CN116426487A

CN116426487A - Porcine epidemic diarrhea virus strain and application thereof in vaccine preparation

Info

Publication number: CN116426487A
Application number: CN202310303863.5A
Authority: CN
Inventors: 王麒文; 徐宏军; 李义星; 侯晓岚; 张艳平; 董婷婷; 杨杰华; 赵玉惠; 王少沛; 李文华; 张孝智; 高杰; 王宗祥; 韩娜
Original assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-07-14

Abstract

The invention provides a porcine epidemic diarrhea virus strain and application thereof in vaccine preparation, wherein the antigen protein gene of the porcine epidemic diarrhea virus strain is mutated to form a novel mutated virus strain, and the virus strain can be used as an antigen for preparing vaccines. The preservation number of the porcine epidemic diarrhea virus PEDV-AQ strain provided by the invention is CCTCC NO: V202164. The inactivated vaccine prepared from the porcine epidemic diarrhea virus PEDV-AQ strain obtained by separation can obviously reduce the diarrhea rate and death rate of piglets, and has a protective effect on the piglets which is obviously superior to that of commercially available vaccines.

Description

Porcine epidemic diarrhea virus strain and application thereof in vaccine preparation

Technical Field

The invention relates to the technical field of biological products for livestock, in particular to a porcine epidemic diarrhea virus strain PEDV-AQ strain and a vaccine composition prepared from the porcine epidemic diarrhea virus strain PEDV-AQ strain.

Background

Porcine epidemic diarrhea (Porcine Epidemic Diarrhea, PED) was first discovered in europe and gradually transferred into america and asia. The porcine epidemic diarrhea virus is mainly used as a host, pigs at various ages are susceptible, especially the loss of the suckling piglets after infection is the largest, and the digestive system and the immune system of the piglets just born are imperfect, so that the diarrhea rate and the diarrhea death rate are very high after infection, and the production order and the economic benefit of a pig farm are seriously influenced. In 2010, PED was successively outbreaked in southern provinces such as Guangdong, guangxi, sichuan and the like, which caused very serious economic losses for the pig industry in China. Since 2019 African swine fever is completely exploded in China, the disease still has no tendency to be lightened under the background of greatly improving the biosafety control level, and the epidemic effect of various porcine epidemic diarrhea related vaccines which are applied to the market at present is not ideal, and the porcine epidemic diarrhea is the most troublesome problem in the prevention and control process of the porcine epidemic disease.

The pathogen of PED is porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV), belonging to the genus coronavirus of the family coronaviridae, the genetic material is RNA, and the genome is about 28kb. Structural proteins of porcine epidemic diarrhea virus mainly include S protein, E protein, M protein and N protein. Wherein the S protein is a fiber glycoprotein on the surface of the virus particle, and is involved in the adsorption and fusion of viruses and host cells, and is also a main antigen molecule for inducing the host to generate neutralizing antibodies. The S protein of porcine epidemic diarrhea virus can be divided into two domains, S1 and S2. The S protein is also the main structural protein of mutation of porcine epidemic diarrhea virus, and mutation comparison of genes encoding the S protein is the main target gene for dividing strains. Viruses with RNA as genetic material have fewer or less active polymerases to correct errors during replication, and RNA has lower efficiency of correcting errors during replication, which increases the variation of RNA viruses.

Since 2010, epidemic strains of PEDV in China have changed greatly due to various factors such as foreign afferent, self-variation of PEDV, and immune pressure of long-term use of vaccine. Recent epidemiological investigation and research results indicate that strains popular in China are mainly variant strain G2 subgroup and G1 subgroup exists. Although the G1 subpopulation has cross-protective power with the G2 subpopulation, it is weaker. Commercial PEDV vaccine strains are applied in China earlier to be the G1 subgroup vaccine strain, a better effect is obtained clinically, the G2 subgroup vaccine strain begins to prevail in China after 2010, and the G2 subgroup vaccine strain is gradually marketed, so that a better immune effect is achieved. However, as the recombination and mutation are continued among wild strains with different genotypes, the vaccine for pig farm immunization is not shaped, the generated antibody can not effectively exert the specific neutralization effect, and the clinical manifestation is that the protection capability of the existing commercial vaccine is insufficient.

