Dengue virus serum type 3 epidemic strain GZ14D3 replicon and application thereof

A technology of dengue virus and replicons, applied in the field of genetic engineering

Pending Publication Date: 2021-11-05
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific antiviral drug, and there are several vaccines used in some countries or regions or in clinical trials. However, due to the phenomenon of dengue antibody-dependent enhancement (Antibody-Dependent Enhancement, ADE), the development and Promotion presents a formidable challenge

Method used

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  • Dengue virus serum type 3 epidemic strain GZ14D3 replicon and application thereof
  • Dengue virus serum type 3 epidemic strain GZ14D3 replicon and application thereof
  • Dengue virus serum type 3 epidemic strain GZ14D3 replicon and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Phylogenetic tree of DENV-3 epidemic strain GZ14D3

[0051] First, inoculate clinical dengue virus samples on Vero cells for culture and amplification to obtain higher titer viruses, extract viral RNA, reverse to obtain cDNA, and amplify sequences covering most of the genome by PCR in three segments , Sequencing identification of strains. According to the Rico-Hesse dengue virus genotype nomenclature, we downloaded the E protein gene sequences of DENV-3 genotypes and representative strains of DENV1-4 from GenBank, and compared them with MEGA7 software, neighbor-joining (NJ) to build a phylogenetic tree. According to the phylogenetic tree, it can be seen that the epidemic strain ZG14D3 belongs to the dengue virus serotype III type, and the genotype belongs to the Indian subcontinent type ( figure 1 ).

Embodiment 2

[0052] Construction steps of embodiment 2 DENV-3 replicon

[0053] The vector we use is the pTight vector, which contains a high-copy origin of replication derived from pBR322, a seven-time tetracycline response element (7xTRE), a cytomegalovirus (CMVmin) eukaryotic promoter, 3'UTR, and the first 402 of the capsid protein Base sequence, renilla luciferase reporter gene (Gluc), FMDV2A protein cleavage sequence, neomycin resistance gene (Neo), EMCV IRES element, base sequence of the first 24 amino acids of E protein, the entire nonstructural protein sequence, the 3'UTR of DENV-2 16681, the antigenome sequence of hepatitis delta virus ribozyme (HDVr), and the polyadenylation signal sequence of Simian Virus 40 (SV40). When constructing the replicon, the method of homologous recombination was adopted. Since the 3'UTR of this strain was cloned into the vector, there was a deletion phenomenon, so the 3'UTR of the DENV-2-16681 strain was used to replace it, and finally a chimeric DEN...

Embodiment 3

[0054] Example 3 DENV-3 replicon effectively replicates in cells

[0055] The replicon plasmid was transfected into BHK-21 cells and screened with neomycin, and the DENV-3 monoclonal replicon cells containing stable expression of high luciferase reporter gene signal were screened out after 3 weeks ( image 3 ).

[0056] Extract the RNA of the replicon cells that have been continuously passaged for 120 days, and sequence and analyze the DENV-3 RNA sequence. It is found that there are 3 synonymous mutations in NS2B, NS3 and NS5 respectively (Table 1), and the luciferase activity of the replicon during passage has remained at a high and stable level.

[0057] Table 1 The sequence of the RNA extracted from the BHK-21 replicon cells of 120 days of continuous passage and analyzed by reverse sequencing

[0058]

[0059]

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Abstract

The invention relates to the field of gene engineering, in particular to a dengue virus serum type 3 epidemic strain GZ14D3 replicon and application thereof. A direct-early promoter of cytomegalovirus which can be transcribed in eukaryotic cells is utilized to construct a subgenome replicon of a dengue virus serum type 3 clinical isolate with a luciferase reporter gene, and the subgenome replicon comprises 5 'UTR of the virus, a partial capsid protein sequence and an NS1-NS5 sequence. The replicon plasmid is transfected to a BHK-21 cell, and the BHK-21 cell is screened by using neomycin, so that a DENV-3 monoclonal replicon cell containing a stable expression luciferase reporter gene signal is screened out. The known DENV inhibitor mycophenolic acid is used for verifying that the medicine shows a good virus replication inhibition effect on replicon cells, and the medicine of methylclocycline sulfosalicylate with an inhibition effect on DENV-3 replicon is found.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a dengue virus serotype 3 epidemic strain GZ14D3 replicon and application thereof. Background technique [0002] A series of diseases caused by dengue virus (DENV) infection are important mosquito-borne viral diseases in tropical areas at present, and the vectors are Aedes aegypti and Aedes albopictus. Before 1970, 9 countries had serious dengue fever epidemics, and dengue has affected more than 100 countries in tropical and subtropical regions at present, and about 390 million dengue fever infection cases occur every year, and about 70% of the cases occur in Asia. The World Health Organization estimates a 30-fold increase in the observed global incidence over the past 50 years. Today, dengue virus poses a major threat to public health worldwide, with approximately two-fifths of the world's population at risk of dengue infection. [0003] Globally, there are four serotypes of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/85C12N15/65C12N15/66C12N5/10C12Q1/02A61K31/65A61K31/60A61P31/14
CPCC12N7/00C12N15/85C12N15/65G01N33/5008A61K31/65A61K31/60A61P31/14C12N2770/24121A61K2300/00Y02A50/30
Inventor 李义平杨炀
Owner SUN YAT SEN UNIV
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