人凝血因子Ⅷ cDNA在鼠体内的转染与表达

The transfection and expression of human coagulation factor Ⅷ in rats

目的逆转录病毒载体介导的人凝血因子Ⅷ(FⅧ)鼠肝细胞BRL-3A,通过门静脉途径输入后,观察其在鼠体内的表达。方法将一B区缺失(760～1639aa)的人FⅧcDNA(FⅧBDcDNA)克隆至逆转录病毒载体pLNCX2,构建重组表达载体pLNCX2-FⅧBD,感染BRL-3A细胞。将转染后的靶细胞经门静脉输注到大鼠体内,通过免疫组化法和ELISA法检测人FⅧ在大鼠体内的表达。结果在输注后第1天可检测到人FⅧ抗原,平均为7.2ng/mL大鼠血浆,可持续表达20天;输注的细胞主要分布在肝、脾,在第30天细胞的数目没有明显减少。结论 pLNCX2-FⅧBD转染BRL-3A细胞输注Buffalo大鼠体内后,获得短暂的人FⅧ的生理水平表达,可持续20天左右,为基因治疗血友病A奠定了基础。

Objective To observe the expression of retroviral-mediated high efficient expression system of human coagulation factor Ⅷ in rat,which was inputted through portal vein.Methods The retroviral vector pLNC-FⅧBD was generated by cloning a B-domain-deleted FⅧ cDNA（760～1 639 aa）into retroviral vector pLNCX2,then the BRL-3A cells were transfected.The transfected cell was inputted through portal vein in rat,then the expression of human coagulation factor Ⅷ was measured by ELISA assay and immunohistochemistry method,respectively.Results The FⅧ：Ag was 7.2 ng/mL in rat plasma at 1 day after infusion,which lasted to 20 days and there was no reduction in quantity obviously at 30 days.The target cells mainly distributed in liver and spleen.Conclusion The transfected BRL-3A cells by pLNCX2-FⅧBD are able to generate physiology level expression of human FⅧ after infusion in Buffalo rats and last about 20 days.It may have potential utility in the gene therapy for Hemophilia A.