Pig PRRS purified vaccine and preparation method thereof

A technology for porcine PRRS and vaccines, applied in biochemical equipment and methods, recovery/purification, viruses, etc., can solve the problems of low virus content in vaccines, short cell maintenance time, and the impact of porcine PRRS immune prevention and control work, etc.

Active Publication Date: 2019-12-06
QILU ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing porcine PRRS vaccine mainly has the following three problems: 1. Immunity protection is unstable, mainly manifested in: after immunization, no neutralizing antibody or antibody level is very low, or the immunization period is shorter, only 2-2 3 months; 2. Large immune side effects, mainly: the downstream production process is still the traditional method, without purification treatment, there are some impurities and allergic substances in the serum and host cells in the vaccine, and the effective antigen content is low
3. The virus content of the vaccine is low. The existing large-scale production of PRRS virus mostly adopts the rotary bottle process, but there are problems such as intensive manual operation, short cell maintenance time, low density and vitality in the production process, which limits the output and stability of the product. sex
This has a great impact on the immune prevention and control of porcine PRRS

Method used

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  • Pig PRRS purified vaccine and preparation method thereof
  • Pig PRRS purified vaccine and preparation method thereof
  • Pig PRRS purified vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] ——Full suspension culture of Marc-145 cells

[0036] Use shake flasks to resuscitate the cells frozen in liquid nitrogen, and when the cells grow to the required amount, inoculate the cell bioreactor for culture.

[0037] Specific steps are as follows:

[0038] (1) The Marc145-QLS strain cells frozen in liquid nitrogen were taken and melted in a water bath at 37°C, then inoculated into the shake flask culture medium, and cultured at 37°C, with the rotation speed set at 120r / min. Cells were cultured for 3 to 4 days after recovery by 0.5×10 6 cells / ml seeding density for subculture expansion.

[0039] (2) Dilute the cells expanded in the above step (1) to 1.0×10 6 The bioreactor was inoculated at a density of 1 / ml, and the temperature was 27° C., pH 7.2, DO 50%, and the rotation speed was 120 r / min. The growth of the cells meets the requirements of cell proliferation, and is close to the proliferation speed, cell viability, and plateau phase of the cells cultured in s...

Embodiment 2

[0043] ——Marc145-QLS Cell Culture Attenuated PRRS Virus Strain

[0044] The cell density at the time of inoculation was 2.5×10 6 Individuals / ml, 0.1% inoculation of porcine reproductive and respiratory syndrome virus JXA1-R strain, when the cell viability is less than 70%, take a sample to determine the virus titer (TCID 50 / ml), the results are shown in Table 2.

[0045] Table 2 Virus titers inoculated with different cell densities

[0046]

Embodiment 3

[0048] ——Purification of PRRS virus cultured in Marc145-QLS cells

[0049] The harvested cell suspension is clarified or centrifuged by continuous flow to remove impurities such as cell debris. According to the size of the PRRS virus, an ultrafiltration membrane with a molecular cut-off of 100KD can be selected to concentrate the virus solution 50-100 times. The high-power concentrated solution is placed on a Separate6FF chromatography column, equilibrated with pH 7.0-7.4, 0.01M PBS buffer, and then subjected to column chromatography. The eluate is detected with a UV spectrophotometer A280nm, and the virus flows out. Collect and store at 4°C , to obtain purified virus liquid.

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Abstract

The invention relates to a porcine reproductive and respiratory syndrome purified vaccine and a production method thereof and also relates to a full-suspension cell strain of Marc-145 and an application thereof in preparing a porcine reproductive and respiratory syndrome vaccine. The porcine reproductive and respiratory syndrome purified vaccine is prepared by the following steps: 1) suspension domestication for obtaining a full-suspension cell strain of Marc-145; 2) serum-free suspension culture of vaccine preparation cells; 3) culture and harvesting of a porcine reproductive and respiratory syndrome virus; 4) purification of virus liquid; and 5) vaccine preparation. By adopting the method to prepare the vaccine, the problems such as low antigen content, serum residue and cell residue of the existing vaccine are solved, the immune efficacy of the vaccine can be effectively improved, and the allergic reaction is reduced. In the method, Marc-145 cells are subjected to full-suspension culture by a serum-free medium to produce the porcine reproductive and respiratory syndrome virus, and the virus is concentrated and purified in combination with ultrafiltration equipment; and the method is a simple, cheap and efficient purifying method.

Description

technical field [0001] The invention relates to a purified vaccine against porcine blue-ear disease and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Pig blue-ear disease, also known as porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS), is a viral infectious disease that endangers the pig industry. It is a new type of infectious disease that broke out in the late 1980s. The occurrence of the disease was first reported in the United States in 1987. It was called Mysteryswinedisease (MSD) at that time. (Yan Yulin, Gao Hong. Research progress in diagnostic techniques for porcine reproductive and respiratory syndrome [J]. Advances in Veterinary Medicine, 2008, 29(7): 81-85) (Grebennikova T V, Clouser D F, Vorwald A C, et al. Genomic characterization of viral , attenuated, and revertant passages of a North American porcine reproductive and respiratory syndrom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12A61P31/14C12N7/00C12N7/02
CPCA61K39/12A61K2039/5252C12N7/00C12N2770/10034C12N2770/10051
Inventor 王蕾巩志宏于宗幸魏联果高小龙
Owner QILU ANIMAL HEALTH PROD
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