大鼠GLUT3基因重组腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus vector carrying rat GLUT3 gene

目的构建携带大鼠葡萄糖转运体3(GLUT3)基因的复制缺陷型重组腺病毒载体,为研究GLUT3的生物学功能提供工具。方法采用逆转录-聚合酶链反应(RT-PCR)的方法获取大鼠GLUT3基因全长cDNA片段,将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-GLUT3,经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α,构建重组腺病毒质粒pAd-GLUT3,酶切线性化重组腺病毒质粒后转染293细胞包装成重组病毒颗粒,重组腺病毒在293细胞中反复扩增数代后,分别用聚合酶链反应(PCR)及免疫印记法(westernblot)的方法从基因和蛋白表达水平鉴定重组的腺病毒。结果经DNA测序和PCR分析显示GLUT3cDNA序列正确。成功筛选出重组腺病毒质粒pAd-GLUT3后在HEK293细胞中成功包装出重组病毒,包装后冻融细胞行PCR及westernblot检测表明重组腺病毒包装成功。结论本研究成功构建了携带大鼠GLUT3基因的复制缺陷型重组腺病毒载体。

Objective To construct the recombinant adenovirus vector carrying rat GLUT3 gene so as to further investigate the biological functions of GLUT3. Methods The full-length cDNA of rat GLUT3 gene was amplified by RT-PCR technique from rat cerebral tissue and was inserted into pShuttle vector to construct shuttle particles （pShuttle-GLUT3）. After identification with restriction enzymes, GLUT3 was ligated to linearized pAdeno-X viral DNA to form a recombinant adenoviral plasmid （pAd-GLUT3）, and subsequently transformed into DH5α, which was packaged into infectious recombinant adenoviral particles （Ad-GLUT3） by transfecting human embryonic kidney （HEK） 293 cells. After amplifying several times in HEK 293 cells, the recombinant adenovirus Ad-GLUT3 was identified at gene and protein levels with PCR and western blot methods respectively. Results DNA sequencing and PCR assays showed that the sequence of the GLUT3 cDNA was correct, and the amplified GLUT3 gene was 1 600 bp in length. The linearized recombinant adenoviral plasmid pAd-GLUT3 was acquired successfully, and then, was packed into recombinant adenovirus Ad-GLUT3 in HEK 293 cells after transfection. Conclusion The recombinant adenovirus vector carrying rat GLUT3 gene was successfully constructed.