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Porcine epidemic diarrhea virus vaccine strain and preparation method thereof

A porcine epidemic diarrhea and vaccine strain technology, applied in the field of microbiology, can solve the problems of limited cross-protection ability of mutant epidemic strains, unsatisfactory clinical effect, mutation, etc.

Active Publication Date: 2021-05-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the existing commercial attenuated vaccines in China are mainly porcine epidemic diarrhea, porcine transmissible gastroenteritis dual live vaccine and porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine rotavirus triple live vaccine. The vaccines used The strain is mainly derived from the classic porcine epidemic diarrhea virus CV777 strain, and the classic strain has a large gene variation with the current domestic epidemic strain, and its nucleotide homology is less than 97%. Among them, the S protein is in The nucleotide homology between classic strains and epidemic strains is 91.9%-93.5%, while the amino acid homology is 90.1%-92.0%. The ability of cross-protection is obviously limited, and it can't resist the infection of popular strains well
Therefore, although the above-mentioned vaccines have been widely used, due to the continuous variation of the antigens of the re-emerging PEDV strains, the existing attenuated vaccines developed based on classic strains have insufficient cross-protection against the mutated epidemic strains, resulting in unsatisfactory clinical effects. , leading to piglet diarrhea epidemics in my country are still endemic and seasonal

Method used

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  • Porcine epidemic diarrhea virus vaccine strain and preparation method thereof
  • Porcine epidemic diarrhea virus vaccine strain and preparation method thereof
  • Porcine epidemic diarrhea virus vaccine strain and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Screening and preparation of FJzz1 vaccine strain

[0034] Materials and methods

[0035] Cells, Viruses and Antibodies

[0036] Put Vero E6 cells (African green monkey kidney cells) in DMEM medium containing 10% FBS at 37°C in 5% CO 2 cultivated under conditions. FJzz1 strain (GenBank accession number: MK288006) was isolated from Vero E6 cells in the previous period by our laboratory. The PEDV G2 subgroup strain (Chen et al. 2019) mouse-derived anti-PEDV N protein monoclonal antibody was prepared by our laboratory. The mouse fluorescent secondary antibody was purchased from Invitrogen Company, and DAPI was purchased from Beyontian Company.

[0037] Wash well-cultured Vero cells that have grown to more than 90% of the bottom area of ​​the T25 cell culture flask 2-3 times with sterile PBS, and dilute FJzz1 fifth-generation virus (FJzz1-F5) in 1 mL containing 10 μg / mL Serum-free culture solution of trypsin was added to cells for 2 h, discarded as solution, ...

Embodiment 2

[0045] Example 2. Full-length genome sequencing and genetic variation analysis of different generations of FJzz1 strains

[0046] RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract the PEDV RNA of different generations of FJzz1 strains such as F20, F50, F100, F150 and F200, and the RNA was extracted by Revert Aid First Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). ) into cDNA, and then using the cDNA as a template and 16 pairs of overlapping primers as amplification primers (Table 1), the full-length gene was amplified by PCR using LA Taq. The above PCR products were sequenced by Shanghai Bioengineering Co., Ltd. The sequencing results were spliced ​​by the SeqMan program in Lasergene 6.0 software, and the full-length gene sequences of FJzz1 strain PEDV of different generations such as F20, F50, F100, F150 and F200 were respectively obtained. Select 60 strains from GenBank to publicize the S gene sequences of PEDV representative referenc...

Embodiment 3

[0063] Embodiment 3. Different generations of FJzz1 strain clinical trials

[0064] Fifteen 5-day-old healthy suckling piglets negative for PEDV, TGEV, PoRV, PDCoV, PRRSV, PRV and CSFV antigens and negative for PEDV antibodies were selected and randomly divided into three groups: A, B and C. Piglets in group A were inoculated with low-generation virus FJzz1-F20, and the challenge dose was 1×10 5 TCID 50 / 1 mL / head, piglets in group B were inoculated with high-generation virus FJzz1-F200, and the challenge dose was 1×10 5 TCID 50 / 1 mL / head, group C was inoculated with 1 mL DMEM as the control group. The route of inoculation in the above three groups was neck intramuscular injection. During the experiment, the clinical manifestations of the experimental pigs were observed and recorded every day, including spirit, appetite, diarrhea and vomiting. The body temperature and body weight were measured regularly every day, and the feces viscosity of the piglets were scored as fol...

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Abstract

The invention discloses a porcine epidemic diarrhea virus vaccine strain and a preparation method thereof. The vaccine strain is obtained by continuously transmitting FJzz1 to 200 generations in Vero cells. Low-generation FJzz1-F20 and high-generation FJzz1-F200 strains are selected to perform pathogenicity evaluation on piglets, and the result shows that the clinical manifestation of the piglets in a high-generation FJzz1-F200 infection group is obviously lighter than that of the piglets in an FJzz1-F20 infection group, and the diseased pigs do not die. Compared with the FJzz1-F20 infection group, the excrement toxin expelling amount of the piglets in the FJzz1-F200 infection group and the viral load in intestinal tissues are remarkably reduced, and the intestinal tissues are not subjected to obvious histopathological changes. Meanwhile, detection finds that the FJzz1-F200 infection group can induce high-level I-type and III-type interferon generation in the intestinal tissues. The research result shows that the FJzz1-F200 is weak in pathogenicity and has the potential of serving as a PEDV candidate attenuated vaccine strain.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to the preparation and application of an attenuated vaccine strain of porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease that causes severe diarrhea, high morbidity and high mortality in suckling piglets. It is caused by porcine epidemic diarrhea virus (PEDV) infection. Even the world's pig industry has caused huge economic losses (Wood 1977; Coussement et al. 1982; Shibata et al. 2000; Lee 2015). PED was first reported in the United Kingdom in 1971 and subsequently became prevalent in other Eurasian countries (Pensaert and de Bouck 1978; Horvath and Mocsari 1981; Pospischil et al. 1981; Takahashi et al. 1983; Kusanagi et al. 1992; Pijpers et al. 1993; Smid et al.1993; Martelli et al. 2008; Zhou et al.2012), and with the widespread application of the CV777 vaccine strain, t...

Claims

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Application Information

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IPC IPC(8): C12N7/08A61K39/215A61P31/14
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2770/20021C12N2770/20022C12N2770/20064C12N2770/20034A61K2039/552A61K2039/5254Y02A50/30
Inventor 周艳君童光志陈鹏飞王康于家荣童武虞凌雪高飞姜一峰
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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