Foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain as well as construction method and application thereof

A technology of foot-and-mouth disease vaccine and construction method, which is applied in the field of foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain and its construction, which can solve the problems that the recombinant virus cannot be successfully rescued and affects the virus replication ability, etc.

Active Publication Date: 2021-09-03
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inappropriate gene recombination in the existing technology will affect the replication ability of the recombined virus, or cause the recombined virus to fail to be rescued successfully, which brings great technical difficulties to the research and development of FMDV vaccine.

Method used

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  • Foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain as well as construction method and application thereof
  • Foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain as well as construction method and application thereof
  • Foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Construction method of FMDV recombination full length cloning

[0062] PSK-Z123 in FMDV O / HN / CHA / 93 vaccine strains (this plasmid is open in article Evaluation of Agenetical Modified Foot-Mouth Disease Virus Vacine Candidate GeneratedBy Reverse Genetics, Li et al.bmc Verinary Research 2012 8: 57) For the skeleton, it is designed to synthesize plasmids PSK-Z123 / Tur09VP1 containing FMDV O / Tur / 5 / 2009 strain VP1 gene. The above recombinant plasmids were digested with SPE I and BGLII, respectively, and the target belt of approximately 5400 bp was recovered, and the plasmid of digestible plasmid was inserted with the same enzyme (the plasmid Evaluation of a genetically modified foot-mouthdisease Vircine Candidate generated). By Reverse Genetics Open, Li ETAL.BMC Veterinary Research 2012, 8: 57), is obtained, and yang-type POFS-TUR09 / VP1 is obtained. Structure of the whole genome structure of FMDV full length recombinant plasmid figure 1 .

[0063] The recombinant pl...

Embodiment 2

[0066] Rescue of restructuring of viruses

[0067] Using NOT I enzyme linear plasmid POFS-TUR09 / VP1, then purified as a transfection template with a DNA fragment recovery kit. Conventional cultured single-layer BSR / T7 cells grow to 70% to 80%, liposome Lipofectamine TM 2000 mediated transfection (see the operating manual). Fully medium was added 5 hours after transfection, and 37 ° C 5% CO was placed. 2 The incubator continues to cultivate, and the transfection of cells have caused cytoplasions daily. After the transfection of 72h, the cells were harvested, and after repeatedly frozen 2 to 3 times, it was continuously passed in BHK21, and the various viruses were stored at -70 ° C.

[0068] The results showed that the linear plasmid POFS-TUR09 / VP1 transfected with BSR / T7 cells for 70 hours, the cells showed a typical cell lesion, that is, the cells became large, the circle, and became the distribution of grapes (see image 3 And the control cell morphology is intact and is ...

Embodiment 3

[0070] Identification of recombinant viruses

[0071] 3.1, indirect immunofluorescence

[0072] The collected transfection supernatant and parent virus RHN growth growth to 70% to 80% of the full single-layer BHK-21 cells, incubated for 6 h, rinsed with PBS buffer 3 times, 3.7% polyformaldehyde room temperature fixed 20 min, PBS buffer Wash 3 times, 50 mmol / L ammonium chloride, thermostat 10min, PBS buffer washing 3 times, add anti-FMDV non-structural protein 3a, a monoclonal antibody 3A24 (3A24 in the literature "foot-and-mouth disease, non-structural protein 3A monoclonal antibody And Identification, China Veterinary Science 2010, 40 (04): 331-336 is disclosed) 37 ° C for 1 h, PBS rinsing 3 times, then add FITC-labeled goat anti-mouse antibody 37 ° C for 1 h, PBS rinsing After 3 times, 0.5 μg / ml DAPI (PBS preparation) was added to 10 minutes, and PBS washed three times, and the excess DAPI was removed, and the photo was placed under the coke fluorescence microscope.

[0073]...

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Abstract

The invention provides a foot-and-mouth disease O-type PanAsia-2 pedigree reserve vaccine strain as well as a construction method and application thereof, and belongs to the technical field of veterinary drug biological products. The foot-and-mouth disease recombinant virus rHN / TUR09 / VP1 is obtained by taking an O / HN / CHA / 93 virus strain as a skeleton and embedding a VP1 gene of O / TURR / 5 / 2009. The rHN / TUR09 / VP1 not only is highly matched with antigens of a foot-and-mouth disease O-type PanAsia pedigree strain and an Ind-2001 pedigree strain which are relatively close to the genetic relationship, but also is matched with antigens of a foot-and-mouth disease O-type Cathay pedigree strain and a Mya-98 pedigree strain which are relatively far from the genetic relationship, so that the rHN / TUR09 / VP1 can be used for well immunizing, preventing and controlling the prevalence of the current O-type multi-pedigree FMD virus strain in China; and the strain can be used as a strategic reserve vaccine strain for effective prevention and control of O / Panaa-2 pedigree foot-and-mouth disease in border areas of China.

Description

Technical field [0001] The present invention belongs to the technical field of veterinary drug biological products, and in particular, the present invention relates to a foot-and-mouth disease O-type Panasia-2 spectrum type reserve vaccine poison strain and its construction methods and applications. Background technique [0002] FMD (FOOT-AND-MOTH DISEASE, FMD) is a strong infectious disease with major livestock such as foot-and-mouth diseasevirus, FMDV, sheep and pigs. The outbreak of this disease is seriously harming the productivity of the animal group and the quality of animal husbandry, and the animal husbandry and its related industries gab to the disease areas have huge economic losses. Therefore, the disease is one of the animal diseases in my country's focus prevention. [0003] my country is a country with severe foot-and-mouth disease, especially the harm of O-mouth-and-mouth disease to my country's livestock farming industry. In addition to the historical O-Type Catha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/42C12N15/63A61K39/295A61K39/135A61P31/14
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2770/32121C12N2770/32134C12N2770/32122A61K2039/5256A61K2039/70Y02A40/70
Inventor 李平花刘在新卢曾军黄书伦孙普查晶晶李冬白兴文曹轶梅马雪青李坤袁红付元芳
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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