CN112280748A

CN112280748A - Sheep-derived sheep parainfluenza virus type 3 vaccine strain, vaccine composition prepared from vaccine strain, and preparation method and application of vaccine composition

Info

Publication number: CN112280748A
Application number: CN202011061189.7A
Authority: CN
Inventors: 王炜; 马艳华
Original assignee: Inner Mongolia University
Current assignee: Inner Mongolia University
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-01-29
Anticipated expiration: 2040-09-30
Also published as: CN112280748B

Abstract

The invention discloses a sheep parainfluenza virus 3 type vaccine strain, a vaccine composition prepared from the vaccine strain, and a preparation method and application thereof. The sheep parainfluenza virus 3 type vaccine strain is obtained by separating sheep and belongs to a different pedigree with the prior goat source parainfluenza virus strain. The invention also provides a vaccine composition prepared by using the strain as an antigen and a preparation method thereof. The inactivated vaccine immunogenicity test proves that the parainfluenza virus 3 type inactivated vaccine has good immune efficacy. The invention provides an effective technical means for preventing and treating the infection of the sheep parainfluenza virus type 3.

Description

Sheep-derived sheep parainfluenza virus type 3 vaccine strain, vaccine composition prepared from vaccine strain, and preparation method and application of vaccine composition

Technical Field

The invention relates to a sheep derived sheep parainfluenza virus type 3 vaccine strain, and also relates to a vaccine composition prepared from the vaccine strain, and a preparation method and application thereof. The invention belongs to the technical field of biological products for livestock.

Background

China is a big sheep-raising country, the number of sheep raised is the first world, wherein about 1.6 hundred million sheep stocks, about 1.4 hundred million goats stocks and about 3 hundred million sheep and goats are released in the field every year. Epidemic diseases are an important bottleneck restricting the green, healthy and high-quality development of sheep raising industry in China. With the development of intensive breeding of sheep, the harm of epidemic diseases is gradually increased.

Clinical research finds that besides the major epidemic diseases of sheep such as foot-and-mouth disease, peste des petits ruminants, sheep pox and the like, the respiratory disease of sheep is the most important epidemic disease which harms sheep, and huge economic loss is caused to the sheep breeding industry. Parainfluenza virus type 3 (PIV 3) is one of the most important pathogens causing respiratory diseases in sheep and goats, and can cause severe pneumonia in goats and sheep. Epidemiological investigation shows that the disease has wide epidemiology in Chinese respiratory disease sheep. Researches prove that the virus is also one of important pathogens causing the respiratory diseases of sheep, and the virus is often mixed with mycoplasma to infect clinically, so that the respiratory diseases are aggravated.

Parainfluenza virus type 3 is one of the important etiological agents of sheep respiratory diseases (Fischman 1967, Belak and Palfi 1974, Lyon, Leroux et al 1997). The etiological agent of the respiratory disease of sheep is an important viral disease of infected sheep, and PIV3 infected sheep can cause obvious respiratory symptoms, produce viremia and expel toxin and cause lesions in the lung (Yener, Saglam et al 2005).

The positive rate of the antibody against parainfluenza virus type 3 of Hokkaido, Japan was 14.92% (Giangaspero, Savini et al 2013). Mexico investigated parainfluenza virus type 3 (Contreras-Luna, Ramirez-Martinez et al 2017). In 2013, the separation of the goat parainfluenza virus from goats suffering from serious respiratory diseases is reported for the first time in China. Epidemiological investigation finds that the goat and sheep in China have high antibody positive rate which reaches 60% at most and 20% of antigen positive rate (Mao, Yang et al 2019). In 2019, the subject group to which the present inventors belong isolated the ovine parainfluenza virus type 3 from sheep suffering from respiratory diseases. Further epidemiological investigation proves that the parainfluenza virus type 3 has high antibody positive rate in goats and sheep, and has important effect in the respiratory tract disease occurrence and other pathogen cooperative pathogenesis of the goats.

Currently, some studies on the pathogenicity of goats have been carried out by goat parainfluenza. The research shows that after the sheep parainfluenza virus is artificially attacked, the attacked sheep has clinical symptoms of temperature rise, cough, rhinorrhea and the like. Detoxification can be detected 1d after challenge. The goat lung tissue in the toxin attacking group is subjected to autopsy observation to show hyperplasia, consolidation and swelling, and histopathological detection shows that the lung tissue has widened alveolar space, disappearance of alveolar structure and inflammatory cell infiltration. It was demonstrated that goat parainfluenza challenge can cause pneumonia in goats (hogfei, leville et al 2016). The genetic relationship and pathogenicity of sheep isolated parainfluenza and goat parainfluenza has not been studied. Therefore, it is necessary to conduct intensive studies on the isolation of parainfluenza virus in sheep.

