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Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence

A bovine viral diarrhea, polypeptide sequence technology, applied in the fields of peptides, material testing products, instruments, etc., can solve the problems of cattle industry loss, persistent infection, etc., and achieve the effect of low detection cost and good affinity

Active Publication Date: 2015-05-06
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It mainly causes symptoms such as cattle diarrhea, persistent infection, and abortion. The virus is widespread worldwide and has caused huge losses to the cattle industry.

Method used

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  • Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence
  • Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence
  • Polypeptide sequence combined with bovine viral diarrhea E2 protein and application of polypeptide sequence

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0020] Example 1 Screening of phage random peptide library

[0021] Coat the polyvinyl chloride plate with the purified expressed BVD E2 protein, 100 μl per well (100 μl / ml), place it at 4°C overnight, wash with PBST and then block with 5% BSA solution.

[0022] Add 100 μl of the diluted phage peptide library to the corresponding wells, incubate at room temperature for 2 hours, wash with TBST solution 8-10 times, add 200 μl of eluent to each well, shake for 5-8 minutes at room temperature, aspirate and elute liquid and neutralized to neutral.

[0023] The eluate after neutralization can be used for titer determination and the next round of culture; the phage after LB culture are collected by centrifugation for the next round of screening.

[0024] The enrichment and identification of phages in different screening rounds are shown in Table 1, and the titer determination results of the eluate are shown in Table 2.

example 2

[0025] Example 2 Identification of Screening Peptides

[0026] (1) Sonicate the inoculated MDBK cell culture solution of bovine viral diarrhea virus, and then coat the ELISA plate at 2 μg / ml (protein amount); in the same way, artificially express the bovine viral diarrhea virus E2 protein Coated with MDBK not inoculated with virus as a control. When performing specificity tests, different viral proteins were coated.

[0027] (2) Add the artificially synthesized polypeptide KRLREL coupled with horseradish peroxidase at an amount of 100 ng / ml to 50 μl per well of the above ELISA plate, mix well on a shaker, and incubate at 37°C in the dark for 30 minutes;

[0028] (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;

[0029] (4) Add TMB solution to the corresponding microwell according to the required amount, add 100 μl to...

example 3

[0036] Example 3 Affinity Identification of Screening Peptides

[0037] (1) Sonicate the inoculated MDBK cell culture solution of bovine viral diarrhea virus, and then coat the ELISA plate at 2 μg / ml (protein amount); in the same way, artificially express the bovine viral diarrhea virus E2 protein Coated with MDBK not inoculated with virus as a control. When performing specificity tests, different viral proteins were coated.

[0038] (2) Add the artificially synthesized polypeptide KRLREL coupled with horseradish peroxidase, and add 50 μl of the enzyme-labeled polypeptide at different concentrations to the above ELISA plate, mix well on a shaker, and incubate at 37°C in the dark for 30 minutes;

[0039] (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;

[0040] (4) Add TMB solution to the corresponding microwell accor...

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Abstract

The invention mainly relates to a polypeptide sequence combined with bovine viral diarrhea E2 protein and application of the polypeptide sequence. The polypeptide sequence is KRLREL and is a linear combined polypeptide, the polypeptide sequence can be prolonged and modified by taking the polypeptide sequence as a core, and modification materials can be but not limited to nano materials, fluorescent materials, enzymes, biotin and specific protein. By adopting a phage peptide library screening technique, the expressed and purified bovine viral diarrhea E2 protein can be screened for multiple rounds, and polypeptide clone strains which can be specifically combined with the bovine viral diarrhea E2 protein can be ultimately obtained; the polypeptide clone strains can be selected for sequencing, the core sequence of the polypeptide is KRLREL, the manually synthesized polypeptide used for ELISA combination experiment shows that the synthesized polypeptide can be well combined with the bovine viral diarrhea E2 protein; the process is simple, and the operation is relatively convenient compared with the operation that expression protein is manually expressed and is further immunized to obtain a protein antibody; by marking the polypeptide, a kit or a test paper bar (card) for quantitative and qualitative detection on the bovine viral diarrhea E2 protein can be rapidly produced.

Description

technical field [0001] The invention belongs to the field of virus detection, and mainly relates to bovine viral diarrhea virus E2 binding polypeptide and application thereof. Background technique [0002] Bovine viral diarrhea virus (Bovine viral diarrhea, BVD) is the main pathogen that causes bovine viral diarrhea. It mainly causes symptoms such as cattle diarrhea, persistent infection, and abortion. The virus is widespread worldwide and has caused huge losses to the cattle industry. BVD belongs to the Pestivirus genus of the Flaviviridae family and is a single-stranded positive-sense RNA virus. E2 is located at positions 2077-3198 of BVDV ORF; the protein consists of 374 amino acids and has 5 very conserved glycosylation sites. E2 is a structural protein of the virus that forms surface protrusions. Among the structural proteins of BVDV, the E2 protein has the strongest immunogenicity and is the most important antigenic site of BVDV. It is also the binding region for an...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCC07K7/06G01N33/56983G01N33/68G01N2333/183
Inventor 王方雨邓瑞广邢广旭罗俊胡骁飞赵东余秋颖
Owner HENAN ACAD OF AGRI SCI
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