The existing PEDV vaccine strain is mostly obtained from pig farm separated strain mode, and the PEDV virus is fast in mutation, so that the mutant PEDV strain is obtained from pig farm, and the method has important significance for preparation and application of PEDV vaccine and is also a relatively difficult task.

Disclosure of Invention

The invention aims to provide a porcine epidemic diarrhea virus strain and application thereof in vaccine preparation, wherein the antigen protein gene of the porcine epidemic diarrhea virus strain is mutated to form a novel mutated virus strain, and the virus strain can be used as an antigen to prepare a vaccine.

The invention firstly provides a porcine epidemic diarrhea virus PEDV-AQ strain which is preserved in China center for type culture collection (CCTCC NO: V202164) of university of Wuhan in China on 8 th month 5 of 2021.

The amino acid sequence of the S gene of the porcine epidemic diarrhea virus PEDV-AQ strain provided by the invention is SEQ ID NO. 1;

the porcine epidemic diarrhea virus PEDV-AQ strain provided by the invention can be used for preparing vaccines;

the vaccine is specifically described as an embodiment, and is an inactivated vaccine;

in a further aspect, the present invention provides an inactivated vaccine wherein the antigen used is an inactivated porcine epidemic diarrhea virus PEDV-AQ strain;

the inactivation is described as an example, and is performed using formaldehyde.

Another use of the strains of the invention is in the preparation of neutralizing antibodies.

The inactivated vaccine prepared from the porcine epidemic diarrhea virus PEDV-AQ strain obtained by separation can obviously reduce the diarrhea rate and death rate of piglets, and has a protective effect on the piglets which is obviously superior to that of commercially available vaccines.

Drawings

FIG. 1 shows the results of exogenous detection of mycoplasma;

FIG. 2 shows exogenous detection results of transmissible gastroenteritis virus, porcine rotavirus and blue ear virus;

FIG. 3 shows RT-PCR detection of porcine epidemic diarrhea virus G2 subtype cultures of different generations; wherein: 1 is a negative control, 2 is a positive control, 3-7 are culture supernatants of porcine epidemic diarrhea virus of

generation

2, 4, 6, 8 and 10;

FIG. 4 is an analysis chart of an amino acid homology evolutionary tree encoded by the S gene;

FIG. 5 is a transmission electron micrograph of PEDV-AQ strain infected Vero cells;

FIG. 6 is a photograph of an indirect immunofluorescence assay.

Detailed Description

The inactivated vaccine in the embodiments of the present invention refers to a virus or bacteria that is cultured and then inactivated with heat or chemicals (usually beta-propiolactone or formalin). Inactivated vaccines may consist of either whole viruses or bacteria or of split fragments thereof as split vaccines.

The antibody titer in the embodiment of the invention is a reaction which expresses the immune effect of the antibody by the physical state of the antibody and the residence time of the antibody in the body and the reaction quantity of the antibody and the antigen.

The present invention will be described in detail with reference to the following examples and the accompanying drawings.

Example 1 isolation and identification of viruses

1. Sample of

After commercial vaccine of porcine epidemic diarrhea virus is used in a large scale pig farm in Jiangxi province in 2020, epidemic situation of porcine diarrhea still occurs in 4 months. Part of intestinal tissues and contents of diarrhea-dead piglets were obtained by aseptic dissection.

2. Isolation of viruses and detection of foreign pathogens

The intestinal tract sample is firstly treated by liquid nitrogen, ground by a mortar, continuously homogenized by adding sterile PBS (0.1 mol/L, pH 7.2) according to the proportion of 1:5 (w/v), repeatedly freeze thawing for 3 times, centrifuging at 6000r/min for 10min, filtering and sterilizing the supernatant by a 0.22um filter, obtaining tissue filtrate, and preserving the tissue filtrate for standby after the sterility test is qualified.

Resuscitates Vero cells, cultures them in culture flask with DMEM medium containing 10% fetal bovine serum, and when the cells grow into compact monolayer, inputs PBS buffer solution, infiltrates for 20min, then pours out, repeatedly uses PBS to wash for 3 times, and is used.

