CN107184969B

CN107184969B - A-type cenecar valley virus inactivated vaccine and preparation method and application thereof

Info

Publication number: CN107184969B
Application number: CN201710254431.4A
Authority: CN
Inventors: 董金杰; 田波; 吕宏亮; 刘萍; 陈苗苗; 邓瑞雪; 张涛; 王会宝; 王凡; 刘西兰; 景志忠; 高世杰; 王超英; 张云德; 殷宏; 张永光
Original assignee: China Agricultural Vet Bio Science And Technology Co ltd
Current assignee: China Agricultural Vet Bio Science And Technology Co ltd
Priority date: 2017-04-18
Filing date: 2017-04-18
Publication date: 2020-07-10
Anticipated expiration: 2037-04-18
Also published as: CN107184969A

Abstract

The invention discloses an A-type cenecar valley virus inactivated vaccine, and a preparation method and application thereof. The A-type cenecar valley virus inactivated vaccine contains an inactivated swine A-type cenecar valley virus whole virus particle antigen and an adjuvant, wherein the swine A-type cenecar valley virus whole virus particle antigen is obtained by virus rescue of a swine A-type cenecar valley virus full-length infectious cDNA clone, and the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID NO. 1. The inactivated porcine A-type Seneca valley virus complete virus particle antigen is compatible with veterinary acceptable adjuvants, particularly the veterinary acceptable polylysine hydroxymethyl cellulose polyinosinic cell, so that the immunogenicity and effect of the inactivated complete virus vaccine can be improved, complete protection can be realized by one part, and the protection duration is 1 year. Compared with subunit and virus-like particle vaccines, the vaccine has low production cost and is safer compared with attenuated live vaccines.

Description

A-type cenecar valley virus inactivated vaccine and preparation method and application thereof

Technical Field

The invention relates to an inactivated vaccine of A-type Seneca valley virus, a preparation method and application thereof, and also relates to a full-length infectious cDNA clone of pig A-type Seneca virus and pig A-type Seneca virus obtained by the rescue of the full-length infectious cDNA clone. The invention belongs to the technical field of biological medicines.

Background

Seneca Valley virus a (SVA) type a is the only member of the genus Seneca Valley virus of the picornaviridae family, and can cause severe vesiculopathy in sows and death in newborn piglets, possibly transmitted through the pig urine. In 2014 and 2015, the U.S. and Brazilian sows suffer from vesiculosis and the piglets die and outbreak epidemic, which causes huge economic loss.

The key point of preventing and controlling the Seneca valley virus lies in preparing the Seneca valley virus inactivated vaccine, the preparation method of the traditional Seneca valley virus inactivated vaccine is that the pig A-type Seneca valley virus is adopted to proliferate in the diploid cells of the pig testis or the passage cells of the pig kidney, and then the inactivated vaccine is prepared, but the virus has the problems of slow growth, low titer and the like in the proliferation process, and the cells have endogenous virus pollution, thus greatly limiting the large-scale industrial production.

Therefore, the invention provides the full-length infectious clone of the swine A-type Seneca valley virus, overcomes the problems of slow growth and low titer of the swine A-type Seneca valley virus on a swine testicular diploid cell or a swine kidney passage cell, rescues the continuous passage of the obtained A-type Seneca valley virus on MRC-5 and BHK-21 cells, purifies the virus, improves the growth performance of the virus, and provides an effective technical means for the prevention and control of Seneca valley virus infection.

Disclosure of Invention

The invention aims to solve the technical problem of providing an A-type cenecar valley virus inactivated vaccine as well as a preparation method and application thereof. The vaccine can enable pigs to generate protective immunity against Seneca valley virus infection and diseases caused by Seneca valley virus.

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

the A-type cenecar valley virus inactivated vaccine contains an inactivated swine A-type cenecar valley virus whole virus particle antigen and an adjuvant, wherein the swine A-type cenecar valley virus whole virus particle antigen is obtained by virus rescue of a swine A-type cenecar valley virus full-length infectious cDNA clone, and the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID NO. 1.

In the present invention, it is preferable that the adjuvant is a phosphate buffer containing 20-50 g/L polylysine hydroxymethylcellulose polyinosinic cells (Poly IC L C), 0.1-0.5 g/L EDTA and 0.01-0.1 g/L Tween-20, and the pH is adjusted to 8.0, and it is preferable that the adjuvant is a 50mM phosphate buffer containing 25 g/L polylysine hydroxymethylcellulose polyinosinic cells (Poly IC L C), 0.2 g/L EDTA and 0.05 g/L Tween-20, and the pH is adjusted to 8.0.

Furthermore, the invention also provides a method for preparing the inactivated vaccine of the A-type Seneca valley virus, which comprises the following steps:

1) preparation of porcine A-type seneca virus whole virus particle antigen

(1) Rescue of porcine type a seneca valley virus

Constructing an expression vector containing a full-length infectious cDNA clone of the porcine Seneca valley virus A, wherein the cDNA clone has a nucleotide sequence shown in SED ID NO.1, preferably, the expression vector is constructed by cloning the nucleotide sequence shown in SED ID NO.1 into a pSV L vector;

(2) culturing porcine kidney cell line IBRS-2 in MEM (minimum essential medium) containing 10v/v% fetal calf serum until the cell line IBRS-2 is 70-90% confluent, transfecting the expression vector constructed in the step (1) with IBRS-2 at 37 ℃ and 5% CO₂Culturing in an incubator, transferring the supernatant after culturing for 48 hours to ST cells for continuous culture, observing cytopathic effect, and harvesting virus when obvious cytopathic effect appears after infection, namely the rescued porcine A-type Seneca valley virus;

(3) passage adaptation of viruses

The rescued pig A-type seneca valley virus is subjected to subculture of ST cells and MRC-5 cells to obtain a pig A-type seneca valley virus adapting to the MRC-5 cells, and then BHK-21 cells are inoculated for subculture to obtain a virus culture solution of the pig A-type seneca valley virus adapting to BHK-21 cell culture;

(4) clarification

Filtering the virus culture solution by using a polypropylene prefilter with the particle size of 8 mu m, filtering and clarifying by using filters with the particle sizes of 0.45 mu m and 0.2 mu m, and determining the virus titer;

(5) ultrafiltration dialysis

The clear virus liquid is ultrafiltered and dialyzed by a 100KD membrane;

(6) purification of

Adding the virus solution after dialysisInto MgCl₂Setting the final concentration to be 2mM, adding nuclease for 2 hours according to 15U/ml, centrifuging, carrying out discontinuous cesium chloride density gradient centrifugation, then carrying out continuous cesium chloride density gradient centrifugation, collecting a grating zone after each step of centrifugation is finished, and dialyzing by 200mM Tris-HCl cold dialysis buffer solution containing 10v/v% of glycerol and 50mM HEPES pH 8.0;

(7) inactivating

Diluting the purified virus with RPMI 1640, inactivating with bisethyleneimine, and neutralizing with sodium thiophosphate;

(8) dialysis

Concentrating to 1/6 with original volume by using a 100KDa membrane, and dialyzing by using PBS buffer solution containing 150mM NaCl to obtain the A-type Seneca valley virus whole virus particle antigen;

2) preparation of adjuvants

Adding polylysine hydroxymethyl cellulose polyinosinic cell (Poly IC L C), EDTA and Tween-20 into phosphate buffer solution to make the final concentration reach 20-50 g/L, 0.1-0.5 g/L and 0.01-0.1 g/L respectively, and adjusting the pH value to 8.0, preferably adding polylysine hydroxymethyl cellulose polyinosinic cell (Poly IC L C), EDTA and Tween-20 into 50mM phosphate buffer solution to make the final concentration reach 25 g/L, 0.2 g/L and 0.05 g/L respectively, and adjusting the pH value to 8.0;

3) vaccine preparation

Adding the obtained A-type Seneca valley virus whole virus particle antigen into the prepared adjuvant to make the amount of virus protein reach 5-10 μ g/m L, and subpackaging according to 0.5-1.0 ml/head.

In the method of the present invention, preferably, the concentration of cesium chloride used in the discontinuous cesium chloride density gradient centrifugation is 1.24g/ml and 1.4g/ml, and the concentration of cesium chloride used in the continuous cesium chloride density gradient centrifugation is 1.33 g/ml.

