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目的克隆白纹伊蚊主要过敏原AL-100 30ku蛋白基因,并构建其原核表达载体。方法RT-PCR克隆白纹伊蚊主要过敏原AL-100 30ku的全长基因,设计简并引物,扩增白蚊伊蚊30ku的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果成功克隆白纹伊蚊主要过敏原30ku基因并构建其原核表达载体。该基因含有长度为816bp的开放阅读框,编码271个氨基酸。该蛋白质的分子质量单位为28.33ku,等电点为4.04,编码序列与数据库中已知的白纹伊蚊30ku基因的同源性为99%。结论成功克隆了白蚊伊蚊主要过敏原30ku基因并构建了原核表达载体,为白纹伊蚊主要过敏原30ku蛋白的重组表达和免疫活性鉴定等奠定了基础。
Objective To clone the 30 ku protein gene of AL-100, the major allergen of Aedes albopictus, and construct its prokaryotic expression vector. Methods RT-PCR was used to clone the full-length gene of AL-100 30ku, a major allergen of Aedes albopictus. The degenerate primers were designed and a complete open reading frame of Aedes albopictus 30ku was amplified and ligated with pET-28a vector to construct prokaryotic expression vector . Results The 30ku gene, the major allergen of Aedes albopictus, was successfully cloned and the prokaryotic expression vector was constructed. The gene contains an open reading frame of 816 bp, encoding 271 amino acids. The molecular mass unit of the protein was 28.33 ku and the isoelectric point was 4.04. The homology between the coding sequence and the 30 ku gene of Aedes albopictus known in the database was 99%. Conclusion The 30ku gene of Aedes albopictus, a major allergen of Aedes albopictus, was successfully cloned and a prokaryotic expression vector was constructed. It laid the foundation for the recombinant expression of 30ku protein and identification of immunological activity of Aedes albopictus allergen.