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Recombinant human adenovirus 3, and preparation method and application thereof

An adenovirus and genome technology, applied in the field of human type 3 and type 7 adenovirus bivalent vaccine candidate strains and their preparation

Active Publication Date: 2014-08-06
GUANGZHOU GIR MEDICINE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, multivalent vaccines that simply mix different types of adenoviruses may be limited by recombination in vivo to produce highly pathogenic viruses

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: Construction of recombinant virus vector

[0018] Shuttle vector construction: HAdv3-gz01 virus strain genome (Genbank accession no.DQ099432) was used as a template to PCR amplify the 26,782 to 27,736, 973bp E3L fragment (KpnI+ClaI) and the genome 30,900 To 31,901, 1020bp long E3R fragment (SpeI+NotI), use pEGFP C2 vector as a template to PCR amplify CMV-eGFP-SV40 expression cassette (ClaI+SpeI), and sequentially digest and ligate to pBluescript II SK(+) vector Above, the vector pSKE3LCMV-eGFP-SV40E3R (deleting the 3203 bp sequence of the E3 region) including the left and right arm sequences of the E3 pressure was obtained. With primer pair A7Hu(5'-aat cccggg ccaccatggccacccccatcgatgatgcc-3', AvaI site was introduced) and A7Hr (5'-taaaagcttttatgtggtggcgttgccg-3', HindIII site was introduced) PCR amplified HAdv7 hexon whole gene, AvaI+HindIII double digestion followed by AgeI+HindIIl double digestion The vector pSKE3LCMV-eGFP-SV40E3R was connected, and t...

Embodiment 2

[0021] Example 2: Identification of Capsid Composition of Recombinant Human Adenovirus Type 3 Particles

[0022] In order to verify the composition of the capsid hexon of the obtained recombinant virus particles, Western Blot detection was performed with HAdv3 and HAdv7 hexon type-specific antibodies. Antiserum immunized with Ad3-R4 epitope (VKPTTEGGVET) GST fusion protein mainly reacted with HAdv3 hexon, but only weakly reacted with HAdv7 hexon; antiserum immunized with Ad7-R4 epitope (VKPTEGDVET) GST fusion protein Only reacts with HAdv7 hexon, but not with HAdv3 hexon. Western Blot test results see figure 1 , figure 1a is the result of detection of anti-Ad3-R4-GST antiserum and purified Ad3dE3Ad7H virus particles, figure 1 b is the graph of detection results of anti-Ad7-R4-GST antiserum and purified Ad3dE3Ad7H virus particles. The results showed that the prepared and purified Ad3dE3Ad7H virus particles were chimeras containing two types of hexons, HAdv3 and HAdv7.

Embodiment 3

[0023] Example 3: Immunogenicity of Recombinant Viral Vectors and Recombinant Viral Particles

[0024] Antiserum was prepared and serum neutralization test was performed to verify its immunogenicity.

[0025] Recombinant virus vector: the purified Ad3dE3Ad7H virus vector was intramuscularly injected into mice (1×10 10 VPs / mouse), obtain polyclonal serum, and then carry out micro-neutralization test to detect whether the antibody produced by the recombinant virus vector has the ability to neutralize HAdv3 and HAdv7 viruses in vitro.

[0026] Recombinant virus particles: After inactivating the purified Ad3dE3Ad7H virus particles with β-propiolactone or formalin, immunize mice (1×10 10 VPs / mouse), obtain polyclonal serum, and then perform microneutralization test to detect whether the antibodies induced by recombinant virus particles have the ability to neutralize HAdv3 and HAdv7 viruses in vitro.

[0027] The results are shown in Table 1, which is the neutralization titer of t...

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Abstract

The invention discloses a novel human adenovirus 3 and 7 bivalent vaccine candidate strain with human adenovirus 3 (HAdv3) as a carrier, and a preparation method thereof. The capsid of the HAdv3 includes an HAdv3 hexon and an HAdv7 hexon. An HAdv7 hexon protein expression cassette is inserted to human adenovirus 3 genome E3 region, rescued packaging is carried out in in-vitro cells to obtain a hexon chimeric recombinant adenovirus, and the capsid of the hexon chimeric recombinant adenovirus includes an HAdv3 hexon and an HAdv7 hexon. The inactivated or non-inactivated vaccine candidate strain can induce an anti-HAdv3 and anti-HAdv 7 neutralization effect antibody, 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 human type 3 and type 7 adenovirus bivalent vaccine candidate strain using human type 3 adenovirus (HAdv3) as a carrier and a preparation method thereof. Background technique [0002] Human adenovirus (Adenovirus) was isolated and identified from patients with acute respiratory diseases in 1953. It is a kind of pathogen that mainly infects the human respiratory tract and digestive tract. So far, humans have discovered 6 species and 52 different serotypes of adenoviruses; about 1 / 3 of them can cause acute respiratory infection, epidemic keratitis, acute hemorrhagic conjunctivitis, pharyngeal conjunctivitis, acute Pharyngitis, cystitis, fatal pneumonia in infants and young children and other diseases; patients are mainly infants and young children and frail people. In my country, about 5% of acute respiratory diseases in children under t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/04A61K39/235A61P31/20C12R1/93
Inventor 周荣田新贵苏晓波薛春燕钟南山
Owner GUANGZHOU GIR MEDICINE CO LTD
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