摘要
目的构建真核共表达载体pNKG2A-IRES-CD94。方法提取人外周血淋巴细胞总RNA,逆转录-聚合酶链反应(RT-PCR)扩增NKG2A、CD94基因,分别克隆pMD-NKG2A、pMD-CD94,对克隆基因进行DNA序列分析。pMD-CD94经XbaⅠ和SalⅠ酶切,插入相应酶切的真核表达载体pIRES(多克隆位点B),NKG2A经XhoⅠ和MluⅠ酶切,插入相应酶切的pIRES-CD94(多克隆位点A)。结果扩增的DNA片段和预期的人NKG2A和CD94大小一致;DNA序列分析显示,克隆的基因序列和GenBank数据库中人NKG2A和CD94基因序列一致。酶谱分析显示,NKG2A和CD94正向插入表达载体pIRES。结论成功构建了含人NKG2A和CD94的真核共表达质粒pNKG2A-IRES-CD94,为研究NKG2A和CD94基因的功能奠定了基础。
Objective To construct a eukaryotic co-expression plasmid, pNKG2A- IRES-CD94, which is capable of stable co-expression of NKG2A and CD94 gene. Methods The human NKG2A and CD94 gene fragments were amplified by reverse transcription polymerase chain reaction（RT-PCR） from the total RNA isolated from human peripheral blood lymphocyte and cloned into the pMD18-T vector to construct pMD-NKG2A and pMD-CD94 repectively, which were identified by sequence analysis. The plasmid pMD-CD94 was digested with Xba Ⅰ and Sal Ⅰ and inserted into eukaryotic co-expression vector pIRES to obtain recombinant human CD94 expression plasmid pIRES-CD94. Similarly, pMDNKG2A was digested with Xho Ⅰ and Mlu Ⅰ and then cloned into plasmid pIRES-CD94 to establish a recombinant human coexpression vector eventually, defined as pNKG2A-IRES-CD94. Results NKG2A and CD94 genes were cloned, the DNA sequencing analysis demonstrated that the NKG2A and CD94 genes were exactly consistent with the sequence recorded in GenBank. Restriction analysis indicated that NKG2A and CD94 genes were inserted expression vector pIRES correctly. Conclusions The NKG2A and CD94 coexpression plasmid is successfully constructed. It provides a novel expression system, which enable to further study on the functions of NKG2A and CD94 genes.
出处
《检验医学》
CAS
北大核心
2007年第2期159-162,共4页
Laboratory Medicine
基金
国家自然科学基金资助项目(30371304)
