类橡胶蛋白AbAMP1的重组表达及其抑菌活性研究

Recombinant Expression and Antimicrobial Assay of Hevein-like Peptide AbAMP1

摘要:

将类橡胶蛋白AbAMP1基因克隆至pPIC9, 获得重组载体pPIC9-Ab, 线性化后转化Pichia pastoris SMD1163, 筛选获得阳性转化子。取表型为Mut*的转化子AS16进行诱导表达, 上清经Tricine-SDS-PAGE分析, 在约4.7 kD处有一条较强主带, 与AbAMP1的预期大小相符, 表明获得高效表达。上清经酸性非变性电泳后, 用凝胶琼脂糖弥散法测定其抑菌活性, 在含Bacillus thuringiensis琼脂糖平板AbAMP1对应处有一明显抑菌带, 说明重组表达的AbAMP1具有天然活性。上清液抑菌活性试验显示, AbAMP1对Fusarium oxysporum f. sp. cubense以及B. thuringiensis, B.

关键词:类橡胶蛋白, AbAMP1, 重组表达, 抑菌活性

Abstract:

The gene of hevein-like peptide AbAMP1 was cloned into the vector pPIC9 successfully. After linearized with restriction enzymes, the recombinant plasmid pPIC9-Ab was transformed into Pichia pastoris SMD1163 by electrotransformation. The transformants were screened on MD plate medium, and then identified using PCR method. The transformant AS16 with Mut＋ phenotype was used for inducing expression and other assays. A protein band of about 4.7 kD was detected on the gel by a tricine-SDS-PAGE analysis of induced culture supernatant, which indicates the protein content of the supernatant of culture after 72 h induction are almost the target peptide AbAMP1. A conspicuous inhibition band formed on a double-layered B. thuringiensis-containing plate where AbAMP1 gel band located after acid-native PAGE. This suggested the recombinant expressed AbAMP1 retains its natural antimicrobial activity. The supernatant of induced AS16 culture showed remarkable inhibitory effect on Fusarium oxysporum f. sp. cubense and Bacillus thuringiensis, B.

Key words:hevein-like peptide, AbAMP1, recombinant expression, antimicrobial assay