Porcine epidemic diarrhea virus as well as separation method and application thereof
A porcine epidemic diarrhea and virus technology, applied in the directions of virus antigen components, viruses, antiviral agents, etc., can solve the problems of low virus price, difficult separation, affecting the protective effect of porcine epidemic diarrhea vaccine, etc., and achieves simple operation and immune effect. Good and practical effect
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Embodiment 1
[0064] The present embodiment provides a method for separating and purifying porcine epidemic diarrhea virus, comprising the steps of:
[0065] (a), take the small intestine tissue and contents of the diseased piglets, grind them with a grinder, freeze and thaw repeatedly at -20°C for 3 times, then add 4 times the volume of PBS to mix, let stand at 4°C for 2 hours, extract the supernatant, and put the above Centrifuge at 5000r / min at 4°C for 30min, and take the centrifuged supernatant for later use.
[0066] (b), 40% PEG 6000 solution is added to the centrifugal supernatant obtained in step (a), so that its final concentration is 8%, the pH is adjusted to 7.2, NaCl is added to make its final concentration 1%, and stirred at 150r / min After 20 minutes, stand at 4°C for 2 hours.
[0067] (c) Take the resting solution obtained in step (b), centrifuge once at 10,000 r / min for 20 minutes at 4°C, and discard the supernatant.
[0068] (d) Wash the precipitate obtained in step (c) on...
Embodiment 2
[0071] The present embodiment provides a method for separating and purifying porcine epidemic diarrhea virus, comprising the steps of:
[0072] (a), take the small intestine tissue and contents of the diseased piglets, grind them with a grinder, freeze and thaw twice at -20°C, then add 5 times the volume of PBS to mix, let stand at 4°C for 1 hour, extract the supernatant, and Centrifuge at 5500r / min at 4°C for 25min, and take the centrifuged supernatant for later use.
[0073] (b), 40% PEG 6000 solution is added to the centrifuged supernatant obtained in step (a) to make its final concentration 10%, adjust the pH to 6.5, add NaCl to make its final concentration 2%, and stir at 100r / min After 25 minutes, stand at 4°C for 1 hour.
[0074] (c) Take the resting solution obtained in step (b), centrifuge once at 12000r / min for 15 minutes at 4°C, and discard the supernatant.
[0075] (d) Wash the precipitate obtained in step (c) once with PBS, and then add 60% of the total volume o...
Embodiment 3
[0078] The present embodiment provides a method for separating and purifying porcine epidemic diarrhea virus, comprising the steps of:
[0079] (a), take the small intestine tissue and contents of the diseased piglets, grind them with a grinder, freeze and thaw repeatedly 4 times at -20°C, then add 3 times the volume of PBS to mix, let stand at 4°C for 3 hours, extract the supernatant, and Centrifuge at 4500r / min at 4°C for 35min, and take the centrifuged supernatant for later use.
[0080] (b), 40% PEG 6000 solution is added to the centrifuged supernatant obtained in step (a), making its final concentration 6%, adjusting the pH to 8, adding NaCl to make its final concentration 0.1%, stirring at 200r / min After 15 minutes, stand at 4°C for 3 hours.
[0081] (c) Take the resting solution obtained in step (b), centrifuge once at 8000r / min for 25 minutes at 4°C, and discard the supernatant.
[0082] (d) Wash the precipitate obtained in step (c) once with PBS, and then add 40% of...
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