At present, no commercial application report of the sheep parainfluenza virus type 3 vaccine exists at home and abroad. Therefore, in view of the prevalence and harm of the parainfluenza virus type 3 in China, a vaccine for preventing the parainfluenza virus type 3 in sheep is urgently needed to be developed.

In view of the above, the invention provides an inactivated vaccine capable of preventing the sheep parainfluenza virus type 3, and the product is used for immunization, so that the sheep can be effectively prevented from being infected by the sheep parainfluenza virus type 3.

Disclosure of Invention

One of the purposes of the invention is to provide a sheep derived sheep parainfluenza virus type 3 vaccine strain;

the invention also aims to provide a sheep parainfluenza virus 3 inactivated vaccine composition prepared from the sheep parainfluenza virus 3 vaccine strain and a preparation method thereof.

The invention also aims to provide application of the sheep parainfluenza virus 3 inactivated vaccine in preparation of a medicament for preventing or treating sheep parainfluenza virus 3.

In order to achieve the purpose, the invention adopts the following technical means:

the inventor of the invention separates a sheep-derived sheep parainfluenza virus type 3 vaccine strain from nasal swabs and serum samples of sheep with respiratory diseases, and the strain is named as CRV3-TX01 strain and is classified and named as goat parainfluenza virus. The virus was a Paramyxoviridae (Paramyxoviridae), a respiratory virus (Respirovirus), a Caprine respiratory virus type 3 (Caprine respiratory virus 3) according to the latest classification report by the international committee for classification of viruses in 2019. This provirus is also known as ovine parainfluenza virus type 3 virus (Caprine parainfluenza virus 3, CPIV 3). Thus, the CRV3 and CPIV3 referred to in this invention are the same virus. The vaccine strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is addressed in the North Cheng, and the strain preservation numbers are as follows: CGMCC No.19970, and the preservation time is 8 months and 21 days in 2020.

Furthermore, the invention also provides application of the sheep parainfluenza virus type 3 vaccine strain in preparing a vaccine composition for preventing and treating sheep parainfluenza virus type 3 infection.

Wherein, preferably, the vaccine composition is an inactivated vaccine composition.

A vaccine composition comprises the sheep parainfluenza virus type 3 vaccine strain.

Preferably, the vaccine composition comprises the inactivated sheep parainfluenza virus type 3 vaccine strain and an adjuvant.

Further, the invention also provides a method for preparing the vaccine composition, which comprises the following steps:

(1) inoculating the sheep parainfluenza virus type 3 vaccine strain to a passage cell line, and carrying out passage and culture;

(2) inoculating the cultured 3-type vaccine strain CRV3-TX01 of the sheep parainfluenza virus to a medium full of 90-100% cells, and culturing by using a cell maintenance solution to prepare a production seed virus;

(3) inoculating the prepared production seed virus into a medium full of 90-100% of cells, and culturing by using a cell maintenance solution to obtain a virus antigen solution of the sheep parainfluenza virus 3 type vaccine strain CRV3-TX 01;

(4) adding an inactivating agent into a virus antigen solution of the sheep parainfluenza virus type 3 vaccine strain CRV3-TX01 for inactivation, adding a neutralizing agent for neutralization, adding an adjuvant, and emulsifying to obtain the product;

wherein the cell maintenance solution is DMEM containing 3.5% v/v horse serum and has a pH of 7.0-7.2.

Wherein, preferably, the continuous cell lines in step (1) include MDBK cell lines, BT cell lines and other cattle and sheep continuous cell lines or primary cell lines, and the passaging and culturing in step (1) includes: digesting the cell line by EDTA-pancreatin cell dispersion liquidPassage, continuously culturing by using a cell growth liquid, and continuously passing or inoculating the virus when the cell growth liquid is full of 90-100%; wherein the cell growth solution contains 92-94% v/vDMEM solution and 6-8% v/v bovine serum, and the pH value is 7.0-7.2; the culture method is any one of the following methods: culturing in a roller bottle to reach a cell density of 2 × 10⁵～6×10⁵Per ml; or adding an attachment carrier into a bioreactor for suspension culture or pure suspension culture to make the cell density reach 2 × 10⁶～5×10⁶And/ml, the attaching carrier is a microcarrier or a paper sheet.

