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PEDV (porcine epidemic diarrhea virus), inactivated vaccine and preparation method of inactivated vaccine

A porcine epidemic diarrhea and inactivated vaccine technology, which is applied in the field of bioengineering, can solve the problems of difficult adaptation of porcine epidemic diarrhea virus cells, unsatisfactory protection effect of newly circulating viruses, and inability to protect newborn suckling piglets, etc. The effect of virus protection is good, the survival rate of piglets is improved, and the effect of increasing daily weight gain

Active Publication Date: 2017-04-26
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, vaccine immunization is the main method of prevention and treatment of the disease, but the existing commercial vaccines can only be used to immunize weaned piglets (about 30 days old), and cannot provide protection for newborn suckling piglets (newborn to two-week-old piglets) In addition, there are certain differences in gene sequence between the existing vaccine strains and the newly epidemic strains in China, and the protective effect on the challenge of newly epidemic viruses is not ideal; it is difficult for porcine epidemic diarrhea virus cells to adapt, and the breeding conditions are complicated. The low titer of culture and reproduction in cells has seriously hindered the prevention and control of PED disease

Method used

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  • PEDV (porcine epidemic diarrhea virus), inactivated vaccine and preparation method of inactivated vaccine
  • PEDV (porcine epidemic diarrhea virus), inactivated vaccine and preparation method of inactivated vaccine
  • PEDV (porcine epidemic diarrhea virus), inactivated vaccine and preparation method of inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0028] Isolation and Identification of Example 1 Porcine Epidemic Diarrhea Virus NJ Strain

[0029] 1. Isolation of NJ strain

[0030] (1) disease material

[0031] The disease material is the small intestine and its contents of the suspected porcine epidemic diarrhea disease dead piglet collected aseptically. The M gene was amplified by RT-PCR to obtain an amplified product with a length of 476bp, which was completely consistent with the size of the designed fragment, and the sequence was shown in SEQ ID NO:1. At the same time, the PCR detection method was used to detect whether the disease material was infected with other pig common viruses, and the results were as follows: figure 1 , no other exogenous viruses were detected.

[0032] (2) Disease material treatment

[0033] Cut the above disease material into pieces in a sterile container, grind, dilute 1:5 with sterilized PBS buffer containing penicillin, freeze and thaw three times, centrifuge at 3000rpm / min for 20min,...

Embodiment 2

[0054] The virulence test of embodiment 2 porcine epidemic diarrhea virus NJ strain

[0055] 7 piglets at the age of 5, 25 and 45 days were purchased, and 5 piglets of the same age were randomly selected as the test group, and the other two were used as the control group. Piglets of all ages were artificially fed with milk, and 100 IU penicillin and streptomycin were orally administered to each piglet 8 hours before challenge. Attacking method: Each piglet in the test group was orally administered 0.25 mL of porcine epidemic diarrhea virus NJ strain virus liquid (poison price 10 5 TCID 50 / mL) to challenge the virus, isolate and raise after the challenge, observe the clinical symptoms within 10 days after the challenge, dissect the piglets that died of diarrhea, observe the pathological changes, collect the small intestine disease material and use RT-PCR to amplify the M gene after grinding. To detect whether they are infected with porcine epidemic diarrhea virus NJ strain. ...

Embodiment 3

[0057] Embodiment 3 prepares porcine epidemic diarrhea virus NJ strain inactivated vaccine

[0058] Prepare porcine epidemic diarrhea virus NJ strain inactivated vaccine (abbreviated as NJ strain inactivated vaccine), concrete method is as follows:

[0059] (1) Preparation of porcine epidemic diarrhea virus NJ strain virus liquid

[0060] Inoculate the Vero cell fermenter with porcine epidemic diarrhea virus NJ strain, harvest the cytotoxic antigen solution, and take the virus content as 10 8.0 TCID 50 / mL or more of porcine epidemic diarrhea virus NJ strain virus liquid to prepare NJ strain inactivated vaccine. The preparation method of porcine epidemic diarrhea virus NJ strain virus liquid is specifically as follows:

[0061] Cell recovery: Prepare cell growth medium: add 7 mL of calf serum to 100 mL of DMEM medium, and also add penicillin and streptomycin; each ml of cell growth liquid contains 100 units of penicillin and 100 units of streptomycin. Take the Vero cells o...

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Abstract

The invention provides a PEDV (porcine epidemic diarrhea virus), an inactivated vaccine and a preparation method of the inactivated vaccine and belongs to the field of bioengineering. The collection registration number of a PEDV NJ strain is CGMCC NO.13283. The invention provides an application of the virus strain in preparation of a PED vaccine, a preparation method of a virus solution of the virus strain as well as the obtained virus solution and the inactivated vaccine. The preparation method of the inactivated vaccine comprises steps as follows: the inactivated virus solution and tween-80 in the volume ratio being 96:4 are mixed uniformly, and a water phase is obtained; white oil for injection and span-80 are mixed uniformly in the volume ratio being 96:4 and an oil phase is obtained; the water phase and the oil phase are mixed in the volume ratio being 1:(2-3) and then emulsified. The NJ strain is a recent prevalent virus, has good immunogenicity, adapts to cell culture and can be subjected to large-scale fermenting culture, and pregnant sows inoculated with the inactivated vaccine of the virus strain can effectively protect newly-born suckling pigs during toxicity attacking by the recent prevalent virus.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a porcine epidemic diarrhea virus, an inactivated vaccine and a preparation method thereof. Background technique [0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by PED virus (PEDV), characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. Since 2000, the disease has been prevalent throughout the country, especially since 2010, the disease has re-emerged in immunized pigs, characterized by a high incidence of suckling piglets, and has brought great harm to my country's pig industry. Porcine epidemic diarrhea has high mortality in piglets from birth to two weeks of age. At present, vaccine immunization is the main method of prevention and treatment of the disease, but the existing commercial vaccines can only be used to immunize weaned piglets (about 30 days old), and cannot provide prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/215A61P31/14
Inventor 陈瑾郑其升于晓明侯立婷侯继波
Owner JIANGSU ACAD OF AGRI SCI
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