HLA-A胞外区成熟肽基因的克隆及其原核表达载体的构建

根据GenBank基因库中HLA-A胞外区成熟肽基因序列设计2对引物,用RT-PCR法从健康人血液中扩增HLA-A胞外区基因,扩增产物进行T-A克隆、测序.结果表明,获得的HLA-A胞外区基因大小为819 bp,与模板序列的同源性为96%.利用基因重组技术,将HLA-A胞外区基因亚克隆入pET-21a(+) 载体中.经PCR、酶切和测序鉴定,证实所获重组表达质粒pET-21/ HLA-A中含有目的片段,且连接、构建正确,表明成功构建了重组表达质粒pET-21/ HLA-A.

Gene cloning and prokaryotic expression vector construction of HLA-A mature peptide of extracellular domain gene

Two pairs of primers were designed according to the reference extracellular domain of HLA-A α chain mature peptide genes from GenBank. The extracellular domain gene was amplified from the blood of healthy people by using RT-PCR. PCR product was cloned into the T easy vector and sequenced. The sequencing result showed that the target gene was 819 bp and the homology between the extracellular domain gene and the template reached 96%. The extracellular domain gene was subcloned into pET-21a (+) vector. The recombinant plasmid pET-21/ HLA-A was identified by PCR, DNA restriction and sequencing. The result showed that the recombinant plasmid pET-21/ HLA-A was connected and tedconstructed correctly, which paved the way of the preparation of HLA-A -Peptide Tetramer and the research on its role in antigen identification and immune response.