Recombinant human adenovirus 3, and preparation method and application thereof

An adenovirus and genome technology, applied in the field of novel enterovirus type 71-recombinant human type 3 adenovirus vaccine candidate strains and their preparation, can solve problems such as application limitations and achieve the effect of preventing infection

Inactive Publication Date: 2014-08-06
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT) +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the pre-existing immunity against human type 5 adenovirus vectors in the population, and the strong immunogenicity of adenovirus vectors, the immune system clears th

Method used

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  • Recombinant human adenovirus 3, and preparation method and application thereof
  • Recombinant human adenovirus 3, and preparation method and application thereof
  • Recombinant human adenovirus 3, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Bioinformatics analysis predicts the surface exposure region of human adenovirus type 3 hexon

[0021] The template structure of HAd3 hexon homology modeling was searched in Protein Data Bank (PDB) using BLAST-P program, and Modeller9.9 was used for homology modeling to construct a 3D model of HAd3 hexon protein. Experimental Pymol processing of image files. Select the hypervariable region sequence exposed on the surface of the capsid for the next experiment.

[0022] By analyzing the 3D model, the potential neutralizing epitope on the human adenovirus type 3 hexon can be located, and the sequence exposed on the capsid surface is most likely to be replaced without affecting the stability of the hexon structure . 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 t...

Embodiment 2

[0025] Embodiment 2: Construction of recombinant virus vector

[0026]The E3 region deletion shuttle vector was constructed using the HAdv3-gz01 virus strain genome (Genbank accession no. DQ099432) as a template to PCR amplify the 26,782 to 27,736, 973bp E3L fragment (KpnI+ClaI) and the 30 , 900 to 31, 901, the E3R fragment (SpeI+NotI) with a length of 1020bp, the CMV-eGFP-SV40 expression cassette (ClaI+SpeI) was amplified by PCR using the pEGFP C2 vector as a template, and ligated to pBluescript II SK (+ ) vector, the vector pSKE3LCMV-eGFP-SV40E3R (deleting the 3203bp sequence of the E3 region) including the left and right arm sequences of the E3 region was obtained.

[0027] Construction of human type 3 adenovirus backbone plasmid: AdLU1 (5'-GAATTCGCGATCGCTATCTATATAATATACCTTATAGATGG-3', introducing EcoRI and AsisI restriction sites) and AdLD1 (5'-CTGCTGTGGATAAGCTTGAG-3', including HindIII restriction site) were amplified Increase the fragment A3L of 1391bp at the left end o...

Embodiment 3

[0035] Embodiment 3: Recombinant viral vector immunogenicity test

[0036] Antiserum was prepared and serum neutralization test was performed to verify the immunogenicity of the recombinant virus vector. The purified recombinant virus AdSP70R1SP55R2△E3VP1 was injected intramuscularly to immunize mice to obtain polyclonal serum, and then a microneutralization test was performed to detect whether the antibodies produced by the recombinant virus had the ability to neutralize EV71 virus and HAdv3 virus in vitro.

[0037] The experimental results of the neutralization reaction of mouse serum collected 21 days after a single immunization are shown in Table 2. A single immunization can induce high-titer neutralizing antibodies against EV71 virus and anti-HAdv3 virus; The experimental results of the neutralization reaction of the mouse serum collected on the 14th day are shown in Table 3. Table 3 is the neutralization titer of the mouse serum for multiple immunizations. be strengthen...

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Abstract

The invention discloses a novel enterovirus 71-recombinant human adenovirus 3 vaccine candidate strain with human adenovirus 3 (HAdv3) as a carrier, and a preparation method thereof. Two EV71 neutralizing epitopes are embedded to the hexon of the human adenovirus 3, and the VP1 protein cassette of EV71 is inserted to the genome E3 region of the human adenovirus 3. The vaccine candidate strain can induce a strong anti-EV71 infection and anti-HAdv3 infection immunization reaction, and can be used for making bivalent vaccines for preventing the EV71 infection and the HAdv3 infection.

Description

technical field [0001] The invention belongs to the technical field of recombinant virus genetic engineering, and in particular relates to a novel enterovirus type 71-recombinant human type 3 adenovirus vaccine candidate strain using human type 3 adenovirus (HAdv3) as a carrier and a preparation method thereof. Background technique [0002] Enterovirus 71 (EV71) is one of the main pathogens causing hand-foot-mouth disease (HFMD), and often causes aseptic meningitis, encephalitis, poliomyelitis-like paralysis and neurogenic Severe central nervous system complications such as pulmonary edema and death. The global outbreak of EV71 infection was first reported in 1969. In recent years, reports of EV71 outbreaks have increased, and the prevalence has shown a clear upward trend. It has become an important pathogen that threatens public health in the Asia-Pacific region, including China. EV71 is transmitted through air droplets and other channels, and has high pathogenicity and re...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66C12R1/93
Inventor 周荣田新贵苏晓波李潇钟南山白培胜
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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