Wherein, preferably, the culture temperature in the step (1), (2) or (3) is 33-37 ℃, and the cell culture environment is 5% CO₂(ii) a The virus culture time is 30-72 hours; the virus inoculation MOI of the sheep parainfluenza type 3 virus TX01 strain in the step (2) or (3) is 0.01-0.05.

Wherein, preferably, the inactivating agent in the step (4) is diethylene imine (BEI) with the final concentration of 0.001-0.003M; the neutralizing agent is sodium thiosulfate, and the final concentration of the sodium thiosulfate is 0.03M.

Wherein, preferably, the virus antigen liquid of the sheep parainfluenza virus type 3 vaccine strain CRV3-TX01 strain in the step (4) is 54 volume parts, the adjuvant is 206 adjuvant, the adjuvant 206 is 46 volume parts, and preferably, the virus antigen liquid of the sheep parainfluenza virus type 3 vaccine strain CRV3-TX01 strain in each dose contains 10 virus content before inactivation^7.5TCID₅₀。

Compared with the prior art, the invention has the beneficial effects that:

the invention separates a sheep parainfluenza virus type 3 strain from sheep bodies for the first time, and the strain and the current goat-derived parainfluenza virus strain belong to different pedigrees. The invention also provides a vaccine composition prepared by using the strain as an antigen and a preparation method thereof. The inactivated vaccine immunogenicity test proves that the parainfluenza virus 3 type inactivated vaccine has good immune efficacy, and the protection rate to virulent attack is 100%.

Drawings

FIG. 1 is a cytopathic result of a cell culture;

wherein: A. inoculating the virus with the cells; B. normal cells

FIG. 2 shows the result of PCR amplification of MDBK cell isolate;

wherein: 1. negative control; 2. separating the MDBK cell from the virus; m, Marker DL2000

FIG. 3 is a phylogenetic tree based on the whole genome of strain CRV3-TX01 of parainfluenza virus type 3 and the M gene;

wherein: (a) evolutionary analysis of the whole genome sequence shows that TX01 has remarkable aggregative property with CPIV3, but forms a branch; (b) (ii) an evolutionary analysis based on the M gene sequence; circles represent newly isolated sheep parainfluenza virus sequences;

FIG. 4 shows pathological changes in lung after virus challenge;

wherein: a normal group B, C, D challenge group; the attacking group shows alveolar cytosis, alveolar wall thickening, alveolar epithelial hyperplasia, lymphocyte and plasma cell infiltration, bronchial epithelial papillary hyperplasia and compensatory emphysema;

FIG. 5 is a gross anatomy after virus challenge;

wherein: A. e is a normal group, and the other groups are a toxic counteracting group, wherein the toxic counteracting group is characterized by cellulose pneumonia, all pathological changes (left and right lung, lobular and septal) lobar pneumonia and severe lung adhesion;

FIG. 6 is the body temperature change after challenge in the experimental animals.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 isolation and identification of ovine parainfluenza Virus type 3 CRV3-TX01 Strain

(1) Virus isolation

The inventor collects nasal swab and serum samples of sheep with respiratory diseases, centrifuges the samples at 4 ℃ and 6000 r/min for 10min, takes supernatant fluid, filters and sterilizes the supernatant fluid through a filter with the diameter of 0.45 mu m, inoculates the supernatant fluid on MDBK cells growing in 80% of single layer, and cultures the cells in a culture box with the temperature of 37 ℃ and 5% CO 2. Microscopic observation shows that the virus can generate typical lesions (CPE) on MDBK cells, and the typical lesions are expressed as cell shedding, aggregation and shrinkage to form meshes. As shown in fig. 1.

(2) RT-PCR identification

Specific primers for synthesizing CPIV3 are designed according to the CPIV 3M gene sequence and used for detecting CPIV3 virus.

An upstream primer: 5'-CATTGAATTCATACTCAGCAC-3', respectively;

a downstream primer: 5 '-AGATTGTCGCATTT(AG) CCTC 3-3',

the result shows that the 6 th generation cell culture of the MDBK cell can obtain a specific target fragment of about 400bp through PCR amplification, and the negative control has no band.

