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II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using spike

A rabies virus and protective antigen technology, which is applied in the development and application of new animal rabies vaccines, and can solve problems that have not yet been reported.

Inactive Publication Date: 2012-02-01
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

In order to improve the practical application value of recombinant adenovirus vaccine, at present, the display vaccine technology based on human adenovirus type 5 has been studied, but the technology of display vaccine based on canine adenovirus type II has not been reported at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cloning of the complete genome of recombinant vaccines displaying rabies virus protective antigens with six neighbor particles

[0028]Eco52I and SnaB I double digested pPOLYII-CAV2, recovered a 12516bp fragment by agarose gel electrophoresis, and cloned it into the pET-28a vector to obtain the recombinant plasmid pET-LH. Then pET-LH was double-digested with Nde I and Mlu I, and the 4325bp fragment was recovered by agarose gel electrophoresis, and cloned into pET-28a vector to obtain the recombinant plasmid pET-SH. The neutralizing epitopes of rabies virus were inserted into four different sites in the hexon by overlapping PCR, and the PCR products were cloned into pMD18-T vector for sequence determination. After the sequencing was correct, the modified hexosome gene was cloned into pET-SH using the two natural restriction sites of hexosome Kpn I and Dra I. Finally, according to the above route, four kinds of recombinant canine adenovirus type II whole genom...

Embodiment 2

[0055] Example 2: Cloning of the complete genome of recombinant vaccines displaying rabies virus protective antigens with fibers

[0056] Using the enzyme cutting sites Sac I and Kpn I in pBluescriptII KS (+ / -), insert the artificially synthesized sequence Linker SK (containing multiple enzyme cutting sites of Sal I, Xma I, Spe I and Pac I in sequence) to obtain recombinant Plasmid pBS-SKI. According to the filament gene sequence, artificially synthesized canine adenovirus type II genome sequence 28050bp ~ 28106bp segment, and inserted it into Linker SK between Sal I ~ Xma I, and obtained the sequence of 30182bp ~ 29358bp in the whole genome of canine adenovirus type II virus by PCR , Insert the two fragments between Spe I~Pac I in Linker SK to obtain the recombinant plasmid pBS-SKII. Then insert the rabies virus neutralizing antigenic epitope between Xma I and Spe I sites in the Linker SK of pBS-SKII according to the transcriptional reading frame of the spike gene to obtain ...

Embodiment 3

[0072] Embodiment 3: the cloning of the complete genome of the recombinant vaccine that displays rabies virus protective antigen with IX protein

[0073]EcoR I and Cla I double-digested pPOLYII-CAV2, recovered a 3610bp fragment by agarose gel electrophoresis, and cloned it into pBluescriptII KS (+ / -) to obtain the recombinant plasmid pBS-LIX. Mlu I and EcoR I double digested pBS-LIX, agarose gel electrophoresis recovered a 340bp fragment, cloned into pEGFP-C1, and obtained the recombinant plasmid pEGFP-SIX. At the stop codon of the IX protein, a single restriction site Ase I was used to insert the rabies virus glycoprotein outer region gene (G') and the SV40 poly A transcription termination signal sequence to realize the fusion expression of the IX protein and the glycoprotein outer region . Finally, according to the above-mentioned route, reverse cloning to obtain the whole genome plasmid pPOLYII-CAV2 / IX-G' of recombinant canine type II adenovirus.

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Abstract

The invention discloses an II type adenovirus live vector recombinant vaccine of a dog for showing a lyssa virus protective antigen by using a spike and relates to a recombinant vaccine for respectively showing the lyssa virus protective antigen or an epitope by using characteristics of different structure proteins of the II type adenovirus of the dog. A lyssa virus protective antigen gene or a nucleotide sequence for expressing the epitope is inserted in a hydrophobic locus spike of the structure protein gene of the II type adenovirus of the dog, so that an exogenous antigen or epitope is subjected to fusion expression with the structure proteins of the II type adenovirus of the dog on the surface of the adenovirus. After toxic substances are eliminated from the recombinant vaccine, the closed isolated feeding for a test animal is carried out and the food searching and pathogenic conditions of the test animal are observed and recorded. The result shows that the dog subjected to oral administration and intramuscular injection of any recombinant vaccine can resist attack of virulent strain and has a survival rate of over 90 percent.

Description

Technical field: [0001] The invention relates to a recombinant vaccine which utilizes the characteristics of different structural proteins of canine type II adenovirus to respectively display protective antigens or antigenic epitopes of rabies virus, and can be used for the development and application of novel animal rabies vaccines. Background technique: [0002] Rabies is an important zoonotic infectious disease caused by rabies virus and characterized by non-suppurative encephalomyelitis. After infecting humans and animals, once the onset occurs, the mortality rate is almost 100%. my country is a place where rabies frequently occurs. Thousands of people die of rabies every year, mainly caused by the bite of a virus-carrying dog. Although commercial vaccines for humans are safe and effective at present, the existence of a large number of infected animals still poses a direct threat to human life. Especially in recent years, the number of people who die from rabies is incr...

Claims

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Application Information

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IPC IPC(8): A61P31/14C12N15/66A61K39/205C12N15/861
Inventor 扈荣良张守峰刘晔李忠
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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