Diluting the tissue filtrate with sterile DMEM at a volume ratio of 10%; then adding porcine trypsin into the diluted tissue filtrate according to three gradients of 20 mug/ml, 30 mug/ml and 40 mug/ml, and incubating for 2 hours at 37 ℃ in a carbon dioxide incubator; adding the nurturing virus liquid into the compact monolayer Vero cells, and then placing the Vero cells at 37 ℃ for culturing in a carbon dioxide incubator.

Negative control group: 20 mug/ml, 30 mug/ml and 40 mug/ml of porcine trypsin are added into aseptic DMEM, then added into compact monolayer Vero cells respectively, inoculated into the Vero cells, and then placed at 37 ℃ for culture in a carbon dioxide incubator. The culture was continued for 72 hours, the cell state was observed at 24 hours intervals, and the culture was stored at-80 ℃.

The first generation cultures were freeze-thawed 4 times and then added to DMEM containing three gradient porcine trypsin at a ratio of 10%, 20 μg/ml, 30 μg/ml, 40 μg/ml, respectively, and after incubation in a 37 ℃ incubator for 2 hours, all three groups were added to 2% fetal bovine serum DMEM. Adding the nurturing virus liquid into the compact monolayer Vero cells, and then placing the Vero cells at 37 ℃ for culturing in a carbon dioxide incubator. A negative control group was also set. Cell status was observed at 24h intervals, culture was continued for 72h, and the culture was kept at-80 ℃.

The procedure was blinded to passage 10 and the observation was gradually started to produce plaque lesions. Blank cells were also set as controls. Serial subculturing was performed according to the above method until obvious plaque lesions appeared in 20 passages, while cells of the negative control group were normal. And after blind transmission, picking out the virus liquid with obvious lesions, sub-packaging, and freezing at-80 ℃ to obtain the separated strain.

And further carrying out exogenous pathogen detection on the obtained isolated strain. And (3) blind transferring the separated strain to 100 generations on Vero cells, and detecting whether mycoplasma, transmissible gastroenteritis viruses of pigs, rotaviruses of pigs and blue ear viruses are mixed in the separated strain by a PCR method.

As a result, as shown in FIGS. 1 and 2, no mycoplasma, transmissible gastroenteritis virus, porcine rotavirus and blue ear virus were detected in the 90 th, 95 th and 100 th generation secondary cells of the isolated strain of the present invention.

3. Identification of viruses

(1) PCR identification

The isolated strain was passaged on Vero cells for 10 passages, and in the process, cell cultures of

passages

2, 4, 6, 8, and 10 were selected to obtain 250. Mu.L each, and RNA was extracted using Total RNA Kit II Kit (OMEGA). The extracted RNA is subjected to reverse transcription, and the reaction system is as follows: 6 mu L of RNA was mixed with 1 mu L of Oligo (dT) uniformly, and cooled at 70℃for 10min. To this mixture was added 2. Mu.L of 5 XBuffer, 0.5. Mu.L of 10mM dNTP mix, 0.25. Mu.L of MLV reverse transcriptase and 0.25. Mu. L RNase Inhibitor. After mixing evenly, the mixture reacts according to the following conditions: 42 ℃ for 60min;70 ℃ for 15min. The cDNA after reverse transcription was PCR amplified using the following primers.

Identification primers

Upstream primer NF:5'-CGGTTCTCACAGATAGTGAG-3';

downstream primer NR:5'-CGCTAGAAAAACACTCAGTAAT-3'.

The reaction system is as follows: 1. Mu.L of cDNA, 12.5. Mu.L of 2 XTaq Mix, 0.5. Mu.L of each of 10. Mu.M/mL upstream and downstream primers, and 25. Mu.L of deionized water were added thereto and mixed uniformly. The PCR conditions were as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 2min, and reaction for 30 cycles; extending at 72℃for 10min. After the PCR was completed, the PCR amplification product was subjected to 1% agarose gel electrophoresis.

As a result, as shown in FIG. 3, the isolated strain of the present invention detected specific positive bands from the cell culture samples of different generations starting from the generation 2 up to the generation 10. Thus, the isolated strain was described as porcine epidemic diarrhea virus.

The isolated strains were serially passaged, and the cell cultures of

passages

2, 4, 6, 8 and 10 were selected for PCR identification. As shown in FIG. 5, the

generation

2, 4, 6, 8 and 10 cell culture supernatants were all positive for PCR amplification.

