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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 mouse Parkin co-regulated gene (Pacrg) / GFP-p Fast Bac1 recombinant baculovirus vector and express it in Sf9 insect cells. Methods: The full-length Pacrg encoding c DNA sequence of mouse was amplified by PCR. The gene was inserted into the donor plasmid p Fast Bac1 carrying e GFP by TA cloning and ligation to obtain the recombinant vector rp FBac-Pacrg-GFP. Transformed into DH10Bac host bacteria. The recombinant baculovirus vector r Bacmid-Pacrg-e GFP obtained by screening was transfected into Sf9 insect cells by lipofectamine. The recombinant protein was analyzed by fluorescence microscopy and Western blotting. Results: Sequencing and restriction endonuclease digestion showed that the constructed PacBg / GFP-p Fast Bac1 recombinant baculovirus vector was correct. Western blotting results showed that the PACRG / e GFP fusion protein was expressed in Sf9 insect cells and carried green fluorescence. CONCLUSION: Pacrg / GFP-p Fast Bac1 recombinant baculovirus vector was successfully constructed and the recombinant protein was abundantly expressed in Sf9 insect cells, which laid the foundation for further study on the structure of PACRG protein and its regulation in spermatogenesis.