Cloning Of Porcine IFITM Gene And Its Role In The Proliferation Of PRRSV

Interferon (IFN) is cytokine, produced by monocytes and lymphocytes. Interferon has a variety of biological activities, including antiviral activity, influence on cell growth, differentiation, and immune regulation. The antiviral activity of interferon dose not occur by directly killing or inactivating the virus. However, it can inhibit virus replication by inducing the expression of multiple proteins, through cell surface receptors. The gene which expressed functional protein was called interferon stimulated genes (ISGs).IFITM gene family (interferon induced transmembrane protein) is the earliest established interferon-inducible genes, composed of three functional members IFITM1, IFITM2and IFITM3. They encoded respectively14.6kDa,14.5kDa and13.9kDa proteins. IFITM proteins were induced by IFN, they played a significant role in antivirus progress, induction of cell differentiation, anti tumor, immune monitoring.PRRSV is a member of the Nido virales, Arteriviridae, Arterivirus, can cause swine fever, anorexia, difficulty in breathing, abortion, premature delivery, stillbirth, weak fetus and mummified. The disease spreads quickly, causing huge losses to the pig industry. Therefore, a method of anti PRRSV is urgently needed. It can be beneficial to the clinical diagnosis and treatment of porcine reproductive and respiratory syndrome (PRRS) by researching the IFITM gene family, and provide the necessary basis for production of clinical medicine. Therefore, this study was mainly aim to the cloning of porcine IFITM gene and the effect on the proliferation of PRRSV. The main contents were as follows:1. The Cloning, sequence and phylogenetic tree analysis of the porcine IFITM gene familyThrough the analysis of the porcine EST database and Nucleotide sequence of other known species IFITM gene, we predicted nucleotide sequence of porcine IFITM and designed the specific primers. The RNA of PK-15cells was used as the template and the porcine IFITM gene sequence was amplified via RT-PCR. The result of sequencing analysis demonstrated that sequences of porcine IFITM gene family were respectively375bp、435bp and438bp, and their cDNA encoded respectively124aa、144aa and 145aa residues. IFITM1had a amino acid homology of71%,64%,57%and56%with pig, man, rat and monkey; IFITM2had a amino acid homology of76%,68%and54%with pig, human and rat; IFITM3also had73%、69%、62%、56%of amino acid sequence homology compared with bovine, human, rat and monkey. Phylogenetic tree analysis exhibited that there were differences among the amino acid sequence of different species IFITM. IFITM gene family was changeable in evolution, and porcine IFITM had the closest relationship with cattle IFITM.2. The subcellular localization of porcine IFITM gene familyThe porcine IFITM gene family was inserted into the vector pEGFP-C2to construct the eukaryotic expression plasmid of pEGFP-C2-IFITMs, and then transfected into the PK-15cells and293cells. Via Confocal immunofluorescence analysis, we found that porcine IFITM was located in the plasmalemma3. The influence of porcine IFITM gene family on PRRSV proliferationThe eukaryotic expression plasmid of pCAGGS-HA-IFITMs was transfected into SJPL cells and CD163-293cells which were then infected with PRRSV. By absolute quantitative PCR and barren spot, we detected that porcine IFITM inhibited the proliferation of PRRSV4. The influence of PRRSV infection on the porcine IFITM gene familypEGFP-C2-IFITMs were transfected into Marc-145cells,12hours later, the cells were infected with PRRSV. By laser confocal microscopy after DAPI staining, we observed that the subcellular localization of porcine IFITM was not changed by PRRSV infection.By relative quantitative PCR, we investigated the influence of PRRSV infection on the expression of the porcine IFITM gene family in PAM cells, and found that the expression of the porcine IFITM gene was induced in prophase of PRRSV infection in PAM cells.