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Subunit influenza vaccine split agent and application thereof

A technology of influenza vaccine and subunit, which is applied in antiviral agents, antisense single-stranded RNA viruses, medical preparations containing active ingredients, etc., and can solve problems such as vaccine side effects

Active Publication Date: 2020-10-23
天津中逸安健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now most of the lysing agents of influenza virus vaccine products are TRITON-X100, TWEEN-80, etc., and the lysed products contain more miscellaneous proteins, and the purity of HA in the products is often lower than 80%, especially the obvious M protein or N protein recovery, and M protein or N protein can cause significant side effects of the vaccine

Method used

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  • Subunit influenza vaccine split agent and application thereof
  • Subunit influenza vaccine split agent and application thereof
  • Subunit influenza vaccine split agent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Preparation of virus particles: take 9-11 day-old chicken embryos and inoculate them with influenza virus virus seeds (the virus type A H1N1 is purchased from NIBSC in the UK, and the virus strains are determined by the annual epidemic strains, according to the seasonal influenza virus distribution experiment stipulated by WHO room), the purchased viruses were thawed in a 37°C water bath, and then inoculated into the allantoic cavity of chicken embryos; cultivated at 35°C for 72 hours, harvested the allantoic fluid, and passed through 0.1m 2 Ultrafiltration membrane bag (product of PALL company, molecular weight cut-off: 100 KDa) was concentrated to one-tenth of the original volume by ultrafiltration, and set aside.

Embodiment 2

[0036] Preparation of virus particles: take 9-11 day-old chicken embryos and inoculate them with influenza virus virus seeds (the virus type B YAMAGATA was purchased from NIBSC in the UK, and the virus strains are determined by the annual epidemic strains, according to the seasonal influenza virus distribution experiment stipulated by WHO room), the purchased viruses were thawed in a 37°C water bath, and then inoculated into the allantoic cavity of chicken embryos; cultivated at 35°C for 72 hours, harvested the allantoic fluid, and passed through 0.1m 2 The ultrafiltration membrane bag (product of PALL company, molecular weight cut-off 1000 KDa) was concentrated to one-eighth of the original volume by ultrafiltration, and was set aside.

Embodiment 3

[0038] Preparation of virus lysate:

[0039] Take 2.9 g of sodium hydroxide and 13 g of sodium dihydrogen phosphate, stir and dissolve in 500 ml of distilled water, then weigh nonoxynol-9 by 1.5 g, stir and dissolve, add 600 g of sucrose, after dissolving, add water to 1000 ml (liquid A);

[0040] Another 2.9 g of sodium hydroxide and 13 g of sodium dihydrogen phosphate were stirred and dissolved in 500 ml of distilled water, then 1.5 g of nonoxynol-9 was weighed, stirred and dissolved, and 100 g of sucrose was added. After dissolving, add water to 1000 ml ml (liquid B);

[0041] Add solution A to a zonal centrifuge rotor with a rotational speed of 10000×g and centrifuge for 5 minutes, then add solution B; continue to centrifuge at 25000×g for 45 minutes to form a density gradient and then continuously add 5000 mL of the A / H1N1 virus concentrate prepared in Example 1 After adding the sample, continue to centrifuge at 4°C for 2 h; then add 100 ml of 60% sucrose solution, and ...

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Abstract

The invention discloses a subunit influenza vaccine split agent and application thereof, and belongs to the technical field of vaccine production. The subunit influenza vaccine split agent comprises nonoxynol-9 with a mass concentration of 0.1-5%, sodium hydroxide with a mass concentration of 1-5 g / L, sodium dihydrogen phosphate with a mass concentration of 5-20 g / L, sucrose with a mass concentration of 10-60% and the balance distilled water. According to the subunit influenza vaccine split agent and application thereof, the split agent is subjected to a density gradient centrifugation process, due to the fact that the split agent with the formula has the character of efficiently splitting influenza viruses, virus particles do not need to be split in advance, and are split in the centrifugation process instead, that is to say, one-step splitting is achieved, thus a purification treatment process is shortened, no obvious M protein bands or N protein bands appear in a product PAGE electrophoresis, and the purity of a main antigen component HA of a vaccine is higher than 90%, and is higher than those of products which are obtained by adopting aforehand splitting and other split agentformulas.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to a subunit influenza vaccine cracking agent and its application. Background technique [0002] After the influenza virus is purified, it will be cracked with a lysing agent. In fact, the simple point of cleavage is to use the chemical action of the lysing agent to "decompose" the virus. The complete virus is dissociated into "fragments". The order in which the influenza virus is dissociated For bilayer lipid membrane & hemagglutinin, neuraminidase, matrix protein, nucleoprotein. Because hemagglutinin has the largest molecular weight, lysing agents and other impurity proteins can be removed by ultracentrifugation and ultrafiltration after lysis. [0003] In the production process of subunit influenza vaccine, in order to ensure the purity of the antigen, only the lipid membrane on the surface of influenza virus must be lysed, and the HA on the surface must be separated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16
CPCA61K39/12A61K2039/5258A61P31/16C12N2760/16234
Inventor 高辉阮承迈李凤凯赵斌
Owner 天津中逸安健生物科技有限公司
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