人NKp30基因克隆及其真核表达载体的构建

Cloning of Human NKp30 gene and construction of its eukaryotic expression vector

目的对人NKp30(hNKp30)进行基因克隆并在大肠埃希菌中重组表达,为进一步研究NK细胞抗肿瘤作用奠定基础。方法提取人外周静脉血单个核细胞,分离纯化外周血总RNA。用PCR扩增hNKp30片段,克隆至质粒载体pMD18-T,对克隆的DNA片段行序列分析。用限制酶XhoI、EcoRI消化pMD18-T-hNKp30重组质粒,分离hNKp30片段,插入真核表达载体pIRES2-EGFP相应限制酶位点,酶谱分析鉴定重组表达载体pIRES2-EG-FP-hNKp30,并转染COS-7细胞。结果PCR扩增DNA片段与hNKp30 cDNA大小一致。重组质粒pMD18-T-hNKp30的DNA序列分析显示,克隆DNA序列与文献报道hNKp30的cDNA序列一致。重组表达质粒pIRES2-EGFP-hNKp30转染COS-7细胞后,实现了hNKp30基因在COS-7细胞中的体外转染及瞬时表达。结论采用重组技术成功构建了hNKp30真核表达载体,为探讨NK细胞受体的特性及其信号转导机制奠定了基础。

Objective To clone the gene of human NKp30 and construct its eukaryotic expression vector in order to provide a basis for further study of anti-tumor immune response of NK cells. Methods Peripheral blood mononuelear cells （ PBMC）were extracted from a health volunteer＇s peripheral venous blood. Total RNA was isolated from PBMC. A hNKp30 DNA fragment , about 606 bp was amplified from the total RNA by PCR and cloned to plasmid pMD18-T, and the cloned DNA fragment was sequenced. The recombinant plasmid pMD18-T-hNKp30 was digested with XhoI,EeoRI, then hNKp44 fragmentwas isolated and inserted to the corresponding restriction site on eukaryotic expression vector pIRES2-EGFP. The recombinant expression plasmid was transfected into COS-7 cells. Results The length of DNA fragment amplified by PCR was consistent with that of hNKp30 cDNA. DNA sequencing of pMD18-T-hNKp30 revealed that the cloned DNA sequence was identical to that of reported hNKp30 eDNA. After the recombinant expression plasmid was transfected into COS-7 cells, hNKp30 was instantaneous expressed and transfeeted in vitro. Conclusion The eukaryotic expression vector can be construtted successfully by reconstruction technology, which may provide basis for the study of biological activity.