The primary purpose of discontinuous cesium chloride density gradient centrifugation is to remove the major cellular contaminants and defective viral particles. The main purpose of continuous cesium chloride density gradient centrifugation is to separate infectious and defective viral particles.

Still further, the invention also provides application of the A-type cenecarbal virus inactivated vaccine in preparation of a medicament for preventing and treating diseases caused by A-type cenecarbal virus infection.

The full-length infectious cDNA clone of the porcine Seneca virus A has a nucleotide sequence shown in SEQ ID No. 1.

ZNFT-01-2015 cDNA clone is constructed as shown in FIG. 1, the 5 ' end of the genome is SV40 promoter, the full length of the genome is 7293nt, the 3 ' end of PolyA contains 11nt, compared with gene bank SVA (gene bank accession No. KC667560.1), the 5 ' end contains additional T, the 12 site of the 5 ' UTR region generates T-to-C mutation, the 7236 site generates T-to-C mutation, and the 3 ' UTR region 7265 site generates C-to-T mutation. In the 2C region, a SacI restriction site is introduced, 4216 introduces a C to T mutation, and 4216 a mutation from C to T is a silent mutation without changing the coding amino acid sequence.

An expression vector contains the full-length infectious cDNA clone of the porcine Seneca virus A, preferably, the expression vector is constructed by cloning the nucleotide sequence shown in SED ID No.1 into pSV L vector.

Furthermore, the invention also provides the full-length infectious cDNA clone of the porcine type A cenecar valley virus and the application of the expression vector in the rescue of the porcine type A cenecar valley virus.

A method of rescuing porcine savirus type a comprising the steps of:

(1) constructing an expression vector containing a full-length infectious cDNA clone of the porcine Seneca valley virus A, wherein the cDNA clone has a nucleotide sequence shown in SED ID No.1, and preferably, the expression vector is constructed by cloning the nucleotide sequence shown in SED ID No.1 into a pSV L vector;

(2) culturing porcine kidney cell line IBRS-2 in MEM (minimum essential medium) containing 10v/v% fetal calf serum until the cell line IBRS-2 is 70-90% confluent, transfecting the expression vector constructed in the step (1) with IBRS-2 at 37 ℃ and 5% CO₂Culturing in incubator, transferring supernatant after 48 hr culture to ST cell, continuously culturing, observing cytopathic effect, and harvesting virus when obvious cytopathic effect appears after infection, i.e. porcine type A Seneca valley virus。

Furthermore, the invention also provides the porcine Seneca valley virus A prepared by the method. And

the application of the porcine A-type cenecar valley virus in preparing a reagent for detecting a neutralizing antibody of the porcine A-type cenecar valley virus and preparing an inactivated vaccine of the A-type cenecar valley virus is provided.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a porcine type A cenecar valley virus full-length infectious cDNA clone, which solves the problems of slow growth and low titer of the porcine type A cenecar valley virus on porcine testicular diploid cells or porcine kidney passage cells;

2. the rescued pig A-type seneca valley virus is subjected to subculture of ST cells and MRC-5 cells to obtain the pig A-type seneca valley virus adaptive to the MRC-5 cells, and then the pig A-type seneca valley virus is inoculated to BHK-21 cells for subculture, so that the virus is purified and the growth of the virus is improved. The BHK-12 cells are standard vaccine substrates of animal vaccines, are easy to produce in a large scale and have controllable vaccine quality.

2. Polylysine hydroxymethyl cellulose polyinosinic cells are adopted as an adjuvant to prepare the inactivated vaccine, 1 part of the inactivated vaccine is efficient in immunity, completely protected and durable in immunity, complete protection can be achieved by one part of the inactivated vaccine, and the protection duration is 1 year. Compared with subunit and virus-like particle vaccines, the production cost is low, and compared with attenuated live vaccines, the vaccine is safe.

Drawings

FIG. 1 is a schematic diagram of construction of ZNFT-01-2015 full-length infectious clone.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 cloning and rescue of Seneca Valley Virus type A

1. Materials and methods

1.1. Cell and virus culture

MEM minimal medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100mg/ml streptomycin for ST, PK-15, IBRS-2 cell culture was purchased from Gibco, USA, at 37 deg.C and 5% CO 2.

1.2 construction and cloning

RT-PCR of FMDV and SVDV was used for detecting negative, SVA RT-PCR was used for detecting positive porcine vesicular fluid, total RNA was extracted with highly purified viral RNA kit (purchased from Roche Diagnostics, China), first strand cDNA was synthesized with random primers and transcription fidelity cDNA synthesis kit (Roche Diagnostics, China) to construct full length infectious cDNA clone, platinum Taq high fidelity polymerase (L domains Technologies, China) was used, 20152015 virus (Gene accession No. KC667560) sequence in the gene bank was used as template design primer (see Table 1), A-E5 fragments obtained by RT-PCR with unique restriction enzyme site primer were used, fragment A-E, poly (A) was inserted into pSV L vector (pharmacia Biotech, WI, USA 2015 20152015 with promoter 40 (USA plasmid named SphI/pSpSpSpScI plasmid), subsequent fragment B, fragment C, SaNEE fragment is inserted into BucSV 4251 promoter, cDNA clone A-4218 was inserted into DNA sequence, WT-4213 was inserted into DNA cloned into the DNA of the DNA clone DNA sequence named as cDNA clone No. 4218, WT-4213.

The 5 'and 3' ends of ZWT-01-2015 were sequenced using a GeneRacer core kit (purchased from Invitrogen, USA) and the sequencing primers are shown in Table 1.

TABLE 1 construction of full-Length cDNA cloning and viral genome sequencing primers

1.3. Rescue of virus

Pig kidney cellLine IBRS-2 was cultured to 70-90% confluency on a 12-well plate using a basal medium (MEM) containing 10v/v% fetal bovine serum (manufactured by GIBCO, USA), pSV L SD-01-2015 was transfected at 0.1-1000 ng/ml with L ipofectamine3000 (available from L ies Technologies) at 37 ℃ with 5% CO₂And (5) culturing in an incubator. The supernatant after 48 hours of transfection and culture was transferred to ST cells and cultured continuously, the cytopathic effect was observed every day, and the virus was harvested when a significant cytopathic effect appeared after infection (18-48 hours of infection), and named as VZWT-01-2015 clone.

1.4. Detection of viruses

The V ZNFT-01-2015 clone was passaged 2 times on ST cells, RNA was extracted using a QIAamp viral RNA min ikit kit (purchased from Qiagen, USA) as described, cDNA was synthesized using Superscript III reverse transcriptase (purchased from Invitrogen, USA), and PCR was performed using SVA-F3547/SVA-R5200 (Table 1). The PCR product was purified with gel and confirmed by digestion with SacI and sequencing.

1.5. Viral growth kinetics and plaque assay

PK-15 was cultured in 24-well plates and infected with V ZNFT-01-2015 clone at 100% confluence, with MOI 0.1. After 1 hour incubation the supernatant was removed, the cells were washed with MEM, fresh medium was added, the supernatant was harvested 6, 12, 24, 36, 48h after infection, and titration was performed on PK-15 cells to express viral titer as TCID 50/ml.

1.6 Swine infection test

A total of 8 pigs aged 3 weeks were randomized into 2 groups, 1 group (n ═ 5) was inoculated intranasally with 5ml of V ZNFT-01-2015 virus solution at a titer of 10⁸TCID₅₀And/ml, inoculating 2 groups (n ═ 3) of the basic culture solution. Clinical symptoms and temperature were observed daily for 14 days. Serum, oral fluid, nasal swabs, fecal swabs were collected at 0, 3, 7, 14 days post infection. The experiment was terminated on 14 days, and macroscopic pathology assessments were performed and tissue and skin samples were collected.

