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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

Inactive Publication Date: 2018-06-08
TECON BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first object of the present invention is to provide a method for separating and purifying porcine epidemic diarrhea virus, so as to alleviate the difficulty in separating the porcine epidemic diarrhea mutant strains existing in the prior art, and to produce low toxicity on cells, Technical issues used to prepare vaccines that affect the protective effect of porcine epidemic diarrhea vaccines

Method used

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  • Porcine epidemic diarrhea virus as well as separation method and application thereof
  • Porcine epidemic diarrhea virus as well as separation method and application thereof
  • Porcine epidemic diarrhea virus as well as separation method and application thereof

Examples

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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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Abstract

The invention provides a porcine epidemic diarrhea virus as well as a separation method and application thereof, relating to the technical field of biology. According to a separation purification method of the porcine epidemic diarrhea virus, the porcine epidemic diarrhea virus is separated by virtue of polyethylene glycol 6000. The method is simple in operation and strong in practicability, the porcine epidemic diarrhea virus can be effectively separated, and the separated porcine epidemic diarrhea virus has relatively high purity and virus titer. A tissue inactivated vaccine prepared from the porcine epidemic diarrhea virus has the beneficial effects that impure proteins are few, the batch difference of the vaccines is small, the immune effect is good, and the side effect is slight.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a porcine epidemic diarrhea virus and its isolation method and application. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae and belongs to the genus Coronaviridae. Porcine epidemic diarrhea is an acute intestinal infectious disease of piglets and finishing pigs caused by a virus. The disease is very similar to infectious gastroenteritis. In my country, it mostly occurs from December to January-February of the next year, and it is also reported in summer. It can occur in pigs of any age, the younger the age, the more serious the symptoms and the higher the mortality rate. Pigs of all ages can be infected. Suckling pigs, shelf pigs or fattening pigs have a high incidence rate, especially suckling pigs suffer the most, and the incidence rate of sows varies greatly, about 15-90%. Sick pigs are the main source of infection. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2770/20034C12N2770/20051
Inventor 杜久斌李延涛贺笋李俊辉
Owner TECON BIOLOGY CO LTD
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