Cloning of IL-18 gene and research on the expression of IL-18 gene in cervical cancer Cell.

Objective To research the expression of interleukin-18(IL18)gene in the cervical cells(Hela cells) Methods IL18 gene was amplified by Reverse Transcription PCR And the eukarayotic gene targeting vector(pcDNA3 1(+)-IL18)was constructed by molecular biolgical techenique Cell supernants was collected 24～48 hours after the plasmid pcDNA3 1(+)-IL18 was transducted into Hela cells by Lipofectamine 2000 The transient expression of IL18 gene was examined by Western-Blotting The single clone cell of stable expression of IL18 gene was screened by G418 RT-PCR and Western-Blotting was applied for testing the stable expression of IL18 gene Hela cells were divided into 3 groups:(1)control group;(2)Hela cells transfected by pcDNA3 1(+);(3)Hela cells transfected by pcDNA3 1(+)-IL18 Results (1)IL18 gene was cloned successfully (2)eukaryotic gene targeting vecter(pcDNA3 1(+)-IL18) was constructed correctly (3)the result of transient expression of IL18 gene examined by Western-Blotting was that there was a specific protein band(18 3KDa) in the supernants of the Hela cells transfected by pcDNA3 1(+)-IL18 But there wasn's any protein band in the supernants of the control group and the Hela cells transfected by pcDNA3 1(+) (4)the result of stable expression of IL18:first,a specific DNA band which is between 750bp and 1000bp was amplified by RT-PCR in the Hela cells transfected by pcDNA3 1(+)-IL18 But there was not any DNA band in the control group and the Hela cells transfected by pcDNA3 1(+) Second,the result of Western-blotting was that there was a specific protein band (18 3KDa) in the supernants of the Hela cells transfected by pcDNA3 1(+)-IL18 But there wasn't any protein band in the supernants of the control group and the Hela cells transfected by pcDNA3 1(+) Conclusion (1)IL18 gene was cloned successfully and the eukaryotic gene targeting vector (pcDNA3 1(+)-IL18) was constructed correctly (2)IL18 gene was transducted into cervical cancer cells (Hela cells) successfully and was expressed