ratio of 2:1, and emulsifying to obtain the inactivated vaccine strain of goat parainfluenza virus 3JS 14-2. Subpackaging the emulsified qualified vaccine into sterilized vaccine bottles, and storing at 2-8 deg.C.
Example 7 CPIV3JS14-2 Strain inactivated vaccine Immunity test
1. Test grouping and sampling
15 healthy negative goats of 2-3 months of age were selected and randomly divided into three groups of 5: immunization challenge group (VC, challenge after immunization), challenge control group (CC, non-immune challenge), blank control group (NC, non-immune and non-challenge). VC groups are subjected to neck intramuscular injection immunization twice with interval of 14d, the vaccine prepared in the example 5 is injected into the neck, 1ml of vaccine is injected into the neck, and blood is collected before and after immunization respectively to detect the HI antibody. Challenge was performed 14 days after the second immunization on the VC and CC sheep (10)7TCID50mL, 2 mL/head, nasal drip), NC groups were inoculated with MDBK cell culture supernatant at the same dose and method. Clinical symptoms were observed daily after challenge, nasal swabs and serum samples were collected from each group of goats on days 1, 3, 5, 7, 9, 11, and 14, and stored at-80 ℃. RT-PCR detects viremia and nasal detoxification. The virus was killed 14 days after challenge (14dpc) and pathologically observed.
Detection of HI antibody and indexes after challenge
Hemagglutination-inhibiting antibody detection after immunization was performed as in example 5. The results are shown in FIG. 2: HI antibody can be detected 14 days after immunization, and the HI antibody titer is 27.2±0.8(ii) a After secondary immunization, the antibody titer is rapidly increased to 212.2±0.5(ii) a The remaining two groups were negative. The inactivated vaccine has a good immune effect. After challenge, group CC antibodies were rapidly produced, reaching 2 at 14 days8.8±1.5Indicating successful counteracting toxic substance. The VC group HI antibody titer remained unchanged and did not change much.
Clinical symptoms such as rhinorrhea, cough, increase of eye secretion and the like begin to appear in the CC group 3 days after the toxin attack, and the clinical symptoms of the CC group are obvious, serous/purulent rhinorrhea appears and last for the 14 th day at most. In the VC group, 2 (2/5) had mild rhinorrhea symptoms on days 5-7, with a degree and duration that were significantly less than in the offending group (Table 3). All the CC groups (5/5) have obvious toxin expelling in 3-11 days, and 4/5 has viremia in 5-9 days; in the VC group 2 (2/5) had transient detoxification on days 5 and 7 (table 4).
Pathological histological changes were observed by dissection, and the lungs in group CC showed hyperplasia, excess change, and adhesion to the chest wall. The lung of the immune attacking group is normal. The results show that the inactivated vaccine has better immune protection effect.
TABLE 3 post-challenge clinical symptoms
Note: -, normal; +, light; + +, medium; + + + + +, severe degree
TABLE 4 post-challenge viremia and detoxification status test
Example 8 CPIV3JS14-2 Strain inactivated vaccine safety test
20 healthy 2-3 month old lambs negative for CPIV3 were selected and randomized into 4 groups (single dose, double dose, single dose repeat and control). In the single dose group, 1mL of the CPIV3JS14-2 inactivated vaccine prepared in example 6 is injected into the neck part of the patient through muscle; double dose group, neck intramuscular injection of inactivated vaccine, 2 mL/one; the single dose is repeated for each group, 1mL of the first immunization is needed, and the injection is repeated once in 14 days after the first immunization according to the same method of the first immunization; control group, immune PBS buffer. Observing whether anaphylaxis and excessive thermal reaction exist after injection; measuring the rectal temperature every day, and observing the mental status, drinking water, eating and other conditions of the goats in each group; checking whether local inflammatory reactions such as red swelling and hot pain exist at the injection part of each goat by touch; weighing the body weight before immunization and 21 days after immunization respectively, wherein the immune group and the non-immune group have obvious difference on daily weight gain in average; after 28 days of immunization, test goats were killed and examined for vaccine absorption and pathological changes at the injection sites.
