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Method of avian infectious laryngotracheitis live vaccine using cell line

A technology of tracheitis and cell lines, applied in biochemical equipment and methods, microorganisms, pharmaceutical formulas, etc., can solve the problems of SPF embryo purity, chickens are prone to latent infection, and users cannot be obtained, so as to reduce immune side effects Response problems, reduced batch-to-batch variability, and clear-cut effects

Inactive Publication Date: 2016-01-27
河北远征药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical application, the virus used in the attenuated vaccine has the disadvantages of strong virulence, easy return to the ancestors, and easy formation of latent infection in chickens; in the actual production of vaccines, SPF chicken embryos are mainly used for the production of ILT live vaccines, while Since 2006, the Ministry of Agriculture has stipulated that all poultry live vaccines and several pig live vaccines use SPF embryos, which makes SPF chicken embryos accept a greater challenge, and the use of chicken embryos far exceeds its supply capacity. , there is a gap in the supply of SPF embryos; there are also certain defects in the quality monitoring of SPF chicken embryos, which is because it is judged by detecting the microbial results of SPF chicken flocks, but because the detection is still immature, it is still difficult to monitor, thus There are certain problems in the purity of SPF embryos, which cannot be fully recognized by users; in addition, because the breeding level of SPF chickens is in the development stage, there are uncertain factors in the cultivation of SPF chickens, the daily operation and management of breeders, etc., resulting in SPF The existence of differences between embryo batches becomes inevitable
There are so many shortcomings in SPF chicken embryos, we have to seek new culture methods

Method used

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  • Method of avian infectious laryngotracheitis live vaccine using cell line
  • Method of avian infectious laryngotracheitis live vaccine using cell line
  • Method of avian infectious laryngotracheitis live vaccine using cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of virus-adapted cells

[0033] 1 selection of cells

[0034] AILTV and HSV belong to the α subfamily of Herpesvirus genus, and their characteristics have many similarities. According to relevant reports, HSV can grow well in various cells such as BHK cells and Vero cells. For screening AILTV adaptive cells, the present invention selects BHK-21 and Vero cells were used for propagation of AILTV. The selection of DF1, primary CEF cells, chicken embryonic liver and kidney cells is mainly based on the strong culture characteristics of ILTV host specificity.

[0035] 2 Preparation and passage of cells

[0036] CEF, chicken embryo liver, chicken embryo kidney three kinds of cells are chicken embryo primary cell, use respectively the whole chicken embryo that removes the eyeball, the shredded chicken embryo liver, the smashed chicken embryo kidney through 2.5% trypsin (100 units / ml double antibody, pH 7.5-7.8) was digested, and prepared by adding M199 g...

Embodiment 2

[0046] Example 2 Cloning of Chicken Embryo Hepatocytes

[0047] 1 Cloning of chicken embryo liver cells

[0048] 1.1 Selection of chicken embryo liver cells

[0049] Take the prepared chicken embryo liver cells, count the cells, inoculate them in a cell bottle with a cell number of 3 million / ml, shake well, place them in a 5% CO2 incubator for 10 minutes, discard the culture medium, and add 12 % fetal bovine serum M199 growth medium, reset in the incubator and cultivated until monolayer cells.

[0050] 1.2 Chicken embryo liver cell cloning

[0051] Trypsinize and resuspend the chicken embryonic hepatocytes covered with a single layer in step 1.1, add 8% to 12% fetal bovine serum M199 growth medium for dilution, the dilution concentration is 2 cells / ml, and mix well , add to a 48-well cell plate, 0.5ml / well, place in a 5% CO2 incubator for 10 minutes after adsorption, discard the culture medium, add 1ml of growth medium / well, and record the content of Cultivate for 1 to 2 day...

Embodiment 3

[0084] Embodiment 3 chicken infectious laryngotracheitis virus clone purification

[0085] 1 Experimental materials

[0086] Agar powder, cell maintenance solution, 6-well cell culture plate, seed virus of AILTVK317 strain.

[0087] 2 Experimental methods

[0088] 2.1 Selection of virus dilution factor

[0089] Take the seed virus of AILTVK317 strain, use the cloned chicken embryo liver cells to measure the virus titer, and carry out 10-fold serial dilution of the virus according to the titer test results, and dilute to 10 -8 , take 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Virus dilutions were inoculated into 6-well cell plates full of monolayer cells, and three replicate cell wells were inoculated for each dilution factor, adsorbed in a 5% CO2 incubator for 1.5 hours, discarded the adsorption solution, and washed with 1×PBS (PH is 7.2) wash 2 times, add 0.5-1% agar powder cell maintenance solution, place it upside down in the incubator for culture, after 4-5 days, select dil...

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Abstract

A method of an avian infectious laryngotracheitis live vaccine using a cell line includes: replacing chick embryos with the cell line to proliferate avian infectious laryngotracheitis virus liquid, and performing cell line culturing, cloning and purifying, passage, culturing, cloning and culturing of a viral strain, preparing of vaccines, collecting of vaccine preparation virus liquid, subpackaging, freeze-drying and the like. The avian infectious laryngotracheitis live vaccine is produced using passage cells, the production cost is lowered, vaccine immunity side effect is reduced, inter-batch difference is diminished, quality stability of the vaccine is guaranteed, and basis is laid for the large-scale culturing of the virus. Experiments show that bright, round, and vividly outlined cells free of intra-cell impurity can be obtained by only three times of cloning, while an existing method needs seven times of cloning, the cloning time of the present method is shortened, and high activity and high cloning rate are given to cloning.

Description

technical field [0001] The invention relates to a method for producing a live vaccine, in particular to a method for producing a chicken infectious laryngotracheitis live vaccine with a cell line, and belongs to the technical field of veterinary biological products. Background technique [0002] Avian infectious laryngotracheitis (AILT) is a contagious respiratory infectious disease, the pathogen is laryngotracheitis virus, which can infect chickens of different ages. At present, the age of onset tends to be advanced. The earliest visible chickens are 20 days old, and it is endemic. The incidence rate is as high as 90% and the mortality rate is 10-40%. It is more harmful to the development of poultry industry. [0003] Avian infectious laryngotracheitis virus (AILTV) belongs to the α-subfamily of the genus Herpesvirus, and its biological characteristics and gene structure are similar to those of human herpes simplex virus (HSV), such as gene structure characteristics, molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/255C12N7/00A61P31/22
Inventor 毛雅园魏丽娟刘毅郭鸿志杨国辉王德功周岩谢艳芳孔瑞岗魏占勇刘欣
Owner 河北远征药业有限公司
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