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Long-acting animal rabies vaccine and preparing method thereof

A technology of rabies vaccine and rabies virus, which is applied in the field of long-acting rabies vaccine for animals and its preparation, can solve the problems of short duration and achieve the effects of safe use, wide range of animals, and long duration of immunity

Inactive Publication Date: 2012-07-25
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the antibody immune response induced by these vaccines is often short-lived, and due to the long life cycle of dogs, multiple re-immunizations or booster immunizations are required

Method used

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  • Long-acting animal rabies vaccine and preparing method thereof
  • Long-acting animal rabies vaccine and preparing method thereof
  • Long-acting animal rabies vaccine and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Construction of rabies virus glycoprotein-recombinant retroviral vector

[0029] The pLNCL vector containing the lacZ gene and the plasmid vector pVAX-G were constructed and preserved by our laboratory; the packaging cell line PA 317, Escherichia coli (E.coli) JM109, was purchased commercially and preserved by our laboratory.

[0030] The main process of recombinant plasmid construction is as follows: (1) After BamH I digests pLNCL, the linearized fragment of 5812bp is recovered, filled with Klenow enzyme, and then dephosphorylated by alkaline phosphatase (CIAP) to obtain both ends Both are blunt-ended pLNCX empty vectors. (2) BspHI, Not I and Mlu I three enzymes are combined to digest pVAX-G, reclaim the 2326bp fragment, and fill it up with Klenow enzyme, so as to obtain a nucleic acid with both ends being blunt and containing the G protein gene of rabies virus SRV9 strain fragment. (3) The 2326bp nucleic acid fragment containing the G protein gene of the rabies viru...

Embodiment 2

[0073] Packaging of recombinant virus

[0074] The minimum lethal dose of G418 to the packaging cell line PA 317 was determined with different concentrations of G418 ranging from 0.2 to 0.7 g / L. Referring to the instructions of Lipofectamine 2000, the recombinant virus vector was introduced into the packaging cell line PA 317 with liposome as the medium. And screened with the least lethal dose of G418.

[0075] (1) Determination of the minimum lethal dose of G418 by PA317 and its results

[0076] Inoculate an appropriate amount of PA317 cells in a 24-well plate, and when the cells grow into 75% confluent pieces, add selection medium containing different concentrations of G418 (0.2, 0.3, 0.35, 0.4, 0.5, 0.6, 0.7g / L), each gradient Three repetitions were set up, and a blank control was set at the same time. The medium was changed every 3 days, and the minimum lethal concentration of G418 for PA317 cells was determined according to the cell death situation in 10-14 days. On t...

Embodiment 3

[0085] Cloning of Toxigenic Cells and Expansion of Recombinant Viruses

[0086] Add feeder cells to each well of the 96-well plate, mark the position of the clone with a marker pen on the 12-well plate where cell clones grow, pick the cell clone with a sterile tip on the ultra-clean workbench, and inject it into the 96-well plate After the cells adhere to the wall, replace half of the liquid in the 96-well plate with DMEM nutrient solution with a final concentration of 400 μg / L G418, and then replace all the liquid with DMEM nutrient solution with a final concentration of 400 μg / L G418 after the cells grow Carry out culture, after overgrown, transfer into 24-well plate, 6-well plate, 30ml cell culture bottle, 50ml cell culture bottle successively, expand culture gradually. When expanding the culture, the concentration of G418 was 400 μg / ml, and the amount of serum added was 10%.

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Abstract

The invention relates to a long-acting animal rabies vaccine and a preparing method thereof. Replication-defective retrovirus is used as a carrier. After forming a virus infection organism, the carrier can be integrated into a chromosome of a host cell. An expression cassette for expressing a rabies virus glycosidoprotein is used as an exogenous target gene, and the protein expressed by the target gene can stimulate the organism to produce the immune response to the rabies virus. By converting the packaging cell of the retrovirus, recombinant viruses can be continuously produced to recombineviruses and immune animals, and therefore the animals can obtain long-term immune protection effect on the rabies. The recombinant vaccine has the advantages of wide applicable range for animals, safe use, and long duration time of the induced immune effective period, and is a novel long-effective vaccine for preventing rabies.

Description

Technical field: [0001] The invention belongs to the technical field of preparation of animal immune vaccines, and in particular provides a long-acting rabies vaccine for animals and a preparation method thereof, which uses a replication-deficient retrovirus as a carrier and is cloned into an expression cassette of a rabies virus glycoprotein gene through transformation, The recombinant virus obtained by transfecting packaging cells and immunizing animals with the recombinant virus solution can make the immunized animals produce a new type of rabies vaccine with long-term immune protection. Background technique: [0002] Rabies is an important zoonotic infectious disease caused by rabies virus infection. Once it occurs, it can cause 100% death. The number of deaths due to rabies in my country is between 2000 and 3000 cases every year. The occurrence of human rabies is caused by animal bites. The current vaccines used to prevent animal rabies include inactivated vaccines and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/205A61P31/14C12N15/47C12N15/867
Inventor 扈荣良张守峰刘晔张菲赵娜
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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