1.7 quantitative RT-PCR

Quantitative RT-PCR assessment of serum, oral fluid, nasal swab, fecal swab viral load Virus VZNFT-01-2015 genome was performed using a MagMAX-96viralRNA isolation kit (purchased from L ife Technologies, USA) according to the instructions provided, using Path-ID^TMMultiplex One-Step RT-PCR Kit (available from Applied Biosystems, USA) performs qRT-PCR on CFX96Touch Real-TimePCR detection system, with parameters: at 48 ℃ for 10min, at 95 ℃ for 10min, 45 cycles of 95 ℃ for 15s and 60 ℃ for 60s were carried out. Clone series 10-fold dilution to 10⁶TCID50/ml—1×10¹TCID50/ml, and a detection standard curve is established. The viral load TCID50/ml was determined from a standard curve.

1.8. Neutralizing antibody assay

Immunofluorescent-oven analysis was used, briefly described: the serum samples were inactivated by heating at 56 deg.C for 30min, diluted 2-fold with 2% horse serum in MEM, added to each well in 100. mu.l, and mixed with 100. mu.l of 200TCID₅₀The virus VZNWT-01-2015 was mixed, incubated at 37 ℃ for 1 hour, 150 microliters was added to 90-100% confluent PK-15 cells, incubated at 37 ℃ after 18 hours of infection, the cells were fixed with cold methanol at 20 ℃ for 30min, stained with specific VP2 affinity chromatography purified polyclonal antibody, AlexaFluors488affinipur sheep-rabbit IgG (H + L) antibody (purchased from Jackson Immuno Research Inc. usa) as the secondary antibody, diluted per well with EVOSF L cell imaging system (purchased from L if technologies.,) neutralizing antibody was the highest serum level capable of preventing 90% virus growth.

1.9 SVA-specific antibody response

After 100% confluency, infection with virus VZNFT-01-2015 at MOI 0.01, after infection for 14 hours, fixation with ice-cold methanol at 20 ℃ for 30min, pig serum was initially diluted 1:5 and added to the fixed cells for incubation at 37 ℃ for 1 hour, the cells were washed three times with PBS, FITC-conjugated secondary antibody mixture (goat anti-pig IgM, goat anti-pig IgA, goat anti-pig IgG) was diluted 1: 200 and incubated with the cells, PBS was washed 3 times, and the cells in each well were observed with EVOSF L cell imaging system (L iftech, USA).

2. Results

Construction of infectious cDNA clone of Selenecar valley virus type A

The cDNA sequencing result of infectious clone ZNFT-01-2015 of Seinivirus A is shown in SEQ ID NO.1, and the nucleotide homology with the GenBank SVA virus (GenBank accession No. KC667560) is 93.4%. cDNA clone construction of full-length infectious clone ZNFT-01-2015 is shown in FIG. 1, the 5 ' end of the genome is SV40 promoter, the full-length of the genome is 7293nt, the 3 ' end of PolyA contains 11nt, compared with the gene bank SVA (gene bank accession No. KC667560.1), the 5 ' end contains additional T, the 12 site of the 5 ' UTR region has T-C mutation, the 7236 site has T-C mutation, and the 3 ' UTR region 7265 site has C-T mutation. In the 2C region, a C to T mutation was introduced at the restriction site 4216 of SacI, and the mutation at the 4216 site from C to T was a silent mutation without changing the coding amino acid sequence.

In vitro recovery and viral characterization of SVA full-Length cDNA clone ZNFT-01-2015

IBRS-2 monolayers transfected with 1000ng/ml and 100ng/ml pSV L ZNFT-01-2015 exhibited cytopathic effects 2 days after transfection, 0.1-10 ng/ml of transfected IBRS-2 cells exhibited lesions 3 days after transfection, supernatant of transfected cells 48 hours after transfection was collected for passage on ST cells, 12 hours after infection, stained with polyclonal antibody purified by VP1 affinity chromatography, VP1 protein detectable in ST cells, supernatant of transfected cultures of IBRS-2 cells was passaged 2 times on ST cells, and cytopathic effects were exhibited on ST cells 18-48 hours after infection, viral titer reached 1 × 10⁸TCID₅₀And/ml. These results indicate that the virus VZNFT-01-2015 restored rescue from the full length infectious clone ZNFT-01-2015.

The SacI digestion and sequencing results show that: the SacI restriction site of the 2C region of 3551-5227 nt of the genome is inactivated. That is, the 1677bp fragment of the RT-PCR product of the 2C region was not digested with SacI.

VZNWT-01-2015 virus infected PK-15 cells at MOI ═ 0.01, harvested at 6, 12, 24, 36, 48h post infection, respectively. The viral titer was 10 at 36 hours after infection with peak viral titer^8.6TCID₅₀/ml。

2.3 Swine infection test

The V ZNFT-01-2015 virus infects nursery pigs, and 10 pigs at 3 weeks are divided into 2 groups. Group 1 was infected with clone virus VZNWT-01-2015(n ═ 5), and group 2 was mock-infected cell culture broth (n ═ 5). Group 1 4/5 pigs had clinical symptoms: respiratory distress, lethargy. 3/5 pigs developed high fever 6 days after infection, with anal temperature up to 105 ℉. 4/5 blisters appeared on the lips of the pigs and lasted for 3-4 days. 2/5 pigs were infected 7 days later, with obvious ulcerated lesions in the hind leg trotters and particularly in the crown. No clinical symptoms were observed in group 2. After 14 days of infection, 1 pig blister ulcer in 1 group healed, and the other 4 pigs had round erosive lesions and had limb lesions in the coronal zones. Dissection was performed after 14 days, and group 1, 2/5, pigs had enlarged mesenteric lymph nodes with mild lung injury in 3/5 pigs. There were no pathological phenomena observed by naked eyes in tonsil, heart, liver, spleen, kidney and intestine.

2.4 growth characteristics of VZNFT-01-2015 Virus in pigs

Serum, nasal swabs, oral fluid were collected from VZNWT-01-2015 virus infected pigs to determine whether the virus replicated and was toxin-expelling. In the 1 group of pigs, the qRT-PCR results of the serum samples of only 1 pig infected with 3 days and 7 days show that the virus RNA exists, the qRT-PCR results of 4/5 pig nose swabs are positive, and the qRT-PCR results of all pig oral fluid and excrement are positive. Viral RNA could not be detected in serum and nasal swab samples 14 days after infection. However, viral RNA, equivalent to 10, was detected in the oral fluid sample^1.0TCID₅₀And/ml. All samples of group 2 were tested for virus-free RNA by qRT-PCR.

Example 2 passage and Adaptation of porcine Seneca Valley Virus type A

1.1 materials

VZMWT-01-2015 is the virus cloned and rescued in example 1, and the characteristics of the cloned and rescued virus are shown in example 1. piglet 1-3 weeks old, SVA antibody E L ISA kit, ST cell, MRC-5 cell and BHK-21 cell are purchased from American ATCC.

1.2 passage and culture methods

75cm²ST cells are cultured in a square flask, a cell culture solution is a DMEM culture solution containing 10% v/v fetal calf serum, the culture is carried out at 37 ℃, 1ml of pig vesicular fluid which is detected as positive VZNFT-01-2015 by SVA antibody is inoculated after cell monolayer, the culture is carried out at 32 ℃ in a DMEM culture solution containing 2 v/v% fetal calf serum, and cytopathic effect (CPE) is observed every day. After 3 days, the cells were cultured80% slough off, collect virus fluid, inoculate 1ml to another 75cm monolayer of prepared ST cells²The virus liquid is stored in a square bottle and the rest virus liquid is stored in a refrigerator at minus 80 ℃ for standby. The passage was repeated 5 times, wherein VZNFT-01-2015 virus obtained by twice passage on ST cells was designated as an offending strain SVAA-ST-p 2.

MRC-5 cells at 75cm²The square bottle of (1) is full of a monolayer, the cell culture solution is DMEM culture solution containing 8-10 v/v% fetal calf serum, 1ml of VZNFT-01-2015 virus solution cultured and passaged by ST cells is inoculated according to 0.01-0.1MOI, cultured for 6-8 days at 37 ℃, and blind transferred for 2-3 generations, and virus with pathological changes on cells is separated. The separated virus is continuously transmitted to MRC-5 cells for multiple generations to generate a continuous and stable cytopathic effect, and VZWT-01-2015 virus adapting to MRC-5 cell culture is obtained.

