冻干BMP-2重组基因慢病毒载体的制备  

Preparation of lyophilized lentiviral vector of bone morphogenetic protein 2

作　　者：韦晓玲 陈荣敬[2] 赵君[2] 胡铮[2] 

机构地区：[1]上海市口腔病防治院牙体牙髓病科,上海200001 [2]上海交通大学医学院附属第九人民医院.口腔医学院口腔正畸科,上海200011

出　　处：《上海口腔医学》2015年第5期525-529,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81200815)~~

摘　　要：目的:构建BMP-2重组基因慢病毒载体,并制备成稳定的冻干剂型。方法:利用基因重组技术构建BMP-2基因重组慢病毒载体,并将重组基因慢病毒与不同配比的保护剂相混合后冻干。根据冻干后外观、病毒感染性滴度测定筛选合适的冻干保护剂组分,并通过病毒热稳定性测定、PCR和基因测序评价冻干品的质量。采用SPSS13.0软件包对数据进行统计学分析。结果:经PCR方法鉴定,BMP-2基因重组慢病毒载体构建成功,以10%海藻糖、1%牛血白蛋白、3%甘露醇、0.5%明胶为组分的B组保护剂对BMP-2基因重组慢病毒显示了较好的保护作用,冻干前、后病毒感染性滴度下降0.42 Lg PFU/m L,优于A、C组以及对照组,差异显著(P<0.05);B组保护剂制成的冻干慢病毒在37℃放置28 d后,仍维持良好外观,病毒滴度下降0.63 Lg PFU/m L,液体慢病毒对照组感染性滴度下降较快,1周后病毒滴度下降2.37 Lg PFU/m L,2组间差异显著(P<0.05);PCR以及基因测序结果显示,复溶后的冻干慢病毒所携带的目的基因信息无丢失或变异。结论:选用合适的冻干保护剂,可以有效保护BMP-2重组基因慢病毒载体的生物稳定性。PURPOSE： To construct recombinant lentiviral vectors of bone morphogenetic protein 2 （BMP-2） gene and prepare a stable lyophilized state. METHODS： The BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and mixed with different stabilizers in an appropriate proportion and then lyophilized. The optimum stabilizer was selected according to the appearance and virus titer after being lyophilized. The quality of lyophilized product was measured by thermal stability of the virus, PCR and gene sequencing. SPSS13.0 software package was used for statistical analysis. RESULTS： Recombinant BMP-2 lentiviral vector was successfully constructed. 10% trehalose, 1% bovine serum albumin, 3%mannitol and 0.5% gelatin in group B showed good protection on BMP-2 gene lentiviral vector. The virus titer decreased 0.42 LgPFU/mL after being lyophilized, which was better than group A, C and the control group and the difference was statistically significant （P〈0.05）; Lyophilized protective agent made from group B still maintained a good appearance at 37~C after 28 days and the virus titer decreased 0.63 LgPFU/mL. The virus titer in liquid lentiviral infection control group decreased rapidly to 2.37 LgPFU/mL 1 week later and the difference between the two groups was statistically significant （P〈0.05）; PCR and gene sequencing showed that the target gene information after redissolve had no loss or mutation. CONCLUSIONS： Appropriate selection of lyoprotectants can effectively protect the biological stability of recombinant BMP-2 gene lentiviral vector.

关 键 词：BMP-2 慢病毒载体 冷冻干燥 保护剂 

分 类 号：R450[医药卫生—治疗学]