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Novel rabies vaccine and preparation method thereof

A rabies vaccine, adenovirus technology, applied in the field of biotechnology and virology, can solve the problem of weakening the immune effect of vaccines

Active Publication Date: 2013-12-25
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since commonly used human serotype adenoviruses such as AdHu5 and AdHu2 generally infect humans, 60-80% of individuals in the population have corresponding neutralizing antibodies, which will weaken the immune effect of vaccines based on AdHu5, AdHu2, etc.

Method used

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  • Novel rabies vaccine and preparation method thereof
  • Novel rabies vaccine and preparation method thereof
  • Novel rabies vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Embodiment 1, the cloning of rabies virus Gp coding region

[0108] Agarose gel electrophoresis showed that the digested PUC57-0 / Gp was consistent with the expected size (bp), such as figure 2 ; Since Nhe I and Xba I are homologous enzymes, reverse cloning may occur when screening the recombinant shuttle plasmid pAdshuttle-CMV / gp. The single restriction site EcoR I in the region other than the target fragment was re-identified, and the recombinant adenovirus shuttle vector pAdshuttle-CMV / Gp was digested, and the results were consistent with expectations. Sequencing results showed that the obtained nucleic acid sequence was identical to the nucleic acid sequence of the target fragment after codon optimization.

Embodiment 2

[0109] Embodiment 2, identification of recombinant adenovirus

[0110] PI-Sce I and I-Ceu I double digest pAdshuttle-CMV / gp and pAdC68, and use T4 DNA ligase to ligate the above products. After selecting ampicillin-resistant clones, digest and identify the plasmid pAdC68-Gp, such as image 3 , which is the expected size. Sequencing results showed that the obtained nucleic acid sequence was identical to the nucleic acid sequence of the target fragment after codon optimization.

[0111] A large amount of recombinant adenovirus plasmid DNA was prepared, and the recombinant plasmid after digestion with Pac I was transfected into HEK293 cells. Visible cytopathic changes appear after 6-8 days. In the early stage of the lesion, plaques appeared in the cells, which gradually became larger, and in the later stage, they appeared in a net state, and finally all the cells floated. Collect 20 bottles of 150ml cells that have been infected with the virus, and use cesium chloride density...

Embodiment 3

[0112] Example 3, Determination of Recombinant Adenovirus Infection Titer and Genetic Stability Analysis

[0113] HEK293 cells and the liver cancer cell line Huh7 were infected with different amounts of virus, such as Figure 4 . After 24 hours of infection, plaques appeared in HEK293 cells (the amount of virus was 10 8 vps); and infected Huh7 cells (the amount of virus was 10 8 vps) had no significant difference with the control group, when the amount of virus reached 10 10 Plaques also appeared in vps, Huh7 cells. The purified virus was continuously passed on for 15 generations in HEK293 cells, a small amount of the virus was collected and purified, and the genomes of the 5th and 15th generation viruses were identified by enzyme digestion. The results proved that the recombinant virus had no mutation and maintained the infection of the primary virus ability, such as Figure 5 .

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Abstract

The invention relates to a novel rabies vaccine and a preparation method thereof. Based on a vaccine carrier for chimpanzee type adenovirus AdC68 genome, an inventor constructs a novel rabies vaccine carrier. The invention further provides a virus vaccine prepared from the vaccine carrier, which can efficiently express and has excellent immunogenicity.

Description

technical field [0001] The invention belongs to the fields of biotechnology and virology; more specifically, the invention relates to a novel rabies vaccine and a preparation method thereof. Background technique [0002] Rabies is a fatal zoonotic infectious disease caused by rabies virus and characterized by invading the central nervous system. Rabies kills more than 50,000 people every year worldwide, and has a huge impact on life, health and social economy. Rabies virus is a single-stranded negative-sense RNA virus belonging to Rhabdoviridae, Rhabdovirus genus. The glycoprotein (Gp) antigen on the outer membrane of the rabies virus is a component of the spikes on the surface of the virus, which has the ability to agglutinate cells and can bind to the acetylcholine receptor to make the virus neurotropic; the glycoprotein can induce the body to produce neutralizing antibodies and induce Cellular immunity, neutralizing antibodies have complete protection against virus infe...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K39/205A61P31/14
CPCA61K39/12A61K39/205A61K2039/5256A61P31/14C12N15/86C12N2710/10343C12N2760/20134C12N2799/022
Inventor 周东明迟喻丹邓飞蓝柯
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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