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Objective To determine the region of human transmembrane tumor necrosis factor-alpha (TM-TNFa) , essential for cytotoxic activity a-gainst human breast cancer cell line MCF-7.Methods Single amino-acid-substituted TM-TNFa mutant proteins (muteins) were produced by in vitro transcription linked translation techniques. The cDNA of TM-TNFa was site-directed mutagenized by recombinant PCR.Results 13 single ammo-acid substituted TM-TNFa muteins were generated and assayed for cytotoxic activity. The cytotoxic activities of TM-TNFa muteins, eg, TM-TNFa -71/Lys, -28/Phe and 117/Leu were significantly decreased (P < 0.01) compared to that of parent TM-TNFa, 143/Tyr decreased 4-folds, and -17/Thr, -39/Ser, 119/His, 35/Gly, 95/Cys and 147/Phe decreased 1.5-2.5-folds, respectively. However, the cytotoxic activities of TM-TNFa-8/Arg, 31/Gly and 87/Phe showed no significant change. Conclusion These results indicate that the regions associated with cytotoxic-activity of TM-TNFa are different with that of secretory TNF-
Objective To determine the region of human transmembrane tumor necrosis factor-alpha (TM-TNFa), essential for cytotoxic activity a-gainst human breast cancer cell line MCF-7.Methods Single amino-acid- substituted TM-TNFa mutant proteins were produced by in vitro transcription linked translation techniques. The cDNA of TM-TNFa was site-directed mutagenized by recombinant PCR. Results 13 single ammo-acid substituted TM-TNFa muteins were generated and assayed for cytotoxic activity. The cytotoxic activities of TM- TNFa muteins, eg, TM-TNFa-71 / Lys, -28 / Phe and 117 / Leu were significantly decreased (P <0.01) compared to that of parent TM- TNFa, 143 / Tyr decreased 4-folds, Thr, -39 / Ser, 119 / His, 35 / Gly, 95 / Cys and 147 / Phe decreased 1.5-2.5-folds, respectively. However, the cytotoxic activities of TM-TNFa-8 / Arg, 31 / Gly and 87 Conclusion These results indicate that the regions associated with cytotoxic-activity of TM-TNFa are different with that of secretory TNF-