复合通用CD4^+T辅助细胞表位基因克隆载体的构建及测序

Construction and Sequencing of Cloning Vector for Recombinant Universal CD4^+ T Helper Multi-epitope Gene

目的构建复合通用CD4+T辅助细胞表位基因克隆载体,并进行测序。方法由DNA work2.0软件设计并人工合成20条55个碱基的寡核苷酸序列,利用套叠PCR技术人工合成全基因序列,并克隆至pUC19载体,转化大肠杆菌DH5α,提取质粒,酶切鉴定并进行测序。结果经PCR扩增出645bp的目的DNA片段。酶切鉴定筛选出8个阳性克隆,经测序获得一个序列完全正确的克隆。结论已成功构建了复合通用CD4+T辅助细胞表位基因克隆载体,为研究表位疫苗和细菌多糖结合疫苗提供了新的载体表位。

Objective To construct and sequence the cloning vector for recombinant universal CD4 ^＋ T helper multi-epitope gene. bieth- ods Design and synthesize 20 oligonucleotide fragments,each at a length of 55 bp,by DNA work 2.0 software.Synthesize the full-length of gene by overlap PCR and clone into vector pUC19. Transform the recombinant plasmid to E. coli DH5a,then extract the plasmids,identify by restriction analysis and screen by sequencing.Remits A DNA fragment at length of 645 bp was amplified by PCR. Eight positive clones were screened by restriction analysis, of which one clone with completely correct sequence was obtained. Conclusion The cloning vector for recombinant universal CD4^＋ T helper multi-epitope gene was successfully constructed, which provided new carrier epitopes for the development of epitope vaccine and bacterial polysaecharide conjugate vaccine.