CAJ | 학술논문

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72 h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FⅧ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72 h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.
목적 응용만병독재체계통건립고효은정표체인응혈인자Ⅷ(FⅧ)적세포주병평고해표체계통적생물안전성.방법 구건휴대인B구결실FⅧ(BDDhFⅧ)화록색형광단백기인적만병독전이질립BDDhFⅧ/pXZ9급대조pXZ9,포장병농축만병독과립.체외감염중국지서란소(CHO)세포후72 h취배양상청,ELISA법측정FⅧ항원표체수평,일기촉응법측정FⅧ활성,RT-PCR검측감염후CHO세포중FⅧ적전록.검측재체복제능력이평고안전성.결과 성공구건휴대BDDhFⅧ화록색형광단백기인적만병독전이질립BDDhFⅧ/pXZ9급대조질립pXZ9,병제비출출고적도적만병독.해만병독재체가고효감염CHO세포,감염후72 h배양상청중FⅧ항원농도위(1724.9±283.7)mU/ml,FⅧ활성위(10.58±1.55)％,RT-PCR검측도BDDhFⅧ기인적전록.감염후적CHO세포미검측도gag기인적표체,배양상청중야미검측도병독.결론 만병독가이개도인FⅧ재CHO세포내고효표체；해표체계통불산생자대병독,구유량호적생물안전성.
Objective To establish a high efficient human coagulation factor Ⅷ (FⅧ) eukaryotic stable expression system using lentiviral vector,and determine its biosafety.Methods Lentiviral transfer plasmid carrying human B-domain-deleted FⅧ (BDDhFⅧ)-IRES-GFP (BDDhFⅧ/pXZ9) or IRES-GFP (pXZ9) was constructed.Lentivirus 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 Ⅷ antigen (FⅧ:Ag) concentration in the medium was examined by ELISA,the activity was detected via onestage coagulantation,and the transcription of F Ⅷ in the infected CHO cells was determined by RT-PCR.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 F Ⅷ 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 FⅧ: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 supemant was detected.Conclusion Lentivirus-mediated human coagulation factor Ⅷ could be expressed efficiently in CHO cells.The system couldn' t produce offspring virus,proving a good biosafety.