After immunization, continuous observation shows that all groups of lambs are normal in mental state, ingestion and drinking water, no anaphylactic reaction and an over-heat reaction appear, and no swelling is found when the inoculated part is touched; the weights of the immunized group and the non-immunized group were not significantly different. After immunization for 28 days, each test sheep group is killed by dissection, and the vaccine absorption condition is observed, so that the injection part is well absorbed, and no pathological changes such as edema, exudation, hemorrhage and the like exist. Therefore, the inactivated vaccine of the goat parainfluenza 3 type JS14-2 strain is safe for lambs of 2-3 months of age.
Example 9 immune antibody duration test
20 healthy lambs of 2-3 months of age were selected and randomized into 2 groups of 10 lambs each. Group 1 was immunized with vaccine, 1 ml/vaccine of the CPIV3JS14-2 strain inactivated vaccine prepared in example 6, twice at intervals of 14d by intramuscular injection at the neck; group 2 was a blank control group. Serum HI antibody levels were measured as described in example 5, from the first immunization (-0.5 months) and from blood collected at 0, 1, 2, 3, 5, 6, 7, 9, 12 months after the second immunization.
The results are shown in FIG. 3: the HI antibody of the goat parainfluenza virus 3 inactivated vaccine can reach 2 after the first immunization8.1 ±0.7Peak value of 2 after 1 month of secondary immunization11.3±0.8Followed by a slow decline with antibody titers of greater than 64 (2) over 9 months6) Remains positive by 12 months (2)5.1±1). The duration of the inactivated vaccine can reach 12 months.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. The inactivated vaccine is characterized in that the goat parainfluenza virus 3 strain is named as goat parainfluenza virus 3JS14-2 strain, and the preservation number of the inactivated vaccine is as follows: CCTCC NO: the number of the V201832 (V),
the preservation date is as follows: 26/6/2018, deposit address: china, wuhan university, china type culture collection.
2. An inactivated vaccine prepared from the goat parainfluenza virus type 3 strain as claimed in claim 1, wherein the vaccine comprises an immunological adjuvant and a carrier.
3. An inactivated vaccine prepared from the goat parainfluenza virus type 3 strain according to claim 1, wherein the vaccine is a monovalent vaccine, a multivalent vaccine or a combined vaccine.
4. The method for preparing the inactivated vaccine prepared from the goat parainfluenza virus type 3 strain of claim 1 is characterized by comprising the steps of inactivating the goat parainfluenza virus type 3JS14-2 strain, uniformly mixing the inactivated virus with Tween-80 to prepare a water phase, uniformly mixing white oil for injection with span-80 to prepare an oil phase, and mixing and emulsifying the oil phase and the water phase to prepare the goat parainfluenza virus type 3 vaccine.
5. Use of an inactivated vaccine prepared from the goat parainfluenza virus type 3 strain of claim 1 in the preparation of a therapeutic agent for preventing diseases caused by the goat parainfluenza virus type 3.
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| CN113736799B (en) * | 2021-09-17 | 2024-02-06 | 江苏省农业科学院 | Construction method and application of goat parainfluenza virus 3-type infectious cDNA clone |
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| CN105838678A (en) * | 2016-04-11 | 2016-08-10 | 江苏省农业科学院 | Hybridoma cell strain capable of secreting CPIV3 antibody and ELISA kit |
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| CN105838678A (en) * | 2016-04-11 | 2016-08-10 | 江苏省农业科学院 | Hybridoma cell strain capable of secreting CPIV3 antibody and ELISA kit |
Non-Patent Citations (2)
| Title |
|---|
| 不同条件对山羊副流感病毒3型灭活效果及免疫原性的影响;李文良等;《中国畜牧兽医学会动物传染病学分会第九次全国会员代表大会暨第十七次全国学术研讨会论文集》;20170820;第178页第2-3段 * |
| 山羊副流感病毒3型人工感染山羊的致病性;郝飞等;《中国兽医学报》;20161231;第36卷(第12期);摘要 * |
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