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Recombinant antigenic protein for detecting dengue virus antibody, kit and application thereof

A technology of recombinant antigenic protein and detection kit, applied in the field of genetic engineering, can solve problems such as difficulties in immunological diagnosis and differential diagnosis, difficulty in infection with dengue virus, and limited coverage, so as to facilitate quality control, ensure sensitivity and specificity, highly specific effect

Inactive Publication Date: 2012-05-02
中国人民解放军广州军区疾病预防控制中心
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] From the data of dengue virus serodiagnosis disclosed in the prior art, there are mainly problems in the following aspects: (1) there is serious serological cross-reaction between different flaviviruses, and there are multiple flavivirus epidemics in those Due to the mixed infection, there are cross-antibodies in the patient's body, which makes immunological diagnosis and differential diagnosis very difficult
(2) Since IgG antibodies can exist in the host body for a long time, and some will last more than 10 months, or even carry them for life, it is very difficult to identify past, recent or present infection with dengue virus
(3) Dengue virus is a small RNA virus with a high mutation rate, so its coverage is often limited when using single antigen detection

Method used

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  • Recombinant antigenic protein for detecting dengue virus antibody, kit and application thereof
  • Recombinant antigenic protein for detecting dengue virus antibody, kit and application thereof
  • Recombinant antigenic protein for detecting dengue virus antibody, kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1, detect the preparation of the recombinant antigenic protein of dengue virus

[0046] Prepare the recombinant antigen protein for detecting dengue virus according to the following steps:

[0047] Step 1, select the coding gene presenting the highly specific peptide of the virus in the dengue virus protein as the target gene fragment, which includes the extended segment of the flexible arm of the recombinant antigen protein, and the flexible arm can eliminate the expression vector. The masking of the polypeptide epitope by the fusion protein enables the correct folding of the expressed recombinant antigen protein. Wherein, the target gene fragment includes the coding sequence of Den-Ag5, the coding sequence of Den-Ag1 and the coding sequence of Den-Ag2, and the coding gene sequence of Den-Ag1 has the nucleotide sequence shown in SEQ ID NO.1; The coding gene sequence of Den-Ag2 has the nucleotide sequence shown in SEQ ID NO.3; the coding gene sequence of Den...

Embodiment 2

[0075] Embodiment 2, the coating experiment of recombinant antigenic protein

[0076] In this example, it is necessary to optimize the coating conditions and concentration of the recombinant antigenic protein of the present invention; in order to achieve this purpose, it is also necessary to first explain the components of the ELISA reaction solution in the kit:

[0077] Enzyme conjugate working solution: horseradish peroxidase-labeled anti-human IgG polyclonal antibody;

[0078] Positive control: Dengue fever patient serum.

[0079] Negative control: dengue virus antibody negative human serum.

[0080] Sample diluent: 10mmol / L pH7.4 phosphate buffer containing 1% BSA, 0.05% Tween-20 and 1mg / L gentamicin;

[0081] Concentrated washing solution: 0.1mol / L pH7.4 phosphate buffer containing 1% fetal bovine serum, 0.5% Tween-20 and 20mg / L gentamicin;

[0082] Chromogenic solution A: 0.02% H2O2; dilute with 0.1M citric acid-0.2M disodium hydrogen phosphate, pH4.5~5.0.

[0083] C...

Embodiment 3

[0088] Embodiment 3, ELISA reaction condition optimization

[0089] The antigen coating conditions and ELISA reaction conditions were systematically optimized, and the best ELISA reaction conditions were finally determined as follows: recombinant antigen 100ul (concentration: 10ug / ml) coated overnight at 4°C, washed with PBST for 3 minutes, patted dry, 5% BSA 37 Block at ℃ for 2 hours, add 100ul of the serum to be tested into the reaction well after 1:100 times dilution, react at 37°C for 1 hour, wash with PBST 4 times, each time for 1 minute, pat dry, after the HRP-labeled secondary antibody is diluted 1:10000 times, Add 100ul to each well, react at 37°C for 40 minutes, wash with PBST 4 times, 1 minute each time, pat dry, add 100ul of TMB substrate solution to each well, react at 37°C for 15 minutes, add 50ul 2M H 2 SO 4 The reaction was terminated, and the OD value was measured with a microplate reader at a wavelength of 450 nm.

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Abstract

The invention relates to a recombinant antigenic protein for detecting a dengue virus antibody. The recombinant antigenic protein is formed by mixing prokaryotic expression antigens of the dengue virus, namely Den-Ag5, Den-Ag1 and Den-Ag2, in same amount. The invention further discloses a preparation method of the recombinant antigenic protein. Furthermore, a detection kit of the dengue virus disclosed by the invention comprises an antibody detection plate and an ELISA reaction solution. The recombination antigenic protein formed by mixing the prokaryotic expression antigens of the dengue virus, namely Den-Ag5, Den-Ag1 and Den-Ag2, in same amount is coated on the antibody detection plate. The recombinant antigenic protein disclosed by the invention has the advantages of being strong in specificity and high in affinity. Serological cross reaction does not exist among the recombinant antigenic protein and other similar entomoplily spreading viruses. The recombination antigenic protein has ultra-high affinity with the dengue virus antibody. The dengue virus antibody can be detected rapidly and accurately, so that the infection condition of the dengue virus can be diagnosed.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant antigenic protein for diagnosing dengue virus antibodies and a preparation method thereof, and a dengue virus detection kit using the antigenic protein as a coated antigen. Background technique [0002] Dengue virus (DV) belongs to the Flaviviridae family, including 1 to 4 serotypes. According to the statistics of the World Health Organization, 2.5 billion people in more than 100 countries around the world are currently threatened by dengue virus, and more people are infected every year. It has reached 50 million to 100 million, of which 250,000 to 500,000 people belong to severe dengue hemorrhagic fever, with an average mortality rate of 5%. It is an important tropical disease second only to malaria. The southern provinces of my country are high-incidence areas of dengue fever. Monitoring shows that dengue fever has been prevalent in my country every ye...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/40C12N15/70G01N33/569
CPCY02A50/30
Inventor 任瑞文唐博恒张培洪文艳于德宪
Owner 中国人民解放军广州军区疾病预防控制中心
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