Gene Cloning,Expression And Antibody Preparation Of Interferon Induced Protein IFIT1s From Homo Sapiens And Mus Musculus

Interferon is a kind of cytokines produced by mononuclear cells and lymphocytes, it's an important part of the innate immune system as it has many kinds of biological activities such as broad-spectrum antiviral, antibiosis and antitumor abilities, and regulate immune functions and so on. IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1) was the first discovered ISGs (Interferon stimulated genes) protein whose gene expression is regulated by the interferon, it will participate in and regulate the body's physiological activities through a variety of means. HIFIT1 (GenBank Accession number:BC007091.1) is a IFIT1 gene from Homo sapiens, the open reading frame (ORF) of which is 1437 bp, encoding HIFIT1 protein containing 478 amino acids with the predicted molecular weight of 55.36 kDa. mifit1(GenBank Accession number:CT010226.1) gene is that from Mus musculus, the ORF of which is 1392 bp, encoding MIFIT1 protein containing 463 amino acids with the predicted molecular weight of 53.74 kDa. After total RNAs were extracted from human 293T cells and mouse 3T3 cells, the ORFs of HIFIT1 and mifitl genes were amplified by using reverse transcriptase PCR (RT-PCR) and conventional PCR, and inserted into bacterial expression vector pGEX-4T-1, respectively. Then, the recombinant plasmids were transformed to Escherichia coli DH5a, which was subsequently induced to express foreign genes by the addition of isopropyl (3-D-l-thiogalactopyranoside. As a result, the recombinant HIFIT1 and MIFIT1 were successfully expressed in E. coli as fusion proteins, rHIFIT1 and rMIFIT1, the molecular masses being 81 kDa and 79 kDa, respectively, which were consistent with the theoretically predicted molecular weights. rHIFITl and rMIFITl were used to immunize BALB/c mouses and New Zealand rabbits respectively. The antibody titers of the immunized mice and rabbits were detected by using indirect enzyme-linked immunosorbent assay (ELISA), the result showed that the antibody titers in antisera from immunized animals were all reached to 105 and above 10 d post 3 immunizations. Western blotting analysis showed that the mouse antisera against rHIFIT1 recognized the native HIFIT1 protein in 293T cells and the rabbit antiserum against rMIFIT1 recognized the native MIFIT1 in 3T3 cells. It suggested that both rHIFITl and rMIFIT1 proteins had good immunogenicity. The localizations of native HIFIT1 protein in 293T cells and native MIFIT1 protein in 3T3 cells were detected respectively by using indirect immunofluorescent antibody test (IFAT). The result indicated that both native HIFIT1 and MIFIT1 proteins are mainly distributed in the cytoplasm. Murine spleen cells from BALB/c mice immunized with rHIFIT1 protein were fused with myeloma cells SP2/0,9 positive hybridoma cell lines of HIFIT1 resistance were selected after 3 screenings and clonizations. They were injected to abdominal cavities of BALB/c mice to produce ascites containing monoclonal antibodies against rHIFIT1, eventually got 9 ascites. In a conclusion, prokaryotic expression system of HIFIT1 and MIFIT1 was successfully constructed, and both polyclonal and monoclonal antibodies against rHIFIT1 as well as anti-rMIFIT1 polyclonal antibody were prepared, which would promote the furthe studies on the functions of IFIT1 protein, and laid a foundation for immunotherapy of related diseases.