BHK-21 cells at 75cm²The square bottle is full of a monolayer, a cell culture solution is a DMEM culture solution containing 8-10 v/v% fetal calf serum, 1ml of VZNFT-01-2015 virus solution suitable for MRC-5 cell culture is inoculated, the VZNFT-01-2015 virus solution is adsorbed at 32 ℃ for 4 hours, the VZNFT-2015 virus solution is cultured at 32 ℃, the VZNFT-01-2015 virus solution is subjected to liquid change and then is cultured, the VZNFT-2015 virus solution is subjected to primary passage. After 8-10 passages, BHK-21 cells begin to have cytopathic effect, continue passages, and perform titer determination to obtain continuous and stable cytopathic effect, thereby obtaining VZWT-01-2015 virus suitable for BHK-21 cell culture.

1.3 detection and identification method:

1.3.1 detection of Seneca Valley virus type A molecules:

a conserved sequence design primer of a D region of a genome ((DQ641257, KC667560, KR063107, KR063108, KR063109, KT757280, KT757281, KT757282, KT321458 and KX349733) disclosed by a gene library Sernica virus is SVA3D-F1: 5'-AGAATTTGGAAGCCATGCTCT-3' SVA3D-R1:5 '-GAGCCAACATAGARACAGATTGC-3' probe and SVV3D-Pr1:5 '-FAM-TTCAAACC AGGAACACTACTCGAGA-BHQ 1-3' is used for amplifying the sequence of the 3D region of the BHK-21 cell adaptive virus genome by real-time RT-PCR and sequencing.

1.3.2 detection of vesiculitis virus E L ISA, detection of porcine vesicular stomatitis E L ISA, and detection of porcine foot-and-mouth disease virus are carried out according to the kit instructions.

1.3.3 Gene stability testing:

the primer is designed by taking the cloned A-type Seikaga virus original virus genome sequence shown in SEQ ID NO.1 as a template. And (3) performing RT-PCR amplification to separate toxic and different generation cell adaptive toxic genome 2B-2C regions, and sequencing.

1.3.4 isolated virus whole genome sequencing:

after virus adaptation, RNA is extracted by concentration, primers are designed by taking the cloned A-type seneca virus original virus genome sequence shown in SEQ ID NO.1 as a template, and RT-PCR and sequencing are carried out.

2. Results

2.1 Virus passage and characterization

2.1.1 amplification of viruses in MRC-5 diploid cells

The VZNFT-01-2015 virus cultured and passaged by the ST cells is inoculated to a monolayer diploid MRC-5 cell according to 0.01-0.1MOI and transmitted to 8-10 generations, and the virus titer can reach 10^9.0logTCID₅₀And/ml. The primers are designed by taking the cloned A-type seneca virus original virus genome sequence 2B-2C interval shown in SEQ ID NO.1 as a template, and the RT-PCR amplification is carried out on P10 generation adaptive virus on MRC-5, the result is positive, and the sequence sequencing of the amplified fragment and the homology of the isolated virus are 100%. The real-time PCR detection of the P103D area on the type A Seika virus MRC-5 is positive.

2.1.2 passaging and Adaptation of viruses on BHK-21 cells

Using 75cm²After culturing a BHK-21 cell monolayer in the square flask, washing the cell monolayer for 3 times by using DMEM culture solution without fetal calf serum, inoculating A-type Seneca valley virus VZNFT-01-20151 ml adapted to the diploid MRC-5 cell, adsorbing the cell by 32 ℃ for 4 hours, culturing the cell at 32 ℃ after changing the solution, carrying out passage once for 6 weeks at first, and carrying out freeze-thaw ultrasound on the cell at each passage. After 8-10 passages, BHK-21 cells begin to have cytopathic effect, continue passages, and carry out titer determination until the virus titer reaches the maximum and maintains for 10 passages. The final virus titer reaches 10^7.8logTCID₅₀And/ml. Results of sequencing of BHK-adapted viral genome 2B-2C intervals of P10, P20: compared with ZWT-01-2015 clone, the nucleotide homology is 99.9%, and the amino acid homology is 99.8%. The P30 subsoxicity is detected to contain 7297 base pairs by whole gene sequencing(containing 3-terminal Poly A40bp), the homology with ZWT-01-2015 clone nucleotide and amino acid is 99.0% and 99.9%, respectively. The A-type Seikaga virus BHK-21-P30 generation secondary 3D region real-time PCR detection is positive. The inactivated swine Seneca valley virus vaccine primary seed batch virus is named as 30-generation BHK-21 adaptive strain of swine Seneca valley virus A (SVA-BHK-21-p 30).

Example 3 preparation of viral vaccines and antibodies

1. Preparation of antigens

1.1 Virus culture

BHK-2137 deg.C, culturing at pH7.2 + -0.2, infecting SVA-BHK-21-p30 with 0.01MOI, and culturing at 32 deg.C for 3-4 days to obtain virus culture solution.

1.2 clarification

The virus culture broth was filtered through an 8 μm polypropylene prefilter and clarified by filtration through 0.45 μm and 0.2 μm filters to determine the virus titer (TCID 50).

1.3 Ultrafiltration dialysis

The clarified virus fluid was dialyzed against 100KD membrane ultrafiltration (20mM Tris, 150mM NaCl, pH 7.5).

1.4 purification

Adding MgCl into the virus liquid after dialysis₂To a final concentration of 2mM, nuclease was added at 15U/ml for 2 hours, centrifugation was carried out at 1500g for 10 minutes in a 277K centrifuge, discontinuous cesium chloride density gradient centrifugation (1.24-1.4g/ml) was carried out, continuous cesium chloride density gradient centrifugation (1.33g/ml) was carried out, and after completion of each centrifugation step, the grating zone was collected and dialyzed against 1L cold dialysis buffer [200mM Tris-HCl, 50mM HEPES pH 8.0, 10% (v/v) glycerol]The light absorption value at 260nm was measured by an ultraviolet spectrophotometer, and the OD260nm was 9.5 × 10¹²The virus particles are detected by SDS-PAGE electrophoresis to see electrophoresis bands of VP1, VP2, VP3 and VP4, and the relative molecular weights are 33kDa, 40kDa, 28kDa and 6 kDa.

1.5 inactivation

The purified virus was diluted to 10 with RPMI 1640⁸TCID50/M L, inactivated with 0.1M bisethyleneimine at 37 ℃ for 1 hour, neutralized with 0.1mM sodium thiophosphate at 37 ℃ for 2 hours⁸TCID50/M L, inactivated with 0.1M bisethyleneimine at 37 ℃ for 1 hour0.1mM sodium thiophosphate was neutralized at 37 ℃ for 2 hours.

Inactivation verification test 1m L inactivation solution was added to 150cm of 50m L medium²The cells are cultured in a cell bottle at 37 ℃ for 1 week, fresh culture solution is replaced for continuous culture for 1 week, and meanwhile, 1m of inactivated virus solution of L is inoculated to the same cell bottle for observing cytopathic effect.

1.6 dialysis

The mixture was concentrated to 1/6 in the original volume using a 100kDa membrane and dialyzed against PBS buffer (50mM phosphate buffer, 150mM NaCl, pH 7.5).

2. Preparation of adjuvant (100ml)

To 100ml of 50mM phosphate buffer, polylysine hydroxymethylcellulose polyinosinic cell (PolyIC L C, average molecular weight 500000Da) 2.5g, EDTA 0.02g, and Tween-20 0.005g were added to adjust pH to 8.0.

The compound polyIC L C was synthesized by the present inventors, polylysine Poly-L-L ysine (molecular weight 1000-4000Da, Cat. No. P0879), polyinosinic acid-polycytidylic acid (Poly IC, Cat. No. P0913) and hydroxymethylcellulose (carboxmethyl hyicellulose, low viscosity, Cat. No. C5678) were all purchased from Sigma-Aldrich (St. L ouis, Mo.) polyinosinic acid-polycytidylic acid (Poly IC, average molecular weight 200000-500000Da, 500m L; 4.0mg/m L3), polylysine (polylysine-L-lysine, 250 ml; 6.0mg/m L) and 2% hydroxymethylcellulose (carboxmymethylicellulose, 250m L) were prepared from 0.85% of a prepared solution of a source, stabilized with polylysine, 250 mg/m L C, and the polylysine was prepared by slowly adding the polylysine at a concentration of polylysine, after annealing at 19720 mg/5, the polylysine C, the final concentration of polylysine was calculated as polylysine, and the polylysine was adjusted to give a stable solution containing polylysine, and the concentration of polylysine, and the polylysine was added to the physiological concentration of polylysine C, and the final polylysine was adjusted to give a physiological concentration of polylysine, and the final concentration of polylysine was adjusted to give a stable, and the polylysine, and the final concentration of polylysine, and the concentration of polylysine, the polylysine, and the concentration of the polylysine was adjusted to give a stable, the concentration of polyl.

