阅读说明：本技术 一种禽流感h9亚型疫苗生产中的灭活病毒的方法 (Method for inactivating virus in production of avian influenza H9 subtype vaccine ) 是由 张文炎 刘之文 詹烜子 刘锦容 高艳 张云丽 王土活 于 2020-06-30 设计创作，主要内容包括：本发明提供一种禽流感H9亚型疫苗生产中的灭活病毒的方法,通过对病毒灭活工艺的优化,可以显著提高疫苗制剂的安全性,减少应激反应,提高免疫效果。(The invention provides a method for inactivating virus in the production of avian influenza H9 subtype vaccine, which can obviously improve the safety of vaccine preparation, reduce stress reaction and improve immune effect by optimizing the virus inactivation process.)

1. A method for inactivating virus in the production of avian influenza H9 subtype vaccine is characterized in that: the method comprises the following steps:

(1) inoculating and culturing virus, selecting LMH cell for preparing vaccine, subculturing until LMH cell density reaches 1.0 × 10⁶-1.5×10⁶Respectively inoculating avian influenza virus H9 subtype in each/mL, and adding cell maintenance liquid for propagation culture;

(2) virus collection, concentration and purification: freezing and thawing the cell culture solution in the step (1) for 3 times, centrifuging for 10-20min at 4 ℃, 6000-8000rpm, collecting avian influenza virus H9 subtype supernatant, and performing ultrafiltration concentration on the supernatant to obtain avian influenza virus H9 subtype virus venom;

(3) virus inactivation: adding BPL (beta-propiolactone) with the final concentration of 0.025-0.1% in a negative pressure production area according to the amount of virus liquid, putting the mixture into a constant temperature shaking table at the temperature of 2-8 ℃, inactivating the mixture for 16-48H, and stopping the reaction to obtain a solution containing inactivated H9 subtype avian influenza virus;

(4) preparing a vaccine: mixing white oil, tween and aluminum stearate to prepare an oil phase, and mixing and emulsifying the inactivated avian influenza virus solution in the step (3) and the oil phase according to the volume ratio of 1:2 to prepare the avian influenza H9 subtype vaccine.

2. The method for inactivating viruses in the production of avian influenza H9 subtype vaccine according to claim 1, wherein: the centrifugation speed in the step (2) is 7000rpm, and the centrifugation time is 15 min.

3. The method for inactivating viruses in the production of avian influenza H9 subtype vaccine according to claim 1, wherein: in the step (3), the inactivation temperature is 2-4 ℃, and the inactivation time is 24-48 h.

4. The method for inactivating viruses in the production of avian influenza H9 subtype vaccine according to claim 3, wherein: in the step (3), the inactivation temperature is 4 ℃, and the inactivation time is 24 hours.

5. The method for inactivating viruses in the production of avian influenza H9 subtype vaccine according to claim 1, wherein: in the step (3), BPL with the final concentration of 0.05% is added into the virus solution, and the virus solution is inactivated for 24 hours in a constant temperature shaking table at 4 ℃.

6. The method for inactivating viruses in the production of avian influenza H9 subtype vaccine according to claim 1, wherein: the method for terminating the reaction comprises the following steps: hydrolysis was carried out at 37 ℃ for 2 hours to hydrolyze propiolactone and terminate the reaction.

7. The vaccine preparation obtained by the method for inactivating viruses in the production of the avian influenza H9 subtype vaccine as claimed in any one of claims 1 to 6.

Technical Field

The invention belongs to the field of animal vaccines, and particularly relates to a method for inactivating viruses in the production of an avian influenza H9 subtype vaccine.

Background

According to the difference of pathogenicity, avian influenza can be divided into low-pathogenicity avian influenza and high-pathogenicity avian influenza. Now, high-disease avian influenza is represented by the H7N9 subtype, and low-pathogenicity avian influenza is represented by the H9N2 subtype. The H9N2 subtype avian influenza is the avian influenza which has high propagation speed and wide epidemic range in poultry in China; chickens of various ages in days are susceptible, the clinical severity degree of the chickens has larger difference, but the economic loss is large, the egg laying rate of laying hens is reduced by 10-60% or the laying hens are dead, and the death and culling rate of broilers and brooding or bred chickens is different from 10-80%; can be used for treating Escherichia coli, avian infectious bronchitis, infectious laryngotracheitis, newcastle disease, and various immunosuppressive pathogens, especially secondary infection with Escherichia coli.

