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Zika virus NSI antigen and application of Zika virus NSI antigen in preparing fluorescence immunochromatographic reagent

A fluorescent immunochromatography and Zika virus technology, which is applied in the field of fluorescent immunochromatography in medical immunology, can solve the problems of false positive detection results, low detection sensitivity, low antigen specificity, etc., and achieve accurate and reliable detection results , Expand the detection range, and the effect of short time consumption

Active Publication Date: 2019-12-13
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the enzyme-linked immunosorbent assay method requires special detection equipment, the detection time is long, and the detection sensitivity is not high. The antigen used in the detection is not specific. When used in immunological detection, there are disadvantages of false positive detection results
The current method of Zika virus antigen preparation, its expression level is not high, mainly because the codons for amino acid translation of Zika virus antigen are not applicable

Method used

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  • Zika virus NSI antigen and application of Zika virus NSI antigen in preparing fluorescence immunochromatographic reagent
  • Zika virus NSI antigen and application of Zika virus NSI antigen in preparing fluorescence immunochromatographic reagent
  • Zika virus NSI antigen and application of Zika virus NSI antigen in preparing fluorescence immunochromatographic reagent

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Experimental program
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Embodiment 1

[0034] The preparation of embodiment 1 Zika virus E antigen

[0035] 1. Construction of recombinant plasmids

[0036] According to the Escherichia coli O127:H6 preferred codons, the gene sequence of the Zika virus NS1 protein was selected, and the secondary structure of the recombinant protein was screened through bioinformatics. After testing, the nucleotide sequence of the amino acid encoding the NS1 protein Connect it into the expression vector PGEX-4T-2, obtain the connection product, namely the recombinant plasmid, transform it into DH5α competent cells, and check whether the corresponding protein can be obtained.

[0037] After a large number of experimental verifications, the amino acid sequence of the Zika virus NS1 protein was selected as shown in SEQ ID NO.1, and 25 corresponding gene sequences were connected to the expression vector PGEX-4T-2, and the corresponding 25 gene sequences were selected. Clone expression, and finally select the single clone with the large...

Embodiment 2

[0050] Example 2 Preparation of Fluorescence Immunochromatography Reagent for Detecting Zika Virus NS1 Antibody

[0051] 1. Antibody protein labeled with fluorescent latex microspheres

[0052] (1) 1. Take 200 μL of fluorescent latex microspheres (microspheres with carboxyl groups), add 800 μL of 0.15 mol / L PBS, and centrifuge at 12000 rpm for 10 minutes. Aspirate the supernatant, repeat centrifugation twice, add 500 μL 0.2mol / L PBS to resuspend for use, take 200 μL 8 mg / mL EDC (N-hydroxysuccinimide) solution, 200 μL 5 mg / mL NHS (N-hydroxyl succinimide) solution. Add the fluorescent latex microspheres obtained in the previous step, vortex and mix well, and place them on the reaction rack in the dark for 30 minutes to obtain activated fluorescent latex microspheres.

[0053] (2) The above-mentioned obtained activated fluorescent latex microspheres were centrifuged at 12000rpm for 10min, discarded the supernatant and redissolved with labeling buffer, and simultaneously added m...

Embodiment 3

[0070] The repeatability of embodiment 3 fluorescent immunochromatography reagents

[0071] The fluorescent immunochromatography reagent prepared in Example 2 was used to detect three concentrations of 2×, 10×, and 50× (representing a 50-fold diluted positive sample, and the positive sample was provided by Changchun Customs Inspection and Quarantine Center) in the linear range, and detected At the same concentration, T fluctuates obviously, and C basically remains unchanged, such as Figure five ; Each concentration is detected 15 times, and the ratio of the variance and the mean value of the obtained T, C, T / C is used as the coefficient of variation, i.e. (CV), as a result, it is found that the CV of the signal values ​​of T, C≥15%T The CV of / C≤8%, as shown in Table 2, so T / C can correct T. Using the fitting curve of T / C to calculate the variation coefficients of the intra-assay and inter-assay concentrations of the three batches of test strips, the intra-assay and inter-as...

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Abstract

The invention relates to a Zika virus NSI antigen and the application of Zika virus NSI antigen in preparing a fluorescence immunochromatographic reagent. The amino acid sequence of the Zika virus NS1antigen is shown as SEQ ID NO. 1. The prepared fluorescence immunochromatographic reagent is a fluorescence immunochromatographic reagent for detecting a Zika virus antibody. A sample pad, a marker pad, a coating pad and an absorption pad are sequentially overlapped and adhered to a bottom lining card, and the marker pad is a glass fiber coated with a fluorescent latex microsphere labeled mouse anti-human IgG monoclonal antibody, and the coating pad is a nitrocellulose membrane coated with a goat anti-rabbit-mouse IgG antibody as a quality control line and a detection line coated with the Zika virus NS1 antigen. The Zika virus NS1 antigen provided by the invention has a better expression effect and is used for preparing the fluorescence immunochromatographic reagent for detecting a Zika virus IgG antibody, and a detection result can be observed by naked eyes after irradiation by ultraviolet rays.

Description

technical field [0001] The invention relates to a method for detecting Zika virus NS1 antigen and its application in the preparation of fluorescent immunochromatography reagents, belonging to the technical field of fluorescent immune chromatography in medical immunology. Background technique [0002] After people are infected with Zika virus (Zika Virus), 1 / 4-1 / 5 of the infected people will develop the disease. The clinical features are mild and mostly flu-like, mainly fever, arthralgia, rash or conjunctivitis, which lasts for several days. However, some patients will develop Guillain-Barré syndrome, which poses a great threat to pregnant women, leading to fetal miscarriage, neonatal microcephaly and even death. Zika virus disease urgently needs prevention and control. [0003] After Zika virus infection, its symptoms are mild, and about 80% of Zika virus-infected patients will not show any symptoms, which has greatly interfered with clinical diagnosis and the prevention an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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