3. Vaccine preparation

4. Preparation of purified polyclonal antibody of A-type Seneca valley virus

Purifying inactivated whole virus particles of the rabbit by a classical method to obtain high-titer antibody serum, purifying the serum by saturated ammonium sulfate, G protein chromatography and Sepharose-4B column chromatography coupled with purified virus to obtain an antibody purified by affinity chromatography, and testing to show that the antibody reacts specifically and sensitively with the A-type Seneca valley virus.

5. Determination of antigen content of whole virus particle of A-type Seneca valley virus

The method is characterized in that the content of the A-type Seneca valley virus is quantified by an indirect E L ISA method based on the polyclonal antibody of the A-type Seneca valley virus purified by affinity chromatography, the relationship between the level of the neutralizing antibody of a pig body and the antigen amount of purified whole virus is determined by a parallel line method, protective neutralizing antibody can be generated when the purified A-type Seneca valley virus is 5 mu g, the protective neutralizing antibody is determined to be 5 units, and the titer of the inactivated porcine Seneca valley virus vaccine can be estimated by the indirect E L ISA method.

Example 4 safety and immunogenicity of inactivated Seneca valley Virus vaccine on gilts

1. Method of producing a composite material

1.1 Experimental groups

70 healthy SVA-free antibodies 1-3 week pigs were randomized into 7 groups containing: the test group A of 10 mug inactivated whole virus particle antigen of the A-type Seneca valley virus only by nasal drip, the test group B of 25mg polylysine hydroxymethyl cellulose polyinosinic component only by nasal drip, the test group C-F of 5 mug, 10 mug, 20 mug, 50 mug inactivated whole virus particle antigen of the A-type Seneca valley virus and 25mg polylysine hydroxymethyl cellulose polyinosinic component respectively by nasal drip, the control group instills nasal phosphate buffer solution, 10 pieces of each group. All experimental groups and control groups were immunized once with 5.0TCID 30 days after immunization₅₀SVAA-ST-p2 challenge with virus blood samples taken at 0, 3, 6, 9, 14, 21, 28, 35, 42, 49, 56, 70 days post-immunization and at 0, 3, 4, 7, 10, 15, 22 days post-challenge were subjected to E L ISA, neutralization test, safety observations stored at-70 ℃ prior to virus quantification in piglets included temperature, appetite, weight gain observations in piglets.

1.2 detection method

SVA-specific antibodies are detected by indirect E L ISA method, collecting and separating serum from pig bodies and storing the serum at-20 ℃ for later use, coating with purified SVA antigen, diluting the pig serum, detecting with horseradish peroxidase-labeled mouse anti-pig antibodies, operating at room temperature, measuring the light absorbance at 650nm (Bio-Rad 680 microplate reader), and determining whether SVA antibodies are positive or not by the ratio of positive and negative samples.

Serum neutralizing antibody detection method, serum dilution by multiple times, and 5 × 10³TCID50/m L, incubated at 37 ℃ for 1 hour, transferred to a full monolayer of ST cell 96-well plates, analyzed for cytopathic effects 3 days later, and the reciprocal of the serum dilution that prevented 50% of the cytopathic effects was the neutralizing antibody titer.

Viremia Detection, SVA antibody positive pig serum is subjected to Real-Time fluorescence System Detection PCR (Bio-Rad iQ5Real Time PCR Detection System),20 mu L (iQ SYBR Green Supermix; Bio-Rad) reaction volume contains 10 mu L mixture, 1 mu L pig serum sample cDNA extract and 8.5 mu L RNase-free water, and the short sequence most conserved in a primer Sernica Valley virus gene library VP1 is adopted as a template to design and synthesize:

a forward primer 5'-GGGTAACACTGACACCGATTT-3';

reverse primer 5'-TCGAGATCGATCAAACAGGAAC-3'

Taking 2.5 mu L, setting a SVA 1: 10 diluted standard curve for each reaction, setting up reaction conditions of 48 ℃ for 5min, 95 ℃ for 10min, then 40 cycles, each cycle, 95 ℃ for 5s of denaturation, 60 ℃ for 1min, amplifying to 87bp fragments, converting the PRRSV content evaluated in each reaction into a cycle threshold Ct, and determining the virus content (copy/ml) by Bio-Rad iQ5 qPCR software and a standard curve correlation coefficient.

1.3 challenge virus culture: SVAA-ST-p2 was cultured by the ST cell culture method described in example 1, ST was purified from triplicate plaque clones, and cultured in DMEM containing 10v/v% fetal bovine serum at 37 ℃ under 5% CO2 for 3 days. Viral titers were determined by ST cell culture. Dilute to 6.0log TCID50/ml with MEM containing 4% calf serum, and drip 2ml for counteracting toxic substance in oral cavity of piglet.

2. As a result:

as shown in table 1, there was no significant difference in body weight gain at 56 days after receiving immunization with 1 dose. No fever, anorexia and the like are observed, which indicates that the vaccine of the invention is safe.

TABLE 1 weight gain of piglets (days) after vaccine immunization

TABLE 2 vaccine induced neutralizing SVA antibody (log2) changes in piglets

Table 2 shows that the inactivated and purified vaccine containing the inactivated whole virus particle antigen of the A-type Seneca valley virus and polylysine hydroxymethyl cellulose polyinosinic cell induces the production of the anti-SVA neutralizing antibody, and the neutralizing antibody is fast to produce and high in titer.

Table 2 results show that group A pigs neutralized antibody positive conversion by SVA 7-14 days after immunization and all pigs were antibody positive conversion 3 days after challenge. SVA neutralizing antibodies were not detected in the adjuvant-vaccinated group B before challenge and were detected 14-28 days after challenge. The antibody of the pigs in the C-F group is positive transferred within 1 week after the inoculation immunization, all the pigs are positive transferred within 3 days after the challenge, the antibody titer of the A group is higher than that of the B group, the antibody titer of the C-F group within 1 week after the immunization and the antibody titer of the C-F group within 1 week after the challenge are higher than that of the A group, and the A group has a significant difference (P is less than 0.05), but the C-F group has no significant difference (P is more than 0.05). Before challenge, no SVA neutralizing antibody was detected in group B pigs, and after challenge, neutralizing antibodies were detected with mean titers between 4.0-8.9log 2. After challenge, neutralizing antibodies in pigs in group A were positive-converted in 14-21 days. Neutralizing antibody titers in groups C-F remained high between 5-7 post challenge, and in group A, the highest antibody titers were at day 5 post challenge. There was a significant difference in neutralizing antibody titers and peak times for both group B and C-F (p < 0.05).

Before challenge, the sera of pigs in experimental group A and experimental group C-F do not check viruses, which indicates that SVA in the vaccine is completely inactivated, the vaccine is safe, after challenge, all immunized pigs do not include group B, and the viremia occurrence time is shortened, after 4 days of challenge, the virus titer reaches a peak and averagely reaches 1.4log10TCID50/m L, and then the virus titer starts to decline, after 3 weeks of challenge, no virus is detected, after 14 days of challenge, the virus titer reaches a peak and averagely reaches 4.9log10TCID50/m L, then the virus titer starts to decline, after 28 days of challenge, no virus is detected, the viremia appears in groups A and B, and the peak is obviously different, after 17-21 days of challenge, the viremia is cleared, after 4 days of challenge, the virus titer reaches 2.3log10 TCID50/m L, after 7 days, the virus titer does not reach the viremia is reduced, after 3 weeks, the viremia is not detected in groups C-F, the viremia is cleared, and the virus titer reaches 2.A-F group, and the difference is obviously, and the virus titer does not reach the peak and the peak after 3 weeks.

