A dual-cell attenuated vaccine for sheep pox and aphthous ulcers and its preparation method and application

Abstract

The invention discloses a sheep pox and aphtha bigeminy cell attenuated vaccine and a preparation method and application thereof. The effective ingredient of the sheep pox and aphtha bigeminy cell attenuated vaccine is sheep pox virus and contagious pustular dermatitis virus cell passage attenuate virus or passage virus thereof which are separated through the inventor and obtained after passage attenuate culture, wherein the sheep pox virus cell passage attenuate virus is Sheeppox Virus GS-WW 2010 F44 strain, the preservation number of CCTCC NO: V201504, the sheep pox virus (contagious pustular dermatitis virus) cell passage attenuate virus is Orf Virus HB-TS09F65 strain, and the preservation number is CCTCC NO:V201406. Toxin attacking protection test results show that the sheep pox and aphtha bigeminy cell attenuated vaccine is safe and capable of producing effective immune protection force. Accordingly, the sheep pox and aphtha bigeminy cell attenuated vaccine is of an important practical significance in researching safe and efficient attenuated vaccine and preventing and controlling sheep pox and aphtha bigeminy.

Description

technical field

[0001] The invention relates to an attenuated vaccine and its preparation method and use, in particular to a sheep pox and aphthous double cell attenuated vaccine and its preparation method and use. The invention belongs to the field of veterinary biological products. Background technique

[0002] Capripoxvirus (CPV) is a member of the Capripoxvirus genus of the Poxviridae Vertebratepoxvirinae subfamily. Members of this genus include goat pox virus (GTPV), sheep pox virus (SPPV) and bovine nodular rash virus (Lumpy skin disease virus, LSDV). Goatpox is an acute, contagious infectious disease caused by goatpox virus. It is listed as one of the 93 animal diseases that must be reported by the World Organization for Animal Health (FAO / OIE), and it is listed as a first-class infectious disease in my country. Its morbidity rate can reach more than 75%, the mortality rate can reach more than 50%, and the lamb mortality rate can reach 100%, and it can also cause abo...

Examples

Embodiment 1

[0033] Example 1 Isolation, cultivation and identification of Sheep Sertoli cells

[0034] 1.1 Preparation of primary sheep testis cells

[0035] Select healthy male lambs born to healthy ewes (whose physique is healthy and negative for foot-and-mouth disease, brucellosis, and tuberculosis), and collect the scrotum after slaughter (the root of the scrotum is ligated, and the outside of the scrotum is sterilized with 75% alcohol), in an incubator, for 2 hours sent back to the laboratory. Use ophthalmic tweezers and scissors to cut the testicular parenchyma into small pieces of about 1mm×3mm, gently pipette several times in D-hanks solution, remove the supernatant, put the small tissue pieces into a 50mL centrifuge tube and add 10 times the volume of The digestion solution containing 0.1wt% type IV collagenase (GIBCO) and 0.25wt% trypsin (GIBCO) was digested at 4°C for 12h or overnight, and cultured with an equal volume of DMEM containing 10% (v / v) newborn bovine serum (GIBCO) ...

Embodiment 2

[0051] Embodiment 2 Proliferation of sheep pox virus

[0052] Sheep pox virus can grow on tissue cells derived from cattle, sheep, and goats, among which primary or secondary lamb testicular cells (LT) and lamb kidney cells (LK) are the most sensitive; the disease material can appear as early as 3 days after infection A small amount of cytopathic effect (CPE), after 4-6 days CPE rapidly spread to the entire cell layer, observe whether CPE appears and last up to 14 days. Typical CPE is cell rounding, cell aggregation in a fascicular structure, cell shrinkage or swelling, nuclei vacuolation, chromatin fragmentation, loss of coherence in the cell monolayer, and inclusion bodies in the cytoplasm (Adla et al., 1971; Joshi et al., 1994). In addition, sheeppox virus can also propagate on BHK-21 cells and Vero cells. Some strains also grow in fetal bovine muscle cells, calf kidney cells, and bovine kidney epithelial (MDBK) cell lines.

[0053] Therefore, the present inventor select...

