Method for preparing thermal-stability vaccine

A virus and calcium phosphate technology, applied in biochemical equipment and methods, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve the problems of limited protective effect and complicated operation process, and achieve simple methods, improve immune prototype, The effect of enhanced stability

Active Publication Date: 2012-11-21
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the above steps have the limitations of complicated operation process and limited protection effect

Method used

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  • Method for preparing thermal-stability vaccine
  • Method for preparing thermal-stability vaccine
  • Method for preparing thermal-stability vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, preparation JEV vaccine liquid

[0045] 1. JEV vaccine solution

[0046] 1. Infect BHK-21 cells with Japanese encephalitis virus SA14-14-2 strain (using low-sugar DMEM medium), and incubate at 37°C for 6 hours (in practical applications, incubation for 2-8 hours is acceptable).

[0047] 2. Centrifuge the culture system obtained in step 1 (16000g, 10min), collect the supernatant and filter it (using a filter membrane with a pore size of 0.22 microns), and collect the filtrate, which is the virus liquid (after testing, the pH of the virus liquid is 7.0 ), named it JEV vaccine liquid.

[0048] 2. Zeta potential of JEV vaccine solution

[0049] Take the JEV vaccine liquid obtained in step 1, adjust it to reach different pHs, and then measure the surface Zeta potential of virus particles. The results showed that within the pH range of 6.0-9.5, the surface of the virus particles was negatively charged and could absorb cations.

Embodiment 2

[0050] Example 2, using the JEV vaccine solution prepared in Example 1 to prepare the JEV-CaPi vaccine solution (that is, to prepare a heat-stable vaccine by in situ mineralization)

[0051] One, the preparation of JEV-CaPi vaccine liquid-A

[0052] In the JEV vaccine solution (the virus concentration is 2×10 7 PFU / mL, that is, 2×10 10 PFU / L) by adding CaCl 2 , so that Ca 2+ The ion concentration reaches 5.55839mM (including the newly added Ca 2+ Ions and Ca in JEV vaccine solution 2+ Ions; Ca in JEV vaccine solution 2+ The ion is theoretically equal to the Ca in the low-sugar DMEM medium 2+ ions, that is, the concentration is 1.80839mM), which is the initial system; incubate the initial system at 37°C for 2 hours, which is the terminated system; add polyacrylic acid to the terminated system to make the concentration reach 10ug / mL, which is the JEV-CaPi vaccine Liquid-A-I; Centrifuge JEV-CaPi Vaccine Liquid-A-I (16000g, 10min), collect the supernatant and precipitate re...

Embodiment 3

[0096] The biological characteristic experiment of embodiment 3, JEV-CaPi vaccine liquid

[0097] 1. Plaque characteristics

[0098] The JEV-CaPi vaccine solution-C-II and the JEV vaccine solution were respectively subjected to the following experiments:

[0099] Diluted 10 times by volume (10 -1 、10 -2 、10 -3 、10 -4 , 10 -5 、10 -6 and 10 -7 ), and then inoculated at 500 μl / well on a monolayer of BHK-21 cells spread on a 6-well plate, placed at 37°C, 5% CO 2 Incubate in an incubator for 2 hours; then discard the virus solution, cover the cells with agar cover (that is, low-sugar DMEM medium containing 1% agar and 2% FBS), and place at 37°C, 5% CO 2 Incubate in an incubator for 3 days; then fix with 4% formaldehyde solution at room temperature for 1 hour, discard the agar cover, and stain with crystal violet for 10 minutes at room temperature; observe the plaque morphology and take pictures, and calculate plaque forming units (PFU); vaccine titer = (empty Plaque formin...

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Abstract

The invention discloses a method for preparing thermal-stability vaccine and particularly relates to a method for preparing thermal-stability vaccine based on calcium phosphate mineralization. The invention provides a method for preparing calcium phosphate coated viruses. The method comprises the following steps that (1) compounds with Ca<2+> ions are added into virus liquid, an initial system is obtained, and PO4<3-> ions are contained in the virus liquid; (3) the initial system is subjected to incubation; and (3) precipitation is centrifugally collected, and the calcium phosphate coated viruses are obtained. The method has the advantages that through virus surface mineralization modification, the problem of heat inactivation phenomenon in the vaccine transportation and storage process can be effectively solved, and the financial expenditure of the attenuated live vaccine is obviously reduced. The method can be widely applied to thermal-stability vaccine production and preparation.

Description

technical field [0001] The invention relates to a method for preparing a thermostable vaccine, in particular to a method for preparing a thermostable vaccine based on calcium phosphate mineralization. Background technique [0002] Infectious diseases pose a serious threat to public health and cause millions of deaths every year. Vaccination, as the most effective medical treatment against infectious diseases, has saved countless lives. Live attenuated vaccines (that is, attenuated strains of the virus) are the most important form of vaccine and have played a huge role in the eradication of smallpox and polio. Live attenuated vaccines still in clinical use include polio vaccine, varicella vaccine, influenza vaccine, yellow fever vaccine, measles vaccine, mumps vaccine, rubella vaccine, Japanese encephalitis vaccine, rotavirus vaccine, hepatitis and adenovirus vaccines. The activity of the attenuated live vaccine becomes the pathogen of (1) or (2) or (3): (1) After the path...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/00A61K47/02C12N7/00A61P31/14
Inventor 王广川秦成峰唐睿康
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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