Zika virus E antigen and application of Zika virus E antigen in fluorescence immunochromatographic reagent

A fluorescence immunochromatography and Zika virus technology, which is applied in the field of fluorescence immunochromatography in medical immunology, can solve the problems of low preparation and expression of Zika virus E antigen protein, false positive detection results, weak specificity, etc. Achieve the effect of simple result determination, wide detection range and strong detection specificity

Active Publication Date: 2019-12-13
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Zika virus has strong serological cross-reactivity with other viruses of the flavivirus genus, so the selection of antigens is particularly important. At present, the existing antigens are not specific

Method used

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  • Zika virus E antigen and application of Zika virus E antigen in fluorescence immunochromatographic reagent
  • Zika virus E antigen and application of Zika virus E antigen in fluorescence immunochromatographic reagent
  • Zika virus E antigen and application of Zika virus E antigen in fluorescence immunochromatographic reagent

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 Zika virus E antigen

[0034] 1. Construction of recombinant plasmids

[0035] Select the Zika virus E protein, its nucleotide sequence (GenBank sequence number: HM133639.1), modify the gene sequence according to the preferred codons of Escherichia coli O127:H6, and analyze the secondary structure of the recombinant protein by bioinformatics After screening, the nucleotide sequence shown in GenBank sequence number: HM133639.1 was ligated into the expression vector PGEX-4T-2 after testing, and the ligated product was obtained as a recombinant plasmid, which was transformed into DH5α competent cells, but the corresponding protein could not be obtained .

[0036] After a large number of experimental verifications, 20 clones were selected for expression, and finally the single clone with the largest expression was selected. The determined gene sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2...

Embodiment 2

[0041] Example 2 Preparation of Fluorescence Immunochromatography Reagent for Detecting Zika Virus Antibody

[0042] 1. Antibody protein labeled with fluorescent microspheres

[0043] (1) Microsphere activation: 1. Take 200 μL of fluorescent microspheres (bangs carboxyl microspheres), add 1 mL of 0.2 mol / L PBS, and centrifuge at 12000 rpm for 10 minutes. Aspirate and discard the supernatant, repeat centrifugation twice, add 600 μL 0.2mol / LPBS to resuspend for use, take 200 μL 10 mg / mL EDC (N-hydroxysuccinimide) solution and 200 μL 6 mg / mL NHS (N-hydroxysuccinimide) imine) solution. Add the resuspended fluorescent microspheres, vortex and mix well, and place on a rotating reaction rack to react in the dark for 30 minutes.

[0044] (2) Preparation of activated fluorescent microspheres labeled mouse anti-human IgG monoclonal antibody: the activated fluorescent microspheres obtained by the above preparation method were centrifuged at 12000g for 5min, and the supernatant was disc...

Embodiment 3

[0057] The detection of embodiment 3 fluorescence immunochromatography reagent sensitivity

[0058] The reagents prepared in Example 2 were used to detect Zika IgG positive samples (provided by Changchun Customs Inspection and Quarantine Center) respectively, and diluted with diluent PBS to 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, at the same time, use negative serum as a negative control, take 80 μL of serum samples and add sample pads, and judge the results within 8 minutes. The test result is a positive sample with a detection sensitivity of 1:100000 dilution.

[0059] The E antigen prepared in Example 1 was used to prepare colloidal gold immunochromatography test paper, prepared by conventional methods, using SPA to mark colloidal gold particles, the detection zone on the nitrocellulose membrane was coated with NS1 antigen protein, and the quality control zone Coated goat anti-mouse IgG; the obtained colloidal gold immunochromatographic test paper was diluted to...

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Abstract

The invention relates to a Zika virus E antigen and application of the Zika virus E antigen in a fluorescence immunochromatography reagent. The amino acid sequence of the Zika virus E antigen is shownas SEQ ID NO.2. The invention also relates to the application of the Zika virus E antigen in the fluorescence immunochromatography reagent. According to the application, the fluorescence immunochromatographic reagent for detecting a Zika virus antibody is prepared by using the Zika virus E antigen, a sample pad, a marker pad, a coating pad and an absorption pad are sequentially overlapped and adhered to a bottom lining card, the marker pad is a glass fiber coated with a fluorescent microsphere labeled mouse anti-human IgG monoclonal antibody, and the coating pad is a nitrocellulose membrane which is coated with a goat anti-mouse IgG antibody as a quality control line and coated with a detection line of Zika virus E protein. The fluorescence immunochromatography reagent for detecting the Zika virus IgG antibody provided by the invention has the advantages of strong specificity, high detection sensitivity and simplicity and convenience in detection result judgment.

Description

technical field [0001] The invention relates to a Zika virus E antigen and its application in fluorescent immunochromatography reagents, belonging to the technical field of fluorescent immune chromatography in medical immunology. Background technique [0002] Zika virus disease (Zika Virus Disease) is a self-limiting acute infectious disease caused by Zika virus (Zika), the clinical features are mainly fever, rash, arthralgia or conjunctivitis, and rarely cause death. It is mainly transmitted through the bite of Aedes aegypti mosquito, and may be transmitted sexually. The population is generally susceptible, and the prognosis is good. Severe cases and deaths are rare. However, the disease poses a great threat to pregnant women, leading to fetal miscarriage, neonatal microcephaly and even death. Zika virus infection not only causes symptoms such as fever and microcephaly in newborns, but also Guillain-Barré syndrome (GBS), an autoimmune peripheral neuropathy that causes prog...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/533G01N33/543G01N33/569G01N33/577
CPCG01N33/577G01N33/533G01N33/53G01N33/543G01N33/56983Y02A50/30
Inventor 杨宇聂聪侯宝翠王静
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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