H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof

A technology for antigenic epitopes and influenza virus, applied in the field of biomedicine, can solve the problems of inability to obtain spatial conformational epitopes, time-consuming and labor-intensive, etc.

Inactive Publication Date: 2008-05-28
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Conventional epitope mapping methods are time-consuming

Method used

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  • H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof
  • H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof
  • H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Prokaryotic expression of influenza virus hemagglutinin fragment HA1

[0046] 1) Cloning of influenza virus hemagglutinin fragment HA1 gene ha1: On MDCK cells growing into a single layer, inoculate H3 influenza virus A / Swine / Guangdong / 6 / 2004 (H3N2) strain, 37 ° C 5% CO 2 Culture in an incubator for 48 hours. When obvious cell lesions appear, the cells are collected, the cells are lysed by repeated freezing and thawing, centrifuged to remove the precipitate, and the supernatant is taken. Total RNA was extracted with Trizol reagent (product of Invitrogen Company), and a pair of primers were designed according to the hemagglutinin gene sequence of influenza virus A / Swine / Guangdong / 6 / 2004 (H3N2) strain (Gene ID: AY852277), followed by reverse transcription RT-PCR was carried out under the action of enzyme (product of Invitrogen Company) and Taq enzyme (product of Takara Company) to obtain the ha1 gene, which was identified by DNA sequencing. The two primer seque...

Embodiment 2

[0051] Example 2, Purification and Identification of Anti-recombinant HA1 Rabbit Serum IgG

[0052] 1) Purified recombinant HA1 was used to immunize New Zealand rabbits: the purified recombinant HA1 was adjusted to 200 μg / mL with 0.01 M, pH 7.4 PBS, mixed with an equal volume of Freund's adjuvant, emulsified, and subcutaneously multi-pointed on the back to immunize New Zealand rabbits For rabbits, the second immunization is performed two weeks after the first immunization, and the third immunization is performed two weeks after the second immunization. The second and third doses were emulsified with an equal volume of Freund's incomplete adjuvant, and were injected subcutaneously at multiple points on the back. After three immunizations, the titer of the antibody was tested. When the titer reached 1:32, blood was collected from the heart, and the serum was separated.

[0053] 2) Purification of anti-recombinant HA1 rabbit serum IgG: use saturated ammonium sulfate fractional p...

Embodiment 3

[0054] Embodiment 3, phage random peptide library screening

[0055] 1) The phage random peptide library is the M13 phage twelve peptide library (Ph.D.-12TMPhage Display Peptide Library Kit) commercialized by BioLabs

[0056] 2) Screening method: Coat the ELISA plate with anti-recombinant HA1 rabbit serum IgG (100 μg / mL) diluted in the coating solution (carbonate buffer at pH 9.6), and add blocking solution (3% BSA) at 4°C for 2-3 hours, wash with TBST, and spin dry the washing liquid. Add diluted phage random peptide library to each well, and the number of phage added in each round should be 2×10 10-11 CFU, incubate at room temperature for 1 hour, shake off the liquid, rinse with TBST buffer containing 0.1% tween-20 (v / v), and finally use 100 μl pH 2.2 glycine buffer to elute the IgG that specifically binds to anti-recombinant HA1 rabbit serum phage, and immediately neutralized with 10 μl pH 9.8 Tris buffer, 10 μl was taken for phage titer determination, and the remaining p...

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Abstract

The invention relates to polypeptide biological technical field, which discloses a spatial conformation mimic antigen epitope of H3-type influenza virus hemagglutinin, and relative application. The sequence of the spatial conformation mimic antigen epitope is SEQ ID No. 1. The invention utilizes anti-recombination HA1 rabbit serum IgG selection phage 12 peptide library, uses phage ELISA, DNA sequencing, western bolt, and software analysis to analyze selected colony, and selects the small peptide from random phage 12 peptide library, which can be specially combined with anti-recombination HA1 rabbit serum IgG. The inventive spatial conformation mimic antigen epitope can prepare diagnostic reagent box of H3-type influenza virus.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an H3 type influenza virus hemagglutinin space conformation mimic antigen epitope and application thereof. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family, which is a single-strand negative-sense, segmented RNA virus. The genome consists of 8 segments and encodes 10 viral proteins. According to the different antigenic types of the internal proteins of influenza virus, it is divided into A (A), There are three types of B (B) and C (C), and the type A influenza virus is divided into different serotypes according to the antigenicity of the influenza virus surface protein hemagglutinin (HA) and neuraminidase. Now 15 different HA subtypes (H1-H15). H3 influenza virus can infect humans, poultry, pigs, horses and other mammals. In 1968, H3N2 influenza virus caused a sudden influenza pandemic and seriously endangered public safety. [0003] Hemagg...

Claims

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Application Information

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IPC IPC(8): C07K14/705G01N33/569
Inventor 钟辉邓小昭姚文娟董莉莉于娟储春丽刁振宇张云
Owner CHINA PHARM UNIV
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