(2) Identification of S protein

Extracting genomic RNA of the isolated strain; and performing reverse transcription and PCR amplification (RNA extraction and reverse transcription system and reaction conditions are the same as in the step 2). The cDNA after reverse transcription was subjected to PCR using the following primers: s gene is amplified in a segmented way.

Primer S1F:5'-TAGTGATGTTGTGTTAG-3';

primer S1R:5'-CGCTGCACAGCAGCTC-3';

primer S2F:5'-CATACCAGAAGGTTTTAG-3';

primer S2R:5'-GTAATCAACTCACCCTT-3';

primer S3F:5'-TTACCCTGAGTTTGGTAGTG-3';

primer S3R:5'-TCCGTCTGTAGAGCAAGAT-3';

primer S4F:5'-CTTCACATGTATAGTGCGTCT-3';

primer S4R:5'-CAGACTTTGAGACATCTTTGAC-3'.

Sequencing results show that the amino acid sequence encoded by the S gene of the isolated strain is SEQ ID NO. 1, and the specific sequence is as follows:

MTPLIYFWLFLPVLLTLSLPQDVTRCQSTINFRRFFSKFNVQAPAVVVLGGYL

PSMNSSSWYCGTGIETDSGVHGIFLSYIDSGQGFEIGISQEPFDPSGYQLYLHKATN

GNTSAIARLRICQFPDNKTLGPTVNDVTTGRNCLSNKAIPALQDGKNIFIGITWDN

DRVTVFADKIYHFYIKNDWSRVATRCYNKRSCAMQYVYTPTYYMLNVTSAGED

GIYYEPCTANCSGYAANVFATDSNGHIPEGFSFNNWFLLSNDSTTAFGKVVSNQPL

LVNCLWAIPRIYGLGQFFSFNQTMDGVCNGAAAQRAPEALRFNINDTFVILAEGSI

VLHTALGTNLSFVCSNSSDPHKAIFTIPLGVTEVPYYCFLKVDTYKSTVYKFLAVL

PPTVKEIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTVASTNF

VDALIEVQGTAIQRILYCDDPVSQLKCSQVSFDLDDGFYPISSRNLLSHEQPISFVT

LPSFNDHSFVNITVSAAFGGHSGANLIASDTTINGFSSFCVDTRQFTITLFYNVTNS

YGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLAGACTIDLFGYPEFGSGVKFT

SLYFQFTKGELITGTPKPLQGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAG

VYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSNSTFNNTR

ELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPLQDGQVKIAPMVTGNISIP

TNFSMSIRTEYLQLYNTPVSVDCVTYVCNGNSRCKQLLTQYTAACKTIESALQLS

ARLESVEVNSMLTISEEALQLATISSFNGDGYNFTNVLGVSVYDPASGRVVQKGSF

IEDLLFNKVVTNGLGTVDEDYKRCSNGRSVADLVCAQYYSGVMVLPGVVDAEK

LHMYSASLIGGMALGGLTTAAALPFSHAVQARLNYLALQTDVLQRNQQLLAESF

NSAIGNITSAFESVKEAISQTSNGLNTVAHALTKVQEVVNSQGSALTQLTIQLQHN

FQAISSSIDDIYSRLDILSADVQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQ

QKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVNVIAIDG

LCVNGDIALTLREPGLVLFTHELQTYTATEYLVSSRRMFEPRKPTVSDFVQIESCV

VTYVNLTSDQLPDVIPDYIDVNKTLDEILASLPNRIGPSLPLDVFNATYLNLTGEIA

DLEQRSESLRNTTEELRSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFV

VSLLVFCCISTGCCGCCGCCGACFSGCCRGPRLQPYEAFEKVHVQ。

amino acid homology analysis was further performed on the S protein sequences of SEQ ID NO. 1 and other porcine epidemic diarrhea virus strains (FIG. 4), and homology comparison results are shown in Table 1.

Table 1: pig epidemic diarrhea virus S protein homology comparison table

As can be seen from FIG. 4, the isolate belongs to group G1. Thus, the isolated strain of the invention is a porcine epidemic diarrhea virus subtype G1 epidemic strain.

