Cloning And Expression Of Porcine P58^IPK Gene And Regulation Of Its Expression During Influenza A Virus Infection

P58^IPK is a cellular inhibitor of PKR that is activated at the post-translational level in response to influenza A virus infection. The activation of P58^IPK results in reduced level of PKR-mediated eIF2αphosphorylation, which has long been thought to benefit influenza virus by maintaining a high rate of viral protein translation. However, it was found that influenza A virus infection was more lethal in mice lacking P58^IPK, due to increased lung pathology and inflammation. In order to confirm the function of porcine P58^IPK in influenza A virus infection and further explore the mechanisms, we made the experiments as follows:1. The swine gene P58^IPK was cloned through homology searching in the swine EST database. Then this gene was cloned using reverse transcription polymerase chain reaction (RT-PCR). The cDNA of the gene contains the complete open reading frame (ORF) of 1518 bp, encoding 505 amino acid residues. The gene was preliminarily analyzed using bioinformatics methods. The N-terminal and C-terminal were highly conservative, and the similarity of P58^IPK protein between swine and human was 99.21%.2. P58^IPK was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid pET-P58^IPK. The fusion protein His-P58^IPK was expressed in E.coli BL21 and purified using a His-tag protein purification column. Subsequently, New Zealand white rabbits were immunized with the purified protein. Specific polyclonal antibodies against the fusion protein His-P58^IPK was obtained. The titer of the antibody was 1: 100000 detected by ELISA. The protein p58^IPK could be specifically detected by Western blotting and immunofluorescence assay using the polyclonal antibodies.3. We inserted porcine P58^IPK into eukaryotic expression vetor pEGFP-C1 and pEGFP-N1, which were named as EGFP-P58^IPK-C1 and P58^IPK-EGFP-N1, respectively. Each recombinant plasmid containing a green fluorescence reporter gene was transfected into SUVECs (Swine Umbilical Vein Endothelial Cells) by lipofectamine-2000. Fluorescence microscope showed that fusion protein EGFP-P58^IPK-C1 and P58^IPK-EGFP-N1 were at the best expression level 24 hours post transfection. According to the fluorescence position of the cells, we concluded that porcine P58^IPK was localized to the cytoplasm. Western blotting analysis confirmed the expression of P58^IPK in vitro and in vivo. 4. Healthy piglets were infected with H1N1 and H3N2 influenza A virus strains, respectively. Seven days later, lung tissues were collected from the healthy controls and infected piglets, respectively. RNA was extracted from a part of lung tissue samples and reversely transcripted for qRT-PCR detection. Another part of lung tissue samples were made for pathological slices and P58^IPK immunohistochemical assay. Pathological changes were observed on the slices. These results showed that the expression of porcine P58^IPK in the levels of mRNA and protein were up-regulated in infected piglets.