论文部分内容阅读
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 ,为血友病A的基因治疗奠定了基础
Objective To establish a retroviral vector-mediated human factor Ⅷ (FⅧ) in vitro expression system. Methods Human FⅧ cDNA (FⅧBD cDNA), which was deleted in region B (76 0aa ~ 16 39aa), was cloned into retrovirus vector pLNCX to construct recombinant expression vector pLNC FⅧBD. After PA317 cells are packaged, a variety of target cells are infected. The coagulation activity (F Ⅷ: C) and antigen content (F Ⅷ: Ag) of human FⅧ and the expression of FⅧBD cDNA in the cell culture supernatants were detected by one-phase method, ELISA and RT-PCR respectively. Results The expression of pLNC FⅧBD in NIH3T3 cells was the highest among the four target cells. FⅧ: C was 1.6U for 106 cells per ml in 24 h, and the FⅧ:Ag was 500 ng. Conclusion The retroviral vector can mediate the high expression of human FⅧ in vitro and lay a foundation for the gene therapy of hemophilia A.