As shown in Table 1, the homology of the S protein sequence of the isolated strain with the classical strain of porcine epidemic diarrhea virus CV777 was 99.6%. The comparison result shows that: compared with CV777 strain, the neutralizing epitope of the isolated strain S protein COE has the following mutation: the 159 th amino acid is mutated from V to F, the 169 th amino acid is mutated from V to I, the 264 th amino acid is mutated from L to T, the 265 th amino acid is mutated from L to A, the 266 th amino acid is mutated from H to F, and the 285 th amino acid is mutated from R to K.

(3) Electron microscope observation and identification

After the isolated strain is infected with the Vero cell monolayer, the cells are scraped and collected by the cells at 24h, 36h and 48h respectively, and after centrifugation at 800r/min, the cells are collected and fixed by a 2.5% glutaraldehyde electron microscope fixing solution, ultrathin sections are manufactured and stained by uranium acetate and lead citrate, and the virus morphology is observed by an electron microscope.

As a result, typical Porcine Epidemic Diarrhea Virus (PEDV) particles were observed in Vero cells infected with the isolated strain, as shown in FIG. 5. Thus, the isolated strain of the invention is porcine epidemic diarrhea virus and can be stably proliferated in Vero cells.

(4) Indirect immunofluorescence assay

Vero cells were cultured using 12-well plates, grown to a monolayer, and inoculated with isolated strain 5 th generation virus cultures, while non-virus-receiving Vero cells were established as negative controls. After 36h incubation, cytopathic effect was observed under a microscope, the culture broth was gently aspirated, and the cells were washed 3 times with 4℃pre-chilled PBS. 500. Mu.L of pre-chilled acetone at 20 ℃ is added to each well, and the culture plate is fixed in a refrigerator at 4 ℃ for 15min. The PBS was washed 3 times for 5min each. Each well was blocked for 30min with 500 μl of 5% nonfat dry milk. The skim milk powder was discarded and gently washed with PBS 2 times for 3min each. mu.L of each of the appropriately diluted primary antibodies (mouse anti-PEDV 6C8 monoclonal antibodies (BioNote, seoul, korea)) was added to Kong Nadi, and the mixture was allowed to act at 37℃for 1 hour. Washing with PBS for 5min for 3 times; FITC (fluorescein isothiocyanate) labeled goat anti-mouse IgG secondary antibody (1:20000) was added dropwise, and the mixture was allowed to act at 37℃for 30min, and washed with PBS 3 times for 5min each. 500 μLPBS was added to each well and observed under an inverted fluorescence microscope and photographed. In the experimental process, negative control which only adds primary antibody and secondary antibody and only adds secondary antibody and secondary antibody is needed to be set.

The results are shown in FIG. 6, where none of the negative control wells were fluorescent; the positive control wells and the wells inoculated with the isolated strain all exhibit fluorescence in the cell plasma; negative control wells with primary only and secondary only had no fluorescent background. Thus, the isolated strain of the invention is porcine epidemic diarrhea virus, which has good antigenicity.

(5) Effect of passaging on viral virulence

Use of viral titre TCID ₅₀ The detection method detects the effect of serial passage on the titer of the isolated strain: the titer of the isolated strain is determined by a microtitration method, which is specifically as follows: the virus titer was determined using Vero cells. Vero cells were cultured in 96-well cell culture plates and isolated strains were isolated from 10 with serum-free DMEM ^-1 Dilution of the ratio to 10 ^-10 100. Mu.L of each dilution of virus solution was inoculated into culture wells filled with Vero cells, respectively, 8 wells were inoculated for each dilution, and negative cell control wells were set. Culturing in a 5% CO2 incubator at 37 ℃ for 4 days, observing the virus infection condition of cells, and calculating the virus titer. The results show that the virus values of the cultured cell cultures of the 2 nd generation, the 4 th generation, the 6 th generation, the 8 th generation and the 10 th generation can reach 10 ^8.5 TCID ₅₀ And/ml or more.

The above results indicate that passage did not affect virulence of the isolated strains of the invention.

(6) Regression experiments on animals

The porcine epidemic diarrhea virus neutralizing antibodies are all smaller than 1:8 of 3-day-old piglets produced by sows, and each of the piglets is orally taken with the 5 th generation 10 of the isolated strain ³ TCID(s) ₅₀ Observation for 7 days, statistical inoculationAnd (3) the disease condition of the pigs, and the experimental pigs are subjected to section inspection to observe pathological changes. The diseased porcine intestinal mucosa and contents were taken, RNA was extracted, RT-PCR was performed as described in example 1, and the PCR products were sequenced.

