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Dog type II adenovirus live vector recombinant vaccine for displaying protective antigen of rabies virus

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: 2012-05-23
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 SalI, Xma I, Spe I and Pac I in sequence) to obtain a recombinant plasmid pBS-SKI. According to the spike gene sequence, the 28050bp-28106bp segment of the whole genome sequence of canine adenovirus type II was artificially synthesized, and inserted between SalI-XmaI in Linker SK, and the sequence of 30182bp-29358bp in the whole genome of canine adenovirus type II was obtained by PCR. The two fragments were inserted between Spe I~Pac I in Linker SK to obtain 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 the modified spike gene....

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 a recombinant vaccine with canine adenovirus type-2 as a live vector for displaying a protective antigen of rabies virus, which relates to recombinant vaccines displaying the protective antigen or an antigenic epitope of the rabies virus respectively by utilizing the characteristics of different structural proteins of the canine adenovirus type-2. The protective antigen ofthe rabies virus or a ribonucleotide sequence expressing the antigenic epitope are inserted at a hydrophobic locus of a structural protein gene to ensure that an exogenous antigen or an antigenic epitope is subject to fusion expression with structural protein of the exogenous antigen or the antigenic epitope on the surfaces of adenoviruses. After the recombinant vaccines challenge, test animals are subject to closed and isolated rearing, feeding and disease conditions of the test animals are observed and recorded; and the results show that dogs taking any kind of recombinant vaccine by oral administration and intramuscular injection all can resist the attack of strong viruses, and the survival rate is more than 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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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/205A61P31/14C12N15/861C12N15/11C12N15/47C12N15/62
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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