重组小鼠Parkin共同调节基因在昆虫细胞系统中的表达

Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells

目的:构建小鼠Parkin co-regulated gene(Pacrg)/GFP-p Fast Bac1重组杆状病毒载体,并在Sf9昆虫细胞中表达。方法:PCR扩增小鼠全长Pacrg编码c DNA序列,通过TA克隆、连接等方法将该基因插入到携带e GFP的供体质粒p Fast Bac1中,获得重组载体rp FBac-Pacrg-GFP,然后转化到DH10Bac宿主菌中。筛选获得的重组杆状病毒载体r Bacmid-Pacrg-e GFP经脂质体转染至Sf9昆虫细胞中,荧光显微镜及Western印迹检测分析重组蛋白。结果:经测序及酶切鉴定显示构建的Pacrg/GFP-p Fast Bac1重组杆状病毒载体正确,Western印迹结果显示PACRG/e GFP融合蛋白在Sf9昆虫细胞中表达且携带绿色荧光。结论:成功构建了Pacrg/GFP-p Fast Bac1重组杆状病毒载体,且该重组蛋白在Sf9昆虫细胞中大量表达,为进一步研究PACRG蛋白的结构及其在精子发生中的调控作用奠定了基础。

Objective： To construct a GFP-fused mouse Parkin co-regulated gene （PACRG） baculovirus recombinant PACRG/ GFP-pFastBacl vector and express the fusion protein in Sf9 insect cells. Methods： Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBacl with eGFP （rpFBac-PACRG-GFP recombinant vector）. The plasmid was trans- formed into DHIOBac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the ex- pressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy. Results.. The construction of the PACRG/GFP-pFastBacl baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells. Conclusion ： A PACRG/GFP-pFastBacl recom-binant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.