(3) Determination of the Virus content (TCID50)

Diluting the isolate F6 virus generation solution to 10 times by using a maintenance culture solution^-8The dilution was repeated in 8 wells, and MDBK cells were seeded in monolayer-grown 96-well cell culture plates, with a normal cell control of 100. mu.L/well, 37 ℃ and 5% CO₂The cells were cultured, the lesions were observed daily, the number of CPE wells was recorded for 4 days, and the TCID50 of the virus was calculated according to the Reed-Muench method. The virus titer was 10^7.8TCID50/mL。

(4) Whole gene sequencing

Genome scanning sequencing of the strains is completed by adopting an Illumina sequencing technology, wherein the nucleotide sequence of the M gene of the CRV3-TX01 strain is shown as SEQ ID NO. 1. Sequencing results show that the total length of the virus is 15738bp, and 6 structural proteins are coded and respectively: hemagglutinin-neuraminidase (HN), Fusion protein (F), matrix protein (Matrx protein, M), Large protein (Large protein, L), Nucleoprotein (N), phosphoprotein (P).

The gene sequence of the virus strain and the sequence of a representative strain published on GenBank are analyzed for nucleotide and amino acid homology by using DNAStar software, and the analysis result is shown in Table 1.

The strain is subjected to homology analysis with all genes of goat parainfluenza (CPIV3), bovine parainfluenza type 3 (BPIV3) and human parainfluenza type 3 (HPIV3) in GenBank, and the analysis result shows that the homology of the strain with the CPIV3 strain is 97.5-97.7.0%, the homology with the BPIV3 is 74.2-75.4%, and the homology with the human parainfluenza type 3 is 72.6-73.0%. The MEGA 7 software was used to map the whole gene system and M gene evolutionary trees, as shown in fig. 3. The relatives are far away and are in independent branches on the phylogenetic tree. The sources of the PIV3 strains for the whole gene sequence analysis are shown in Table 2, and the sources of the PIV3 strains for the M gene sequence analysis are shown in Table 3.

TABLE 1 CRV3-TX01 Strain nucleotide and amino acid homology analysis

Note: 1: a nucleotide; 2: an amino acid.

TABLE 2 PIV3 Strain complete gene source record table

TABLE 3 PIV3 Strain M Gene Source record Table

(5) Identification of virulence

4ml of CRV3-TX01 strain culture (10 ml) was injected into 5 healthy susceptible sheep of 3-4 months of age, each trachea^7.5TCID₅₀Ml) as a test group, 5 normal sheep cultured under the same conditions as a normal control, and the lungs were sampled 21 days after inoculationPathological as well as anatomical examinations were performed.

Fig. 4 is a pathological change of lung after virus challenge, wherein: a normal group B, C, D experimental group; the experimental group shows that the lung foam cells are increased, the alveolar wall is thickened, the alveolar epithelium is proliferated, the lymphocyte and plasma cell infiltration is carried out, the papillary proliferation of the bronchial epithelium is proliferated, and the compensatory emphysema is caused. Fig. 5 is a gross anatomy after virus challenge, wherein: A. and E is a normal group, and the other groups are experimental groups, wherein the experimental groups show cellulose pneumonia, and all pathological changes (left and right lung, lobular and septal lobe) lobar pneumonia have serious lung adhesion.

The above experimental results show that the isolated strain is sheep parainfluenza virus type 3, named as CRV3-TX01 strain, which is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, and is addressed to the institute of microbiology, Zhongkou academy of sciences, No.1, North Chen West Lu, Yangyang, Beijing, and has the strain preservation number: CGMCC No.19970, and the preservation time is 8 months and 21 days in 2020.

EXAMPLE 2 production of inactivated vaccine against sheep parainfluenza Using MDBK cell line

(1) The goat parainfluenza virus 3 CRV3-TX01 strain is inoculated to full MDBK cells according to the inoculation dose of MOI of 0.01, cell culture is harvested after about 40h, freezing and thawing are carried out for 1 time at-70 ℃, continuous propagation and plaque cloning purification are carried out on the MDBK cells, and pure seed virus is obtained.

