人类GAD_(67)基因扩增及pEGFP-C2-GAD_(67)真核表达载体的构建

Amplification of human GAD_(67) gene and construction of its eukaryotic expression vector pEGFP-C2-GAD_(67)

目的扩增人类谷氨酸脱羧酶(GAD)67基因,并构建pEGFP-C2-GAD67真核表达载体。方法应用基因重组技术,采用反转录PCR方法对来自人类脑组织cDNA中编码GAD67的基因片段进行扩增,插入PUC57克隆载体,经XhoⅠ和BamHⅠ双酶切出GAD67基因片段,再克隆入真核表达载体pEGFP-C2后构建pEGFP-C2-GAD67重组载体,经PCR扩增、酶切后测序鉴定。结果人类GAD67基因扩增产物大小为1785bp,编码594个氨基酸残基。重组真核表达载体经酶切鉴定表明为正确重组子,PCR正确扩增出目的条带。与GenBank中人类GAD67基因序列比较,核苷酸和氨基酸水平的同源性分别达99.78%和99.66%。结论人类GAD67基因克隆后,可成功构建真核表达载体pEGFP-C2-GAD67,为进一步该基因转染神经干细胞移植治疗癫痫提供了实验基础。

Objective To amplify human glutamic acid decarboxylase （GAD）67 gene fragments and to construct its recombinant eukaryotic expression vector pEGFP-C2-GAD67. Methods With the technology of gene re-arrangement, the target sequence of human GAD67 gene from human brain cDNA was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The amplified fragment of GAD67 gene was inserted into cloning vector PUC57, digested with Xho Ⅰ and BamH Ⅰ, and then cloned into eukaryotic expression vector pEGFP-C2 to obtain pEGFP-C2-GAD67 plasmid. The recombinant plasmid was identified by restriction enzyme analysis and sequencing. Results The amplified human GAD67 cDNA fragment was about 1 785 bp in length and encoded 594 amino acids. Enzyme digestion and PCR analysis showed that GAD67 gene had been successfully cloned into the vectors of PUC57 and pEGFP-C2, which was confirmed by restriction enzyme. By means of sequencing, it shared 99.78% homology with the sequence of nucleotide for human GAD67 in GenBank, and got 99.66% homology at the level of amino acid. Conclusion Human GAD67 gene fragments can be successfully cloned and eukaryotic expression vector pEGFP-C2-GAD67 can be successfully constructed, which may provide an experimental basis for the application of GAD67 gene-transfected neural stem cell transplantation in the treatment of epilepsy.