人芳香族氨基酸脱羧酶基因的克隆及测序

Cloning and Sequencing of Human-l-aromatic-amino Acid Decarboxylase Gene

【目的】用RT PCR法获得人类神经元性芳香族氨基酸脱羧酶 (AADC)基因全序列 ,并建立优化方法。【方法】从人嗜铬细胞瘤中提取组织总RNA ,自行设计引物 ,采用两步RT PCR法扩增出长约 15 5 0bp的基因片断 ,凝胶回收后与高效克隆PCR产物的新型载体T 载体连接 ,并转化入大肠杆菌 ,通过蓝白斑反应筛选出重组体 ,质粒提取后进行重组体的酶切鉴定及基因测序。【结果】BamHⅠ和HindⅢ酶切证实了目的基因同克隆载体进行了有效的连接 ,基因测序证实了所克隆的片断为人AADC基因。【结论】本实验通过对引物 ,Taq酶的选择及反应条件的优化 ,保证了扩增的有效性和特异性。新型的克隆载体明显提高了克隆效率。基因测序证实了AADC基因扩增的成功。本研究为AADC基因治疗帕金森病等多巴胺能缺乏疾病的研究打下了基础。

To clone human neuronal aromatic l amino acid decarboxylase (AADC)gene by using RT PCR, and establish an optimal reaction condition for RT PCR. Total tissue RNA was extracted from human pheochromocytoma, primers were designed by computer and amplified with a segment of 1 550 bp by two steps RT PCR, the products were purified and then linked with the new clone vector, the recombinants were screened by blue white plaque test. After extraction of the recombinant plasmids, enzyme cutting reaction and gene sequencing were used to identified them. Enzymes cut by BamHⅠ and HindⅢ demonstrated the high efficient ligation between target gene and vector, gene sequencing confirmed the former was just the human AADC gene. [Conclusion] Optimal primers and reaction conditions, Hi Fi Taq enzyme guarantee the fidelity and specifity of the PCR. New clong vector promotes the effiency of cloning and gene sequencing demonstrated the success of human AADC gene amplification. All these make preparation for gene therapy for Parkinson's disease and other dopaminergic deficiency diseases.