The results show that the incidence rate of all 5 piglets is 100%, the incidence rate of the piglets is 100%, the affected pigs are diarrhea, dehydration and vomiting of individual pigs, and typical pathological changes of the stomach and the small intestine are observed through section inspection. A1326 bp fragment can be amplified from intestinal mucosa and contents of the diseased pigs, and the fragment is analyzed by a sequence to obtain the specific gene of the isolated strain.

In summary, the isolated strain obtained by the invention is porcine epidemic diarrhea virus, named porcine epidemic diarrhea virus PEDV-AQ strain, and is preserved in China center for type culture Collection (CCTCC NO: V202164) of university of Wuhan, china, 8 months and 5 days in 2021.

Example 2 preparation and testing of porcine epidemic diarrhea Virus PEDV-AQ strain inactivated vaccine

The Vero cell freezing tube is taken out from the liquid nitrogen tank and immediately put into a water bath with the temperature of 37 ℃ for melting. The cell suspension was added to a centrifuge tube of 5-10ml DMEM solution and centrifuged at 800r/min for 5 minutes. The supernatant was discarded, and cells were suspended in DMEM solution containing 10% serum, added to a cell culture flask, and incubated at 37 ℃. After growing a good monolayer of Vero cells, digesting the monolayer with pancreatin solution, and carrying out cell subculture. After continuous passage, the Vero cells with the growth density of 100% are discarded from the culture medium, and the cell maintenance solution of the epidemic diarrhea virus PEDV-AQ strain is added according to the volume ratio of 1% for culture at 37 ℃. After detoxification, cytopathic effect was observed daily, and when cytopathic effect was 80% or more, cell cultures were harvested 3 times by freeze thawing. The titer of the porcine epidemic diarrhea virus PEDV-AQ strain virus liquid obtained by inspection can reach 10 ^8.5 TCID ₅₀ /ml。

The virus solution of the porcine epidemic diarrhea virus PEDV-AQ strain obtained by culturing is added into formaldehyde solution according to the volume ratio of 0.1 percent, and stirred for 30 minutes. The virus solution containing 0.1% formaldehyde was put into another inactivation vessel and inactivated at 37 ℃ for 12 hours, during which it was stirred every 3-6 hours for 60 minutes. The inactivated virus liquid is preserved at 2-8deg.C. And (3) sampling the inactivated virus liquid, and checking according to the annex of the current Chinese animal pharmacopoeia to determine the sterile growth.

The inactivated virus solution was diluted 10 times with the maintenance solution and inoculated into Vero cells in 1ml per bottle. Adsorbing at 37deg.C for 2 hr, discarding the inoculating solution, adding cell maintenance solution, and setting Vero cell control without virus. Culturing at 37deg.C for 5 days without cytopathy. The disease is no cytopathy after 3 generations of blind transmission in the above way.

Uniformly mixing the virus liquid which is qualified in the inactivation test with an equivalent amount of 20% of inactivated aluminum hydroxide brine diluent, precipitating for 24 hours at room temperature, sucking out more than 2/5 supernatant, concentrating to 3/5 according to the total amount, adding 1/1.5 ten thousand of merthiolate solution, and fully mixing to obtain the PEDV-AQ strain inactivated vaccine.

EXAMPLE 3 examination of inactivated vaccine against PEDV-AQ strain

1. Inspection of PEDV-AQ strain inactivated vaccine

The inactivated vaccine of the porcine epidemic diarrhea virus PEDV-AQ strain prepared in example 2 is tested by referring to the related test method of the current Chinese animal pharmacopoeia. The inactivated vaccine is subjected to sterile test and other character test according to the current annex method of Chinese animal pharmacopoeia.

2. Safety test of PEDV-AQ strain inactivated vaccine

The test selects the PEDV-AQ strain inactivated vaccine which is qualified in test and prepared by the invention.

(1) Safety test of piglets

Cervical muscle vaccinates 5 piglets at 8-10 day age negative for PEDV antigen and antibody, at 15 day intervals. The first immunization and the second immunization were performed with a double dose of 4 ml/head, and the piglets were observed for 14 days after the second immunization, and the inoculation sites and clinical symptoms of the piglets were observed. And meanwhile, the control group 2 heads are set up without any treatment. The test results are shown in Table 2.

