Study On The Expression, Purification And Preliminary Crystallization Of Polymerase Domain On PB1 Subunit (PB1_P) Of Avian Influenza Virus Subtype H5N1

Avian influenza (AI) is an acute infection caused by the influenza A virus, and it has caused huge losses to poultry industry in the world. AI has been classfied as A-type animal disease by International Organization for Animal Health (OIE). Since AI was first found in Hong Kong in 1997, hundreds of people infected by AI have already been killed by avian influenza virus (AIV). Nowadays AI has become one of the major public epidemics in the world, and the researches on AI are of great practical significance.The middle region of PB1 subunit of the influenza virus polymerase, namely, polymerase domain on PB1 (PB1_P),which has the RNA polymerase activity, is directly responsible for virus transcription and replication. PB1_P is the polymerase core area. This study succeed in expressing the H5N1 subtype avian influenza virus PB1_P subunit fragment (197-554aa) in E. coli and obtaining the high purity recombinant proteins, and we also carried out the initial crystallization of target proteins.According to the cDNA sequence of the avian influenza PB1 of H5N1 subtype in Genbank, we designed a pair of specific primers. The PB1_P genes of AIV(H5N1) were amplified by RT-PCR, and then were Cloned into the Pet-28a vector. We obtained the positive recombinant pET-28a/PB1_P plasmid by using PCR, restriction enzyme digestion and sequencing. Homologous comparison analysis indicated that PB1 gene sequence was highly conserved, type-specific, and sharing nearly 99% homology with that of the same avian subtype. Positive recombinant plasmid was transformed into E. coli BL21 to obtain the stable expressing of positive engineering bacteria .The optimal express conditions about pET-28a/PB1_Precombinant protein could be found as follows: IPTG concentration is 0.5mM; induced temperature is 37℃; induction time is 4h .The recombinant protein was identified by Western-blot. The expressed fusion protein was purified by nickel affinity chromatography and Gel Filtration Chromatography. Protein concentration was determined by Lowry method.We obtained protein crystals in two conditions by the hanging drop vapor diffusion method .Results and Conclusions:(1) The pET-28a / PB1_P recombinant plasmid was successfully reconstructed;(2) The recombinant protein PB1_P was purified with high concentration and the screening crystals were obtained. This lay a solid foundation for the further study on the structure and function of the influenza virus polymerase, and also promote the development of the new diagnostic kits.