AIM: To construct the recombinant vectors carrying interleukin (IL)-28A, IL-28B and IL-29 cDNAs and express them in human A549 cells, and analyze their antiviral activity in vesicular stomatitis virus (VSV)-infected human immortalized amnion epithelial cell line (WISH cells). METHODS: Total cell RNA was extracted from human peripheral blood mononuclear cells (PBMC) activated with poly I:C. The cDNAs encoding human IL-28A, IL-28B, and IL-29 were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pcDNA3.1/V5-His-TOPO vectors. These recombinant vectors were transfected into human A549 cells by a liposome-mediated gene transfer method. Semiquantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression of IL-28A, IL-28B, and IL-29. The antiviral activity of IL-28A, IL-28B, and IL-29 was determined by a cytopathic effect reduction assay on WISH cells using VSV as a challenge virus. RESULTS: The DNA sequences of the inserts were identical to the published sequences encoding IL-28A, IL-28B, and IL-29 in GenBank. It was demonstrated that IL-28A, IL-28B, and IL-29 genes were markedly transcribed in transfected cells. Expression of all 3 interleukins in A549 cells was confirmed by Western blot analysis. IL-28 and IL-29 expressed by A549 cells, like interferon (IFN) alpha-2b, were able to protect WISH cells against VSV infection. CONCLUSION: IL-28 and IL-29 cDNAs were successfully cloned and expressed in eukaryotic cells via transfection with pcDNA3.1/V5-His-TOPO-IL-28/IL-29. Transfection with this vector produced a specific antiviral activity similar to that of IFN-alpha, which will provide a new tool for the functional study of these cytokines in humans.
AIM: To construct the recombinant vectors carrying interleukin (IL)-28A, IL-28B and IL-29 cDNAs and express them in human A549 cells, and analyze their antiviral activity in vesicular stomatitis virus (VSV)-infectedhuman immortalized amnion epithelial cell line (WISH cells). METHODS: Total cell RNA was extracted from human peripheral blood mononuclear cells (PBMC) activated with poly I:C. The cDNAs encoding humanIL-28A, IL-28B, and IL-29 were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pcDNA3.1/V5-His-TOPO vectors. These recombinant vectors were transfected into human A549 cells by a liposome-mediated gene transfer method. Semiquantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression of IL-28A, IL-28B, and IL-29. The antiviral activity of IL-28A, IL-28B, and IL-29 was determined by a cytopathic effect reduction assay on WISH cells using VSV as a challenge virus. RESULTS: The DNA sequences of the inserts were identical to the published sequences encoding IL-28A, IL-28B, and IL-29 in GenBank. It was demonstrated that IL-28A, IL-28B, and IL-29 genes were markedly transcribed in transfected cells. Expression of all 3 interleukins in A549 cells was confirmed by Western blot analysis. IL-28 and IL-29 expressed by A549 cells, like interferon (IFN) alpha-2b, were able to protect WISH cells against VSV infection. CONCLUSION:IL-28 and IL-29 cDNAs were successfully cloned and expressed in eukaryotic cells via transfection with pcDNA3.1/V5-His-TOPO-IL-28/IL-29. Transfection with this vector produced a specific antiviral activity similar to that of IFN-alpha, which will provide a new tool for the functional study of these cytokines in humans.
Authors: Simone Regina Souza da Silva Conde; Luciana L Rocha; Vanessa M Ferreira; Julius Caesar Mendes Soares Monteiro; Nathália Karla Fonseca Filgueiras; Pedro Alves de Almeida Lins; Bruna Tereza Silva dos Santos; Felipe Bonfim Freitas; Ednelza da Silva Graça; Sâmia Demachki; Marialva Tereza Ferreira de Araújo; Ricardo Ishak; Antonio C R Vallinoto Journal: Dis Markers Date: 2014-04-10 Impact factor: 3.434