逆转录病毒载体介导人凝血因子Ⅷ的体外高效表达  

Retroviral mediated high efficient in vitro expression of human coagulation factor Ⅷ

作　　者：郭雪梅[1] 王鸿利[1] 储海燕[1] 王学锋[1] 璩斌[1] 李志广[1] 戚正武[2] 王振义[1] 

机构地区：[1]上海第二医科大学附属瑞金医院,上海血液学研究所,200025 [2]中国科学院上海生物化学研究所

出　　处：《中华血液学杂志》2000年第9期457-459,共3页Chinese Journal of Hematology

基　　金：上海市新药发展基金资助项目!(975419001);上海血液学研究所胡应洲基金资助项目

摘　　要：目的　建立逆转录病毒载体介导的人凝血因子Ⅷ (FⅧ )体外高效表达体系。方法　将一B区缺失 (76 0aa～ 16 39aa)的人FⅧcDNA(FⅧBDcDNA)克隆至逆转录病毒载体pLNCX ,构建了重组表达载体pLNC FⅧBD。经PA317细胞包装后 ,感染多种靶细胞。分别采用一期法、ELISA法和RT PCR检测细胞培养上清中人FⅧ的凝血活性 (FⅧ∶C)和抗原含量 (FⅧ∶Ag)以及细胞中FⅧBDcDNA的转录。结果　pLNC FⅧBD在 4种靶细胞中以NIH3T3细胞的表达最高 ,在 2 4h内每毫升中 10 6细胞表达FⅧ∶C为 1.6U ,FⅧ∶Ag为 5 0 0ng。结论　逆转录病毒载体能够介导人FⅧ的体外高效表达 。Objective To develop a retroviral mediated high efficient expression system of human coagulation factor Ⅷ. Methods The retroviral vector LNC ⅧBD was generated by cloning a B domain deleted FⅧ cDNA (760aa～1639aa) into retroviral vector pLNCX. Several cell lines including NIH3T3, CHO, COS 7 and human hepatic cell line L 02 were infected with viral supernatant from the highest productive PA317 clones. The antigen and procoagulant activity of human FⅧ in the cell culture medium were measured by ELISA assay and one stage method, respectively. RT PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells. The highest expression was observed in NIH3T3, the procoagulant activity of secreted FⅧ was up to 1.6U, and the FⅧ antigen was 500ng by 10 6 cells/ml in 24 hours,respectively. Conclusion The constructed retroviral vector was able to generate high level expression of human FⅧ in some cell lines, and it might have potential utility in the gene therapy for Hemophilia A.

关 键 词：基因表达 逆转录病毒载体介导 血友病 FⅧ 

分 类 号：R394[医药卫生—医学遗传学]