重组hIFN-α-2b-BCG的冻干制备及生物特性的研究  

Lyophilization-Preparation and Biological Characteristics of Recombinant hIFN-α-2b-BCG

作　　者：孙二琳[1] 赵杰[1] 范晓东[1] 韩瑞发[1] 

机构地区：[1]天津医科大学第二医院、天津市泌尿外科研究所,300211

出　　处：《天津医药》2010年第11期929-932,共4页Tianjin Medical Journal

基　　金：国家科技部“十一五”重大专项新药创建课题(项目编号:2009ZX09103-699);国家自然科学基金资助项目(项目编号:30872587);天津市科技支撑计划重大项目(项目编号:07ZCKFSH03200)

摘　　要：目的:研究重组hIFN-α-2b-BCG真空冷冻干燥的制备工艺,研究冻干后生物特性的变化。方法:采用真空冷冻干燥法制备重组BCG(rBCG),筛选最佳工艺参数。平板计数法比较冷冻干前后的活菌数,计算存活率。比较冻干前后rBCG的抗酸染色特征与形态,连续测定OD值,绘制生长曲线,酶联免疫吸附测定(ELISA)IFN-α-2b表达水平。PCR和抗性培养分析冻干后的质粒稳定性。结果:冻干后重组BCG为(6.51±0.33)×106CFU/mL,冻干前为(1.02±0.11)×107CFU/mL,存活率约65%,达到BCG生物免疫治疗所需的活菌标准。冻干前后的菌落形态、生长曲线及抗酸染色特征无明显差异。质粒插入基因稳定率91.7%,丢失率为8.3%。冻干前后分泌IFN-α-2b水平差异无统计学意义(P>0.05)。结论:成功制备了重组hIFN-α-2bBCG冻干制剂,活菌数达到生物免疫治疗标准,其生物学特性和质粒稳定性均可耐受冷冻、低温与真空干燥过程中的不利影响。Objective： To explore the preparation process of lyophilization of recombinant hIFN-α-2b-BCG （rBCG）, and the biological characteristics of lyophilized rBCG thereof. Methods： It was used to determine the optimum process parameters that preparation by vacuum freeze-drying recombinant rBCG. The number of viable cells was compared with the plate count before and after lyophilization to calculate the survival rate. The acid-fast staining characteristics and morphology of the lyophilized rBCG were analyzed. The OD value of freeze-dried rBCG was continuously measured for drawing the growth curve. The level of IFN-α-2b expression was determined by ELISA. The plasmid stability was analysed by PCR. Results： The number of viable rBCG reached （6.51±0.33）×106 CFU/mL after lyophilization. The value was （1.02±0.11）×107 CFU/mL before freeze-drying, and the survival rate was about 65%. The viable number achieved the required standard of bio-immunotherapy. There were no significant differences in morphology, growth rate and acid-fast staining characteristics of lyophilized rBCG compared with those of un-lyophilized. The stability rate of gene inserted （hIFN-α-2b） was 91.7%, loss rate 8.3%. There was no significant difference in the level of secretion of rIFN-α-2b by rBCG after lyophilization （P 0.05）. Conclusion： Freezedried powder of hIFN-α-2b-rBCG was successfully prepared, which was without significant adverse effects on the hIFN-α-2b expression, plasmid insert and bio-characteristics.

关 键 词：干扰素α-2b卡介苗重组 遗传质粒冷冻干燥法酶联免疫吸附测定 

分 类 号：R737.14[医药卫生—肿瘤]