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Method for producing avian adenovirus inactivated vaccine through LMH clone line

An inactivated vaccine, avian adenovirus technology, applied in veterinary vaccines, biochemical equipment and methods, vaccines, etc., can solve the problems of low vaccine protection rate, low virus titer, poor cell viability, etc. High, reduce production costs, improve the effect of valence

Inactive Publication Date: 2017-06-06
广州博恒生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Digested cells with traditional trypsin-EDTA concentration are used for subculture, and the cell activity is poor and easy to age. The cells in this state are used to prepare avian adenovirus vaccine under different production conditions, and the animal immune efficacy test is carried out. The conclusions drawn It is the low rate of protection of the vaccine
In addition, the use of traditional cell culture time, virus inoculation concentration, and virus harvest time all have the problem of low virus titer and low protection rate of the vaccine produced.

Method used

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  • Method for producing avian adenovirus inactivated vaccine through LMH clone line
  • Method for producing avian adenovirus inactivated vaccine through LMH clone line
  • Method for producing avian adenovirus inactivated vaccine through LMH clone line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of liposome complex:

[0037] (1) Weigh 0.2g phosphatidylcholine, 0.08g cholesterol, and 0.04g distearoylphosphatidylethanolamine, mix, dissolve in 50mL ethanol, and ultrasonic treatment for 5min;

[0038] (2) Then, the ethanol was removed by rotary evaporation under the conditions of 28℃ and 0.1MPa, and it was allowed to stand for 30min. Then, add 100mL pH 7.4 and 0.5% (m / v) catalase-containing phosphate buffer solution, and put it in a 35℃ water bath. The hydration reaction is about 1h by shaking to obtain liposome suspension;

[0039] (3) Use a syringe filter to adjust the particle size of the liposome suspension to 150 nm to obtain a liposome containing catalase inside.

Embodiment 2

[0040] Example 2 Selection of the best trypsin-EDTA concentration for digesting LMH cells and the selection of the best culture time for LMH cells

[0041] The LMH cells (ATCC LMH-CRL-2117) were digested and dispersed with trypsin-EDTA (0.02%) with mass-volume ratios of 0.25%, 0.05%, and 0.025%; added with 5-10% (v / v) newborn Bovine serum, 1.0-1.2% (v / v) bi-antibody and 0.5-2.5% (m / v) hydroxyethylpiperazine ethanesulfonic acid in DMEM culture medium at 37℃, 5% CO 2 Cultivate the cells to a monolayer in an incubator for passage.

[0042] The LMH cells digested with three different concentrations of trypsin-EDTA were passaged separately, and the cell growth of different passages is shown in Table 1. Digest LMH cells with 0.025% pancreatin-EDTA (0.02%) in a mass-volume ratio at 37℃, 5% CO 2 After culturing in an incubator for different times, the cells were tested for cell viability by MTT method and counted with a red blood cell counter. The results are shown in Table 2.

[0043] Tabl...

Embodiment 3

[0049] Example 3 Screening of the optimal inoculation concentration of avian adenovirus type 4

[0050] The monolayer of LMH cells were diluted at different concentrations for avian adenovirus type 4 inoculation. After inoculation to about 80% of the cells, the cell venom was harvested and freeze-thawed twice at -20°C, 5000rpm, After centrifugation at 4°C for 10 minutes, collect the supernatant, which is the virus stock, and determine its TCID 50 , And the results are shown in Table 3.

[0051] Table 3 Screening of avian adenovirus type 4 inoculation concentration

[0052]

[0053] The results show that when the virus solution with different dilution concentrations is used for inoculation, the time when the cells appear lesions are very different and the titer of the cytovenom is very different. From the above table, it can be seen that the 200-fold diluted virus was used for inoculation and cultured for 58 hours. The virus has the highest titer.

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Abstract

The invention relates to a method for producing avian adenovirus inactivated vaccine through an LMH clone line. The method comprises the following steps: firstly, preparation continuous cell line LMH cell; secondly, virus-seed reproduction; thirdly, virus collection, concentration and purification; fourthly, virus content determination; fifthly, inactivation and emulsification of virus, and forming emulsion inactivated vaccine. Compared with the traditional technical method for producing avian adenovirus through cell culture, the method has the advantages that high-quality inactivated vaccine with higher antigen titer through the optimization of the concentration of digestive LMH cell pancreatin-EDTA, the culture time of LMH cell and the inoculation concentration and harvest time of virus, injection immunization is performed at different doses, and all the vaccine protection rates reach 100 percent, so that the immune efficiency of the avian adenovirus type IV inactivated vaccine produced through the method is high, and the vaccine has complete immune protection effect on avian adenovirus type IV.

Description

Technical field [0001] The invention belongs to the technical field of veterinary biological products, and specifically relates to a method for producing an avian adenovirus inactivated vaccine by using an LMH cell line. Background technique [0002] Avian group I adenovirus is a common infectious pathogen of poultry, and it has a worldwide distribution. It has been found that poultry of all ages are susceptible, but the pathogenicity is different in view of the breed and age of poultry. At present, it has been found that avian group I adenovirus is highly pathogenic to chickens. The main lesions are inclusion body hepatitis and pericardial effusion-hepatitis syndrome. The disease can directly cause more than 30% of chickens at 20-40 days old in chicken farms. In addition, it is also clinically often concurrently infected with infectious bronchitis virus, reovirus, H9 subtype avian influenza virus, etc., resulting in obvious respiratory diseases, arthritis, and severe egg product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/235A61P31/20C12N7/04
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2710/10234C12N2710/10251C12N2710/10263
Inventor 张毓金严悌昆谢秉超敖艳华
Owner 广州博恒生物科技有限公司
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