美洲商陆中新发现的一种抗菌蛋白基因的克隆和表达

Cloning and Expression of a New Gene Encoding an Antifungal Protein in Phytolacca americana

自美洲商陆(PhytolaccaamericanaL.)的种子中发现了一种具有38个氨基酸残基的蛋白(PaAFP),它有明显抑制立枯丝核菌(RhizoctoniasolaniKiihn)作用。依此蛋白的氨基酸序列合成引物,从该种子的mRNA中,通过逆转录PCR,获得了这一基因的具有65个氨基酸的前体蛋白cDNA序列(已在GenBank注册),将这一成熟蛋白的cDNA克隆在pGEX4T1上,并转化到大肠杆菌(E.coli),融合蛋白被大量表达。表达产物经谷胱甘肽Sepharose4B亲合层析,融合蛋白被纯化。纯化后的融合蛋白经凝血酶(thrombin)作用,将谷胱甘肽S转移酶降解。

A protein (Pa_AFP) with molecular weight about 4 kD was purified from the seeds of Phytolacca americana L., which obviously inhibits the growth of Rhizoctonia solani Kiihn in vitro . The authors isolated mRNA from the seeds of pokeberry and designed a degenerate PCR primer according to the N_terminal sequence of the purified protein. The full_length cDNA encoding Pa_AFP was cloned by RT_PCR and 5′_RACE and sequenced. The deduced amino acid sequence indicates that a preprotein with 65 amino acid residues is firstly translated and then processed to a mature protein with 38 amino acids. The DNA encoding the mature protein was subcloned into expression vector pGEX_4T1,and expressed efficiently in E.coli BL21 as a GST_Pa_AFP fusion protein. The fusion protein was purified by glutathione_Sepharose 4B affinity column chromatography. The purified fusion protein was specifically digested by thrombin and the Pa_AFP was further purified by filtration column chromatography.