摘要
[目的]评价重组牛粒细胞集落刺激因子(recombinant bovine granulocyte colony stimulating factor,rbG-CSF)的安全性,为后期rbG-CSF临床应用价值的研究打下基础。[方法]采用rbG-CSF重组大肠杆菌,经过发酵、纯化后获得rbG-CSF;利用SDS-PAGE法对目的蛋白进行鉴定;应用BCA法测定目的蛋白浓度。急性毒性试验使用健康昆明小鼠112只,分为8组,雌雄各半,将8组小鼠分为肌肉注射组和腹腔注射组,肌肉注射组和腹腔注射组分别设有1个对照组和3个剂量组,3个剂量组注射rbG-CSF的剂量分别是150、75、37.5μg/只,对照组注射生理盐水,给药后每隔3 d进行1次称重,持续观察14 d,给药后14 d对各组小鼠进行剖检,取肝、脾、肾、生殖器官进行脏器系数测定和病理切片观察。慢性毒性试验使用健康昆明小鼠160只,分组与急性毒性试验相同,注射rbG-CSF的剂量分别是15、10、5μg/只,每周注射1次,每周注射的第6天进行1次称重,持续注射12周,第12周给药后7 d对小鼠进行剖检,处理方法同急性毒性试验。[结果]rbG-CSF纯化成功,经SDS-PAGE法鉴定目的蛋白大小为21.5 kDa,与设计大小一致;测得目的蛋白浓度为302.19μg/mL,可满足后期安全性试验所需浓度要求;急性毒性试验和慢性毒性试验中,小鼠的体重和脏器指数经SPSS 20.0单因素方差分析,剂量组与对照组相比均无显著差别;剖检各器官未出现明显病变;HE染色切片观察,各剂量组与对照组不存在显著变化。[结论]该试验建立的纯化鉴定rbG-CSF的方法可行,rbG-CSF在试验浓度下对小鼠并未造成器质性的伤害。
[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 37.5μg 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 14th 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 6th day post injection weekly.The mice were dissected 7 days after the injection on the 12th 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 302.19μg/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.
作者
石青青
孙珊珊
李丽琴
宋秀梅
于海超
程雪娇
王建
SHI Qing-qing;SUN Shan-shan;LI Li-qin;SONG Xiu-mei;YU Hai-chao;CHENG Xue-jiao;WANG Jian(Tianjin Speerise Challenge Biotechnology Co.,Ltd.,Tianjin300380,China)
出处
《畜牧与饲料科学》
2022年第3期8-15,共8页
Animal Husbandry and Feed Science
基金
天津市科技计划项目(19YFZCSN00090)。
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
recombinant bovine granulocyte colony stimulating factor
purification and identification
safety
toxicity test
