慢病毒介导的人凝血因子Ⅷ高效真核表达系统的建立  

Establishment of a high efficient human coagulation factorⅧ eukaryotic expression system using len- tiviral vector

作　　者：宋旭光[1] 曹江[1] 曾令宇[1] 张焕新[1] 程海[1] 王缦[1] 王力[1] 陈翀[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,221002

出　　处：《中华血液学杂志》2013年第9期757-761,共5页Chinese Journal of Hematology

基　　金：基金项目：国家自然科学基金（30370606）

摘　　要：目的应用慢病毒载体系统建立高效稳定表达人凝血因子Ⅷ（FⅧ）的细胞株并评估该表达系统的生物安全性。方法构建携带人B区缺失FⅧ（BDDhFⅧ）和绿色荧光蛋白基因的慢病毒转移质粒BDDhFⅧ／pXZ9及对照pXZ9，包装并浓缩慢病毒颗粒。体外感染中国地鼠卵巢（CHO）细胞后72h取培养上清，ELISA法测定FⅧ抗原表达水平，一期促凝法测定FⅧ活性，RT-PCR检测感染后CHO细胞中FN的转录。检测载体复制能力以评估安全性。结果成功构建携带BDDhFⅧ和绿色荧光蛋白基因的慢病毒转移质粒BDDhFⅧ／pXZ9及对照质粒pXZ9，并制备出高滴度的慢病毒。该慢病毒载体可高效感染CHO细胞，感染后72h培养上清中FⅧ抗原浓度为（1724．9±283．7）mU／ml，FⅧ活性为（10．58±1．55）％，RT-PCR检测到BDDhFⅧ基因的转录。感染后的CHO细胞未检测到gag基因的表达，培养上清中也未检测到病毒。结论慢病毒可以介导人FⅧ在CHO细胞内高效表达；该表达系统不产生子代病毒，具有良好的生物安全性。Objective To establish a high efficient human coagulation factor Ⅷ（FVS） eukaryotic stable expression system using lentiviral vector, and determine its biosafety. Methods Lentiviral transfer plasmid carrying human B-domain-deleted FVS （BDDhFⅧ）-IRES-GFP （BDDhFⅧ/pXZ9） or IRES-GFP （pXZ9） was constructed. Lentivims particles were produced by transiently co-transfected 3-plasmids into 293FT cells and further concentrated via ultracentrifugation. CHO cells were infected, 72h later, the F VII antigen （FⅧ： Ag） concentration in the medium was examined by ELISA, the activity was detected via one- stage coagulantation,and the transcription of F VIII in the infected CHO cells was determined by RTPCR. Virus infection ability in the medium and the gag gene in CHO cells were determined to evaluate the modle＇ s biosafety. Results Lentiviral transfer plasmid BDDhFⅧ-IRES-GFP （BDDhFⅧ/pXZ9） carrying human B-domain-deleted FⅧ or IRES-GFP （pXZ9） was successfully constructed, and high titer lentiviruses has been prepared. The lentivirus could infect CHO cells efficiently, after an additional 72 h, the FⅧ： Ag concentration had up to （ 1724.9±283.7 ） mU/ml, the FVS ： C level increased to （ 10.58±1.55 ） %, and transcripts of BDDhFⅧ mRNA could be measured by RT-PCR. Neither the gag gene nor the virus in the supernant was detected. Conclusion Lentivirus-mediated human coagulation factor VS could be expressed efficiently in CHO cells. The system couldn＇ t produce offspring virus, proving a good biosafety.

关 键 词：慢病毒属 因子Ⅷ 真核表达 中国地鼠卵巢细胞 

分 类 号：R554.1[医药卫生—血液循环系统疾病]