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Zika virus (ZIKV) E antigen and application thereof in detecting anti-ZIKV antibody

A Zika virus and antigen technology, which is applied in the field of immune detection of anti-Zika virus antibodies, can solve the problems of lack of immune detection reagents, etc., and achieve the effects of easy promotion and application, easy operation, and reduced risk

Active Publication Date: 2017-06-23
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] my country's Center for Disease Control and Prevention and some companies have developed Zika virus nucleic acid diagnostic reagents, but there is still a lack of immune detection reagents

Method used

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  • Zika virus (ZIKV) E antigen and application thereof in detecting anti-ZIKV antibody
  • Zika virus (ZIKV) E antigen and application thereof in detecting anti-ZIKV antibody
  • Zika virus (ZIKV) E antigen and application thereof in detecting anti-ZIKV antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning, expression and activity identification of zika virus E antigen

[0040] 1. Design and synthesis of E antigen gene

[0041] In order to obtain efficient expression of the zika virus E gene in E. coli, based on the determined amino acid sequence of the zika virus E antigen, the E. gene was designed by selecting the biased codons of E. coli and entrusted with Shanghai Invitech Biotech Service Co., Ltd. The company synthesizes the gene of the above sequence, and the sequence is shown in SEQ ID No.2.

[0042] 2. Construction and transformation of E antigen expression plasmid

[0043] In order to facilitate the cloning of the gene into the expression vector, two restriction sites of Xho I and Xba I were introduced at both ends of the connecting arm to suit the expression vector pBVIL1 (see Chinese Patent ZL00100695.9: Expression Vector pBVIL1 and its construction method and use). After double enzyme digestion, it was inserted into the vector pBVIL1 to construct ...

Embodiment 2

[0080] Example 2: Anti-ZIKV antibody detection

[0081] Use the following steps: Dilute the purified candidate antigen with pH 9.6 carbonate buffer to 2.5μg / ml, take 100μl in an enzyme-linked plate, coat overnight at 4°C; wash the plate twice, add 120μl blocking solution, room temperature Block for 6h, shake off the blocking solution, pat dry, and dry at room temperature; add sample diluent 90μl, sample 10μl, react at 37°C for 30min; wash the plate 5 times, add 100μl goat anti-human IgG-HRP, react at 37°C for 20min; wash the plate 5 times , Add 50μl each of display solution A and B, react for 10min at 37℃; add 50μl stop solution, and measure the OD value at 450nm wavelength within 10min after termination.

[0082] Anti-ZIKV antibodies were detected in the serum of 17 Zika patients and 53 healthy controls. The test results and analysis results are shown in Table 2: E antigen was positive in 17 cases with a positive rate of 100%; in 53 healthy control sera, all were negative with a ...

Embodiment 3

[0085] Example 3: Preparation of an enzyme-linked immunosorbent assay kit

[0086] 1. Configuration of reagents

[0087] The configurations of carbonate buffer, PBST washing buffer, blocking solution, sample diluent, developer A solution, developer B solution, and stop solution are the same as in Example 1.

[0088] 2. Antigen coating

[0089] Dilute the candidate antigen purified in Example 1 with pH 9.6 carbonate buffer to 2.5μg / ml, take 100μl in an enzyme-linked plate and coat overnight at 4°C; wash the plate twice and add 120μl blocking solution, Seal at room temperature for 6 hours, shake off the blocking solution, pat dry, and dry at room temperature.

[0090] 3. Enzyme-labeled secondary antibody

[0091] Label HPR enzyme on goat anti-human antibody IgG, and dilute with PBST buffer to make the final detection titer 1:2000.

[0092] 4. Subpackage

[0093] Take PBST washing buffer, sample diluent, developer A solution, developer B solution, stop solution, enzyme-labeled secondary anti...

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Abstract

The invention belongs to the field of immunity detection of an anti-zika virus (ZIKV) antibody, and particularly relates to a ZIKV E antigen and application thereof in detecting the anti-ZIKV antibody. After a ZIKV gene group and the expression protein thereof are analyzed, the E antigen for specifically detecting the anti-ZIKV antibody is found, the anti-ZIKV antibody in human blood is specifically detected by the founded E antigen, and a kit for detecting the anti-ZIKV antibody is prepared. The method for detecting the anti-ZIKV antibody has the advantages that the use of ZIKV with strong infection ability is effectively avoided in the detection process, so that the risk of a whole experiment is decreased; by adopting the technical scheme, the operation is simple and easy, the repeatability is good, and the like; the method can be easily popularized and applied.

Description

Technical field [0001] The invention belongs to the field of immune detection of anti-Zika virus antibodies, and specifically relates to a Zika virus E antigen and its application in detecting anti-Zika virus antibodies. Background technique [0002] Zika virus (ZIKV) is an arbovirus transmitted by the Aedes mosquito. It was first isolated from rhesus monkeys in the Zika forest in Uganda in 1947. Before 2005, there were sporadic human infections of Zika virus in Africa and Southeast Asia. In 2007, the first outbreak of human infection with Zika virus occurred on Yap Island in the Federated States of Micronesia in the Western Pacific. With the spread of Zika virus worldwide and the worsening of the epidemic, the World Health Organization (WHO) Both countries have issued Zika virus disease prevention and control plans, and my country has also released the 2016 Zika virus disease diagnosis and treatment plan in time to guide the diagnosis and treatment of clinical cases. Among thos...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 杨锡琴冯晓燕张贺秋修冰水刘志强张旭辉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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