Embodiment 3

[0067] Example 3 Laboratory trial production of sheep pox and aphthous dual cell attenuated vaccine

[0068] 3.1 Preparation of sheep pox and sheep infectious impetigo virus attenuated antigens

[0069] Select the newborn bovine testis Sertoli cell line NBSC (CCTCC NO: C201438) and the lamb testis Sertoli cell line that grow well and cover the monolayer as the lamb testis Sertoli cell natural passage cell line (LSC) (CCTCC NO: C2016120), and pour off the nutrient solution , respectively inoculate the virus solution (CCTCC NO: V201406) of Goat Infectious Pustular Virus cells passaged to attenuate Orf Virus HB-TS09F65 by 5% of the original nutrient solution volume and Sheeppox Virus cells passaged to attenuate Sheeppox Virus GS-WW 2010 F44 (CCTCC NO: V201504), after 10 minutes of adsorption, add DMEM maintenance solution, adjust the pH to 7.0, add 100 units / ml of penicillin and streptomycin, and cultivate in a 37°C, 5% CO2 concentration incubator. Cultivate for 48 hours, harves...

technical field

[0001] The invention relates to an attenuated vaccine and its preparation method and use, in particular to a sheep pox and aphthous double cell attenuated vaccine and its preparation method and use. The invention belongs to the field of veterinary biological products. Background technique

[0002] Capripoxvirus (CPV) is a member of the Capripoxvirus genus of the Poxviridae Vertebratepoxvirinae subfamily. Members of this genus include goat pox virus (GTPV), sheep pox virus (SPPV) and bovine nodular rash virus (Lumpy skin disease virus, LSDV). Goatpox is an acute, contagious infectious disease caused by goatpox virus. It is listed as one of the 93 animal diseases that must be reported by the World Organization for Animal Health (FAO / OIE), and it is listed as a first-class infectious disease in my country. Its morbidity rate can reach more than 75%, the mortality rate can reach more than 50%, and the lamb mortality rate can reach 100%, and it can also cause abo...

Examples

Embodiment 1

[0033] Example 1 Isolation, cultivation and identification of Sheep Sertoli cells

[0034] 1.1 Preparation of primary sheep testis cells

[0035] Select healthy male lambs born to healthy ewes (whose physique is healthy and negative for foot-and-mouth disease, brucellosis, and tuberculosis), and collect the scrotum after slaughter (the root of the scrotum is ligated, and the outside of the scrotum is sterilized with 75% alcohol), in an incubator, for 2 hours sent back to the laboratory. Use ophthalmic tweezers and scissors to cut the testicular parenchyma into small pieces of about 1mm×3mm, gently pipette several times in D-hanks solution, remove the supernatant, put the small tissue pieces into a 50mL centrifuge tube and add 10 times the volume of The digestion solution containing 0.1wt% type IV collagenase (GIBCO) and 0.25wt% trypsin (GIBCO) was digested at 4°C for 12h or overnight, and cultured with an equal volume of DMEM containing 10% (v / v) newborn bovine serum (GIBCO) ...

Embodiment 2

[0051] Embodiment 2 Proliferation of sheep pox virus

[0052] Sheep pox virus can grow on tissue cells derived from cattle, sheep, and goats, among which primary or secondary lamb testicular cells (LT) and lamb kidney cells (LK) are the most sensitive; the disease material can appear as early as 3 days after infection A small amount of cytopathic effect (CPE), after 4-6 days CPE rapidly spread to the entire cell layer, observe whether CPE appears and last up to 14 days. Typical CPE is cell rounding, cell aggregation in a fascicular structure, cell shrinkage or swelling, nuclei vacuolation, chromatin fragmentation, loss of coherence in the cell monolayer, and inclusion bodies in the cytoplasm (Adla et al., 1971; Joshi et al., 1994). In addition, sheeppox virus can also propagate on BHK-21 cells and Vero cells. Some strains also grow in fetal bovine muscle cells, calf kidney cells, and bovine kidney epithelial (MDBK) cell lines.

[0053] Therefore, the present inventor select...

Embodiment 3

[0067] Example 3 Laboratory trial production of sheep pox and aphthous dual cell attenuated vaccine

[0068] 3.1 Preparation of sheep pox and sheep infectious impetigo virus attenuated antigens

[0069] Select the newborn bovine testis Sertoli cell line NBSC (CCTCC NO: C201438) and the lamb testis Sertoli cell line that grow well and cover the monolayer as the lamb testis Sertoli cell natural passage cell line (LSC) (CCTCC NO: C2016120), and pour off the nutrient solution , respectively inoculate the virus solution (CCTCC NO: V201406) of Goat Infectious Pustular Virus cells passaged to attenuate Orf Virus HB-TS09F65 by 5% of the original nutrient solution volume and Sheeppox Virus cells passaged to attenuate Sheeppox Virus GS-WW 2010 F44 (CCTCC NO: V201504), after 10 minutes of adsorption, add DMEM maintenance solution, adjust the pH to 7.0, add 100 units / ml of penicillin and streptomycin, and cultivate in a 37°C, 5% CO2 concentration incubator. Cultivate for 48 hours, harves...

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