[Stable and efficient expression of human interleukin-6 cDNA in mammalian cells after gene transfer].
Following reverse transcription and PCR amplification, the human interleukin-6 (IL-6) cDNA was cloned from total RNA of activated human tonsillar mononuclear cells. Southern blot showed the presence of specific band, and its DNA sequence demonstrated that the fragment was 650 bp in length, spanning the complete coding region and part of 5' and 3'-untranslated regions. The human IL-6 sequence seemed to be well conserved. One nucleotide change at 429 position was observed, but this change did not affect the amino acid composition. After inserting the cloned cDNA into retroviral vector XM-6, the recombinant was packaged in PA317 cells and the amphotropic virus titer reached 5 x 10(5) CFU/ml. Human IL-6 was expressed in mammalian cell line SP2/0 cells, after being infected by the constructed retrovirus. The G418 resistant SP2/0 cells secreted IL-6. Its supernatant was able to maintain in vitro growth of the IL-6 dependent T1165 cell line. Northern blot analysis of the transfected SP2/0 cells showed significantly elevated IL-6 message, being consistent with the result of T 1165 bioassay. The expression was stable during the 12 month period of observation. The hybridoma cells, formed after fusion of the transfected SP2/0 cells and lymphocytes, exhibited accelerated growth.
Chen
Q Y
QY
Cancer Institute, Chinese Academy of Medical Sciences, Beijing.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000473:pathology; D014066:Palatine Tonsil / Q000166:cytology; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D004247:DNA