大豆主要过敏原Gly m Bd 30K基因的克隆及其原核表达载体的构建

Cloning and Prokaryotic Expression Vector Construction of Gly m Bd 30K Gene from Soybean(Glycine max)

利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆Gly m Bd 30K的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体。该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸。该蛋白质的相对分子质量为42 758,等电点为5.08。序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600。克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础。

The RT- PCR was applied to clone the full- length allergen genes from soybean and the sequences were analyzed. The specific primers were designed. The ORF of Gly m Bd 30K of soybean was subcloned into the expression vector pET 28a. Results showed that the open reading frame（ORF） of cloned eDNA contained 1 140 bp and encoded 379 amino acids, and the estimated molecular mass of the encoded protein was 42 758 with pI 5.08. Sequence analysis showed that this clone shared high identities with Gly m Bd 30K from soybean. The deduced protein was therefore regarded as the major allergen of Gly m Bd 30K of soybean（ GenBank database entry No. EU883600）. Cloning and prokaryotie Expression Vector Construction of Gly m Bd 30K gene from soybean,which will be used as base for the expression and identification of allergen protein.