关键词: 重组牛粒细胞集落刺激因子, 纯化鉴定, 安全性, 毒性试验

Abstract: [Objective] To assess the safety of recombinant bovine granulocyte colony stimulating factor （rbG-CSF）, so as to set the groundwork for its future clinical trials. [Method] Using rbG-CSF recombinant Escherichia coli, rbG-CSF was obtained after fermentation and purification. The target protein was identified by SDS-PAGE, and its concentration was determined by BCA method. In acute toxicity test, a total of 112 healthy Kunming mice were divided into eight groups, each with half males and half females. The eight groups were then evenly divided into intramuscular injection groups and intraperitoneal injection groups, with one control group and three rbG-CSF treatment groups each. The treatment groups intramuscularly or intraperitoneally received 150, 75, and 37.5 μg per mice of rbG-CSF, while the corresponding control groups both received a saline injection. Following administration, the mice were weighed every 3 days for consecutive 14 days. On the 14^th day post injection, the liver, spleen, kidney, and reproductive organs of mice in each group were dissected for organ coefficient assessment and pathological section observation. In chronic toxicity test, a total of 160 healthy Kunming mice were used. The grouping protocol was identical to that of acute toxicity test. The rbG-CSF were intramuscularly or intraperitoneally administrated once a week with dosages of 15, 10, and 5 μg per mice for consecutive 12 weeks. The mice were weighed on the 6^th day post injection weekly. The mice were dissected 7 days after the injection on the 12^th week. The subsequent treatment approaches were identical to those used in acute toxicity test. [Result] The target protein rbG-CSF was successfully purified and was found to be 21.5 kDa identified by SDS-PAGE, which was in line with the anticipating size. The concentration of the target protein was 302.19 μg /mL, which met the concentration requirement for the subsequent safety test. In both acute and chronic toxicity tests, one-way ANOVA analysis （SPSS 20.0） revealed that there were no significant differences in body weight and organ coefficient between rbG-CSF intramuscular or intraperitoneal injection groups and corresponding control groups. No macroscopic and histopathological changes were observed in mice received rbG-CSF injection. [Conclusion] The developed methods for purifying and identifying rbG-CSF are feasible. At the concentrations observed in this study, rbG-CSF has no organic damage in mice.

Key words: recombinant bovine granulocyte colony stimulating factor, purification and identification, safety, toxicity test