Porcine rotavirus strain and inactivated vaccine prepared from strain as well as application

A porcine rotavirus inactivation technology, applied in vaccines, viruses, antiviral agents, etc., can solve the problems of product batch differences, high production costs, poor safety, etc., and achieve small differences between product batches and good immunity The effect of originality and low production cost

Active Publication Date: 2018-05-04
JILIN ZHENGYE BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the porcine rotavirus vaccines available on the market are live vaccines prepared by the spinner bottle cultu

Method used

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  • Porcine rotavirus strain and inactivated vaccine prepared from strain as well as application
  • Porcine rotavirus strain and inactivated vaccine prepared from strain as well as application
  • Porcine rotavirus strain and inactivated vaccine prepared from strain as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Isolation and identification of porcine rotavirus zyh25 strain

[0023] 1. Experimental method

[0024] 1.1 Isolation of virus

[0025] In 2016, from pigs suspected of having porcine rotavirus in a pig farm in Jilin Province, China, the small intestine tissue of sick pigs with typical clinical symptoms was aseptically cut into pieces, and added to sterilized pigs at a ratio of 1:5 (W / V) After normal saline and mortar grinding, freeze-thaw three times at -40°C / room temperature, centrifuge at 3000r / min for 15min, and take the supernatant for later use.

[0026] The supernatant of the above-mentioned disease material was sterilized through a 0.22 μm microporous filter, and inoculated with the cell maintenance solution containing trypsin at a final concentration of 10 μg / ml (serum-free DMEM medium containing 20,000 units of double antibody), and the Marc-145 For the T25 square flask of monolayer cells, inoculate 4 flasks of cells with each disease material, and ...

experiment example 1

[0046] Pathogenicity determination of the isolated porcine rotavirus zyh25 strain of Experimental Example 1

[0047] 1. Experimental method

[0048] 1.1 Proliferation of virus zyh25 strain in serum-free full suspension Marc-145 cells

[0049] After diluting the purified virus zyh25 strain with sterilized physiological saline, inoculate the cell density at 3-5*10 6 Cells / ml of serum-free fully suspended Marc-145 cells, in a 5% carbon dioxide incubator at 37°C, suspended on a shaker at 110rpm for 60-72 hours, when the number of dead diseased cells exceeds 75%, harvest the cell suspension and put it into- Freeze and thaw three times in a 70°C refrigerator to harvest the virus liquid. Passage 10 times until the virus proliferates well in Marc-145 cells suspended in serum-free whole.

[0050] 1.2 Virus median infective dose (TCID 50 ) determination

[0051] Marc-145 cells in good condition were mixed with 2~3×10 4 Inoculate a 96-well cell culture plate per well at 37°C, 5% CO...

experiment example 2

[0062] Experimental example 2 Immune protection experiment of porcine rotavirus zyh25 strain

[0063] 1. Experimental method

[0064] 1.1 Amplification of virus

[0065] The isolated virus zyh25 strain was amplified by Marc-145 cell roller bottle culture (collecting the cell culture supernatant) and inoculated with serum-free full suspension culture Marc-145 cells (collecting the culture supernatant), and the culture supernatant was harvested and separated. Packed and stored at -70°C for later use.

[0066] 1.2 Preparation of inactivated vaccine

[0067] The two virus liquids amplified by the spinner bottle process and the serum-free full suspension process were centrifuged at 5 000 r / min for 30 min, the supernatant was taken, and formalin solution with a final concentration of 0.1% (v / v) was added to inactivate at 37°C 48h. After the sterility test, the inactivated virus liquid supernatant was mixed with Marcol 52 white oil adjuvant at a volume ratio of 1:2.5, emulsified ...

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Abstract

The invention discloses a porcine rotavirus strain and an inactivated vaccine prepared from the strain as well as application. The invention firstly discloses a separated porcine rotavirus zyh25 strain, of which the microbial preservation number is CGMCC No. 13792. The invention further discloses application of the porcine rotavirus zyh25 strain in the preparation of a vaccine for preventing porcine rotavirus diseases. Moreover, the invention discloses a vaccine composition for preventing the porcine rotavirus diseases, and the vaccine composition is prepared from an effective quantity of inactivated porcine rotavirus zyh25 strain used for prevention and treatment and an adjuvant which can be accepted in pharmacy. The porcine rotavirus zyh25 strain separated in the invention has good immunogenicity when serving as a vaccine strain, can generate a relatively high antibody level after used for immunizing an animal and can tolerate the attack of a virulent strain. The porcine rotavirus zyh25 strain can be used for serum-free full-suspended continuous production, and the prepared inactivated vaccine has the advantages of low production cost, small difference between product batches andthe like.

Description

technical field [0001] The present invention relates to an isolated porcine rotavirus strain, and also relates to an inactivated vaccine prepared from said porcine rotavirus strain and its application in preventing and treating porcine rotavirus infection, belonging to the isolation of porcine rotavirus strains and application fields. Background technique [0002] Porcine Rotavirus (Porcine Rota Virus, PoRV) disease is a diarrheal disease of piglets caused by rotavirus infection. There are many types of rotaviruses, with different serotypes and genotypes, and the host spectrum of infection is very wide. Humans, chickens, pigs, cattle, horses and other animals are all infected at the young stage, and the main clinical manifestations are diarrhea and diarrhea. It can synergize with other pathogenic microorganisms to cause secondary infection, thereby causing high morbidity and mortality in pig herds, which is very harmful to the pig industry in various countries. [0003] Th...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/15A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2720/12321C12N2720/12334C12N2720/12351
Inventor 王国辉董雅文贾小营张微杨泽斌李长艳朱俊辉于志刚王英祺廉唯王石何玉友
Owner JILIN ZHENGYE BIOLOGICAL PROD
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