GFP评价Non-stop变异对蛋白的影响

Study of non-stop mutation on GFP protein

目的通过构建的non-stop绿色荧光蛋白(GFP)质粒,了解non-stop变异对GFP的影响。方法使用T4连接酶将合成的含有点突变的DNA短片段插入质粒pEGFP-C1生成non-stop GFP。通过荧光显微镜及Western blot观察non-stop GFP的变化。结果 non-stop GFP蛋白量少于正常GFP蛋白量,只有正常GFP条带蛋白量的12.6%。同时,non-stop GFP蛋白分子量约大于正常GFP 8 000 Mr。结论 non-stop变异使GFP持续翻译至poly(A),同时加速了蛋白的衰减。

Objective To construct a recombinant green fluorescent protein （GFP） plasmid to observe the effect of non-stop mutation on GFP. Methods The DNA fragment containing point mutation was inserted into the plasmid pEGFP-C 1 to form a non-stop GFP. Observation of the non-stop mutated GFP was performed by fluorescence microscopy and western blot. Results The quantity of non-stop mutated GFP was less than the normal GFP, while the non-stop mutated GFP had a molecular weight about 8 000 Mr more than normal GFP. Conclusion It suggested the non-stop mutation leading to the translation of the codons up to poly （ A）, and accelerate the decay of protein.