Preparation method and application of human adenovirus type 3 displaying human adenovirus type 55 neutralizing epitope vaccine candidate strain

An adenovirus and candidate strain technology, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problems of no vaccine and market launch, and achieve the effect of strengthening immunity and preventing infection

Inactive Publication Date: 2018-10-19
东莞市第八人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Vaccination against adenovirus is one of the most effective ways to prevent adenovirus type 55 infection, but there is currently no vaccine on the market

Method used

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  • Preparation method and application of human adenovirus type 3 displaying human adenovirus type 55 neutralizing epitope vaccine candidate strain
  • Preparation method and application of human adenovirus type 3 displaying human adenovirus type 55 neutralizing epitope vaccine candidate strain
  • Preparation method and application of human adenovirus type 3 displaying human adenovirus type 55 neutralizing epitope vaccine candidate strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Bioinformatics analysis predicts the surface exposure region of human adenovirus type 3 hexon

[0022] The template structure of HAdv3 hexon homology modeling was searched in the Protein Data Bank (PDB) using the BLAST-P program, and Modeller 9.9 was used for homology modeling to construct a 3D model of the HAdv3 hexon protein. Select the hypervariable region sequence exposed on the surface of the capsid for the next experiment.

[0023] By analyzing the 3D model, the neutralizing epitope on the hexon of human adenovirus type 3 can be located, and the sequence exposed on the surface of the capsid that is most likely to be substituted without affecting the stability of the hexon structure can be analyzed. HVR1, HVR2, HVR4, HVR5, and HVR7 are potential neutralizing epitope regions, in which some amino acids may be substituted without affecting the stability of the hexon structure. The sequences of the two hypervariable regions are shown below, where the underli...

Embodiment 2

[0025] Embodiment 2: Construction of recombinant virus vector

[0026] The modified hexon gene was amplified by overlapping PCR (overlapping PCR). First, the DNA sequence of A55R2 (5'-TTGAAAGTTTCAGATGAAGAAAGT-3') was obtained by overlapping PCR using the HAdv3 genome as a template to replace the corresponding HVR2 of the HAdv3 hexon gene. The mutant hexon fragment of the sequence is connected to the T vector and identified by sequencing; Bam HI+ cla After double enzyme digestion, I was connected to the pBRHLR vector to obtain the shuttle vector pBRHLRA55R2. use EcoR I+ Sal I double enzyme cut shuttle vector, with vR II+ Pac The backbone plasmid pBRAd△E3GFP cut with double restriction enzymes was subjected to homologous recombination in Escherichia coli BJ5183 to obtain the recombinant plasmid pAd△E3GFPA55R2. through Bam H I / Sa l I / Hin d III enzyme digestion identification, no mutation occurred in the recombinant plasmid ( figure 1 ). The primers used are ...

Embodiment 3

[0030] Embodiment 3: Recombinant viral vector immunogenicity test

[0031] Antiserum was prepared and serum neutralization test was performed to verify the immunogenicity of the recombinant virus vector. The purified recombinant virus Ad3A55R2 was injected intramuscularly to immunize mice to obtain polyclonal serum, and then an in vitro neutralization test was performed to detect whether the antibodies produced by the recombinant virus had the ability to neutralize HAdv55 virus and HAdv3 virus in vitro.

[0032] The experimental results of the neutralization reaction of the mouse serum collected 35 days after the two immunizations are shown in Table 2, and high titers of neutralizing antibodies against HAdv55 virus and anti-HAdv3 virus can be induced.

[0033] Table 2 Neutralization titer of immune serum

[0034]

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Abstract

The invention relates to the technical field of genetic engineering of recombinant viruses, in particular to an application and preparation method of a candidate strain of a human type-3 adenovirus expressed human type-55 adenovirus neutralization epitope vaccine. Two human type-55 adenovirus neutralization epitopes are respectively embedded to hexons of human type-3 adenoviruses to be displayed on the surfaces of the hexons. The candidate strain of the vaccine is capable of inducing strong anti-HAdv55 and anti-HAdv3 infection immunoreactions and can be used for preparation of bivalent vaccines for prevention of HAdv55 and HAdv3 infection.

Description

technical field [0001] The invention relates to the technical field of recombinant virus genetic engineering, in particular to a preparation method and application of a human type 3 adenovirus displaying a human type 55 adenovirus neutralizing antigen epitope vaccine candidate strain. Background technique [0002] First discovered in 1953, adenoviruses are capable of causing many diseases, including acute respiratory disease (ARD), pneumonia, epidemic keratoconjunctivitis, and acute gastroenteritis. According to the homology and difference of their genomic DNA, human adenoviruses (HAdVs) are divided into seven categories (A-G), more than 65 serotypes, and different adenoviruses can cause different diseases. In recent years, the reports on new alloadenoviruses mainly focus on gene recombination, including human adenovirus type 55 (HAdV-55), which were reported in Singapore and Shaanxi, China in 2005 and 2006, respectively. Five years later, in 2011, an outbreak occurred in B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/861A61K39/235A61P31/20
Inventor 马强田新贵蒋再学刘倩陆小梅骆庆明
Owner 东莞市第八人民医院
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