The practice of preventing and treating avian influenza at home and abroad shows that vaccine immunity is a main measure for preventing the outbreak of avian influenza and avoiding causing huge loss. At present, the conventional vaccine applied to avian influenza is mainly a full-virus inactivated vaccine which has many advantages, is convenient to store, and can be immediately used for emergency vaccination after the virus subtype of avian influenza outbreak is determined. Meanwhile, the whole virus inactivated vaccine can be prepared into a multivalent vaccine aiming at several different subtype viruses, and the subtype antigens do not generate immune interference. The whole virus inactivated vaccine can induce an organism to generate an effective immune response reaction, can control further spreading and diffusion of epidemic situation in the past avian influenza generation process, and reduces economic loss.

The production process of inactivated virus vaccines is generally divided into amplification, inactivation, purification and/or lysis of the virus. In the domestic production process of the avian influenza inactivated vaccine, a formaldehyde solution is used as an inactivating agent in the inactivating process, the inactivating effect can be good, but the inactivating of the formaldehyde not only inactivates viruses but also destroys the immunogenicity of pathogens, the inactivating time of the formaldehyde is long, the inactivating process is easily influenced by factors such as temperature, pH value, concentration, existence of organic matters, types of the pathogens, nitrogen content and the like, and residual free formaldehyde can generate stress reaction after being injected into organisms along with the vaccine. Therefore, a safer and more reliable inactivator is needed to inactivate viruses. The invention optimizes the inactivation condition by selecting a proper inactivating agent, and determines the optimal inactivation process parameters.

Disclosure of Invention

The invention provides a method for inactivating virus in the production of avian influenza H9 subtype vaccine, and by adopting the virus inactivation treatment, the safety of vaccine preparations can be obviously improved, the stress reaction is reduced, and the immune effect is improved.

The invention provides the following technical scheme:

a method for inactivating virus in the production of avian influenza H9 subtype vaccine comprises the following steps:

(1) inoculating and culturing virus, selecting LMH cell for preparing vaccine, subculturing until LMH cell density reaches 1.0 × 10⁶-1.5×10⁶Inoculating avian influenza virus H9 subtype at one/mL, and adding cell maintenance liquid for propagation culture;

(2) virus collection, concentration and purification: freezing and thawing the cell culture solution in the step (1) for 3 times, centrifuging for 10-20min at 4 ℃, 6000-8000rpm, collecting avian influenza virus H9 subtype supernatant, and performing ultrafiltration concentration on the supernatant to obtain avian influenza virus H9 subtype virus venom;

(3) virus inactivation: in a negative pressure production area, BPL (beta-propiolactone) with the final concentration of 0.025-0.1 percent is respectively added according to the amount of virus liquid, and the mixture is put into a constant temperature shaking table at the temperature of 2-8 ℃ to be respectively inactivated for 16-48H, and then the reaction is stopped, so as to obtain the solution containing the inactivated H9 subtype avian influenza virus.

(4) Preparing a vaccine: mixing white oil, tween and aluminum stearate to prepare an oil phase, and mixing and emulsifying the inactivated avian influenza virus solution in the step 3 and the oil phase according to the volume ratio of 1:2 to prepare the avian influenza H9 subtype vaccine.

In the method for inactivating viruses in the production of the avian influenza H9 subtype vaccine, the centrifugation speed of 7000rpm and the centrifugation time of 15min in the step (2) are preferred.

In the method for inactivating the virus in the production of the avian influenza H9 subtype vaccine, the inactivation temperature in the step (3) is preferably 2-4 ℃ and the inactivation time is 24-48H. More preferably, the inactivation temperature is 4 ℃ and the inactivation time is 24 h.

In the method for inactivating the virus in the production of the avian influenza H9 subtype vaccine, the method for terminating the reaction comprises the following steps: adding BPL with 5 percent of sodium sulfite to terminate the reaction; or hydrolyzing at 37 deg.C for 2 hr to hydrolyze propiolactone and terminate the reaction.

Compared with the prior art, the invention has the following beneficial effects:

(1) the BPL inactivated vaccine has better capability of inducing the chicken to generate antibodies than the traditional formaldehyde inactivated vaccine. (2) And (3) screening out the optimal inactivation process conditions by optimizing the inactivation process conditions, wherein the inactivation BPL with the BPL concentration of 0.05 percent is carried out at the inactivation temperature of 4 ℃ for 24-48 h.

Detailed Description

The following detailed description of specific embodiments of the present invention is provided in conjunction with examples, but it should be understood that the scope of the present invention is not limited to the specific embodiments.