Purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography

An ion exchange chromatography and rotavirus technology, applied in the field of preparing virus inactivated vaccines, can solve the problems of virus particle damage, high cost, high equipment requirements, etc., and achieve the effects of good stability and low cost

Active Publication Date: 2012-07-11
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

In recent related literature, the purification of rotavirus generally adopts CsCl density gradient centrifugation or sucrose density gradient centrifugation to obtain high-purity virus particles, "Cesium Chloride Density Gradient Centrifugation Purification of Rotavirus" (Wen Yuling, Zhao Qinghuan, Yu Yang, Chen Yuanding. Chinese Journal of Modern Internal Medicine. Issue 3, 2005. Pages 195-197), "Immunogenicity of a thermally inactivated rotavirus vaccine in mice" (Baoming Jiang, Yuhuan Wang, Jean-Francois Saluzzo, et al. Human Vaccines. 2008 March / April; 4(2): 143-147.) However, the former CsCl density gradient centrifugation requires high equipment and is expensive; the latter centrifugation in sucrose medium will cause damage to virus particles and easily lead to loss of virus infection sucrose is highly viscous and highly permeable, which can be toxic to cells, and it needs to be removed before the sample is analyzed for infectious titer
In addition to the above problems, the two methods are cumbersome and time-consuming to operate, and their purification is not easy to scale up, and both of them are on the scale of laboratory research.

Method used

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  • Purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography
  • Purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography
  • Purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography

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Experimental program
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Effect test

Embodiment 1

[0039] 1. Preparation and concentration of virus liquid

[0040] The method of spinner bottle culture was used to amplify the virus on Vero cells, and 45 spinner flasks were cultivated on a large scale. When the cells were a dense monolayer, the virus was seeded, and the cell density was as follows: 1.33×10 5 cells / cm 2 ×850cm 2 / Rotary bottle=1.1×10 8 cells / spinner bottle; harvest the virus when the virus in the Vero cell culture medium multiplies for 72 hours, after repeated freezing and thawing 3 times, centrifuge at 8000rpm for 20min to get the supernatant and harvest 9.8 L virus stock solution containing virus particles, the protein content of the virus stock solution is between 600ug / mL~700ug / mL, the virus infectivity titer is above 4.0lgCCID50 / mL; use the Pellicon tangential flow ultrafiltration system with a molecular weight cut-off of 100KDa to concentrate the 9.8L virus stock solution by ultrafiltration 36 times to 0.27L for further analysis. Purified by Q Sepharo...

Embodiment 2

[0053] Except for the following steps of detecting antigenicity and total protein removal rate before and after virus purification, the remaining steps are the same as in Example 1

[0054] unanimous.

[0055] (a) Detection of infectious titer and antigenicity before and after virus purification

[0056] Fluorescence focus assays (FFA) were used to measure the infectious titer and antigenicity before and after virus purification:

[0057] Take a 10-fold serial dilution of the virus and inoculate MA104 monolayer cells for CCID 50 detection. Divide MA104 cells into 1.5×10 4 cells / well were seeded on a 96-well plate at 37°C, 5% CO 2 Cultivate in the incubator until it grows into a dense monolayer, add 10-fold serially diluted virus, the dilution is from 10 -1 ~10 -8 , 10 wells were set for each dilution, after adsorption at 37°C for 1 hour, DMEM cell maintenance solution without calf serum was added, after incubation at 37°C for 16 hours, an equal volume of formaldehyde sol...

Embodiment 3

[0070] Except for the SDS-PAGE and Western-blot detection steps of the virus liquid in the following purification process, the remaining steps

[0071] Consistent with embodiment 1 or embodiment 2. The detection steps are as follows:

[0072] Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis: 5% stacking gel, 10% separating gel. The purified rotavirus sample was mixed with 6× loading buffer, lysed by heating at 95°C for 5 min, loaded with 15 μl of sample, and electrophoresed at 90V. Coomassie brilliant blue staining was used to detect the purification effect.

[0073] Western-blot: Purified rotavirus protein was separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. First, the membrane was blocked for 2 h with TBST solution containing 5% skimmed milk powder. Then use 1:500 dilution of guinea pig anti-rotavirus immune serum in TBST buffer to bind to the protein; finally use 1:2000 horseradish peroxidase-labeled goat...

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Abstract

A purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography relates to a preparation method of virus inactivated vaccines. The purification preparation method comprises concentrating virus harvest liquid, performing chromatographic purification on the concentrated virus harvest liquid through a Qsepharose Fast Flow ion exchange column, performing dialysis desalting on a purified product, and filtering, degerming, inactivating and the like to obtain the human rotavirus inactivated vaccines; and further detecting the purification, the totalprotein removal rate and the infectious titer, and then detecting the antigenicity, the immunogenicity and the genome banding pattern stability. The total protein removal rate of the purified virus harvest liquid is 99.69%, and the infectious titer of the virus before purification and the infectious titer of the virus after purification are respectively 4.25 lg CCID 50 / ml and 7.0 lg CCID 50 / ml, the genome banding pattern of purified virus after inactivation does not mutate, and antigenicity and immunogenicity are kept to be good.

Description

technical field [0001] The invention belongs to the method for preparing virus inactivated vaccine, especially the method for preparing human rotavirus inactivated vaccine. Background technique [0002] Rotavirus ( Rotavirus , RV ) is the most important pathogen causing viral diarrhea in infants and young children. Since there is no specific treatment method, vaccination has become the most cost-effective method, but there are still some problems in the prevention of rotavirus infection with the currently used attenuated live vaccines. Therefore, the injection inactivated rotavirus vaccine used for medical purposes has its special significance. In recent related literature, the purification of rotavirus generally adopts CsCl density gradient centrifugation or sucrose density gradient centrifugation to obtain high-purity virus particles, "Cesium Chloride Density Gradient Centrifugation Purification of Rotavirus" (Wen Yuling, Zhao Qinghuan, Yu Yang, Chen Yuanding. Chinese J...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/15G01N33/569C12N7/02A61P31/14C12R1/93
Inventor 孙茂盛李鸿钧吴晋元杨星易山张光明
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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