花烟草NaD1基因的原核表达及纯化

花烟草的花中特异地存在一种植物防御素Nicotiala alata defensin 1(NaD1),对多种致病性真菌具有防御活性,在天然免疫系统中起着重要作用。该蛋白还对哺乳动物肿瘤细胞具有杀伤作用。本研究构建了原核表达载体p ET28a-NaD1,将其转化到Rosetta菌株中进行诱导表达并优化了诱导表达条件,发现在25℃下,0.5 mmol/L IPTG诱导表达过夜后,目的蛋白积累量达到最高,并主要以可溶性蛋白形式存在。利用Ni柱对NaD1重组蛋白进行亲和层析纯化,产物经Western Blot鉴定发现在16.8 kD处有特异性条带,与预期的NaD1重组蛋白大小一致。本研究结果为进一步研究NaD1蛋白的作用机制提供实验依据。

Prokaryotic Expression and Purification of NaD1 Gene from Nicotiana alata

A plant defensin,Nicotiala alata defensin 1(NaD1),is uniquely present in the flowers of Nicotiana alata,and has a defensive activity against a variety of pathogenic fungi and plays an important role in the natural defense system.The protein also has killing effect on a variety of mammalian tumor cells.In this study,the prokaryotic expression vector p ET28 a-NaD1 was constructed and transformed into Rosetta strain for expression and optimization of the expression conditions.It was found that the accumulation of target protein reached the highest after induction of 0.5 mmol/L IPTG overnig ht at 25℃.NaD1 recombinant protein is mainly present as a soluble protein.The NaD1 recombinant protein was purified by affinity chromatography on a Ni column.Western Blot analysis showed a specific band at 16.8 kD,which was consistent with the expected NaD1 recombinant protein size.The results of this study provide experimental evidence for further research on the mechanism of action of NaD1 protein.