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目的构建携带GHS-R1a基因的重组腺病毒,为转基因做准备。方法采用PCR法扩增GHS-R1a基因片段,随后亚克隆至腺病毒穿梭质粒pAdTrack-CMV,形成重组质粒pAdTrack-CMV-GHS-R1a。线性化的pAdTrack-CMV-GHS-R1a和腺病毒骨架质粒pAdEasy1在感受态大肠杆菌BJ5183内同源重组。将线性化重组质粒pAdGHS-R1a转染进HEK293细胞,并用激光共聚焦显微镜、RT-PCR和Western blot技术检测绿色荧光蛋白及GHS-R1a的表达。结果重组质粒pAdGHS-R1a经Pac I酶切后得到一个大于30kb的大片段和一个4.5 kb的小片段,证明重组腺病毒质粒pAdGHS-R1a构建成功。Ad-GHS-R1a转染293细胞后24h能观察到绿色荧光蛋白的表达。RT-PCR和Westernblot均证实GHS-R1a基因能在293细胞中有效表达。结论携带GHS-R1a基因的重组腺病毒构建成功,为进一步研究GHS-R1a的功能提供了基础。
Objective To construct a recombinant adenovirus carrying GHS-R1a gene and prepare for transgene. Methods The GHS-R1a gene fragment was amplified by PCR and then subcloned into shuttle adenovirus shuttle plasmid pAdTrack-CMV to form recombinant plasmid pAdTrack-CMV-GHS-R1a. The linearized pAdTrack-CMV-GHS-R1a and adenovirus backbone plasmid pAdEasy1 were homologously recombined in competent E. coli BJ5183. The recombinant plasmid pAdGHS-R1a was transfected into HEK293 cells. The expression of green fluorescent protein (GFP) and GHS-R1a was detected by laser confocal microscopy, RT-PCR and Western blot. Results The recombinant plasmid pAdGHS-R1a was digested with Pac I to obtain a large fragment of more than 30 kb and a small fragment of 4.5 kb. The recombinant plasmid pAdGHS-R1a was successfully constructed. The expression of green fluorescent protein (GFP) was observed 24h after Ad-GHS-R1a transfection. Both RT-PCR and Western blot confirmed that GHS-R1a gene was efficiently expressed in 293 cells. Conclusion The successful construction of recombinant adenovirus carrying GHS-R1a gene provides the basis for further study on the function of GHS-R1a.