TABLE 3 viremia following challenge of vaccine immunized piglets

The results show that the vaccine provided by the invention can reduce the viremia time and protect the piglet from the blister symptoms and death by carrying out intramuscular injection immunization on the piglet once, and the vaccine provided by the invention is safe and effective for the piglet.

Example 5 nasal drip Seneca Valley Virus vaccine for sow safety and immune protective period

1. The method comprises the following steps:

1.1 challenge virus culture: SVAA-ST-p2 was cultured by the ST cell culture method described in example 1, ST was purified by triple plaque cloning, and DMEM containing 10% fetal bovine serum was used at 37 ℃ with 5% CO₂Cultured under the conditions for 3 days. Viral titers were determined by ST cell culture. Diluting to 6.0log TCID50/ml with MEM containing 4% calf serum, diluting to 8.0TCID50/ml with MEM containing 4% calf serum, and orally instilling 5ml to challenge the sow.

1.2 grouping, immunization, counteracting toxic substances, Observation

Sows with different body weights and ages of at least 6 months are selected, 90 pigs which are negative to SVA antibody detected and screened by a kit are divided into 9 groups, 10 in each group, 1 group is subjected to nasal drip of the vaccine prepared in example 3 weeks before delivery, the nasal drip of the vaccine is subjected to secondary immunization at intervals of 2 weeks, the volume is 2ml (10 mu g of antigen +25mg of P L CIC/m L), 2 groups of sows which are pregnant are confirmed by ultrasonic detection, 2 doses of the vaccine prepared in example 3 are subjected to nasal drip at 30-45 days of gestation period, and 3 groups of sows are subjected to nasal drip of water for injection for 0 and 14 days respectively.

4-9 groups of sows are injected by nasal cavity drip the vaccine prepared in example 3 weeks before delivery, the nasal cavity drip vaccine is injected for secondary immunization at the interval of 2 weeks, the volume is 2ml (10 mug antigen +25mg P L CIC/m L), after the last 1 dose of immunization is completed, the pigs are respectively attacked on 30, 60, 90, 120, 180 and 210 days, the health condition, the weight change and the body temperature change of the pigs are observed before and after the attack and the immunization, the neutralizing antibody titer of serum is detected, and the health condition of the pigs after the attack and the infection and the occurrence time of viremia are observed.

1.3 serum neutralizing antibody assay:

serum fold-rate dilutions, each 5 × 10³TCID50/m L, incubated at 37 ℃ for 1 hour, transferred to a full monolayer of ST cell 96-well plates, analyzed for cytopathic effects 3 days later, and the reciprocal of the serum dilution that prevented 50% of the cytopathic effects was the neutralizing antibody titer.

2. Results

All sows immunized 2 times by nasal drip had normal body temperature and average weight gain greater than that of the pigs instilled with water for injection, and no systemic and local side effects were observed. The vaccine does not influence the growth and development of the sow, and the nasal cavity immunizes the sow safely.

The pregnant sows which are immunized by nasal drip for 2 times have normal average body temperature and average weight gain larger than that of the sows which are injected with water, and the sows immunized by nasal drip have no fever and nasal cavity symptoms and have no other side effects. The vaccine nose drop immunization is safe for pregnant sows.

2 groups of 10 pregnant sows are immunized by intranasal instillation for 2 times, the average litter size is 9.8, the body temperature and weight gain of piglets are normal after 28 days of delivery, and the phenomena of stillbirth and hypoplasia of the piglets do not exist.

2 groups of ultrasonic tests confirm that 10 sows are pregnant, the vaccine of the invention is instilled intranasally for 2 times in 30-45 days of gestation period, the immunization is carried out at 2-week intervals, and 10 pregnant sows all produce neutralizing antibodies with the average titer of 512.

30 days after 3 groups of sows are immunized, 10/10 generates SVV specific neutralizing antibodies before challenge, 10/10 pigs have no viremia after challenge, and sows generate neutralizing antibodies after challenge, and the average titer is 512; the 4 groups are attacked 60 days after immunization, 1/10 produces viremia, the viremia time is 1 day, and the average titer of neutralizing antibodies is 512; after 90 days of virus attack after group 5 immunization, 1/10 produced viremia, the viremia time was 2 days, 10/10 gilts produced neutralizing antibodies, and the titer was 512; the 6 groups are attacked 120 days after immunization, 1/10 parvoviremia of the sows occurs, the occurrence time is 2 days, 10/10 sows generate neutralizing antibodies, and the titer is 256; 7 groups are attacked 150 days after immunization, 9/10 sows generate neutralizing antibodies, the average neutralizing antibody titer is 128, and 10/10 sows all survive; group 8 had 180 days post-immunization infection, 1/10 had viral hematopathy for 2 days, and the level of neutralizing antibodies in the 10/10 gilts remained at 128; the 10/10 pigs all produced neutralizing antibodies after 210 days of infection after immunization of 9 groups, the antibody titer was 32, and 1/10 pigs had viral hematopathy for 3 days.

The results show that the vaccine of the invention has 180 days for the immune protection period of the sow, 2 times of immunization times, 2 weeks for nasal drip immunization and 180 days for the immune validity period.

The vaccine of the invention can induce the generation of serum SVA neutralizing antibody by intranasal instillation immunization for 2 times, reduce the occurrence of viremia and the death of infection of gilts, has 100 percent of death protection rate caused by virus infection and 180 days of immune protection period.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Zhongnong Witt Biotech Ltd

<120> A-type Seneca valley virus inactivated vaccine, preparation method and application thereof

