Method for preparing swine epidemic encephalitis B inactivated vaccine

A technology of Japanese encephalitis and inactivated vaccines, applied in the field of vaccines, can solve problems affecting the quality of hamster primary cell batch-to-batch stability, concerns about vaccine side effects and immunity, and incomplete product safety guarantees, and achieve improved Batch-to-batch stability, small destructive effects, and stable immunogenicity

Inactive Publication Date: 2014-12-17
TIANJIN RINGPU BIO TECH
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The inactivated vaccine is mainly rat brain tissue inactivated vaccine (HW1 strain), which contains more rat brain tissue components, and the side effects and immunity of the vaccine are worrying; hamster kidney primary cell culture attenuated virus (JEVSA14-14- 2 strains) prepared live vaccines with small side effects and good effects, but the primary cells of hamsters come from the living tissues of clean experimental animals, and the complexity of the animals affects the safety of the cells and increases the chance of exogenous virus contamination. A large number of experimental animals must be purchased in each production cycle, and the batch-to-batch variance of experimental animals has seriously affected the quality stability of the hamster primary cells used for vaccine production.
Both of the above two production methods have the disadvantages of high production cost, poor product stability, incomplete guarantee of product safety, and restrictions on large-scale production.

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  • Method for preparing swine epidemic encephalitis B inactivated vaccine
  • Method for preparing swine epidemic encephalitis B inactivated vaccine
  • Method for preparing swine epidemic encephalitis B inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 The comparison experiment of different cell culture porcine encephalitis virus

[0030] 1. Experimental method:

[0031] Cell preparation: Prepare primary hamster kidney cells according to the "Trial Procedures for the Manufacture and Inspection of Live Attenuated Japanese Encephalitis Vaccine of Porcine Japanese Encephalitis", 96-well culture plate, Chinese hamster lung cells (V79 strain) plated at a concentration of 100,000 to 200,000 / mL .

[0032] Toxic value determination: take the venom for 10-fold serial dilution, take 10 -5 、10 -6 、10 -7 、10 -8 Inoculate plated cells with 4 dilutions of virus liquid, inoculate 5-8 wells for each dilution, and set up non-inoculated control cell wells at the same time, culture at 36-37°C for 7 days, observe cell lesions, and calculate TCID 50 .

[0033] 2. Test results:

[0034] (1) Comparison of cultured virus titers

[0035] Result analysis: The titer of JEV virus cultured by Chinese hamster lung cells (V79 s...

Embodiment 2

[0040] Example 2 Pathogenicity comparison test of JEV virus after continuous passage on Chinese hamster lung cells

[0041] Take Chinese hamster lung cells (V79 strain) to culture JEV virus, and measure cytopathic TCID respectively 50 and PFU, LD causing mouse death 50 , to compare the virulence changes of the virus after serial passage on the cells.

[0042] The results showed that: JEV virus was continuously cultured in Chinese hamster lung cells for 20 generations, and the virulence changed little, and the virulence did not return to strong (see Table 2).

[0043] Table 2 Changes in virulence of JEV virus after continuous passage on Chinese hamster lung cells (V79 strain)

[0044]

Embodiment 3

[0046] A method for preparing porcine epidemic Japanese encephalitis inactivated vaccine, comprising the following steps:

[0047] (1) Virus seed preparation: select the virus in the hamster lung cell line to adapt to the cell virus seed (JEV P3 strain) identified by cloning, select the first 3 generations of virus as the virus seed for production, and the virus content per milliliter is ≥10 7.5 TCID 50 , Store below -20°C.

[0048] (2) Cell culture and inoculation: Chinese hamster lung cells (V79 strain) were passaged at a ratio of 1:2 to 1:6. When the cells grew into a dense monolayer, the Chinese hamster lung cells that had grown into a dense monolayer ( V79 strain) was washed twice with Hank's solution, then the virus seed diluted with serum-free RPMI 1640 was added, inoculated at an MOI of 0.1, and adsorbed and cultured for 30 min to 1 h, then replaced with RPMI 1640 containing 4% bovine serum, adding 0.05% Arginine and 200IU antibiotics, pH adjusted to 7.4-7.6, culture...

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Abstract

The invention provides a method for preparing a swine epidemic encephalitis B inactivated vaccine. According to the method, a Chinese hamster lung cell adaptive virus screened by bacteriophage plaque cloning and purification is capable of generating cytopathic effect, the virulence is stabilized at 107.0-108.0TCID50/mL, and the immunogenicity is stable; the swine epidemic encephalitis B virus is multiplied by utilizing the hamster pulmonary cell system has good virus adaptability and high virus virulence of prepared vaccine. According to the method, a virus concentrating process technology which sequentially performs separating and concentrating is adopted, and virus solution and cell debris can be separated by utilizing a hollow fibrous membrane clarifying and purifying system, so that the problems that the membrane is easily blocked, is sensitive to shearing force and easily aggregated when virus particles are concentrated can be solved, side effects of an inactivated vaccine finished product can be effectively reduced, and the safety of the inactivated vaccine can be improved. Binary ethyleneimine is used as an inactivator instead of formaldehyde and has small destruction on virus antigen, so that the completeness of effective immunogen in the antigen can be guaranteed.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a method for preparing an inactivated vaccine against porcine epidemic Japanese encephalitis. Background technique [0002] Porcine Japanese encephalitis is a zoonotic infectious disease caused by Japanese encephalitis virus. After infection, pigs manifested as high fever, abortion, stillbirth and boar orchitis. The disease is transmitted by mosquitoes and is often prevalent in late summer and early autumn, with obvious seasonality. From the first discovery in Japan in 1871 to the isolation of Japanese encephalitis virus in Japan in 1934, there have been more than a dozen pandemics. The disease is widely distributed, mainly occurs in Asian countries, and occurs in many areas of my country. It is a zoonotic disease and is very harmful. It is also one of the infectious diseases that our country focuses on prevention and control. [0003] At present, there are two types of dome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12C12N7/00C12N7/02A61P31/14
Inventor 吕茂杰刘涛杨保收盛长忠梁武
Owner TIANJIN RINGPU BIO TECH
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