奶牛主要过敏原β-乳球蛋白基因的克隆及其原核表达载体的构建

Cloning and Sequence Analysis of the Major Allergen β-Lactoglobulin in Dairy Cow (Bos taurus)

目的克隆奶牛主要过敏原β-乳球蛋白(BLG)基因及其原核表达载体的构建。方法RT-PCR克隆奶牛主要过敏原BLG的全长基因,根据序列设计带有酶切位点的特异性引物,扩增奶牛BLG基因的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果克隆了奶牛主要过敏原BLG基因,且构建了其原核表达载体。该基因含有长度为537bp的开放阅读框,编码178个氨基酸。该蛋白质的相对分子质量为19883,等电点为5.14(Gen Bank数据库中的登录号为EU883598)。序列同源性分析发现其与数据库中已知的BLG基因同源性很高。结论成功克隆了奶牛主要过敏原BLG基因及其原核表达载体的构建,为进一步奶牛主要过敏原BLG的重组表达和免疫活性鉴定等奠定基础。

Objective Cloning and prokaryotic expression vector construction of β-1actoglobulin （BLG） gene from dairy cow （Bos taurus ）. Methods The RTPCR was applied to clone the full-length allergen genes from dairy cow and the sequences were analyzed. The specific primers were designed. The ORF of BLG of dairy cow was subcloned into the expression vector pET28a. Results BLG gene of dairy cow was cloned into expression vector pET28a. The cloned eDNA ORF sequence contained 537 bp and encoded 178 amino acids, with the molecular mass of 19 883, and pI 5.14 （GenBank database entry No. EU883600）. Sequence analysis showed that this clone shared high identities with BLG from Bos taurus. Conclusion The cow BLG gene was cloned into prokaryotic expression vector. The construct will be used for the expression of the milk allergic protein for further research.