| 摘要 |
PURPOSE:To prepare a reverse transcriptase having high specific activity with simple procedure, by separating an easily maintainable tumor cell strain capable of releasing a large amount of C-particles in proliferation from the spleen of a wild leukemia pig, and using the cell system as the starting material. CONSTITUTION:The cells separated from the spleen of a wild leukemia pig are suspended and cultured for a definite period without agitation, proliferated for a definite period by the symbiotic culture with the thymocyte of healthy pigling, subjected to subculture at intervals of 3 days, and after 60 days, cloned with soft agar to obtain a tumor cell strain. The cell is cultured in a roller bottle and the culture liquid is collected and centrifuged at a low speed to remove the cells. The obtained concentrate is ultracentrifuged with 20% glycerol, and the obtained pellet is dispersed in a TNE buffer solution and subjected repeatedly to the density gradient centrifugal separation in sucrose solution to obtain a fraction having a density of 1.15-1.16g/mol and containing purified C- particles. The particles are solubilized with NP-40 and ultrasonic treatment. The dissolved C-particle is subjected successively to DEAE cellulose treatment, cellulose phosphate treatment and poly-C-agarose treatment to obtain a reverse transcriptase almost free of liponuclease contamination. |
