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Method for producing aviadenovirus 4 type vaccine by using LMH cell line, and vaccine

A poultry adenovirus and cell line technology, applied in the direction of microorganism-based methods, veterinary vaccines, biochemical equipment and methods, etc., can solve the problems of no obvious advantages and high production costs, and achieve the effect of improving immunity

Active Publication Date: 2018-12-11
广州渔跃生物技术有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are dual vaccines against avian adenovirus 4 (FAV-4) and chicken infectious bursal disease produced by LMH cell lines on the market, as well as against chicken city new disease, avian adenovirus 4 (FAV-4) and H9 subtype avian influenza virus triple vaccine, but there is no obvious advantage over the existing technology, generally has the disadvantage of high production cost, and there is still room for improvement in the protection of the vaccine against viruses

Method used

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  • Method for producing aviadenovirus 4 type vaccine by using LMH cell line, and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of LMH cell

[0035] S1. LMH cells were cultured with cell culture medium containing 0.8% (v / v) newborn bovine serum, 100IU / ml penicillin and 0.25% (v / v) N-acetyl-D-glucosamine, 37°C, 5% CO 2 Culture until the cells grow to a density of 30-55%, and use 0.025% trypsin-EDTA (0.02%) to digest and disperse the cells;

[0036] S2. Culture the LMH cells obtained in step S1 with a cell culture solution containing 0.8% (v / v) neonatal bovine serum, 100 IU / ml penicillin and 1.25% (v / v) N-acetyl-D-glucosamine, 37 °C, 5% CO 2 Culture until the cells grow to a density of 60-75%, and use 0.025% trypsin-EDTA (0.02%) to digest and disperse the cells;

[0037] S3, using the cell culture medium containing 0.8% (v / v) newborn calf serum, 100IU / ml penicillin and 2.5% (v / v) N-acetyl-D-glucosamine to culture the LMH cells obtained in step S2, 37 °C, 5% CO 2 Incubate until cells form a well-grown cell monolayer.

Embodiment 2

[0038] Embodiment 2, preparation of poultry adenovirus type 4 virus liquid

[0039] A) LMH cells were prepared according to the method described in Example 1;

[0040] B) Inoculate avian adenovirus type 4 into the LMH cells prepared in step A according to the virus inoculum MOT of 0.1, absorb the virus solution at 37°C for 60 minutes, and add 0.5% (v / v) newborn bovine serum and 100IU / ml penicillin cell maintenance solution, at 37°C, 5% CO 2 After culturing for 60 hours, when the cytopathic CPE reached more than 80%, the cytovenom was harvested.

[0041] C) The harvested cell venom was repeatedly frozen and thawed at -20°C for 3 times, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected to obtain a virus stock solution, which was concentrated 10 times by ultrafiltration to obtain a seedling virus solution;

[0042] D) the seedling virus solution obtained above is diluted serially by 10 times with DMEM culture fluid, and 10 -5 、10 -6 、10 -7 、10 -8 、10...

Embodiment 3

[0050] Embodiment 3, optimal virus harvesting time

[0051] Observe the TCID of the virus liquid harvested at different times by preparing the poultry adenovirus type 4 vaccine preparation virus liquid by the method described in Example 2 and Comparative Examples 1 to 3 50 , and screen out the best virus harvesting time, the results are shown in Table 1 below.

[0052] Table 1 The TCID of the virus liquid harvested at different times 50

[0053]

[0054] As can be seen from Table 1 above, compared with Example 2, in Comparative Example 1, N-acetyl-D-glucosamine was not added to the LMH cell culture, and the LMH cells obtained from the prepared venom were used to prepare the seedling virus solution and reached the maximum in 60 h. value 10 7.0 TCID 50 / 0.1ml, the virus titer was significantly lower than in Example 2; in Comparative Example 2, the LMH cells obtained without setting the content of N-acetyl-D-glucosamine in a gradually increasing gradient were used to prepa...

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Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to a method for producing an aviadenovirus 4 type vaccine by using an LMH cell line, and the vaccine. The LMH cell cultured by utilizing the method provided by the invention is used for culturing an aviadenovirus 4 type, so that the production cost can be greatly reduced, and meanwhile, the titer of the virus is ensured; through the process, within the shortest time 60h, the aviadenovirus 4 type virus with the titer being more than 108.5TCID50 / 0.1ml can be stably obtained, so that comparedwith the prior art, the remarkable technological advantage is realized. According to the vaccine prepared from a vaccine antigen with high titer, the immunity of the vaccine can be remarkably improved, and the immune challenge test result proves that 100 percent of protection can be realized on the attack of the aviadenovirus 4 type.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for producing poultry adenovirus type 4 vaccine with LMH cell lines and the vaccine. Background technique [0002] Avian adenovirus 4 (FAV-4) is highly pathogenic and can directly cause pericardial effusion-hepatitis syndrome. Currently, vaccines are effective prevention and control measures for highly pathogenic FAV-4. [0003] Conventional culture of FAV-4 virus mostly uses four primary cells: chicken embryo fibroblasts, chicken liver and kidney cells, chick kidney cells, and chicken embryo liver cells. However, primary liver and kidney cells are cumbersome to prepare, easy to contaminate, and difficult to transfect. , and chicken embryo fibroblasts are easily mixed in during the preparation process, which is not conducive to the test operation. [0004] In 1981, a Japanese scholar induced liver cancer in male chickens by using dieth...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61P31/20A61K39/12A61P31/14A61K39/17A61K39/145A61P31/16C12N7/00C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/70A61P31/14A61P31/16A61P31/20C12N7/00C12N2710/10234C12N2710/10251C12N2720/10034C12N2760/16134C12N2760/18134
Inventor 张毓金严悌昆谢秉超敖艳华
Owner 广州渔跃生物技术有限公司
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