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Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody

A technology of recombinant antigen protein and Japanese encephalitis virus, applied in the field of genetic engineering, can solve the problems of difficulty in immunological diagnosis and differential diagnosis, and achieve the effects of high specificity, high sensitivity and high-efficiency expression effect.

Active Publication Date: 2015-04-08
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] From the data of Japanese encephalitis 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. In areas where flaviviruses are prevalent, due to mixed infection, cross-antibodies exist in patients, making immunological diagnosis and differential diagnosis very difficult

Method used

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  • Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody
  • Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody
  • Recombinant antigen protein and kit for detecting epidemic encephalitis B virus antibody

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Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of the recombinant antigenic protein that detects Japanese encephalitis virus

[0036] Prepare the recombinant antigen protein for detecting Japanese encephalitis virus according to the following steps:

[0037] Step 1, select the coding gene presenting the highly specific peptide segment of the virus in the Japanese encephalitis 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 It includes an extended segment encoding a flexible arm of the recombinant antigen protein, and the flexible arm can eliminate the masking of the polypeptide epitope by the fusion protein of the expression vector, so that the expressed recombinant antigen protein can be folded correctly;

[0038] Step 2, designing PCR amplification primers, and amplifying the target gene fragment by PCR;

[0039] Step 3. Ligate the target gene fragment into the prokaryotic expression vect...

Embodiment 2

[0056] Embodiment 2, the optimization of detection test strip

[0057] In this embodiment, it is necessary to optimize the coating conditions and concentration of the recombinant antigen protein of the present invention; in order to achieve this purpose, it is also necessary to explain the composition of the kit first: including antibody detection test strips and sample diluent, the anti The body detection test strip is coated with a recombinant antigen protein for detecting Japanese encephalitis virus antibody, and the recombinant antigen protein is the recombinant antigen protein disclosed in the above scheme of the present invention.

[0058] Among them, the kit includes: detection test strips containing recombinant Japanese encephalitis virus antigen protein, sample diluent, and dry pack.

[0059] Test strips: the T line is coated with Japanese encephalitis virus recombinant antigen protein, and the C line is coated with specific antibodies;

[0060] Sample diluent: physi...

Embodiment 3

[0063] Embodiment 3, preparation of gold-labeled SPA probe

[0064] Add 30ml of colloidal gold solution into a 50ml beaker, adjust the pH of the colloidal gold to the optimum pH value, add 1ml of the optimal concentration of SPA protein solution dropwise under stirring at a speed of 250r / min, continue stirring for 30min, and finally add dropwise 10% PEG20000 solution, so that the concentration of PEG20000 in the system is 0.25%, centrifuge at 3000rpm at room temperature for 15min, discard the precipitate, take the red supernatant and centrifuge at 11000rpm at 4°C for 35min, gently suck and discard the supernatant, and use 1% Suspend the pellet to the original volume with BSA, and repeat the centrifugation twice to completely remove unbound protein. The last washed and centrifuged precipitate was suspended in PB containing 1% BSA and 0.02mol / L NaN3 to 1 / 10 of the original volume, and stored at 4°C for future use.

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Abstract

The invention discloses a recombinant antigen protein and a kit for detecting an epidemic encephalitis B virus antibody. The recombinant antigen protein has an amino acid sequence as shown in SEQ ID NO. 1. The kit comprises a colloidal gold test strip for rapid detection, wherein the test strip is prepared from a prokaryotic expression antigen JEV-Ag45 of the epidemic encephalitis B virus. The invention further discloses a preparation method of the recombinant antigen protein. The recombinant antigen protein disclosed by the invention has the advantages of high specificity and high affinity, can not be subjected to serological cross reaction with other similar insect-borne viruses, is highly affiliative to the epidemic encephalitis B virus antibody and can be used for detecting the epidemic encephalitis B virus antibody rapidly and accurately and further diagnosing epidemic encephalitis B virus infection.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant antigenic protein for diagnosing Japanese encephalitis virus antibodies and a preparation method thereof, and a Japanese encephalitis virus detection kit using the antigenic protein as a coated antigen . Background technique [0002] Japanese encephalitis, referred to as Japanese encephalitis, is the most common viral encephalitis caused by Japanese encephalitis virus (JEV). In order to distinguish it from the lethargic encephalitis (also known as Japanese encephalitis) that was prevalent in Europe at the beginning of this century, the Ministry of Health of my country named it Japanese encephalitis after liberation. It was once called the "Plague of the East". Japanese encephalitis virus belongs to the flavivirus genus of the flaviviridae family of arboviruses. The genome is a single-stranded positive-strand RNA with non-coding regions at both ends. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/40C12N15/70G01N33/68G01N33/569G01N33/543
CPCC07K14/005C12N2770/24122G01N33/558G01N33/56983G01N33/68G01N2333/185G01N2469/20
Inventor 任瑞文梁克峰沓世远杨柳张培胡文龙唐博恒
Owner 中国人民解放军南部战区疾病预防控制中心
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