PiggyBac Transposon Construction Of Pig IFITM3 Gene And Its Expression In PK15 Cell

Interferon(Interferon, IFN) is a kind of cytokines that have a variety of biological activities of antiviral, inhibit cell division, regulating immunity and promote apoptosis. Interferon(IFN) induce Interferon Stimulated Genes(ISGs) through its receptor(IFNR) in cells, expressing the corresponding protein so as to complete a variety of biological functions. Interferon induced transmembrane protein 3(IFITM3) gene is an important kind of ISGs. IFITM3 gene can influence cell growth differentiation, affect cell proliferation, against virus invasion and so on the many kinds of biological functions. At present for IFITM3 gene research mainly concentrated in human and in mice, and researches of IFITM3 genes mainly about expression in tumor tissues and cells changes in abundance. For the porcine IFITM3 gene,researches are focused on the antiviral effect.To figure out further discussion of IFITM3 in the cell test, carrier of construct eukaryotic expression vector is needed. Piggy Bac is a kind of DNA transposons, under the action of the enzymes in turn, the swivel mount, follow the "cut off- paste" insert genes can be quickly and efficiently, providing the convenience for evaluation of gene functions in cell.This study is to construct eukaryotic expression vector Piggy Bac carrier of porcine IFITM3 gene, and evaluate its expression in PK15 cells, Preparing experimental basis for further study of the antiviral mechanism in cell level. Porcine IFITM3 gene ORF area was cloned from pig spleen tissues by RT-PCR, and the porcine IFITM3 gene expression in pig tissues was analyzed. p ET-21b(+) was builded as the carrier to a prokaryotic expression vector, and analyzes the porcine IFITM3 gene fusion expression in E.coli with SDS-PAGE and Western Blot methods. Eukaryotic expression vector PB-IFITM3 was constructed with PB transposon. Porcine IFITM3 genes was integrated into pig kidney epithelial cells PK15 cells. To evaluate the influence of IFITM3 gene to the inflammatory signaling molecules expression mediated by LPS and detect the porcine IFITM3 gene m RNA expression level in PK15 cell, RT-q PCR was carried out. Results show that the cloning of porcine IFITM3 gene was 438 bp, encoding 145 amino acids; Organization, according to the distribution of porcine IFITM3 gene expression abundance is higher in spleen and lungs; In the E.coli BL21(DE3) expression analysis showed that protein expressed by the prokaryotic expression vector size is 22.3 ku, some of the protein was soluble protein, another part was in the form of inclusion body; Q-PCR analysis showed, the porcine IFITM3 genes are expressed in PK15 cells implemented. With LPS induced, IFITM3 gene expression was further raised after porcine IFITM3 gene overexpression, and rely on time. TLR4, NFκB, IFN-α, IFN-β, TNF-α gene expression decreased,relying on the time of LPS inducing. TBK1, p38 MAPK gene expression decreased slightly,but not significant in 4 hour and 8 hour,and decreased in 12 hour. This results revealed that the IFITM3 overexpression inhibited the TLR4 signaling pathways activated by LPS in a certain extent.