Open AccessLiposome‐mediated IL‐28 and IL‐29 expression in A549 cells and anti viral effect of IL‐28 and IL‐29 on WISH cells 1
Author(s) -
LI Mingcai,
WANG Haoyang,
WANG Haiyan,
LI Tao,
HE Shaoheng
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00292.x
Subject(s) - microbiology and biotechnology , transfection , biology , recombinant dna , virology , a549 cell , vesicular stomatitis virus , cell culture , peripheral blood mononuclear cell , virus , gene , in vitro , genetics
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‐28 A, 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) α‐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‐α, which will provide a new tool for the functional study of these cytokines in humans.