Table 2: double dose safety test table (piglet) for PEDV-AQ strain inactivated vaccine

After the immunization group 5 piglets are inoculated with the PEDV-AQ strain inactivated vaccine prepared by the invention, the continuous observation is carried out for 14 days, the spirit and diet are normal, the body temperature is not increased, and the diarrhea-related clinical symptoms are not seen. Cutting off and killing after 14 days of the second time, taking an inoculation part for pathological section observation, and ensuring that no abnormality is found; the vaccine is safe for piglets.

(2) Safety test of sow

Neck muscle vaccinates 5 pregnant sows negative for PEDV antigen and antibody, first immunization at 6 weeks before birth, second immunization at 3 weeks before birth, first immunization and second immunization at double dose of 4 ml/head. And (5) observing the condition of the inoculated part, and counting the number and the quality of the farrowing of the sow. While the control group 2 head was set up without any treatment. The test results are shown in Table 3.

Table 3: PEDV-AQ strain inactivated vaccine double dose safety test table (sow)

After 5 pregnant sows are inoculated with the PEDV-AQ strain inactivated vaccine prepared by the invention, no obvious clinical symptoms are seen, and the inoculated part has no abnormal condition. Sow delivery was observed, and both immunized and control piglets had no weaning, abortion and stillbirth. The average parity of the immunized group and the control group is not obviously different, which indicates that the vaccine is safe for the sow.

Example 4: immune efficacy comparison test of porcine epidemic diarrhea virus PEDV-AQ strain inactivated vaccine

1. Piglet immunopotency test of PEDV-AQ strain inactivated vaccine

7 PEDV antigens and 8-10 day old piglets negative for antibodies were selected for the test. Randomly dividing piglets into 2 groups, wherein the first group is PEDV-AQ strain inactivated vaccine immunization group, and the second group is 5-head and neck muscle inoculation, and the first group is 2 ml/head; the second group is a blank group, 2 heads, without any treatment. The second immunization is carried out 15 days after the first immunization, the inflammation condition of the inoculated part and the clinical symptoms of piglets are observed after the immunization, blood is collected every week after 2 weeks of the second immunization, and the neutralizing antibody titer of PEDV in serum is detected. The results are shown in Table 4.

Table 4: results table of neutralizing antibodies after immunization of piglets with PEDV-AQ strain inactivated vaccine

No inflammatory reaction such as red swelling, hard lumps, crusting and the like is seen at the vaccinated part of the immunized group piglet, and no obvious abnormal clinical symptoms are seen. The result of neutralizing antibody of PEDV in serum shows that the neutralizing antibody of PEDV-AQ strain inactivated vaccine immune group is larger than 1:32 in the 2 nd week after the immunization, reaches the highest peak in the 4 th week after the immunization, and gradually descends after the fifth week after the immunization. The test result shows that the PEDV-AQ strain inactivated vaccine has good immune efficacy and long immune duration for 8-10-day-old piglets.

2. Sow immunopotency test of PEDV-AQ strain inactivated vaccine

11 replacement gilts negative for PEDV antigen and antibody were selected for this test. Randomly dividing the backup sows into 3 groups, wherein the first group is vaccine 1 immunization group, 5 head and neck muscle inoculation, and 2 ml/head; the second group is vaccine 2 immunization group, 5 head, neck muscle inoculation, 2 ml/head; the third group is a blank group, 2 heads, without any treatment. The first 4 weeks before the birth, the second 4 weeks before the birth, the inflammation condition of the inoculated part and the clinical symptoms of the sow are observed after the immunization, blood is collected weekly after 2 weeks after the second immunization, and the titer of the PEDV neutralizing antibodies in serum is detected. The results are shown in Table 5.

