Liposome-mediated IL-28 and IL-29 expression in A549 cells and anti-viral effect of IL-28 and IL-29 on WISH cells.
OBJECTIVE
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.


CONCLUSIONS
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.
Li
Ming-Cai
MC
Allergy and Inflammation Research Institute, Shantou University Medical College, Shantou 515041, China.
Wang
Hao-Yang
HY
Wang
Hai-Yan
HY
Li
Tao
T
He
Shao-Heng
SH
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells / Q000821:virology; D005822:Genetic Vectors; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology; D007372:Interferons; D007378:Interleukins / Q000096:biosynthesis* / Q000235:genetics; D008081:Liposomes; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects*; D000095022:Interferon Lambda
D000998:Antiviral Agents; D016207:Cytokines; D018076:DNA, Complementary; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D007372:Interferons; D007378:Interleukins; D008081:Liposomes; D012333:RNA, Messenger; D011994:Recombinant Proteins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda