A kind of attenuated vaccine of goat infectious impetigo virus cell and its preparation method and application

Abstract

The invention discloses an attenuated vaccine of contagious ecthyma virocyte as well as a preparation method and an application thereof. In the attenuated vaccine of the contagious ecthyma virocyte disclosed by the invention, the effective component is the attenuated vaccine caused by passage of the contagious ecthyma virocyte which is obtained by automatic separation and passage-attenuated cultivation of the inventor, and the preservation number is CCTCC NO:V201406. The attacking protection test result shows that the prepared orf virus (ORFV) cell attenuated vaccine is safe, and can generate effective immune protection force. Therefore, the attenuated vaccine has important practical significance on development of the safe and efficient attenuated vaccine and large-scale production of the attenuated vaccine of the contagious ecthyma virocyte by using the disclosed attenuated strain to prevent and control contagious ecthyma.

Description

Technical field

[0001] The invention relates to an attenuated vaccine and a preparation method and application thereof, in particular to an attenuated goat infectious pustular virus cell vaccine and a preparation method and application thereof. The invention belongs to the field of veterinary biological products. Background technique

[0002] Sheep contagious pustulosis (Contagiousecthyma, CE), also known as contagious pustulardermatitis (Contagiouspustulardermatitis), commonly known as “Orf”, is caused by a member of the parapox virus, Sheep contagious pustular virus (Orfvirus, ORFV) infection causes an acute, contact and epithelial zoonotic disease in goats, sheep and humans, which causes erythema and papules on the skin and mucous membranes of the lips, hooves, breasts, vulva, etc. of the affected sheep , Abscesses, ulcers and verrucous thick scabs. The World Organization for Animal Health (OIE) lists the disease as an animal disease requiring notification, and my country lis...

Examples

Embodiment 1

[0025] Example 1 Isolation and attenuation of sheep infectious pustular virus strain (OrfVirusHB-TS09 strain)

[0026] 1.1 Isolation and identification of sheep infectious pustular virus strain (OrfVirusHB-TS09 strain)

[0027] In mid-November 2009, more than 300 black goat lambs from 1 month to 3 months of age were onset in a sheep farm in Tongshan County, Hubei Province, with an incidence rate of 80%, mainly manifested by typical symptoms of aphthous ulcers, treated with multiple antibiotics and deworming The medication was ineffective, and more than 40 sick sheep died one week later. The inventors of the present invention carried out case replication of the disease material, virus isolation, and finally obtained a virus strain. The virus strain was verified by identification of the physical and chemical characteristics of the virus, specific PCR detection and sequencing of the B2L gene of sheep infectious pustulosis virus. The strain is Sheep Infectious Pustular Virus (Wang Gua...

Embodiment 2

[0037] Example 2 Preparation of sheep infectious pustular cell attenuated vaccine

[0038] 2.1 Preparation of sheep infectious pustule attenuated antigen

[0039] Select the newborn bovine testicular primary cells (BT) that grow well and cover a single layer, pour out the nutrient solution, and inoculate the sheep infectious pustule virus cells at 5% of the original nutrient solution volume to pass down the virus solution (CCTCCNO : V201406), add DMEM maintenance solution after 10 minutes of adsorption, adjust the pH to 7.0, add 100 units / ml of penicillin and streptomycin, and cultivate in an incubator at 37°C and 5% CO2 concentration. Cultivate for 48 hours, harvest when the cytopathic effect (CPE) reaches 80%, put it in the refrigerator at -20℃ and freeze and thaw twice, then carry out sterility inspection, safety inspection, and mycoplasma inspection. It meets the requirements of "The People's Republic of China Veterinary Biological Products Quality Standard" The Reed-Muench me...

Embodiment 3

[0042] Example 3 Inspection of finished sheep infectious pustular cell attenuated vaccine

[0043] 3.1 Physical properties

[0044] The product is a light yellow spongy loose mass, which is easily separated from the bottle wall and dissolves quickly after adding diluent.

[0045] 3.2 Sterility test

[0046] Randomly take three bottles from each batch of the prepared and preserved three batches of sheep infectious pustular cell attenuated vaccines 20130526, 20130822, 20130829, and proceed according to the current "Chinese Veterinary Pharmacopoeia", 2010 edition, appendix page 42. The finished vaccine has no bacterial growth.

