人神经生长因子基因真核细胞表达载体的构建与鉴定

Construction and Identification of Eukaryotic Cell Expression Vector Carrying Hu man Nerve Growth Factor Gene

目的：构建携带人神经生长因子（ｈ Ｎ Ｇ Ｆ） 基因的真核细胞表达载体，为应用 Ｎ Ｇ Ｆ 进行老年性痴呆（ Ａ Ｄ） 等疾病基因治疗打基础。方法：应用基因重组技术将ｈ Ｎ Ｇ Ｆｃ Ｄ Ｎ Ａ 克隆到逆转录病毒载体ｐ Ｌ Ｘ Ｓ Ｎ 中，用限制性内切酶酶切分析重组质粒ｐ Ｌ Ｎ Ｇ Ｆ Ｓ Ｎ 中ｈ Ｎ Ｇ Ｆ基因的插入方向，用 Ｐ Ｃ Ｒ、斑点杂交和 Ｓｏｕｔｈｅｒｎ 杂交对重组质粒ｐ Ｌ Ｎ Ｇ Ｆ Ｓ Ｎ 作进一步鉴定。结果：ｈ Ｎ Ｇ Ｆｃ Ｄ Ｎ Ａ已正确地克隆到逆转录病毒载体ｐ Ｌ Ｘ Ｓ Ｎ 中，而构建成重组逆转录病毒载体ｐ Ｌ Ｎ Ｇ Ｆ Ｓ Ｎ。结论：真核细胞表达载体ｐ Ｌ Ｎ Ｇ Ｆ Ｓ Ｎ 的成功构建，为进一步开展 Ｎ Ｇ Ｆ基因治疗 Ａ Ｄ 等中枢神经系统疾病奠定了基础。

Objective : To construct eukaryotic cell expression vector carrying human nerve growth factor complemen tary D N A(h N G Fc D N A) for genetherapy . Methods : Through genetic recombination technique ,h N G Fc D N Awas insert ed into polylinker site of retroviral vector p L X S N. The recombinant plasmid p L N G F S N was verified with restriction analy sis , P C R, Dot blot hybridization and Southern blot hybridization . Results :h N G Fc D N Awas cloned correctly into retro viral vector p L X S N. Recombinant retroviral vector p L N G F S N was constructed . Conclusion : The eukaryotic cell expres sion vector p L N G F S Nwas constructed successfully for gene therapy of Alzheimer′s and other central nervous system dis eases .