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Influenza virus RNA polymerase purifying or crystallizing method

An RNA polymerase, influenza virus technology, applied in the biological field, can solve the problems of reports, no influenza virus polymerase structure, structural biology research crystal structure obstacles, etc.

Inactive Publication Date: 2015-10-28
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • Influenza virus RNA polymerase purifying or crystallizing method
  • Influenza virus RNA polymerase purifying or crystallizing method
  • Influenza virus RNA polymerase purifying or crystallizing method

Examples

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Embodiment 1

[0048] Embodiment 1, the crystallization of the influenza virus RNA polymerase that contains PB2-126 truncated body

[0049] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-126 truncated body;

[0050] The amino acid sequence of the PB2-126 truncated body is the 1-126th amino acid from the N-terminal of the sequence 2, and the coding gene thereof is the 1-378th nucleotide from the 5' end of the sequence 5.

[0051] 1. Obtaining influenza virus RNA polymerase

[0052] 1. Expression of each subunit of influenza virus RNA polymerase

[0053] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0054] 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 respe...

Embodiment 2

[0142] Embodiment 2, the crystallization (comparative example) containing the influenza virus RNA polymerase of PB2-130 truncated body

[0143] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-130 truncated body;

[0144] 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.

[0145] 1. Obtaining influenza virus RNA polymerase

[0146] 1. Expression of each subunit of influenza virus RNA polymerase

[0147] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0148] 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 ...

Embodiment 3

[0195] Embodiment 3, the crystallization (comparative example) containing the influenza virus RNA polymerase of PB2-103 truncated body

[0196] The three subunits of the influenza virus RNA polymerase polymer precrystallized in this embodiment include PB1 protein, PA protein and PB2-103 truncated body;

[0197] 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.

[0198] 1. Obtaining influenza virus RNA polymerase

[0199] 1. Expression of each subunit of influenza virus RNA polymerase

[0200] 1), the construction of the recombinant vector expressing each subunit of influenza virus RNA polymerase

[0201] 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 ...

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Abstract

The invention discloses an influenza virus RNA polymerase purifying or crystallizing method. The influenza virus RNA polymerase crystallizing method comprises the following steps: co-expressing and purifying PB1 subunit encoding gene and PA subunit encoding gene of influenza virus RNA polymerase and encoding gene of PB2 subunit truncation in insect cells to obtain RNA polymerase, combining the RNA polymerase with RNA to form a complex, and crystallizing the complex to obtain influenza virus RNA polymerase crystals, wherein the PB2 subunit truncation is a protein formed by amino acid residues from N end to 126th position in a PB2 subunit amino acid sequence. Experiments prove that after different PB2 subunit truncations are used, the expression level of the influenza virus polymerase complex containing the 126 amino acid fragments of the amino end of PB2 is high, and 10mg of the protein can be purified through 1L of an insect cell culture solution, so the expression level of the influenza virus polymerase complex containing the 126 amino acid fragments of the amino end of PB2 is higher than that of other truncations; and the crystals obtained in the invention have the advantages of good purity, slight degradation, good crystallization repeatability, good quality, large size and strong diffraction.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying or crystallizing influenza virus RNA polymerase. 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 target influenza virus surface proteins, such as HA, NA and M2. Because influenza viruses can chan...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/866
CPCC12N9/1247C12Y207/07006
Inventor 刘迎芳
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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