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Method for crystallizing influenza virus RNA polymase

A technology of RNA polymerase and influenza virus, applied in the biological field, can solve the problems of lack of influenza virus polymerase structure, structural biology research crystal structure obstacles, reports, etc.

Inactive Publication Date: 2015-11-04
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve these problems, it is very necessary to have an overall structure of the polymerase at atomic resolution. However, since the influenza virus polymerase is a 250KD complex, in order to perform its various functions, it may have multiple conformations. , especially the acquisition of the crystal structure has caused great obstacles, so even after decades of efforts, there is still no structure of the overall complex of influenza virus polymerase reported

Method used

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  • Method for crystallizing influenza virus RNA polymase
  • Method for crystallizing influenza virus RNA polymase
  • Method for crystallizing influenza virus RNA polymase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the crystallization of the influenza virus RNA polymerase that contains PB2-37 truncated body

[0053] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-37 truncated body;

[0054] The amino acid sequence of the PB2-37 truncated body is the 1st-37th amino acid from the N-terminal of sequence 2, and the encoding gene is the 1st-111th nucleotide from the 5' end of sequence 5.

[0055] 1. Obtaining influenza virus RNA polymerase

[0056] 1. Expression of each subunit of influenza virus RNA polymerase

[0057] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0058] Generally speaking, H5N1, WSN, PR8, 1918, and H3N2 gene DNAs were synthesized by gene synthesis methods according to the gene sequences published on the NCBI website. According to the respective sequences, DNA primers were designed respectively, and P...

Embodiment 2

[0146] Embodiment 2, the crystallization of the influenza virus RNA polymerase that contains PB2-130 truncated body

[0147] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-130 truncated body;

[0148] The amino acid sequence of the PB2-130 truncated body is the 1-130th amino acid from the 5' end of sequence 2, and the corresponding nucleotide sequence is the 1-390th nucleotide from the 5' end of sequence 5.

[0149] 1. Obtaining influenza virus RNA polymerase

[0150] 1. Expression of each subunit of influenza virus RNA polymerase

[0151] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0152] Due to the need for nickel column purification in the later stage, it is necessary to add a HIS tag to the carboxyl end of the subunit PB2-130 or PB1. Experiments were carried out on both of them. Any one of PB2-130 and PB1 subunits is...

Embodiment 3

[0198] Embodiment 3, the crystallization of the influenza virus RNA polymerase that contains PB2-103 truncated body

[0199] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-103 truncated body;

[0200] The amino acid sequence of the PB2-103 truncated body is the 1-103 amino acid from the 5' end of sequence 2, and the corresponding nucleotide sequence is the 1-309 nucleotide from the 5' end of sequence 5.

[0201] 1. Obtaining influenza virus RNA polymerase

[0202] 1. Expression of each subunit of influenza virus RNA polymerase

[0203] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0204] Due to the need for nickel column purification in the later stage, it is necessary to add a HIS tag to the carboxyl end of the subunit PB2-130 or PB1. Experiments were carried out on both of them. Any one of PB2-103 and PB1 subunits is suf...

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Abstract

The invention discloses a method for crystallizing influenza virus RNA polymase, which comprises the steps of carrying out coexpression on a PB1 subunit encoding gene, a PA subunit encoding gene and a PB2 truncated subunit encoding gene of influenza virus RNA polymase in an insect cell to obtain RNA polymase; and purifying and crystallizing to obtain a influenza virus RNA polymase crystal. An experiment proves that PB2 truncated protein segments which have different lengths and amino ends retained and other two subunit full-length proteins are subjected to coexpression purification and complex assembling, a small truncated complex can use different strains to obtain crystal at multiple truncating circumstances. The method lays a foundation for structure analysis of influenza virus RNA polymase and also has important meanings for anti-influenza virus drug screening and design and research and development process of broad spectrum vaccines and drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a method for crystallizing influenza virus RNA polymerase, in particular to a method for expressing, purifying and crystallizing an influenza virus RNA polymerase complex. Background technique [0002] Influenza virus, especially influenza A virus, has always been a big problem threatening human society. It has a wide range of hosts including humans, pigs, horses and poultry, etc., and can cause upper respiratory tract infections in humans and animals, namely influenza. It can spread rapidly through the air, causing several worldwide pandemics in the last century. Influenza viruses belong to the Orthomyxoviridae family and are negative-sense single-stranded RNA segmented genome viruses. Its genome is divided into 8 segments, and it has been found that at least 16 proteins can be encoded. Although there are many anti-influenza virus drugs and vaccines, these drugs and vaccines mainly t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/866
Inventor 刘迎芳
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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