【摘要】 AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。更多还原

【Abstract】 AFP (antifugal protein) secreted by the filamentous fungi Aspergillus giganteus MDH18894 is a strong basic protein (pI>10.65). Its mRNA has an 430 bp open reading frame encoding the 94 amino acids’ precursor of AFP. The precursor of AFP is processed to form the mature AFP of 51 amino acids by a putative two-step processing. A 285 bp AFP-like gene (i.e. AFP cDNA) lacking introns was reported to exist in the genome of Trichoderma viride, another species of filamentous fungi, but the gene was silent. In order to investigate whether the genomic AFP gene from A. giganteus could be expressed in T. viride, an expression plasmid with the open reading frame of AFP gene fused between the promoter and terminator of trpC gene was constructed and was transformed into T. viride. SDS-PAGE and Western-blot analysis showed that AFP was expressed and secreted into the culture supernatant of the transformant of T. viride. Our study provides a basis for further secretory expression of heterologous eukaryotic genes of important value in the system of T. viride.更多还原