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Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein

A technology of recombinant antigen protein and detection kit, which is applied in the field of genetic engineering, can solve the problems of immunological diagnosis and differential diagnosis, and infection identification, and achieve high specificity, high expression effect and high sensitivity effect

Active Publication Date: 2012-12-19
中国人民解放军南部战区疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, there have been many reports in China on the research on the immunological diagnosis method of yellow fever virus. In endemic areas, due to mixed infections, cross-antibodies exist in patients, making immunological diagnosis and differential diagnosis very difficult
At the same time, since IgG antibodies can exist in the host for a long time, some for more than 10 months, or even carry them for life, it is very difficult to distinguish past, recent or present infections, and higher requirements are placed on the quality of antigens

Method used

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  • Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein
  • Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein
  • Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, detect the preparation of the recombinant antigenic protein of yellow fever virus

[0039] Prepare the recombinant antigen protein for detecting yellow fever virus according to the following steps:

[0040] Step 1, select the coding gene presenting the highly specific peptide segment of the virus in the yellow fever virus protein as the target gene segment, the target gene segment has a nucleotide sequence as shown in SEQ ID NO.2, and the target gene segment includes the encoding The extended segment of the flexible arm of the recombinant antigenic protein, the flexible arm can eliminate the masking of the polypeptide epitope by the fusion protein of the expression vector, so that the expressed recombinant antigenic protein can be folded correctly;

[0041] Step 2, designing PCR amplification primers, and amplifying the target gene fragment by PCR;

[0042] Step 3. Ligate the target gene fragment into the prokaryotic expression vector by restriction enzym...

Embodiment 2

[0065] Embodiment 2, the coating experiment of recombinant antigenic protein

[0066] 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:

[0067] Enzyme conjugate working solution: horseradish peroxidase-labeled goat anti-human IgG;

[0068] Positive control: yellow fever virus antibody positive mouse serum.

[0069] Negative control: Yellow fever virus antibody negative human serum.

[0070] Concentrated washing solution: 0.1mol / L pH7.4 phosphate buffer containing 1% fetal bovine serum, 0.5% Tween-20 and 20mg / L gentamicin;

[0071] Chromogenic solution A: 0.02% H2O2; dilute with 0.1M citric acid-0.2M disodium hydrogen phosphate, pH4.5~5.0.

[0072] Chromogenic solution B: 0.4‰TMB-HCl: dissolve with 50mM sodium citrate and concentrated HCl to adjust ...

Embodiment 3

[0075] Embodiment 3, ELISA reaction condition optimization

[0076] 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℃ for 1 hour, wash with PBST 4 times, 1 minute each time, pat dry, add 100ul of HRP-labeled IgG to each well, at 37℃ React for 40 minutes, wash with PBST 4 times, each time for 1 minute, pat dry, add 100ul of TMB substrate solution to each well, react at 37°C for 15 minutes, add 50ul of 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 discloses a recombinant antigen protein for detecting a yellow fever virus antibody. The sequence of the recombinant protein is shown as SEQ ID NO.1; the sequence of an encoding gene of the recombinant antigen protein is shown as SEQ ID NO.2, and comprises an extension segment for encoding a flexible arm of the recombinant antigen protein; and the flexible arm can be used for eliminating masking of a peptide epitope by a fusion protein of an expression carrier, so that the expressed recombinant antigen protein is folded correctly. The invention further discloses a preparation method of the recombinant antigen protein. Moreover, a yellow fever virus kit disclosed by the invention comprises an antigen detection plate coated with the recombinant antigen protein and an ELISA (Enzyme-Linked Immunosorbent Assay) reaction liquid. The recombinant antigen protein provided by the invention has the advantages of high specificity and high affinity, does not undergo any serological cross reaction with other congenial arthropod-borne viruses, has ultrahigh affinity with the yellow fever virus antibody, and can be used for detecting the yellow fever virus antibody rapidly and accurately for diagnosing the infection condition of a yellow fever virus.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant antigen protein for diagnosing yellow fever virus antibody and a preparation method thereof, and a yellow fever virus detection kit using the antigen protein as a coated antigen. Background technique [0002] Yellow fever virus (Yellow Fever Virus, YFV) belongs to the Flavivirus genus of the Flaviviridae family. The viral genome is a single-stranded positive-sense RNA without segments. There are several different genotypes, but only one serotype, and the antigenicity is conserved. . Yellow fever is an acute infectious disease caused by the yellow fever virus, which is transmitted by the Aedes aegypti mosquito. Clinical features include fever, severe headache, jaundice, hemorrhage, and proteinuria. The disease is mainly prevalent in tropical regions such as South America, Central America, and Africa. According to WHO estimates, there are 20 cases of mor...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/40C12N15/70G01N33/68G01N33/569G01N33/543
Inventor 唐博恒任瑞文胡文龙沓世远梁克峰
Owner 中国人民解放军南部战区疾病预防控制中心
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