Study On Expression And Preliminary Crystallographic Analysis Of H5N1 Highly Pathogenic Avian Influenza RNA Polymerase PB1B Subuint

Avian influenza (AI) is a highly communicable and economically devastating viral disease, which mainly infects avian. H5N1 infections in humans were seen in Hong Kong in a small outbreak in 1997 that resulted in 18 human infections and six fatalities, and since 2003, 384 human cases of H5N1 have been confirmed with a 62.8% fatality rate.The genome of influenza A virus consists of eight negative-sense RNA segments (called vRNAs) that together encode the 11 known viral proteins. The PB1 subunit plays a central role in the catalytic activities of the RNA polymerase. It contains the conserved motifs characteristic of RNA-dependent RNA polymerases and is directly involved in RNA chain elongation. It binds to the promoter sequences of vRNA and cRNA and, depending on its interaction with RNA, performs the endonucleolytic cleavage of capped RNA.Because of the lack of structural information, there is no precise illustration of the catalysis mechanism of PB1 till now. In this article, we focused on the recombination, purification, crystallization and preliminary crystallographic analysis of PB1B.We overproduced the putative PB1B protein in E.coli. and purified it by Ni2+-chelating affinity chromatography, Resource Q ion-exchange chromatography and Superdex-75 size-exclusion chromatography. We will use hanging drop vapor diffusion method of protein crystallization. Screening and optimization of crystallization conditions to obtain suitable X-ray diffraction of different crystal space group, and collected high-quality X-ray diffraction data.The experimental results show that the recombinant constructed plasmid pET-28a-PB1B successfully, and their inducible expression, access to soluble proteins. Through the optimization of purification conditions, we obtain the high purity of the target protein. Purified protein derived from the molecular weight of about 47kDa. In the crystallization solution, the purified target protein PB1B has a good homogeneity for protein crystallization. By X-ray irradiation, to obtain two different crystallization conditions for X-ray diffraction of the two different crystal space group.