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Polypeptide combined with Japanese encephalitis virus envelope protein and application of polypeptide

A Japanese encephalitis virus and protein technology, which is applied in the field of polypeptides combined with Japanese encephalitis virus E protein, can solve the problem of ineffective pathogenic mechanism, and achieve the effects of low detection cost, rapid acquisition and high specificity.

Inactive Publication Date: 2015-11-25
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

E protein is the main antigenic component of the virus, and it has specific neutralization and hemagglutination functional antigenic determinants. Although M and C proteins also have antigenicity, they play little role in the pathogenic mechanism

Method used

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  • Polypeptide combined with Japanese encephalitis virus envelope protein and application of polypeptide
  • Polypeptide combined with Japanese encephalitis virus envelope protein and application of polypeptide
  • Polypeptide combined with Japanese encephalitis virus envelope protein and application of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0022] The screening of example 1 phage random peptide library

[0023] Coat the polyvinyl chloride plate with purified and expressed Japanese encephalitis virus E protein (JEVE protein), coat each well with 100 μl (100 μl / ml), place it at 4°C overnight, wash with PBST, and then use 5% BSA solution closed;

[0024] 100 μl of the diluted phage peptide library (containing about phage 2×10 11 pfu)) into the corresponding wells, incubated at room temperature for 2 hours, washed 8-10 times with TBST solution, and added 200 μl of eluent (TBST buffer, containing 50mM Tris-HCl pH7.5, 150mM NaCl, 0.2% Tween-20), shake at room temperature for 5-8min, suck out the eluate and neutralize to neutral;

[0025] The eluate after neutralization can be used for titer determination and the next round of culture (pick a single colony on LB-Tet medium and put it in 5-10mL LB culture medium, culture it with shaking at 37°C until logarithmic phase (OD 600 =0.5)); the phage cultured in LB were co...

example 2

[0031] Example 2 Identification of Screening Polypeptides

[0032] (1) Sonicate the inoculated PK15 cell culture solution of JE virus, and then coat the ELISA plate at 2 μg / ml (protein amount); The PK15 was used as a control for coating; in the specificity test, different viral proteins were coated;

[0033] (2) Add the HRHHV-coupled horseradish peroxidase (commercialized coupling kit) of the artificially synthesized polypeptide to the above-mentioned ELISA plate, 50 μl per well, the concentration is 100 ng / ml, add Put it into the above ELISA plate, mix it on a shaker, and incubate at 37°C in the dark for 30min;

[0034] (3) Wash 6 times with a microplate washer, or manually shake off the liquid in the wells of the microplate, fill each well with diluted buffer solution, and then spin dry, repeat the washing process 6 times;

[0035] (4) Add TMB solution to the corresponding microwell according to the required amount, add 100 μl to each well, shake on the microplate shaker...

example 3

[0038] Example 3 Affinity Identification of Screening Polypeptide

[0039] (1) Sonicate the inoculated PK15 cell culture solution of JE virus, and then coat the ELISA plate at 2 μg / ml (protein amount); The PK15 was used as a control for coating. When performing specificity tests, different viral proteins are coated;

[0040] (2) Add the synthetic peptide HRHHV coupled with horseradish peroxidase and enzyme-labeled peptides of different concentrations to 50 μl of the above-mentioned ELISA plate, mix well on a shaker, and incubate at 37°C in the dark for 30 minutes;

[0041] (3) Wash 6 times with a microplate washer, or manually shake off the liquid in the wells of the microplate, fill each well with diluted buffer solution, and then spin dry, repeat the washing process 6 times;

[0042] (4) Add TMB solution to the corresponding microwell according to the required amount, add 100 μl to each well, shake on the microplate shaker for 30 seconds, and fully develop the color at r...

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Abstract

The invention mainly relates to polypeptide combined with Japanese encephalitis virus envelope protein and an application of the polypeptide. The sequence of the polypeptide is HRHHV, and is linear binding polypeptide, a polypeptide sequence serves as a core, the polypeptide sequence can be prolonged and modified, and modification materials comprise but are not limited to nanometer materials, fluorescent materials, enzymes, biotin and specific protein. Based on the phage peptide library screening technology, several rounds of screening are carried out through expressed and purified Japanese encephalitis virus envelope protein, and finally a polypeptide clone strain capable of being specifically combined with Japanese encephalitis virus envelope protein can be obtained. The clone strain is selected for sequence determination, and the core sequence of the polypeptide is HRHHV. The polypeptide is artificially synthesized for an ELISA binding experiment, and a result proves that the synthesized polypeptide can be combined with Japanese encephalitis virus envelope protein well. Compared with the process of artificially expressing protein and then carrying out immunization to obtain a protein antibody, the polypeptide is simple and convenient, and operation is easier. As the polypeptide is marked, and qualitative and quantitative detection can be fast carried out on Japanese encephalitis virus envelope protein.

Description

technical field [0001] The invention belongs to the field of virus detection, and mainly relates to a polypeptide combined with E protein of Japanese encephalitis virus and its application. Background technique [0002] Japanese encephalitis virus is called Japanese encephalitis virus for short, and it is the pathogen of Japanese encephalitis. The virus is single-stranded RNA with an outer envelope and hemagglutinin on the surface of the envelope. Young pigs are the main source of infection and intermediate host of JE virus, and mosquitoes are the medium of JE virus transmission. When a person is bitten by a mosquito carrying the virus, the JE virus enters the human body, proliferates in phagocytic cells such as vascular endothelial cells, lymph nodes, liver, and spleen, and reaches the brain through blood circulation to cause inflammation. The core part of the virus is composed of core protein C and RNA, and the outer membrane part has glycosylated protein E and non-glyco...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/68
Inventor 樊剑鸣张晓峰冯斐斐胡梦华张巧
Owner ZHENGZHOU UNIV
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