A triple subunit vaccine for porcine epidemic diarrhea, porcine transmissible gastroenteritis and porcine D-coronavirus disease
A technology for porcine epidemic diarrhea and coronavirus, which is applied to viruses, vaccines, antiviral agents, etc., can solve the problems of the epidemic situation of recombinant mutant viruses in the field, limited clinical application effect, and increased difficulty in purification, so as to achieve good immunogenicity, High safety and small immune dose
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Embodiment 1
[0023] Example 1: Screening of Porcine Epidemic Diarrhea Virus (PEDV) S1 Gene, Porcine Transmissible Gastroenteritis Virus (TGEV) S1 Gene and Porcine Dicoronavirus (PDCoV) S1 Gene
[0024] Since 2010, a large number of piglets with diarrhea have appeared in many provinces and cities in mainland China, and some affected pigs have been injected with the diarrhea vaccine that was available in the market at that time. Therefore, it is speculated that the field virus is a mutated strain. After the detection of clinical samples in 2016, different porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine D-coronavirus were isolated from the diseased pigs for whole genome sequencing and genetic evolution analysis ( image 3 , Figure 5 and Figure 7 ), the PEDV KB2013-4 strain, the TGEV BD strain and the PDCoV SD strain were selected as reference strains of the current field strains; among them, the PEDV KB2013-4 strain (Porcine epidemic diarrhea virus)...
Embodiment 2
[0028] Embodiment 2: Preparation of recombinant porcine epidemic diarrhea virus S1 protein, porcine transmissible gastroenteritis virus S1 protein and porcine D-coronavirus S1 protein,
[0029] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying recombinant porcine epidemic diarrhea virus S1 protein, porcine transmissible gastroenteritis virus S1 protein and porcine D-coronavirus S1 protein. The details are as follows: the positive clone plasmid pEASY-T1-PEDV-S1 or pEASY-T1-TGEV-S1 or pEASY-T1-PDCoV-S1 and the expression vector pPICZα vector were digested with Bam HI and Hind III respectively, and passed through 1.0% agar After sugar gel electrophoresis, purify and recover with a DNA gel recovery kit to obtain 1.9 kb or 1.9 kb or 1.6 kb and 3.3 kb fragments, respectively, and construct pPICZα-PEDV-S1 or pPICZα-TGEV-S1 or pPICZα by directional ligation at 16°C - PDCoV-S1 expr...
Embodiment 3
[0040] Embodiment 3: the preparation of porcine epidemic diarrhea, porcine transmissible gastroenteritis, porcine D-type coronavirus disease triple subunit vaccine
[0041] The semi-finished PEDV-S1, TGEV-S1 and PDCoV-S1 protein antigens after passing the inspection are used for vaccine preparation (the liquid components in the following preparations are calculated by volume ratio).
[0042] 1 Adjuvant Preparation Disperse 0.5% volume Span-85 in 5% volume squalene phase, mix with 0.5% volume Tween-80 into 94% volume 10mM sodium citrate buffer, then mix at high speed A crude emulsion is obtained. This coarse emulsion is then repeatedly passed through a high-pressure homogenizer (1200bar, 6-8 cycles) to produce an emulsion with uniform oil droplet size; the emulsion is filtered through a 0.22μm filter membrane to remove large oil droplets to obtain an average Droplet size remained unchanged for at least 3 years at 4°C.
[0043] 2 Antigen preparation Porcine epidemic diarrhea v...
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