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Recombinant antigen protein for detecting dengue 2 type virus antibody, kit and application of recombinant antigen protein

A technology of recombinant antigen protein and type 2 virus, applied in the field of genetic engineering, can solve the problems of global national threat, lack of dengue virus, increase of cases of secondary heterotypic infection, etc., to achieve high sensitivity, high detection range, and high-efficiency expression effect Effect

Active Publication Date: 2019-11-08
中国人民解放军南部战区疾病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] According to the data of dengue virus serodiagnosis disclosed in the prior art, there are mainly problems in the following aspects: (1) secondary heterotype of dengue virus Infection can lead to severe Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). As the infected population base increases, the probability of secondary heterogeneous infection cases is increasing, which has posed a serious threat to countries around the world
However, there is still a lack of diagnostic products for dengue virus immunology typing. Therefore, such typing kits are developed for differential diagnosis of dengue virus infection, assessment of the immune status of the population and patients, early warning of severe dengue patients, formulation of clinical treatment plans and epidemic prevention. Pathological investigations are of great significance
(2) There are serious serological cross-reactions between various types of dengue viruses. In those areas where multiple dengue virus serotypes are prevalent, due to mixed infection, there are cross-antibodies in patients, which makes immunological diagnosis and differential diagnosis very difficult. Difficult, put forward higher quality requirements for diagnostic antigens

Method used

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  • Recombinant antigen protein for detecting dengue 2 type virus antibody, kit and application of recombinant antigen protein
  • Recombinant antigen protein for detecting dengue 2 type virus antibody, kit and application of recombinant antigen protein
  • Recombinant antigen protein for detecting dengue 2 type virus antibody, kit and application of recombinant antigen protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the preparation of detection dengue type 2 virus antibody recombinant antigenic protein (DENV-Ag2)

[0046] Prepare the recombinant antigen protein for detection of dengue type 2 virus antibody according to the following steps:

[0047] Step 1, select the coding gene presenting the highly specific peptide of the virus in the dengue type 2 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 The masking of the polypeptide epitope by the fusion protein of the carrier enables the correct folding of the expressed recombinant antigen protein. Wherein, the nucleotide sequence of the coding gene sequence of the target gene fragment DENV-Ag2 is shown in SEQ ID NO.1.

[0048] Step 2, designing PCR amplification primers, and amplifying the target gene fragment by PCR. Wherein, the PCR amplification primer sequence that adopts in descr...

Embodiment 2

[0068] Embodiment 2, the coating experiment of recombinant antigenic protein

[0069] In this example, it is necessary to optimize the coating conditions and concentration of the recombinant antigen protein DENV-Ag2 provided by 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:

[0070] Enzyme conjugate working solution: horseradish peroxidase-labeled anti-human IgG polyclonal antibody solution (200ng / mL);

[0071] Positive control: serum from patients with dengue type 2 virus;

[0072] Negative control: dengue virus antibody negative human serum;

[0073] Sample diluent: phosphate buffer (pH 7.4) containing 10mmol / L, 1% BSA by mass percentage concentration, 0.05% Tween-20 by volume percentage concentration and 1 mg / L gentamicin;

[0074] Concentrated washing solution: containing 0.2mol / L phosphate buffer (pH is 7.4), volume percent concentration is 1% Tween-20 and concentra...

Embodiment 3

[0079] Embodiment 3, ELISA reaction condition optimization

[0080] The antigen coating conditions and ELISA reaction conditions were systematically optimized, and finally the best ELISA reaction conditions were determined as follows: recombinant antigen 100 μl (concentration: 10 μg / ml) was coated at 4°C for 16 hours, PBST buffer (its pH value was 7.4, containing Concentration is 10mmol / L phosphate buffer solution and volume percentage concentration is 0.05% Tween-20) Wash for 3 minutes, pat dry, block in 37 ℃ 2 hours in 5% BSA solution with mass percentage concentration, the serum to be tested is 1:100 After doubling dilution, 100 μl was added to the reaction well, reacted at 37°C for 1 hour, and washed 4 times with PBST buffer (its pH value was 7.4, containing phosphate buffer solution with a concentration of 10 mmol / L and a concentration of 0.05% Tween-20 by volume). , 1 minute each time, pat dry, after the HRP-labeled secondary antibody was diluted 1:10000 times, add 100 μ...

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Abstract

The invention discloses a recombinant antigen protein for detecting a dengue 2 type virus antibody, a kit and an application of the recombinant antigen protein. The preparation method of the recombinant antigen protein comprises the following steps of screening and detecting a target coding gene, then connecting the coding gene into an prokaryotic expression vector to construct a recombinant expression vector; and converting the recombinant expression vector into a host cell, and carrying out inducing expression to obtain the recombinant antigen protein DENV-Ag2, wherein the recombinant antigen protein is a dengue 2 type virus specific antigen, and is obtained by prokaryotic expression and purification. The recombinant antigen protein disclosed by the invention has the advantages of beingstrong in specificity and high in affinity, has very high affinity with dengue 2 type virus antibody, and does not have a serum cross reaction with other dengue viruses and similar arboviruses. The kit provided by the invention has the advantages of simplicity and convenience in operation, high sensitivity, strong specificity and the like, can rapidly and accurately identify the dengue virus 2 type virus antibody, so as to diagnose the infection condition of dengue virus, thereby providing favorable support for subsequent treatment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant antigen protein for detecting dengue type 2 virus antibody, a kit and an application thereof. Background technique [0002] Dengue virus (Dengμe virμs, DENV) belongs to the Flaviviridae family, including 1-4 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. Reaching 50-100 million, of which 250,000-500,000 people belong to severe dengue hemorrhagic fever, with an average mortality rate of 5%, which 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 year from 1990 to 2010. There are various signs that my country has become the source...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983Y02A50/30
Inventor 任瑞文李硕李春缘陈月李以圣陈迪佳刘军
Owner 中国人民解放军南部战区疾病预防控制中心
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