<130>KLPI161405

<160>1

<170>PatentIn 3.5

<210>1

<211>7293

<212>DNA

<213>Seneca valley virus A

<400>1

ttttgagttg gcgtctgggc cctcatgccc agtccttcct ttccccttcc ggggggtaaa 60

ccggctgtgt ttgctagagg cacagaggag caacatccaa cctgctcttg tggggaacgg 120

tgcggctcca attcctgcgt cgccaaaggt gttagcgcac ccaaacggcg catctaccaa 180

tgctattggt gtggtctgcg agttctagcc tactcgtttc tcccctatcc actcactcac 240

gcacaaaaag tgtgttgtaa ctacaagact tagccctcgc acgagatgtg cgataaccgc 300

aagattgact caagcgcgga aagcgctgta accacatgct gttagtccct ttatggctgc 360

gagatggcta tccacctcgg atcactgaac tggagctcga ccctccttag taagggaacc 420

gagaggcctt cttgcaacaa gctccgacac agagtccacg tgattgctac caccatgagt 480

acatggttct cccctctcga cccaggactt ctttttgaat atccacggct cgatccagag 540

ggtggggcat gatcccccta gcatagcgag ctacagcggg aactgtagct aggccttagc 600

gtgctttgga tactgcctga tagggcgacg gcctagtcgt gtcggttcta taggtagcac 660

atacaaatat gcagaactct catttttctt tcgatacagc ctctggcacc tttgaagacg 720

taaccggaac aaaagtcaag atcgttgaat accctagatc ggtgaacaat ggtgtttacg 780

attcgtccac tcatttagag atactgaacc tacagggtga aattgaaatt ttaaggtctt 840

tcaatgaata ccaaattcgc gccgccaaac aacaacttgg actggacatc gtatatgaac 900

tacagggtaa tgttcagaca acctcaaaga atgattttga ttcccgcggc aataatggta 960

acatgacctt caattactac gcaaacactt atcagaattc agtagacttc tcgacctcct 1020

cgtcggcgtc aggcgccgga cccgggaact cccggggcgg attagcgggt ctcctcacaa 1080

atttcagtgg aatcttgaac cctcttggct acctcaaaga ccacaatacc gaagaaatgg 1140

aaaactctgc tgatcgagtc ataacacaaa cggcgggcaa cactgccata aacacgcaat 1200

catcactggg tgtgttgtgt gcctacgttg aagacccgac caaatctgac cctccgtcca 1260

gcagcacaga tcaacccacc accactttta ctgccatcga caggtggtac actggacgcc 1320

tcaattcttg gacaaaagct gtaaaaacct tctcttttca ggccgtcccg ctccctggag 1380

ccttcctgtc tagacaggga ggcctcaatg gaggggcctt cacggctacc ctacatagac 1440

atttcttaat gaagtgcggg tggcaggtgc aggtccaatg caatttgaca caattccacc 1500

aaggtgctct tcttgttgcc atggtccccg aaaccaccct tgatgtcaag cccgacggca 1560

aggcaaagag cctacaggag ctgaatgaag agcagtgggt agaaatgtct gacgattacc 1620

ggaccgggaa aaacatgcct tttcagtctc ttggcacata ctatcggccc cctaactgga 1680

cttggggccc taatttcatc aacccctatc aagtaacagt tttcccacac caaattctga 1740

acgcgagaac ctctacctcg gtagacataa gtgtcccata catcggggag actcctacac 1800

aatcctcaga gacacagaac tcctggaccc tcctcgttat ggtgcttgtc cccctggact 1860

acaaggaggg agccacaact gacccagaaa ttacattttc cgtaaggcct acaagtcctt 1920

acttcaatgg gcttcgtaac cgctacacga ccgggacgga cgaggaacag gggcccattc 1980

ccacagcacc cagagaaaat tcgcttatgt ttctctcgac catccctgac gacactgtcc 2040

ctgcttacgg gaatgtgcgt acccctcccg tcaattacct tcctggtgaa ataaccgacc 2100

tcttacaact ggcccgtata cccactctca tggcgtttgg gcgggtgcct gaacctgaac2160

ctgcctcaga cgcttatgtg ccctacgttg ccgttcccac ccagttcgat gacaagcctc 2220

tcatctcctt cccgatcacc ctttcagatc ctgtctacca gaatactctg gtaggcgcca 2280

tcagttcaaa tttcgccaac taccgggggt gtatccaaat cactctgaca ttttgtggac 2340

ctatgatggc aagagggaaa ttcctactct cgtattctcc cccaaatgga acacaaccac 2400

agaccctttc tgaagccatg cagtgcacat attctatttg ggatataggc ttaaactcta 2460

gttggacctt tgtcatcccc tacatctcgc ccagtgacta ccgtgaaact cgggccatta 2520

ccaattcggt ttattctgct gatggctggt ttagcctgca caagctgacc aaaattactc 2580

taccacctga ttgcccacag agcccctgta ttctcttttt cgcctctgct ggtgaggatt 2640

acaccctccg tctccccgtt gattgtaatc cttcttatgt gttccactcc accgacaacg 2700

ccgagactgg ggttattgag gcgggtaaca ctgacaccga tttctctggt gaattggcgg 2760

ctcctggctc taaccacact aatgtcaagt tcctgtttga ccgatctcga ttactgaatg 2820

taattaaggt actggagaag gacgccgtct tcccccgccc tttccccaca gcaactggta 2880

cacaacagga cgatggttac ttttgtcttc taacaccccg cccaacagtc gcctcccgac 2940

ccgccactcg tttcggcctg tacgtcagtc cgtctgacag tggcgttctc gccaacactt 3000

cactggattt caatttttac agcttggcct gtttcactta ctttagatca gaccttgaag 3060

tcacggtggt ctcactggag ccagatctgg aattcgctgt agggtggttc ccctctggca 3120

gtgagtacca ggcttccagc tttgtctacg accaactgca tgtaccctac cactttactg 3180

ggcgcactcc ccgcgctttc gccagcaagg gtgggaaggt atctttcgtg ctcccttgga 3240

actctgtctc atccgtgctt cccgtgcgct gggggggcgc ttccaagctt tcttctgcca 3300

cgcggggtct gccggctcat gctgactggg ggactattta cgcctttatc ccccgtccca 3360

acgagaagaa aagcaccgct gtaaagcatg tggccgtgta cgttcggtac aagaacgcgc 3420

gtgcctggtg ccccagcatg cttccctttc gcagctacaa gcagaagatg ctgatgcaat 3480

caggcgacgt cgagaccaac ccaggccctg cttctgacaa cccaatcttg gaatttcttg 3540

aagcagaaaa tgatctagtc actctggcct ctctctggaa gatggtacac tctgttcaac 3600

agacctggag aaagtatgtg aagaacgaca atttttggcc caatttactc agtgagttag 3660

tgggggaagg ctccatcgcc ttggccgcca cgctgtctaa ccaagcttca gtaaaagctc 3720

tcttgggcct gcattttctc tctcgagggc tcaattacac agacttttac tctttactga 3780

tagagaaatg ctctagtttc tttactgtag aaccgcctcc tccaccagct gaaaatctga 3840

tgaccaagcc ctccgtgaag tcgaaattcc gaaagctgtt taagatgcaa ggacccatgg 3900

acaaagtcaa agactggaac caaatagccg ccggcttgaa gaattttcaa tttgttcgtg 3960

acctagtcaa agaggtggtc gactggctcc aggcctggat caacaaagag aaagccagcc 4020

ctgtcctcca gtaccagttg gagatgaaga agctcgggcc cgtggctttg gctcatgatg 4080

ccttcatggc cggttccggg ccccctctta gtgacgacca gattgaatac ctccaaaacc 4140

tcaaatctct tgccctaaca ctggggaaga ctaatttggc ccaaagtctc accactatga 4200

tcaatgccaa gcgagctccc gcccaacgag tcgaacccgt tgtggtggtc ctcagaggca 4260

agccgggatg cggcaagagc ttggcctcca cgttgattgc ccaggctgtg tccaagcgtc 4320

tctacggctc acaaagtgtg tattctcttc ctccggaccc agacttcttc gacggataca 4380

aaggacagtt tgtaaccttg atggatgatt tgggacaaaa cccggatggg caagatttct 4440

ccaccttttg tcagatggtg tcgaccgccc aatttcttcc caacatggcg gaccttgcag 4500

agaaggggcg cccctttacc tccaatctta tcattgcaac tacaaacctc ccccacttca 4560

gccctgtcac cattgctgat ccttctgcgg tctctcgtcg gatcaactac gacttgactc 4620

tagaagtatc tgaggcctac aaaaagcaca cacggctgaa ttttgacctg gctttcaggc 4680

gcacagacgc cccccccatt tatccttttg ctgcccatgt gcctttcgtg gacgtggctg 4740

tgcgcttcaa aaatggccac cagagcttca atctcctaga gttggtcgac tctatttgtg 4800

cagacattcg agccaagcaa caaggtgccc gaaatatgca gactctagtt ctacagagcc 4860

ctaacgagaa tgatgacacc cccgtcgacg aggcgctggg tagagttctc actcccgctg 4920

cggtcgacga ggcgcttgtc gacctcgctc aagaggccga tccggttggc cgcttggcta 4980

ttcttgccaa actaggtctt gctctagctg cggtcacccc cggcttgata atcttggcag 5040

tgggactcta taggtacttc tctggctctg atgcagacca agaagaaacg gaaagtgagg 5100

aacctgctaa agcgcctagg agcgagaatg cttatgacgg cccgaagaaa aactctaagc 5160

cccctggagc gctctctctt atggaaatgc aacagcccaa cgtggacatg ggctttgagg 5220

ctgcggtcgc taagaaagtg gtcgtcccca ttacattcat ggttcccaac agaccttctg 5280

gacttacaca gtccgccctt cttgtggccg gccggacctt cctaattaat gagcatacat 5340

ggtccaaccc ctcctggacc agtttcacaa tccgtggtga ggtgcacact cgtgatgagc 5400

ctttccaaac ggttcatttt actcaccatg gtgttcccac agacctgatg atggtacgtc 5460

tcggaccggg caactctttc cctaacaatc tagacaagtt tggacttgac cagatgccgg 5520

cacgtaactc ccgtgtggtt ggcgtttcgg ctagttacgg taatttcttc ttctctggga 5580

atttccttgg gtttgttgac tccatcacct ctgaacaagg aacttatgca agacttttta 5640

ggtacagggt gaccacctac aagggatggt gcggttcggc cctggtctgt gaggccggtg 5700

gtgtccggcg catcattggc ctgcattctg ctggtgccgc tggtatcggc gccgggacct 5760

acatctcaaa attaggactg atcaaagccc ttaaacacct cggtgaacct ctggctacaa 5820

tgcaaggact gatgactgag ctagagcctg gagtcaccgt gcatgtaccc cggaaatcta 5880

aattgagaaa gacgaccgca cacgcggtgt acaaaccgga gtttgaacct gctgtgttgt 5940

caaaatttga tcccagactg aacaaggatg ttgacctaga tgaggtaatt tggtctaaac 6000

