Prokaryotic Expression Of Swine Interferon Induced Transmembrane Proteins And Preparation Of Polyclonal Antibody And The Effect Of PRRSV Infection On Ifitm

Interference induced transmembrane protein(IFITM) is a kind of antiviral protein found by an American scientists through test in poultry and swines, the protein can stimulate the body’s own suppression with or without cell membrane virus replication and anti viral effect. The first reported of Cell in 2009 IFITMs significantly inhibit viral replication early in the influenza a virus infection, make people start antiviral activity of the members of the family have a new understanding and more profound understanding.This thesis mainly studies the swine IFITM gene sequences, prokaryotic expression, the preparation of polyclonal antibody, at the same time, preliminary study the different tissues of PRRSV infection swine IFITMs mRNA expression level. The results of this study are as follows:1. IFITM Swine gene sequence published with reference to GenBank, with Primer Premier software design three swine specific primers of interferon induced transmembrane protein gene, extraction of RNA from peripheral blood lymphocytes of swines, cDNA and target genes were obtained by RT-PCR. After transformation and connection step, objective gene eventually was connected to the pMD18-T Vector(simple) and the formation of recombinant plasmid.Nucleic acid sequence alignment was performed by sequencing results, recombinant plasmid of sequence alignment correct marked pMD18 IFITM1/2/3.2. IFITM gene was connected to the prokaryotic expression vector pColdⅠin this test, constructed prokaryotic expression vector pColdⅠ- IFITM, transformed into E.coli BL21 cells, the inducer IPTG inducing expression, after ultrasonic broken bacteria by SDS-PAGE and Western blot analysis, shows that the induced expression of the corresponding size recombinant protein, it exists in supernatant in the form of soluble.3. Choosing Ni- NT Resin affinity chromatography medium for inducing expression of protein purification, got high purity of target protein.The purified target protein was injected into mice abdominal cavity, after three times immunization, removing eye,collecting blood, serum was isolated, detection of indirect ELISA antibody titer.The results showed that IFITM1, IFITM2, IFITM3 antibody titer reached the highest 1:10 24000 in serum.4. In this experiment, fluorescence quantitative PCR technique was used to detect IFITM expression levels in the tissues of normal swine and infected PRRSV swine.The results showed that IFITM was expressed in all the detected tissues, but expression levels have obvious difference.Compared the same tissue, IFITM gene in PRRSV infection organization is more than the number of expression quantity in normal tissue.