[0047] 3.3 Mycoplasma test

[0048] Randomly take three bottles from each batch of the prepared and preserved 20130526, 20130822, 20130829 three batches of sheep infectious pustular cell attenuated vaccine, and conduct mycoplasma test according to the current "Chinese Veterinary Pharmacopoeia", 2010 edition, appendix page 49. No mycoplasma growth of cytotoxicity in...

Technical field

[0001] The invention relates to an attenuated vaccine and a preparation method and application thereof, in particular to an attenuated goat infectious pustular virus cell vaccine and a preparation method and application thereof. The invention belongs to the field of veterinary biological products. Background technique

[0002] Sheep contagious pustulosis (Contagiousecthyma, CE), also known as contagious pustulardermatitis (Contagiouspustulardermatitis), commonly known as “Orf”, is caused by a member of the parapox virus, Sheep contagious pustular virus (Orfvirus, ORFV) infection causes an acute, contact and epithelial zoonotic disease in goats, sheep and humans, which causes erythema and papules on the skin and mucous membranes of the lips, hooves, breasts, vulva, etc. of the affected sheep , Abscesses, ulcers and verrucous thick scabs. The World Organization for Animal Health (OIE) lists the disease as an animal disease requiring notification, and my country lis...

Examples

Embodiment 1

[0025] Example 1 Isolation and attenuation of sheep infectious pustular virus strain (OrfVirusHB-TS09 strain)

[0026] 1.1 Isolation and identification of sheep infectious pustular virus strain (OrfVirusHB-TS09 strain)

[0027] In mid-November 2009, more than 300 black goat lambs from 1 month to 3 months of age were onset in a sheep farm in Tongshan County, Hubei Province, with an incidence rate of 80%, mainly manifested by typical symptoms of aphthous ulcers, treated with multiple antibiotics and deworming The medication was ineffective, and more than 40 sick sheep died one week later. The inventors of the present invention carried out case replication of the disease material, virus isolation, and finally obtained a virus strain. The virus strain was verified by identification of the physical and chemical characteristics of the virus, specific PCR detection and sequencing of the B2L gene of sheep infectious pustulosis virus. The strain is Sheep Infectious Pustular Virus (Wang Gua...

Embodiment 2

[0037] Example 2 Preparation of sheep infectious pustular cell attenuated vaccine

[0038] 2.1 Preparation of sheep infectious pustule attenuated antigen

[0039] Select the newborn bovine testicular primary cells (BT) that grow well and cover a single layer, pour out the nutrient solution, and inoculate the sheep infectious pustule virus cells at 5% of the original nutrient solution volume to pass down the virus solution (CCTCCNO : V201406), add DMEM maintenance solution after 10 minutes of adsorption, adjust the pH to 7.0, add 100 units / ml of penicillin and streptomycin, and cultivate in an incubator at 37°C and 5% CO2 concentration. Cultivate for 48 hours, harvest when the cytopathic effect (CPE) reaches 80%, put it in the refrigerator at -20℃ and freeze and thaw twice, then carry out sterility inspection, safety inspection, and mycoplasma inspection. It meets the requirements of "The People's Republic of China Veterinary Biological Products Quality Standard" The Reed-Muench me...

Embodiment 3

[0042] Example 3 Inspection of finished sheep infectious pustular cell attenuated vaccine

[0043] 3.1 Physical properties

[0044] The product is a light yellow spongy loose mass, which is easily separated from the bottle wall and dissolves quickly after adding diluent.

[0045] 3.2 Sterility test

[0046] Randomly take three bottles from each batch of the prepared and preserved three batches of sheep infectious pustular cell attenuated vaccines 20130526, 20130822, 20130829, and proceed according to the current "Chinese Veterinary Pharmacopoeia", 2010 edition, appendix page 42. The finished vaccine has no bacterial growth.

[0047] 3.3 Mycoplasma test

[0048] Randomly take three bottles from each batch of the prepared and preserved 20130526, 20130822, 20130829 three batches of sheep infectious pustular cell attenuated vaccine, and conduct mycoplasma test according to the current "Chinese Veterinary Pharmacopoeia", 2010 edition, appendix page 49. No mycoplasma growth of cytotoxicity in...