(2) Selecting an MDBK cell line as a cell for preparing a seedling;

(3) subjecting MDBK cells to trypsinization and passage, adding cell growth liquid, and treating at 37 deg.C with 5% CO₂Culturing in an incubator, and when the cells grow to 90-100%, using the cells for passage or virus inoculation; when the microcarrier or the paper sheet is used for suspension culture, 1 × 10 seeds are inoculated into a cell culture reactor⁵-2×10⁵cells/mL, 80-90% of which are attached with the cells, so as to realize continuous passage or virus inoculation of the cells;

(4) breeding of the virulent seeds: taking well-grown MDBK cells, washing with PBS for 1 time, and inoculating the seed virus according to the inoculation amount of MOI 0.01; adding maintenance solution (DMEM containing 3.5% v/v horse serum, pH 7.0) for 30-48 hr, collecting cell culture venom when 80% of cells have CPE as production virus;

poison seedAnd (3) identification: performing sterile and mycoplasma inspection according to appendix of pharmacopoeia of people's republic of China, wherein the virus seed should be pure and has a toxicity value of not less than 10^7.5TCID₅₀。

(5) And (3) breeding the vaccine preparation venom: when the cells are full of 90-100%, discarding cell growth solution, washing with PBS for 2 times, inoculating virus seeds according to the inoculation amount of MOI 0.01, adding maintenance solution (DMEM with 3.5% v/v horse serum, pH 7.0), and collecting cell culture virus solution as virus solution for preparing vaccine when 80% of cells have CPE in 30-48 hours; storing the harvested venom at a temperature below-20 ℃;

and (3) testing the vaccine preparation venom: the test is carried out according to the appendix of the pharmacopoeia of the people's republic of China, and no bacteria, mold and mycoplasma grow.

(6) Inactivation and emulsification: mixing 0.2mol/L BEA and 0.4mol/L NaOH in a volume ratio of 2: 1 ratio in a suitable container. Cyclizing for 1h at 37 +/-2 ℃ to generate BEI, and adjusting the pH value to 7.2-7.6. And (3) placing the qualified virus culture solution into a container, adding a BEI inactivator with the final concentration of 3mM, inactivating for 36-40 hours, fully shaking, and adding sodium thiosulfate with the final concentration of 0.03M for neutralization. Slowly injecting the inactivated antigen into 206 adjuvant, keeping the temperature at 30 ℃, and mixing and emulsifying according to 54/100 volume parts of antigen and 46/100 volume parts of 206 adjuvant. Rapidly cooling to 15 deg.C, standing at 4 deg.C for 24 hr without shaking, and packaging to obtain the final product. The content of two antigens in each dose of vaccine is not less than 10^7.5TCID₅₀。

And (4) inspecting a finished product: the test is carried out according to the appendix of the pharmacopoeia of the people's republic of China, and no bacteria, mold and mycoplasma grow.

The inactivated vaccine prepared by the embodiment is qualified in character inspection, safety inspection, efficacy inspection and the like.

Example 3 safety testing of vaccines

The test uses 15 healthy susceptible sheep of 1-2 months of age, divided into 3 groups, each sheep was injected intramuscularly with 4ml of the inactivated vaccine prepared in example 2, and 5 sheep raised under the same conditions as the immunized sheep were used as controls. There should be no local and systemic adverse reactions observed for 14 days.

The results are shown in table 4 and indicate that all the test sheep had no local and systemic adverse reactions after vaccination.

TABLE 4 safety test

Example 4 immunogenicity assay

15 healthy susceptible sheep of 1-2 months of age were divided into 3 groups, and 2ml of the inactivated vaccine prepared in example 2 was injected subcutaneously or intramuscularly to the neck of each group, and 5 sheep raised under the same conditions as the immunized sheep were used as controls, and 4ml of the CRV3-TX01 strain culture (10 ml) was injected intratracheally 21 days after the inoculation^7.5TCID₅₀Ml), continuously observing for 21 days. The control sheep should be totally attacked, the immune sheep should be protected by at least 4, or the control sheep should be totally protected by at least 4.

The results are shown in table 5 and fig. 6, and show that the inactivated vaccine can generate better protection effect on virulent attack after being used for immunizing experimental animals, and the protection rate is 100%.

TABLE 5 immunogenicity assays

Sequence listing

<110> university of inner Mongolia

<120> sheep derived sheep parainfluenza virus type 3 vaccine strain, vaccine composition prepared from vaccine strain, preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1059

