[Construction of recombinant adenovirus carrying human IFNgamma gene by in vitro ligation and its expression].
OBJECTIVE
Interferon-gamma (IFNgamma) can promote directly the apoptosis of some kinds of tumor cells and regulate cellular immunity, therefore, it may play an important role in gene therapy for tumors. This study was to construct recombinant adenovirus carrying human IFNgamma cDNA, and detect its expression profile in eukaryotic cells.


METHODS
IFNgamma cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from human peripheral blood mononuclear cells, and cloned into adenoviral shuttle plasmid pShuttle to construct recombinant adenovirus carrying human IFNgamma cDNA (Ad-IFNgamma) by in vitro ligation. Hepatocellular carcinoma cell line HepG2 was infected with Ad-IFNgamma, and the expression of IFNgamma was detected by RT-PCR and immunohistochemistry.


RESULTS
The sequence of the cloned IFNgamma cDNA was completely consistent with that reported in GenBank. PCR analysis confirmed the existence of IFNgamma gene in Ad-IFNgamma without wild adenovirus contamination, and the genome of Ad-IFNgamma showed the predicted map of restricted endonuclease enzymes analysis. IFNgamma mRNA and protein were detected in HepG2 cells infected with Ad-IFNgamma.


CONCLUSIONS
In vitro ligation is a simple and convenient method for construction of recombination adenovirus vector. We have constructed a functional recombinant adenovirus expressing human IFNgamma, which could be a potential antivirus and antitumor agent in vitro or in vivo.
Xue
Gang
G
Department of General Surgery, Chengdu Army General Hospital, Chengdu, Sichuan, 610083, P. R. China.
Li
Yan
Y
Liu
Ran-Yi
RY
Tian
Fu-Zhou
FZ
Huang
Wen-Lin
WL
D000256:Adenoviridae / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma