复合T辅助细胞表位基因质粒的构建及测序

Construction and sequencing of recombinant T helper multi-epitope gene plasmid

目的制备复合T辅助细胞表位基因。方法复合T辅助细胞表位由5个T辅助细胞表位组成(结核杆菌热休克蛋白65p120、风疹蛋白E2-4p5465、人工合成T辅助细胞表位PADRE、沙眼衣原体热休克蛋白60p3548、破伤风类毒素p830843),其基因序列由219个核苷酸组成。人工合成4条69bp的DAN片段,各片段首尾之间有19bp的互补序列,应用DNA互补延伸拼接技术将4条DNA片段拼接成复合T辅助细胞表位,通过克隆、测序进行筛选。结果通过人工合成基因片段,应用DNA互补延伸拼接技术,成功制备了219个碱基的复合T辅助细胞表位基因,构建了复合T辅助细胞表位基因质粒。结论复合T辅助细胞表位基因的成功制备为研究复合T辅助细胞表位高效疫苗奠定了基础。

Objective To make Th multi-epitope gene.Methods Th multi-epitope peptide was made up of 5 Th epitopes(heat shock protein 65 of Mycobacterium tuberculosis p1-20,E_2 protein of Rubella virus p54-65,engineering epitope PADRE,heat shock protein 60 of Chlamydia trachomatis p35-48,and tetanus toxin p830-843),and its gene sequence including of 219bp.Four 69bp oligonucleotides overlapping by 19bp and a pair of primers were synthesized according to the sequence of the Th multi-epitope.The four oligonucleotides were spliced together by using splicing by overlap extension(SOE).The splicing products were cloned into PUC18 and checked for mutations by sequencing.Results The Th multi-epitope gene was made by using SOE.A recombinant plasmid was constructed.Conclusion The successful construction of the Th multi-epitope gene lays a foundation for constructing high effective Th multi-epitope vaccines.