<212> DNA

<213> Parainfluenza virus type 3

<400> 1

atggagaata taacaagccc agcaatatac acttttccta agtcatcatt cactgagaat 60

ggtgatttgg aaccactacc acttaaagtc aatgaaaata agaaagctat tcctcatata 120

agagttgtca aggtaggcaa tccaccaaag catggggtaa gataccttga tgttttctta 180

cttggcttct ttgaaatgga aaggactaaa gataagtatg gaagtattag tgatctagat 240

gaggacctag gatacaagat ctgtggatca ggttctttac caatcgggct agccaagtat 300

ggtgggagcg acaaggaact tctacaggct gcaaccaaac tagacataga agtaaggagg 360

acagttaagg ctacagaaat gattgtatat actgtacaaa acataaaacc tgagttatat 420

ccatgggcca gtagattgag aaaagggatg atctttgatg ctcaaaaagt agcacttgct 480

cctcaatgtc ttccgttgga cagaggaata aaattcagag tcatattcgt taattgtacg 540

gctattggct cagtaacact atttaagatc ccaaaatcaa tggcgatgtt gtcattacca 600

aatacaatat caataaatct acaagttcac attaaaacag gggtacagac agattcaaag 660

ggggttgttc agatactaga tgaaaagggc gaaaaatcac ttaatttcat gattcatctt 720

ggcttgatca agcgaaaaat tgggaagatg tattcaattg aatactgcaa gcaaaaaatt 780

gaaaagatga gattgatatt ctcattagga ttgattggag gaatcagctt ccatatcaat 840

gcaactggat caatatccaa aaccttagca agccagttaa cattcaaacg agagatttgc 900

tatcctctta tggatcttaa tccacattta aacattgtta tatgggcatc ctcggttgag 960

attacaagag tagatgcaat tttccaaccc tcattacctg gcgaattcag atactaccca 1020

aatataatag caaagggggt cggcaagatt aaacaataa 1059

Claims

1. Sheep derived sheep parainfluenza virus type 3 vaccine strain is named as CRV3-TX01 strain, is preserved in China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and has the culture preservation number as follows: CGMCC No. 19970.

2. Use of the vaccine strain of sheep parainfluenza virus type 3 according to claim 1 for the preparation of a vaccine composition for the prevention and treatment of sheep parainfluenza virus type 3 infection.

3. The use according to claim 2, wherein the vaccine composition is an inactivated vaccine composition.

4. A vaccine composition comprising the ovine parainfluenza virus type 3 vaccine strain of claim 1.

5. The vaccine composition of claim 4, wherein said vaccine composition comprises inactivated sheep parainfluenza virus type 3 vaccine strain of claim 1 and a suitable oil adjuvant or water adjuvant.

6. A method of preparing the vaccine composition of claim 5, comprising the steps of:

(1) inoculating the sheep parainfluenza virus type 3 vaccine strain of claim 1 into a continuous cell line, and carrying out passage and culture;

7. As claimed in claim6, wherein the continuous cell lines in step (1) include MDBK cell lines, BT cell lines and other cattle and sheep continuous cell lines or primary cell lines, and the passaging and culturing in step (1) includes: digesting and passaging the cell line by EDTA-pancreatin cell dispersion liquid, continuously culturing by using cell growth liquid, and when the cell growth is full of 90-100%, using the cell growth liquid for continuous passage or virus inoculation; wherein the cell growth solution contains 92-94% v/v DMEM solution and 6-8% v/v bovine serum, and the pH value is 7.0-7.2; the culture method is any one of the following methods: culturing in a roller bottle to reach a cell density of 2 × 10⁵～6×10⁵Per ml; or adding an attachment carrier into a bioreactor for suspension culture or pure suspension culture to make the cell density reach 2 × 10⁶～5×10⁶And/ml, the attaching carrier is a microcarrier or a paper sheet.

8. The method of claim 6, wherein the culture temperature in step (1), (2) or (3) is 33-37 ℃ and the cell culture environment is 5% CO₂(ii) a The virus culture time is 30-72 hours; the virus inoculation MOI of the sheep parainfluenza type 3 virus TX01 strain in the step (2) or (3) is 0.01-0.05.

9. The method of claim 6, wherein the inactivating agent in step (4) is diethylene imine (BEI) at a final concentration of 0.001-0.003M; the neutralizing agent is sodium thiosulfate, and the final concentration of the sodium thiosulfate is 0.03M.

10. The method according to claim 6, wherein the virus antigen solution of the sheep parainfluenza virus type 3 vaccine strain CRV3-TX01 in the step (4) is 54 volume parts, the adjuvant is 206 adjuvant, the adjuvant is 46 volume parts, and preferably, the virus antigen solution of the sheep parainfluenza virus type 3 vaccine strain CRV3-TX01 contains 10 volume parts of virus before inactivation in each dose^7.5TCID₅₀。