表达载体pEGFP—C3一PENK的构建以及在NIH3T3细胞中的表达

pEGFP-C3-PENK construction and expression in eukaryotic cells

目的：构建质粒pEGFP-C3-PENK,并观察其在真核细胞中表达的时效.方法：构建质粒pEGFP-C3-PENK;低浓度裸质粒、高浓度裸质粒、脂质体介导分别转染NIH3T3细胞,观察转染率、绿色荧光蛋白表达情况;放射免疫法和免疫细胞化学法检测脑啡肽的表达情况.结果：质粒pEGFP-C3-PENK成功构建;低浓度裸质粒转染率5%;高浓度裸质粒转染率8%,脂质体介导转染率为20%;转染5周后仍有绿色荧光蛋白表达.结论：成功构建质粒pEGFP-C3-PENK,可在NIH3T3细胞中表达超过5周.

Objective： To construct recombinant plasmid of pEGFP-C3-PENK, and identify the position and time that the plasmid was expressed. Methods： Link PENK with FL eukaryotic expression vector to construct recombinant plasmid （pEG- FP-C3-PENK）, and transfect it into NIH3T3 cells with low concentration of naked plasmid and high concentration of naked plasmid. Mediated by Liposome 2000, transfection rate and the expression of enkephalin protein were observed by cell immunochemist and radioimmunity. Results： Recombinant plasmid pEGFP-C3-PENK was constructed successfully; the transfection rate of low concentration of naked plasmid was about 5%; the transfection rate of high concentration of naked plasmid was about 8 %; transfection rate by Liposome 2000 was about 20%; and green-fluorescence protein was expressed for 5 weeks. Conclusion： Recombinant plasmid pEGFP-C3-PENK was constructed successfully, which can express in NIH3T3 cells for 5 weeks.