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RV (rabies virus) dominant-epitope peptide antigen and application thereof

A rabies virus and dominant epitope technology, applied in the field of rabies vaccine, can solve the problems of low sensitivity, high false negative rate, poor specificity, etc., and achieve the effect of improving sensitivity

Active Publication Date: 2012-01-25
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

The clinically used ELISA antibody detection methods are all coated with whole virus lysed antigen or full-length G protein antigen, which has problems such as high false negative rate, low sensitivity, and poor specificity.

Method used

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  • RV (rabies virus) dominant-epitope peptide antigen and application thereof
  • RV (rabies virus) dominant-epitope peptide antigen and application thereof
  • RV (rabies virus) dominant-epitope peptide antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Determination of the dominant epitope antigen segment of rabies virus

[0032] Using BIOSUN biological analysis software to analyze the epitope distribution of rabies virus G protein and N protein, first input their amino acid sequence, and then search for B cell epitope, the obtained epitope distribution map is as follows figure 1 with figure 2 shown. Determine the specific dominant epitope antigen segment according to the epitope distribution, which are: the dominant epitope segment GP-1 (1-200aa), the amino acid sequence is SEQ ID NO: 1; the dominant epitope segment GP-2 ( 301-400aa), the amino acid sequence is SEQ ID NO: 2; the dominant epitope segment GP-3 (60-230aa), the amino acid sequence is SEQ ID NO: 3; the dominant epitope segment GP-4 (231-345aa) , the amino acid sequence is SEQ ID NO: 4; the dominant epitope segment NP-1 (301-420aa), the amino acid sequence is SEQ ID NO: 5; the dominant epitope segment NP-2 (20-180aa), the amino acid sequence ...

Embodiment 2

[0033] Example 2: Cloning of rabies virus dominant epitope peptide antigen coding gene

[0034] 1. Primer design

[0035] According to the full-length nucleotide sequences of G protein and N protein and the nucleotide sequences of 8 dominant epitope peptide antigens, the upstream and downstream primers were designed and synthesized respectively, and the restriction enzymes used were Xho I and Xba respectively. I, BamH I or EcoR I.

[0036] The primers used to amplify the full-length G protein gene are GF and GR, and the nucleotide sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively;

[0037] The primers used to amplify the full-length gene of N protein are NF and NR, and the nucleotide sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0038] The primers used to amplify the gene encoding the dominant epitope segment GP-1 are GF-1 and GR-200, and the nucleotide sequences are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively;

[0039]...

Embodiment 3

[0052] Example 3: Expression and purification of rabies virus dominant epitope peptide antigen

[0053] Transform the Escherichia coli strain with the obtained recombinant plasmid, heat-induced expression, collect the thalline, weigh the wet weight of the precipitate, suspend the precipitate with 10 times the volume of 20mmol / L pH8.0TE buffer, add lysozyme (1mg / ml suspension), stirred magnetically for 10 minutes at room temperature. Sonicate the bacteria in an ice bath for 30 seconds each time, with an interval of 30 seconds, a total of 10 times. Centrifuge at 1,2000 rpm at 8°C for 20 minutes, discard the supernatant, wash the precipitate once with 1 mol / L NaCl (prepared with TE), wash twice with TE, and collect the precipitate. Dissolve the precipitate with a dissolving solution (25mM Tirs-HCl, 1% β-mercaptoethanol, 8M urea, pH 7.8), put it on a Ni-Sepharose Fast Flow column, and use 3 column volumes of binding buffer (20mM Tirs-HCl , 0.1% β-mercaptoethanol, 6M urea, pH 7.8...

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Abstract

The invention discloses an RV (rabies virus) dominant-epitope peptide antigen and an application thereof, and belongs to the technical field of rabies vaccines. In the invention, dominant-epitope antigen segments are screened by using bioinformatics software, coding genes of the dominant-epitope antigen segments are amplified through PCR (polymerase chain reaction), and the coding genes are efficiently expressed in escherichia coli, thereby obtaining a high-purity antigen; and an indirect ELISA (enzyme-linked immuno sorbent assay) test shows that the obtained antigen can detect protective antibodies in immune human or dog serums. A dominant-epitope peptide segment antigen expressed by genetic engineering is adopted to replace a whole-virus antigen so as to improve the detection specificity, and through carrying out detection by compositely using various specific antigens, the detection sensitivity is significantly improved.

Description

technical field [0001] The invention belongs to the technical field of rabies vaccines, and in particular relates to a rabies virus dominant epitope peptide antigen and its application. Background technique [0002] Rabies is a natural foci, highly lethal zoonotic disease caused by rabies virus (RV), which is distributed globally. Rabies virus can cause fatal central nervous system infection in humans and various animals. The clinical manifestations of rabies are mainly hydrophobia, photophobia, dysphagia, mania, etc., and the case fatality rate is almost 100%. About 55,000 people die from rabies every year in the world, and it is by far the acute infectious disease with the highest fatality rate. India is the country with the most severe rabies epidemic, and the number of rabies cases in China is second only to India, ranking second in the world. [0003] The rabies virus belongs to the rhabdoviridae family (rhabdoviridae), lyssa virus (lyssa virus), and is bullet-shaped....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/145C12N15/47C12N15/70C12N1/21G01N33/68G01N33/543C12R1/19
Inventor 冯晓燕张贺秋宋晓国王国华陈堃修冰水何竞朱翠侠杨锡琴
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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