acactgccaa cgtcccttat caacctcctt tgttctacac atacatgtca gagtacgctc 6060

atcgggtttt ctcctttttg ggaaaagaca atgacattct gaccgttaaa gaagcaatcc 6120

tgggcatccc tggactagac cctatggatc cccacacagc tccgggtctg ccctacgcca 6180

ttagcggcct tcgacgtact gatctcgtcg attttgtgaa cggtacggta gacgcagcac 6240

tggccatgca aatccagaaa ttcttagacg gtgactactc tgatcatgtc ttccaaactt 6300

ttctgaaaga tgaaatcaga ccctcagaga aggtccgagc gggaaaaacc cgcattgtcg 6360

atgtgccctc cctggcacac tgcattgtgg gcagaatgct gctcgggcgt ttcgccgcca 6420

agtttcaatc ccatcccggc tttcttcttg gttctgctat cgggtccgac cctgatgtct 6480

tctggaccgt cataggggct cagctcgagg gaagaaagaa cacgtacgac gtggactaca 6540

gtgcctttga ctcttcacac ggcactggct ccttcgaggc tctcatctct cactttttca 6600

ccgttgacaa tggttttagc cctgcgctgg gaccgtatct cagatccctg gctgtctcgg 6660

tgcacgctta cggcgagcgt cgcatcaaga ttaccggagg ccttccctct ggttgtgccg 6720

cgaccagcct tctgaacaca gtgctcaaca atgtgatcat caggactgct ctggcattga 6780

cttacaagga atttgagtat gacatggttg atatcatcgc ctacggtgac gaccttttgg 6840

ttggtacgga ttatgatctg gacttcaatg aagtggcgcg gcgcgctgcc aaactggggt 6900

ataagatgac tcctgccaac aagggttccg tcttccctcc gacttcctct ctctccgatg 6960

ctgtttttct aaaacgcaaa ttcgtccaaa acaatgacgg cttatataaa ccagttatgg 7020

atttaaagaa tttggaagcc atgctctcct acttcaaacc aggaacacta ctcgagaagc 7080

tgcaatctgt ttctatgttg gctcaacatt ctggaaaaga agaatatgat agattgatgc 7140

accccttcgc tgactacggt gccgtaccga gtcacgagta cctgcaggca agatggaggg 7200

ccttgttcga ttgacctaga tagcccaacg cgcttcggtg ccgccggcga ttctgggaga 7260

actctgtcgg aacagaaaag ggaaaaaaaa aaa 7293

Claims

1. An inactivated vaccine of A-type Seneca valley virus is characterized by containing inactivated whole virus particle antigen of pig A-type Seneca valley virus and adjuvant, wherein the whole virus particle antigen of pig A-type Seneca valley virus is obtained by virus rescue of full-length infectious cDNA clone of pig A-type Seneca valley virus, and the nucleotide sequence of the infectious cDNA clone is shown in SEQ ID NO. 1.

2. The inactivated Seneca valley virus vaccine type A according to claim 1, wherein the adjuvant is phosphate buffer containing polylysine, hydroxymethyl cellulose, polyinosinic acid (Poly IC L C) 20-50 g/L, EDTA 0.1-0.5 g/L and Tween-20 0.01-0.1 g/L, and the pH is adjusted to 8.0.

3. The inactivated Seneca valley virus vaccine type A according to claim 1, wherein the adjuvant is 50mM phosphate buffer containing 25 g/L polylysine hydroxymethyl cellulose polyinosinic cell (Poly IC L C), 0.2 g/L EDTA and 0.05 g/L Tween-20, adjusted to pH 8.0.

4. A method for preparing the inactivated vaccine for seneca valley virus type a according to any one of claims 1 to 3, comprising the steps of:

1) preparation of porcine A-type Seneca valley virus whole virus particle antigen

(1) Rescue of porcine type a seneca valley virus

Constructing an expression vector containing a full-length infectious cDNA clone of the porcine Seneca valley virus A, wherein the nucleotide sequence of the cDNA clone is shown as SED ID NO. 1;

(2) culturing porcine kidney cell line IBRS-2 in MEM (minimum essential medium) containing 10v/v% fetal calf serum until the cell line IBRS-2 is 70-90% confluent, and transfecting the expression vector constructed in the step (1) with IBRS-2 at 37 ℃ and 5% CO₂Culturing in an incubator, transferring the supernatant after culturing for 48 hours to ST cells for continuous culture, observing cytopathic effect, and harvesting virus when obvious cytopathic effect appears after infection, namely the rescued porcine A-type Seneca valley virus;

(3) passage adaptation of viruses

(4) clarification

(5) ultrafiltration dialysis

The clear virus liquid is ultrafiltered and dialyzed by a 100KD membrane;

(6) purification of

Adding MgCl into the virus liquid after dialysis₂To a final concentration of 2mM, 15U/ml of the solution was addedPerforming nuclease action for 2 hours, centrifuging, performing discontinuous cesium chloride density gradient centrifugation, then performing continuous cesium chloride density gradient centrifugation, collecting a grating zone after each step of centrifugation is finished, and dialyzing by 200mM Tris-HCl cold dialysis buffer containing 10v/v% of glycerol and 50mM HEPES pH 8.0;

(7) inactivating

(8) dialysis

2) preparation of adjuvants

Adding polylysine hydroxymethyl cellulose polyinosinic cell (Poly IC L C), EDTA and Tween-20 into phosphate buffer solution to make the final concentration reach 20-50 g/L, 0.1-0.5 g/L and 0.01-0.1 g/L respectively, and adjusting pH to 8.0;

3) vaccine preparation

5. The method of claim 4, wherein the expression vector is constructed by cloning the nucleotide sequence set forth in SED ID No.1 into the pSV L vector.

6. The method of claim 4, wherein the concentration of cesium chloride used in said discontinuous cesium chloride density gradient centrifugation is 1.24g/ml and 1.4g/ml and the concentration of cesium chloride used in said continuous cesium chloride density gradient centrifugation is 1.33 g/ml.

7. The method according to claim 4, wherein polylysine hydroxymethylcellulose polyinosinic cells (Poly IC L C), EDTA and Tween-20 are added to a 50mM phosphate buffer to a final concentration of 25 g/L, 0.2 g/L and 0.05 g/L, respectively, and the pH is adjusted to 8.0.

8. Use of the inactivated vaccine for the virus of Seneca valley A as claimed in any one of claims 1 to 3 for the preparation of a medicament for preventing diseases caused by Seneca valley A virus infection.

9. A pig A-type Seneca valley virus full-length infectious cDNA clone is characterized in that the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID NO. 1.

10. An expression vector comprising the full-length infectious cDNA clone of porcine Seneca valley virus type A of claim 9.

11. The expression vector of claim 10, wherein the expression vector is constructed by cloning the nucleotide sequence set forth in SED ID No.1 into the vector pSV L.

12. A method for rescuing a porcine Seneca valley virus type A, comprising the steps of:

(1) constructing an expression vector containing a full-length infectious cDNA clone of the porcine Seneca valley virus A, wherein the nucleotide sequence of the cDNA clone is shown as SED ID NO. 1;

(2) culturing porcine kidney cell line IBRS-2 in MEM (minimum essential medium) containing 10v/v% fetal calf serum until the cell line IBRS-2 is 70-90% confluent, and transfecting the expression vector constructed in the step (1) with IBRS-2 at 37 ℃ and 5% CO₂Culturing in an incubator, transferring the supernatant after 48 hours of culture to ST cells for continuous culture, observing cytopathic effect, and harvesting virus when obvious cytopathic effect appears after infection, namely the porcine Seneca valley virus A.

13. The method of claim 12, wherein the expression vector is constructed by cloning the nucleotide sequence set forth in SED ID No.1 into the pSV L vector.

14. Porcine seneca valley virus type a prepared according to the method of claim 12 or 13.

15. Use of the porcine savirus a of claim 14 for the preparation of a reagent for detecting neutralizing antibodies against porcine savirus a and for the preparation of an inactivated vaccine against savirus a.