Table 5: results table of neutralizing antibodies after immunization of replacement gilts with PEDV-AQ strain inactivated vaccine

The vaccinated parts of the immunized group sows have no inflammatory reaction such as red swelling, hard lumps, crusting and the like, and have no obvious abnormal clinical symptoms. The result of neutralizing antibodies of PEDV in serum shows that the neutralizing antibodies of all sows are larger than 1:32 at the 2 nd week after the immunization of the PEDV-AQ strain inactivated vaccine group, reach the highest peak at the 4 th week after the immunization, and gradually decline after the fifth week after the immunization. The test result shows that the PEDV-AQ strain can generate higher neutralizing antibodies after being used for inoculating the sow with the inactivated vaccine, can be maintained at a relatively higher level, and has good immune efficacy.

Example 5 comparative toxicity protection test of inactivated vaccine against PEDV-AQ strain

The PEDV antibody antigen was negative for 4 healthy sows of the same birth period. Wherein 2 sows are inoculated with the PEDV-AQ strain inactivated vaccine prepared by the invention, and 2 sows are inoculated with the commercial Porcine Epidemic Diarrhea Virus (PEDV) inactivated vaccine. The first immunization was performed for all 4 sows 7 weeks before the birth, the neck muscle was inoculated, 2 ml/head, and the second immunization was performed in the same manner for 4 weeks before the birth.

After the production of the PEDV-AQ strain inactivated vaccine and the PEDV commercial vaccine immunized sow, 5 piglets with good states are selected for the toxicity test. The piglets are all fed by breast milk. At 7 days of age, the piglets were orally challenged (0.5×)

10 ^3.0 TCID ₅₀ Per ml,2 ml/head), the grouping of piglets and the toxicity attack are shown in Table 6.

Table 6: comparison result table of toxicity attack protection after immunization of piglet with PEDV inactivated vaccine

As shown in the results of Table 6, compared with the commercial PEDV inactivated vaccine, the PEDV-AQ strain inactivated vaccine provided by the invention has better protection performance on the attack of epidemic strains. The piglets produced by the sow immunized by the PEDV-AQ strain inactivated vaccine are protected after being challenged, and the attack protection rate of the commercial PEDV inactivated vaccine on epidemic strains is only 2/5. Therefore, the PEDV-AQ strain inactivated vaccine prepared by the invention has better immune protection effect on PEDV-AQ strain.

Example 6 clinical trials of porcine epidemic diarrhea Virus PEDV-AQ strain inactivated vaccine

In the sample separation area, experiments are carried out on large-scale pig farms with porcine epidemic diarrhea virus infection, each pig farm is divided into 40 healthy pregnant sows with the same birth period, the healthy pregnant sows are randomly divided into 1, 2, 3 and 4 groups, immunization is respectively carried out according to the method shown in table 7, average diarrhea rate and average death rate of piglets produced by sows in each pig farm are counted after immunization, and the protection condition of different porcine epidemic diarrhea virus vaccines on the piglets is compared, wherein the specific results are shown in table 8.

Table 7: pregnant sow immunization schedule table

/>

Table 8: piglet diarrhea rate and mortality statistics table for pregnant sow production after immunization

From the statistical results in tables 7 and 8, the average diarrhea rate of piglets produced by 1 group pregnant sows immunized by the porcine epidemic diarrhea virus PEDV-AQ strain inactivated vaccine of the invention is only 12%, the average death rate is only 8%, and the average diarrhea rate of piglets is far lower than that of piglets produced by 2, 3 and 4 group pregnant sows immunized by injecting commercial vaccine. Therefore, the inactivated vaccine prepared from the porcine epidemic diarrhea virus PEDV-AQ strain obtained by separation can obviously reduce diarrhea rate and death rate of piglets, and has a better protective effect on piglets than that of commercially available vaccines.

Claims

1. A porcine epidemic diarrhea virus is characterized in that the preservation number of the virus is CCTCCNO: V202164.

2. The porcine epidemic diarrhea virus according to claim 1, wherein the amino acid sequence of the S protein of the virus is SEQ ID NO. 1.

3. Use of the porcine epidemic diarrhea virus of claim 1 in the manufacture of a vaccine.

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

5. An inactivated vaccine, wherein the antigen in the inactivated vaccine is the porcine epidemic diarrhea virus of claim 1.

6. The inactivated vaccine of claim 5, wherein said inactivation is performed using formaldehyde.

7. The inactivated vaccine of claim 5, wherein said inactivated vaccine further comprises a vaccine strain of another virus.

8. Use of the porcine epidemic diarrhea virus of claim 1 for the preparation of a neutralizing antibody.