Method for purifying or crystallizing influenza virus rna polymerase

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 at least 16 proteins have been found to be encoded so far. 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 chang...

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, which is of great importance to structural biology research. , especially the acquisition of crystal structures poses a great obstacle, so even after decades of efforts, no structure of the overall complex of influenza virus polymerase has been reported

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