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A kind of rabies vaccine and preparation method thereof

A rabies vaccine and adenovirus technology, applied in the fields of biotechnology and virology, can solve problems such as weakening the immune effect of vaccines

Active Publication Date: 2015-07-29
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 and AdHu2.

Method used

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  • A kind of rabies vaccine and preparation method thereof
  • A kind of rabies vaccine and preparation method thereof
  • A kind of rabies vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Cloning of rabies virus Gp coding region

[0108] Agarose gel electrophoresis shows that the digestion of PUC57-0 / Gp is ​​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. First, use Nhe I and Xba I to identify positive clones, and then use pAdshuttle-CMV / gp The single restriction site EcoRI in the region outside the target fragment was identified again, and the recombinant adenovirus shuttle vector pAdshuttle-CMV / Gp was cut with restriction enzymes, and the results were consistent with expectations. The sequencing results show that the obtained nucleic acid sequence is the same as the nucleic acid sequence after the optimized codon of the target fragment.

Embodiment 2

[0109] Example 2. Identification of recombinant adenovirus

[0110] PI-Sce I and I-Ceu I double digested pAdshuttle-CMV / gp and pAdC68, and ligated the above products with T4DNA ligase. After selecting the ampicillin resistant clones, restriction enzyme digestion to identify the plasmid pAdC68-Gp, such as image 3 , Consistent with the expected size. The sequencing results show that the obtained nucleic acid sequence is the same as the nucleic acid sequence after the optimized codon of the target fragment.

[0111] A large amount of recombinant adenovirus plasmid DNA was prepared, and the recombinant plasmid digested with Pac I was transfected into HEK293 cells. Obvious cytopathic changes appeared after 6-8 days. Plaques appear in the cells in the early stage of the disease, and gradually become larger, and in the later stage, they are in a net state, and finally all the cells float. Collect 20 bottles of 150ml cells that have been infected with the virus. The mixture of repeated...

Embodiment 3

[0112] Example 3. Determination of recombinant adenovirus infection titer and analysis of genetic stability

[0113] Infect HEK293 cells and liver cancer cell line Huh7 with different numbers of viruses, such as Figure 4 . After 24 hours of infection, plaques appeared in HEK293 cells (the amount of virus was 10 8 vps); and Huh7 cells after infection (with a viral load of 10 8 vps) has no significant difference with the control group, when the virus amount reaches 10 10 Plaques also appeared in vps and Huh7 cells. Pass the purified virus in HEK293 cells for 15 consecutive generations, collect and purify the virus in a small amount, and identify the genomes of the 5th and 15th generation viruses by restriction enzyme digestion. The results prove that the recombinant virus has no mutations and maintains the original virus infection. Ability, such as Figure 5 .

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Abstract

The present invention relates to a new rabies vaccine and method for preparation thereof. The present inventor has constructed, on the basis of a vaccine vector of the genome of chimpanzee adenovirus AdC68, a new type of rabies vaccine vector. The present invention also provides a viral vaccine prepared using said vaccine vector, highly expressed, and having good immunogenicity.

Description

Technical field [0001] The present invention belongs to the fields of biotechnology and virology; more specifically, the present invention relates to a novel rabies vaccine and a preparation method thereof. Background technique [0002] Rabies is a fatal zoonotic disease that is caused by rabies virus and is characterized by invading the central nervous system. Rabies kills more than 50,000 people worldwide every year, and has a huge impact on life, health and social economy. Rabies virus is a single-stranded negative-strand RNA virus, belonging to the Rhabdovirus family Rhabdovirus, with a rhabdo-like appearance, a spirally symmetrical nucleocapsid, and an envelope on the surface. The glycoprotein (Gp) antigen on the outer membrane of rabies virus is a component of the spinous processes on the surface of the virus. It has the ability to agglutinate cells and binds to acetylcholine receptors to make the virus neurotropic; glycoproteins can induce the body to produce neutralizing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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