摘要
目的 探讨不同载体结构在成肌细胞中表达hFⅨ蛋白的效果 ,优化载体结构 ,为血友病B基因治疗提供实验依据。方法 制备带有两拷贝肌酸激酶增强子 (Me2 )和内含子m2 的三种逆转录病毒载体pLMe2Ⅸm2 SN (Me2正向插入 3′LTR的增强子区域 )、pLMe2Ⅸm2 SN(Me2反向插入 )及pLⅨm2 SN ,筛选高效表达hFⅨ的PA317细胞克隆 ,稳定转染成肌细胞 ,分别对单克隆及混合克隆的上清和细胞进行ELISA、PCR等检测及鉴定 ,并与pLⅨSN载体相比较。结果 不同逆转录病毒载体转染靶细胞后hFⅨ表达水平依次为LMe2Ⅸm2 SN(正向 ) >LMe2Ⅸm2 SN(反向 ) >LⅨm2 SN >LⅨSN ;增强子方向不同的两个载体表达水平差异有显著性 ,正向插入者蛋白表达量较反向插入者高 ;靶细胞中反向插入的Me2在 5′LTR中有不同程度的缺失 ,其方向未发生变化。结论 选择组织特异性增强子及优化FⅨ微小基因结构是提高表达水平的有效手段 。
Objective To explore the effect of different vector structure on hFⅨ protein expression in murine myoblasts in order to construct more efficient expression vector for myoblast mediated gene therapy for hemophilia B. Methods Three types of retroviral vectors pLMe2Ⅸm 2SN(forward Me2:Me2 inserted into enhancer region of 3′LTR in forward orientation)、pLMe2Ⅸm 2SN(reversed Me2) and pLⅨm 2SN which contained intron m 2 and two copies of MCK enhancer (Me2)were transferred into packaging cell line PA317. The hFⅨ expression level in selected single and mixed clones of stably transfected myoblasts were determined by ELISA and PCR. Results There was statistically significant difference in hFⅨ expression levels among the four vectors tested. The expression order was LMe2Ⅸm 2SN(forward)> LMe2Ⅸm 2SN(reversed)> LⅨm 2SN> LⅨSN. The hFⅨ expression level of forward Me2 vector was higher than that of reversed one, because of one or two copies of reversed Me in the 5′LTR being deleted in the genome of myoblasts, but the orientation of Me or Me2 remained unchanged. Conclusions To select a tissue specific enhancer and optimize FⅨ minigene construction are effective methods for increasing the expression level. Forward Me2 vector can express hFⅨ more stably than the reversed one.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2001年第3期121-124,共4页
Chinese Journal of Hematology
基金
国家自然科学基金资助项目!(39370 32 1)
上海科委留学回国人员启动基金资助项目!(1997 2 4)
