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A kit for detecting whether isolated serum samples are infected with Japanese encephalitis virus

A Japanese encephalitis virus and kit technology, applied in the field of microorganisms, can solve the problems of small molecular weight of EIII protein, low detection sensitivity, and few antigenic epitopes

Active Publication Date: 2022-02-25
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the EIII protein has a small molecular weight and fewer antigenic epitopes, so the detection sensitivity is low, especially when used to detect serum with low antibody titer, and it is easy to miss the detection.

Method used

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  • A kit for detecting whether isolated serum samples are infected with Japanese encephalitis virus
  • A kit for detecting whether isolated serum samples are infected with Japanese encephalitis virus
  • A kit for detecting whether isolated serum samples are infected with Japanese encephalitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, the preparation of fusion protein Nano-EIII-opti

[0070] 1. Optimization of the gene encoding EIII protein

[0071] The EIII protein of JEV (hereinafter referred to as EIII protein) was selected as the diagnostic antigen for serological detection of JEV infection. The amino acid sequence of the EIII protein is shown in sequence 3 in the sequence listing. In JEV, the nucleotide sequence of the gene encoding EIII protein (EIII gene) is shown as sequence 1 in the sequence listing.

[0072] In order to make the nucleotide sequence of the EIII gene more suitable for expression in eukaryotic cells, the inventor of the present invention has optimized the nucleotide sequence of the EIII gene through a large number of experiments, and the optimized EIII gene (hereinafter referred to as the EIII-opti gene) The nucleotide sequence of is shown in sequence 2 in the sequence listing. The amino acid sequence of the EIII-opti protein encoded by the EIII-opti gene is s...

Embodiment 2

[0096] Example 2, the optimization of serum conditions for detection of Japanese encephalitis by luciferase immunocapture method

[0097] Buffer A: containing 20mM Tris, 150mM NaCl, 5mM MgCl 2 and 1% (v / v) Triton X-100 in water.

[0098] Antigen use solution: take the cell culture supernatant (that is, the solution containing the fusion protein Nano-EIII-opti) of the recombinant plasmid pNLF-JEV-EIII-opti collected in Example 1 transfected for 48 hours, add buffer A to dilute luciferase Intensity to 1×10 7 luc / 50 μL.

[0099] 1. Detection of JE serum by different incubation methods

[0100] Detection sample use solution: obtained by mixing 1 volume part of the detection sample and 50 volume parts of buffer A. The test samples were 3 positive human sera of clinically confirmed JEV infection (named positive sample 1, positive sample 2 and positive sample 3 in sequence) and 1 healthy human serum (named negative sample).

[0101] 1. Separate incubation

[0102] (1) Take the ...

Embodiment 3

[0133] Example 3. Specificity and sensitivity of luciferase immunocapture method for detection of Japanese encephalitis clinical serum samples

[0134] Buffer A: containing 20mM Tris, 150mM NaCl, 5mM MgCl 2 and 1% (v / v) Triton X-100 in water.

[0135] Serum sample to be tested solution: obtained by mixing 1 volume part of serum sample to be tested and 50 volume parts of buffer A. Serum samples to be tested were 20 JEV-positive serum samples (including 10 clinically confirmed JEV-infected patient serum samples and 10 healthy human serum samples vaccinated with JEV vaccine), 10 healthy human serum samples, and 10 clinically confirmed Serum samples from patients with tick-borne encephalitis virus infection (TBEV serum samples), 2 serum samples from WNV rabbits (WNV serum samples), 10 serum samples from patients with clinically confirmed dengue virus infection (dengue serum samples) and 3 Serum samples from patients with clinically confirmed Zika virus infection (ZIKV serum samp...

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Abstract

The invention discloses a kit for detecting whether an isolated serum sample is infected with Japanese encephalitis virus. The kit includes fusion protein composed of EIII protein and Nano luciferase, Nano luciferase substrate, protein A / G conjugate and isolated serum samples not infected with Japanese encephalitis virus. Experiments have proved that the kit provided by the invention can detect whether the isolated serum sample is infected with Japanese encephalitis virus, and the specificity reaches 100%, and there is no cross-reaction with other virus-infected sera; the sensitivity also reaches 100%. The invention has great application value.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a kit for detecting whether an isolated serum sample is infected with Japanese encephalitis virus. Background technique [0002] Japanese encephalitis virus (JEV) belongs to the family Flaviviridae and belongs to the genus Flavivirus. It is a virus that is transmitted through mosquito bites. It can cause encephalitis and meningitis, and can cause severe neurological sequelae. my country used to be a high-incidence area of ​​Japanese encephalitis. Since the large-scale vaccination of Japanese encephalitis vaccine in the 1980s, the incidence of Japanese encephalitis has dropped significantly. The diagnosis of JEV infection mainly relies on serology and pathogenic nucleic acid detection. Serological methods are not only an effective means of virus infection detection, but also can be used for epidemiological investigation of JEV infection and evaluation of immune effect of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26C07K19/00
CPCC12Q1/26C07K14/005C07K2319/61G01N2333/185C12N2770/24122
Inventor 康晓平李裕昌吴晓燕户义李靖张森
Owner ACADEMY OF MILITARY MEDICAL SCI