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Recombined human A3 type influenza viruse stromatin and its preparation process and application thereof

An influenza virus and antigen protein technology, which is applied in the field of recombinant human influenza A3 virus matrix protein, can solve the problems of inefficient and rapid diagnosis of reagents

Inactive Publication Date: 2006-06-28
首都儿科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the shortcomings of the existing reagents on the market that cannot be diagnosed efficiently and quickly, the inventors analyzed the nucleotide sequence of the influenza virus M1 protein, and invented an antibody and its corresponding antigen for the efficient and rapid diagnosis of influenza disease

Method used

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  • Recombined human A3 type influenza viruse stromatin and its preparation process and application thereof
  • Recombined human A3 type influenza viruse stromatin and its preparation process and application thereof
  • Recombined human A3 type influenza viruse stromatin and its preparation process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Acquisition of M1 antigenic fragment gene

[0062] 1. Design and synthesis of primers

[0063] According to the analysis of the antigenicity of M1 protein by Antheprot software, the results are as follows figure 1 As shown, the M1P2 (439-756 base pair) region contains high antigenicity. Select M1P2 as the research object, use Primer Premier software to design and synthesize the following primers (including BamH I, Xho I restriction endonuclease sites) :

[0064] Upstream primer, 5’cgg gat cct agt atg cgc aac ctg tg 3’

[0065] Downstream primer, 5’tcc gct cga gct tga atc ​​dtt gca tc 3’

[0066] 2. Obtain the target gene fragment by RT-PCR reaction

[0067] Use Trizol to extract viral RNA from MDCK cells infected with the human influenza A 3 standard strain. Add 1 μL of Oligo dT at a concentration of 40 μg / μL to 9 μL RNA, react at 70°C for 5 minutes, then cool to room temperature, and add 5 μL 5× MLV buffer, 2.5μL 10mmol / L dNTP, 3.75μL 25mmol / LMgCl 2 , 0.5μL Rnas...

Embodiment 2

[0070] Example 2 Construction of recombinant pET-28c(+) / IFV.m1 prokaryotic expression plasmid

Embodiment 2-1

[0071] Example 2-1 Construction of recombinant pET-28c(+) / IFV.m1 prokaryotic expression plasmid

[0072] 1. Put 15μL of SEQ ID NO.1 fragment into a 2μL Xho I, 2μL 10×buffer, 37°C reaction system and digest for 12 hours, then add 2μL BamH I and continue digestion at 37°C for 2 hours;

[0073] 2. Put 6μL pET-28c(+) into 2μL BamH I, 2μL Xho I, 4μL 10×buffer, 26μL dH 2 O, 37°C reaction system for enzyme digestion, let stand for 2 hours;

[0074] 3. Take 5μL of digested SEQ ID NO.1 fragment and 3μL of digested pET-28c(+) in 1μL T4DNA ligase, 1μL 10×buffer, 16℃ reaction system for ligation reaction for 12 hours to obtain Recombinant pET-28c(+) / IFV.m1 prokaryotic expression plasmid; The recombinant plasmid was extracted from the kanamycin-resistant transformed strain, and the recombinant plasmid was cut by BamH I and Xho I restriction enzymes, and 1.5% Agarose gel electrophoresis identification. The result is Figure 4 As shown, Figure 4 Lane5 is pBR322 / HinfImarker, Lane1-4 is the targe...

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Abstract

The invention relates to bioengineering product manufacturing and application technique field, concretely is recombination human 3 influenza a virus stromatin and its manufacturing method and application field. It includes the following steps: analyzing the human 3 influenza a virus M1 protein antigenicity; selecting high antigenicity area; using molecular biology method to do antigen gene segment in vitro recombination and manufacture its corresponding antigen protein and the antibody. It solves the defect that the influenza a virus infection can not be quickly diagnosed at early stage in current clinic.

Description

Technical field [0001] The present invention relates to the technical field of production and application of bioengineering products, in particular to a recombinant human influenza virus type 3 matrix protein (matrix protein, M1), a production method of the matrix protein and the technical field of application thereof. Background technique [0002] Influenza virus infection has not been well controlled, and it is still one of the main causes of human death. Influenza virus (IFV), abbreviated as influenza virus, belongs to Orthomyxoviridae (Orthomyxoviridae). According to the antigenicity of virus nucleoprotein (NP) and matrix protein, it is divided into type A, B, and C; Type influenza viruses are prone to mutate and form new subtypes, such as Type A1 and Type A3. Influenza A virus is an important pathogen that has the most frequent repeated epidemics and causes the global epidemic of influenza. Matrix protein (M1 protein) is a protein that is located under the lipid envelope of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/18C12N1/21C12N15/70
Inventor 包怡华辛若雷钱渊王芳张霆
Owner 首都儿科研究所
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